Immunogenetic Profiling of Dupilumab for the Treatment of Atopic Dermatitis
Phase: PHASE4
Conditions: Atopic Dermatitis, Atopic Dermatitis Eczema, Eczema, Atopic Dermatitis and Related Conditions
Sponsor: University of California, San Francisco

Immunogenetic Profiling of Dupilumab for the Treatment of Atopic Dermatitis 
 
NCT03293030 
 
Protocol Version: November 8, 2020 

 
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Clinical Research Protocol 
 
Immunogenetic profiling of dupilumab for the treatment of atopic dermatitis 
 
 
 
Version Date: 
November 8, 2020 
Investigational Product: 
Dupilumab 
Sponsor: 
Regeneron Pharmaceuticals and Sanofi-Genzyme 
Principal Investigator: 
Wilson Liao, MD 
2340 Sutter St. Rm N426 
San Francisco, CA 94115 
Telephone: (415) 476-8364  
Fax: (415) 476-8837  
E-mail: wilson.liao@ucsf.edu  
Funding Organization: 
Regeneron Pharmaceuticals and Sanofi-Genzyme 
 
 
Approval: 
 
 
 
PI or Sponsor Signature (Name and Title)  
 
Date 
This confidential information about an investigational product is provided for the exclusive use of 
investigators of this product and is subject to recall at any time.  The information in this document 
may not be disclosed unless federal or state law or regulations require such disclosure.  Subject to the 
foregoing, this information may be disclosed only to those persons involved in the study who have a 
need to know, with the obligation not to further disseminate this information.   

 
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PROTOCOL AGREEMENT 
 
I have read the protocol specified below. In my formal capacity as Investigator, my duties 
include ensuring the safety of the study subjects enrolled under my supervision and 
providing Regeneron and Sanofi-Genzyme with complete and timely information, as 
outlined in the protocol.  It is understood that all information pertaining to the study will 
be held strictly confidential and that this confidentiality requirement applies to all study 
staff at this site. Furthermore, on behalf of the study staff and myself, I agree to maintain 
the procedures required to carry out the study in accordance with accepted GCP principles 
and to abide by the terms of this protocol. 
 
Protocol Title: Immunogenetic profiling of dupilumab for the treatment of atopic 
dermatitis 
 
Protocol Date:  November 8, 2020 
 
 
 
 
Investigator Signature  
 
Date 
 
Print Name and Title 

 
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TABLE OF CONTENTS 
1 
BACKGROUND .................................................................................................................. 11 
2 
STUDY RATIONALE ........................................................................................................ 11 
2.1 
Impact of Targeted Biologics on Atopic Dermatitis Skin……………………..……..12 
2.2 
Risk / Benefit Assessment ........................................................................................... 18 
3 
STUDY OBJECTIVES ....................................................................................................... 18 
3.1 
Primary Objective ........................................................................................................ 18 
3.2 
Secondary Objectives .................................................................................................. 19 
4 
STUDY DESIGN ................................................................................................................. 19 
4.1 
Study Overview ........................................................................................................... 19 
5 
CRITERIA FOR EVALUATION ...................................................................................... 19 
5.1 
Primary Efficacy Endpoint .......................................................................................... 19 
      5.2    Secondary Efficacy Endpoint…………………………………………………….......19 
5.3 
Safety Evaluations ....................................................................................................... 19 
6 
SUBJECT SELECTION ..................................................................................................... 20 
6.1 
Study Population .......................................................................................................... 20 
6.2 
Inclusion Criteria ......................................................................................................... 20 
6.3 
Exclusion Criteria ........................................................................................................ 20 
7 
CONCURRENT MEDICATIONS .................................................................................... 22 
7.1 
Allowed Medications and Treatments ......................................................................... 22 
7.2    Prohibited Medications and Treatments.....………………...………….………..……22 
8 
STUDY TREATMENTS ..................................................................................................... 22 
8.1 
Supply of Study Drug at the Site ................................................................................. 22 
8.2 
Storage ......................................................................................................................... 23 
8.3 
Dosage/Dosage Regimen……………………………………………………..……...23 
8.4 
Administration instructions .......................................................................................... 23 
       8.5    Study Drug Accountability…………………………………………………..…….....23 
       8.6    Measures of Treatment Compliance..………………………………………...……....23 
9 
STUDY PROCEDURES AND GUIDELINES ................................................................. 24 
9.1 
Clinical Assessments ................................................................................................... 24 
9.2 
Clinical Laboratory Measurements  ............................................................................. 25 
10 EVALUATIONS BY VISIT ............................................................................................... 28 
10.1 Visit 0 (Screening) ....................................................................................................... 28 
10.2   Visit 1 (Patch testing) ………………………………………………………………..29 
10.3 Visit 2 (Week 0) ........................................................................................................... 29 
10.4 Visit 3 (Week 2) ........................................................................................................... 29 
10.5 Visit 4 (Week 4) ........................................................................................................... 30 
10.6 Visit 5 (Week 8) ........................................................................................................... 30 
10.7 Visit 6 (Week 12) ......................................................................................................... 30 
10.8 Visit 7 (Week 16) ......................................................................................................... 30 

 
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10.9 Visit 8 (Week 24) ......................................................................................................... 30 
10.10 Visit 9 (Patch testing)  ................................................................................................. 31 
10.11 Visit 10 (Week 36) ....................................................................................................... 31 
10.11 Visit 11 (Week 44) ....................................................................................................... 31 
10.12 Visit 12 (Week 52) ....................................................................................................... 31 
10.14 Early Withdrawal Visit ................................................................................................ 32 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION ........................ 32 
11.1 Adverse Events ............................................................................................................ 32 
11.2 Serious Adverse Experiences (SAE) ........................................................................... 35 
11.3 Serious Adverse Experiences Reporting………………………………………………35 
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS ................................... 36 
12.1 Early Discontinuation of Study Drug……………………………………...…….…...36 
12.2 Withdrawal of Subjects ................................................................................................ 37 
13 PROTOCOL VIOLATIONS……………………………………………………………..37 
14 STATISTICAL METHODS AND CONSIDERATIONS ................................................ 37 
14.1 Analysis of Primary Endpoint ...................................................................................... 38 
      14.3   Analysis of Secondary Endpoint……………………………………………………..38 
      14.2 Sample Size .................................................................................................................. 38 
15 DATA COLLECTION, RETENTION AND MONITORING ........................................ 38 
15.1 Data Collection Instruments ........................................................................................ 39 
15.2 Data Management Procedures ..................................................................................... 39 
15.3 Data Quality Control and Reporting ............................................................................ 39 
15.4 Archival of Data ........................................................................................................... 39 
15.5 Availability and Retention of Investigational Records ................................................ 40 
15.6 Subject Confidentiality ................................................................................................ 40 
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS ................... 40 
16.1 Protocol Amendments .................................................................................................. 40 
16.2 Institutional Review Boards and Independent Ethics Committees .............................. 40 
16.3 Informed Consent Form ............................................................................................... 41 
16.4 Publications .................................................................................................................. 42 
16.5 Investigator Responsibilities ........................................................................................ 42 
17      References………………………………………………………………………………..43 
List of Figures 
    Fig 1. Multi-parameter flow cytometric analysis of human skin and atopic dermatitis……......15 
    Fig 2A. Workflow of a cytometry experiment………………………………………………....16 
    Fig 2B. Immune cells in human skin analyzed by mass cytometry (CyTOF)…..……..………17 
    Fig 3. RNA-sequencing of distinct cell populations within human skin .……..…….…….…..17 
    Fig 4. Power to detect differentially expressed genes using RNA-Seq………………………..39 
List of Tables 
     Table 1. AE Severity Grading……………………………………………………………..…33 

 
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     Table 2. AE Relationship to Study Drug……………...………………………………….…..33 
Appendix 
     Appendix 1. Evaluation Schedule ……………………………………………………………44 

 
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LIST OF ABBREVIATIONS 
AE 
adverse event 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BUN 
blood urea nitrogen 
CBC 
CFR 
Complete blood count 
Code of Federal Regulations 
CyTOF 
Cytometry by Time of Flight 
DMC 
Data Monitoring Committee 
DSMB 
Data Safety Monitoring Board 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICF 
informed consent form 
ICH 
International Conference on Harmonization 
IEC 
Independent Ethics Committee 
IgG4 
Immunoglobulin-G4 
IL-4 
Interleukin-4 
IL-13 
Interleukin-13 
IRB 
IUD 
Institutional Review Board 
Intrauterine device 
EASI 
IGA 
Eczema Area and Severity Index 
Investigator Global Assessment 
PI 
Principal Investigator 
PK 
QOL 
RNA-seq 
RT-PCR 
Pharmacokinetic 
Quality of life 
RNA-sequencing 
Reverse transcriptase polymerase chain reaction 
SAE 
serious adverse experience 
SGOT 
serum glutamic oxaloacetic transaminase 
SGPT 
SC 
 
serum glutamate pyruvate transaminase 
subcutaneously 
 

 
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PROTOCOL SYNOPSIS 
TITLE 
Immunogenetic profiling of dupilumab for the treatment of atopic 
dermatitis 
SPONSOR 
Regeneron Pharmaceuticals and Sanofi-Genzyme 
FUNDING 
Regeneron Pharmaceuticals and Sanofi-Genzyme 
STUDY DESIGN 
& OVERVIEW 
This is a single-arm, open-label study, which will examine the effect of 
dupilumab on the immunologic and genetic environment within atopic 
dermatitis lesions.  
PRIMARY 
OBJECTIVE 
Perform quantitative analysis of the immunologic and genetic changes in 
immune cell populations in lesional and non-lesional skin after dupilumab 
treatment (15 AD patients) at weeks 2, 4, 12 compared to baseline week 0. 
The immunologic and genetic profiles in AD patients will also be 
compared to healthy control skin surgical discard specimens (n=15). The 
primary endpoint is the percent change from pre-treatment baseline of 
CD4+ T effector cells expressing IL-4 at weeks 2, 4, 12. 
 
SECONDARY 
OBJECTIVES 
Use RNA-seq to identify differentially expressed genes and pathways in 
each cell population at weeks 2, 4, 12 compared pre-treatment baseline in 
dupilumab-treated patients. We will explore whether early molecular 
changes are predictive of the clinical response to dupilumab at 52 weeks 
(reduction of EASI and improvement in pruritus score), which could lead 
to the identification of novel biomarkers. Microbiome samples from skin 
and stool at weeks 0, 2, 4, 12, and 52 will be banked for future analysis. 
Allergic contact dermatitis patch testing will be performed prior to and 
during treatment with dupilumab in order to gain a better understanding of 
how dupilumab affects contact allergy. 
NUMBER OF 
SUBJECTS 
15 

 
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SUBJECT 
SELECTION 
CRITERIA 
Inclusion Criteria: 
For subjects with atopic dermatitis:  
1. Ability to provide written informed consent and comply with the 
protocol.  
2. At least 18 years of age. 
3. Diagnosis of chronic atopic dermatitis for at least 3 years prior to 
enrollment. 
4. Subject is considered a candidate for phototherapy or systemic therapy 
5. Eczema Area and Severity Index (EASI) score ≥ 12. 
6. Investigator Global Assessment (IGA) ≥ 3. 
7. 10% body surface area (BSA) or greater. 
8. Subject is unlikely to conceive due to male, post-menopausal, or using 
adequate contraceptive (barrier, hormonal, implant, or permanent 
sterilization methods). 
9. Physical exam within clinically acceptable limits. 
 
For healthy control subjects:  
1. Sample of non-inflamed skin. 
2. No history of autoimmune disease. 
3. At least 18 years of age.  
 
Exclusion Criteria: 
1. Subject is unable to provide written informed consent or comply with 
the protocol. 
2. Subject is younger than 18 years of age. 
3. Subject has had atopic dermatitis for less than 3 years prior to 
enrollment. 
4. Subject with mild atopic dermatitis (EASI<12 and IGA<3) or is not a 
candidate for phototherapy or systemic treatments. 
5. Subject with current, or a history of, severe atopic dermatitis well 
controlled on current therapy. 
6. Serious known infection. 
7. History of immunosuppression (including human immunodeficiency 
virus (HIV)). 
8. History of malignancy within 5 years before the screening visit, except 
completely treated in situ carcinoma of the cervix, completely treated 
and resolved non-metastatic squamous or basal cell carcinoma of the 
skin. 
9. Severe concomitant illnesses. 
10. Having used immunosuppressive/immunomodulating drugs (eg, 
systemic corticosteroids, cyclosporine, mycophenolate-mofetil, IFN-γ, 
Janus kinase inhibitors, azathioprine, methotrexate, etc.) or 
phototherapy within 4 weeks before the baseline visit. 
11. Treatment with topical corticosteroid or topical calcineurin inhibitor 
within 1 week before the baseline visit. 

 
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12. Treatment with oral antibiotics within 2 weeks of the baseline visit.  
13. Treatment with any cell-depleting agents including but not limited to 
rituximab: within 6 months before the baseline visit, or until lymphocyte 
count returns to normal, whichever is longer, or use of other biologics: 
within 5 half-lives (if known) or 16 weeks prior to baseline visit, 
whichever is longer. 
14. Physical or laboratory exam not within clinically acceptable limits.  
15. Subjects possess other diagnoses that, in the investigator’s opinion, 
preclude him/her from safely participating in this study or interfere 
with the evaluation of the subject’s atopic dermatitis. 
16. At baseline visit, the subject does not have sufficient clear skin for 
which to perform patch testing as determined by the investigator’s 
opinion, which is also defined as approximately 50-75% of skin on the 
back clear of atopic dermatitis.  
17. History of known or suspected intolerance to any of the ingredients of 
the investigational study product. 
18. Pregnant or nursing (lactating) women, where pregnancy is defined as 
the state of a female after conception and until the termination of 
gestation, confirmed by a positive hCG laboratory test (>10 mIU/mL). 
19. Women of child-bearing potential, defined as all women 
physiologically capable of becoming pregnant, unwilling to use 
effective contraception during the study and for 6 weeks after stopping 
treatment. 
For healthy control subjects: 
1. No areas of non-inflamed skin. 
2. History of autoimmune disease. 
3. Subject is younger than 18 years of age.  
STUDY 
TREATMENT 
Dupilumab (Dupixent ®) 600 mg SC at week 0, then 300 mg SC every 
other week. 
STUDY 
DURATION 
The total duration of the study is expected to be 104 weeks: 52 weeks for 
recruitment and 52 weeks for site visits. 
PRIMARY 
ENDPOINT 
Percentage change from pre-treatment baseline of CD4+ T effector cells in 
lesional and non-lesional skin expressing IL4 at weeks 2, 4, 12 in 
dupilumab-treated patients.  
SECNDARY 
ENDPOINT 
Differentially expressed genes and pathways in each cell population at 
weeks 2, 4, 12 compared to pre-treatment baseline using RNA-seq. 
SAFETY 
EVALUATIONS 
Safety and tolerability will be assessed by adverse events, vital signs, 
physical examinations (including skin examinations and injection-site 
evaluations), and concomitant medication review. Laboratory assessments 

 
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will be performed at screening.  
STATISTICS 
Primary Analysis 
Plan 
CyTOF: One sample paired t test comparing mean change in lesional and 
non-lesional IL-4 positive, CD4+ T effector cells compared to baseline. 
RNA-seq: Identification of differentially expressed genes compared to 
baseline within each immune population studied using the statistical 
program Cuffdiff (paired analysis). 
ETHICAL 
CONSIDER-
ATIONS 
This study will be conducted in accordance with applicable laws and 
regulations and according to the recommendations of International 
Conference on Harmonization Good Clinical Practice (ICH GCP) 
guidelines and those of the Declaration of Helsinki (Edinburgh, 2000); 
only after approval for the study has been obtained from the relevant 
regulatory authority and relevant independent ethics committee (IEC). The 
institutional review board (IRB)/IEC must review and approve the protocol 
and informed consent form (ICF) before any subjects are enrolled. The 
subject must be consented using the approved ICF before any procedures 
specified in the protocol are performed. 

 
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1 BACKGROUND 
Atopic dermatitis is a chronic, relapsing inflammatory skin disease characterized by 
intense pruritus and skin xerosis. Dupilumab is the first FDA-approved therapy for adult 
patients with moderate-to-severe atopic dermatitis in the class of recombinant high-
affinity, fully human immunoglobulin-G4 (IgG4) monoclonal antibody that selectively 
targets the receptor alpha subunit of Interleukin-4 (IL-4). By binding to the IL-4 receptor 
alpha subunit, dupilumab modulates both the IL4 and Interleukin-13 (IL-13) pathways, 
which type 2/Th2 cytokines implicated in numerous allergic diseases.  Through this 
blockade, dupilumab inhibits the inflammatory response that plays a role in the 
development of atopic dermatitis.  
Non-clinical studies have not shown any impediment to using dupilumab administered 
subcutaneously in man. 
The approval of dupilumab (Dupixent ®) was based on the safety and efficacy outcomes 
from 3 Phase III studies known as SOLO 1, SOLO 2, and CHRONOS. These trials 
included over 2,119 patients with moderate-to-severe atopic dermatitis not adequately 
controlled by topical medications.  
For more detail refer to the Prescribing Information. 
2 STUDY RATIONALE 
The University of California San Francisco (UCSF) Department of Dermatology has 
expertise in immunologic/genetic analyses of inflammatory skin conditions such as atopic 
dermatitis (AD) and psoriasis. This unique proposal will examine the effect of dupilumab 
on the immunologic and genetic environment within atopic dermatitis lesional and non-
lesional skin. While dupilumab is a targeted biologic that is expected to have a rapid and 
sustained impact on the Th2 axis, the full scope of dupilumab’s molecular mechanism of 
action on multiple cell types is not yet known.  
This proposal represents a translational collaborative effort between a nationally 
recognized clinical center specializing in atopic dermatitis and strong research enterprise. 
The UCSF Psoriasis and Skin Treatment Center, directed by the principal investigator Dr. 
Liao, is a high-volume atopic dermatitis referral center whose activities include outpatient 
care, a phototherapy unit, a Goeckerman tar treatment program, and over 20 clinical trials 
conducted over the past three years. Staff for the center includes 6 attending physicians, 8 
nurses, 3 administrators, and 2 full time research fellows. Over 500 atopic dermatitis 
patients are seen each year. The infrastructure at the Center will facilitate ready 
recruitment of subjects and will ensure accurate, detailed AD phenotypes are ascertained 
for this study. Dr. Michael Rosenblum is a board-certified dermatologist and basic 
scientist with expertise in cutaneous immunology. His lab has identified a new population 
of regulatory cells in the skin, termed memory Tregs, which play an important role in 
chronic inflammatory skin diseases such as psoriasis. Dr. Rosenblum has helped pioneer a 
novel technology which allows for immunologic profiling of freshly isolated human skin 
leukocytes as detailed in this application. Dr. Wilson Liao is a board-certified 
dermatologist and geneticist who has contributed to the discovery of over 20 psoriasis 
genes. Dr. Liao’s group has expertise in the analysis of genetic and genomic datasets and 

 
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has collaborated extensively with Dr. Rosenblum. 
2.1 
Impact of Targeted Biologics on Atopic Dermatitis Skin 
Regarding understanding the impact of biologics on the molecular profile of AD skin, a 
fundamental question that has not yet been adequately answered is: What is the response 
of specific cell populations in lesional skin to biologic treatment?  
A number of recent studies have used techniques such as RT-PCR, gene microarrays, and 
immunohistochemistry to evaluate the immunologic environment of AD lesions and its 
changes with treatments such as ultraviolet light, cyclosporine, and dupilumab (Ungar, 
Tintle, Rozenblit, Hamilton). However, these studies are limited by the use of bulk 
skin tissue, which does not permit precise quantitative analysis of individual cell 
types, and by the reliance on limited gene probe libraries used in RT-PCR or 
microarray analysis. A more recent study of AD used the more sensitive technique of 
RNA-sequencing (Suárez-Fariñas), but also used bulk tissue. Ideally, in order to 
understand the fundamental biology of how a biologic medication improves AD, one 
would like to be able to isolate the different cell populations and study their individual 
characteristics.  
At UCSF, we have developed innovative technologies to characterize individual skin cell 
populations at the protein and RNA level. Using an optimized flow cytometry protocol, 
we are able to separately analyze distinct cell populations in human skin (Figure 1). 
Distinct skin populations such as CD4+ T effector cells, CD8+ T cells, T regulatory cells, 
and innate lymphoid type 2 cells can be separated and the production of proteins such as 
IL-4, IL-13, IL-17, IFN-g, and IL-22 can be measured in parallel. Because this technique 
is based on protein expression, it offers a truly functional view of the activity of cell 
populations in AD and healthy skin. We have used this technique to publish on the 
analysis of psoriasis and hidradenitis suppurativa skin in high impact journals such as the 
Journal of the American Academy of Dermatology, JAMA Dermatology, and Journal 
of Clinical Investigation (Cordoro et al; Debbaneh et al; Leslie et al; Sanchez Rodriguez 
et al).  
Moreover, we have recently achieved a major breakthrough in upgrading this 
technology to the Cytometry by Time-Of-Flight (CyTOF), which allows for the 
simultaneous measurement of 40+ molecular markers as compared to 15-16 markers with 
flow cytometry (Figures 2A and 2B). CyTOF is a fusion of flow cytometry and mass 
spectrometry whereby cells of interest are stained with antibodies conjugated to stable 
heavy metal isotopes. Cells are then passed in a single-cell suspension into a nebulizer, 
which places cells into droplets for ionization by argon plasma, which allows the heavy 
metals to be detected by their mass-to-charge ratio in a time-of-flight mass spectrometer. 
The measurement of over 40 simultaneous cellular parameters at single-cell resolution 
significantly augments our ability of cytometry to evaluate complex cellular systems and 
processes.  
To our knowledge, we are the first group to adapt CyTOF to human skin samples and 
biopsies. Tissue is finely minced, resuspended in a cocktail of enzymes (collagenase and 
DNase) and incubated overnight at 37˚C to yield a single-cell suspension. A viability 

 
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stain, cisplatin, is added to mark dead cells for exclusion from the downstream analysis. 
Cells are then fixed in 1.5% paraformaldehyde to preserve the cell state and to enable 
banking of samples. Samples are then stained with a cocktail of antibodies targeting 
extracellular molecules of interest, permeabilized, and stained again with antibodies, this 
time targeting intracellular molecules. Finally, samples are incubated with a DNA 
intercalator containing iridium ions in fixative before analysis on the instrument. Data 
generation on the mass cytometer requires approximately 1 hr per 106 cells. Upon data 
collection, data are normalized and de-barcoded into their respective conditions using 
methods we have previously published and open-source software in MATLAB. After data 
pre-processing, we utilize an analytical pipeline we have previously developed to create 
maps of the immune cells from each sample called Scaffold maps (Figure 2B). Using this 
approach, groups of similar cells are identified in the data using an unsupervised 
clustering algorithm. The relationship between these cell subsets is visualized as a force- 
directed graph, in which related cell types form an edge between them and appear adjacent 
to one another in the display. These graphs also incorporate classical immune cell 
populations identified manually in the data as reference points to help orient the 
researcher. The result is a robust and thorough evaluation of all immune cells present in 
different samples, which has resulted in powerful mechanistic insights in a number of 
contexts. 
In addition, we are able to perform RNA-sequencing on distinct cell populations. RNA-
seq is a novel technique of quantifying gene expression in which RNA species are 
sequenced rather than hybridized as in microarrays. RNA-seq provides greater 
sensitivity than microarrays, particularly for low-expressed transcripts (Li et al).  
At UCSF, the Liao Lab has developed expertise in the analysis of RNA-seq data. 
First, our group has shown that RNA-seq can also identify the expression of noncoding 
transcripts in inflammatory skin disease that are absent on microarray platforms (Gupta et 
al). Our group is one of the few that has published on non-coding RNA in human skin. 
These non-coding transcripts are important for the regulation of coding transcripts and 
may play an important role in the pathogenesis of inflammatory skin disease.  
Second, in addition to performing standard differential gene analysis (e.g. between 
lesional and healthy skin), we apply state-of-the-art gene network analysis, which can 
identify modules of significantly correlated genes and define key driver genes. At UCSF 
we conducted whole transcriptome RNA-seq analysis on 18 psoriasis patients (pre-
treatment and post-treatment with the TNF-α inhibitor adalimumab) and 16 healthy 
controls, generating an average of 52.3 million 100-bp paired-end reads per sample. Using 
Weighted Gene Co-expression Network Analysis (WGCNA) (Horvath et al), we 
identified 3 network modules that were significantly associated with psoriasis and 6 
network modules significantly associated with biologic treatment, with only 16% of the 
psoriasis-associated and 5% of the treatment-associated co-expressed genes being 
identified by differential expression analysis. Dissection of the network modules identified 
short-chain fatty acid metabolism as a pathway highly associated with psoriasis, while 
regulation of leukocyte mediated cytotoxicity was highly associated with biologic 
treatment (Ahn et al, 2016).  

 
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Third, our group has expertise in the analysis of RNA-seq data derived from specific 
cell populations in human skin (Ahn et al, 2017). A limitation of all previously 
published RNA-seq studies is that they have relied on bulk skin biopsies, which 
contain many cell types, thus making it difficult to discern the contributions of 
individual cell populations.  We performed RNA-seq on keratinocytes, dendritic cell, 
CD4+ T effector cells, and CD8+ T effector cells (Figure 3) from human skin and 
demonstrated that RNA-seq of bulk skin primarily captures gene expression from 
keratinocytes, but is less sensitive in detecting gene expression from immune cell 
populations such as T cells. Thus, previous gene expression studies of bulk skin (e.g. 
following dupilumab treatment) are skewed towards studying keratinocyte biology, while 
potentially missing out on important biology occurring within T cells. We are able to 
identify “transcriptional signatures” within each cell type, rather than in bulk skin. 
Importantly, the cell type-level RNA-seq analysis proposed here has never previously 
been utilized to evaluate patients with AD undergoing biologic therapy. 
 
CyTOF panel for AD: 
CD3, CD19, CD117, CD11b, CD4, CD8a, CD11c, CD14, IL-4, TNFa, CD123, gdTCR, 
GATA3, IFNg, IL-13, IL-10, IL-4Ra, CD27, IL-33, Tbet, CD152 (CTLA-4), Foxp3, 
CD33, CD45RO, CD127, IL-25, IL-17A, Ki-67, CD25, RORgt, TSLP, CD38, IL-2, HLA-
DR, PD-1, CD56, CD16 
 
RNA-seq cell types for AD 
CD4+ T effectors, CD4+ T Regs, CD8+ T effectors 
 

 
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Figure 1. Multi-parameter flow cytometric analysis of human skin and atopic dermatitis markers. (A) 
Example of functional immunoflow cytometry on 4 mm punch biopsy specimen of normal human skin. 
Tissue was minced and digested for 12 hrs in a digestion cocktail containing collagenase. Cells were 
washed, stimulated with PMA/ionomycin, and stained for 13-color flow cytometry to detect cell surface 
proteins and intracellular cytokine production. In contrast to other methods, our protocol does not rely on 
cell culture or addition of exogenous growth factors such as IL-2, which can substantially alter the 
phenotype of cells. Foxp3 is a transcription factor that drives the differentiation of regulatory T cells. (B) 
Gating strategy for atopic dermatitis flow panel. Live CD45+ leukocytes are further subsetted as CD4+ T 
effector cells, CD4+ T regulatory cells, and CD8+ T effector cells, or innate lymphoid cells type 2 (ILC2). 
(C). This technique is extremely sensitive, as a biopsy specimen from normal skin (having no appreciable 
inflammatory infiltrate by routine histology) shows a defined CD4+ and CD8+ T cell population capable of 
producing specific effector cytokines such as IL-4, IL-17, IFN-gamma, and IL-22. 
 

 
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Figure 2A. Workflow of a cytometry experiment. Cells are placed into a single cell suspension, and a 
viability stain is added to exclude dead cells from the analysis. Cells are then fixed to preserve the 
endogenous cell state. Barcodes are applied to different samples to enable pooling, which minimizes 
technical variability between samples. Pooled samples are then stained with a panel of antibodies targeting 
molecules of interest as well as a DNA intercalator.  Samples are then introduced into the mass cytometer by 
a nebulizer, which creates fine droplets containing cells. Cells travel though an inductively coupled argon 
plasma heated to 7500K, which breaks covalent bonds to form an ion cloud. Low molecular weight ions are 
filtered out, enriching for heavy metal reporter ions that were bound to antibodies. Ions are quantified using 
a time-of-flight mass spectrometer and signal is integrated for each cell event. Data are stored as a 
conventional “flow cytometry standard” file, which can be separated into samples based on the unique 
barcodes and then analyzed. From Spitzer et al., Cell, 2016. 

 
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Figure 2B. Immune cells in human skin analyzed by mass cytometry (CyTOF). We have recently 
pioneered and optimized comprehensive analysis of immune cell subsets from 4mm skin punch biopsy 
specimens (inset) using CyTOF. Cell subsets in skin are visualized as a Scaffold map (Spitzer et al., 
Science, 2015). Left: Red nodes in the graph represent manually identified landmark immune cell types 
from human peripheral blood (PBMC). Blue nodes represent unsupervised clusters of immune cells 
identified in healthy human skin. Right: Scaffold map colored by CD117, highlighting a connected group of 
clusters exhibiting high expression. These cells also express FceR1 (data not shown) and represent skin-
resident mast cells that are distinct from any cell type found in peripheral blood. 
 
A) 
B) 
 
 
Figure 3. RNA-sequencing of distinct cell populations within human skin (Ahn et al, 2017). (A) As 
opposed to examining the transcriptome of bulk skin, we have pioneered the capability to perform 
transcriptome profiling in specific cell populations. (A) A comparison of bulk vs cell population-specific 
RNA-seq was performed. Unsupervised hierarchical clustering reveals that the bulk skin samples (green) are 
most similar to keratinocytes (blue), whereas dendritic cells (beige) and T cells (purple) cluster distinctly. 
This suggests that prior gene expression studies of bulk skin to not adequately capture immune cell profiles. 
(B) Gene network analysis of cell population-specific RNA-seq data. Each vertical line represents a 
different gene. We are able to identify gene modules that are “signatures” of each cell population (black 
boxes). 

 
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2.2 
Risk / Benefit Assessment 
Details of the risk and benefits of dupilumab are outlined in the Prescribing Information. 
The most common adverse effects (incidence ≥ 1%) of dupilumab include injection site 
reactions, conjunctivitis, blepharitis, oral herpes, keratitis, eye pruritus, other herpes 
simplex infection, and dry eye. Other adverse effects include hypersensitivity reactions, 
including generalized urticarial and serum sickness or serum sickness-like reactions.  
The risk to subjects in this trial will be minimized by compliance with the 
inclusion/exclusion criteria, proper study design, and close clinical monitoring. 
In regards to skin biopsy procedures, as in any surgical procedure, there are certain 
inherent risks including bleeding, post-operative pain, infection, reactions to sutures, 
anesthetics or topical antibiotics, and scarring. All reasonable efforts will be made to 
minimize the possibility of these potential complications. 
In regard to patch testing, in rare cases there may be blistering reactions, in highly allergic 
patients. It is possible in these instances for scarring to occur at the site of the offending 
allergen. Sometimes, particularly in patients with photodamaged skin temporary bruising 
can occur with removal of the tape from the patches. Patch testing, in rare circumstances, 
can lead to excited skin syndrome (typically in patients who have significant active 
dermatitis at the time of testing). To mitigate this, patients deemed to have no clear areas 
for testing due to significant active dermatitis will not be patch tested. Patients who are 
pregnant will also be excluded, as patch testing has not been studied in this population. 
We believe there is only minimal risk to those participating in any and all parts of this 
study. We also believe that the minimal risk is reasonable for the benefits gained by a 
better understanding of how dupilumab affects contact allergy. Additionally, patients will 
receive patch testing and allergen avoidance which is of immense benefit to improving 
their skin disease and quality of life going forward.  
The benefit to subjects is that they will be receiving an approved therapy for moderate-to-
severe atopic dermatitis. Although it cannot be guaranteed, dupilumab has shown to be 
safe and effective in moderate-to-severe atopic dermatitis. An additional benefit to 
patients is that they are contributing to society by helping the enhancement of science.  
3 STUDY OBJECTIVES 
3.1 
Primary Objective 
Perform quantitative analysis of the immunologic and genetic changes in immune cell 
populations in lesional and non-lesional skin after dupilumab treatment (15 AD patients) 
at weeks 2, 4, 12 compared to baseline week 0. The immunologic and genetic profiles in 
AD patients will also be compared to healthy control skin surgical discard specimens 
(n=15). The primary endpoint is the percent change from pre-treatment baseline of CD4+ 
T effector cells expressing IL-4 at weeks 2, 4, 12 in dupilumab-treated patients. 

 
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3.2 
Secondary Objectives 
Use RNA-seq to identify differentially expressed genes and pathways in each cell 
population at weeks 2, 4, 12 compared to pre-treatment baseline in dupilumab-treated 
patients. We will explore whether early molecular changes are predictive of the clinical 
response to dupilumab (reduction of EASI/IGA and improvement in pruritus score), 
which could lead to the identification of novel biomarkers. Microbiome samples from skin 
and stool at weeks 0, 2, 4, 12, and 52 will be banked for future analysis. Allergic contact 
dermatitis patch testing will be performed prior to and after 6 months of treatment with 
dupilumab to gain a better understanding of how dupilumab affects contact allergy.  
4 STUDY DESIGN 
4.1 
Study Overview 
This is a single center, open-label study. Fifteen subjects with moderate to severe AD will 
receive dupilumab for a treatment period of 52 weeks (i.e. last injection on week 48). 
Surgical discard specimens will also be collected from fifteen healthy subjects. Biopsy 
samples from AD subjects and surgical discard samples will undergo molecular profiling. 
Skin swabs and stool samples will be collected and banked for future analysis. The reason 
to treat patients for 52 weeks is to have the ability to correlate early molecular events 
with clinical outcomes (EASI, IGA, itch score) at week 52. In addition, collection of 
microbiome samples will be performed at week 52. See Flowchart for timeline of study 
activities. 
5 CRITERIA FOR EVALUATION 
5.1 
Primary Analysis Endpoint 
Percentage change from pre-treatment baseline of CD4+ T effector cells expressing IL-4 
at weeks 2, 4, 12 in dupilumab-treated patients. 
5.2 
Secondary Analysis Endpoint 
Number of differentially expressed genes and pathways in each cell population at weeks 
2, 4, 12 compared to pre-treatment baseline using RNA-seq. 
We will also explore whether early molecular changes are predictive of the clinical response 
to dupilumab at 52 weeks (reduction of EASI and improvement in pruritus score), which 
could lead to the identification of novel biomarkers.  
5.3 
Safety Evaluations 
Safety and tolerability to dupilumab will be assessed by adverse events, vital signs, 
physical examinations (including skin examinations and injection-site evaluations), and 
concomitant medication review. Laboratory assessments will be performed at screening. 

 
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6 SUBJECT SELECTION 
6.1 
Study Population 
Subjects with a diagnosis of moderate to severe atopic dermatitis for at least 3 years prior 
to enrollment who meet the inclusion and exclusion criteria will be eligible for 
participation in this study.   
6.2 
Inclusion Criteria 
Inclusion criteria for AD subjects:  
1. Ability to provide written informed consent and comply with the protocol.  
2. At least 18 years of age. 
3. Diagnosis of chronic atopic dermatitis for at least 3 years prior to enrollment. 
4. Subject is considered a candidate for phototherapy or systemic therapy 
5. Eczema Area and Severity Index (EASI) score ≥ 12 
6. Investigator Global Assessment (IGA) ≥ 3 
7. 10% body surface area (BSA) or greater 
8. Subject is unlikely to conceive due to male, post-menopausal, or using adequate 
contraceptive (barrier, hormonal, implant, or permanent sterilization methods). 
9. Physical exam within clinically acceptable limits. 
 
Inclusion criteria for healthy controls obtained by surgeons (surgical specimens): 
4. Sample of non-inflamed skin. 
5. No history of autoimmune disease. 
6. At least 18 years of age.  
6.3 Exclusion Criteria 
Exclusion criteria for AD subjects:  
1. Subject is unable to provide written informed consent or comply with the protocol. 
2. Subject is younger than 18 years of age. 
3. Subject with mild atopic dermatitis (EASI<12 and IGA<3) or is not a candidate for 
phototherapy or systemic treatments. 
4. Subject with current, or a history of, severe atopic dermatitis well controlled on 
current therapy. 
5. Serious known infection. 
6. History of immunosuppression (including human immunodeficiency virus (HIV)). 
7. History of malignancy within 5 years before the screening visit, except completely 
treated in situ carcinoma of the cervix, completely treated and resolved non-metastatic 
squamous or basal cell carcinoma of the skin. 
8. Severe concomitant illnesses. 
9. Having used immunosuppressive/immunomodulating drugs (e.g., systemic 
corticosteroids, cyclosporine, mycophenolate-mofetil, IFN-γ, Janus kinase inhibitors, 
azathioprine, methotrexate, etc.) or phototherapy within 4 weeks before the baseline 
visit. 
10. Treatment with topical corticosteroid or topical calcineurin inhibitor within 1 week 

 
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before the baseline visit.  
11. Treatment with any cell-depleting agents including but not limited to rituximab: within 
6 months before the baseline visit, or until lymphocyte count returns to normal, 
whichever is longer, or use of other biologics: within 5 half-lives (if known) or 16 weeks 
prior to baseline visit, whichever is longer. 
12. Treatment with oral antibiotics within 2 weeks of the baseline visit.  
13. Physical exam not within clinically acceptable limits.  
14. Subjects possess other diagnoses that, in the investigator’s opinion, preclude him/her 
from safely participating in this study or interfere with the evaluation of the subject’s 
atopic dermatitis. 
15. Subjects possess other diagnoses that, in the investigator’s opinion, preclude him/her 
from safely participating in this study or interfere with the evaluation of the subject’s 
atopic dermatitis. 
16. History of known or suspected intolerance to any of the ingredients of the 
investigational study product. 
17. Pregnant or nursing (lactating) women, where pregnancy is defined as the state of a 
female after conception and until the termination of gestation, confirmed by a positive 
hCG laboratory test (>10 mIU/mL). 
18. Women of child-bearing potential, defined as all women physiologically capable of 
becoming pregnant, unwilling to use effective contraception during the study and for 6 
weeks after stopping treatment. Effective contraception is defined as either: 
• Barrier method: Condom or occlusive cap (diaphragm or cervical/vault caps) with 
spermicide (where available). Spermicides alone are not a barrier method of 
contraception and should not be used alone. 
 
The following methods are considered more effective than the barrier method and are also 
acceptable: 
• Total abstinence: When this is in line with the preferred and usual lifestyle of the 
subject (Periodic abstinence [e.g. calendar, ovulation, symptothermal, post-
ovulation methods] and withdrawal are not acceptable methods of contraception) 
• Female sterilization: have had a surgical bilateral oophorectomy (with or without 
hysterectomy) or tubal ligation at least six weeks before taking study treatment. In 
case of oophorectomy alone, only when the reproductive status of the woman has 
been confirmed by follow up hormone level assessment. 
• Male partner sterilization (at least 6 months prior to screening). For female 
subjects on the study, the vasectomized male partner should be the sole partner for 
that subject. 
• Use of established oral, injected or implanted hormonal methods of contraception, 
intrauterine device (IUD) or intrauterine system (IUS) 
 
NOTE: Women are considered post-menopausal and not of child bearing potential if 
they have had: 12 months of natural (spontaneous) amenorrhea with an appropriate 
clinical profile (e.g. age appropriate, history of vasomotor symptoms) or six months of 

 
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spontaneous amenorrhea with serum FSH levels >40 mIU/mL and estradiol <20 
pg/mL or surgical bilateral oophorectomy (with or without hysterectomy) or tubal 
ligation at least six weeks before taking study treatment. In case of oophorectomy 
alone - only when the reproductive status of the woman has been confirmed. 
 
Exclusion criteria for healthy controls obtained by surgeons (surgical specimens): 
1. No areas of non-inflamed skin. 
2. History of autoimmune disease. 
3. Subject is younger than 18 years of age.  
 
7 CONCURRENT MEDICATIONS 
7.1 
Allowed Medications and Treatments 
All prior concomitant medications must be on a stable dose for at least 4 weeks before the 
first study treatment administration. All subjects should be maintained on the same 
medications throughout the entire study period, as medically feasible. Any changes in 
medications and medication dosing will be recorded on the CRF. After the screening 
period, the use of concomitant medication for atopic dermatitis in all body regions is 
restricted to bland emollients and other non-medicated interventions. 
7.2 
Prohibited Medications and Treatments 
The following are prohibited during the study and prior to the study for a set duration as 
follows:  
• Systemic immunosuppressive or immunomodulatory treatments: 4 weeks. 
• Phototherapy: 4 weeks. 
• Cell-depleting agents including but not limited to rituximab: 6 months or until 
lymphocyte count returns to normal, whichever is longer. 
• Biologics: 16 weeks or within 5 half-lives (if known), whichever is longer. 
• Topical treatment that is likely to impact signs and symptoms of atopic dermatitis 
such as corticosteroids, vitamin D analogues, pimecrolimus, retinoids, salicylic acid, 
lactic acid, tacrolimus, tar, urea, α-hydroxy or fruit acids: 1 week. 
• Live vaccinations: 6 weeks. 
• Any investigational treatment or participation in any interventional trial: 4 weeks or 
5 half-lives, whichever is longer. 
• Any oral antibiotic: 2 weeks.  
• Subjects will be asked to avoid bathing areas of microbiome skin swabbing for a 24 
hour period prior to swabbing. 
8 STUDY TREATMENTS   
Dupilumab (Dupixent ®)  
8.1 
Supply of Study Drug at the Site 
Regeneron and Sanofi-Genzyme will ship the study drug to the investigational sites. The 

 
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study drug is supplied in cartons containing either two 300 mg/2 mL solution pre-filled 
syringes with needle shield or 2 pre-filled syringes. Each kit of study drug will be labeled 
with the required FDA warning statement, the protocol number, and directions for use and 
storage. 
8.2 
Storage 
Dupilumab must be refrigerated at 36°F to 46°F (2°C to 8°C) in the original carton to 
protect from light. The medication will be kept in a temperature-monitored refrigerator at 
the study site. If necessary, pre-filled syringes may be kept at room temperature up to 
77°F (25°C) for a maximum of 14 days. The product will be kept in the original carton 
until the time of use. 
8.3 
Dosage/Dosage Regimen 
Dupilumab will be administered at an initial dose of 600 mg subcutaneously followed by 
300mg subcutaneously through the study, which is the FDA-approved and recommended 
dose for dupilumab. There is no weight-based dosing. The dosing schedule will be as 
follows: dupilumab 600mg subcutaneously once at week 0, followed by dupilumab 300 
mg subcutaneously every other week until week 50.  
8.4 
Administration Instructions  
All doses of study treatment will be administered at the study site after the study 
assessments for the visit have been completed at visits occurring between week 0 and 4, as 
well as weeks 8, 12, 16, 24, 36, and 44. After week 4 subjects will administer the 
medication independently every other week (weeks 6, 10, 14, 18, 20 22, 26, 28, 30, 32, 
34, 38, 40, 42, 46, 48, and 50) only after they have shown the competency to self-
administer the treatment outside of the study site. The medication will be administered via 
subcutaneous injection into the thigh or abdomen, except for the 2 inches (5 cm) around 
the navel. The upper arm can also be used if a caregiver administers the injection. There 
will be no medication given after week 50.  
The subject should be instructed to contact the investigator if he/she is unable for any 
reason to attend a study visit as scheduled. All dates and times of injections done to the 
subject during the study must be recorded on the Dosage Administration Record CRF. 
The investigator will promote compliance by instructing the subject to attend the study 
visits as scheduled and by stating that compliance is necessary for the subject’s safety and 
the validity of the study. 
8.5 
Study Drug Accountability 
The qualified site personnel will maintain an accurate record of the shipment and 
dispensing of study treatment in a treatment accountability log. All study treatment kits 
assigned to the subject during the study will be recorded.  
8.6 
Measures of Treatment Compliance 
Subjects will be given scheduled doses of dupilumab at each visit from visit 1 (week 0) 

 
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until visit 9 (week 44). After week 4, subjects will be given a dosing diary to record their 
scheduled injections administered at home every other week. 
9 STUDY PROCEDURES AND GUIDELINES 
A Schedule of Events representing the required testing procedures to be performed for the 
duration of the study is diagrammed in Appendix 1. 
Prior to conducting any study-related activities, written informed consent and the Health 
Insurance Portability and Accountability Act (HIPAA) authorization must be signed and 
dated by the subject. 
All clinical assessments will take place at the study site: 515 Spruce St., San Francisco, 
CCA 94118 
All blood draws will take place at the UCSF Lab at Mount Zion: 2330 Post Street, San 
Francisco, CA 94115  
All patch testing will take place at UCSF Department of Dermatology, 1701 Divisadero 
4th floor, San Francisco, CA 94115. 
9.1 
Clinical Assessments 
For subjects with a diagnosis of atopic dermatitis:  
9.1.1 
Medical History  
Relevant medical history, including history of current disease, and information regarding 
underlying diseases will be recorded at Screening. 
1.1.2 
Concomitant Medications 
All concomitant medication and concurrent therapies will be documented at Screening, at 
every site visit, and at early termination when applicable.  Dose, route, unit frequency of 
administration, and indication for administration and dates of medication will be captured. 
9.1.3 
Demographics  
Demographic information (date of birth/age, gender, ethnicity, age of onset, vital signs, 
body mass index, body surface area, EASI, IGA, pruritus numerical rating, laboratory 
values (CBC with differential, BUN, Cr, AST/ALT/Alk Phos), baseline medications)) will 
be recorded at Screening. 
9.1.4 Physical Examination 
A complete physical examination will be performed by either the investigator or a sub-
investigator who is a physician at screening and weeks 0, 12, 24, and 52. New abnormal 
physical exam findings must be documented and will be followed by a physician or other 
qualified staff at the next scheduled visit. 
9.1.5 Vital Signs 
Body temperature, blood pressure, pulse and respirations will be performed after resting 
for 5 minutes at all site visits.  

 
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9.1.6 Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study. 
Duration (start and stop dates and times), severity/grade, outcome, treatment and relation 
to study drug will be recorded on the case report form (CRF). 
 
9.1.7 EASI and IGA 
Eczema Area and Severity Index (EASI) and Investigator Global Assessment (IGA) will 
be completed at screening and weeks 0, 2, 4, 8, 12, 16, 24, 36, 44, and 52. 
9.1.8 Pruritus Numerical Rating 
Pruritus numerical rating will be completed at weeks 0, 2, 4, 8, 12, 16, 24, 36, 44, and 52. 
9.1.9 Photography 
Photographs (full front and back) will be taken at weeks 0, 2, 4, 12, and 52.  
 
9.2 Clinical Laboratory Measurements 
9.2.1 Hematology 
Blood will be obtained and sent to the clinical hematology lab for a complete blood count 
(hemoglobin, hematocrit, red blood cell count, white blood cell count, white blood cell 
differential, and platelet count) determinations for assessment of systemic evidence for 
infection and/or inflammation at Screening. 
9.2.2 Blood Chemistry Profile 
Blood will be obtained and sent to the clinical chemistry lab for determination of 
electrolyte levels, serum BUN, creatinine, AST, ALT, and alkaline phosphatase at 
screening. 
9.2.3 Pregnancy Test 
A urine pregnancy test will be obtained at the study site for female subjects of 
childbearing age at screening and weeks 0, 4, 8, 12, 16, 24, 36, 44, and 52. 
 
9.2.4   Microbiome Sampling 
 
Sterile-technique skin swabbing and collection of stool samples will be performed at 
weeks 0, 2, 4, 12, and 52. Specimens will be stored at -80 C for future analysis. 
 
9.2.5   Skin biopsy procedure 
 
At weeks 0, 2, 4, and 12, four standard 5mm punch biopsies (the approximate size of a 
pencil-end eraser) will be performed. Of the biopsies performed at each visit, 3 will be 
from atopic dermatitis lesional skin and 1 will be from non-lesional (clear) skin. After 
selecting biopsy sites on trunk or extremities (subject preference), the area will be 

 
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cleansed with an antiseptic solution and a numbing medication (lidocaine or lidocaine 
with epinephrine) will be injected to the biopsy area. Each 5mm piece of skin will be 
removed via punch biopsy and a sterile gauze pad will be placed over the site to control 
bleeding. The biopsy site may be closed with a stitch. The parents will be provided with 
post-biopsy care instructions. All standard of care guidelines and sterile technique will be 
used. These specimens will be analyzed by CyTOF and RNAseq.  
 
9.2.6   Pre-treatment and post-treatment dupilumab testing  
Pre-dupilumab patch testing: 
Each of the 15 patients will be patch tested to a core series of allergens (see below). The 
allergens will be applied to the back of the patient using Finn Chambers (SmartPractice, 
Phoenix, AZ) on Scanpor tape (Norgesplaster Alpharma AS, Vennesla, Norway). More 
Scanpor tape will be applied over the patches to secure them to the back. 48 hours later 
the patient will return for patch removal and photography. Readings will be graded as 
follows: ? (weak/doubtful), + (mild), ++ (strong), or +++ (very strong) on the basis of 
degrees of induration, papules, vesicles, and/or spreading beyond the disc space. A final 
determination of ‘‘allergic/positive’’ or ‘‘not allergic’’ will be determined on the basis of 
the pattern (crescendo/decrescendo), patch test appearance, and known characteristics of 
that allergen. For example, a weak/doubtful (macular erythema) reaction to a 
formaldehyde-releasing agent could be determined to be an allergic/positive reaction in 
the setting of multiple or stronger reactions to related formaldehyde-releasing allergens.  
The clinical relevance of the positive patch test reaction will be determined using the 
patient’s history and clinical skin examination. Current relevance is defined as definite (a 
use test with the suspected item containing the putative allergen was positive, or a patch 
test to the object or product was positive), probable (the allergen can be verified as present 
in known skin contactants, and the clinical presentation was consistent), or possible (the 
patient is exposed to circumstances in which skin contact with materials known to contain 
the allergen was likely to occur). Past relevance will also be indicated. At the final visit 
the patient will be counseled regarding allergen avoidance. 
 
Post-dupilumab patch testing: 
After 6 months of dupilumab therapy the patient will undergo a second round of patch 
testing, to the same core series of allergens and using the same procedures described 
above. If new allergens are identified, additional avoidance counseling will occur. 
 
Timing and logistics: 
Each patch test will require 2 three-part patient visits. Application of allergens will take 
approximately 20 minutes. Removal and photography (48 hours later) approximately 10 
minutes. Final reading (120 hours after placement) and allergen avoidance counseling will 
occur at the final visit (approximately 30 minutes).  
 
ACD Core 80 Series 

 
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1. Benzocaine, 5.0% pet. 
   
2. 2-Mercaptobenzothiazole, 1.0% pet. 
   
3. Colophonium (rosin), 20.0% pet. 
   
4. 4-phenylenediamine base, 1.0% pet. 
   
5. Benzyl Alcohol 10.0% 
   
6. Fragrance mix II, 14.0% pet. 
   
7. Lanolin alcohol (Amerchol L101), 50% pet. 
   
8. Carba mix, 3.0% pet. 
   
9. Neomycin sulfate, 20.0% pet. 
   
10. Thiuram mix, 1.0% pet. 
   
11. Oleamidopropyl Dimethylamine 0.1% aq. 
   
12. Ethylenediamine dihydrochloride, 1.0% pet. 
   
13. Bisphenol A epoxy resin, 1.0% pet. 
   
14. Quaternium-15, 2.0% pet. 
   
15. 4-tert-butylphenol formaldehyde resin, 1.0%  
   
16. Ethylhexyl Salicylate, 5.0% pet. 
   
17. Fusidic acid sodium salt, 2% pet. 
   
18. Potassium dichromate, 0.25% pet. 
   
19. Myroxylon Pereirae resin (Balsam of Peru) 
   
20. Nickel sulfate hexahydrate, 2.5% pet. 
   
21. Diazolidinyl urea (Germall II), 1.0% pet. 
   
22. DMDM hydantoin (Germall 115), 1.0% pet. 
   
23. Imidazolidinyl urea, 2.0% pet. 
   
24. Bacitracin, 20.0% pet. 
   
25. Mixed dialkyl thioureas, 1.0% pet. 
   
26.Methylchloroisothiazolinone/methylisothiazolinone,  
   
27. Paraben mix, 12.0% pet. 
   
28. Cinnamal (Cinnamic aldehyde), 1.0% pet. 
   
29. Fragrance mix I, 8.0% pet. 
   
30. Amidoamine(stearamidopropyl dimethylamine) 
   
31. 2-Bromo-2-nitropropane -1,3-diol (Bronopol) 
   
32. Sesquiterpenelactone mix, 0.1% pet. 
   
33. 2-Hydroxyethyl methacrylate, 2.0% pet. 
   
34. Propylene glycol, 30.0% aq. 
   
35. Benzophenone-3 (Oxybenzone) 
   
36. Chloroxylenol (4-Chloro-3,5-xylenol), 1.0% pet. 
   
37. Isopropyl myristate, 20.0% pet. 
   
38. Methylisothiazolinone, 0.2% aq. (2000 ppm) 
   
39. Desoximetasone, 1.0% pet. 
   
40. Methyldibromo  glutaronitrile/Phenoxyethanol, (MDBGN/PE) (Euxyl K 400), 2.0% pet   
41. Triethanolamine, 2% pet. 
   
42. Tocopherol (DL-a-Tocopherol),, 100.0% 
   
43. Iodopropynyl butylcarbamate, 0.5% pet. 
   
44. Ethyl acrylate, 0.1% pet. 
   
45. Polysorbate 40, 5% pet. 
   
46. Tosylamide /formaldehyde resin, 10.0% pet. 
   
47. Methyl methacrylate, 2.0% pet. 
   
48. Cobalt (ii) chloride hexahydrate, 1.0% pet. 
   
49. Tixocortol-21-pivalate, 1.0% pet. 
   

 
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50. Budesonide, 0.1% pet. 
   
51. Hydroperoxide of linalool, 1% pet. 
   
52. Disperse blue mix 124/106, 1.0% pet. 
   
53. Propolis, 10.0% pet. 
   
54. Lidocaine-HCI, 15.0% pet. 
   
55. Hydroperoxied of limonene, 0.3 pet. 
   
56. Clobetasol-17-propionate, 1.0% pet. 
   
57. Cocamidopropyl betaine, 1.0% aq. 
   
58. Formaldehyde, 2% aq. 
   
59. Disperse orange 3, 1% pet. 
61. Cocamide DEA 
   
62. Compositae mix, 6.0% pet. 
   
63. 2-n-octyl-4-isothiazolin-3-one, 0.1% pet.  
   
64. Melaleuca Alternifolia (tea tree leaf oil) 
   
65. Cananga Odorata Flower oil (ylang –ylang oil) 
   
66. Benzyl salicylate, 10% pet. 
   
67. Disperse yellow 3, 1% pet. 
   
68. Decyl glucoside, 5.0% pet. 
   
69. Mercapto mix, 1.0% pet. 
   
70. Glyceryl thiogycolate, 1% pet.  
   
71.  Benzoyl peroxide, 1% pet. 
   
72. Dibucaine hydrochloride, 2.5% pet. 
   
73. 2-tert-buytl-4-methoxylphenol, 2% pet. 
   
74. Isoamyl-p-methoxycinnamate, 10% pet. 
   
75. Ethyleneurea, melamine formaldehyde mix, 5%  
   
76. Gold sodium thiosulfate, 0.5% pet. 
   
77. Hydroxyisohexyl 3-cyclohexene carboxaldehyde, 5% pet. 
   
78. Texile mix, 6.6% pet. 
   
79. Glutural, 0.5% pet. 
   
80. Diphenylguanidine, 1.0% pet. 
   
 
10 EVALUATIONS BY VISIT 
See Appendix 1.  
10.1  Visit 0 (Screening) 
1. 
Review inclusion/exclusion criteria. 
2. 
Review the study with the subject and obtain written informed consent and 
HIPAA authorization and assent, if appropriate. 
3. 
Assign the subject a unique screening number. 
4. 
Record demographics data. 
5. 
Record medical history, including a history of atopic dermatitis, diagnosis date, 
and prior treatments for atopic dermatitis. 
6. 
Record concomitant medications.  
7. 
Perform and record vital signs. 
8. 
Perform a complete physical examination. 
9. 
Perform EASI and IGA. 

 
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10. Perform pruritus numerical rating. 
11. Collect blood for clinical laboratory tests (CBC, serum electrolytes, BUN, 
Creatinine, AST, ALT, alkaline phosphatase). 
12. Perform urine pregnancy test (if applicable). 
 
10.2 Visit 1: PRE-TREATMENT PATCH TESTING 
1. Application of contact allergens (20 minutes). 
2. 48 hours later: removal of allergens and photography (30 minutes). 
3. 120 hours later: final reading of allergens (10 minutes) and allergen avoidance 
counseling (approximately 30 minutes).  
 
10.3 Visit 2 (week 0) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs.  
4. Perform a complete physical examination. 
5. Perform EASI and IGA. 
6. Perform pruritus numerical rating.  
7. Perform urine pregnancy test (if applicable). 
8. Perform skin biopsy procedure. 
9. Obtain photographs. 
10. Dispense study medication. 
11. Collect skin swabs and stool sample. 
 
10.4 Visit 3 (week 2) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA. 
5. Perform pruritus numerical rating. 
6. Perform skin biopsy procedure. 
7. Obtain photographs. 
8. Dispense study medication. 
9. Collect skin swabs and stool sample. 
 
10.5 
Visit 4 (week 4)  
1. Record any Adverse Experiences 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA. 
5. Perform pruritus numerical rating. 
6. Perform urine pregnancy test (if applicable).  

 
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7. Perform skin biopsy procedure. 
8. Obtain photographs. 
9. Dispense study medication. 
10. Collect skin swabs and stool sample. 
 
10.6  Visit 5 (week 8)  
1. Record any Adverse Experiences. 
2. Concomitant medication review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA.  
5. Perform pruritus numerical rating.  
6. Perform urine pregnancy test (if applicable).  
7. Dispense study medication. 
 
10.7 
Visit 6 (week 12) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform a complete physical examination.  
5. Perform EASI and IGA. 
6. Perform pruritus numerical rating.  
7. Perform urine pregnancy test (if applicable). 
8. Perform skin biopsy procedure. 
9. Obtain photographs. 
10. Dispense study medication. 
11. Collect skin swabs and stool sample. 
 
10.8 
Visit 7 (week 16) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA assessments. 
5. Perform pruritus numerical rating.  
6. Perform urine pregnancy test (if applicable). 
7. Dispense study medication. 
 
10.9 
Visit 8 (week 24) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs.  
4. Perform a complete physical examination. 
5. Perform EASI and IGA. 

 
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6. Perform pruritus numerical rating.  
7. Perform urine pregnancy test (if applicable). 
8. Dispense study medication 
 
10.10 Visit 9 POST-TREATMENT PATCH TESTING (between weeks 24 and 36) 
1. Application of contact allergens (20 minutes). 
2. 48 hours later: removal of allergens and photography (30 minutes). 
3. 120 hours later: final reading of allergens (10 minutes) and allergen avoidance 
counseling (approximately 30 minutes).  
 
10.11 Visit 10 (week 36) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA. 
5. Perform pruritus numerical rating. 
6. Perform urine pregnancy test (if applicable). 
7. Dispense study medication. 
 
10.12 Visit 11 (week 44) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform EASI and IGA. 
5. Perform pruritus numerical rating.  
6. Perform urine pregnancy test (if applicable). 
7. Dispense study medication. 
 
10.13 Visit 12: END OF TREATMENT (week 52) 
1. Record any Adverse Experiences. 
2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform a complete physical examination. 
5. Perform EASI and IGA. 
6. Perform pruritus numerical rating. 
7. Perform urine pregnancy test (if applicable). 
8. Obtain photographs. 
9. Collect skin swabs and stool sample. 
 
10.14 EARLY WITHDRAWAL / TERMINATION VISIT 
1. Record any Adverse Experiences. 

 
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2. Concomitant medications review. 
3. Perform and record vital signs. 
4. Perform a complete physical examination. 
5. Perform EASI and IGA. 
6. Perform pruritus numerical rating. 
7. Perform urine pregnancy test (if applicable). 
8. Obtain photographs. 
 
For healthy controls: 
1) Clinical consent will be obtained prior to surgery by the surgeon or their research 
coordinator for surgical tissue that will be resected for clinical purposes and will otherwise 
be discarded. 
2) The surgeon will notify our technician the day before samples are collected 
3) On the day of surgery, redundant skin will be obtained in the operating room. 
4) The samples will be de-identified at the time of harvest. 
5) The surgeon will call our technician (after the samples have been harvested and de-
identified) to let them know to come to the operating room suite to pick up the samples. 
6) The samples will be transported by our technician from outside the operating room to 
the lab in a biohazard-labeled container. 
7) In the lab, the samples will be given a unique identifier that is unknown to the surgical 
team. Thus, there will be no way for the surgical team to identify which sample came 
from which patient and there is no way for the laboratory team to know any patient 
information about the samples. This is in accordance with an agreement stating that 
the individual providing the specimen will never provide the recipient research team with 
identifiable information. 
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
11.1  Adverse Events 
Definition of an AE: Any untoward medical occurrence in a subject administered a 
pharmaceutical product that does not necessarily have a causal relationship with the 
treatment. An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not considered related to the investigational 
medicinal product. 
Investigational Medicinal Product (IMP) includes the drug under evaluation and the 
comparator drug(s) if specified as part of the research objective, given at any time during 
the study. Medical conditions/diseases present before starting the drug of interest are only 
considered adverse events if they worsen after starting the drug of interest. 
The occurrence of adverse events will be sought by non-directive questioning of the patient 
at each visit during the study. Adverse events also may be detected when they are 
volunteered by the patient during or between visits or through physical examination, 
laboratory test, or other assessments. All adverse events will be recorded in the study 
database including the following information: 

 
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1. the severity grade (mild, moderate, severe) 
2. its relationship to the drug(s) of interest (suspected/not suspected) 
3. its duration (start and end dates or if continuing at final exam) 
4. whether it constitutes a serious adverse event (SAE) 
 
AE Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events 
(CTCAE) Version 3.0 should be used to assess and grade AE severity, including laboratory 
abnormalities judged to be clinically significant. The modified criteria can be found in the 
study manual.  If the experience is not covered in the modified criteria, the guidelines shown 
in below should be used to grade severity.  It should be pointed out that the term “severe” 
is a measure of intensity and that a severe AE is not necessarily serious. 
 
Table 1.  AE Severity Grading 
Severity (Toxicity Grade) 
Description 
Mild (1) 
Transient or mild discomfort; no limitation in activity; no medical 
intervention or therapy required. The subject may be aware of the sign 
or symptom but tolerates it reasonably well. 
Moderate (2) 
Mild to moderate limitation in activity, no or minimal medical 
intervention/therapy required. 
Severe (3) 
Marked limitation in activity, medical intervention/therapy required, 
hospitalizations possible. 
Life-threatening (4) 
The subject is at risk of death due to the adverse experience as it 
occurred. This does not refer to an experience that hypothetically 
might have caused death if it were more severe. 
 
Table 2.  AE Relationship to Study Drug 
Relationship 
to Drug 
Comment 
Definitely 
Previously known toxicity of agent; or an event that follows a reasonable temporal 
sequence from administration of the drug; that follows a known or expected response 
pattern to the suspected drug; that is confirmed by stopping or reducing the dosage of 
the drug; and that is not explained by any other reasonable hypothesis. 
Probably 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to the suspected drug; that is 
confirmed by stopping or reducing the dosage of the drug; and that is unlikely to be 
explained by the known characteristics of the subject’s clinical state or by other 
interventions. 

 
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Possibly 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to that suspected drug; but 
that could readily have been produced by a number of other factors. 
Unrelated 
An event that can be determined with certainty to have no relationship to the study 
drug. 
 
 
 

 
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11.2 
Serious Adverse Experiences (SAE) 
A SAE is any untoward medical occurrence that at any dose: 
• results in death, 
• is life-threatening, 
• requires inpatient hospitalization or prolongation of existing hospitalization, 
• results in persistent or significant disability/incapacity, 
• is a congenital anomaly/birth defect,  
• is otherwise a significant medical event. 
This includes any SAEs likely to arise from the trial indication or progression of 
underlying/concomitant illness(es) (e.g. progression of cancer in oncology trials), unless 
specified in the protocol as study specific exemptions. 
Any SAE, irrespective of causality, occurring after the subject has provided informed 
consent and until four weeks after the subject has stopped study participation must be 
reported unless otherwise stated in the protocol. SAEs occurring after four weeks from 
ending study participation should only be reported if considered by the Investigator 
attributable to the exposure to the investigational drug(s) during the trial period. This 
includes the period in which the study protocol interferes with the standard medical 
treatment given to a subject, even if study treatment has not yet started (e.g. withdrawal of 
previous treatment during washout period, change in treatment to a fixed dose of 
concomitant medication). 
11.3 
Serious Adverse Experience Reporting 
Study sites will document all SAEs that occur (whether or not related to study drug) per 
UCSF IRB Guidelines.  The collection period for all SAEs will begin after informed 
consent is obtained and end after procedures for the final study visit have been completed. 
SAEs occurring after four weeks from ending study participation should only be reported if 
considered by the Investigator attributable to the exposure to the investigational drug(s) 
during the trial period. This includes the period in which the study protocol interferes with 
the standard medical treatment given to a subject, even if study treatment has not yet started 
(e.g. withdrawal of previous treatment during washout period, change in treatment to a fixed 
dose of concomitant medication). 
In accordance with the standard operating procedures and policies of the local Institutional 
Review Board (IRB)/Independent Ethics Committee (IEC), the site investigator will report 
SAEs to the IRB/IEC.   
Timelines: All serious adverse events (SAEs) must be reported by the sites to Sponsor 
within 24 hours of occurrence of the SAE. The timelines for investigator initiated trials 
reporting to Regeneron Pharmaceuticals and Sanofi-Genzyme will be done as per Third 
Party Study/Investigator Initiated Trial Agreement.   

 
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Follow-up reports: SAEs will be followed until resolution or until it is judged to be 
permanent, and an assessment will be made at each visit (or more frequently, if necessary) 
of any changes in severity, the suspected relationship to the drug of interest, the 
interventions required to treat it, and the outcome.  
The Sponsor shall support Regeneron and Sanofi-Genzyme in the following-up of all SAEs 
so that complete information is available to maintain patient safety and also as part of any 
commitments by Regeneron and Sanofi-Genzyme to any Health authority OR specific 
Health authority follow-up requests for the product under investigation. 
Pregnancies: Any occurrences of a pregnancy in a patient (or a patients partner) during 
study participation will be collected. All pregnancies will be followed up to determine 
outcome, including spontaneous or voluntary termination, details of the birth, and the 
presence or absence of any birth defects, congenital abnormalities, or maternal and/or 
newborn complications. 
 
12 
DISCONTINUATION AND REPLACEMENT OF SUBJECTS 
12.1 Early Discontinuation of Study Drug  
A subject may be discontinued from study treatment at any time if the subject, the 
investigator, or the Sponsor feels that it is not in the subject’s best interest to continue.  
The following is a list of possible reasons for study treatment discontinuation:  
• Subject withdrawal of consent (or assent) 
• Subject is not compliant with study procedures 
• Adverse event that in the opinion of the investigator would be in the best interest 
of the subject to discontinue study treatment 
• Protocol violation requiring discontinuation of study treatment 
• Lost to follow-up 
• Sponsor request for early termination of study 
• Positive pregnancy test (females) 
If a subject is withdrawn from treatment due to an adverse event, the subject will be 
followed and treated by the Investigator until the abnormal parameter or symptom has 
resolved or stabilized.   
All subjects who discontinue study treatment should come in for an early discontinuation 
visit as soon as possible and then should be encouraged to complete all remaining 
scheduled visits and procedures. 
All subjects are free to withdraw from participation at any time, for any reason, specified 
or unspecified, and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals.  The reason for the subject’s withdrawal from the study will be specified in 

 
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the subject’s source documents Refer to Section 10.13 for early termination procedures. 
12.2 
Withdrawal of Subjects from the Study 
A subject may be withdrawn from the study at any time if the subject, the investigator, or 
the Sponsor feels that it is not in the subject’s best interest to continue.   
All subjects are free to withdraw from participation at any time, for any reason, specified 
or unspecified, and without prejudice. 
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals.  The reason for the subject’s withdrawal from the study will be specified in 
the subject’s source documents. As noted above, subjects who discontinue study treatment 
early (i.e., they withdraw prior to Visit 12) should have an early discontinuation visit. 
Refer to Section 10.13 for early termination procedures. Subjects who withdraw after 
Visit 1 but prior to Visit 10 should be encouraged to come in for a final visit (and the 
procedures to be followed would include those for their next scheduled visit). Subjects 
who sign the informed consent form (ICF) and who are discontinued or withdraw from the 
study before study product administration will be defined as screen failures. No data will 
be collected in the CRFs for screen failure subjects. 
13 
PROTOCOL VIOLATIONS 
A protocol violation occurs when the subject, investigator, or Sponsor fails to adhere to 
significant protocol requirements affecting the inclusion, exclusion, subject safety and 
primary endpoint criteria.  Protocol violations for this study include, but are not limited to, 
the following: 
• Failure to meet inclusion/exclusion criteria. 
• Use of a prohibited concomitant medication. 
• Non-compliance with study drug regimen. 
• Non-compliance with study visit procedures. 
• Failure to comply with Good Clinical Practice (GCP) guidelines will also result in 
a protocol violation. The PI will determine if a protocol violation will result in 
withdrawal of a subject. 
When a protocol violation occurs, it will be discussed with the investigator and a Protocol 
Violation Form detailing the violation will be generated. This form will be signed by a 
Sponsor representative and the Investigator. A copy of the form will be filed in the site’s 
regulatory binder and in the Sponsor’s files. 
14 STATISTICAL METHODS AND CONSIDERATIONS 
Prior to the analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will 
be written describing all analyses that will be performed.  The SAP will contain any 
modifications to the analysis plan described below.   

 
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14.1  Analysis of Primary Endpoint 
CyTOF: One sample paired t-test comparing percentage change in lesional and non-
lesional IL-4 positive, CD4+ T effector cells at weeks 2, 4, 12 compared to baseline. 
14.2 
Analysis Secondary Endpoints 
RNA-seq: Identification of differentially expressed genes at weeks 2, 4, 12 compared to 
pre-treatment baseline in dupilumab-treated patients within each immune population 
studied using the statistical program Cuffdiff (paired analysis). 
We will also explore whether early molecular changes are predictive of the clinical response 
to dupilumab at 52 weeks (reduction of EASI and improvement in pruritus score), which 
could lead to the identification of novel biomarkers.  
 
14.3 
Safety Data 
Safety and tolerability data will be summarized for the treatment group, which is the 15 
subjects who receive treatment with dupilumab.  
Adverse event rates will be coded by body system and MedDra classification term. 
Adverse events will be tabulated and will include the number of patients for whom the 
event occurred, the rate of occurrence, and the severity and relationship to study drug.   
14.4 Sample Size 
15 subjects will receive dupilumab for a treatment period of 52 weeks (i.e. last injection 
on week 50). All subjects will undergo skin biopsies for molecular profiling. 
Sample size justification 
CyTOF: We estimate the minimum effect size that would be clinically relevant would 
be a reduction in lesional IL-4 positive, CD4+ T lymphocytes of 10%. The standard 
deviation of the change in lesional IL-4 positive, CD4+ T lymphocytes in the patient 
population is estimated at 10%. Therefore, our standardized effect size is 1.00. For the 
statistical analysis, we will set alpha at 0.05, beta at 0.20 (power = 0.8). The resulting 
sample size estimate for a one sample paired t test is 9 patients. Our proposal uses a total 
n=15.  
 
RNA-seq: To determine statistical power to identify differentially expressed genes as 
measured by RNA-seq, we used the Scotty (Busby et al.) power calculator which uses a t-
statistic and incorporates the variance attributable to read depth. Performing RNA-seq on 
15 samples before and after treatment to a read depth of 50 million per sample as 
proposed here, we can detect >97% of transcripts displaying 2X fold change and >80% of 
transcripts displaying 1.5X fold change with p<0.01 (Figure 4). 

 
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Fig 4. Power to detect differentially expressed genes using 
RNA-Seq. 
 
15 
DATA COLLECTION, RETENTION 
AND MONITORING 
15.1 
Data Collection Instruments 
The Investigator will prepare and maintain adequate and accurate source documents 
designed to record all observations and other pertinent data for each subject. Study 
personnel at each site will enter data from source documents corresponding to a subject’s 
visit into the protocol-specific electronic or paper Case Report Form (eCRF) when the 
information corresponding to that visit is available. Subjects will not be identified by 
name in the study database or on any study documents, but will be identified by a site 
number, subject number and initials. 
If a correction is required for an eCRF, the time and date stamps track the person entering 
or updating eCRF data and creates an electronic audit trail. If a correction is made on a 
paper CRF, the study staff member will line through the incorrect data, write in the correct 
data and initial and date the change. Changes made to an eCRF should also be made to on 
a paper CRF, and vice versa, if possible. 
The Investigator is responsible for all information collected on subjects enrolled in this 
study.  All data collected during the course of this study must be reviewed and verified for 
completeness and accuracy by the Investigator. A copy of the CRF will remain at the 
Investigator’s site at the completion of the study. 
15.2  Data Management Procedures 
The data will be entered into a validated database.  The Data Management group will be 
responsible for data processing, in accordance with procedural documentation.  Database 
lock will occur once quality assurance procedures have been completed. 
All procedures for the handling and analysis of data will be conducted using good 
computing practices meeting FDA guidelines for the handling and analysis of data for 
clinical trials. 
 
15.3 
Data Quality Control and Reporting 
After data have been entered into the study database, a system of computerized data 
validation checks will be implemented and applied to the database on a regular basis. On 
eCRFs, queries are entered, tracked, and resolved through the EDC system directly. On 

 
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paper, query reports (Data Clarification Requests) pertaining to data omissions and 
discrepancies will be forwarded to the Investigators and study monitors for resolution.  
The study database will be updated in accordance with the resolved queries.  All changes 
to the study database will be documented. 
15.4 
Archival of Data 
The database is safeguarded against unauthorized access by established security 
procedures; appropriate backup copies of the database and related software files will be 
maintained. Databases are backed up by the database administrator in conjunction with 
any updates or changes to the database.   
At critical junctures of the protocol (e.g., production of interim reports and final reports), 
data for analysis is locked and cleaned per established procedures. 
 
15.5 
Availability and Retention of Investigational Records 
The Investigator must make study data accessible to the monitor, other authorized 
representatives of the Sponsor (or designee), IRB/IEC, and Regulatory Agency (e.g., 
FDA) inspectors upon request. A file for each subject must be maintained that includes the 
signed Informed Consent, HIPAA Authorization and Assent Form and copies of all source 
documentation related to that subject. The Investigator must ensure the reliability and 
availability of source documents from which the information on the CRF was derived. 
All study documents (patient files, signed informed consent forms, copies of CRFs, Study 
File Notebook, etc.) must be kept secured for a period of two years following marketing of 
the investigational product or for two years after centers have been notified that the IND 
has been discontinued. There may be other circumstances for which the Sponsor is 
required to maintain study records and, therefore, the Sponsor should be contacted prior to 
removing study records for any reason. 
 
15.6 
Subject Confidentiality 
In order to maintain subject confidentiality, only a site number, subject number and 
subject initials will identify all study subjects on CRFs and other documentation submitted 
to the Sponsor.  Additional subject confidentiality issues (if applicable) are covered in the 
Clinical Study Agreement. 
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of 
Human Volunteers (21 CFR 50), Institutional Review Boards (21 CFR 56), and 
Obligations of Clinical Investigators (21 CFR 312). 
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other 
records will be identified by a coded number and initials only. All study records will be 
kept in a locked file cabinet and code sheets linking a patient’s name to a patient 
identification number will be stored separately in another locked file cabinet.  Clinical 
information will not be released without written permission of the subject, except as 
necessary for monitoring by the FDA.  The Investigator must also comply with all 

 
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applicable privacy regulations (e.g., Health Insurance Portability and Accountability Act 
of 1996, EU Data Protection Directive 95/46/EC). 
16.1 
Protocol Amendments 
Any amendment to the protocol will be written by the PI or a sub-investigator with the 
PI’s approval. Protocol amendments cannot be implemented without prior written 
IRB/IEC approval except as necessary to eliminate immediate safety hazards to patients.  
A protocol amendment intended to eliminate an apparent immediate hazard to patients 
may be implemented immediately, provided the IRBs are notified within five working 
days. 
16.2 
Institutional Review Boards and Independent Ethics Committees 
The protocol and consent form will be reviewed and approved by the IRB/IEC of each 
participating center prior to study initiation.  Serious adverse experiences regardless of 
causality will be reported to the IRB/IEC in accordance with the standard operating 
procedures and policies of the IRB/IEC, and the Investigator will keep the IRB/IEC 
informed as to the progress of the study.  The Investigator will obtain assurance of 
IRB/IEC compliance with regulations. 
Any documents that the IRB/IEC may need to fulfill its responsibilities (such as protocol, 
protocol amendments, Investigator’s Brochure, consent forms, information concerning 
patient recruitment, payment or compensation procedures, or other pertinent information) 
will be submitted to the IRB/IEC.  The IRB/IECs written unconditional approval of the 
study protocol and the informed consent form will be in the possession of the Investigator 
before the study is initiated.  The IRB/IECs unconditional approval statement will be 
transmitted by the Investigator to the designee prior to the shipment of study supplies to 
the site.  This approval must refer to the study by exact protocol title and number and 
should identify the documents reviewed and the date of review. 
Protocol and/or informed consent modifications or changes may not be initiated without 
prior written IRB/IEC approval except when necessary to eliminate immediate hazards to 
the patients or when the change(s) involves only logistical or administrative aspects of the 
study.  Such modifications will be submitted to the IRB/IEC and written verification that 
the modification was submitted and subsequently approved should be obtained.   
The IRB/IEC must be informed of revisions to other documents originally submitted for 
review; serious and/or unexpected adverse experiences occurring during the study in 
accordance with the standard operating procedures and policies of the IRB; new 
information that may affect adversely the safety of the patients of the conduct of the study; 
an annual update and/or request for re-approval; and when the study has been completed. 
16.3 
Informed Consent Form  
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH 
GCP, US Code of Federal Regulations for Protection of Human Subjects (21 CFR 
50.25[a,b], CFR 50.27, and CFR Part 56, Subpart A), the Health Insurance Portability and 
Accountability Act (HIPAA, if applicable), and local regulations. 

 
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The Investigator will prepare the informed consent form, assent and HIPAA authorization 
and provide the documents to the Sponsor or designee for approval prior to submission to 
the IRB/IEC.  The consent form generated by the Investigator must be acceptable to the 
Sponsor and be approved by the IRB/IEC.  The written consent document will embody the 
elements of informed consent as described in the International Conference on 
Harmonization and will also comply with local regulations. The Investigator will send an 
IRB/IEC-approved copy of the Informed Consent Form to the Sponsor (or designee) for 
the study file. 
A properly executed, written, informed consent will be obtained from each subject prior to 
entering the subject into the trial.  Information should be given in both oral and written 
form and subjects (or their legal representatives) must be given ample opportunity to 
inquire about details of the study.  If appropriate and required by the local IRB/IEC, assent 
from the subject will also be obtained.  If a subject is unable to sign the informed consent 
form (ICF) and the HIPAA authorization, a legal representative may sign for the subject.   
A copy of the signed consent form (and assent) will be given to the subject or legal 
representative of the subject and the original will be maintained with the subject’s records. 
16.4 
Publications  
The preparation and submittal for publication of manuscripts containing the study results 
shall be in accordance with a process determined by mutual written agreement among the 
study Sponsor and participating institutions.  The publication or presentation of any study 
results shall comply with all applicable privacy laws, including, but not limited to, the 
Health Insurance Portability and Accountability Act of 1996.  
16.5 
Investigator Responsibilities 
By signing the Agreement of Investigator form, the Investigator agrees to: 
1. Conduct the study in accordance with the protocol and only make changes after 
notifying the Sponsor (or designee), except when to protect the safety, rights or 
welfare of subjects. 
11 Personally conduct or supervise the study (or investigation). 
12 Ensure that the requirements relating to obtaining informed consent and IRB review 
and approval meet federal guidelines, as stated in § 21 CFR, parts 50 and 56. 
13 Report to the Sponsor or designee any AEs that occur in the course of the study, in 
accordance with §21 CFR 312.64. 
14 Ensure that all associates, colleagues and employees assisting in the conduct of the 
study are informed about their obligations in meeting the above commitments. 
15 Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to 
make those records available for inspection with the Sponsor (or designee). 
16 Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be 
responsible for initial and continuing review and approval of the clinical study. 

 
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17 Promptly report to the IRB and the Sponsor (or designee) all changes in the research 
activity and all unanticipated problems involving risks to subjects or others (to 
include amendments and IND safety reports). 
18 Seek IRB approval before any changes are made in the research study, except when 
necessary to eliminate hazards to the patients/subjects. 
19 Comply with all other requirements regarding the obligations of clinical investigators 
and all other pertinent requirements listed in § 21 CFR part 312. 
 
17 
References 
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reveals biological pathways and roles for coding and long non-coding RNAs. BMC 
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APPENDIX 1.  EVALUATION SCHEDULE 
Visit number 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
 
Time of Visit 
Screening 
Patch testing 
wk  
0 
wk  
2 
wk  
4 
wk  
8 
wk  
12 
wk  
16 
wk  
24 
Patch testing 
wk  
36 
wk 
 44 
wk 
 52 
Early termination 
Inclusion/Exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
X 
 
X 
 
 
 
X 
 
X 
 
 
 
X 
X 
Vital Signs 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
EASI and IGA 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Pruritus Numerical Rating 
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Laboratory assessments* 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Pregnancy  
X 
 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Adverse events 
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Review con meds 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
X 
Biopsy Procedures  
 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
Skin swabs and stool sample 
 
 
X 
X 
X 
 
X 
 
 
 
 
 
X 
 
Photography 
 
 
X 
X 
X 
 
X 
 
 
 
 
 
X 
X 
Dispense study medication 
 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
 
 
Patch testing 
 
X  
 
 
 
 
 
 
 
X 
 
 
 
 
*CBCPD, BMP, BUN, Cr, AST/ALT/Alk Phos