A Phase I, Open-Label, Multicenter Trial Exploring the Safety and Tolerability of Autologous Polyclonal Regulatory T Cell Therapy in Adults With Active Pemphigus (APG01)
Phase: PHASE1
Conditions: Pemphigus Foliaceus, Pemphigus Vulgaris
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

 
C O N F I D E N T I A L 
 
 
Autoimmunity Centers of Excellence (ACE) 
 
 
Protocol APG01 
 
A Phase I, Open-Label, Multicenter Trial Exploring the Safety and 
Tolerability of Autologous Polyclonal Regulatory T Cell Therapy in Adults 
with Active Pemphigus 
 
Short Title: PolyTregs for Pemphigus 
 
BB-IND 17351 
 
 
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IND Sponsor:  Division of Allergy, Immunology, and Transplantation (DAIT) 
 
National Institute of Allergy and Infectious Diseases (NIAID) 
 
National Institutes of Health (NIH) 
 
 
 
 
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior permission of 
the Protocol Chairs, or the Division of Allergy, Immunology and Transplantation; the National Institute of Allergy 
and Infectious Diseases; and the National Institutes of Health. 
 

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from Week 8 to Week 12, then quarterly visits from Week 26 to Week 52, then twice a year visits 
until Week 156.  
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FLOW DIAGRAMS OF PROTOCOL 
 
Figure 1, Subject Participation 
 
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Figure 2, Study Design 
 
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Figure 3, Safety Review Committee Process 
 
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ABBREVIATIONS 
 
ACE 
Autoimmunity Centers of Excellence 
ACS 
American Cancer Society 
ADCT 
Autoimmune Diseases Clinical Trials 
AE 
Adverse event 
APGAR 
Appearance, pulse, grimace, activity, and respiration 
ALK 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
ANA 
Anti-Nuclear Antibody 
AST 
Aspartate Aminotransferase 
AUC 
Area Under the Curve 
BCG 
Bacille Calmette Guerin 
BID 
Bis in Die (twice a day) 
BUN 
Blood Urea Nitrogen 
CBC 
Complete Blood Count 
CDC 
Centers for Disease Control and Prevention 
CFDA-SE 
Carboxyfluorescein Diacetate Succinimidyl Ester 
CFR 
Code of Federal Regulations 
CFSE 
Carboxyfluorescein Succinimidyl Ester 
cGMP 
Current Good Manufacturing Practice 
CLE 
Cutaneous Lupus 
CMV 
Cytomegalovirus 
CoA 
Certificate of Analysis 
CRF 
Case Report Form 
CRS 
Cytokine Release Syndrome 
CTCAE 
Common Terminology Criteria for Adverse Events 
d 
Day 
dA 
Deoxyadenosine 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DAIT RMC at PPD 
DAIT Regulatory Management Center at Pharmaceutical Product 
Development, Inc. 
DAIT-SACCC 
DAIT Statistical and Clinical Coordinating Center 
dG 
Deoxyguanosine 
DHHS 
Department of Health and Human Services 
dl 
Deciliter 
DNA 
Deoxyribonucleic Acid 
dR 
Deoxyribose 
Dsg 
Desmoglein 
DSMB 
Data and Safety Monitoring Board 
EBV 
Epstein–Barr virus 
eCRF 
Electronic Case Report Form 
eGFR 
Estimated Glomerular Filtration Rate 
ELISA 
Enzyme-Linked Immunosorbent Assay 
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FACS 
Fluorescence-Activated Cell Sorting 
FDA 
Food and Drug Administration 
FSC 
Forward Scatter 
FWA 
Federal Wide Assurance 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
GvHD 
Graft vs Host Disease 
HICTF 
Human Islet and Cellular Transplantation Facility 
HIV 
Human Immunodeficiency Virus 
HSV 
Herpes Simplex Virus 
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonization 
IgG 
Immunoglobulin G 
IL 
Interleukin 
IND 
Investigational New Drug Application 
IPEX 
Immunodysregulation Polyendocrinopathy Enteropathy X-linked 
syndrome 
IRB 
Institutional Review Board 
ITT 
Intention-to-Treat or Intent-to-Treat 
IU 
International Units 
IV 
Intravenous 
IVIG 
Intravenous Immunoglobulin 
kg 
Kilogram 
L 
Liter 
mAb 
Monoclonal Antibody 
mITT 
Modified Intention-to-Treat or Intent-to-Treat 
mg 
Milligram 
ml 
Milliliter 
MM 
Medical Monitor 
MMF 
Mycophenolate Mofetil 
MMTT 
Mixed-Meal Tolerance Test 
NCI 
National Cancer Institute 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NK 
Natural Killer 
NSAID 
Non-Steroidal Anti-Inflammatory Drug 
OHRP 
Office of Human Research Protection 
PBMC 
Peripheral Blood Mononuclear Cell 
PCR 
Polymerase Chain Reaction 
PDAI 
Pemphigus Disease Area Index  
PF 
Pemphigus Foliaceus 
PGA 
Patient’s Global Assessment 
PhGA 
Physician’s Global Assessment 
PI 
Principal Investigator 
POC 
Point of Care 
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PolyTregs 
Ex Vivo Expanded Autologous CD4+CD127lo/-CD25+ Polyclonal 
Regulatory T Cells  
PP 
Per Protocol 
PPD 
Purified Protein Derivative 
PV 
Pemphigus Vulgaris 
QA 
Quality Assurance 
QoL 
Quality of Life 
qPCR 
Quantitative Polymerase Chain Reaction 
REMS 
Risk Evaluation and Mitigation Strategy 
RhoFED 
Rho Federal Systems Division, Inc. 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Suspected Adverse Reaction 
SLE 
Systemic Lupus Erythematosus 
SMT 
Study Management Team 
SOP 
Standard Operating Procedure 
SP 
Safety Population 
SRC 
Safety Review Committee, consisting of the protocol chair, 
representatives from the Statistical and Clinical Coordinating 
Center (DAIT-SACCC), and representatives of DAIT that will 
review the laboratory data and clinical experience of all subjects in 
the cohort 
SSC 
Side Scatter 
SUSAR 
Serious and Unexpected Suspected Adverse Reaction  
T1DM 
Type 1 Diabetes Mellitus 
TCR 
T Cell Receptor 
Tdap 
Tetanus, Diphtheria, Pertussis 
Teff 
Effector T Cell 
TGFβ 
Transforming Growth Factor Beta 
TH 
T Helper Cell 
Treg 
Regulatory T Cell 
TSDR 
Treg Specific Demethylation Region 
UCSF 
University of California at San Francisco 
ul 
Microliter 
ULN 
Upper Limit of Normal 
UVA 
Ultra Violet A 
UVB 
Ultra Violet B 
WBC 
White Blood Cell 
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TABLE OF CONTENTS 
1 BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE .....................22 
1.1 
DISEASE BACKGROUND ............................................................................................23 
1.2 
SCIENTIFIC RATIONALE.............................................................................................24 
1.3 
SUMMARY OF PRE-CLINICAL AND CLINICAL STUDIES ..............................................25 
 
Pre-Clinical Studies .........................................................................................25 
1.3.1
 
Treg Therapy in Murine Pemphigus Vulgaris ........................................25 
1.3.1.1
 
Expansion of Tregs from Individual Patients .........................................26 
1.3.1.2
 
Expansion of Tregs from Patients with Autoimmune Disease ...............28 
1.3.1.3
 
Documentation of the Functional Capacity of Expanded Tregs ............29 
1.3.1.4
 
Functional Analysis of Tregs in Tissues .................................................30 
1.3.1.5
 
Clinical Studies ................................................................................................31 
1.3.2
 
Prior Experience Using Polyclonal CD4+CD127lo/-CD25+ Tregs in 
1.3.2.1
T1DM .....................................................................................................31 
1.3.2.1.1 
Enrollment .................................................................................... 31 
1.3.2.1.2 
Manufacturing Summary .............................................................. 31 
1.3.2.1.3 
Safety Summary ............................................................................ 32 
1.3.2.1.4 
Pharmacokinetics ......................................................................... 32 
1.3.2.1.5 
Enhanced suppressive function of Polyclonal 
CD4+CD127lo/-CD25+ Tregs .................................................... 35 
1.3.2.1.6 
Metabolic Results ......................................................................... 36 
 
Prior Experience Using Polyclonal CD4+CD127lo/-CD25+ Tregs in 
1.3.2.2
other disease settings .............................................................................37 
1.4 
STUDY PRODUCT BACKGROUND ...............................................................................38 
1.5 
KNOWN AND POTENTIAL RISKS AND BENEFITS OF STUDY PRODUCT ........................38 
 
Known and Potential Benefits of Study Product ..............................................38 
1.5.1
 
Known and Potential Risks of Study Product/Treatment .................................38 
1.5.2
 
Infection ..................................................................................................38 
1.5.2.1
 
T-Cell Suppression .................................................................................39 
1.5.2.2
 
Infusion Reaction ....................................................................................39 
1.5.2.3
 
Lymphoproliferative Disease .................................................................39 
1.5.2.4
 
Loss of Tumor Surveillance ....................................................................39 
1.5.2.5
 
Reproductive risks ..................................................................................39 
1.5.2.6
 
Worsening Disease .................................................................................39 
1.5.2.7
 
Procedure-related risks ..........................................................................40 
1.5.2.8
1.6 
RATIONALE FOR STUDY ............................................................................................40 
 
Rationale for the Phase I Design .....................................................................40 
1.6.1
 
Rationale for the Selection of Doses ................................................................40 
1.6.2
 
Dosing Estimate Based on Rodent Models ............................................41 
1.6.2.1
 
Manufacturing Capacity ........................................................................41 
1.6.2.2
 
Safety of T Cell Therapy .........................................................................41 
1.6.2.3
 
Rationale for the Selection of the Patient Population .....................................42 
1.6.3
 
Rationale for the Mechanistic Studies to be Performed on Skin Biopsies .......42 
1.6.4
2 STUDY OBJECTIVES AND PURPOSE .......................................................................42 
2.1 
PRIMARY OBJECTIVE ................................................................................................43 
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2.2 
SECONDARY OBJECTIVES ..........................................................................................43 
3 STUDY DESIGN ...............................................................................................................43 
3.1 
DESCRIPTION OF STUDY DESIGN ...............................................................................43 
 
Management Plan for Out of Range PolyTreg Doses .....................................44 
3.1.1
 
Stratification, Randomization, and Blinding ...................................................44 
3.1.2
 
Subject Completion and Early Termination ...........................................45 
3.1.2.1
3.2 
DESCRIPTION OF PRIMARY ENDPOINT .......................................................................45 
 
Primary Safety Endpoint ..................................................................................45 
3.2.1
3.3 
DESCRIPTION OF SECONDARY ENDPOINTS ................................................................45 
 
Secondary Safety Endpoints .............................................................................45 
3.3.1
 
Secondary Efficacy Endpoints .........................................................................45 
3.3.2
3.4 
DESCRIPTION OF EXPLORATORY ENDPOINTS ............................................................46 
 
Exploratory Safety Endpoints ..........................................................................46 
3.4.1
 
Exploratory Efficacy Endpoints .......................................................................46 
3.4.2
 
Exploratory Mechanistic Endpoints ................................................................46 
3.4.3
4 SELECTION OF SUBJECTS ..........................................................................................47 
4.1 
INCLUSION CRITERIA ................................................................................................47 
4.2 
EXCLUSION CRITERIA ...............................................................................................47 
 
Co-enrollment Guidelines ................................................................................50 
4.2.1
5 DESCRIPTION OF STUDY PRODUCT .......................................................................50 
 
Product Description .........................................................................................50 
5.1.1
 
Packaging and Labeling of Study Product ......................................................51 
5.1.2
 
Storage and Handling of Study Product ..........................................................51 
5.1.3
5.2 
DOSAGE REGIMEN ....................................................................................................51 
5.3 
ADMINISTRATION OF STUDY PRODUCT .....................................................................51 
 
Preparation for Administration .......................................................................51 
5.3.1
 
Administration..................................................................................................52 
5.3.2
 
Action Plan for Post Release Positive Sterility Results ...................................52 
5.3.3
 
Criteria for Withholding Study Treatment .......................................................53 
5.3.4
5.4 
REPEAT SCREENING WHEN INFUSION IS NOT GIVEN AS SCHEDULED .......................53 
5.5 
TREATMENT INTERRUPTION AND DISCONTINUATION ...............................................53 
 
Criteria for Interruption of Study Treatment ...................................................53 
5.5.1
 
Criteria for Permanent Discontinuation of Study Treatment ..........................54 
5.5.2
 
Procedures for Discontinuation of Protocol-Specified Treatment 
5.5.2.1
Requirements ..........................................................................................54 
 
Subject Withdrawal from the Study..................................................................54 
5.5.3
 
Procedures for Subject Withdrawal from the Study ...............................54 
5.5.3.1
 
Safety Stopping Guidance ................................................................................55 
5.5.4
5.6 
TOXICITY MANAGEMENT PLAN FOR STUDY PRODUCT .............................................56 
 
Infection ...........................................................................................................56 
5.6.1
 
Vaccination .............................................................................................56 
5.6.1.1
 
Herpes Simplex Virus .............................................................................56 
5.6.1.2
 
Epstein-Barr Virus .................................................................................57 
5.6.1.3
 
Cytomegalovirus .....................................................................................57 
5.6.1.4
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Infusion Reaction .............................................................................................58 
5.6.2
 
Reproductive Risks ...........................................................................................58 
5.6.3
 
Malignancies ....................................................................................................58 
5.6.4
 
Skin Biopsies ....................................................................................................59 
5.6.5
 
Digital Photography ........................................................................................59 
5.6.6
5.7 
CONCURRENT MEDICATIONS AND THERAPY .............................................................60 
5.8 
PROHIBITED MEDICATIONS .......................................................................................60 
5.9 
PROCEDURES FOR MONITORING SUBJECT COMPLIANCE ...........................................61 
6 ASSESSMENT OF SAFETY AND EFFICACY ............................................................61 
6.1 
GENERAL ASSESSMENTS ...........................................................................................61 
6.2 
CLINICAL LABORATORY ASSESSMENTS ....................................................................61 
6.3 
DISEASE-SPECIFIC ASSESSMENTS .............................................................................62 
6.4 
STUDY PROCEDURES .................................................................................................62 
6.5 
MECHANISTIC ASSESSMENTS ....................................................................................63 
 
Treg Tracking by Stable Isotope Labeling .......................................................63 
6.5.1
 
Whole Transcriptome RNA-Sequencing ..........................................................64 
6.5.2
 
Functional Immunophenotyping of Skin and Blood .........................................65 
6.5.3
 
Treg Suppression Assays..................................................................................65 
6.5.4
 
Autoantibody Analysis .....................................................................................65 
6.5.5
 
Lymphocyte Subset Analysis ............................................................................65 
6.5.6
 
Serum Cytokine Analysis..................................................................................65 
6.5.7
 
Treg Epigenetic Analysis .................................................................................65 
6.5.8
 
Antigen Specific Studies ...................................................................................66 
6.5.9
6.6 
UNSCHEDULED VISITS ..............................................................................................66 
7 SAFETY MONITORING AND REPORTING .............................................................69 
7.1 
OVERVIEW ................................................................................................................69 
7.2 
DEFINITIONS .............................................................................................................69 
 
Adverse Event (or Adverse Experience) ..........................................................69 
7.2.1
 
Adverse Reaction and Suspected Adverse Reaction ........................................69 
7.2.2
 
“Expected” versus “Unexpected” Suspected Adverse Reaction .....................69 
7.2.3
 
Serious Adverse Event ......................................................................................70 
7.2.4
7.3 
COLLECTION AND RECORDING OF ADVERSE EVENTS ...............................................70 
 
Investigational Product ....................................................................................70 
7.3.1
 
Methods of Collection ......................................................................................70 
7.3.2
 
Methods of Recording ......................................................................................71 
7.3.3
 
Recording Adverse Events ......................................................................71 
7.3.3.1
 
Recording Serious Adverse Events .........................................................71 
7.3.3.2
7.4 
GRADING AND ATTRIBUTION OF ADVERSE EVENTS ..................................................71 
 
Grading Criteria ..............................................................................................71 
7.4.1
 
Attribution Definitions .....................................................................................72 
7.4.2
7.5 
REPORTING OF ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS TO THE 
SPONSOR: DAIT/NIAID ..........................................................................................73 
 
Reporting of Adverse Events ............................................................................73 
7.5.1
 
Reporting of Serious Adverse Events and Grade 3 and Higher Clinical 
7.5.2
Events ...............................................................................................................73 
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DAIT, NIAID Reporting to the Health Authority .............................................74 
7.5.3
 
Reporting of Adverse Events to IRBs ...............................................................75 
7.5.4
7.6 
PREGNANCY REPORTING ...........................................................................................75 
 
Mycophenolate REMS Program ......................................................................76 
7.6.1
7.7 
REPORTING OF OTHER SAFETY INFORMATION ..........................................................76 
7.8 
REVIEW OF SAFETY INFORMATION ...........................................................................76 
 
Study Management Team Review ....................................................................76 
7.8.1
 
Medical Monitor Review ..................................................................................76 
7.8.2
 
DSMB Review ..................................................................................................76 
7.8.3
8 STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ...........................77 
8.1 
SAMPLE SIZE AND POWER .........................................................................................77 
8.2 
ANALYSIS POPULATIONS ..........................................................................................77 
 
Donation Population ........................................................................................77 
8.2.1
 
Safety Population .............................................................................................77 
8.2.2
8.3 
DESCRIPTION OF THE ANALYSES ...............................................................................77 
 
Safety Analysis .................................................................................................77 
8.3.1
 
Efficacy Analysis ..............................................................................................78 
8.3.2
 
Mechanistic Analysis .......................................................................................78 
8.3.3
8.4 
INTERIM ANALYSIS ...................................................................................................78 
 
Interim Analysis of Safety Data .......................................................................79 
8.4.1
8.5 
OTHER STATISTICAL CONSIDERATIONS ....................................................................79 
 
Covariates ........................................................................................................79 
8.5.1
 
Multiple Comparisons and Multiplicity ...........................................................79 
8.5.2
 
Examination of Subgroups ...............................................................................79 
8.5.3
 
Missing Data ....................................................................................................79 
8.5.4
 
Changes to the Statistical Analysis Plan .........................................................79 
8.5.5
9 ACCESS TO SOURCE DATA AND DOCUMENTS ...................................................79 
10 DATA COLLECTION, QUALITY CONTROL AND QUALITY ASSURANCE .....80 
11 ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE .........................................................................................................81 
11.1 
COMPLIANCE WITH GOOD CLINICAL PRACTICES ......................................................81 
11.2 
INSTITUTIONAL REVIEW BOARD ...............................................................................81 
11.3 
INFORMED CONSENT .................................................................................................81 
11.4 
DATA AND SAFETY MONITORING BOARD .................................................................82 
11.5 
STUDY TERMINATION ...............................................................................................82 
12 FINANCING AND INSURANCE ...................................................................................82 
13 PUBLICATION POLICY ................................................................................................82 
14 REFERENCES ..................................................................................................................84 
15 APPENDICES ...................................................................................................................88 
15.1 
PDAI ........................................................................................................................89 
15.2 
SKINDEX-29 (VERSION 2) [62] ..................................................................................90 
15.3 
PATIENT’S GLOBAL ASSESSMENT .............................................................................94 
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15.4 
PHYSICIAN’S GLOBAL ASSESSMENT .........................................................................95 
15.5 
SUBJECT SELF-REPORTED DEMOGRAPHICS OPTIONAL SOURCE DOCUMENT ............96 
15.6 
PRE-INFUSION REQUIRED CHECKLIST .......................................................................97 
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1 BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE 
Pemphigus is a life-threatening autoimmune disease of the skin characterized by loss of 
epidermal cell-cell adhesion due to autoantibodies against desmosomal molecules, 
desmoglein (Dsg)3 and Dsg1 [1]. While broad immunosuppressive regimens have 
significantly improved management of pemphigus over the past several decades [2], current 
therapeutic strategies are associated with significant side effects and toxicities that limit 
effective management [3-9]. As autoantibodies are critical in pemphigus pathogenesis, 
therapeutic strategies aimed at limiting autoantibody production hold great promise for 
effective disease management with minimal adverse effects [10, 11]. Biologic therapy 
targeting B lymphocytes with anti-CD20 monoclonal antibodies has led to successful 
induction of disease remission [12], however, prolonged B cell depletion with rituximab has 
been associated with increased infection risk. An alternate strategy for limiting autoantibody 
production lies in augmenting immune regulation. Autoreactive helper T cells and 
autoreactive B cells play major roles in driving autoantibody formation [13], and both 
populations are controlled by regulatory T cells (Tregs) [14]. Furthermore, recent evidence 
demonstrates reduced peripheral Tregs in pemphigus vulgaris (PV) patients as compared 
with healthy controls regardless of disease status, suggesting an important role for Tregs in 
pemphigus pathogenesis [15, 16]. Taken together, these data suggest that a therapeutic 
strategy targeting Treg depletion may be effective in managing pemphigus by re-establishing 
stable immune tolerance with low toxicity and few side effects. 
The discovery of the importance of regulatory T cells (Tregs) in the effective regulation of 
the basic processes that maintain tolerance has opened an important new opportunity for 
therapeutic intervention in immunology [17]. Tregs have unique and multiple mechanisms of 
action. While requiring specific T cell receptor-mediated activation to develop regulatory 
activity, their effector function appears to work by bystander suppression, regulating local 
inflammatory responses through a combination of cell-cell contact, metabolic effects on 
immunity, and suppressive cytokine production.  
The therapeutic potential of Tregs is now well-established in animal models, but experience 
in humans is very limited. In a pilot study of 23 patients, Brunstein et al. [18] infused ex vivo 
expanded Tregs in leukemia and lymphoma patients who were undergoing transplantation 
with umbilical cord blood. No infusion-related toxicities were observed. Compared with 108 
identically treated historical controls without Treg therapy, there was a lower incidence of 
acute graft-versus-host disease (GvHD). More recently, Marek-Trzonkowska et al. [19] 
infused ex vivo expanded Tregs into 10 children within 2 months of the diagnosis of type I 
diabetes mellitus (T1DM). These investigators also reported no short-term toxicity associated 
with Treg therapy. Compared with matched T1DM controls, there was an increase in Tregs 
in the peripheral blood of the treated children, lower plasma C-peptide levels, and a reduction 
in insulin requirement. These limited but encouraging experiences contribute to the rationale 
for this trial. 
Investigators involved in this project have pioneered the use of Tregs as therapeutic agents 
[20-25]. Some of this work has been conducted with support from the ACE program with the 
intent of laying a sound scientific foundation for the innovative use of Tregs in the treatment 
of autoimmune diseases. The details of the work accomplished to date are included in the 
next section. In brief summary, the following pre-clinical work has been completed: 
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• demonstrated that ex vivo expanded PolyTregs can ameliorate autoimmune disease in 
murine models for systemic lupus erythematosus (SLE) and T1DM [20-22] 
• developed the technology for the production of large quantities of purified PolyTregs 
that meet the rigorous standards for infusion into humans [23] 
• preliminary experience in patients with T1DM that demonstrates the feasibility of the 
proposed trial, including the feasibility of conducting the trial in a multicenter fashion if 
necessary with Treg expansion performed at Univerisity of California, San Francisco 
(UCSF) 
Treg therapy is unlike any other form of therapy currently under investigation for pemphigus. 
Although the development of an antigen-specific approach to Treg therapy is the subject of 
active investigation at UCSF, there is not yet a robust way to expand antigen-specific Tregs 
without risking the simultaneous expansion of potentially autoreactive Teffs. Therefore, at 
present, the use of PolyTregs provides the most feasible and safe approach to Treg therapy. 
PolyTregs have broad self-antigen reactivity, which is a good match for the broad loss of 
self-tolerance that occurs in pemphigus. The pathogenesis of this classic autoimmune disease 
and associated disease effector cells provide strong rationale for study of this novel 
therapeutic strategy in pemphigus. 
 
1.1 Disease Background 
Pemphigus is a severe intraepithelial autoimmune blistering disease resulting in painful 
erosions of the skin and mucous membranes (mouth, genitals, or esophagus). The two classic 
forms of pemphigus are pemphigus foliaceus (PF) and pemphigus vulgaris (PV) [26]. The 
diagnosis of pemphigus can be made by characteristic findings on skin or mucosal 
histopathology and direct immunofluorescence microscopy. Pathogenic anti-desmoglein 3 
(anti-Dsg3) in PV and anti-desmoglein 1(anti-Dsg1) antibodies in PF play a central role in 
these classic, autoantibody-mediated conditions [27]. Clinically, PF is considered a more 
superficial variant of pemphigus because it is limited to body sites where Dsg1 is expressed, 
principally in the granular layer of the epidermis, and does not involve the mucous 
membranes. In contrast, PV is characterized by involvement of body sites where Dsg3 is 
expressed, specifically in the basal and immediate suprabasal layer of the epidermis and 
throughout the oral mucosa. Supporting the significant overlap in features and pathogenesis 
between PV and PF, about one-half to two-thirds of sera from PV patients also contain 
antibodies against Dsg1 [28-32]. In both disease variants, autoantibodies have been shown to 
react with the same Dsg1 ectodomain and share similar binding and disulfide linkage 
properties, supporting that PV and PF are variants of the same disease [33]. Binding of anti-
Dsg antibodies to keratinocytes results in breakdown of intercellular desmosomal bridges and 
failure of the skin barrier. The production of pathogenic anti-Dsg autoantibodies in 
pemphigus requires an orchestrated effort between autoreactive B cells and autoreactive T 
cells [14, 34], implicating dysfunction of both humoral and cell mediated immunity in this 
condition. Furthermore, both quantitative and qualitative dysfunction of Tregs is implicated 
in the pathogenesis of PV [16, 35, 36]. 
In the last 40 years, the availability of broadly immunosuppressive regimens has converted 
pemphigus from a fatal disease to a serious chronic condition. However, patients remain at 
high risk of severe complications and death as a direct result of treatment. Chronic systemic 
corticosteroids are still generally required as a pillar of the therapeutic regimen in pemphigus 
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and are associated with a host of adverse effects (including but not limited to hyperglycemia, 
hypertension, infection risk, glucocorticoid induced osteoporosis, and cataracts) [2, 37]. 
Potent immunosuppressants such as mycophenolic acid and azathioprine have a steroid 
sparing effect but are associated with cytopenias and infection risk. Azathioprine in particular 
may increase cancer risk. While rituximab can induce remission, it results in prolonged B cell 
depletion, which may persist for many years with subsequent associated increased risk of 
adverse events [38, 39]. Intravenous immunoglobulin (IVIG) can be used alone or with 
rituximab for nonresponders [37]; however, the drawbacks of this agent include 
hypercoagulable state, fluid shifts, and a risk for tachyphylaxis over time. Pemphigus patients 
tend to be younger (40-60 years old) than patients with other autoimmune bullous disorders 
(e.g., bullous pemphigoid) and may require decades of potentially toxic therapy. Thus, there 
is a critical need for more effective, less toxic approaches to treat this disease. 
1.2 Scientific Rationale 
An appealing alternative to chronic immunosuppressive therapy rests on the hope that it may 
be possible to harness naturally-occurring regulatory mechanisms to restore self-tolerance in 
people with pemphigus. Under normal circumstances, the immune system relies on multiple 
mechanisms to control untoward immune responses. Recent studies have focused particular 
attention on a small subpopulation of CD4+ T cells, termed Tregs, that play an essential role 
in maintaining self-tolerance [17]. The importance of Tregs is exemplified by an autoimmune 
syndrome called immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) 
syndrome [40]. IPEX is an inherited Treg-deficiency disease that causes severe multiorgan 
autoimmunity and that, left untreated, leads to death within the first few years of life. 
Conversely, recent studies have shown that Tregs can be isolated, expanded, functionally 
defined and, when administered in small animal models of autoimmune disease, provide 
lasting disease-specific therapy in the absence of adjuvant immunosuppression [20-22, 41]. 
These findings suggest that ex vivo expanded autologous Tregs may have the potential to 
stably restore immune homeostasis in the setting of autoimmunity. 
Recent studies in people with other diseases support the feasibility and potential of Treg 
therapy. Indirect evidence in support of this strategy comes from two studies in which 
treatment with low-dose interleukin 2 (IL-2) increased Tregs and ameliorated disease in 
patients with hepatitis C virus-induced vasculitis [42] and in patients with GvHD [43]. More 
direct evidence comes from two trials in which Tregs were harvested from patients, expanded 
ex vivo, and then reinfused. In patients undergoing stem cell transplant for leukemia, 
treatment with Tregs prevented GvHD, promoted lymphoid reconstitution, and reduced the 
frequency of opportunistic infection without weakening the graft-vs-leukemia effect [44]. In 
children with recent-onset T1DM, this approach reduced the insulin requirement in treated 
patients relative to patients who did not receive Tregs [19]. These observations demonstrate 
that the technology now exists to seriously consider autologous Treg therapy in patients with 
pemphigus. 
Against this background, UCSF has devoted considerable effort in recent years to develop an 
alternative approach for the treatment of autoimmune diseases that is based on augmenting 
the beneficial effect of autologous Tregs. The details of this innovative approach are 
described in the following sections of this protocol. In brief, we have developed a Current 
Good Manufacturing Practice (cGMP) process for obtaining >2 x 109 PolyTregs via efficient 
isolation and in vitro-based culture-expansion of Tregs sorted from peripheral blood 
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lymphocytes from a single donor, for the purpose of performing adoptive cell therapy. We 
have used this strategy successfully in mice to treat autoimmune diseases, including murine 
models for diabetes mellitus [22] and systemic lupus erythematosus (SLE) [20, 21]. In 
addition, phase I trials of adoptively transferred PolyTregs in patients with T1DM and SLE 
have occurred. This preliminary experience has established the feasibility of adoptive Treg 
therapy in people and has provided reassuring preliminary information regarding safety. This 
protocol extends this line of investigation to patients suffering from pemphigus, including the 
incorporation of recent approaches designed to enrich for Dsg-specific Tregs during the 
expansion process. By so doing, this trial will be addressing a great unmet need for better and 
safer treatments for patients with pemphigus. This protocol extends this line of investigation 
to patients suffering from pemphigus using polyclonal expansion of autologous Tregs, with 
the intent to follow with a subsequent study using adoptively transferred enriched antigen-
specific Tregs. 
Pemphigus is an ideal disease in which to test this novel approach because: (i) the 
autoantigens driving this disease have been extensively characterized, which will allow 
“head-to-head” eventual assessment of adoptively transferred enriched antigen-specific Tregs 
compared to PolyTregs; (ii) the pathophysiology of pemphigus is well-studied and involves 
aberrant autoreactivity in both the humoral and cell-mediated arms of the immune system 
[14, 34, 45]; (iii) pemphigus is a potentially fatal disease with major shortcomings and 
toxicities of current therapies [2, 37, 46]; (iv) both murine and human data highlight the 
importance of Tregs in both disease pathogenesis and suppression [14, 16, 35, 36, 47]; (v) the 
tissues (i.e., skin and mucous membranes) targeted by this disease are highly accessible, 
allowing for comprehensive cellular and molecular analyses; and (vi) there are clinically 
robust biomarkers (i.e., anti-Dsg3 & anti-Dsg1 Enzyme-Linked Immunosorbent Assays 
[ELISAs]) of disease activity [48].  
1.3 Summary of Pre-Clinical and Clinical Studies 
 
Pre-Clinical Studies 
1.3.1
 
Treg Therapy in Murine Pemphigus Vulgaris 
1.3.1.1
Currently, mouse models that perfectly recapitulate human PV are lacking. However, in one 
of the best-accepted PV mouse models, Tregs have been shown to significantly attenuate 
disease. Using an adoptive transfer approach to generate pathogenic anti-Dsg3 antibodies, 
Yomoyama and colleagues [35] have shown that co-adoptive transfer of PolyTregs resulted 
in reduced anti-Dsg3 antibodies in serum and reduced disease activity as measured by weight 
loss and PV clinical score (Figure 4). In addition to this study, there are numerous reports 
providing evidence that Tregs potently suppress humoral immune responses in mice [14]. 
Taken together, this data provides a strong scientific rationale to explore the potential of 
adoptive Treg therapy for human PV.  
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Figure 4, Tregs suppress anti-Dsg3 autoantibody production and clinical disease in a PV 
mouse model 
Production of anti-Dsg3 IgG in Rag2-/- recipient mice that were adoptively transferred with 
immunized Dsg3-/- splenocytes together with Tregs or Tconvs obtained from wild type mice. The time 
course of changes in the titer of anti-Dsg3 IgG examined by Dsg3 ELISA assay score (A), body 
weight loss (B) and PV score (C) are shown [35].  
 
Expansion of Tregs from Individual Patients 
1.3.1.2
A robust selection and expansion method for Tregs from individual patients has been 
established [23]. Preclinical and clinical studies have been in agreement in showing that 
expression of the transcription factor, FOXP3, is found in Tregs. The combined use of three 
markers (CD4, CD25, and CD127) identifies the majority of FOXP3+ Tregs present in the 
peripheral blood as illustrated in Figure 5. The fluorescence-activated cell sorting (FACS) 
plots illustrate the selection strategy in which the lymphocyte population is gated based on 
forward scatter vs. side scatter (upper left panel), followed by CD4+ T cells (upper right 
panel) and subsequently on CD127lo/-CD25+ T cells (lower left panel). A representative 
histogram of percent FOXP3 expression (lower right panel) is shown for the final 
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protein expression in blue and percent demethylation in red. The top panel represents 
expanded Effector T Cell (Teffs) compared to the bottom panel illustrating expanded Tregs. 
FOXP3 protein levels correlate very well with overall methylation status at the FOXP3 Treg 
Specific Demethylation Region (TSDR) indicating overall stability of the expanded Tregs 
compared to the Teffs. 
 
 
Figure 6, Correlation of protein expression and methylation patterns of FOXP3 in  
Teff and Tregs 
Expanded effector T cells (Teff, top panel) and expanded Tregs (bottom panel) were analyzed for 
protein levels of FOXP3 by flow cytometry and DNA demethylation. FOXP3 expression is shown in 
blue bars as the present of cells positive of all CD4+ cells in culture. Demethylation is shown in red 
bars as the percentage of demethylation seen in the FOXP3 locus. Representative plots from both 
autoimmune patients and healthy controls are shown, where autoimmune subjects are the second and 
third sets of bars and all other samples are from healthy controls. 
 
Expansion of Tregs from Patients with Autoimmune Disease 
1.3.1.3
Overall, the expansion technique has been tested in healthy controls, organ transplant 
recipients, and in patients with autoimmune diseases, including SLE and T1DM; the current 
trial is the first experience in pemphigus patients. Among subjects with autoimmune disease, 
the sorted population expansion has ranged from 30-fold to greater than 2000-fold in 14 
days. The mean expansion observed in patients with SLE and T1DM is approximately 650-
fold; among two patients with pemphigus the mean expansion is 43-fold. Based on this 
experience, a dose has been selected that is believed to be achievable in patients with 
pemphigus.   
 
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Documentation of the Functional Capacity of Expanded Tregs 
1.3.1.4
Extensive analyses of ex vivo expanded Tregs have been conducted in healthy subjects and in 
patients with autoimmune diseases (T1DM and SLE). To assess the capacity of expanded 
Tregs to suppress proliferation of CD8+ T cells (responders) when stimulated with anti-CD3 
and anti-CD28, carboxyfluorescein succinimidyl ester (CFSE)-labeled responders were 
incubated with varying ratios of Tregs. A representative example of the suppression of 
proliferation of CD8+ T cells by an expanded Treg population as assessed by CFSE assay is 
shown in Figure 7, left panel. The right panel of Figure 7 illustrates the mean percent 
suppression from 12 individuals with decreasing ratios of Tregs to responders as measured 
by 3[H]-thymidine incorporation. Overall, expanded Tregs suppressed responder proliferation 
within a small range of activity with approximately 50% suppression at a 1:8 
(Treg:responder) ratio.  
Figure 7, Functional Treg suppression assays 
Suppression was assessed by the capacity of expanded Treg to suppress proliferation of CFSE-
labeled CD8+ T cells (responders) when stimulated with anti-CD3 and anti-CD28. (Left Panel) A 
representative example of suppression by expanded Tregs from a subject with autoimmunity, where 
CFSE florescence is plotted on the x-axis. Overlaid is a titration of Treg to CD8+ responder rations, 
as labeled. The top histogram (0:1) indicates proliferation of responders when incubated alone in 
the presence of anti-CD3/anti-CD28 whereas the bottom histogram represents unstimulated 
responders. Decreased proliferation of responders with increasing ratio of Treg is shown. (Right 
Panel) The mean percent suppression from 12 individuals (non-diabetic healthy controls and T1DM 
subjects within one year of diagnosis) with decreasing ratios of Tregs to responders as measured by 
3[H]-thymidine incorporation following stimulation with anti-CD3/anti-CD28. 
In summary, Tregs from individual subjects, including subjects with active autoimmune 
disease, have been shown to be able to be expanded to sufficient numbers for in vivo therapy. 
Importantly, although the percentage of FOXP3+ cells at the end of culture ranged from as 
low as 79.2% upward to 98.4% in these experiments, all the cultures of expanded Tregs 
suppressed in vitro, were anergic and retained low levels of CD127 expression, suggesting 
that these cells were not contaminated with significant numbers of Teffs. Furthermore, it has 
been illustrated that FOXP3 expression by FACS directly correlates with percent 
demethylation indicating that the FOXP3+ expression in these cells is stable following a 14-
day ex vivo expansion period. Last, we have demonstrated the suppressive function of these 
expanded Tregs. Together, these results suggest that the expansion protocol of CD4+127lo/-
25+ FACS-sorted T cells, expanded with anti-CD3/anti-CD28-coated beads (1:1) plus IL-2 
(300 U/ml) and restimulated on day 9 in X-vivo media/10% human sera led to efficient 
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expansion of FOXP3+ T cells with demonstrated regulatory function in vitro via functional 
suppression assays.  
 
Functional Analysis of Tregs in Tissues 
1.3.1.5
A new protocol for comprehensively studying leukocyte populations isolated from human 
skin has recently been developed and optimized at UCSF. Traditional approaches have relied 
on culturing skin biopsy specimens for 10-20 days in the presence of growth factors (i.e., 
recombinant IL-2), to allow infiltrating leukocytes to migrate out of the skin and expand in 
response to these factors. It is becoming increasingly appreciated that lymphocytes cultured 
for this length of time can alter their functional capabilities and may not accurately reflect in 
vivo differentiation and function. Using a single 4mm punch biopsy of freshly isolated human 
skin, lymphocytes and monocytes may be isolated and assessed for their functional activities 
after a 12-hour culture in the absence of growth factors (Figure 8). A 13-color flow 
cytometry is performed on these specimens, allowing for the quantification of multiple 
cytokine, chemokine, and cell surface and intracellular differentiation/activation markers 
(Figure 8A-C). This technique is extremely sensitive, as a biopsy specimen from normal skin 
(having no appreciable inflammatory infiltrate by routine histology) shows a defined memory 
T cell population capable of producing specific effector cytokines (Figure 8C). 
 
Figure 8, Functional immunoflow cytometry of human skin 
(A) A single 4mm punch biopsy was obtained from normal skin. Tissue was minced and digested for 
12hrs in a digestion cocktail. Cutaneous T cells were assayed for Foxp3 expression and cell surface 
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markers (B) as well as intracellular cytokine production after stimulation with PMA/ionomycin (C). 
Panels B and C are gated on live CD3+ T cells. (D) This approach was used on 4mm skin biopsies 
from a patient with pemphigus (lesional and non-lesional skin). Cells in panel D are pre-gated on live 
CD3+CD4+cells [51].  
This technique is being used to define the major inflammatory pathways active in the skin of 
patients with diverse inflammatory and autoimmune diseases as well as to functionally 
characterize Tregs in both normal and diseased skin, including pemphigus (Figure 8D). 
Using this approach UCSF has discovered that Tregs are activated, proliferate, and secrete 
small amounts of interleukin-17 (IL-17) in lesions of psoriasis compared to non-lesional skin 
from the same individual. In contrast, absolute numbers and percentages of Tregs are reduced 
in both non-lesional and lesional skin in patients with systemic sclerosis. Isolation of 
leukocytes and assessment of their in vivo function using this approach will for the first time 
allow both Treg and Teff cell function in inflamed tissue before and after adoptive Treg 
therapy to be functionally characterized.  
 
Clinical Studies 
1.3.2
 
Prior Experience Using Polyclonal CD4+CD127lo/-CD25+ Tregs in T1DM  
1.3.2.1
1.3.2.1.1 Enrollment  
UCSF and Yale have completed the study “A Phase 1 Safety Trial of CD4+CD127lo/-CD25+ 
Polyclonal Treg Adoptive Immunotherapy for the Treatment of Type 1 Diabetes” conducted 
under IND 14462 (NCT01210664) [52]. In this study, we tested the safety and feasibility of 
treating adults with recent onset (within 3-24 months of diagnosis) T1DM with a single 
infusion of Polyclonal Tregs and obtained data on the pharmacokinetics of the transferred 
cells. A total of 26 potential participants signed consent and underwent screening 
assessments. Sixteen subjects were determined to be eligible and were enrolled. Two of these 
sixteen subjects were withdrawn from the study prior to infusion due to failure of PolyTreg 
product to meet release criteria. The other fourteen subjects were treated with a single dose of 
Polyclonal Tregs. Three subjects were treated in each of the first and second dosing cohorts 
with 0.05x108 and 0.4x108 cells, respectively, and four subjects were treated in each of the 
third and fourth dosing cohorts with approximately 3.2x108 and 26 x108 cells, respectively. 
The highest dose represents approximately 20% of the total number of Tregs (13 x 109) that 
are predicted to exist in normal individuals [53]. At the time of this update, all 14 subjects 
have completed follow-up up to 5 years.  
1.3.2.1.2 Manufacturing Summary  
Our experience in this dose escalation trial has shown that we can consistently expand 
Polyclonal Tregs in 14 days, averaging 748-fold for cohorts 3 and 4 combined and 555-fold 
overall. Fourteen of 16 cell products met release criteria for identity, purity, viability, and 
sterility. 
The two subjects who were withdrawn from the study were withdrawn due to failure of the 
manufactured Treg product to meet release criteria. The first failure was due to unanticipated 
expression of CD8 on a small subset of CD4+FOXP3+ cells. This subset was pre-existing in 
this patient prior to expansion and the proportion was not markedly altered by the ex vivo 
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expansion. Further investigation demonstrated that this subset expressed both CD4 and CD8 
markers but had all other features of Tregs and was equally suppressive in vitro when 
compared with the subset that did not express CD8. The second product did not pass 
specified FOXP3 release criteria: 20.9% (less than 60%) of the cells were FOXP3+ by flow 
cytometry. This was ultimately due to a suboptimal anti-CD127 antibody staining for sorting 
the initial Treg population on the clinical sorter. This resulted in the contamination of non-
Treg CD4+ T cells in the initial sort leading to selective expansion of the contaminating cells. 
1.3.2.1.3 Safety Summary  
At the conclusion of the study, 166 adverse events were reported in 15 subjects since the 
beginning of the trial. All 14 treated subjects reported at least one Adverse Event (AE). One 
subject who underwent phlebotomy but was withdrawn before treated, reported one adverse 
event before withdrawal from the trial. One hundred and five events were judged as mild in 
severity, 48 were judged as moderate, 11 were judged as severe, and 2 were judged as life-
threatening. Thirty events were judged to be possibly related, 40 unlikely related and 96 
unrelated to study therapy.  
The most common SOC affected was “Infections and Infestations” followed by 
“Gastrointestinal Disorders” and “General Disorders and Administration Site Conditions.” Of 
36 infections recorded, 24 were upper respiratory infections. Of those, 19 were judged grade 
1 (CTCAE category: Infections and Infestations Other, Specify) and 5 were judge grade 2 in 
severity (CTCAE designation; Upper Respiratory Infection). One infection, initially reported 
as grade 2 pharyngitis, was subsequently demonstrated to reflect a new cytomegalovirus 
(CMV) infection that occurred prior to treatment with Tregs.  
 
Of the 11 AEs judged as severe (grade 3), all were judged unlikely related or unrelated to the 
investigational agent. Two occurred prior to Treg infusion and were judged unrelated to the 
investigational agent. Four events were hypoglycemia, of which three were judged unrelated 
and one judged unlikely related to the investigational agent. One event was syncope 
occurring 35 weeks after Treg infusion. One event was hyperglycemia occurring 10 weeks 
after Treg infusion. One was depressed level of consciousness due to inebriation. One was 
vomiting occurring 156 weeks after Treg infusion. One was elevated D-dimers occurring 104 
weeks after Treg infusion.  
 
Five serious adverse events (SAE) have been reported since the beginning of the trial. Three 
severe (grade 3) hypoglycemic SAEs, one judged unlikely related and two judged unrelated 
to the investigational product have been reported. One severe (grade 3) hyperglycemic SAE 
judged unrelated to the investigational product has been reported. One severe (grade 3) 
vomiting SAE judged unrelated to the investigational product has been reported. 
 
Two events were life-threatening (grade 4) events of hypoglycemia in one subject occurring 
59 and 62 weeks after Treg infusion. Grade 4 hypoglycemia is defined with a glucose < 30 
mg/dL. Both of these events were judged unrelated to the investigational product. 
1.3.2.1.4 Pharmacokinetics 
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After infusion of the product, the team used stable isotope labeling to track CD4+CD127lo/-
CD25+ polyclonal Tregs in the peripheral vascular space in humans. During ex vivo 
expansion, the 2H label from deuterated glucose (2H2-glucose) contained in the cell culture 
medium is incorporated into the deoxyribose moiety in replicating DNA through the de novo 
purine nucleotide synthesis pathway. Following infusion of stable isotope-labeled Tregs, the 
total number of Tregs in peripheral blood can be measured by flow cytometry and stable-
isotope enrichment in purified Tregs can be determined. Following isolation and hydrolysis 
of genomic DNA, the isotopic enrichment of the purine deoxyribonucleosides in Tregs sorted 
from whole blood can be assessed by gas chromatography/mass spectrometry. The change in 
2H enrichment in the total Treg pool can be assessed at specified time points post-infusion. 
Three subjects were treated with a target dose of 3.2x108 cells and 4 subjects were treated 
with a target dose of 2.6x109 that were approximately 60% enriched for the 2H-label in the 
study “A Phase 1 Safety Trial of CD4+CD127lo/-CD25+ Polyclonal Treg Adoptive 
Immunotherapy for the Treatment of Type 1 Diabetes” conducted under IND 14462. 2H-
labeled Tregs were detectable in the peripheral blood up to 365 days post-infusion in cohort 4 
patients as shown in Figure 9. A peak enrichment of 6% indicates that approximately 0.2% 
Tregs of the total Treg pool were ex vivo expanded infused Tregs. The delayed peak 
enrichments in several individuals suggest that the infused Tregs may initially migrate to the 
tissues. The majority of the tissue-blood exchange and/or cell death occurs by day 28, after 
which the kinetics appear stable: based on a two-phase decay curve, the average half-life of 
the fast decay phase is about 19.6 days (range 4.7 to 32.5 days) and the second slow decay 
phase has a half-life of a year or more in 4 of 7 patients studied (Figure 9). 
 
 
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Figure 9, Treg Tracking by Stable Isotope Labeling 
 2H label Tregs (target dose of 3.2 x108 cells) from cohort three subjects (002-015, 007-102, and 002-
017) and 2H-labeled Tregs (target dose of 26 x108 cells) from cohort four subjects (002-018, 002-019, 
007-103, and 002-022) were infused as part of study treatment. All infused Tregs were approximately 
60% enriched for the 2H-label. (Top Panel) Peripheral blood was collected on days 1, 4, 7, 14, 28, 
91, 182, and 364 days post infusion, and Tregs were isolated via FACS. 2H isotopic enrichment of the 
purine deoxyribonucleosides in Tregs is plated on the y-axis. Background enrichment of unlabeled 
Tregs was ≤0.1% for each of the seven subjects. (Bottom Panel) Isotope dilution and Treg die away is 
shown for each subject.  
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immune reconstitution, reduced Cytomegalovirus (CMV) reactivation, and lower incidence 
of tumor relapse with robust graft vs. leukemia effect in patients who received polyclonal 
donor Treg therapy when compared to historical controls. These subjects received no adjunct 
immunosuppression for GvHD prevention [44]. In another trial, PolyTreg therapy was well-
tolerated in children with recent-onset T1DM who had reduced insulin requirement relative 
to patients who did not receive PolyTregs [19]. These experiences provide a solid foundation 
for this trial. 
1.4 Study Product Background 
The study product under investigation in this trial consists of purified autologous PolyTregs 
that have been expanded ex vivo from the subject’s own peripheral blood. The manufacturing 
facility, the cGMP process for isolation and expansion of the Tregs, and the final formulation 
and release testing are described in detail in Section 5, Description of Study Product. 
1.5 Known and Potential Risks and Benefits of Study Product 
 
Known and Potential Benefits of Study Product 
1.5.1
The potential benefit is that treatment with autologous PolyTregs might provide a means to 
reduce the symptoms and long-term complications of pemphigus without requiring chronic 
immune suppression. This goal is important, because current approaches to the treatment of 
pemphigus are incompletely effective and often cause adverse effects that can be severe and, 
occasionally, fatal. If this treatment strategy is effective, it has the potential to restore self-
tolerance and spare patients the risks of chronic immunosuppressive therapy. 
 
Known and Potential Risks of Study Product/Treatment 
1.5.2
Therapy with autologous PolyTregs carries the potential risk that the expanded PolyTregs 
might be contaminated with Teffs that could conceivably cause exacerbation rather than 
suppression of disease. This protocol describes a detailed set of preliminary experiments that 
provides reassurance that the transferred cells will be suppressive, not stimulatory, in effect 
(Section 1.3.1, Pre-Clinical Studies).  
Safety, especially with regard to protection against infection, is a paramount concern in 
studies of novel approaches to immunotherapy. Although prior studies have been 
encouraging about the safety of infused PolyTregs, the data are still quite limited. This trial 
has been designed to proceed slowly and carefully through the dose-escalation process in 
order to limit the risk to study subjects.  
 
Infection  
1.5.2.1
As with any therapy that suppresses the immune system, there is a risk of developing 
infections. It should be noted that on a theoretical basis, this risk is minimal, since the total 
input of Tregs is far below the resident population.  
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T-Cell Suppression  
1.5.2.2
Tregs are known to suppress naïve T cell responses to a variety of antigens. Less is known 
about ongoing immune responses especially to viruses and bacteria. It is not known whether 
Tregs will alter protective immunity.  
 
Infusion Reaction  
1.5.2.3
Side effects reported from previous human trials involving T cell infusions include transient 
fever, chills, and/or nausea. Appropriate pre-medications (see Section 5.3.1, Preparation for 
Administration) have mitigated the effects of these reactions. Infusion reactions have been 
limited. In the previous T1DM study, only four AEs in four subjects were reported, in the 24 
hours following infusion of PolyTregs. Two were mild headache, one was mild nausea and 
one was mild abdominal pain.  
With any T cell therapy there is a theoretical concern for cytokine release syndrome (CRS). 
However, to date, there have been no documented cases. 
 
Lymphoproliferative Disease  
1.5.2.4
Treg immunosuppression has been shown to enhance growth of already present tumors in 
some small animal model systems. Thus, complications such as lymphoproliferative disease 
are possible on a theoretical basis. Clinical experience in transplant recipients suggests that 
the risk of lymphoproliferative disease is highest in those who develop a primary Epstein-
Barr virus (EBV) infection while immunosuppressed. Thus individuals without prior 
exposure to EBV are excluded from this study. In previously exposed individuals, EBV 
reactivation is associated with a degree of immunosuppression higher than that likely to be 
observed in this study since the total input of Tregs is far below the resident population. 
Nonetheless, careful attention will be paid to the potential for this complication.  
 
Loss of Tumor Surveillance  
1.5.2.5
T lymphocytes are one major component of tumor surveillance and it is possible that cells 
that inhibit T lymphocytes could impair this function. There has not been evidence of de 
novo development of tumors in preclinical models. No issues have been noted in previous 
clinical trial experience, which includes subjects that have been followed for five years, as of 
the time of this submission.  
 
Reproductive risks  
1.5.2.6
There may be an unexpected risk to an unborn or nursing child as described in Section 5.6.3, 
Reproductive Risks.  
 
Worsening Disease 
1.5.2.7
It is theoretically possible that some of the PolyTreg cells could convert to Teffs, which 
could increase pemphigus disease activity. No issues have been noted in previous clinical 
trial experience in other autoimmune indications, which includes subjects having been 
followed for five years, at of the time of this submission. 
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Procedure-related risks  
1.5.2.8
In addition to the risk that might be associated with PolyTreg infusion, study subjects also 
will be exposed to the risks of multiple skin biopsies. In general, the risks of skin biopsy are 
similar to blood draw or any other procedure that punctures the skin. Discreet, hidden sites 
are chosen whenever possible, and infectious complications are very rare even in 
immunocompromised hosts.  
Other potential risks of participation in this study are those associated with study-related 
procedures including blood draws and intravenous access. 
1.6 Rationale for Study 
 
Rationale for the Phase I Design 
1.6.1
This phase I dose-escalation study design exposes only small numbers of subjects to the cell 
infusions, separated by defined intervals. The built-in delays in enrollment avoid exposing 
multiple subjects simultaneously as potential toxicities are assessed in a step-by-step fashion. 
The cohort size is chosen to allow detection of high frequency Serious Adverse Events (SAE) 
at a given dose. The proposed inclusion of two cohorts ensures that the total safety 
experience will include a large enough number of subjects exposed over the two doses in 
order to support the optimal design for a phase II trial. 
 
Rationale for the Selection of Doses 
1.6.2
Two dosing groups will be employed in this trial: 1 x 108 and 2.5 x 108 polyclonal Tregs. The 
lower dose will aid in detection of biomarkers and labeling of cells, and also allow us to 
compare Treg dosing to dosing regimens used in other Treg trials.  
The dose of PolyTregs is selected based on a combination of practical considerations of 
manufacturing capacity, expected Treg numbers in pemphigus patients, the available safety 
data and a predicted efficacious dose of the PolyTreg product currently in clinical trials for 
the treatment of T1DM and cutaneous lupus/SLE.  
In previous versions of this protocol, we chose to forego the low-dose cohort that had been 
included in the ALE08 Protocol (1.0 x 108) based on (i) increasing evidence in other trials 
that a higher dose would be safe, and (ii) the assumption that a higher dose might be more 
likely to demonstrate the desired biologic affect (successful trafficking to affected skin).  
However new data is now available from mechanistic studies in one SLE patient [54]  
enrolled in the ALE08 Protocol and in the first pemphigus patient enrolled in APG01 
Protocol. The data indicates that a lower cell dose is sufficient to observe an impact on skin 
inflammation (Figure 13) [54].  
At the same time, we have encountered challenges in the low yield of PolyTregs that appear 
to be pemphigus-specific. For these reasons, we believe that the feasibility of the study will 
be enhanced by reducing the target number of the PolyTreg to be infused without 
compromising our ability to demonstrate the desired biologic activity. The choice of 
PolyTregs dose in earlier cases of SLE, pemphigus, and other diseases was somewhat 
arbitrary, based in large part on what was technically feasible. We now have evidence that 
the use of a lower dose is probably sufficient for the proof-of-concept purpose of this study, 
and this change is very likely to enhance feasibility. An inclusion criterion for absolute Treg 
count at screening has also been added to increase the likelihood of meeting the target dose.     
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Figure 13, Activated Tregs Accumulated in Skin with Time After Adoptive Transfer for the first 
subject on APG01 
Treg adoptive transfer resulted in a 2-fold increase in activated Tregs in skin from baseline to week 
12 (A and B), and increased aTreg to Teff and aTreg to CD8 ratios (B). There was also increased IL-
17 production from Teffs after Treg adoptive transfer (C). These are similar to the results observed in 
our SLE patient. 
 
Dosing Estimate Based on Rodent Models 
1.6.2.1
The effective dose of Tregs for controlling autoimmunity in humans is currently unknown. 
Data from animal models demonstrate that a high percentage of Tregs, 600% of that found in 
the steady state, is needed to prevent transplant rejection and autoimmunity in a non-
manipulated setting. This is likely due to the very high frequency of autoreactive 
conventional T cells present in these settings. Thus, a significant number of Tregs is likely to 
be needed for efficacy. These considerations support the idea of maximizing Treg dosing to 
the extent permitted by manufacturing capacity and available safety data when Tregs are 
used in the setting of appropriate immune suppression. 
 
Manufacturing Capacity 
1.6.2.2
In the phase I Treg study in T1DM conducted under IND 14462, UCSF has shown that 2.6 x 
109 expanded Tregs can be produced for infusion in the majority of patients. In recent trials 
we have focused on a maximum dose of 10 x 108 for feasibility reasons and in order to 
compare between disease cohorts.  
 
Safety of T Cell Therapy 
1.6.2.3
The standard dose of 10 x 109 cells per infusion of non-Treg autologous polyclonal T cells 
has been tested and found to be safe in hundreds of subjects to date [55]. 
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The highest dose tested for safety in the phase I Treg study in T1DM conducted under IND 
14462 was 2.6 x 109 (range 2.35-2.94 x 109). Adverse events in the trial have generally been 
mild or moderate in severity as described in Section 1.3.2.1, Prior Experience Using 
Polyclonal CD4+CD127lo/-CD25+ Tregs in T1DM.  
Based on the information summarized above, it is estimated that hundreds of millions to 
billions of PolyTregs will be needed to control pemphigus. Because the safety profile of 
PolyTreg therapy in this patient population has not been established, APG01 will begin with 
a dose of 1.0 x 108 cells to determine its safety and impact on Treg trafficking to the skin.  
In the above T1DM study, the minimum interval between subject doses was 6 days and 
serious adverse events reported were assessed to be ‘unlikely’ or ‘unrelated’ to study therapy. 
Based on this data, the APG01 protocol will require the previous subject to reach Week 2 
prior to initiating the next subject’s blood donation.  
 
 
Rationale for the Selection of the Patient Population 
1.6.3
This phase I trial is being conducted in subjects with pemphigus for two principal reasons. 
First, the selection of this population allows us to examine the impact of PolyTreg therapy 
not only in the peripheral blood, but also in the skin in patients with pemphigus. Mild to 
moderately active disease is being targeted in this phase I safety study, with a PDAI severity 
score of 3-10 being required for screening (with activity score no higher than 12 at time of 
PolyTreg infusion) [56, 57]. Second, cutaneous manifestations of pemphigus can be severe, 
refractory, and life-threatening, with significant insensible losses and impaired barrier 
function leaving patients susceptible to serious infections. Therefore, this patient population 
warrants examination of a novel therapy that would be of utility if the treatment proves to 
have a favorable risk:benefit ratio. 
 
Rationale for the Mechanistic Studies to be Performed on Skin Biopsies 
1.6.4
Skin biopsies will enable the determination of whether the infused PolyTregs migrate to 
active lesions in an affected organ, whether they persist and/or proliferate there, whether they 
maintain their function, and how they affect local mechanisms that contribute to 
immunopathology. Three (4 mm) punch samples are needed at each time point to allow: (1) 
tracking of deuterium 2H2-labeled Tregs and Treg clonality via T Cell Receptor (TCR) 
sequencing; (2) whole transcriptome RNA sequencing to determine the effects PolyTreg 
infusion on inflammatory and immunoregulatory pathways in the target tissue; and (3) flow 
cytometric quantification of inflammatory cell subsets and immunologic biomarkers in skin, 
allowing comparison to blood. These mechanistic studies will be critical to the understanding 
of the biologic effects of PolyTreg therapy. 
2 STUDY OBJECTIVES AND PURPOSE  
The objective of this trial is to determine the safety profile of PolyTregs in subjects with 
pemphigus. 
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Two dosing groups will be employed in this trial: 1.0 x 108 and 2.5 x 108 PolyTregs. Each 
cohort will enroll 4-6 subjects with pemphigus. Each subject in cohorts 1 and 2 will receive 
an infusion and must reach Week 2 prior to the subsequent subject’s blood donation. A 
schematic representation of an individual’s participation is shown in Figure 1. The study dose 
can be escalated after the DSMB and SRC evaluates the safety data collected when the fourth 
subject in cohort 1 completes Week 8. After reviewing the data from the first four subjects, 
the DSMB and SRC will recommend adding additional subject(s) to the first cohort, 
advancing enrollment in the second cohort, or stopping the trial. A schematic representation 
the study’s cohort design is shown in Figure 2. 
Since a significant event in this trial can occur from toxicity from the study drug or disease 
worsening (e.g., a pemphigus flare), the standard 3 + 3 design rules for dose escalation will 
not be employed. Therefore, if a Grade 3 or higher AE or any SAE occurs in a subject during 
his/her first year of study participation, enrollment will be suspended until the SRC can 
convene to review the event. If this occurs, the SRC may decide at that time to proceed with 
enrollment, adjust the dose as described above, continue the suspended enrollment until the 
Data and Safety Monitoring Board (DSMB) can review the data, or place the trial on hold. 
The SRC will review all Grade 3 or higher AEs or any SAEs that occur throughout the study. 
However, since the likelihood of events being related to the investigational treatment 
diminishes over time, Grade 3 or higher AEs or any SAEs occurring during a subject’s 
second or third year of the study will not result in an immediate suspension of enrollment, 
donation, and treatment, unless the SRC determines that such action is indicated. Provided 
the SRC can review the event within 5 business days of awareness, events judged to be 
significant will drive protocol management in terms of proceeding, suspending 
enrollment/treatment pending ad hoc DSMB input, or placing the study on hold pending 
further evaluation. 
DSMB input will be required for the evaluation of some specific events. Description of these 
events and additional directives are outlined in Section 5.5.4, Safety Stopping Guidance. 
Accrual of all subjects is expected to take approximately 1.5 years.  
 
Management Plan for Out of Range PolyTreg Doses 
3.1.1
For subjects in cohort 1, any Treg product with less than 0.7 x 108 PolyTregs will not be 
infused and subject will be replaced. In cohort 2, products falling short of the minimal 
defined PolyTreg dose may be infused for up to maximum dose of cohort 1 in up to 2 
subjects (not to exceed 6 total subjects in cohort 1). The SRC will review available 
information after the first occurrence of the dose falling short of target range. Occurrence of a 
second product not meeting the specified dose will result in a comprehensive SRC review of 
the manufacturing issues prior to the trial proceeding.  
If a higher dose of PolyTregs is attained than specified for a given cohort, the maximum 
defined dose for that cohort will be infused. Unused product will be stored for future 
unspecified research commensurate with subject informed consent.  
 
Stratification, Randomization, and Blinding 
3.1.2
This is an open-label, phase I trial. Randomization will not be performed and site staff will 
not be masked to the subject’s dosage. 
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Subject Completion and Early Termination  
3.1.2.1
A subject is considered to have completed the study if he/she has completed the Week 156 
visit. 
In order to better understand safety through the peri- and proximal post-Treg infusion period, 
subjects who withdrew for non-safety issues prior to Week 12 will be replaced. 
3.2 Description of Primary Endpoint 
 
Primary Safety Endpoint 
3.2.1
The primary study endpoint is the number of significant events in each cohort through Week 
52, defined as any related NCI-CTCAE Grade 3 or higher AE or any related SAE. 
3.3 Description of Secondary Endpoints 
 
Secondary Safety Endpoints 
3.3.1
The secondary safety endpoints include the following: 
• Number of significant events in each cohort through Week 156 
• All AEs through Week 156  
• All NCI-CTCAE Grade 3-5 AEs through Week 52 
• All NCI-CTCAE Grade 3-5 AEs through Week 156 
• All SAEs through Week 156 
• All infection related events through Week 156  
• All infusion reactions, defined as any adverse reaction of NCI-CTCAE Grade 1 and 
higher within 24 hours of infusion 
 
Secondary Efficacy Endpoints 
3.3.2
The secondary efficacy endpoints include the following: 
• Change in pemphigus disease area index (PDAI) score from baseline to Weeks 1, 2, 8, 
12, 26, 39, 52, 78, 104, 130, and 156 
• Change in Desmoglein 1 and 3 titers by ELISA from baseline to Weeks 1, 2, 8, 12, 26, 
39, 52, 78, 104, 130, and 156 
• Time to relapse (flare) 
• Number of participants on prednisone dose ≤10 mg/day at Weeks 12, 26, 39, 52, 78, 
104, 130, and 156 
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3.4 Description of Exploratory Endpoints 
 
Exploratory Safety Endpoints 
3.4.1
• All pemphigus relapses/flares, as defined by consensus definition of 3 or more new 
lesions a month that do not heal spontaneously within 1 week or by the extension of 
established lesions in a patient who has achieved disease control through Week 156 
• Absolute and change from baseline in clinical chemistry, hematology, and urinalysis 
after treatment  
 
Exploratory Efficacy Endpoints 
3.4.2
The exploratory efficacy endpoints include the following: 
• Change in Skindex-29 scores (symptoms domain, emotions domain, functioning 
domain, overall score) from baseline to Weeks 1, 2, 8, 12, 26, 39, 52, 78, 104, 130, and 
156 
• Change in patient global assessment (PGA) from baseline to Weeks 1, 2, 8, 12, 26, 39, 
52, 78, 104, 130, and 156 
• Change in physician global assessment (PhGA) from baseline to Weeks 1, 2, 8, 12, 26, 
39, 52, 78, 104, 130, and 156 
• Number of participants on prednisone dose 0 mg/day at Weeks 12, 26, 39, 52, 78, 104, 
130, and 156 
• Number of participants requiring an increase in prednisone dose relative to last 
recorded prednisone dose at any time prior to Weeks 12, 26, 39, 52, 78, 104, 130, and 
156 
 
Exploratory Mechanistic Endpoints 
3.4.3
The exploratory mechanistic endpoints include the following: 
• Determine the relative frequency of adoptively transferred Tregs in blood and skin by 
quantifying transferred deuterium-labeled Tregs in peripheral blood and skin from 
Week 1 to 12. In addition, TCR sequencing may be performed on sorted Tregs from 
either skin or blood. 
• Perform whole transcriptome RNA sequencing to determine the effects of PolyTreg 
infusion on inflammatory and immunoregulatory pathways in the target tissue (skin) 
• Quantify inflammatory cell subsets and immunologic biomarkers in blood and skin, 
including changes in lymphocyte subsets, cytokine profile, and autoantibody production 
• Changes in disease specific and immunologic biomarkers in peripheral blood including 
lymphocyte subsets, cytokine profiles, polyclonal and antigen-specific Treg function 
(flow cytometry with MHC II-tetramer staining), and autoantibody production from 
baseline to Weeks 1, 2, 8, and 12 
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• Changes in disease specific and immunologic biomarkers in skin including lymphocyte 
subsets, polyclonal and antigen-specific Treg function (flow cytometry with MHC II-
tetramer staining), and cytokine profiles from baseline to Weeks 1 and 12 
4 SELECTION OF SUBJECTS 
Subjects with pemphigus must grant written informed consent prior to the subject undergoing 
any study-related procedure, including screening tests and medication adjustments, when 
applicable. 
4.1 Inclusion Criteria 
Subjects who meet all of the following criteria are eligible for enrollment into the study: 
1. Ability to provide informed consent 
2. Age 18-75 years at Screening Visit 
3. Diagnosis of PV or PF, defined by H&E and direct immunofluorescence staining of 
skin biopsy at any time prior to enrollment 
4. Pemphigus treated with systemic corticosteroids within the 2 years prior to screening 
(historic or current) or treated with rituximab at least 12 months prior. 
5. Presence of: 
a. anti-Dsg3 antibodies (>20.0 U/ml) at screening visit consistent with diagnosis 
of pemphigus vulgaris, or 
b. anti-Dsg1 antibodies (>20.0 U/ml) at screening visit consistent with diagnosis 
of pemphigus foliaceus 
6. Active PV or PF as defined by PDAI overall activity score 3-10 at screening visit, and 
PDAI overall activity score 1-12 at baseline visit  
7. Positive test for EBV antibody  
8. Adequate venous access to support draw of 400 ml whole blood and infusion of 
investigational therapy 
9. An absolute Treg count of ≥42 cells/µL within 6 weeks prior to whole blood collection 
at Week -2  
 
4.2 Exclusion Criteria 
Subjects who meet any of the following criteria are ineligible to participate in the study: 
1. Initiation of systemic corticosteroid therapy, prednisone dose > 25 mg/d (or equivalent) 
or change in prednisone dose within 4 weeks prior to screening 
2. Addition of a new medication, or change in the dose of any background medication 
used to treat any aspect of pemphigus within the timeframes listed below. Specifically:  
a. methotrexate, mycophenolate mofetil, mycophenolic acid, azathioprine, 
cyclosporine or dapsone within the 6 weeks prior to screening or in the time 
between screening and study drug infusion  
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b. IVIG within 12 weeks prior to screening or in the time between screening and 
study drug infusion (subjects on IVIG must be on stable dose for at least 12 
weeks prior to screening) 
c. treatment with cyclophosphamide within 12 weeks prior to screening or in the 
time between screening and study drug infusion  
3. Doses of background medications at screening:  
a. methotrexate > 25 mg/week 
b. mycophenolate mofetil > 3000 mg/d 
c. mycophenolic acid > 1080 mg/bid 
d. azathioprine > 200 mg/d 
e. cyclosporine > 2 mg/kg/d 
f. dapsone >250 mg/d 
g. IVIG > 4mg/kg IV monthly 
4. Use of rituximab within the 12 months prior to screening 
5. Change in dosing frequency, concentration, or applied surface area of topical steroids 
and/or topical calcineurin inhibitors within 2 weeks prior to screening 
6. Paraneoplastic pemphigus 
7. Pemphigus erythematosus 
8. Pemphigus vegetans 
9. IgA pemphigus 
10. Drug-induced pemphigus 
11. Blood donation within 10 weeks prior to baseline visit (Day 0) 
12. Hemoglobin < 10 g/dl 
13. White blood cell (WBC) count < 3,000/ mm3 (equivalent to < 3 x109/L) 
14. Absolute lymphocyte count < 800/mm3 (equivalent to < 0.8 x109/L) 
15. Absolute neutrophil count < 1,500/mm3 (equivalent to < 1.5 x109/L) 
16. Platelets < 100,000/mm3 (equivalent to < 100 x 109/L) 
17. Liver function test (aspartate aminotransferase [AST], alanine aminotransferase [ALT], 
or alkaline phosphatase [ALK]) results that are ≥ 2 times the upper limit of normal 
(ULN) 
18. Direct bilirubin > ULN 
19. End stage renal disease (estimated glomerular filtration rate [eGFR] < 20 
ml/min/1.73m2 using the CKD-EPI equation [59]) 
20. At or within three months of screening:  
a. positive QuantiFERON®-TB Gold test or positive purified protein derivative 
tuberculin skin test (PPD) (>5mm induration, regardless of Bacille Calmette 
Guerin [BCG] vaccine administration) unless completion of treatment has 
been documented for active TB 
b. an indeterminate QuantiFERON®-TB Gold test unless followed by a 
subsequent negative PPD or negative QuantiFERON®-TB Gold test as well 
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as a consultation with and clearance by local infectious disease (ID) 
department  
21. Recent or ongoing active bacterial, viral, fungal, or opportunistic infections requiring 
systemic anti-infective therapy 
22. Evidence of current or prior infection with human immunodeficiency virus (HIV), 
hepatitis B (as assessed by HBsAg and anti-HBc Ab) or hepatitis C (as assessed by 
anti- HCV Ab)  
23. Detectable circulating EBV or CMV genomes or active infection  
24. Chronic infection that is currently being treated with suppressive anti-infective therapy, 
including but not limited to tuberculosis, pneumocystis, CMV, herpes zoster, and 
atypical mycobacteria, with the exception of historical orolabial or localized cutaneous 
herpes simplex infections treated with suppressive anti-viral therapy  
25. Receipt of a live-attenuated vaccine within 12 months prior to screening 
26. Concomitant malignancies or a history of malignancy, with the exception of completely 
treated basal cell carcinoma of the skin 
27. Pregnancy 
28. Lactating or breastfeeding 
29. Unwilling or unable to use reliable method(s) of contraception (as described in Section 
5.6.3, Reproductive Risks) 
a. For females of child-bearing potential, from four weeks prior to Day 0 
through 1 year after Treg dosing 
b. For males, from the day of Treg infusion (baseline visit) to three months after 
Treg infusion  
30. Use of an investigational therapeutic medication, or other biologic medications except 
rituximab, within the past 90 days, or 5 half-lives prior to screening, whichever is 
greater  
31. Concomitant medical condition that places the subject at risk by participating in this 
study, including but not limited to: 
a. another severe, systemic autoimmune disease or condition (besides 
pemphigus) requiring systemic immunosuppressive therapy (e.g., rheumatoid 
arthritis, SLE, systemic sclerosis, primary Sjorgen’s syndrome, primary 
vasculitis, psoriasis, multiple sclerosis, ankylosing spondylitis, and 
inflammatory bowel disease), or 
b. severe, progressive, or poorly controlled renal, hepatic, hematological, 
gastrointestinal, pulmonary, cardiac, or neurological disease, or  
c. history of significant infection or recurrent infection that, in the investigator’s 
opinion, places the subject at risk by participating in this study, or 
d. any other concomitant medical condition that, in the investigator’s opinion, 
places the subject at risk by participating in this study 
32. Comorbidities requiring glucocorticoid therapy, including those which have required 
three or more courses of systemic glucocorticoids within the previous 12 months 
33. Current or history within the past year of substance abuse 
34. Inability to comply with study and follow-up procedures 
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Co-enrollment Guidelines 
4.2.1
Subject may be in observational registries or cohorts as long as the combined blood draw 
totals do not exceed the limits of the local institutional review boards and objectives do not 
confound the current study.  
5 DESCRIPTION OF STUDY PRODUCT 
 
Product Description 
5.1.1
The name of the drug product is: Ex Vivo Expanded Autologous CD4+CD127lo/-CD25+ 
Polyclonal Regulatory T Cells (PolyTregs). PolyTregs are cells that have been collected from 
the donor by phlebotomy, purified by Ficoll density gradient, and subsequently selected for 
the target CD4+CD127lo/-CD25+ T cell subset by FACS, using three monoclonal antibodies 
specific for the cell surface antigens CD4, CD127, and CD25. The isolated cells are then 
expanded using anti-CD3/anti-CD28-coated magnetic microbeads in cell culture medium 
containing deuterated glucose ([6,6-2H2]-Glucose) (see Section 6.5.1 for more information on 
deuterium labeling) for a total of 14 days before harvesting. Following expansion, anti-
CD3/anti-CD28 beads are removed via a magnetic separation system, and cell cultures are 
combined, washed and adjusted to desired concentration.  
The CD4+CD127lo/-CD25+ PolyTreg product is a sterile cell suspension of fresh, non-
cryopreserved cells in a mixture of 75% FDA approved infusion solution conforming to USP 
standards (49.02% (v/v) Plasma-Lyte A, 49.02% (v/v), Dextrose 5% with 0.45% NaCl, and 
1.96% (v/v)) and 25% Human Serum Albumin (HSA). The final product is administered via 
IV over a 10-30 minute period by gravity.  
The product will be manufactured at UCSF Human Islet and Cellular Transplantation Facility 
(HICTF) and GMP Facility (San Francisco, CA). 
Ex Vivo Expansion of Human Autologous PolyTregs  
Study subjects will provide up to a target of 400 ml of whole blood via phlebotomy to obtain 
an adequate number of cells for product manufacture. Whole blood will be transported to the 
manufacturing facility for processing. Previous studies [60] have shown that this process can 
be conducted successfully using cells obtained locally or at other sites that ship to UCSF.  
PBMC will be isolated by Ficoll gradient density centrifugation. The pooled lymphocytes 
will be stained with clinical-grade fluorescently conjugated antibodies and sorted for 
CD4+CD127lo/-CD25+ T cells using a commercial cell sorter. Tregs will be expanded per 
standard operating procedures adapted from optimized methods previously described [23]. 
This method uses co-stimulation of the Tregs with anti-CD3 and anti-CD28 immobilized on 
magnetic beads. Enriched Tregs will be cultured in growth medium containing human serum 
and deuterated glucose and stimulated to proliferate over a 14-day period in the presence of 
IL-2 and above-mentioned beads. Cultures will be maintained in sterile culture vessels or 
bags in a humidified 37°C 5% CO2 incubator throughout the culture period. Cells will be 
counted every 2 to 3 days and transferred to vessels of increasing size to maintain appropriate 
cell density as cells proliferate, and fresh medium and IL-2 will be added at each cell 
passage. Cells will be restimulated with additional anti-CD3/anti-CD28 beads at day 9 of the 
culture period. Cultures will be tested for presence of bacteria, fungus, mycoplasma, and 
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endotoxin at several interim time points during the expansion period. Following expansion, 
the beads will be removed via a magnetic separation and the cells will be concentrated, 
consolidated, and resuspended in sterile infusion solution at the required concentration. 
The investigational product will be quarantine refrigerated and will be released with an 
Interim CoA. The final CoA will be provided once all sterility test results are available.  
Studies will be run on the final product for infusion to identify the dominant Treg clones. 
Results from bacterial and fungal testing of the final product will be available 14 days and 4 
weeks post-infusion, respectively.  
 
Packaging and Labeling of Study Product 
5.1.2
The Tregs product will be manufactured for subjects at UCSF and the remote sites. It will be 
shipped to the remote clinical sites via next-day service using validated conditions and 
containers. Qualified staff at the remote sites will receive the PolyTregs, verify the recorded 
shipment temperatures were maintained within the validated range, and assess viability and 
recovery of the product according to UCSF established SOPs. 
PolyTregs will be supplied fresh, in sterile 150 ml infusion bags. Label information will be 
consistent with applicable regulations. All information on the completed label will be 
verified as accurate by at least two individuals prior to transport to the infusion site.  
 
Storage and Handling of Study Product 
5.1.3
The Treg product is delivered to the bedside in a temperature controlled container (2-10°C) 
and is removed from the container just before being administered to the subject. The infusion 
of the product must occur before expiration time as indicated on the CoA. 
Unused product will be disposed in accordance with established standard operating 
procedures or placed in archive if authorized by the subjects on the informed consent form. 
5.2 Dosage Regimen  
A single infusion of 1.0 x 108 or 2.5 x 108 PolyTregs will be administered. 
5.3 Administration of Study Product 
 
Preparation for Administration 
5.3.1
On the day of scheduled infusion, blood chemistries, a complete blood count (CBC) with 
differential blood count, and a pregnancy test (for women of childbearing potential) will be 
obtained locally and reviewed prior to infusion of the PolyTregs. A pre-infusion checklist 
(see Section 15.6, Pre-Infusion Required Checklist) will be used to review relevant clinical 
and laboratory data prior to infusion. Cell infusion may be held based on the pre-infusion 
checklist at the discretion of the site investigator. A history of any recent illness or fever will 
be obtained. The subject should be both asymptomatic and the illness should be resolved 
before infusion. See Section 5.3.4 for additional criteria for withholding study treatment.  
 
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Subjects will be pre-medicated with acetaminophen 650 mg by mouth and diphenhydramine 
hydrochloride 25-50 mg by mouth or intravenous (IV), approximately 30 minutes prior to the 
infusion of Tregs. These medications may be repeated every six hours as needed. A course of 
non-steroidal anti-inflammatory (NSAID) medication may be prescribed if the patient 
exhibits a fever not relieved by acetaminophen. Patients will not receive systemic 
glucocorticoids higher than their usual dose such as hydrocortisone, prednisone, prednisolone 
(Solu-Medrol) or dexamethasone (Decadron).  
Prior to administration, the identity of the subject and of the lot are verified by two individuals at 
the clinical site.  
 
Administration 
5.3.2
After double verification of cell therapy product and recipient identification, the cells will be 
infused via a peripheral IV line with an 18-gauge needle primed with saline per established 
standard operating procedures. All doses will be administered by peripheral IV line via 
gravity in approximately 20 to 30 minutes. Following administration of the PolyTregs, the 
product bag, tubing, and peripheral IV line will be flushed with normal saline to ensure 
complete dose is infused.  
Vital signs (temperature, respiratory rate, pulse, and blood pressure) will be taken 
immediately prior, midway, and at completion of the infusion, and then every 15 minutes (+/- 
3 minutes) for at least one hour and longer at this interval as indicated to confirm vital signs 
are stable. From then on, vital signs will be monitored approximately every hour until four 
hours post-infusion and approximately every four hours thereafter until discharge. 
The IV catheter will be maintained after the infusion until discharge and the subject will 
remain in the clinical research unit for 24 hours. The subject will be contacted by telephone 
daily for the next 2 days to assess condition and AEs. 
If a study subject experiences symptoms suggestive of an infusion reaction during 
administration, study treatment will be slowed or temporarily stopped. The subject will be 
monitored until symptoms resolve. If symptoms can be resolved with acetaminophen, 
diphenhydramine, or other medication (other than glucocorticoids) at the discretion of the 
investigator, the infusion may be restarted at the original rate. (Section 5.5.1 Criteria for 
Interruption of Study Treatment) 
Emergency medical equipment (i.e., emergency crash code cart) will be made available for 
the infusion in case the subject has an allergic response, severe hypotensive crisis, or any 
other reaction to the infusion.  
 
Action Plan for Post Release Positive Sterility Results 
5.3.3
The results of aerobic bacterial culture, anaerobic bacterial culture, and fungal culture on the 
final product will not be available prior to product release. In the event of a post-release 
bacterial or fungal positive result, the finding will be reported to the clinical investigator, 
institutional review board (IRB), and FDA and a temporary suspension in enrollment will be 
in effect until a cause is identified and remedied, if required (see Section 5.5.4, Safety 
Stopping Guidance).  
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The clinical team (site PI[s]) will consult with local infectious disease specialists, and anti-
microbial therapy will be considered for the subject as deemed appropriate by the clinical 
team. The cause of the sterility failure will be investigated and necessary corrective actions 
will be implemented. The sterility failure, results of investigation of the cause, and corrective 
actions will be reported to the FDA within 30 calendar days of the receipt of the positive 
culture test result. 
 
Criteria for Withholding Study Treatment 
5.3.4
Infusion of study product is to be withheld if the subject is experiencing any of the following 
on the day of infusion: 
• Fever > 38.0°C 
• Acute Diarrhea and/or Vomiting 
• Other signs/symptoms of an intercurrent NCI-CTCAE Grade 2 or higher infection. 
• Significant pemphigus relapse/flare (PDAI >12)  
• Other elements precluding infusion as per the study pre-infusion checklist (see 
Section 15.6, Pre-Infusion Required Checklist) 
• Other comorbid conditions which in the opinion of the investigator would pose risk to 
the subject with administration of the study product 
5.4 
Repeat Screening When Infusion is Not Given as Scheduled  
If the participant completes the Week -2 visit (400ml blood donation) and does not receive 
the infusion on Day 0 as scheduled, the participant may be eligible to return to repeat 
screening no sooner than 8 weeks following the preceding blood donation. If the repeat 
Week -2 visit (blood donation) is scheduled:  
• Less than or equal to 12 weeks after initial blood donation, only Hematology and 
whole blood quantitative PCR for EBV/CMV viral load must be repeated, with other 
screening tests repeated as clinically indicated  
• Greater than 12 weeks after initial blood donation, all screening assessments must be 
repeated 
5.5 Treatment Interruption and Discontinuation  
Treatment will consist of a single infusion of ex vivo expanded PolyTregs. Once a PolyTreg 
infusion has concluded, the treatment phase of the protocol is complete. Thus, the question of 
subsequent withdrawal from treatment will not arise. Therefore, all subjects who undergo an 
infusion will continue per protocol unless they choose to withdraw (see Section 5.5.3, Subject 
Withdrawal from the Study).  
 
Criteria for Interruption of Study Treatment  
5.5.1
If a study subject experiences symptoms suggestive of an infusion reaction during 
administration, study treatment will be slowed or temporarily stopped. The subject will be 
monitored until symptoms resolve. If symptoms can be resolved with acetaminophen, 
diphenhydramine, or other medication (other than glucocorticoids) at the discretion of the 
investigator, the infusion may be restarted at the original rate, provided none of the criteria in 
Section 5.5.2 Criteria for Permanent Discontinuation of Study Treatment have occurred. 
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Criteria for Permanent Discontinuation of Study Treatment  
5.5.2
Study treatment will be discontinued permanently for any individual subject under the 
following conditions:  
1. At any time prior to or during the PolyTreg infusion at the request of the subject  
2. If investigators or NIAID determine that the subject’s health, safety, and/or well-being 
are threatened 
3. If an infusion reaction symptom that caused a slow or temporary stop of the infusion 
cannot be resolved with acetaminophen or diphenhydramine  
4. If administration of glucocorticoids to treat an infusion reaction is required 
5. If a subject experiences any of the following: 
a. NCI-CTCAE Grade 3 or higher infusion-related reaction 
b. NCI-CTCAE Grade 3 or higher cytokine release syndrome 
c. Any other infusion related SAE 
Subjects who discontinue protocol-specified treatment will be treated as medically indicated 
according to physician discretion. Refer to Section 5.5.3.1, Procedures for Subject 
Withdrawal from the Study. 
 
Procedures for Discontinuation of Protocol-Specified Treatment 
5.5.2.1
Requirements  
Whenever possible, subjects who received an abbreviated infusion that was terminated due to 
a possible infusion reaction should complete all remaining scheduled study visits including 
all exams, procedures, assessments, and tests. Furthermore, if discontinuation is due to safety 
concerns, subjects will be given appropriate care under medical supervision beyond the last 
scheduled study visit, if necessary, until the symptoms of any AE resolve or the subject’s 
condition becomes stable. If the site PI determines that completion of these visits is not 
clinically appropriate for the subject or if the subject elects not to complete these visits, the 
subject will be withdrawn from the study per the guidelines in Section 5.5.3.1, Procedures 
for Subject Withdrawal from the Study. 
 
 Subject Withdrawal from the Study 
5.5.3
When a subject is withdrawn from the study, protocol-specified treatment requirements are 
discontinued, and study-related visits, exams, procedures, assessments, tests, and data 
collection are terminated. Individual subjects will be withdrawn from the study under the 
following conditions: 
1. The subject did not receive the planned infusion after two blood donations for PolyTreg 
manufacturing 
2. The subject withdraws consent 
3. The investigator or NIAID believes it is in the best interest of the subject 
4. The subject is lost to follow-up  
 
Procedures for Subject Withdrawal from the Study 
5.5.3.1
Whenever possible, subjects who withdraw from the study will be asked to consent to an 
end-of-study evaluation, in which all scheduled exams, procedures, and laboratory tests 
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planned for Week 12 will be performed; unless the early withdrawal is after that time, in 
which case all scheduled exams, procedures, and laboratory tests scheduled for Week 52 will 
be performed; unless the early withdrawal is after that time, in which case all scheduled 
exams, procedures, and laboratory tests scheduled for Week 156 will be performed. 
Furthermore, if discontinuation is due to safety concerns, subjects will be given appropriate 
care under medical supervision beyond the last scheduled study visit, if necessary, until the 
symptoms of any AE resolve or the subject’s condition becomes stable. After this end-of-
study visit, the PI (or designated treating physician) may continue to follow the subject to 
manage clinical care, but no additional study-related data will be collected. 
 
Safety Stopping Guidance 
5.5.4
The SRC will suspend enrollment after a Grade 3 or higher AE or any SAE (as described in 
Section 3.1, Description of Study Design) in this trial in order to review the event, to review 
any additional events that occurred in the trial, and to make a decision on how to proceed. In 
the event of a temporary suspension in enrollment for a Grade 3 or higher AE or any SAE or 
a post release positive sterility result (see Section 5.3.3, Action Plan for Post Release Positive 
Sterility Results), no new subjects will be consented or started on therapy with PolyTregs; 
subjects who already received PolyTregs will continue to be followed. Subjects in the 
screening phase of the study may continue screening procedures (e.g., blood tests), but a skin 
biopsy to confirm Inclusion Criteria # 3 (see Section 4.1, Inclusion Criteria) should be 
deferred. Enrollment will not occur until the SRC review is complete.  
A significant event in this trial can occur either due to toxicity from the study drug or disease 
worsening (e.g., a pemphigus relapse/flare) assessed as related to the investigational 
treatment.  
The SRC will make this determination, seeking advice from the DSMB, if necessary. After 
SRC review, the study will: 1) proceed with enrollment as originally planned, 2) continue to 
suspend enrollment until the DSMB can review and advise on the event, or 3) be placed on 
hold pending further evaluation of the event, consideration of potential study modifications, 
and review by the DSMB, IRB and FDA.  
For Grade 3 or higher AEs or any SAEs occurring in the first year of the study for each 
participant, DSMB input will be required for the evaluation of the following events unless 
the event was judged clearly unrelated to the investigational treatment: 
• two Grade 3 or higher AEs or SAEs that occurs during the first 12 weeks of each 
participant in each cohort 
• any Grade 4 or 5 AE 
• two or more Grade 3 AEs of the same preferred term in a cohort 
• any Grade 3 AE that is part of a pemphigus relapse/flare using the consensus 
definition of 3 or more new lesions a month that do not heal spontaneously within 1 
week or by the extension of established lesions in a patient who has achieved disease 
control [58] 
For Grade 3 or higher AEs or any SAEs occurring in the second or third years of the study 
for each participant, DSMB input will be required for the evaluation of the following events 
unless the event was judged clearly unrelated to the investigational treatment: 
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• any Grade 4 or 5 AE 
• two or more Grade 3 AEs of the same preferred term in a cohort 
In addition to the pre-scheduled data reviews and planned safety monitoring, the DSMB will 
review any event that potentially impacts safety at the request of the SRC or DAIT, NIAID. 
The DSMB will have the discretion to recommend actions regarding study conduct and 
continuation as a consequence of any planned or unplanned monitoring activity.  
Otherwise, the DSMB will be informed in real time per the following criteria: 
• During Year 1 of each subject’s participation: 
o any decision of the SRC to add additional subjects based on confirmation of a 
significant event (as defined as any related Grade 3 or higher AEs or any 
SAEs) 
o any malignancy 
• During Years 2 and 3 of each subject’s participation: 
o any Grade 3 event that is part of a pemphigus relapse/flare using the 
consensus definition of 3 or more new lesions a month that do not heal 
spontaneously within 1 week or by the extension of established lesions in a 
patient who has achieved disease control [58] 
o any malignancy 
5.6 Toxicity Management Plan for Study Product 
See Section 5.5.2, Criteria for Permanent Discontinuation of Study Treatment for details on 
when to slow, temporarily discontinue, or permanently discontinue the study drug infusion 
for toxicities.  
 
Infection  
5.6.1
Based on the potential for Tregs to have immunosuppressive effects, subjects should be 
closely monitored for the development of signs and symptoms of infection, particularly 
opportunistic infections after treatment including reactivation of EBV and CMV. 
 
Vaccination 
5.6.1.1
Participants will be advised to obtain and will be offered tetanus, diphtheria, and pertussis 
(Tdap) and inactivated seasonal influenza vaccinations prior to treatment. Any such 
vaccination must be completed either prior to day -21, or after Week -2’s whole blood 
donation and before day -7. 
 
Herpes Simplex Virus 
5.6.1.2
Infection with Herpes Simplex Virus (HSV), with predominance of mucocutaneous 
involvement, has been associated with pemphigus [61]. The protocol allows for participation 
of individuals receiving suppressive anti-viral treatment for orolabial or localized cutaneous 
herpes simplex infection (see Section 4.2, Exclusion Criteria #24). Individuals presenting 
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with oral erosions or ulcerations at screening will undergo viral culture to rule out active 
mucocutaneous HSV infection.  
 
Epstein-Barr Virus 
5.6.1.3
Assessment for the presence of EBV genomes will be carried out according to the schedule 
outlined in Table 6.1, Schedule of Events. Management will depend on the presence or 
absence of symptoms and on the viral load.  
For those with no or NCI-CTCAE Grade 1 or 2 symptoms judged at least possibly related to 
EBV reactivation:  
• If EBV viral load is undetectable, study proceeds as per Table 6.1, Schedule of Events  
• If EBV viral load is detectable and < 2000 DNA copies per 106 PBMCs monitoring of 
EBV viral load may be increased as determined clinically necessary by the investigator  
• If EBV viral load is ≥ 2000 DNA copies per 106 PBMCs monitoring of EBV viral load 
will be increased  
For those with NCI-CTCAE Grade 3 or higher symptoms judged at least possibly related to 
EBV reactivation: 
• If EBV viral load is undetectable, monitoring of EBV viral load will be increased as 
determined clinically necessary by the investigator  
• If EBV viral load is detectable, monitoring of EBV viral load will be increased and 
appropriate treatment will be considered as determined clinically necessary by the 
investigator  
 
Cytomegalovirus 
5.6.1.4
Assessment for the presence of CMV genomes will be carried out according to the schedule 
outlined in Table 6.1, Schedule of Events. Management will depend on the presence or 
absence of symptoms and on the viral load.  
For those who were seropositive at study entry:  
• Subjects whose viral load increases 5-fold between scheduled assessments or is > 
10,000 DNA IU/ml at any point during the study will be evaluated for CMV retinitis, 
pneumonia and gastroenteritis/colitis  
• Participants with an active infection may be treated with gancyclovir as determined by 
the investigator. CMV viral load will be monitored by polymerase chain reaction (PCR) 
until it is < 10,000 DNA IU/ml 
For those who were seronegative at study entry:  
• Subjects whose viral load is > 2,000 DNA IU/ml will be evaluated for CMV retinitis as 
clinically indicated, as well as CMV pneumonia and gastroenteritis/colitis  
• Participants with an active infection may be treated with gancyclovir as determined by 
the investigator. CMV viral load will be monitored by PCR until it is < 2,000 DNA 
IU/ml  
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For all subjects:  
• If a participant develops any symptoms of the eye, lungs, or intestines consistent with 
CMV infection, additional blood will be drawn for appropriate laboratory analyses as 
determined by the investigator. Additionally, for those with visual symptoms, an 
ophthalmological exam will be performed. 
 
Infusion Reaction  
5.6.2
Section 5.3.1, Preparation for Administration describes the pre-medications required to help 
prevent infusion reactions and the medications that may be used to treat them. 
Patients will not receive systemic glucocorticoids higher than their usual dose such as 
hydrocortisone, prednisone, prednisolone (Solu-Medrol) or dexamethasone (Decadron) at 
any time, except in the case of a severe or life-threatening emergency since this may have an 
adverse effect on T cells. If glucocorticoids are required for an acute infusion reaction, an 
initial dose of hydrocortisone 100 mg IV is suggested.  
 
Reproductive Risks  
5.6.3
There may be an unexpected risk to an unborn or nursing child. Pregnant and breastfeeding 
women will be excluded from participation in the study. Females must have a negative 
pregnancy test prior to enrolling in the study. Female subjects must agree not to participate in 
a conception process (e.g., active attempt to become pregnant or in vitro fertilization) and 
must agree to use a reliable and effective form of birth control (i.e., oral contraceptives, 
barrier methods, or abstinence) for one year after Treg dosing. Male subjects must agree to 
not participate in a conception process (e.g., active attempt to impregnate or sperm donation) 
and use birth control (i.e., oral contraceptives by female partners, barrier methods, or 
abstinence) for three months after Treg dosing. At every study visit the sexual activity of 
subjects of reproductive age will be re-assessed. If a subject who was previously sexually 
inactive becomes sexually active, s/he will be counseled about the need to use a reliable and 
effective form of birth control. Female subjects will also be required to undergo a urine 
pregnancy test prior to cell administration. A positive pregnancy test will result in holding of 
scheduled cell administration. Pregnancy tests will also be done at subsequent study visits for 
female subjects as described in Table 6.1, Schedule of Events.  
Investigators of female subjects of childbearing potential on concurrent Mycophenolate 
Mofetil (MMF), and those subjects themselves, whether or not they plan to become pregnant, 
are strongly encouraged to participate in Mycophenolate Risk Evaluation and Mitigation 
Strategy (REMS), as described in Section 7.6.1, Mycophenolate REMS Program. 
 
Malignancies 
5.6.4
As the T lymphocytes are one major component of tumor surveillance it is theoretically 
possible that cells that inhibit T lymphocytes could impair this function. Therefore, the 
recommendations for individuals at higher risk for skin cancer apply to this research 
population and exposure to sunlight and UV light should be limited by wearing protective 
clothing and using a sunscreen with high protection factor. Refer to Section 5.7, Concurrent 
Medication and Therapy. 
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Subjects over the age of 50 should have age-appropriate cancer screenings at the 
investigator’s discretion prior to enrollment and throughout the course of the study. For 
additional information, consult the American Cancer Society (ACS) reference below:  
http://www.cancer.org/healthy/toolsandcalculators/reminders/screening-recommendations-
by-age 
 
Skin Biopsies 
5.6.5
Three 4mm skin punch biopsies from affected skin will be collected under local anesthesia 
for proposed mechanistic studies. In addition, APG01 will minimize discomfort and scarring 
by numbing a single site and performing all three 4 mm punch biopsies in a contiguous 
fashion, with a single resultant incision, when possible. In cases where this is not technically 
feasible (e.g., lesion is too small), an alternative approach is to obtain a total of three punch 
biopsies from two or more separate sites; when possible, punches may still be placed in 
contiguous fashion to minimize the total number of individual, smaller incisions. In addition 
to the above biopsies for mechanistic studies, a single standard 4 mm punch biopsy will be 
obtained during screening for subjects without prior histopathologic confirmation of their 
pemphigus.  
In all cases, the following prioritized list of considerations will inform the investigator’s 
clinical judgment when selecting skin biopsy sites: 
1. Wound environment. Investigators will avoid repeat biopsies directly through prior 
biopsy scars or biopsy of any areas that appear to be potentially secondarily infected. 
2. Optimal cosmesis. Preference will be given to more cosmetically favorable areas 
(e.g., avoid central face when possible) with favorable skin mobility to facilitate the 
best possible closure. 
3. Abundance and size of skin lesions. Preference will be given to more numerous and 
larger lesions so that biopsies may be obtained from the same body region. 
4. Activity of lesions. Though the activity of lesions may change throughout study 
duration, preference will be given to active lesions at each time point. 
Skin biopsy on Week 12 may be performed on unaffected skin if no lesional skin present.  
o Please note topicals should be held from the planned biopsy location for the 7 days 
prior to each skin biopsy.  See Section 5.7, Concurrent Medications and Therapies 
and 5.8, Prohibited Medications for specific information on use of topical agents 
prior to skin biopsies. 
Skin biopsy sutures should be removed per local standards. 
 
Digital Photography 
5.6.6
Standardized total body photographs, regional photographs (e.g., torso, extremities, mouth) 
and close-up photographs of selected areas with active disease will be taken to monitor 
disease activity. The subject may decline photographs at any time during the study in 
instances where the subject is sensitive to being photographed. For additional instructions 
regarding Digital Photography, refer to the APG01 Manual of Procedures (MOP). 
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5.7 Concurrent Medications and Therapy 
• If subjects are on prednisone at the Screening visit, subjects may continue to be treated 
with stable doses of prednisone not to exceed <25 mg/d until Week 12. 
o After Week 12, prednisone may be tapered at the discretion of the investigator, at 
a suggested rate of 5-10 mg decrease per month down to 10 mg/d, and then 1-5 
mg decrease per month down to 0 mg/d. 
o Prednisone dose may be increased during the study for flares of pemphigus and 
for conditions that are not related to pemphigus based on the PI’s clinical 
judgment. Increases in prednisone should occur only if absolutely necessary, and 
are discouraged for reasons other than pemphigus flares during the first 12 weeks 
of the trial. Stress doses of glucocorticoids may be used when clinically indicated. 
• If subjects are on background immunosuppressive/immunomodulatory medications at 
the Screening visit, subjects should continue to be treated with stable doses of these 
agents until Week 12 including methotrexate ≤ 25 mg/week, mycophenolate mofetil ≤ 
3000 mg/d, mycophenolic acid ≤ 1080 mg/bid, azathioprine ≤ 200 mg/d, cyclosporine ≤ 
5 mg/kg/d, dapsone ≤ 250 mg/d and IVIG ≤ 4mg/kg monthly. 
o After Week 12, changes in the above background medications may be made per 
the investigator’s judgment. Thus, one medication may be stopped and another 
medication started. The dose of any of the medications must not exceed those 
specified above at any point during the study period. 
• If a subject is having increased pemphigus disease activity at any point during the 
study, they will be treated according to the best clinical judgment of the investigator.  
• If subjects are on topical agents at the Screening visit, subjects must continue to be 
treated with stable amounts (stable percentage and approximate volume) of these 
agents, including steroids and calcineurin inhibitors, until Week 12. After Week 12, the 
amount of the topical medications may be altered per the investigator’s judgment. 
o Of note, topical medications must be held from the planned biopsy location for 
one week prior to each scheduled skin biopsy. Topical medications must be held 7 
days to skin biopsy on Day 0 and may be resumed after the skin biopsy at Week 
1. Topical medications must again be held for 7 days prior to the final biopsy at 
Week 12, and may be resumed after this procedure.  
In addition, iron supplementation and medications for bone health are encouraged as follows: 
• Daily multivitamin containing iron for at least 90 days after the blood donation visit 
• Medications for bone health may include any or all of the following: calcium carbonate 
or citrate (up to 1500 mg/d of elemental calcium), vitamin D (up to 2000 IU/day), and 
bisphosphonates 
In addition, the use of broad spectrum (ultraviolet A [UVA] and ultraviolet B [UVB]) 
sunscreen through the entirety of the trial is encouraged. 
5.8 Prohibited Medications 
Prohibited medications include:  
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• throughout the duration of the trial: 
o immunosuppressive medications other than those specified in Section 5.7, 
Concurrent Medications and Therapy  
o live attenuated vaccines 
• 7 days prior to each skin biopsy at the site of the biopsy: 
o topical steroids, topical calcineurin inhibitors  
5.9 Procedures for Monitoring Subject Compliance 
Subjects will not be self-administering any protocol-specified treatment. Therefore, their 
compliance will be monitored by their appearance for scheduled study visits, including 
acquisition of peripheral blood for expansion of Tregs, a subsequent single PolyTreg 
infusion, and follow-up visits for monitoring. 
6 ASSESSMENT OF SAFETY AND EFFICACY 
6.1 General Assessments 
• Informed consent: written informed consent will be obtained before any study 
assessments or procedures are performed 
• Demographics: date of birth, gender, self-identified race, self-identified ethnicity  
• Medical History: Lifetime history of malignancy and family history of autoimmune 
disease.  All other history should be from within 10 years prior to Screening Visit  
• 
Comprehensive physical examination, at screening visit only 
• Brief physical examination focused on participant’s current complaints and clinical 
status  
• Detailed Skin Examination 
• Adverse Events. Participants will be assessed for adverse events at every visit 
• Adverse Events Phone Assessment. Phone assessment for adverse events will be 
conducted at Day 2 and 3 and Weeks 3, 5 and 14 
• Concomitant medications: Concomitant medications will be documented for 12 weeks 
prior to screening, and subsequently will be recorded at every visit 
• Vital signs: height (at screening only), weight, temperature, heart rate, respiratory rate, 
sitting systolic blood pressure, sitting diastolic blood pressure will be assessed at every 
visit 
6.2 Clinical Laboratory Assessments 
• Chest x-ray: unless chest x-ray has been performed within 3 months of the Screening 
visit and documented test results are available 
• Serum pregnancy test  
• Urine pregnancy test: Stat studies at week -2 and baseline visits 
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• Hematology: CBC with differential and platelets 
• Chemistry: BUN, creatinine, AST, ALT, total bilirubin, direct bilirubin, alkaline 
phosphatase, albumin, sodium, potassium, chloride, and bicarbonate 
• Infectious disease testing: HIV serology, HBsAg, anti-HBc Ab, anti-HBs Ag, and anti-
HCV Ab (and HCV RNA if anti-HCV Ab test is reactive), unless performed within 3 
months of the Screening visit and documented test results are available 
• EBV & CMV IgG, unless performed within 3 months of the Screening visit and 
documented test results are available 
• EBV & CMV viral load. See Section 5.6.1.3, Epstein-Barr Virus and Section 5.6.1.4, 
Cytomegalovirus for management guidelines 
• TB testing: either a PPD or QuantiFERON®-TB Gold In-Tube test, unless performed 
within 3 months of the Screening visit and documented test results are available, or 
unless subject is known to have a positive or indeterminate test and has documentation 
of appropriate therapy 
• Urinalysis with microscopic analysis 
• Desmoglein 1/3 autoantibodies by ELISA 
• Treg absolute count in peripheral blood  
 
6.3 Disease-Specific Assessments 
• Skin biopsy for histopathology and direct immunofluorescence. Not required if results 
are documented prior to screening (see Section 4.1, Inclusion Criteria for details) 
• Pemphigus Disease Area Index (PDAI): see Section 15.1, PDAI  
• Skindex-29: see Section 15.2, Skindex-29 [62, 63] 
• PGA: see Section 15.3, Patient’s Global Assessment 
• PhGA: see Section 15.4, Physician’s Global Assessment 
• Digital photography: see Section 0, Digital Photography for details on types of 
photographs as well as adjustments for subject consent 
6.4 Study Procedures 
• Blood donation: approximately 400 ml will be colleced into a blood bag 
• PolyTreg infusion: the Baseline and Day 1 visits are both a part of the 24-hour 
observation hospitalization for the PolyTreg infusion (see Section 5.3.2, Administration 
for details) 
• Specimen collection for mechanistic studies 
• Skin biopsies: Anatomic location and disease activity will be recorded for each 
biopsy site  
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o See Section 5.6.5, Skin Biopsies, 5.7, Concurrent Medications and Therapies 
and 5.8, Prohibited Medications for information on use of topical agents prior 
to skin biopsies 
o See Section 5.6.5, Skin Biopsies for the considerations to determine location 
of the biopsies as well as procedural steps to minimize discomfort and 
scarring  
o See Section 6.5, Mechanistic Assessments for information on the studies that 
will be conducted on these biopsies 
• Blood: Sodium heparin (green top) tubes 
6.5 Mechanistic Assessments 
These mechanistic studies are designed to demonstrate the presence, persistence, and 
function of adoptively transferred Tregs in the blood and skin. Functionally analyzing both 
the adoptively transferred Tregs as well as endogenous T cell subsets in both the blood and 
skin enables comparison of the influence of adoptively transferred Tregs on the systemic 
immune system as well as on the local immune milieu in inflamed tissue. The following 
mechanistic studies will be performed: (i) mass spectrometry of transferred 2H2-labeled Tregs 
in blood and skin and/or TCR sequencing to demonstrate the presence and assess the 
persistence of adoptively transferred Tregs; (ii) functional immunoflow cytometry on blood 
and skin to assess how transferred Tregs influence the cellular infiltrate and cytokine 
expression both systemically and in the target tissue; and (iii) whole transcriptome RNA-
sequencing to assess gene expression changes in skin after Treg adoptive transfer. 
 
Treg Tracking by Stable Isotope Labeling  
6.5.1
APG01 relies on transferring PolyTregs that have been expanded hundreds-of-fold in vitro, 
which would be indistinguishable from endogenous Tregs by standard surface markers. 
However, because the cells are being expanded ex vivo, it provides a unique opportunity to 
label the cells prior to infusion to monitor engraftment and persistence of the administered 
PolyTregs. For over a decade, the Hellerstein group [64, 65] and others using their 
techniques, have applied stable isotope labeling with mass spectrometric analysis to measure 
the replication of murine and human cells in vitro and in vivo. Importantly, stable isotopes 
are non-radioactive and non-toxic, and they have been safely used as cellular, molecular, and 
metabolic markers in patients and healthy controls for more than six decades.  
APG01 will label Tregs from pemphigus patients with deuterium by including the 2H2 label 
in the culture medium during the entirety of the expansion phase in vitro (Day 0 to Day 14) 
prior to infusion in the subject. In this technique, the 2H2 label is incorporated into 
the deoxyribose (dR) moiety in replicating DNA via the de novo synthesis pathway. Based 
on data from in vitro labeling studies, it is expected for 60% of purine deoxyribonucleosides 
in DNA strands to be labeled by deuterium at the time of infusion. After infusion, both 
PBMCs and single cell suspensions from skin biopsy specimens will be isolated and stored at 
time points as shown in Table 6.1, Schedule of Events. DNA prepared from these samples 
will be analyzed by gas chromatography-mass spectrometry in order to detect isotopic 
enrichment of the purine deoxyribonucleosides (deoxyadenosine [dA] or deoxyguanosine 
[dG]) [64, 66]. This method will identify infused Tregs in the systemic circulation and in the 
target tissue unequivocally. Based on estimates of the number of Tregs in the body (~1 x 
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1010), 1.0 x 108 and 2.5 x 108 transferred autologous Tregs in cohorts 1 and 2, respectively, 
will represent between 2.5% and 10% of total Tregs. Preliminary studies in patients with 
T1DM have confirmed that this method is capable of detecting transferred Tregs in the blood 
(see Figure 9). Based on these calculations, deuterium labeling is predicted to be detectable 
for at least four divisions of the administered Tregs. This method of detection should be 
especially advantageous at early time points (up to 28 days) post-infusion, prior to significant 
division and/or loss of Tregs. Importantly, between 3 x 106 and 5 x 106 live cells will be 
isolated from a single 4 mm punch biopsy of affected skin. Approximately 3-5% of these 
cells are Tregs, enabling a sample between 9 x 104 and 1.5 x 105 Tregs, which is well within 
the sensitivity of this assay [67]. 
 
Whole Transcriptome RNA-Sequencing  
6.5.2
In order to determine if and how adoptively transferred Tregs influence diseased tissue (i.e., 
skin), APG01 will perform whole transcriptome RNA-sequencing of skin biopsy samples 
taken from lesional skin before and after Treg adoptive transfer. This will allow for a 
comprehensive assessment of how adoptively transferred Tregs influence gene expression in 
skin, with a specific focus on pathways known to be important in the pathogenesis of 
pemphigus. As proof of concept, UCSF has recently performed RNA-sequencing on Tregs 
and Teff cells isolated from normal human skin. These populations were purified from 
relatively small samples of skin harvested from normal healthy volunteers (Figure 14A). 
Initial pathway analyses in Tregs compared to Teff cells have revealed IL-27-mediated 
signaling to be significantly increased and the IL-23 pathway to be significantly decreased in 
Tregs (Figure 14B). In a similar fashion, APG01 will comprehensively compare lesional skin 
pre-and post-Treg transfer to elucidate the major pathways that Tregs influence in skin. 
 
 
Figure 14, Whole transcriptome RNA-sequencing of Tregs and Teff cells isolated from 
normal human skin 
(A) Tregs and mTeff cells were sorted from normal skin using expression of CD25 and CD27. RNA 
was isolated from purified cell fractions (from 3 different healthy volunteers) and subjected to whole 
transcriptome RNA-sequencing. Scatter plot shows grouped Treg vs. Teff cell comparisons. (B) 
Ingenuity® Pathway Analyses of the IL-23 and IL-27 pathways showing genes increased (arrows) and 
inhibited (line ends) identified by RNA-seq to have greater than 2-fold changes between mTregs and 
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mTeff cells with log p-values<0.05. Genes in the IL-27 pathway were significantly activated in 
mTregs (activation Z-score of +2.4; p-value 5.4x10-4) and genes in the IL-23 pathway were shown to 
be significantly inhibited in mTregs (inhibition Z-score of -2.4; p-value 3.0x10-7). 
 
Functional Immunophenotyping of Skin and Blood 
6.5.3
Cryopreserved PBMCs and single cell suspensions from fresh skin biopsies will be analyzed 
using multiparameter flow cytometry to assess changes in specific immune subsets following 
administration of expanded Tregs. The functionality of lymphocyte populations in blood and 
skin will be analyzed, specifically focusing on the cellular composition of the inflammatory 
infiltrate, T cell activation and intracellular cytokine expression profiles. T Helper Cell (TH) 
subset differentiation (TH1, TH2, and TH17) and cytokine expression from Teffs and Tregs 
will be compared between blood and skin. Analyses will focus on Treg activation markers 
and proteins known to be involved in Treg function, including but not limited to: CD25, 
CD127, CTLA-4, GARP, CD39, Ki67, and CD27.  
 
Treg Suppression Assays  
6.5.4
Frozen PBMCs will be thawed and Tregs will be isolated by flow-based sorting using CD4, 
CD25 and CD127 (and in some cases CD45RA and CD45RO) antibodies. Tregs will be 
assayed for their ability to suppress polyclonal T cell proliferation using either 
carboxyfluorescein diacetate succinimidyl ester (CFSE) or 3[H]-thymidine in standard mixed 
suppression assays.  
 
Autoantibody Analysis 
6.5.5
Autoantibody titers, including Anti-Dsg1 and 3, will be assessed. 
 
Lymphocyte Subset Analysis  
6.5.6
Lymphocyte subsets include, but are not limited to (CD3 [T] cells, CD4 [helper] T cells, CD8 
[cytotoxic] T cells, CD4+Foxp3+ Tregs, CD19 [B] cells, CD16 and CD56 Natural Killer 
[NK] cells). 
 
Serum Cytokine Analysis 
6.5.7
Collected serum will be stored for the measurement of cytokines. Serum will be assessed for 
pro-inflammatory cytokines (e.g., PDGF, IL-1ß, IL-6, IL-12, TNFα) and anti-inflammatory 
cytokines (e.g., TGF-ß, IL-10) by enzyme-linked immunosorbent assay (ELISA) and/or 
Luminex® bead assay to determine the effect of Treg treatment.  
 
Treg Epigenetic Analysis  
6.5.8
DNA purified from whole blood will be used to measure the frequency of Tregs in peripheral 
blood by means of epigenetic analysis. Analysis will be performed using the methylation-
sensitive quantitative PCR (qPCR) method developed by Epiontis (Berlin, Germany) [68, 
69]. This assay targets the demethylated state of the TSDR of the FOXP3 gene – a highly 
specific marker of Tregs. The proportion of CD4+CD25+CD127- cells as determined by flow 
cytometry strongly correlates with the proportion of demethylation of FOXP3 TSDR as 
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measured by qPCR. The proportion of demethylated FOXP3 will be normalized to 
proportion of demethylated CD3 DNA. The FOXP3 to CD3 ratio as determined by 
epigenetic analysis of methylation state strongly correlates with the ratio of Tregs to CD3+ 
cells as measured by flow cytometry [70].  
 
Antigen Specific Studies 
6.5.9
Because the antigenic targets in pemphigus are well defined, mechanistic studies will include 
attempts at quantifying T cell responses to the pemphigus autoantigens, Dsg1 & Dsg3, before 
and after PolyTreg therapy. These will include, but are not limited to, quantification of Dsg1- 
and/or Dsg3-specific T cells in peripheral blood and skin using MHC class II tetramers. In 
addition, we will determine the magnitude of antigen-specific T cell responses in peripheral 
blood before and after PolyTreg therapy by performing autologous mixed lymphocyte 
reactions with increasing concentrations of exogenous Dsg1 and/or Dsg3 peptides. Together, 
these studies will provide critical information regarding the number of antigen-specific 
pathogenic T cells and the functionality of these cells before and after PolyTreg therapy. 
6.6 Unscheduled Visits 
If disease activity increases or other concerns arise between regularly scheduled visits, 
subjects should be instructed to contact study personnel to determine whether an 
“unscheduled” visit should occur. The following evaluations will be performed at each 
unscheduled visit: 
• Adverse Event Assessment 
• Concomitant medication assessment 
• Physical Examination 
• Other evaluations may be performed at the investigator’s discretion 
If the unscheduled visit is due to an increase in Pemphigus disease activity, these additional 
evaluations should also be performed: 
• PDAI 
• Desmoglein 1/3 autoantibodies by ELISA 
• Skindex-29 (QOL) 
• Physician’s Global Assessment (PhGA) 
• Patient’s Global Assessment (PGA) 
• Hematology Panel 
• Chemistry Panel 
• Digital Photography (optional) 
• Lymphocyte subsets (optional) 
• Mechanistic Blood Draw (optional)
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7 SAFETY MONITORING AND REPORTING 
7.1 Overview 
This section defines the types of safety data that will be collected under this protocol and 
outlines the procedures for appropriately collecting, grading, recording, and reporting that 
data. AEs that are classified as serious according to the definition of health authorities must 
be reported promptly (per Section 7.5, Reporting of Adverse Events and Serious Adverse 
Events to the Sponsor: DAIT/NIAID) and appropriately to the investigational new drug 
application (IND) sponsor (DAIT, NIAID), principal investigators in the trial, IRBs, and 
health authorities. Information in this section complies with International Conference on 
Harmonization (ICH) Guideline E2A: Clinical Safety Data Management: Definitions and 
Standards for Expedited Reporting, ICH Guideline E-6: Guideline for Good Clinical 
Practice, and applies the standards set forth in the NCI-CTCAE, Version 4.0: 
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_40 
7.2 Definitions 
 
Adverse Event (or Adverse Experience) 
7.2.1
Any untoward or unfavorable medical occurrence in a human subject, including any 
abnormal sign, symptom, or disease, temporally associated with the subject’s participation in 
the research, whether or not considered related to the subject’s participation in the research 
(modified from the definition of AEs in the 1996 ICH E-6 Guidelines for Good Clinical 
Practice)." [From Office for Human Research Protections (OHRP) "Guidance on Reviewing 
and Reporting Unanticipated Problems Involving Risks to Subjects or Others and Adverse 
Events (1/15/07)" http://www.hhs.gov/ohrp/policy/advevntguid.html] 
 
Adverse Reaction and Suspected Adverse Reaction 
7.2.2
An adverse reaction means any AE caused by a drug. Adverse reactions are a subset of all 
suspected adverse reactions for which there is reason to conclude that the drug caused the 
event. 
Suspected adverse reaction (SAR) means any AE for which there is a reasonable possibility 
that the drug caused the AE. For the purposes of safety reporting, ‘reasonable possibility’ 
means there is evidence to suggest a causal relationship between the drug and the AE. A 
SAR implies a lesser degree of certainty about causality than adverse reaction, which means 
any AE caused by a drug (21 CFR 312.32(a)). 
 
“Expected” versus “Unexpected” Suspected Adverse Reaction 
7.2.3
A SAR is considered “expected” when it is listed in the investigator brochure, the general 
investigational plan, or the Protocol. A SAR is considered “unexpected” when its nature 
(specificity), severity, or rate of occurrence is not consistent with applicable product 
information as described in the safety information provided in the investigator brochure, the 
general investigational plan, or the protocol (21 CFR 312.32(a) and ICH E2A). A serious 
unexpected suspected adverse drug reaction is referred to as a SUSAR. For this study, 
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expectedness will be determined by product information provided in the investigator 
brochure (IB). 
 
Serious Adverse Event 
7.2.4
An AE or SAR is considered “serious” if, in the view of either the investigator or DAIT, 
NIAID it results in any of the following outcomes (21 CFR 312.32(a) and ICH E2A): 
1. Death 
2. A life-threatening event: An AE or SAR is considered “life-threatening” if, in the view 
of either the investigator or DAIT, NIAID, its occurrence places the subject at 
immediate risk of death. It does not include an AE or SAR that, had it occurred in a 
more severe form, might have caused death.  
3. Inpatient hospitalization or prolongation of existing hospitalization 
4. Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
5. Congenital anomaly or birth defect 
6. Important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 
judgment, it may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed above. 
Elective hospitalizations or hospital admissions for the purpose of conduct of protocol-
mandated procedures (i.e. the 24-hour observation hospitalization for the drug infusion) are 
not to be reported as an SAE unless hospitalization is prolonged due to complications. 
If an event meets any of the above SAE definitions, regardless of the relationship of the event 
to investigational product, the event must be reported to the sponsor as described in Section 
7.5.2, Reporting of Serious Adverse Events and Grade 3 and Higher Clinical Events. 
7.3 Collection and Recording of Adverse Events 
 
Investigational Product 
7.3.1
The investigational product in this protocol is ex vivo Expanded Autologous CD4+CD127lo/-
CD25+ Polyclonal Regulatory T Cells (PolyTregs).  
 
Methods of Collection 
7.3.2
AEs (including SAEs) may be discovered through any of these methods: 
• Observing the subject 
• Questioning the subject in an objective manner 
• Receiving an unsolicited complaint from the subject 
• In addition, an abnormal value or result from a clinical or laboratory evaluation 
(including, but not limited to, a radiograph, an ultrasound, or an electrocardiogram) can 
also indicate an AE, as defined in Section 7.4, Grading and Attribution of Adverse 
Events. The evaluation that produced the result should be followed until that value or 
result returns to normal or can be explained and the subject’s safety in no longer at risk. 
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Methods of Recording 
7.3.3
 
Recording Adverse Events 
7.3.3.1
Throughout the study, the investigator will record all clinical AEs and all laboratory AEs per 
the following criteria on the appropriate AE electronic case report form (eCRF) regardless of 
their severity or relation to study medication or study procedure. 
• From time of signing of informed consent until start of investigational product infusion: 
all SAEs 
• From start of investigational product infusion until 24 hours post infusion: all NCI-
CTCAE Grade 1 and higher AEs 
• From 24 hours post-infusion until Week 52: all NCI-CTCAE Grade 2 and higher AEs 
• From Week 52 until Week 156: all SAEs and all NCI-CTCAE Grade 3 and higher AEs 
Irrespective of study period, all clinical and laboratory events regardless of severity should be 
recorded in the medical research record for the duration of the trial. 
The investigator will treat subjects experiencing AEs appropriately and observe them at 
suitable intervals until their symptoms resolve or their status stabilizes. Once recorded, an 
AE will be followed until it resolves with or without sequelae, or until the end of study 
participation, or after the subject prematurely withdraws (without withdrawing consent)/or is 
withdrawn from the study, whichever occurs first. 
 
Recording Serious Adverse Events 
7.3.3.2
Serious AEs will be recorded on the appropriate AE/SAE eCRF. All requested information 
on the AE eCRF and SAE eCRF should be provided, if available, for submission to the 
DAIT-SACCC, DAIT, NIAID, and health authorities as outlined in Section 7.5, Reporting of 
Adverse Events and Serious Adverse Events to the Sponsor: DAIT/NIAID. 
Once recorded, an SAE will be followed until it resolves with or without sequelae, or until 
the end of study participation, whichever occurs first. End of study participation is the final 
study visit, either at time of study completion or when a subject prematurely withdraws or is 
withdrawn. 
7.4 Grading and Attribution of Adverse Events 
 
Grading Criteria 
7.4.1
The study site will grade the severity of AEs experienced by the study subjects according to 
the criteria set forth in the NCI-CTCAE v4.0. This document (referred to herein as the NCI-
CTCAE manual) provides a common language to describe levels of severity, to analyze and 
interpret data, and to articulate the clinical significance of all AEs. The NCI- CTCAE has 
been reviewed by the Protocol Chairs and has been deemed appropriate for the subject 
population to be studied in this protocol. 
AEs will be graded on a scale from 1 to 5 according to the following standards in the NCI-
CTCAE manual:  
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7.5 Reporting of Adverse Events and Serious Adverse Events to the Sponsor: 
DAIT/NIAID 
 
Reporting of Adverse Events 
7.5.1
This section describes the responsibilities of the site investigator to report AEs to the study 
sponsor (DAIT/NIAID) via the DAIT-SACCC using the AE eCRF. Timely reporting of 
adverse events as specified by study phase in Section 7.3.3.1, Recording Adverse Events is 
expected.   
Unless otherwise noted below in Section 7.5.2, Reporting of Serious Adverse Events and 
Grade 3 and Higher Clinical Events, for serious adverse events and events of special interest, 
AEs should be recorded on the AE eCRF within five (5) business days of the site learning of 
the event(s). Whenever possible, a diagnosis should be provided, rather than compilation of 
signs/symptoms, with grade of the event determined by highest grade of the sign/symptom 
component. 
The exception to this reporting timeframe is the reporting of serious adverse events and 
Grade 3 or higher clinical events which require reporting within 24 hours of discovery as 
described in Section 7.5.2 below. 
 
Reporting of Serious Adverse Events and Grade 3 and Higher Clinical 
7.5.2
Events  
This section describes the responsibilities of the site investigator to report serious adverse 
events and non-serious Grade > 3 events to the sponsor via the electronic CRF (eCRF). 
Timely reporting of adverse events is required by 21 CFR and ICH E6 guidelines.  
The adverse events outlined below must be reported by the site investigators to DAIT/NIAID 
via the DAIT-SACCC regardless of relationship or expectedness to study intervention within 
24 hours of discovery of the event: 
• All SAEs per 21 CFR 312.32 definitions (see Section 7.2.4, Serious Adverse Event) 
• All other Clinical events with a NCI-CTCAE Grade 3 or greater severity  
Note: clinical events include signs/symptoms, diagnoses, and laboratory abnormalities with 
clinical consequence (defined as the requirement for intervention, correction, increased 
monitoring, or further evaluation). Inclusive in this category is any NCI-CTCAE Grade 3 or 
greater clinical/laboratory component associated with a pemphigus relapse/flare per 
protocol Section 3.4.1 Exploratory Safety Endpoints.  
The SAE eCRF will be used to capture all SAEs and non-serious Grade ≥ 3 clinical events 
for submission to the sponsor (DAIT/NIAID). All requested information on the SAE eCRF 
should be provided. Unavailable details of the event at the time of the initial report should 
not delay submission of known information. The initial report should include at a minimum: 
AE term, relationship to PolyTregs, and as applicable, reason why event is serious per the 
definitions in Section 7.2.4 Serious Adverse Event. 
Supplementary CRF pages including medical history, concomitant medications, 
demographics, study drug administration, and death must be provided as applicable. As 
additional details become available, the SAE eCRF should be updated and submitted. With 
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independent of drug therapy) that indicates those events occur more 
frequently in the drug treatment group than in a concurrent or historical 
control group  
o Any findings from studies: The sponsor must report any findings from other 
epidemiological or clinical studies, pooled analysis of multiple studies, or animal 
or in vitro testing that suggest a significant risk in humans exposed to the drug 
that would result in a safety-related change in the protocol, informed consent, 
investigator brochure or other aspects of the overall conduct of the study. 
Safety Reports must be reported by DAIT, NIAID to the appropriate health authorities within 
15 calendar days; fatal or immediately life-threatening, serious, unexpected, SARs must be 
reported within 7 calendar days. 
 
Reporting of Adverse Events to IRBs 
7.5.4
All investigators must report AEs and SAEs in a timely fashion to their respective IRBs in 
accordance with applicable regulations and local reporting guidelines. 
All IND Safety Reports to the FDA will be distributed by the DAIT, NIAID or designee to 
all participating institutions for site IRB submission. 
7.6 Pregnancy Reporting 
Although pregnancy is not an SAE, information about any pregnancy should be reported 
promptly to the DAIT-SACCC on the same timeline as an SAE for tracking purposes 
(Section 7.5.2, Reporting of Serious Adverse Events and Grade 3 and Higher Clinical 
Events). 
All pregnancies identified during the study must be followed to conclusion and the outcome 
of each must be reported. The investigator should be informed immediately of any pregnancy 
in a study subject or a partner of a study subject. A pregnant subject should be instructed to 
stop taking study medication. The investigator should report to the DAIT-SACCC all 
pregnancies within one business day (as described in Section 7.5.2, Reporting of Serious 
Adverse Events and Grade 3 and Higher Clinical Events) using the Pregnancy eCRF. The 
investigator should counsel the subject and discuss the risks of continuing with the pregnancy 
and the possible effects on the fetus. Monitoring of the pregnant subject should continue until 
the conclusion of the pregnancy, and follow-up data detailing the outcome of the pregnancy 
should be submitted to the DAIT-SACCC by updating the Pregnancy eCRF. When possible, 
similar information should be obtained for a pregnancy occurring in a partner of a study 
subject. 
Information requested about the delivery will include: 
• Subject’s enrollment ID 
• Gestational age at delivery 
• Birth weight, length, and head circumference 
• Gender 
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• Appearance, pulse, grimace, activity, and respiration (APGAR) score at one minute, 
five minutes, and 24 hours after birth, if available 
• Any abnormalities 
Should the pregnancy result in a congenital abnormality or birth defect, an SAE must be 
submitted to the DAIT-SACCC using the SAE reporting procedures described above. 
 
Mycophenolate REMS Program 
7.6.1
APG01 investigators of participating subjects taking concurrent MMF are strongly 
encouraged to register with the FDA’s REMS program (www.mycophenolaterems.com). An 
investigator will be strongly encouraged to report to the REMS program any pregnancy 
occurring in an APG01 female subject while she is taking MMF or within the first six weeks 
following discontinuation of MMF treatment. 
7.7 Reporting of Other Safety Information 
An investigator should promptly notify the DAIT-SACCC when an “unanticipated problem 
involving risks to subjects or others” is identified, which is not otherwise reportable as an 
AE. 
7.8 Review of Safety Information 
 
Study Management Team Review 
7.8.1
The study management team (SMT) will receive monthly reports from the DAIT-SACCC 
compiling new and accumulating safety information on, including but not limited to AEs, 
SAEs, and pregnancies recorded by the sites on appropriate eCRFs.  
In addition, the SRC will review safety data collected within dosing cohorts to define 
significant events (see Section 3.1, Description of Study Design). 
 
Medical Monitor Review 
7.8.2
The Medical Monitor (MM), as part of the SMT and SRC, will review all information listed 
above in Section 7.8.1, Study Management Team Review. In addition, the MM will receive 
SAE and pregnancy reports for review and triage after the DAIT-SACCC is made aware of 
these events (see Sections 7.5.2, Reporting of Serious Adverse Events and Grade 3 and 
Higher Clinical Events and 7.6, Pregnancy Reporting). 
 
DSMB Review 
7.8.3
The DSMB will review the safety data collected when the fourth subject in cohort 1 
completes Week 8. In addition, the DSMB will review accumulating safety data at least 
yearly during planned DSMB Data Review Meetings. Data for the planned safety reviews 
will include, at a minimum, a listing of all reported AEs and SAEs. To ensure subject safety 
between Data Review Meetings, the DSMB will be informed of all expedited safety reports 
in a timely manner. 
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AEs including changes in laboratory values will be graded according to the NCI-CTCAE 
Version 4.0 (http://ctep.cancer.gov/reporting/ctc.html). The frequency of AEs will be 
summarized by system organ class, preferred term, severity (grade), and relationship to 
PolyTregs as specified by the SRC and by the site. Relationship to PolyTregs will be 
categorized as either related (possibly, probably, or related) or unrelated (unlikely or not 
related). For the primary safety endpoint defined in Section 3.2.1, Primary Safety Endpoint, 
and each key safety endpoint defined in Section 3.3.1, Secondary Safety Endpoints, the 
number and percentage of subjects experiencing the event will be summarized. 
Pemphigus flares defined in Section 3.4.1, Exploratory Safety Endpoints, will be summarized 
separately through Week 156 using appropriate descriptive statistics. 
Laboratory results by parameter will be displayed in listings and graphically, where 
appropriate, to illustrate changes in laboratory results over time. 
SAEs will also be summarized using the donation population to assess SAEs occurring from 
donation until PolyTreg infusion. 
 
Efficacy Analysis 
8.3.2
All efficacy endpoints will be summarized using appropriate descriptive statistics. For each 
change from baseline efficacy endpoint defined in Section 3.3.2, Secondary Efficacy 
Endpoints, and Section 3.4.2, Exploratory Efficacy Endpoints, change from baseline will be 
summarized at each visit outlined in the respective endpoint. Time to relapse (flare) will be 
measured from the day of PolyTreg infusion. For each endpoint related to prednisone dosing, 
the number of participants meeting the dosing criteria at Weeks 12, 26, 39, 52, 78, 104, 130, 
and 156 will be summarized. The efficacy summaries will be completed using the safety 
population. 
 
Mechanistic Analysis 
8.3.3
Appropriate descriptive statistics will be computed for the change in level of transferred 
deuterium-labeled Tregs in peripheral blood and skin from Week 1 at Week 12, changes in 
disease specific and immunologic biomarkers in peripheral blood and skin from baseline to 
Week 12, and for the relative frequency of adoptively transferred Tregs in blood and skin at 
each visit assessment. The mechanistic summaries will be completed using the safety 
population. 
8.4 Interim Analysis 
Results of interim analyses will be reported to the DSMB for planned Data Review Meetings. 
Reports prepared for these meetings will focus on study conduct and subject safety and may 
include information on enrollment, site activation status, protocol deviations, subject status 
and demographics and safety analyses. Similar reports, with emphasis on the safety analyses, 
will be provided to the SRC each time the study is paused due to a significant event and after 
the completion of each cohort.  
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Interim Analysis of Safety Data 
8.4.1
The SRC will review data on significant events as described in Section 3.1, Description of 
Study Design and Section 5.5.4, Safety Stopping Guidance. Detailed listings of significant 
events, AEs, and safety-related laboratory values will be generated for the SRC review. The 
safety analyses will be completed overall and by dosing group using the safety population.  
The DSMB will periodically review safety data as described in Section 5.5.4, Safety Stopping 
Guidance and Section 7.8.3, DSMB Review. The safety analyses for this trial will be 
descriptive rather than inferential. Detailed listings and summary tabulations of AEs and 
safety-related laboratory values will be generated. The safety analyses will be completed 
overall and by dosing group using the safety population. 
8.5 Other Statistical Considerations 
 
Covariates 
8.5.1
Due to the samples size of this phase I trial, the impact of additional covariates will not be 
explored. 
 
Multiple Comparisons and Multiplicity 
8.5.2
Since this is a phase I study, all safety, efficacy, and mechanistic analyses are exploratory in 
nature. Therefore, no adjustment for multiple comparisons is needed. 
 
Examination of Subgroups 
8.5.3
Due to the sample size of this phase I trial, no additional subgroup analyses will be 
performed. 
 
Missing Data 
8.5.4
Standard procedures will be used to ensure that data are complete and accurate as possible. 
Due to the exploratory nature of this study, no imputation for missing data will be done; 
however per Section 3.1.2.1, Subject Completion and Early Termination, if an infused 
subject does not complete 12 weeks of follow-up, an additional subject will be recruited in 
order to fully understand safety through 12 weeks of treatment. 
 
Changes to the Statistical Analysis Plan 
8.5.5
The principal features of the design of this study and of the plan for statistical analysis of the 
data are outlined in this protocol. Additional details will be included in a Statistical Analysis 
Plan (SAP) before initiating analyses. Any changes to that plan will be documented in the 
Final Study Report and will be approved by the Protocol Chairs before being initiated. 
9 ACCESS TO SOURCE DATA AND DOCUMENTS 
Each participating site will maintain the highest degree of confidentiality permitted for the 
clinical and research information obtained from subjects participating in this clinical trial. 
Medical and research records should be maintained at each site in the strictest confidence. 
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However, as a part of the quality assurance and legal responsibilities of an investigation, each 
site must permit authorized representatives of the IND sponsor, the DAIT-SACCC, and 
health authorities to examine (and when required by applicable law, to copy) clinical records 
for the purposes of quality assurance reviews, audits, and evaluation of the study safety and 
progress. Unless required by the laws permitting copying of records, only the coded identity 
associated with documents or other subject data may be copied (obscuring any personally 
identifying information). Authorized representatives as noted above are bound to maintain 
the strict confidentiality of medical and research information that may be linked to identified 
individuals. Participating sites will normally be notified in advance of auditing visits. 
All subject records and study documentation will be kept after the protocol is completed. 
This will include all documentation of AEs, records of study drug receipt and dispensation, 
and all IRB correspondence. All study records will be kept for at least two years after the 
investigation is completed. 
10 DATA COLLECTION, QUALITY CONTROL AND QUALITY 
ASSURANCE 
The investigator is required to keep accurate records to ensure the conduct of the study is 
fully documented. The period of record retention should be consistent with the record 
retention policies of the sponsoring agency or applicable regulatory agencies. However, in 
certain instances, documents should be retained for a longer period if required by the 
applicable regulatory agency or by the National Institutes of Health. 
The investigator will report all major protocol deviations to DAIT, NIAID and the DAIT-
SACCC per the instructions in the APG01 Manual of Procedures. The DAIT-SACCC will 
forward reports of protocol deviations to the responsible DAIT, NIAID medical officer for 
review as specified in the ACE Manual of Procedures. 
The DAIT-SACCC is responsible for regular inspection of the conduct of the trial, for 
verifying adherence to the protocol, and for confirming the completeness, consistency, and 
accuracy of all documented data. 
Data will be obtained from a variety of sources including, but not limited to laboratory 
notebooks, automated instrument output files, and clinical subject charts. Data from these 
source materials will be transmitted to the DAIT-SACCC via one of two mechanisms. Data 
collected electronically at central laboratories will be transferred electronically directly from 
the laboratory to the DAIT-SACCC using standard secure data transfer procedures. Data 
collected at the clinical sites will be transmitted to the DAIT-SACCC using an internet-based 
remote data entry system. Clinical site personnel use an internet browser to key data into 
eCRFs; each CRF page is submitted to the clinical database electronically as the page is 
completed. Univariate data validation tests are performed as the data are keyed. The clinical 
database is backed up nightly; backup tapes are saved in a secure, off-site location. At any 
time, authorized site personnel may log in to the remote data entry system, review and 
correct previously entered data, or key additional data. The data will be further validated per 
the study data validation plan via a series of computerized and manual edit checks, and all 
relevant data queries will be raised and resolved on an ongoing basis. Complete, clean data 
will be frozen to prevent further inadvertent modifications. All discrepancies will be 
reviewed and any resulting queries will be resolved with the investigators and amended in the 
database. All elements of data entry (i.e., time, date, verbatim text, and the person performing 
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the data entry) will be recorded in an electronic audit trail to allow all data changes in the 
database to be monitored and maintained in accordance with federal regulations. 
The DAIT-SACCC will periodically visit the participating clinical sites and audit the source 
documents in order to validate the data in the DAIT-SACCC central database. Data will be 
provided using the subject’s enrollment number; the DAIT-SACCC will not collect 
personally identifying information such as the subject’s name or social security number. 
Subjects will provide demographic information such as race, ethnicity, and birth date. 
Data collected by the DAIT-SACCC will be held in the strictest confidence, and are 
protected from access that could reveal personally identifying information about any subject 
in the trial. 
11 ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD 
CLINICAL PRACTICE 
The study will be conducted according to Good Clinical Practice (GCP) guidelines, U.S. 21 
CFR Part 50 – Protection of Human Subjects, and Part 56 – Institutional Review Boards. 
11.1 Compliance with Good Clinical Practices 
This trial will be conducted in compliance with the protocol, current GCPs recommended by 
the ICH, and the applicable regulatory requirements for participating institutions. These 
include the tenets of the Declaration of Helsinki and review and approval by the appropriate 
ethics review committee or IRBs of participating organizations. The DAIT-SACCC will 
assure compliance through a program of quality assurance audits performed both at 
participating sites and within the DAIT-SACCC for data quality and adherence to protocol 
requirements. The DAIT-SACCC is operated by Rho Federal Systems Division, Inc. 
(RhoFED), Chapel Hill, North Carolina under a cooperative agreement from NIAID. 
11.2 Institutional Review Board 
Each participating institution must provide for the review and approval of this protocol and 
associated informed consent documents by an appropriate ethics review committee or IRB. 
Any amendments to the protocol or consent materials must be approved by the IRB and 
submitted to the FDA before they are placed into use. In both the United States and in other 
countries, only institutions holding a current Federal Wide Assurance (FWA) issued by the 
OHRP at the Department of Health and Human Services (DHHS) may participate. 
The investigator will inform the IRB of serious or unexpected AEs that might occur during 
the study and are likely to affect the safety of the subjects, or the conduct of the study. The 
investigators will comply fully with all IRB requirements for both the reporting of AEs, 
protocol or consent form changes, as well as any new information pertaining to the use of the 
study medication that might affect the conduct of the study.  
11.3 Informed Consent 
The principles of informed consent in the current edition of the Declaration of Helsinki, as 
well as compliance with all IRB requirements, will be implemented in the study, before any 
protocol-specified procedures are carried out. A standard consent form for subject 
participation will be provided with the protocol to each institution. Any modifications to the 
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standard information in the template will require review and approval by DAIT, NIAID prior 
to IRB submission. Informed consent will be obtained in accordance with 21 CFR 50.52. 
Information may be given to subjects in oral, written, or video form by the investigator. All 
prospective subjects will be given ample time to read the consent form, and ask questions, 
before signing. 
If subjects are to be enrolled who do not speak and read English, the consent and subject 
materials must be translated into the language appropriate for the enrolling subject. 
Translated documents must be certified to contain the complete descriptions provided in the 
English version of the document. If an interpreter is used to provide or assist in describing 
the consent materials to an enrolling subject, the interpreter must also sign the consent 
materials certifying their involvement with the consent process. 
After completion, a copy of the signed consent form will be given to the subject. The original 
signed consent form will be kept on file in the subject’s study chart, available for inspection 
by regulatory authorities, both federal and institutional. 
11.4 Data and Safety Monitoring Board 
The responsibility for reviewing the ethical conduct of the study and for monitoring reports 
of evidence of adverse or beneficial effect is assigned to the DAIT Autoimmunity DSMB. 
The DSMB is an independent group composed of biomedical ethic experts, physicians, and 
other scientists who are responsible for continuing review of study information. The DSMB 
makes recommendations to DAIT, NIAID on issues affecting the course and conduct of this 
clinical study. 
11.5 Study Termination 
In the event that the study is terminated, subjects who have received study treatment will be 
followed for the study’s three years of safety follow-up. 
12 FINANCING AND INSURANCE 
Participating institutions must comply with their institution’s policies on compensation, 
insurance, and indemnity. Institutions must have adequate liability insurance coverage to 
satisfy their local and national requirements for study participation. 
13 PUBLICATION POLICY 
The ACE policy on publication of study results will apply to this study. Authorized 
participants may find details regarding the policy statement on the ACE internet website  
 Study investigators are encouraged to communicate and publish 
study results with prior notification of DAIT, NIAID. The following procedure is suggested: 
1. Manuscripts, abstracts, posters and other material for public distribution will be 
submitted to DAIT, NIAID at least 30 days prior to submission for publication or 
public presentation. 
2. DAIT, NIAID will review and comment on the proposed material within 30 days. 
3. DAIT, NIAID may ask that confidential information be deleted or redacted in this case 
where a patent may be filed or where confidential information is involved. Publication 
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or presentation may be delayed up to 60 additional days in order to file a patent 
application. 
 
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38. 
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15 APPENDICES 
 
• 
15.1: PDAI 
• 
15.2: Skindex-29 (version 2) 
• 
15.3: Patient’s Global Assessment 
• 
15.4: Physician’s Global Assessment 
• 
15.5: Subject Self-Reported Demographics Optional Source Document 
• 
15.6: Pre-Infusion Required Checklist 
 
 
 
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