Pilot Study of Mepolizumab in Episodic Angioedema With Eosinophilia
Phase: PHASE2
Conditions: Eosinophilia, Angioedema
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Pilot Study of Mepolizumab in Episodic Angioedema with Eosinophilia
NIH Protocol Number: 20-I-0002
Conducted by: 
National Institute of Allergy and Infectious Diseases (NIAID)
National Institutes of Health (NIH)
Pharmaceutical Support Provided by: 
GlaxoSmithKline (GSK)
Mepolizumab Provided by:
GSK
Principal Investigator:
Paneez Khoury MD, MHSc
Laboratory of Parasitic Diseases (LPD), NIAID, NIH
Building 10, Room 6D44E
10 Center Drive
Bethesda, MD 20814
301-402-3673
khouryp@niaid.nih.gov
Version Number: 3.0
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Table of Contents
Table of Contents...................................................................................................4
Table of Figures.....................................................................................................8
List of Tables..........................................................................................................9
Statement of Compliance.....................................................................................10
List of Abbreviations.............................................................................................11
Protocol Summary ...............................................................................................12
Précis...................................................................................................................15
1.
Background Information and Scientific Rationale.........................................16
1.1
Background Information..........................................................................16
1.1.1
Description of the Study Agent.........................................................17
1.1.2
Summary of Relevant Clinical Studies.............................................17
1.2
Rationale.................................................................................................17
2.
Study Objectives...........................................................................................18
2.1
Primary Objective ...................................................................................18
2.2
Secondary Objectives.............................................................................18
2.3
Exploratory Objectives............................................................................18
3.
Study Design ................................................................................................18
3.1
Description of the Study Design .............................................................18
3.2
Study Schematic.....................................................................................20
3.3
Study Endpoints......................................................................................22
3.3.1
Primary Endpoint..............................................................................22
3.3.2
Secondary Endpoints.......................................................................22
3.3.3
Exploratory Endpoints......................................................................22
4.
Study Population...........................................................................................22
4.1
Rationale for Subject Selection...............................................................22
4.2
Recruitment Plan ....................................................................................22
4.3
Subject Inclusion Criteria........................................................................23
4.4
Subject Exclusion Criteria.......................................................................24
4.5
Justification for Exclusion of Women, Minorities, Children, and Special 
Populations.......................................................................................................25
5.
Study Agent/Interventions.............................................................................25
5.1
Disposition and Dispensation .................................................................25
5.2
Formulation, Packaging and Labeling ....................................................26
5.3
Study Agent Storage and Stability..........................................................26
5.4
Preparation, Administration, and Dosage of Study Agent ......................26
5.5
Concomitant Medications and Procedures.............................................28
5.6
Prohibited Medications and Procedures.................................................28
6.
Study Schedule.............................................................................................28
6.1
Enrollment (Visit 1) .................................................................................29
6.2
Baseline (Visit 2, Day 0) .........................................................................30
6.3
Primary Study Phase..............................................................................31
6.3.1
AEC peak (Visit 3)............................................................................31
6.3.2
AEC nadir and mepolizumab administration (Visit 4).......................31
6.3.3
Predicted ANC peak (Visit 5) ...........................................................32

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6.3.4
Mepolizumab administration (Visit 6) ...............................................33
6.3.5
Mepolizumab administration (Visit 7) ...............................................34
6.3.6
Completion of primary and secondary endpoint assessments (Visit 
8)
.........................................................................................................34
6.4
Dose De-escalation Phase .....................................................................35
6.4.1
Initiation of de-escalation (Visit 8) ....................................................35
6.4.2
Mepolizumab administration at 500 mg (Visits 9 and 10) ................35
6.4.3
Mepolizumab administration at 300 mg (Visits 11 to 13) .................36
6.4.4
End of de-escalation phase (Visit 14) ..............................................37
6.5
Unscheduled Visit...................................................................................37
6.6
Early Termination Visit............................................................................38
6.7
Pregnancy and Follow-up Visit ...............................................................38
6.8
Recontact of Subjects After Trial Termination ........................................38
7.
Study Procedures/Evaluations......................................................................38
7.1
Clinical Evaluations.................................................................................38
7.1.1
History and Physical Exam ..............................................................38
7.1.2
Photography and Infrared Thermography........................................39
7.1.3
Symptom Logs and Patient-reported Outcomes..............................39
7.1.4
Optional Bone Marrow Biopsy and Aspiration..................................39
7.1.5
Optional Skin Punch Biopsy.............................................................39
7.2
Laboratory Evaluations...........................................................................40
7.2.1
Specimen Preparation, Handling and Shipping ...............................40
8.
Potential Risks and Benefits.........................................................................40
8.1
Potential Risks........................................................................................40
8.1.1
Mepolizumab....................................................................................40
8.1.2
Oral Corticosteroids .........................................................................40
8.1.3
Optional Bone Marrow Biopsy and Aspiration..................................41
8.1.4
Optional Skin Punch Biopsy.............................................................41
8.1.5
Blood Drawing and IV Insertion........................................................41
8.1.6
Photography and Infrared Thermography........................................42
8.2
Potential Benefits....................................................................................42
9.
Research Use of Stored Human Samples, Specimens, and Data ...............42
10.
Data Sharing Plan.....................................................................................43
11.
Remuneration Plan for Subjects ...............................................................44
12.
Assessment of Safety ...............................................................................44
12.1 Toxicity Scale..........................................................................................44
12.2 Recording/Documentation ......................................................................44
12.3 Pregnancy...............................................................................................45
12.4 Type and Duration of the Follow-up of Participants after Adverse Events .
................................................................................................................45
12.5 Pausing Rules for an Individual Participant ............................................45
12.5.1
Reporting a Pause........................................................................46
12.5.2
Resumption of a Paused Participant ............................................46
12.6 Halting Rules for the Protocol.................................................................46
12.6.1
Reporting a Study Halt..................................................................47
12.6.2
Resumption of a Halted Study......................................................47

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12.7 Study Discontinuation.............................................................................47
12.8 Withdrawal Criteria for an Individual Subject..........................................47
12.8.1
Replacement of Withdrawn Participants or Participants Who 
Discontinue Study Agent ..............................................................................48
13.
Reporting Procedures ...............................................................................48
13.1 Reporting to the NIH IRB........................................................................48
13.2 Reporting to the NIAID Clinical Director .................................................48
14.
Clinical Monitoring Structure .....................................................................48
14.1 Quality Management Plan ......................................................................48
14.2 Safety Monitoring Plan............................................................................49
15.
Statistical Considerations..........................................................................49
15.1 Study Hypotheses, Endpoint and Sample Size Justification ..................49
15.2 Description of the Analyses ....................................................................50
16.
Protection of Human Subjects...................................................................51
16.1 Informed Consent Process .....................................................................51
16.2 Subject Confidentiality ............................................................................52
17.
Data Handling and Record Keeping .........................................................53
17.1 Data Capture and Management .............................................................53
17.2 Record Retention....................................................................................53
Appendix A: Scientific References.......................................................................54
Appendix B: Schedule of Procedures/Evaluations ..............................................56
Appendix C: Blood Volumes for Specimen Collection .........................................61
Appendix D: Cycle Length Determination for 3 Patients......................................64
Appendix E: Patient Symptom Log (Daily)...........................................................66
Appendix F: Patient Symptom Log (Weekly) .......................................................67
Appendix G: Quality of Life Scores and Disease Activity Scales.........................68
Appendix H: Research Studies............................................................................75
Table of Figures
Figure 1. Study Schematic...................................................................................21
List of Tables
Table 1. Schedule of Procedures/Evaluations for the Primary Study Phase.......56
Table 2. Schedule of Procedures/Evaluations for the De-escalation Phase. ......59
Table 3. Blood Volumes for Collection in the Primary Study Phase. ...................61
Table 4. Blood Volumes for Collection in the Dose De-escalation Phase. ..........62

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Statement of Compliance
The trial will be carried out in accordance with International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use 
(ICH) Good Clinical Practice (GCP) and the following: 

United States (U.S.) Code of Federal Regulations (CFR) applicable to 
clinical studies (45 CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 
CFR Part 312, and/or 21 CFR Part 812) 
NIH-funded investigators and clinical trial site staff who are responsible for the 
conduct, management, or oversight of NIH-funded clinical trials have completed 
Human Subjects Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all subject 
materials will be submitted to the Institutional Review Board (IRB) for review and 
approval. Approval of both the protocol and the consent form must be obtained 
before any subject is enrolled. Any amendment to the protocol will require review 
and approval by the IRB before the changes are implemented to the study. In 
addition, all changes to the consent form will be IRB-approved; an IRB 
determination will be made regarding whether a new consent needs to be 
obtained from subjects who provided consent, using a previously approved 
consent form.

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List of Abbreviations
AE
Adverse Event
AEC
Absolute Eosinophil Count
ANC
Absolute Neutrophil Count 
AR
Adverse Reaction
CBC
Complete Blood Count
CC
Clinical Center
CFR
Code of Federal Regulations
COVID-19
Coronavirus Disease 2019
CPK
Creatine Kinase
CRIMSON
Clinical Research Information Management System of the NIAID 
CRP
C-reactive Protein
DAES
Daily Adverse Event Score
EAE
Episodic Angioedema with Eosinophilia
ELISA
Enzyme-linked Immunosorbent Assay
FDA
Food and Drug Administration
GCP
Good Clinical Practice
GSK
GlaxoSmithKline
HES
Hypereosinophilic Syndrome
HRPP
Human Research Protections Program
ICH
International Council for Harmonisation of Technical Requirements 
for Pharmaceuticals for Human Use
Ig
Immunoglobulin
IL-5
Interleukin 5
IRB
Institutional Review Board
IV
Intravenous
LDH
Lactate Dehydrogenase
LPD
Laboratory of Parasitic Diseases
N
Number (typically refers to number of subjects/sample size)
NIAID
National Institute of Allergy and Infectious Diseases 
NIH
National Institutes of Health
OHSRP
Office of Human Subjects Research Protections
PBMC
Peripheral Blood Mononuclear Cell
RNAseq
RNA sequencing
SAE
Serious Adverse Event
SC
Subcutaneous
TBNK
T-, B-, and Natural Killer–Cell Analysis
UP
Unanticipated Problem
U.S.
United States

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Protocol Summary
Full Title:
Pilot Study of Mepolizumab in Episodic 
Angioedema with Eosinophilia
Short Title:
Mepolizumab in EAE
Sample Size:
N = 12 
Accrual Ceiling:
15
Study Population:
Adult subjects with confirmed episodic angioedema 
with eosinophilia (EAE)
Accrual Period:
2 years
Study Design:
This will be an open-label pilot study. Following 
completion of eligibility assessments, subjects with 
confirmed EAE will enter a run-in period, during which 
the periodicity of their cycle will be assessed (if not 
known) and the optimal timing of the baseline visit will 
be determined. During this time, subjects will begin 
daily and monthly patient-reported outcome 
measures; the daily measures will be continued for 
about 3 months (through the third mepolizumab 
dose), and the monthly measures will be continued 
through the end of the primary study phase. Also, 
subjects will begin visiting a local lab 2-3X/week for 
collection of blood for complete blood count (CBC) 
with differential and serum and urine to be mailed to 
NIH for research; local lab visits will continue between 
study visits through the second mepolizumab 
administration. Once cycling and periodicity is 
confirmed, the baseline visit will be scheduled at the 
predicted neutrophil peak. Baseline assessments will 
include history and physical examination, routine 
laboratory testing, and whole blood flow cytometric 
analysis. Subjects will again be evaluated and have 
bloodwork during their predicted eosinophil peak (may 
be concurrent with baseline if peak eosinophils and 
neutrophils occur <1 week apart) and at the next 
predicted eosinophil nadir; at the nadir visit, they will 
receive their first administration of mepolizumab 
(700 mg via intravenous [IV] infusion). Subjects will 
return to the NIH at the predicted neutrophil peak for 
clinical evaluation and whole blood flow cytometry. 

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After that visit, subjects will return for additional 
infusions 1 and 2 months after the first, for a total of 
3 monthly mepolizumab infusions at a dose of 
700 mg. 
All subjects will be evaluated 1 month after the third 
mepolizumab infusion at 700 mg for primary and 
secondary endpoint assessments and to assess 
clinical benefit. Subjects who receive benefit from 
mepolizumab will undergo protocol-specified 
de-escalation of drug to monthly doses of 500 mg and 
then 300 mg over the course of 6 months. In-person 
or remote study visits will continue monthly during 
de-escalation to assess symptoms and collect blood 
for clinical and exploratory research evaluations. 
An end of study visit will occur 1 month after the last 
study dose. Clinical responders may be able to 
receive mepolizumab at the completion of the study. 
Study Duration:
Start Date: January 2021 
End Date: September 2023
Subjects will be on study for approximately 6 to 
10 months 
Study Agent/ 
Intervention Description: Mepolizumab 700 mg IV given monthly for a total of 
3 doses. Subjects who undergo dose de-escalation 
will receive 3 additional doses of mepolizumab at 
500 mg (in-person by IV or remotely by subcutaneous 
[SC] injection), followed by 3 additional doses at 
300 mg by SC injection.
Primary Objective:
To determine the effect of mepolizumab on the 
number and severity of symptoms of EAE.
Secondary Objectives:
1) To determine if blocking interleukin 5 (IL-5) with 
mepolizumab will reduce eosinophilia in subjects with 
EAE.
2) To assess the durability of eosinophil reduction 
with approximately monthly mepolizumab 
administration (at Visit 8). 
Exploratory Objectives: 1) To investigate the etiology of cycling in EAE by:
exploring the role of cell lineages in EAE including 
eosinophil reduction on other cell lineages, leukocyte 

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activation markers, serum cytokines and chemokines, 
and other immunologic mediators. 
2) To investigate response to 500 mg and 300 mg of 
mepolizumab during dose de-escalation.
Primary Endpoint:
Reduction in the number and severity of clinical 
symptoms associated with EAE after 3 doses of 
mepolizumab (as compared to symptoms logged in 
the approximately monthly cycle preceding initiation of 
mepolizumab). 
Secondary Endpoints: 
1) A ≥75% reduction in peak blood eosinophil count 
following the first dose of mepolizumab (as compared 
to the peak eosinophil count in the cycle preceding 
initiation of mepolizumab therapy).   
2) Sustained reduction of eosinophilia after 
administration of mepolizumab (at Visit 8). 
Exploratory Endpoints:
1) Effect of mepolizumab on cycling of cells other than 
eosinophils (e.g., neutrophils and lymphocytes) 
following mepolizumab administration. 
2) Effect of mepolizumab on markers of eosinophil 
activation and other biomarkers of disease activity in 
EAE, including whole blood transcriptomics, levels of 
eosinophil and neutrophil granule proteins, serum 
immunoglobulin (Ig) G and IgM levels, serum cytokine 
and chemokine levels, and serum tryptase levels.
3) Reduction of symptoms and severity of clinical 
symptoms of EAE on mepolizumab at doses of 
500 mg and 300 mg SC.   

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Précis
Episodic angioedema with eosinophilia (EAE), also known as Gleich’s Syndrome, 
is a rare disorder characterized by recurrent episodes of urticaria, fever, 
angioedema, weight gain and dramatic eosinophilia that occur at 3- to 6-week 
intervals and resolve with spontaneous diuresis in the absence of therapy. 
Although the syndrome is often classified in the broad category of idiopathic 
hypereosinophilic syndrome (HES), EAE is a distinct eosinophilic syndrome that 
is remarkably homogeneous in clinical presentation. More recently, it has 
become apparent that there is multilineage cycling, involving lymphocytes and 
neutrophils in addition to eosinophils. Early studies described cyclic elevations of 
serum interleukin 5 (IL-5) preceding the rise in eosinophilia, and additional 
studies have shown cyclic elevations in other type II cytokines as well as in 
eosinophilic chemokines. Aberrant T cells with a CD3-CD4+ surface phenotype 
have also been detected in the majority of subjects with EAE. The cyclic nature 
of the disorder and the involvement of multiple cell lineages have made it difficult 
to determine the underlying cause of EAE. 
We hypothesize that IL-5–driven eosinophilia is central to the pathogenesis of 
EAE. Suppression of eosinophil cycling by blocking IL-5 would help determine 
whether eosinophils are indeed the main drivers of the symptoms of angioedema 
and urticaria and pave the way for future mechanistic studies investigating the 
etiology of this unusual disorder. The purpose of this pilot study is to evaluate the 
effect of mepolizumab, a humanized antibody to IL-5, on eosinophil cycling in 
12 subjects with EAE. Subjects with EAE will undergo screening on the National 
Institutes of Health protocol 94-I-0079 to establish the periodicity of their cycling 
(if not previously determined) and the optimal timing for the baseline visit. After 
screening, subjects will be followed closely with signs and symptoms recorded in 
a daily log and daily and monthly questionnaires, as well as complete blood 
counts and research blood collected for one cycle prior to administration of 
mepolizumab. Subjects will receive a total of 3 monthly administrations of 
mepolizumab at 700 mg, followed by drug de-escalation over 6 additional 
monthly administrations for subjects who demonstrate benefit from mepolizumab. 
All subjects will have a follow-up visit about 1 month after the last study 
administration of mepolizumab.
The primary efficacy endpoint will be reduction of symptoms and severity of 
symptoms after mepolizumab. Secondary endpoints will include reduction in 
peak eosinophils after mepolizumab, continued suppression of absolute 
eosinophil count and reduction in symptoms following monthly dosing of 
mepolizumab therapy. 

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1. Background Information and Scientific Rationale
1.1
Background Information
EAE, also known as Gleich’s Syndrome, is characterized by recurrent episodes 
of urticaria, fever, angioedema, weight gain, and dramatic eosinophilia that occur 
at 3- to 6-week intervals and resolve with spontaneous diuresis in the absence of 
therapy (1). The dramatic waxing and waning of clinical manifestations, which 
can include shifts in weight of up to 40 pounds, has a significant impact on the 
lives of patients with EAE. Although conventional HES therapies, including 
glucocorticoids, are effective in reducing symptoms, the toxicity of these 
therapies limits their long-term use in EAE.
EAE is an extremely rare disorder. Among 303 consecutively enrolled subjects 
on our natural history protocol studying eosinophilic syndromes (94-I-0079), 
12 subjects with EAE were identified. The estimated prevalence of HES is 
0.036/100,000 persons based on coding in the SEER (Surveillance, 
Epidemiology, and End Results Program) database, although this is likely 
underestimated due to the lack of specific International Classification of Diseases 
codes for HES (2). Assuming that the proportion of referrals to NIH for EAE 
reflects the proportion of such patients in the general HES population, the 
prevalence of EAE is estimated to be 0.012-0.2/100,000 persons in the United 
States. This low prevalence has precluded clinical treatment studies in EAE to 
date. 
Classified in the broad category of idiopathic HES (3), EAE is a distinct 
eosinophilic syndrome that is remarkably homogeneous in clinical presentation, 
suggesting a common etiology in affected subjects. Early studies of EAE 
demonstrated cyclic elevations of IL-5 and eosinophils with eosinophilic 
deposition and degranulation in the dermis during symptomatic episodes (1,4), 
suggesting that IL-5–driven eosinophils are the primary orchestrators of the 
angioedema in EAE. Although activated T cells and an aberrant T-cell population 
(CD3-CD4+) commonly found in the lymphocytic variant of HES are also present 
in the blood and skin of patients with EAE (5), the role of the T-cell axis in 
regulating eosinophilia and the skin manifestations is unknown. Elevated serum 
IgM levels have also been detected in the majority of subjects with EAE and are 
of unknown significance. More recent studies have demonstrated multilineage 
cycling involving eosinophils, lymphocytes, and neutrophils, as well as cyclic 
elevations in cellular activation markers and a wide variety of cytokines, 
chemokines, and other mediators (5). 
Characterization of the cycles of patients with EAE performed at the NIH have 
revealed characteristics that are reproducible among subjects. Although cycle 
lengths may vary in individual subjects, the duration of time between peak 
neutrophil counts and symptoms to the development of peak eosinophilia is 
approximately 7 days in many individuals. Likewise, the time from the peak 

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eosinophilia to the nadir of counts and cytokines is approximately 9 days (see 
Appendix D: Cycle Length Determination for 3 Patients for examples). As in other 
cyclic hematologic disorders, including cyclic neutropenia, the etiology of the 
cycling in EAE is unknown.
1.1.1
Description of the Study Agent
Mepolizumab is a high-affinity, humanized monoclonal antibody (IgG1, kappa) 
treatment that targets human IL-5, preventing binding to its cognate receptor, 
IL-5 receptor alpha, on human eosinophils and thus preventing downstream 
signaling. Effects of mepolizumab include reduction of eosinophil counts and 
improvement of eosinophil-associated symptoms. 
1.1.2
Summary of Relevant Clinical Studies
Mepolizumab was initially developed for the treatment of severe asthma. 
Although early studies failed their efficacy endpoints despite reduction in blood 
and sputum eosinophilia, more recent studies focusing on eosinophilic asthma 
(6,7) have led to the recent U.S. Food and Drug Administration (FDA)–approval 
of mepolizumab (100 mg subcutaneous injection monthly) for the treatment of 
severe eosinophilic asthma. Phase 3 placebo-controlled, double-blind studies 
have also demonstrated the efficacy of mepolizumab in preventing exacerbations 
in chronic obstructive pulmonary disease and eosinophilic granulomatosis with 
polyangiitis (8). Most relevant to the proposed study, monthly mepolizumab 
(750 mg IV) was well tolerated and effective as a steroid-sparing agent in a 
placebo-controlled, double-blind phase 2 study in 84 subjects with HES (9). An 
open-label extension confirmed the long-term efficacy in this patient population 
(10), and the FDA has approved mepolizumab administered at 300 mg 
(3 separate 100-mg SC injections) once every 4 weeks for HES. There have 
been no published cases of mepolizumab use in EAE to date.
1.2
Rationale
As described above, eosinophils and eosinophil activation have been implicated 
in the pathogenesis of EAE. Furthermore, IL-5, a key mediator in the 
development, maturation, and survival of eosinophils, can be demonstrated by 
flow cytometry intracellularly in CD3+CD4+ populations in subjects with EAE (3), 
and has been found in high levels in the serum just prior to the onset of 
eosinophilia in all EAE subjects studied at NIH, with peak IL-5 levels ranging from 
150-5447 pg/mL (median 2787 pg/mL) in 7 subjects studied. These levels are 
among the highest documented in our cohort of more than 450 subjects with 
HES. Interestingly, the peak cytokine levels occur at the peak of neutrophil 
elevation, raising the question of whether other cell populations (such as 
neutrophils) are involved in the pathogenesis of the disease.  
These findings suggest that blocking IL-5 with mepolizumab could prevent the 
dramatic elevation of eosinophils, trafficking of eosinophils to the skin and soft 

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tissues, and consequently the symptoms of angioedema, weight gain, and 
myalgias associated with EAE. The impact of a reduction of eosinophils and 
eosinophil activation on other features of EAE, including the cycling and 
activation of other cell lineages, immunoglobulins, and serum mediators, could in 
turn provide new insights into the role of eosinophils in the pathogenesis of EAE. 
2. Study Objectives
2.1
Primary Objective
To determine the effect of mepolizumab on the number and severity of symptoms 
of EAE.
2.2
Secondary Objectives 
1) To determine if blocking IL-5 with mepolizumab will reduce eosinophilia in 
subjects with EAE.
2) To assess the durability of eosinophil reduction with approximately monthly 
mepolizumab administration (at Visit 8). 
2.3
Exploratory Objectives 
1) To investigate the etiology of cycling in EAE by: exploring the role of cell 
lineages including the effect of eosinophil reduction on other cell lineages, 
leukocyte activation markers, serum cytokines and chemokines, and other 
immunologic mediators (Appendix H: Research Studies). 
2) To investigate response to 500 mg and 300 mg of mepolizumab during dose 
de-escalation. 
3. Study Design
3.1
Description of the Study Design
Subjects with proven EAE will be invited to participate in the study. The timing of 
visits for each individual subject will be determined based on the periodicity of 
cycles in that subject (section 3.2, Figure 1). Screening will be performed on NIH 
protocol 94-I-0079. Potential study subjects with EAE who have not previously 
been studied at NIH or who are on a new treatment regimen may be asked to 
undergo blood draws 2-3 times per week for several weeks on protocol 94-I-0079 
to a) confirm the diagnosis and cycling with peak absolute eosinophil count 
(AEC) >1500/mm3 and b) to determine presence of symptoms associated with 
EAE since subjects will need to have symptoms to enroll. Subjects who no longer 
cycle or who have controlled disease on prednisone or other agents may taper 
their current therapy 3 months prior to screening under the direction of their 
primary doctor to meet entry criteria before enrolling in this study. 

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If an EAE patient has eosinophilia and symptoms on current therapy and agrees 
to join the study, they will be brought in for an enrollment visit (Visit 1) for initial 
evaluation. After the enrollment visit, subjects will begin a daily symptom and 
weight log (Appendix E: Patient Symptom Log) and daily and monthly 
assessments of angioedema symptoms using validated angioedema activity and 
quality of life scales (Appendix F). Logs and assessments will be collected 
electronically using REDCap and continued as described in section 7.1.2. Also, 
between study visits, subjects will have 2-3X/week collection of blood for CBC 
with differential and serum and urine for cytokine and mediator analysis. These 
blood and urine collections will continue between study visits until Visit 6. 
Once cycling and periodicity is confirmed, the baseline visit (Visit 2) will be 
scheduled at the predicted peak absolute neutrophil count (ANC), and ideally 
while eosinophils have not yet risen. Baseline assessments will include history 
and physical examination, routine laboratory testing, and whole blood flow 
cytometric analysis. 
During their predicted AEC peak subjects will have Visit 3 to receive clinical 
evaluation and laboratory testing including whole blood flow cytometry. If the 
subject has peak eosinophils and neutrophils within a week of each other, Visits 
2 and 3 may be combined. At the time of the next predicted AEC nadir (Visit 4), 
subjects will receive mepolizumab (700 mg IV). History and physical 
examination, routine laboratory testing, and research samples will be repeated at 
this visit. At the predicted ANC peak (Visit 5), subjects will have clinical 
evaluation and laboratory testing research samples. Subjects will also return to 
the NIH for mepolizumab infusions 1 and 2 months after the first (Visits 6 and 7) 
and will undergo clinical and laboratory evaluations and receive mepolizumab. 
All subjects will be evaluated 1 month after the third mepolizumab infusion at 
700 mg for primary and secondary endpoint assessments and to assess clinical 
benefit. Subjects who receive benefit from mepolizumab (clinical improvement as 
determined by the investigator) will undergo protocol-specified de-escalation of 
drug during the Dose De-escalation Phase. Subjects will receive monthly doses 
of 500 mg (in-person by IV or remotely by SC injection) and then 300 mg SC 
over the course of 6 months. In-person or remote study visits will continue 
monthly during de-escalation to assess symptoms and collect blood for clinical 
and exploratory research evaluations. Subjects may additionally be requested to 
obtain bloodwork during a symptomatic flare in between monthly visits for safety 
reasons. Subjects will continue to complete weekly symptom logs, weekly 
angioedema questionnaires, and monthly quality of life questionnaires during 
de-escalation.
For all subjects, the end of study visit will be about 1 month after the final study 
mepolizumab administration and will include safety and research procedures and 
evaluations.

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During the coronavirus disease 2019 (COVID-19) pandemic, subjects may be 
unable to attend study visits due to travel limitations or safety concerns. In these 
circumstances, study visits may be rescheduled or modified to allow for remote 
evaluations (see section 6).
Clinical responders may be eligible to receive compassionate use mepolizumab 
at the discretion of the investigator and GSK (NCT00244686).
Justification for study dosing: This study will begin by administering 
mepolizumab at a higher dose (700 mg) and by a different delivery method (IV 
rather than SC injection) than the FDA-approved administration. The dose and 
administration method was chosen because it was the dose used in prior studies 
of HES (9) as well as in our experience treating HES patients at the NIH CC, 
which involved doses of 500-700 mg IV (11). Pharmacodynamic studies 
performed in asthma studies have not shown differences in maximal eosinophil 
reduction in peripheral blood based on dosing; however, experience with lower 
dosing of mepolizumab in both EAE patients given 100 mg for asthma as well as 
patients receiving 300 mg for eosinophilic granulomatosis with polyangiitis have 
demonstrated continued tissue and peripheral eosinophils in some cases. 
Serologic evaluations of patients with EAE have shown very high levels of IL-5 in 
the bloodstream that are higher than the levels of most HES patients studied at 
NIH, hence the chosen dose of 700 mg IV for this study. Adverse reactions (ARs) 
and side effects from 700 mg IV have not been shown to differ from those noted 
in studies using lower doses (9,11). 
After 3 IV infusions of mepolizumab at 700 mg, subjects who benefit from drug 
administration will undergo de-escalation to monthly doses of 500 mg IV or SC 
and then 300 mg SC, the latter of which is the dosage and administration method 
approved for use in patients with HES.
3.2
Study Schematic 
A general schematic for the primary and secondary assessments on the study is 
given below. Cycle duration varies between subjects, but is approximately 4-6 
weeks (examples of cycle length determination based on data from individual 
subjects are given in Appendix D: Cycle Length Determination for 3 Patients). 
Study dose de-escalation is not shown. 

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Figure 1. Study Schematic. 
The black arrows indicate study visits (V) for blood draws and clinical assessments, the purple arrows 
indicate mepolizumab infusions, and the blue bars indicate interval blood draws being performed locally. The 
estimated timing of visits is indicated in approximate days (~D). The asterisks (* and **) define the 
comparison timepoints for the secondary efficacy endpoint. Subjects will document their weight, 
temperature, and symptoms and severity every day for about 3 months after enrollment. V8 will begin the 
Dose De-escalation Phase for subjects who receive benefit from mepolizumab.
3.3
Study Endpoints
3.3.1
Primary Endpoint
Reduction in the number and severity of clinical symptoms associated with EAE 
after 3 doses of mepolizumab (as compared to symptoms logged in the 
approximately monthly cycle preceding initiation of mepolizumab). 
3.3.2
Secondary Endpoints 
1) A ≥75% reduction in peak blood eosinophil count following the first dose of 
mepolizumab (as compared to the peak eosinophil count in the cycle preceding 
initiation of mepolizumab therapy).
   
2) Sustained reduction of eosinophilia after administration of mepolizumab (at 
Visit 8). 
3.3.3
Exploratory Endpoints 
1) Effect of mepolizumab on cycling of cells other than eosinophils (e.g., 
neutrophils and lymphocytes) following mepolizumab administration.
 

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2) Effect of mepolizumab on markers of eosinophil activation and other 
biomarkers of disease activity in EAE, including whole blood transcriptomics, 
levels of eosinophil and neutrophil granule proteins, serum IgG and IgM levels, 
serum cytokine and chemokine levels, and serum tryptase levels.  
3) Reduction of symptoms and severity of clinical symptoms of EAE on 
mepolizumab at doses of 500 mg and 300 mg. 
4. Study Population
4.1
Rationale for Subject Selection 
Subjects will be selected based on a confirmed diagnosis of EAE and 
demonstration of ongoing symptoms and cycling. Potential subjects whose peak 
eosinophil count is <1500/mm3 in the 3 months prior to enrollment will not be 
eligible. 
4.2
Recruitment Plan
EAE is an extremely rare disorder. A maximum of 12 intention-to-treat subjects 
with EAE will be enrolled with a ceiling of 15 patients for subjects that drop out 
prior to receiving the first dose. Subjects who are withdrawn prior to receiving 
mepolizumab will be replaced. The anticipated recruitment period will be 2 years. 
Subjects will be recruited from those already enrolled on NIH protocol 94-I-0079 
(“Eosinophil activation and function in parasitic infections and other conditions 
with increased tissue or blood eosinophilia in humans”). The NIH is a major 
referral center for HES, with >200 patients actively followed on protocol 
94-I-0079, of which 12 are known to have EAE, and 1-2 new patients referred for 
evaluation of eosinophilia weekly. 
4.3
Subject Inclusion Criteria
A subject will be eligible for participation in the study only if all of the following 
criteria apply:
1) The subject is male or female, aged 18 years or older.
2) The subject has a documented diagnosis of EAE.
3) The subject has symptoms of EAE in the cycle prior to screening, 
including but not limited to fever, swelling, hives or rashes, weight gain, 
muscle pain, and lymphadenopathy.
4) Cycling of eosinophils is ongoing as indicated by a peak AEC ≥1500/mm3 
during at least one cycle in the prior 3 months. 
5) If taking corticosteroids ≤40 mg daily, the subject is able and willing to stay 
on a stable dose for 6 weeks prior to screening. 
6) The subject agrees to storage of study samples.

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7) The subject is able to provide informed consent.
8) Females are eligible for this study if they are:
i. of non-childbearing potential (i.e., women who have had a 
hysterectomy or tubal ligation or are postmenopausal as 
defined by no menses in 1 year); OR 
ii. of childbearing potential but willing to practice effective 
contraception or abstinence during administration of the 
study drug and for 100 days (5 terminal half-lives) after 
administration of the study drug. 
iii. Not breastfeeding.
Participation of Women: 
Pregnancy: The effects of mepolizumab on the developing human fetus are 
unknown. For this reason, women of childbearing potential must agree to use 
adequate contraception (see below for acceptable methods) prior to study entry, 
for the duration of study participation, and for >5 terminal half-lives 
(approximately 100 days) after administration of the last dose of study drug. Non-
reproductive potential is defined as post-menopausal, male partner who has 
azoospermia or is surgically sterile (at least 6 weeks before screening) and is the 
sole sexual partner, surgical sterility, or a congenital or acquired condition that 
definitely prevents conception. Further, postmenopausal is defined as at least 
12 consecutive months with no menses at age 50 or older, or a high 
follicle-stimulating hormone level in the postmenopausal range at ages 45-50 
years in subjects not using hormonal contraception or hormone replacement 
therapy.
Females with reproductive potential must either practice complete and 
uninterrupted abstinence from heterosexual activity or use 2 of the following 
methods of contraception with their partners. The 2 methods must include either 
2 barrier methods, or 1 barrier method and 1 non-barrier method, both of which 
must be consistently used:
Barrier Methods:
1) Diaphragm with spermicide
2) Cervical cap with spermicide or contraceptive sponge (for nulliparous 
subjects only)
3) Male or female condom (cannot be used together)
Non-Barrier Methods
1) An intrauterine device with a documented failure rate of <1%

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2) Hormonal contraception: pill (estrogen/progestin or progestin-only), 
patch, vaginal ring, rod implanted in the skin, or subcutaneous injection 
methods
Females of childbearing-potential must have a negative pregnancy test result 
prior to receiving mepolizumab at each on-site study visit. During the course of 
the study, if a woman becomes pregnant or suspects she is pregnant, she should 
inform the study staff and her primary care physician immediately. Subjects who 
become pregnant will be entered into the mepolizumab pregnancy registry. 
Fertility: There is no fertility data in humans. Animal studies showed no adverse 
effects of anti-IL-5 treatment on fertility.
4.4
Subject Exclusion Criteria
A subject will not be eligible to participate in the study if any of the following 
conditions are fulfilled at the time of screening:
1) Treatment with immunosuppressive or immunomodulatory agents 
including but not limited to cyclosporine, interferon-alpha, azathioprine, 
methotrexate, and cyclophosphamide within the past 3 months. 
2) Treatment with biologics including but not limited to mepolizumab, IVIG, 
anti-TNF agents, rituximab, benralizumab, alemtuzumab, reslizumab, 
dupilumab, lebrikizumab, and omalizumab within 6 months or 5 half-lives 
(whichever is longer). Subjects who received rituximab at any time in the 
past must have normal B-cell numbers to participate. 
3) Co-morbid illness, alcohol or substance abuse, or any other condition 
(e.g., HIV, active hepatitis) that, in the opinion of the investigator, places 
the subject at undue risk by participating in the study. 
4) Treatment with a daily dose of corticosteroids >40 mg.
Co-enrollment Guidelines: Co-enrollment in other trials is restricted, other than 
enrollment on observational studies or those evaluating the use of a licensed 
medication (see section 5.6). Study staff should be notified of co-enrollment as it 
may require the approval of the investigator. 
4.5
Justification for Exclusion of Women, Minorities, 
Children, and Special Populations 
Exclusion of Women:  

Pregnancy: Pregnant women are excluded from this study because the 
effects of mepolizumab on the developing human fetus are unknown (see 
section 4.3). 

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
Breastfeeding: Mepolizumab was excreted into the milk of cynomolgus 
monkeys at concentrations that were less than 0.5% of those detected in 
plasma. Because of the unknown but potential risk for adverse events (AEs) 
in nursing infants secondary to treatment of the mother with mepolizumab, 
subjects who are breastfeeding are not eligible for enrollment. 
Exclusion of Children:  
Although mepolizumab is approved for use in children 6 years and older with 
severe asthma and 12 years and older in HES, there is insufficient data in 
children with EAE to judge the potential risks. Moreover, the amount of blood 
required by this study exceeds the volume permitted for children; therefore, 
children are excluded from this study.
Exclusion of Decisionally Impaired Adults:  
Adults who lack decision-making capacity to consent are not eligible for 
enrollment in this study (section 4.3), and an enrolled subject who permanently 
loses decision-making capacity to consent during the study will be withdrawn 
(section 12.8). NIH Policy 403 Research with Subjects Lacking Capacity to 
Consent will be followed. 
5. Study Agent/Interventions 
5.1
Disposition and Dispensation 
Study agent will be distributed via the NIH Central Pharmacy according to 
standard pharmacy procedures.
5.2
Formulation, Packaging and Labeling 
Study drug will be labeled with the drug name, dose, storage conditions, the 
name and address of the manufacturer, Investigational Use Statement (“Caution: 
New Drug – Limited by Federal [USA] Law to Investigational Use”) if 
investigational study agent is sent. For commercial agent labeled for 
subcutaneous use on the package, the agent may be reconstituted for IV use. 
5.3
Study Agent Storage and Stability 
Mepolizumab will be stored at temperatures of 2°C-8°C in the pharmacy and 
protected from light. Maintenance of a temperature log will be required to ensure 
no excursions. 
5.4
Preparation, Administration, and Dosage of Study Agent
Study Agent: Mepolizumab 700 mg IV; 500 mg IV/SC; and 300 mg SC

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Description
Mepolizumab is a fully humanized monoclonal antibody (IgG1, kappa mAb) 
supplied for IV infusion as 100 mg of lyophilized powder in sterile, single-dose 
vials. Mepolizumab for SC injection is supplied as single-dose prefilled 
autoinjectors containing 100 mg/mL mepolizumab as a clear to opalescent, 
colorless to pale yellow to pale brown solution.
Dosing and Administration
All subjects will receive mepolizumab 700 mg IV x 3 doses at approximately 
one-month intervals at study Visits 4, 6, and 7. Subjects in the Dose 
De-escalation Phase will receive additional monthly doses of 500 mg IV or SC at 
Visits 8 to 10, and 300 mg SC at Visits 11 to 13.
Dose Preparation
For the IV formulation: Mepolizumab 100 mg vials will be reconstituted with 
1.2 mL sterile water for injection (according to the product labeling) and 
secondarily diluted in 100-250 mL of 0.9% normal saline (NaCl) for infusion (to a 
final concentration of 0.3-7.5 mg/mL). The reconstituted solution should be used 
as soon as possible but may be kept for up to 8 hours while refrigerated or at 
controlled room temperature (up to 25°C). Any unused solution remaining after 
8 hours should be discarded.
For the SC formulation: Mepolizumab will be provided in prefilled autoinjectors 
each containing a single 100 mg/mL dose. The autoinjectors should be stored in 
the refrigerator and allowed to sit at room temperature for 30 minutes prior to 
injection. An autoinjector should not be used if it is dropped on a hard surface or 
if the solution appears cloudy, milky, or containing particular matter.
Route of Administration 
For the IV formulation: Mepolizumab will be administered as a peripheral IV 
infusion over 30 minutes.
For the SC formulation: Mepolizumab administration will occur via an autoinjector 
and will be given by the subject or a caregiver as multiple injections of 100-mg 
each. Each injection should be given into the thigh or abdomen (avoiding the 5 
cm [2 inches] around the navel) when given by the subject or caregiver; the 
upper arm may be used if the injection is administered by a caregiver. Each 
injection site should be separated by at least 5 cm (2 inches). Injections should 
not be administered into areas of skin that are tender, bruised, red, or hard. If a 
dose is missed, it should be administered as soon as possible. Thereafter, the 
subject should resume dosing on the usual day of administration. If the next dose 
is already due, then it should be administered as planned.
Dose Adjustments/Modifications/Delays
During the Primary Study Phase administering mepolizumab at 700 mg IV, the 
dose will remain the same without modification unless held or stopped due to a 

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pause (section 12.5), halt (section 12.6), or withdrawal of the subject 
(section 12.8). During the Dose De-escalation Phase, the dose will be reduced 
as outlined in the protocol unless held or stopped due to a pause, halt, or 
withdrawal of the subject.
Duration of Therapy
A total of 3 doses of 700 mg IV will be given on this protocol over a 3-month 
duration for all subjects; subjects in the Dose De-escalation Phase will have 
6 months of additional de-escalating doses at 500 mg (3 doses) and 300 mg 
(3 doses). The total duration of therapy for participation in both the Primary Study 
Phase and the Dose De-escalation Phase will be 9 months.
Tracking of Dose
A record of the number of doses and lots used will be kept in the pharmacy for 
doses administered in-person at the NIH Clinical Center Day Hospital. For 
remote visits during the Dose De-escalation Phase, subjects will either have an 
observed administration via telehealth or will log the dose on a dose tracker. 
Limitations on Prior Therapy
Subjects who have received mepolizumab within 6 months or 5 half-lives 
(whichever is longer) will not be eligible for this protocol.
Use of Ancillary Medications/Over-the-Counter Products/Foods 
Only corticosteroids will be permitted for treatment in addition to mepolizumab. 
There are no restrictions on use of other prescribed medications, 
over-the-counter products or foods. 
Subject Access to Study Agent After End of Study Assessments
All attempts to obtain approved mepolizumab will be pursued by the subject at 
the completion of the study. 
5.5
Concomitant Medications and Procedures 
All concomitant prescription medications taken during study participation will be 
tracked in the Clinical Research Information Management System of the NIAID 
(CRIMSON). For this protocol, a prescription medication is defined as a 
medication that can be prescribed only by a properly authorized/licensed 
clinician. Medications to be reported in CRIMSON are concomitant prescription 
medications, over-the-counter medications and non-prescription medications 
taken at the time of AEs.
5.6
Prohibited Medications and Procedures 
Treatment with other biologics or immunosuppressive or cytotoxic agents will not 
be permitted unless discussed with and approved by the investigator.

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6. Study Schedule
All study assessments/procedures required for each study visit are summarized 
in Appendix B: Schedule of Procedures/Evaluations. Windows for the study 
timepoints are specified where appropriate. Blood volumes are given in Appendix 
C: Blood Volumes for Specimen Collection. Research studies are detailed in 
Appendix H: Research Studies.
If in-person study visits are limited due to the COVID-19 pandemic, study visits 
may be rescheduled or study procedures may be modified to allow for continued 
safety monitoring and data collection for study endpoints remotely. If a visit 
needs to be rescheduled as a remote visit, a member of the study team will 
contact subjects with sufficient time prior to the study timepoint to make 
arrangements. In-person visits may be replaced with or modified to include 
phone calls, virtual telehealth visits or visits with a local healthcare provider for 
assessment and sample collection. Mandatory study procedures to be performed 
at remote visits are indicated for applicable visits throughout section 6. For 
example, EAE symptom logs can be completed electronically, and blood and/or 
urine samples may be collected with a local specialist or healthcare provider and 
sent in for clinical and research analyses. However, some research blood cannot 
be shipped and would be waived based on the discretion of the principal 
investigator. Regular phone or virtual calls will occur (as per the assessment 
schedule or more frequently if needed) for safety monitoring and discussion of 
the subject’s health status until the subject can again visit the site.
6.1
Enrollment (Visit 1)
Screening of subjects for this study will occur under NIH protocol 94-I-0079 
(“Eosinophil activation and function in parasitic infections and other conditions 
with increased tissue or peripheral blood eosinophilia in humans”). 
Subjects with EAE will be brought in for enrollment (Visit 1). Visit 1 may occur 
over several days. 
After signed informed consent is obtained, the following procedures and 
laboratory tests will be performed:

Complete history and physical examination, including limb measurements

Vital signs, including weight 

Photography (with infrared thermography if available)

CBC with differential

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, lactate dehydrogenase (LDH), creatine kinase (CPK), 
C-reactive protein (CRP), and urinalysis 

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
Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Urine pregnancy test (females of childbearing potential only)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for isolation and storage of plasma and cells for 
research studies

Whole blood lymphocyte phenotyping by T-, B-, and natural killer–cell 
analysis (TBNK) and RNA sequencing (RNAseq)

Optional bone marrow biopsy and aspiration, if not available under 
protocol 94-I-0079 or another NIH protocol

Optional skin punch biopsy

AE assessment
The subjects will start a log of symptoms of EAE that they will fill out daily to track 
symptoms, weight, and temperature. They will also start daily and monthly 
patient-reported outcome measures for angioedema symptoms and quality of life 
at this time. These logs and measures will be completed electronically using 
REDCap. Subjects will discontinue daily logs and measures after the third 
mepolizumab visit (Visit 7) but will continue monthly measures through the end of 
the primary study phase (section 7.1.2).
After this visit, subjects will also begin 2-3X/week lab blood draws locally to 
assess cycles and blood counts between visits. Frequency of weekly labs may 
be adjusted to avoid exceeding NIH CC blood volume limits (2X/week for 
subjects with shorter cycles or after peak AEC is reached). Blood will be drawn 
for CBCs (performed at local lab with results sent to NIH), and urine will be 
collected and mailed to the NIH for research evaluations. These local blood and 
urine collections will continue between scheduled study visits through Visit 6. 
Estimated total number of blood draws is approximately 3X/week for 2 months 
(approximately 24 blood draws) while not at NIH.
6.2
Baseline (Visit 2, Day 0)
The baseline visit (Visit 2) will be scheduled to coincide with the estimated peak 
ANC based on data from prior cycles. If the ANC begins to rise prior to the 
scheduled baseline visit, the subject will be brought back to NIH as soon as 
feasible. For subjects whose ANC peak and AEC peak are closer together than a 
week, Visits 2 and 3 may be combined.

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The following procedures and laboratory tests will be performed at the baseline 
visit:

Complete history and physical examination, including limb measurements

Vital signs, including weight 

Photography (with infrared thermography if available)

Review of symptom/weight logs and patient-reported outcome measures 
adherence

CBC with differential 

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, CRP, and urinalysis

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for isolation and storage of plasma and cells for 
research studies

Collection of whole blood for TBNK and RNAseq

Optional bone marrow biopsy and aspiration, if not done at the enrollment 
visit or available under protocol 94-I-0079 or another NIH protocol

Optional skin punch biopsy, if not done at the enrollment visit (Visit 1)

AE assessment
6.3
Primary Study Phase
6.3.1
AEC peak (Visit 3)
Subjects will return to the clinic at the predicted AEC peak (denoted by * in 
Figure 1) as determined by their individual history of EAE cycle times. For a 
4-week EAE cycle time, Visit 3 would occur at about Day 7 (±3 days). For 
subjects whose ANC peak and AEC peak are closer together than a week, Visits 
2 and 3 may be combined.
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

Review of symptom/weight logs and patient-reported outcome measures 
adherence

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
CBC with differential 

Routine laboratory testing, including acute care, hepatic, and mineral 
panels, LDH, CPK, CRP, and urinalysis

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for storage of plasma and cells for research 
studies

Whole blood lymphocyte phenotyping (TBNK) and RNAseq

Optional skin punch biopsy, if not done at Visits 1 or 2

AE assessment
6.3.2
AEC nadir and mepolizumab administration (Visit 4)
Visit 4 will be scheduled to capture the AEC nadir. We anticipate that, for most 
subjects (assuming a 4-week cycle time), this will fall at about Day 15 (±3 days). 
The schedule may be adjusted based on results of at-home CBCs. 
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

Review of symptom/weight logs and patient-reported outcome measures 
adherence

CBC with differential

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, LDH, CPK, CRP, and urinalysis

Urine pregnancy test (females of childbearing potential only)

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for storage of plasma and cells for research 
studies

Whole blood lymphocyte phenotyping (TBNK) and RNAseq

AE assessment

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Following completion of the evaluations and pregnancy test (if applicable), 
mepolizumab 700 mg IV will be administered in the 5SW Day Hospital. The drug 
will be infused over about 30 minutes. Vitals will be checked and subjects will be 
able to leave 30 minutes after the infusion. 
6.3.3
Predicted ANC peak (Visit 5)
Visit 5 will be scheduled based on the individual subject’s cycle length to coincide 
with the predicted ANC peak. It is possible that mepolizumab administration may 
alter cycle times, which could affect the scheduling of this visit; however, the time 
between each infusion will be about 1 month. Every effort will be made to 
schedule Visit 5 in advance of Visit 6; in the event that Visit 5 occurs within 1 day 
of Visit 6, only collection of whole blood for storage will be performed for Visit 5.   
 
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

Photography (with infrared thermography if available)

Review of symptom/weight logs and patient-reported outcome measures 
adherence

CBC with differential

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, LDH, CPK, CRP, and urinalysis

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for storage of plasma and cells for research 
studies

Whole blood lymphocyte phenotyping (TBNK) and RNAseq

AE assessment
In order to capture the peak AEC post-mepolizumab (denoted by ** during blood 
draws in Figure 1), subjects will continue 2-3X/week blood draws for CBC with 
differential until Visit 6. Additionally, at the time of the predicted peak AEC, urine 
and blood will be collected for shipment to the NIH. 
6.3.4
Mepolizumab administration (Visit 6)
Visit 6 will be scheduled 28 days (±5 days) after Visit 4. 

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The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

Review of symptom/weight logs and patient-reported outcome measures 
adherence

CBC with differential

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, LDH, CPK, CRP, and urinalysis

Urine pregnancy test (females of childbearing potential only)

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Serum or plasma and urine for eosinophil granule protein and mediator 
levels

Collection of whole blood for purification and storage for research studies

AE assessment
Following completion of the evaluations and pregnancy testing (if applicable), 
mepolizumab 700 mg IV will be administered in the Day Hospital.
There will not be additional at-home blood or urine collections under this protocol 
after Visit 6. 
6.3.5
Mepolizumab administration (Visit 7)
Visit 7 will be scheduled for 28 days (±5 days) after Visit 6. The following 
procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

Review of monthly patient-reported outcome measures adherence

CBC with differential

Routine laboratory testing, including acute care panel, hepatic panel, 
mineral panel, LDH, CPK, CRP, and urinalysis

Urine pregnancy test (females of childbearing potential only)

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

AE assessment

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Following completion of the evaluation and pregnancy testing (if applicable), 
mepolizumab 700 mg IV will be administered in the Day Hospital.
6.3.6
Completion of primary and secondary endpoint assessments 
(Visit 8)
Visit 8 will again be scheduled 28 days (±7 days) after Visit 7. This visit may 
occur over several days.
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

CBC with differential 

Routine laboratory testing, including acute care, hepatic, and mineral 
panels, CRP, and urinalysis

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for isolation and storage of plasma and cells for 
research studies

Whole blood lymphocyte phenotyping (TBNK) and RNAseq

Optional bone marrow biopsy and aspiration, requested of subjects for 
whom a sample was collected/available prior to study treatment (may be 
done once at any time while on study drug)

AE assessment
After completion of the procedures described above, subjects who have received 
benefit from mepolizumab administration will begin the Dose De-escalation 
Phase (Visits 8-14, as outlined in section 6.4). 
For subjects who do not participate in the Dose De-escalation Phase, Visit 8 will 
be the end of study visit.

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6.4
Dose De-escalation Phase
6.4.1
Initiation of de-escalation (Visit 8)
For subjects who demonstrated clinical benefit from mepolizumab administration, 
Visit 8 will begin the Dose De-escalation Phase. The following additional 
procedures and laboratory tests will be performed:

Urine pregnancy test (females of childbearing potential only)

Teaching for autoinjector drug administration (if applicable)
Following completion of these procedures, mepolizumab 500 mg IV will be 
administered in the Day Hospital. The drug will be infused over about 30 minutes. 
Vitals will be checked and subjects will be able to leave 30 minutes after the 
infusion.
The subjects will start a log of symptoms of EAE that they will fill out weekly 
(rather than daily) to track symptoms and weight during de-escalation. They will 
also continue patient-reported outcome measures: weekly for angioedema 
symptoms and monthly for quality of life. These logs and measures will be 
completed electronically using REDCap (section 7.1.3). Subjects will complete 
the logs and measures through the end of the De-escalation Phase (Visit 14).
If subjects experience a symptomatic flare between visits at any time during the 
dose de-escalation phase, they may be requested to obtain additional bloodwork 
for safety reasons.
6.4.2
Mepolizumab administration at 500 mg (Visits 9 and 10)
Visits 9 and 10 will be scheduled 28 days (±7 days) from the prior visit. In-person 
visits are preferred, but these visits may be performed remotely if needed due to 
COVID-19 restrictions and difficulties with travel. In such cases, the pharmacy 
will provide autoinjectors for SC administration. Visit 10 should be performed at 
NIH if feasible. 
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements. 
If the visit is conducted remotely, a targeted history and physical 
examination will be conducted virtually.

Vital signs, including weight. The subject will measure and report their 
weight and temperature if the visit is performed remotely. 

Pregnancy test (females of childbearing potential; not performed at a 
remote visit)

Review of symptom/weight logs and patient-reported outcome measures 
adherence

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
CBC with differential 

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins (not performed at a remote 
visit)

AE assessment

Visit 10 only:
o
Routine laboratory testing, including acute care, hepatic, and 
mineral panels, LDH, CPK, CRP, and urinalysis (not performed at a 
remote visit)
o
Serum tryptase, and aldolase, serum immunoglobulins and IgE
o
Collection of whole blood for isolation and storage of plasma and 
cells for research studies (not performed at a remote visit)
o
Whole blood lymphocyte phenotyping (TBNK) (not performed at a 
remote visit) and RNAseq
Following completion of the evaluation and pregnancy testing (if applicable), 
mepolizumab 500 mg will be administered by IV infusion or by SC injection.
6.4.3
Mepolizumab administration at 300 mg (Visits 11 to 13) 
Visits 11 to 13 will be scheduled 28 days (±7 days) from the prior visit. These 
visits may be performed remotely if needed due to COVID-19 restrictions and 
difficulties with travel. In such cases, the pharmacy will provide autoinjectors for 
SC mepolizumab administration. 
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements. 
If the visit is conducted remotely, a targeted history and physical 
examination will be conducted virtually.

Vital signs, including weight. The subject will measure and report their 
weight and temperature if the visit is performed remotely. 

Review of symptom/weight logs and patient-reported outcome measures 
adherence

CBC with differential 

Pregnancy test (females of childbearing potential; not performed at a 
remote visit)

AE assessment 
Following completion of the evaluation and pregnancy test (if applicable), 
mepolizumab 300 mg SC will be administered.

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6.4.4
End of de-escalation phase (Visit 14)
The end of study visit for subjects in the Dose De-escalation Phase will be 
scheduled 28 days (±7 days) after Visit 13.  
The following procedures and laboratory tests will be performed:

Targeted history and physical examination, including limb measurements

Vital signs, including weight 

CBC with differential 

Routine laboratory testing, including acute care, hepatic, and mineral 
panels, LDH, CPK, CRP, and urinalysis

Serum tryptase, aldolase, and quantitative immunoglobulin levels 
(including IgE)

Collection of serum or plasma and urine for immunologic analyses and 
quantification of eosinophil granule proteins

Collection of whole blood for isolation and storage of plasma and cells for 
research studies

Whole blood lymphocyte phenotyping (TBNK) and RNAseq

AE assessment
After this visit, participation of subjects in the Dose De-escalation Phase will be 
complete.
6.5
Unscheduled Visit
Extra visits may be scheduled in between any of the scheduled visits due to need 
to re-evaluate the subject or conduct additional studies for clinical care of the 
subject. 
6.6
Early Termination Visit
Subjects who desire to withdraw from study and further administration will be 
brought back for an early termination visit and clinical assessment. The principal 
investigator may also elect to terminate participation of the subject from the study 
for lack of adherence to study visits (according to the withdrawal criteria in 
section 12.8), safety (pausing or halting rules as described in sections 12.5 
and 12.6), or lack of efficacy. Reasons for withdrawal and safety labs will be 
captured during this visit. 
6.7
Pregnancy and Follow-up Visit 
Subjects who become pregnant during the conduct of this study will be 
discontinued from the study. They will return to the NIH CC 4 weeks after 

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discontinuation of mepolizumab for clinical assessment, including physical 
examination and safety laboratory tests. They will be entered into the 
mepolizumab pregnancy registry and followed on 94-I-0079 for their disease and 
eosinophilic process but will be removed from this protocol and will not receive 
further doses of mepolizumab on this study. 
6.8
Recontact of Subjects After Trial Termination
Subjects will not be recontacted on this study after trial termination. Subjects will 
remain on the screening protocol 94-I-0079 on which they may be recontacted if 
there are important findings that result from this study. 
7. Study Procedures/Evaluations
7.1
Clinical Evaluations
7.1.1
History and Physical Exam
A complete medical history and physical examination will be performed as part of 
the enrollment and baseline evaluations. Subsequent clinical evaluations will 
focus on the assessment of new symptoms, signs, or untoward medical events. 
Vital signs, including blood pressure, heart rate, and body temperature, will be 
measured as part of all physical examinations, according to standard nursing 
practice. Weight will be assessed at all visits. Subjects sometimes have 
demonstrable changes in sizes of limbs (forearms, upper arms, or legs). As such, 
limb diameter measurements will be taken at all visits. More specifically, maximal 
forearm and upper arm diameter of both arms will be taken 2 inches above and 
below the cubital fossa. Similarly, for lower extremities, maximal diameter will be 
measured in both legs 3 inches above and below the popliteal fossa in all 
patients. Other measurements may be taken on an individual subject depending 
on symptoms.  
7.1.2
Photography and Infrared Thermography
Full or partial body photographic documentation of skin and musculoskeletal 
involvement will be obtained at enrollment (Visit 1), baseline (Visit 2), and Visit 5 
as well as if clinically indicated. Infrared thermography may be used to assess 
extent of skin involvement using a gradation of colors to demonstrate emission of 
heat, or thermal radiation, emitted from a surface, if available. Subjects will be 
asked to refrain from eating for 4 hours prior to thermography so pictures can be 
compared in the future. The room may also be kept cooler than usual (about 
22°C or 72°F) during infrared thermography. Images will be analyzed, 
transmitted, and stored securely to maintain confidentiality and privacy of 
subjects. 

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7.1.3
Symptom Logs and Patient-reported Outcomes
Patients will fill out a daily log capturing symptoms, weight, and temperature 
(Appendix E: Patient Symptom Log), and fill out validated patient-reported 
outcome measures for angioedema (daily) and quality of life (monthly; 
questionnaires provided as Appendix G). These logs and measures will be 
completed electronically using REDCap. Subjects will also be encouraged to 
notify the investigators of any new signs or symptoms that develop between 
study visits. The daily logs and measures will be completed through the end of 
study endpoint assessments (Visit 8). For subjects in the de-escalation phase, 
after Visit 8 until study completion, weekly angioedema and monthly quality of life 
questionnaires will be continued, and weekly symptoms logs (Appendix F) will be 
completed. 
7.1.4
Optional Bone Marrow Biopsy and Aspiration
Optionally once before starting study treatment or at the time of peak symptoms 
(Visits 1 or 2), and once while on study drug, subjects may be asked to 
participate in bone marrow biopsy and aspiration. If bone marrow was already 
collected at the NIH (e.g., under protocol 94-I-0079), the study team may instead 
request a portion of that sample for analyses under this protocol.
Bone marrow biopsy and aspiration will be performed using standard sterile 
techniques. Local anesthesia (lidocaine) will be administered. Any additional 
anesthetic (e.g., oral benzodiazepine) will be administered at the discretion of the 
investigator. Each aspirate typically provides 2-3 mL of bone marrow, and 2-10 
aspirates will be obtained. 
7.1.5
Optional Skin Punch Biopsy
Optionally once before starting study treatment (Visits 1-3) subjects may be 
asked to undergo skin punch biopsy. 
Skin punch biopsy will be performed using standard sterile techniques after 
administration of local anesthesia (lidocaine). Two to three 3-mm biopsies will be 
collected for histopathology, transcriptomics, and fibroblast cultures. Punch 
biopsies will be obtained by credentialed clinicians following standard 
procedures. One to two sutures will be placed as appropriate. 
7.2
Laboratory Evaluations
Routine laboratory evaluations, including CBC with differential, routine 
chemistries, and urinalysis, will be performed in the Department of Laboratory 
Medicine at the NIH CC or at outside laboratories. Serum, plasma, whole blood, 
and urine will be collected at various timepoints for assessment of eosinophil 
activation, cytokine/chemokine profile, and other immunologic parameters as 
described in Appendix H: Research Studies. Bone marrow and skin punch biopsy 

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samples will be stored for future research, including exploratory research of 
progenitor cells and fibroblasts, respectively.
7.2.1
Specimen Preparation, Handling and Shipping 
Research samples collected at the NIH will be either sent to Clinical Support 
Laboratory, Frederick National Laboratory for Cancer Research, Leidos 
Biomedical Research, Inc. for processing or will be transported from phlebotomy 
to Building 4/B1-28 for processing by laboratory staff after collection. No samples 
will be shipped to other sites. 
8. Potential Risks and Benefits
8.1
Potential Risks
8.1.1
Mepolizumab
The risks of mepolizumab and AE profile have been well described in several 
prior studies of mepolizumab in asthma, eosinophilic granulomatosis with 
polyangiitis, and HES. ARs with a ≥5% incidence include: headache, injection 
site reactions (pain, erythema, swelling, itching, burning), back pain, and fatigue. 
Mepolizumab 100 mg and 300 mg have been approved for SC use. The package 
insert lists the following risks of mepolizumab use: hypersensitivity reactions 
including anaphylaxis, angioedema, bronchospasm, hypotension, urticaria, and 
rash; and herpes zoster infection (12). Other noted findings in studies of 
mepolizumab include the development of anti-drug antibodies; however, 
neutralizing antibodies were only identified in 1 of 260 patients with severe asthma 
receiving mepolizumab 100 mg. Such antibodies are not considered to affect the 
course of treatment. Additionally, there are unknown risks to the fetus in the case 
of pregnancy or nursing infants (see section 4.3). 
8.1.2
Oral Corticosteroids
Prednisone and other oral corticosteroids are commonly used to treat a wide 
variety of disorders, including HES. Some subjects enrolled on this study will be 
on prednisone (or equivalent corticosteroid) for treatment (although subjects 
could be enrolled off all background therapy). Although the protocol does not 
administer corticosteroids specifically, the subjects will be required to maintain 
their current therapy throughout the duration of the protocol without further 
attempts to taper to ensure that changes in counts and cycles are not a result of 
alteration of therapy. As such, the risks of continuing on corticosteroids are listed 
here. The common side effects of corticosteroids are well described and include 
increased appetite, insomnia, fluid retention, indigestion, headache, facial 
swelling, menstrual irregularities, and visual changes. When used over extended 
periods of time, corticosteroids may cause weight gain, cataracts, myopathy, 
osteoporosis, glucose intolerance, increased risk of infections such as 
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for these side effects as per usual care. Additionally, evidence-based approaches 
for glucocorticoid bone loss prevention or treatment will be followed.
8.1.3
Optional Bone Marrow Biopsy and Aspiration
Bone marrow biopsy and aspiration is optional in this protocol. It is a common 
medical procedure associated with low risk. Risks include the potential for 
bleeding and a small possibility of infection at the aspirate site that may require 
antibiotic treatment. There is usually some pressure-like discomfort during needle 
insertion, and the procedure may result in a bruise and/or some discomfort at the 
biopsy site or surrounding tissue for 1-2 days after the anesthetic wears off. 
Standard procedures will be followed for all bone marrow biopsies to minimize 
pain and the possibility of infection.
8.1.4
Optional Skin Punch Biopsy
The risks of skin punch biopsy include local pain, bleeding, infection, and 
potential scar and keloid formation. Antibiotics and oral analgesics will be used to 
manage infection and pain, respectively. The local anesthetic may cause skin 
irritation. These risks should not be substantially different than those experienced 
with diagnostic biopsies routinely done on inflamed or infected skin. Any 
nonroutine complications resulting from the procedure will be addressed in 
consultation with the NIH CC Dermatology service.
8.1.5
Blood Drawing and IV Insertion
The potential risks of IV catheter insertion and the needle stick for blood drawing 
include pain, fainting, infection and bruising, or a small hematoma. The bruising 
may last up to 72 hours. Any hematomas will be treated with local pressure. 
Infection from the needle puncture is rare, but if this does occur, appropriate 
treatment will be given. The total blood drawn during the study is within the 
guidelines of the NIH CC (Medical Administrative Policy 95-9, Guidelines for 
Limits of Blood Drawn for Research Purposes in the Clinical Center: http://cc-
internal.cc.nih.gov/policies/PDF/M95-9.pdf) (see Appendix C: Blood Volumes for 
Specimen Collection for total blood draw volumes).
8.1.6
Photography and Infrared Thermography
Taking pictures of the face and body may be embarrassing to some people. 
Infrared thermography, in contrast, would not risk exposing the patients identity. 
These photographs may be published in medical journals, without identifying the 
subject. We will attempt to preserve the anonymity of the subject as much as 
possible while providing the information needed to support the research being 
published. Subjects may decline photographs or place any restrictions on their 
use. Subjects will be given the opportunity to discuss this with the principal or 
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8.2
Potential Benefits
Since the effect of mepolizumab in EAE is unknown, subjects may or may not 
benefit from the experimental therapy. Findings from this study will help 
researchers understand the underlying mechanisms of EAE and possibly identify 
a new therapeutic option for patients.
9. Research Use of Stored Human Samples, Specimens, 
and Data 

Intended Use: Samples, specimens, and data collected under this protocol 
may be used to study factors related to etiology of EAE. Blood samples from 
subjects may be used for genetic analysis, including RNA expression 
(transcriptomics) and analysis of target variants of interest in EAE by real-time 
polymerase chain reaction. Genetic analyses will be for research purposes 
only, and no individual genetic results will be returned to subjects. Screening 
for diseases unrelated to EAE will not be performed, and findings unrelated to 
eosinophilia and immune function will not be further investigated. Genetic 
data will be coded and stored securely as described below. Deidentified 
genetic data (specifically transcriptomic data) may be shared in public 
databases as described in section 10. The informed consent document will 
describe the possible future use of stored specimens and data.  

Storage: All of the stored study research samples are labeled by a code that 
only the investigators can link to the subject. Serum will be stored in a −80°C 
freezer, cells in liquid nitrogen, and tissue samples at room temperature or 
frozen as appropriate. Data will be kept in password-protected computers. 
Only investigators or their designees will have access to the samples and 
data. 

Tracking: Samples and data will be tracked using Biological Specimen 
Inventory software.

Disposition: In the future, other investigators (both at NIH and outside) may 
wish to use these samples and/or data for research purposes. If the planned 
research falls within the category of “human subjects research” on the part of 
the NIH researchers, NIH IRB review and approval will be obtained. This 
includes the NIH researchers sending out coded and linked samples or data 
and getting results that they can link back to their subjects.

Loss or Destruction:

Any loss or unanticipated destruction of samples (for example, due 
to freezer malfunction) or data (for example, misplacing a printout 
of data with identifiers) that meets the definition of a reportable 
event will be reported to the NIH IRB according to NIH Human 
Research Protections Program (HRPP) Policy 801. 

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
Additionally, subjects may decide at any point not to have their 
samples stored. In this case, the principal investigator will destroy 
all known remaining samples and report what was done to both the 
subject and to the IRB. This decision will not affect the subject’s 
participation in other protocols at NIH.
10.
Data Sharing Plan
Human data generated in this study will be shared for future research as follows: 

Identified data in the Biomedical Translational Research Information 
System (BTRIS) 

De-identified transcriptomic data in a public repository such as the 
National Center for Biotechnology Information Gene Expression Omnibus 
or Sequence Read Archive

De-identified data with approved outside collaborators under appropriate 
agreements

De-identified data will be shared with the public at the time of publication 
and/or public presentations
Data will be shared at the time of publication or shortly thereafter.
If this study and future research using samples and data collected in this study 
are expected to generate genetic data that triggers the NIH Genomic Data 
Sharing Policy, which applies to all NIH-funded research that generates large-
scale human (e.g., transcriptomic data), the NIH Genomic Data Sharing Policy 
(https://osp.od.nih.gov/scientific-sharing/policies/) will be followed for deposition 
of data into the appropriate database (e.g., dbGaP).
11.
Remuneration Plan for Subjects 
Subjects will not be compensated for participation in this study, but will be 
reimbursed for out-of-pocket expenses related to at-home sample collection and 
their transportation costs as is permitted by the NIAID travel policy.
If in-person visits are performed remotely due to the COVID-19 pandemic, 
subjects will be reimbursed for the costs of remote procedures or the costs will 
be paid directly from study funds (e.g., shipping samples collected remotely).
12.
Assessment of Safety
AEs and other reportable events are defined in NIH HRPP Policy 801: Reporting 
Research Events.

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12.1
Toxicity Scale
The investigator will grade the severity of each AE according to the “Common 
Terminology Criteria for Adverse Events (CTCAE)” (v 5.0), which can be found 
at: http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm 
12.2
Recording/Documentation
At each contact with the participant, information regarding AEs will be elicited by 
appropriate questioning and examinations. All events, both expected/unexpected 
and related/unrelated will be recorded on a source document. Source documents 
will include: progress notes, laboratory reports, consult notes, phone call 
summaries, survey tools, and data collection tools. Source documents will be 
reviewed in a timely manner by the research team. All reportable AEs that are 
identified will be recorded in CRIMSON. The start date, the stop date, the 
severity of each reportable event, and the principal investigator’s judgment of the 
AE’s relationship and expectedness to the study agent/intervention will also be 
recorded in CRIMSON. 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis 
rather than the individual signs and symptoms or lab abnormalities will be 
recorded as the AE.
A laboratory abnormality will not be recorded as an AE if ALL of the following 
criteria are met:

It is no more than “Grade 1” or “Mild” per the protocol-specified toxicity 
table (or investigator assessment if not listed on the table); AND

It does NOT require an intervention (e.g., discontinuation of treatment, 
dose reduction/delay, additional assessments, or treatment); AND

It is assessed by the principal investigator as NOT related to the study 
agent(s); AND

It is assessed by the principal investigator as NOT clinically significant 
(e.g., the abnormal value does NOT suggest a disease or organ toxicity).
Laboratory findings that are known to be associated with the disease under study 
(EAE) will not be recorded as AEs (e.g., elevated Ig, aldolase, leukocytes such 
as eosinophils, neutrophils, or lymphocytes, and serum tryptase).
All abnormal laboratory findings will be reviewed on a routine basis by the 
principal investigator to identify potential safety signals. An abnormal lab not 
included on the toxicity table should be assessed in a similar fashion to the 
criteria above.

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12.3
Pregnancy
Although pregnancy itself is not an AE, events occurring during pregnancy, 
delivery, or in the neonate (e.g., congenital anomaly/birth defect) may be AEs or 
serious AEs (SAEs).
In the event of pregnancy, the following steps will be taken:

Discontinue the study agent and procedures and withdraw from this study, 
but continue to follow-up for safety until delivery or termination of the 
pregnancy on 94-I-0079.

Enroll in pregnancy registry.

Report to the IRB.

Advise research participant to notify the obstetrician of study participation 
and study agent exposure.
12.4
Type and Duration of the Follow-up of Participants after 
Adverse Events
AEs that have not resolved by the end of the follow-up period will be followed 
until final outcome is known. If it is not possible to obtain a final outcome for an 
AE (e.g., the participant is lost to follow-up), the reason a final outcome could not 
be obtained will be recorded by the investigator in CRIMSON.
12.5
Pausing Rules for an Individual Participant
Pausing is the suspension of administration of study agent to a single participant 
until a decision is made whether or not to resume administration of the study 
agent.
The pausing criteria for a single participant in this study include any of the 
following:

A participant experiences an SAE that is unexpected and possibly, 
probably, or definitely related to a study agent;

A participant experiences 2 Grade 3 or greater AEs that are unexpected 
and possibly, probably, or definitely related to a study agent;

Any safety issue that the site investigator determines should pause 
administration of a study agent to a single participant.
12.5.1
Reporting a Pause
If a pausing criterion is met, a description of the AE(s) or safety issue must be 
reported by the principal investigator within 1 business day to the IRB by fax or 
email.

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12.5.2
Resumption of a Paused Participant
The principal investigator will determine whether or not it is safe to resume 
administration of the study agent to the participant. The principal investigator will 
notify the IRB of the decision on resumption of the study agent.
12.5.2.1
Discontinuation of Study Agent for an Individual Participant
A participant who does not resume study agent will continue to be followed for 
safety.
12.6
Halting Rules for the Protocol 
Halting the study requires immediate discontinuation of study agent administered 
for all participants and suspension of enrollment until a decision is made whether 
or not to continue enrollment and study agent administration.
The halting rules are:

2 or more participants experience the same or similar SAEs that are 
unexpected and possibly, probably, or definitely related to the study agent;
OR

2 or more of the same or similar AE in different participants that are grade 
3 or above and are unexpected and possibly, probably, or definitely 
related to the study agent;
OR

any safety issue that the principal investigator determines should halt the 
study.
The principal investigator will determine if the study should be halted. 
12.6.1
Reporting a Study Halt
If a halting rule is met, a description of the AE(s) or safety issue must be reported 
by the principal investigator within 1 business day to the IRB by fax or email.
12.6.2
Resumption of a Halted Study
The principal investigator will determine if it is safe to resume the study. The 
principal investigator will notify the IRB of the decision on resumption of the 
study.
12.6.2.1
Discontinuation of Study Agent
Participants who do not resume study agent will continue to be followed for 
safety.

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12.7
Study Discontinuation
The IRB, the NIAID, GSK, and other oversight bodies as applicable, as part of 
their duties to ensure that research participants are protected, may discontinue 
the study at any time. Subsequent review of serious, unexpected, and related 
AEs by the IRB may also result in suspension of enrollment and further trial 
interventions/administration of study agent.
12.8
Withdrawal Criteria for an Individual Subject
A study subject will be withdrawn for any of the following:

An individual subject’s decision. (The investigator should attempt to 
determine the reason for the subject’s decision.)

Non-compliance with study procedures (e.g., changing corticosteroid 
doses without principal investigator discretion, inability to comply with 
study visits) to the extent that it is potentially harmful to the subject or to 
the integrity of the study data. 

Any clinical AE, laboratory abnormality, intercurrent illness, other medical 
condition or situation that occurs such that continued participation in the 
study would not be in the best interest of the subject.

The investigator determines that continued participation in the study would 
not be in the best interest of the participant.

A change in the subject’s baseline condition after enrollment so that the 
subject no longer meets the following eligibility criteria:
o
Development of pregnancy.
o
Development of a new malignancy (with the exception of localized 
skin cancer [basal or squamous] that may be cured by resection).
o
Withdrawal of permission for storage of study samples.
o
Permanent loss of decision-making capacity to provide informed 
consent.
12.8.1
Replacement of Withdrawn Participants or Participants Who 
Discontinue Study Agent
Participants who withdraw or are withdrawn from the study prior to receiving 
mepolizumab will be replaced. If a participant is replaced, all the data collected 
from that participant will still be included for the safety assessment.
13.
Reporting Procedures
13.1
Reporting to the NIH IRB
Reportable events will be tracked and submitted to the NIH IRB according to 
HRPP Policy 801. 

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13.2
Reporting to the NIAID Clinical Director
The principal investigator will report unanticipated problems (UPs), major 
protocol deviations, and deaths to the NIAID clinical director according to 
institutional timelines.
14.
Clinical Monitoring Structure
To help ensure that NIH Office of Research Support and Compliance procedures 
and GCP are being carried out, a Clinical Trials Management designee within the 
Office of Clinical Research Policy and Regulatory Operations, Regulatory 
Compliance and Human Subjects Protection Program will conduct a study 
initiation visit before study enrollment begins. The purpose of this meeting is to 
review with the principal investigator and study team designees the roles and 
responsibilities concerning their commitment to adhere to the requirements of the 
protocol, especially in terms of NIH OHSRP reporting requirements for AEs, 
SAEs, and UPs. In addition, the quality management and data management plan 
for the study will be reviewed. 
14.1
Quality Management Plan 
During the study, the principal investigator and study team will be responsible for 
implementing a quality management plan. Additionally, the study team will be 
responsible for completing and submitting a summary report on the quality plan 
to the NIAID Clinical Director or designee at least annually as detailed in the 
quality management plan. A courtesy copy will also be sent to Clinical Trials 
Management. 
14.2
Safety Monitoring Plan
The data gathered during this study will be monitored by the principal investigator 
for safety and compliance with protocol-specified requirements.
15.
Statistical Considerations
15.1
Study Hypotheses, Endpoint and Sample Size 
Justification
This is a pilot study designed as proof of principle, and the small sample size is 
dictated both by our ability to recruit sufficient numbers of patients in this rare 
disease and the complexity of the study design.  
First, based on the fact that there are n=12 subjects already in the natural history 
protocol, we expect to be able to get n=5 subjects to participate in this study.
For the primary endpoint of change in maximum daily AE score (DAES), we use 
a quasi-Poisson model to estimate the average percent reduction after treatment 
compared to pre-treatment. This model accounts for within-person correlation of 

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the repeated maximum DAES by allowing a different (fixed) person effect for 
each person, and it also allows overdispersion from the Poisson model. Because 
of the small sample sizes and to be conservative, we allow for overdispersion but 
not underdispersion and use a t-distribution for inferences. We calculate power to 
show that the percent reduction is greater than 0. We simulate the maximum 
DAES values using n=5 individuals measured for 4 cycles using a Poisson model 
with random effects for each individual (to induce within-person correlation) and 
additional extra-Poisson variation for each observation. Specifically, for the ith 
individual at the jth cycle, the DAES is distributed Poisson with mean: ai*eij* fj , 
where ai is distributed gamma with mean 3 and variance 3 (this models the 
per-person average baseline maximum DAES such that 8% have values less 
than 1, 58% are less than 3, and 94% are less than 6), eij is distributed gamma 
with mean 1 and variance 0.25, and fj =1 when j=1 for the baseline cycle and fj 
=1-(r/100) for the cycles after treatment, where r is the percent reduction. We first 
simulate 10,000 data sets under the null hypothesis of 0% reduction. The 
simulated the type I error rate for the two-sided test is 4.9% (close to the nominal 
5%); however, the one-sided type I error rate is slightly inflated at 3.4% (for a 
nominal 2.5%). This slight inflation is due to the small sample size and because 
of this, we will make two-sided inferences. Next we simulate 10,000 data sets 
with 87.5% reduction and show that we have over 91% power to show a 
significant effect at the two-sided 5% level. For the analysis we will present 
two-sided 95% confidence intervals which can show larger effects than just the 
difference from 0. For example, the simulations show that with an 87.5% 
reduction we have 80% power to show that the percent reduction is at least 30% 
(i.e., 80% of the simulated 95% confidence intervals show percent reductions of 
at least 30%). 
Now consider the power of the secondary endpoint. Review of cycles occurring in 
subjects collected to date for whom we had 2 consecutive peaks in the absence 
of medication changes shows a typical variation of 4-30% from cycle to cycle. 
One subject had a change of 37% from peak eosinophils from one cycle (AEC 
12100/mm3) to the next (AEC 7600/mm3). A primary endpoint requiring reduction 
of eosinophils by 75% would be double the highest variation in any individual and 
would not be likely to occur without concurrent administration of medications 
such as glucocorticoids in these individuals based on our experience in 
managing these patients. As such, we can provide a rough estimate of the power 
of the study to show that the percent reduction in peak AEC in subjects after 
receiving mepolizumab is more than 75%. 
We model the peak AEC using a linear model on the log transformed peak AEC 
values. Let Yij be the log10(peak AEC) for subject i at cycle j, where j=1 is 
baseline and j=2 is the first cycle after baseline (and the first peak after 
treatment). The model is Yij = mi + b (j-1) + eij where mi is the random effect for 
the ith subject distributed normally with mean u and variance vm, i.e., mi ~ N(u, 
vm) and the errors are eij ~ N(0, v). We use preliminary data on 5 subjects with 2 
peaks to estimate u, vm, and v. We get u=4.2, vm=0.05 and v=0.01. We assume 

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that the effect b=log10(0.125), so that the model predicts a ratio of geometric 
means of post-treatment peak AEC over baseline peak AEC of 12.5%. This 
corresponds to an 87.5% reduction in peak AEC. We simulate from this model 
with 10,000 data sets with n=5 subjects in each data set. The simulated power to 
show at the one-sided 2.5% level that the percent reduction in peak AEC is more 
than 75% (i.e., the ratio of post-treatment to baseline is less than 25%) is 94%. 
15.2
Description of the Analyses 

Safety Analysis: All safety data will be summarized with descriptive 
statistics (number of subjects [N], mean, standard deviation, median, 
minimum, and maximum) for continuous variables, and frequency and 
percentage for discrete variables. The safety summary will include AEs, 
SAEs, and clinical laboratory data.

Efficacy Analysis (change in symptoms): For the primary endpoint we 
will use the quasi-Poisson model as described in section 15.1. As a 
sensitivity analysis, we will model the sum of the DAES during each cycle 
(instead of the maximum), and perform quasi-Poisson analysis on that. 
We may also perform analyses on specific symptoms. For binary 
symptoms (present/absent), we will use a two-sided exact McNemar’s test 
to see if there is a change in the presence of the symptom in the baseline 
cycle as compared to the first subsequent post-treatment cycle. For 
ordinal symptom data (i.e., counts of the event during a cycle), we will use 
a Wilcoxon signed rank tests. For continuous data we can use the 
methods for the percent change in peak AEC (i.e., confidence intervals 
based on the t-distribution of the transformed data). 

Efficacy Analysis (peak AEC): For the percent change in peak AEC, we 
will calculate tests and confidence intervals based on a one-sample t-test 
on the transformed responses. Specifically, we will transform the percent 
change in peak AEC to the log of the ratio of post-treatment peak AEC 
over the baseline peak AEC. Notationally, let xi be the percent change in 
peak AEC for subject i, then the log of the ratio of post-treatment peak 
AEC over baseline peak AEC is Wi = log(1-[xi/100]). Then we will calculate 
the mean of the Wi and the 95% confidence interval about that mean, and 
back-transform the results to the percent change in peak AEC. If the 
resulting confidence interval is completely above 75% reduction, then this 
shows that, at the 2.5% one-sided level, the percent reduction is greater 
than 75%. 
16.
Protection of Human Subjects
16.1
Informed Consent Process
Informed consent is a process where information is presented to enable persons 
to voluntarily decide whether or not to participate as a research subject. It is an 

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ongoing conversation between the human research subject and the researchers 
which begins before consent is given and continues until the end of the subject's 
involvement in the research. Discussions about the research will provide 
essential information about the study and include: purpose, duration, 
experimental procedures, alternatives, risks and benefits. Subjects will be given 
the opportunity to ask questions and have them answered. 
The subjects will sign the informed consent document prior to undergoing any 
research procedures. The subjects may withdraw consent at any time throughout 
the course of the trial. A copy of the informed consent document will be given to 
the subjects for their records. The researcher will document the signing of the 
consent form in the subject’s medical record. The rights and welfare of the 
subjects will be protected by emphasizing to them that the quality of their medical 
care will not be adversely affected if they decline to participate in this study. 
16.2
Subject Confidentiality
All records will be kept confidential to the extent provided by federal, state and 
local law. The study monitors and other authorized individuals may inspect all 
documents and records required to be maintained by the Investigator, including 
but not limited to, medical records. Records will be kept locked and data will be 
coded. Any personally identifiable information maintained for this study will be 
kept on restricted-access computers and networks. Personally identifiable 
information will only be shared with individuals authorized to receive it under this 
protocol. Individuals not authorized to receive personally identifiable information 
will be provided with coded information only, as needed. Clinical information will 
not be released without written permission of the subject, except as necessary 
for monitoring by the IRB, the NIAID, the Office of Human Research Protections, 
or GSK.
To further protect the privacy of study subjects, a Certificate of Confidentiality has 
been issued by the NIH. This certificate protects identifiable research information 
from forced disclosure. It allows the investigator and others who have access to 
research records to refuse to disclose identifying information on research 
participation in any civil, criminal, administrative, legislative, or other proceeding, 
whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify 
research subjects, Certificates of Confidentiality help achieve the research 
objectives and promote participation in studies by helping assure confidentiality 
and privacy to subjects.

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17.
Data Handling and Record Keeping
17.1
Data Capture and Management 
Study data will be maintained in CRIMSON, and collected directly from subjects 
during study visits and telephone or virtual contacts, or will be abstracted from 
subjects’ medical records. Source documents include all recordings of 
observations or notations of clinical activities and all reports and records 
necessary to confirm the data abstracted for this study. Data entry into 
CRIMSON will be performed by authorized individuals. The Investigator is 
responsible for assuring that the data collected are complete, accurate, and 
recorded in a timely manner.
17.2
Record Retention
Study records will be maintained by the principal investigator in accordance with 
regulatory and institutional requirements, ICH GCP guidelines, and the NIH 
Intramural Records Retention Schedule. All stored records will be kept 
confidential to the extent required by federal, state, and local law. No records will 
be destroyed without the written consent of the principal investigator.

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Appendix A: Scientific References
1) Gleich GJ, Schroeter AL, Marcoux JP, Sachs MI, O'Connell EJ, Kohler PF. 
Episodic angioedema associated with eosinophilia. N Engl J Med. 1984 Jun 
21;310(25):1621-1626.
2) Crane MM, Chang CM, Kobayashi MG, Weller PF. Incidence of 
myeloproliferative hypereosinophilic syndrome in the United States and an 
estimate of all hypereosinophilic syndrome incidence. J Allergy Clin Immunol. 
2010 Jul;126(1):179-181.
3) Klion AD, Bochner BS, Gleich GJ, Nutman TB, Rothenberg ME, Simon HU, 
Wechsler ME, Weller PF; The Hypereosinophilic Syndromes Working Group. 
Approaches to the treatment of hypereosinophilic syndromes: a workshop 
summary report. J Allergy Clin Immunol. 2006 Jun;117(6):1292-1302. 
4) Butterfield JH, Leiferman KM, Abrams J, Silver JE, Bower J, Gonchoroff N, 
Gleich GJ. Elevated serum levels of interleukin-5 in patients with the syndrome of 
episodic angioedema and eosinophilia. Blood. 1992 Feb 1;79(3):688-692.
5) Khoury P, Herold J, Alpaugh A, Dinerman E, Holland-Thomas N, Stoddard J, 
Gurprasad S, Maric I, Simakova O, Schwartz LB, Fong J, Lee CC, Xi L, Wang Z, 
Raffeld M, Klion AD. Episodic angioedema with eosinophilia (Gleich’s syndrome) 
is a multilineage cell cycling disorder. Haematologica. 2015 Mar;100(3):300-307.
6) Haldar P, Brightling CE, Hargadon B, Gupta S, Monteiro W, Sousa A, Marshall 
RP, Bradding P, Green RH, Wardlaw AJ, Pavord ID. Mepolizumab and 
exacerbations of refractory eosinophilic asthma. N Engl J Med. 2009 Mar 
5;360(10):973-984.
7) Pavord ID, Korn S, Howarth P, Bleecker ER, Buhl R, Keene ON, Ortega H, 
Chanez P. Mepolizumab for severe eosinophilic asthma (DREAM): a multicentre, 
double-blind, placebo-controlled trial. Lancet. 2012 Aug 18;380(9842):651-659.
8) Wechsler ME, Akuthota P, Jayne D, Khoury P, Klion A, Langford CA, Merkel 
PA, Moosig F, Specks U, Cid MC, Luqmani R, Brown J, Mallett S, Philipson R, 
Yancey SW, Steinfeld J, Weller PF, Gleich GJ; EGPA Mepolizumab Study Team. 
Mepolizumab or Placebo for Eosinophilic Granulomatosis with Polyangiitis. N 
Engl J Med. 2017 May 18;376(20);1921-1932.
9) Rothenberg ME, Klion AD, Roufosse FE, Kahn JE, Weller PF, Simon HU, 
Schwartz LB, Rosenwasser LJ, Ring J, Griffin EF, Haig AE, Frewer PI, Parkin 
JM, Gleich GJ; Mepolizumab HES Study Group. Treatment of patients with the 
hypereosinophilic syndrome with mepolizumab. N Engl J Med. 2008 Mar 
20;358(12):1215-1228.

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10) Roufosse F, Kahn JE, Gleich GJ, Schwartz LB, Singh AD, Rosenwasser LJ, 
Denburg JA, Ring J, Rothenberg ME, Sheikh J, Haig AE, Mallett SA, Templeton 
DN, Ortega HG, Klion AD. Long-term safety of mepolizumab for the treatment of 
hypereosinophilic syndromes. J Allergy Clin Immunol. 2013 Feb;131(2):461-
467.e1-5.
11) Kuang FL, Fay MP, Ware J, Wetzler L, Holland-Thomas N, Brown T, Ortega 
H, Steinfeld J, Khoury P, Klion AD. Long-term clinical outcomes of high-dose 
mepolizumab treatment for hypereosinophilic syndrome. J Allergy Clin Immunol 
Pract. 2018 Sep-Oct;6(5):1518-1527.e5.
12) NUCALA (Mepolizumab) [package insert]. Philadelphia: GlaxoSmithKline; 
2020.

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Appendix B: Schedule of Procedures/Evaluations 
Table 1. Schedule of Procedures/Evaluations for the Primary Study Phase.
Evaluations ‖
Enrollment
Home 
Labs§
Baseline
(Peak 
ANC)
Home 
Labs§
Peak 
AEC
Home 
Labs§
Study 
Drug 
700mg/ 
AEC 
Nadir
Home 
Labs§
Predict
ed 
ANC
Peak
Home 
Labs§
Study 
Drug  
700mg
Study 
Drug 
700mg 
Primary 
Assess-
ments**
U
V
Visit Number
1
2
3
4
5
6
7
8
Day (assuming 4-week 
cycle)¶
Day -30 to 
-15
Day 0
Day 7
Day 15
Will 
vary
Day 43
Day 71
Day 99
Window (days)
±3
±3
-
±5
±5
±7
Consent and clinical assessments
Informed consent
X
Complete medical history 
and physical examination
X
X
Targeted medical history and 
physical examination
X
X
X
X
X
X
X
Limb measurements 
X
X
X
X
X
X
X
X
X
Vital signs including weight
X
X
X
X
X
X
X
X
X
Photography with/without 
infrared thermography
X
X
X
Symptom log and PROs#
X
X
X
X
X
X
X
X
X
Optional bone marrow biopsy 
and aspiration
X†
X†
X‡
Optional skin punch biopsy
X†
X†
X†

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Study drug administration
Mepolizumab 
X
X
X
Clinical and safety laboratory testing
CBC with differential
X
X
X
X
X
X
X
X
X
X
X
X
X
Acute care panel
X
X
X
X
X
X
X
X
Hepatic panel 
X
X
X
X
X
X
X
X
Mineral panel 
X
X
X
X
X
X
X
X
LDH, CPK
X
X
X
X
X
X
C-reactive protein
X
X
X
X
X
X
X
X
Urinalysis
X
X
X
X
X
X
X
X
Serum tryptase
X
X
X
X
X
X
X
X
Serum aldolase
X
X
X
X
X
X
X
X
Serum immunoglobulins 
X
X
X
X
X
X
X
X
Serum IgE levels
X
X
X
X
X
X
X
X
Urine pregnancy test*
(X)
(X)
(X)
(X)
Research laboratory studies
Eosinophil and PBMC 
purification from whole blood 
and storage
X
X
X
X
X
X
Urine for eosinophil granule 
protein and mediator levels
X
X
X
X
X
X
X
X
X
X
X
X
Whole blood flow for TBNK 
cell subsets and RNAseq
X
X
X
X
X
X
Serum or plasma for 
eosinophil granule protein 
and mediator levels
X
X
X
X
X
X
X

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Study assessments
AE assessments
X
X
X
X
X
X
X
X
Key: AE- adverse event; AEC- absolute eosinophil count; ANC- absolute neutrophil count; CBC- complete blood count; CPK- creatine kinase; IgE- immunoglobulin E; LDH- lactate 
dehydrogenase; PBMC- peripheral blood mononuclear cell; PROs- patient-reported outcome measures; TBNK- T, B, and natural killer cell; UV- unscheduled visits; ‖ Study visits 
may be rescheduled or study procedures may be waived or modified to be performed remotely due to travel limitations or safety concerns during the coronavirus disease 2019 
pandemic (as described in protocol section 6); ** All subjects will complete Visit 8. Subjects who experienced benefit from mepolizumab administration will continue in the 
de-escalation phase (which begins with additional procedures at Visit 8—see Table 2 below); for subjects who do not continue in the de-escalation phase, Visit 8 will be the final 
study visit. ¶ The exact timing of Visit 1 through Visit 5 may vary from this schedule based on subject cycle times. Visits 2 and 3 may be combined if neutrophils and eosinophils peak 
<1 week apart. Every effort will be made to schedule Visit 5 in advance of Visit 6; in the event that Visit 5 occurs within 1 day of Visit 6, only collection of whole blood for storage will 
be performed for Visit 5. Visits 6, 7, and 8 will be scheduled monthly based on the timing of Visit 4. * Urine pregnancy test prior to administration of mepolizumab in women of child-
bearing potential; # Performed electronically via REDCap. Symptom logs to be performed daily, PROs daily and monthly, not only at study visits; daily symptom logs and measures 
will be discontinued after Visit 7, and monthly questionnaires will be continued through Visit 8; § Home labs will be done 2-3X/week, depending on subject cycle times (2X/week in 
some cases or after peak is obtained); † Optional. Bone marrow will be requested to be performed only once at Visit 1 or 2, if a sample is not available from another NIH protocol. 
Skin punch biopsy will be requested to beperformed only once on Visits 1, 2, or 3; ‡ May be requested once at any time while on study drug if a pre-treatment sample was 
collected/available.   

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Table 2. Schedule of Procedures/Evaluations for the De-escalation Phase.
Evaluations ‖
Study Drug 
500 mg
Study Drug 
500 mg
Study Drug 
500 mg
Study Drug 
300 mg 
Study Drug 
300 mg
Study Drug 
300 mg
End of 
De-escalation
UV
Visit Number
8
9
10
11
12
13
14
Day (assuming 4-week cycle)
Day 99
Day 127
Day 155
Day 183
Day 211
Day 239
Day 267
Window (days)
±7
±7
±7
±7
±7
±7
±7
Clinical assessments and laboratory evaluations
Targeted medical history and 
physical examination
X
X
X
X
X
X
X
Limb measurements^ 
X
X
X
X
X
X
X
Vital signs including weight^
X
X
X
X
X
X
X
Symptom log and PROs#
X
X
X
X
X
X
X
Autoinjector training (for 
remote subjects)
X
Mepolizumab 
X
X
X
X
X
X
CBC with differential
X
X
X
X
X
X
X
Acute care panel
X
X
Hepatic panel 
X
X
Mineral panel 
X
X
LDH, CPK
X
X
C-reactive protein
X
X
X
Urinalysis
X
X
X

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Serum tryptase
X
X
X
Serum aldolase
X
X
X
Serum immunoglobulins 
X
X
X
Serum IgE levels
X
X
X
Urine pregnancy test*
(X)
(X)
(X)
(X)
(X)
(X)
Research laboratory studies
Eosinophil and PBMC 
purification from whole blood 
and storage
X
X
Urine for eosinophil granule 
protein and mediator levels
X
X
X
Whole blood flow for TBNK 
cell subsets and RNAseq
X
X
Serum or plasma for 
eosinophil granule protein 
and mediator levels
X
X
X
Study assessments
AE assessments
X
X
X
X
X
X
X
X
Key: AE- adverse event; CBC- complete blood count; CPK- creatine kinase; IgE- immunoglobulin E; LDH- lactate dehydrogenase; PBMC- peripheral blood mononuclear cell; PROs- 
patient-reported outcome measures; TBNK- T, B, and natural killer cell; UV- unscheduled visits; ‖ Study visits may be rescheduled or study procedures may be waived or modified to 
be performed remotely due to travel limitations or safety concerns during the coronavirus disease 2019 pandemic (as described in protocol section 6); ^Some vital signs or 
assessments may not be possible if performed virtually. Visit 8 will begin after completion of procedures listed at Visit 8 in Table 2. Visits 9 to 14 will be scheduled 28 days (±7 days) 
from the prior visit. * Urine pregnancy test prior to administration of mepolizumab in women of child-bearing potential (not performed at remote visits); # Performed electronically via 
REDCap. A weekly symptom log will be completed through Visit 14; PROs will be done weekly and monthly through Visit 14.   

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Appendix C: Blood Volumes for Specimen Collection  
Table 3. Blood Volumes for Collection in the Primary Study Phase.
Evaluations
Enrollment
V1
Home
Labs2
Baseline
V2
Home
Labs2
Peak 
AEC
V3
Home 
Labs2
Study 
Drug/ 
AEC 
Nadir
V4
Home
Labs2
Predicted 
ANC 
Peak
V5
Home 
Labs2
Study 
Drug 
V6
Study 
Drug 
V7
V8 (± 
Study 
Drug)
CBC with 
differential
Light 
Lavender, 
EDTA
3
3
3
3
3
3
3
3
Acute care, 
hepatic, and 
mineral panels 
Green-
Yellow rim
4
4
4
4
4
4
4
4
C-reactive protein
Green-
Yellow rim
X
X
X
X
X
X
X
X
Immunoglobulins 
(quantitative) 
Green-
Yellow rim
X
X
X
X
X
X
X
X
LDH, CPK
Green-
Yellow rim
X
X
X
X
X
X
X
Aldolase, serum
Red-Yellow 
SST
4
4
4
4
4
4
4
4
Tryptase, serum
Red-Yellow 
SST
X
X
X
X
X
X
X
X
IgE
Red-Yellow 
SST
X
X
X
X
X
X
X
X
CBC with 
differential 
Light 
Lavender, 
EDTA
12
12
12
12
12
Whole blood for 
cells and plasma
Light 
Lavender, 
EDTA
 40
 
40
40
 
40
 
40
 
 
 40
Serum for storage
Red-Yellow 
SST
8
8
8
8
8
8
8
TBNK-eos
Light 
Lavender, 
EDTA
3
 
3
3
 
3
 
3
 
 
3

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RNA PAX
PAXgene
2.5
2.5
2.5
2.5
2.5
 
2.5
Daily or Interval Volume (mL)
64.5
12
64.5
12
64.5
12
64.5
12
64.5
12
19
11
64.5
Cumulative Volume (mL)
64.5
76.5
141
153
217.5
229.5
294
306
370.5
382.5
401.5
412.5
477
Abbreviations: AEC- absolute eosinophil count; ANC- absolute neutrophil count; CBC- complete blood count; CPK- creatine kinase; EDTA- ethylenediaminetetraacetic acid; IgE- 
immunoglobulin E; LDH- lactate dehydrogenase; TBNK- T, B, and natural kill cell; V- visit.
1 Specific timing of visits is provided in the Schedule of Evaluations (Appendix B) and Study Schematic (section 3.2). Per NIH MEC Policy M95-9, maximum blood volumes drawn 
for research purposes for an adult subject (aged 18 years or older) may not exceed: 10.5 mL/kg or 550 mL, whichever is smaller, over any 8-week period. This table reflects a 
model 4-week EAE cycle time with Day -15 enrollment, which would result in the highest possible blood volumes per time period. Blood volumes may be adjusted as needed for 
in-person visits that are changed to remote visits during the coronavirus disease 2019 pandemic.
2 Home lab blood volumes reflect total volume over interval. Home lab collections may be performed 2 times per week in subjects with 4-week or shorter cycle times or after peak 
is obtained. For subjects with longer cycle times, home labs will be performed up to 3 times per week without exceeding the NIH CC blood volume limit. 
3 Day 0 evaluations are the baseline for subsequent safety assessments.
Table 4. Blood Volumes for Collection in the Dose De-escalation Phase.
Evaluations (De-escalation phase)
Study Drug V9
Study Drug
V10
Study Drug V11 
Study Drug V12
Study Drug V13
End of 
De-escalation 
V14
CBC with 
differential
Light Lavender, 
EDTA
3
3
3
3
3
3
Acute care, 
hepatic, and 
mineral panels 
Green-Yellow rim
4
4
C-reactive protein
Green-Yellow rim
X
X
Immunoglobulins 
(quantitative) 
Green-Yellow rim
X
X
LDH, CPK
Green-Yellow rim
X
X
Aldolase, serum
Red-Yellow SST
4
4

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Tryptase, serum
Red-Yellow SST
X
X
IgE
Red-Yellow SST
X
X
Whole blood for 
cells and plasma
Light Lavender, 
EDTA
40
40
Serum for storage
Red-Yellow SST
8
8
8
TBNK-eos
Light Lavender, 
EDTA
3
3
RNA PAX
PAXgene
2.5
2.5
Daily or Interval Volume (mL)
22
64.5
3
3
3
64.5
Cumulative Volume (mL)
499
563.5
566.5
569.5
572.5
637
Abbreviations: CBC- complete blood count; CPK- creatine kinase; EDTA- ethylenediaminetetraacetic acid; IgE- immunoglobulin E; LDH- lactate dehydrogenase; TBNK- T, B, and 
natural kill cell; V- visit.
1 Specific timing of visits is provided in the Schedule of Evaluations (Appendix B) and Study Schematic (section 3.2). Per NIH MEC Policy M95-9, maximum blood volumes drawn 
for research purposes for an adult subject (aged 18 years or older) may not exceed: 10.5 mL/kg or 550 mL, whichever is smaller, over any 8-week period. This table reflects a 
model 4-week EAE cycle time with Day -15 enrollment, which would result in the highest possible blood volumes per time period. Blood volumes may be adjusted as needed for 
in-person visits that are changed to remote visits during the coronavirus disease 2019 pandemic.
2 Home lab blood volumes reflect total volume over interval. Home lab collections may be performed 2 times per week in subjects with 4-week or shorter cycle times or after peak 
is obtained. For subjects with longer cycle times, home labs will be performed up to 3 times per week without exceeding the NIH CC blood volume limit. 
3 Day 0 evaluations are the baseline for subsequent safety assessments.

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Appendix D: Cycle Length Determination for 3 Patients

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Appendix E: Patient Symptom Log (Daily)
The following information will be captured on an electronic version of this log through REDCap.

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Appendix F: Patient Symptom Log (Weekly)

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Appendix G: Quality of Life Scores and Disease Activity 
Scales
The following measures will be captured electronically in REDCap.
AE-QoL (Angioedema Quality of Life Assessment)

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AAS (Angioedema Activity Scale)

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Appendix H: Research Studies
Despite eosinophils being touted as central to the etiology of EAE, the 
pathophysiology of EAE is still not fully understood. Multiple cell lineages cycle, 
but the order of cycling and whether various cellular or serum factors influence 
the cycling is not known. Because the etiology of EAE is not fully understood, 
research blood and urine samples will be collected to assess the effects of 
mepolizumab on 1) eosinophil activation, 2) other potential biomarkers of disease 
activity in EAE, and 3) cell lineages other than eosinophils that may play a role. 
With eosinophils reduced or removed from the circulation, this trial will afford an 
opportunity to study the individual effects of other cell populations that may play a 
role in EAE pathogenesis. 
1) Eosinophil activation
Eosinophil activation will be assessed by measurement of serum and 
urine levels of eosinophil degranulation products (MBP, ECP, EDN, EPO) 
using a suspension array assay in multiplex.
2) Biomarkers of disease activity in EAE
Although validated biomarkers of disease activity in EAE have not been 
identified to date, a number of serum mediators, including cytokines (IL-5), 
chemokines (i.e., TARC, eotaxin), soluble receptors (i.e., soluble IL-5 
receptor alpha, soluble CD25), and serum tryptase, have been shown to 
cycle and correlate with disease activity. Blood samples obtained in the 
cycle prior to mepolizumab and those obtained during the cycle following 
the first mepolizumab infusion will be used to explore the effects of 
blocking IL-5 (and hopefully eosinophilia) on production and secretion of 
these mediators using a variety of assays, including enzyme-linked 
immunosorbent assay (ELISA), suspension array assays in multiplex, 
intracellular flow cytometry, and RNA expression analysis. Peripheral 
blood mononuclear cells (PBMCs) will also be viably frozen from the peak 
and nadir pre- and post-mepolizumab for future studies on selected cell 
populations. Finally, PBMCs, eosinophils, and neutrophils will be purified 
and stored in Trizol from the same time points for assessment of changes 
in RNA expression profiles in response to mepolizumab. Whole blood 
RNAseq will be performed at key timepoints.
3) Other lineages

Lymphocytes: In addition to assessing total lymphocyte numbers in 
the CBC with differential, lymphocyte phenotyping will be performed 
in the Department of Laboratory Medicine, NIH on whole blood 
samples using a standard panel that includes CD3, CD4, CD8, 

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CD25, HLA-DR, CD20, and CD16/CD56. In addition to standard 
TBNK cell phenotyping, the numbers of CD3-CD4+ T cells will be 
assessed. Serum TARC levels, an indirect measure of lymphocyte 
activation, will be measured by ELISA. 

Basophils: Due to the low numbers of circulating basophils in 
peripheral blood, basophil numbers and activation will be assessed 
by whole blood flow cytometry using CCR3 and CD203c, a specific 
marker of basophil activation.