Conditions: Liver Transplant Recipient Living Donor (of the Respective Liver Transplant Recipient)
Keywords: Regulatory T Cells ( Treg) Donor Alloantigen Reactive Tregs (darTregs) Liver Transplant Alloantigen Reactive Tregs Calcineurin Inhibitors (CNI) immunosuppressants (IS)
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 15
Healthy volunteers: no

Brief summary:
This research study is for liver transplant recipients and their respective living donors.

The purpose of this study is:

1. To see if it is safe for liver recipients to receive one dose of donor reactive T regulatory cells (Tregs)
2. To see if the Tregs allows a liver recipient to take less, or completely stop medications normally taken after receiving an organ transplant.

Detailed description:
Doctors give drugs called immunosuppressants (IS) to people who receive a liver transplant. IS must be taken every day to prevent the body from injuring the transplanted liver by a process called rejection. Liver transplant recipients usually have to take these drugs for the rest of their lives. These drugs have harmful side effects. Researchers are looking for ways to keep a transplanted liver working normally with as little IS medications as possible. Finding a way to lower and then stop these medications will allow the liver recipient to avoid unwanted side effects.

Another area of research looks at how blood cells work to reject or accept an organ transplant. Studies show that some of the recipient's own cells, called T regulatory cells (Tregs), may play a part in accepting the transplanted liver and preventing rejection.

A recipient's Tregs can be grown in the laboratory to increase their number. Exposing the recipient's Tregs to the liver donor's cells will stimulate the Tregs that recognize the liver donor to grow vigorously. Giving these "donor reactive" Tregs back to the transplant recipient through a vein (intravenously) might allow a liver transplant recipient to take lower doses of IS, or perhaps to stop them altogether, without rejecting the liver.

The study team will collect information about the Treg infusion, liver tests and drug doses during IS withdrawal, and any problems that may arise in the study. Blood, liver tissue, and buccal (cheek) cells will be collected for research tests.

Arms:
arm 1: Donor Alloantigen Reactive Tregs (darTregs). Participants will receive a target dose of 400X10\^6 darTregs (range 300-500 x10\^6) infused intravenously (IV) over an approximate 20-30 minute interval

Interventions: intervention 1: darTregs intervention 2: Acetaminophen intervention 3: Diphenhydramine intervention 4: Immunosuppression (IS) Withdrawal intervention 5: Study Mandated Procedures
Intervention descriptions: intervention 1: A single intravenous infusion as described administered over a 20-30 minute interval with close monitoring prior to, during, and after the infusion. intervention 2: Pre-medication for darTregs infusion. A dose of 15 mg/kg will be administered 30 to 60 minutes prior to the darTregs infusion. intervention 3: Pre-medication for darTregs infusion. A dose of 1-2 mg/kg diphenhydramine will be administered 30 to 60 minutes prior to the darTregs infusion. intervention 4: Subjects:1.) who fulfill study eligibility criteria will withdraw IS 2.) enter the study on calcineurin inhibitor (CNI) monotherapy or a CNI-based regimen with either Prednisone or MMF as a second IS medication 3.) will proceed with changes in CNI dosing according to the protocol's CNI withdrawal algorithm.During the last 2 weeks of IS withdrawal (e.g., Step 2 in algorithm -CNI reduced 25%-"pre darTregs"), a single dose of darTregs will be infused IV. The subject will then, if eligible, proceed with IS withdrawal within 2 weeks after the infusion. Eligible subjects meeting the primary endpoint of 75% reduction in CNI from baseline after darTregs will be offered the opportunity to continue IS withdrawal until complete discontinuation of IS. intervention 5: Blood draws (venipuncture); collection of peripheral blood mononuclear cells (PBMCs) by a procedure known as leukapheresis or venipuncture; buccal (cheek) swab for HLA typing; liver biopsies (per protocol and for cause).

Protocol document:
Prot_000.pdf: 
 
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darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
CLINICAL TRIALS IN ORGAN TRANSPLANTATION IN CHILDREN  
CTOTC-12 
Safety of Donor Alloantigen Reactive Tregs to Facilitate Minimization and/or 
Discontinuation of Immunosuppression in Adult Liver Transplant Recipients 
 Version 7.0 / October 15, 2018 
IND# 16345 
Study Sponsor:  The National Institute of Allergy and Infectious Diseases (NIAID) 
Grant #:  U01AI04347   
PRINCIPAL INVESTIGATOR 
 
Professor of Surgery 
Director, Abdominal Transplant 
Surgery Fellowship 
University of California,  
San Francisco 
 
 
  
CO- INVESTIGATOR  
 
Emeritus Executive Vice 
Chancellor and Provost 
A.W. Clausen Distinguished 
Professor in Medicine 
University of California,  
 
 
 
 
 
CO- INVESTIGATOR  
 
Associate Professor 
Director, UCSF Transplantation 
Research Lab 
University of California, San 
Francisco 
 
0 
 
 
REGULATORY OFFICER 
 
Office of Regulatory Affairs 
Division of Allergy, Immunology, and 
Transplantation 
NIAID 
 
  
 
BIOSTATISTICIAN  
 
Senior Statistical Scientist 
Rho, Inc. 
 
 
 
MEDICAL OFFICER 
 
Transplantation Branch 
Division of Allergy, Immunology, 
and Transplantation 
NIAID 
 
 
  
PROJECT MANAGER 
 
Transplantation Branch 
Division of Allergy, Immunology, and 
Transplantation  
NIAID 
 
  
  
 
 
 
 
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior permission of the Principal Investigator, or the 
Division of Allergy, Immunology and Transplantation, NIAID of the National Institutes of Health. 
 
 
 


 
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darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
INVESTIGATOR SIGNATURE PAGE 
Protocol CTOTC-12: 
darTregs for CNI Reduction  
Version/Date:  
  7.0 / October 15, 2018 
Title:    Safety of Donor Alloantigen Reactive Tregs (darTregs) to Facilitate Minimization 
and/or Discontinuation of Immunosuppression in Adult Liver Transplant Recipients (ARTEMIS) 
Study Sponsor:  The National Institute of Allergy and Infectious Diseases (NIAID) 
INSTRUCTIONS: The site Principal Investigator should print, sign, and date at the indicated location below.  
A copy should be kept for your records and the original signature page sent. After signature, please return 
the original of this form by surface mail to:  
DAIT Regulatory Management Center 
PPD, Inc. 
 
 
 
 
 
 
I confirm that I have read the above protocol in the latest version.  I understand it, and I will work according 
to the principles of Good Clinical Practice (GCP) as described in the United States Code of Federal Regulations 
(CFR) – 45 CFR part 46 and 21 CFR parts 50, 56, and 312, and in the International Conference on 
Harmonization (ICH) document Guidance for Industry: E6 Good Clinical Practice: Consolidated Guidance 
dated April 1996.  Further, I will conduct the study in keeping with local legal and regulatory requirements. 
As the site Principal Investigator, I agree to carry out the study by the criteria written in the protocol and 
understand that no changes can be made to this protocol without the written permission of the IRB and 
NIAID. 
 
____________________________________     
Site Principal Investigator (Print) 
 
____________________________________                                      _________________ 
Site Principal Investigator (Signature)                                                          Date 


 
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darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
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Protocol Synopsis 
Title 
Safety of Donor Alloantigen Reactive Tregs (darTregs) to Facilitate Minimization and/or Discontinuation 
of Immunosuppression in Adult Liver Transplant Recipients (ARTEMIS) 
Short Title 
darTregs for CNI Reduction  
Protocol Number 
CTOTC-12 
 
ClinicalTrials.gov 
Identifier 
NCT02474199 
Clinical Phase 
Phase I/II 
Primary Safety 
Objective 
This study will evaluate the safety and tolerability of a single IV dose of the darTregs product.  
Secondary Safety 
Objective 
This study will evaluate the safety of immunosuppression (IS) 1) minimization and 2) discontinuation 
after one IV dose of darTregs. 
Primary Efficacy 
Objective 
The study will evaluate the ability of a single IV dose of darTregs to reduce baseline, standard of care 
(SOC) calcineurin inhibitor (CNI) dosing by 75% along with discontinuation of either prednisone or 
mycophenolate mofetil (MMF), as applicable.  
Secondary Efficacy 
Objective 
This study will determine whether a single IV dose of darTregs can induce operational tolerance.  
Mechanistic Objectives 
We will assess the pharmacokinetic profile of darTregs by measuring the level of deuterium-labeled Tregs 
in circulation. Potential impact of darTregs therapy on immunological profiles will be assessed by 
comparing leukocyte phenotypes, tissue histology and gene expression in protocol and for-cause biopsies, 
and alloantibody before and after darTregs infusion. 
Study Design 
A multi-center, open-label clinical trial in adult liver transplant recipients with two primary 
interventions:  
• 
administration of a single IV dose of darTregs and  
• 
reduction of IS dosing with possible complete IS discontinuation.   
Adult living donor liver recipients two to six years after LT will initiate IS withdrawal.  Twelve to 14 weeks 
later, they will receive a single dose of darTregs and continue IS withdrawal.    
Primary Safety 
Endpoint 
The safety and tolerability of a single infusion of darTregs administered to LT recipients will be assessed 
24 weeks after darTregs by describing: 
1. Occurrence of CTCAE Grade 3 or higher adverse events (AEs) attributable to the darTregs 
infusion including infusion reaction / cytokine release syndrome 
2. Occurrence of study defined Grade 3 or higher infections  
3. Occurrence of any malignancy, including PTLD  
Secondary Safety 
Endpoint 
The trial will assess the safety of IS withdrawal in the context of darTregs therapy by describing the 
following secondary safety endpoints: 
1. Rate of composite outcome measure including refractory acute rejection, chronic rejection,  re-
transplantation, and death 
2. Incidence of biopsy proven or clinical acute rejection and/or chronic rejection 
3. Timing of biopsy proven or clinical acute rejection and/or chronic rejection 
4. Severity of biopsy proven acute rejection and/or chronic rejection 
 
 
 
 


 
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Primary Efficacy  
Endpoint 
The efficacy of a single IV dose of darTregs will be assessed by the number and proportion of LT subjects 
who are able to reduce CNI dosing by 75% and discontinue a 2nd IS drug (if applicable) with stable liver 
tests for at least 12 weeks.  The frequency of successful CNI minimization will be compared to historical 
cohorts of comparable adult LT recipients undergoing IS withdrawal.   
Secondary Efficacy 
Endpoints 
The efficacy of a single IV dose of darTregs infusion will be assessed by determining the number and 
percentage of subjects who have received darTregs and are identified as operationally tolerant, defined 
by maintaining stable allograft function (assessed by liver tests) and histology (determined by central 
pathologist reading in comparison to screening liver biopsy at study entry) in the absence of IS for one 
year.  The frequency of tolerance will be compared to historical cohorts of adult liver transplant recipients 
undergoing IS withdrawal.   
Primary Mechanistic 
Endpoints 
The level and persistence of deuterium-labeled darTregs in the circulation will be determined by serial 
measurements of deuterium content in DNA from purified peripheral blood Tregs after darTregs 
infusion using gas chromatography-mass spectrometry (GC-MS) testing. 
Secondary Mechanistic 
Endpoints 
The overall increase of darTregs in circulation will be assessed using the alloreactive T cell frequency 
(ATF) assay 
Exploratory 
Mechanistic Endpoints 
The immunologic impact of infused darTregs will be determined by assessing the following: 
• 
Leukocyte phenotypes before and after darTregs infusion using multi-parameter flow cytometry 
(MFC). 
• 
Alloantibody responses before and after darTregs infusion during IS withdrawal. 
• 
Histology and multiplex immunohistochemistry of protocol and for cause biopsies    
• 
The composition of immune infiltrate in liver biopsies post Treg infusion and at the time of 
for-cause biopsies will be profiled using single-cell RNA+TCRseq 
Accrual Objective 
Up to 18 participants will be screened and enrolled to target up to 11 participants eligible for both IS 
withdrawal and darTregs infusion 
Study Duration 
The maximal length of trial participation for an individual subject is anticipated to be 2 years.  The total 
trial duration will be 3 years. 
• 
One year period to accrue nine patients eligible for both IS withdrawal and darTregs infusion 
that will be given approximately 10-11 weeks after initiating IS withdrawal 
• 
Minimum of 52 weeks follow-up after any AR episode or or darTregs infusion  
• 
For all subjects able to discontinue IS, 52 weeks of follow-up after the last IS dose to assess for 
operational tolerance 
The duration of study participation will vary by subject depending on his/her duration of IS withdrawal.  
Accounting for a one year enrollment period, the primary endpoint for all participants will be assessed 
approximately 1.5 years after trial initiation.   
 
 


 
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Treatment Description  
All eligible participants will initiate IS withdrawal.  Approximately 10-12 weeks after initiating IS 
withdrawal, subjects will have autologous Tregs collected for darTregs manufacturing.   
During the last 2 weeks of IS withdrawal Step 2 (CNI reduced by 25%), a single total dose of 400 x 106 ± 
100 deuterium-labeled darTregs will be infused intravenously.  The subject will then, if eligible, resume 
IS withdrawal within 2 weeks after darTregs infusion (see Section entitled “Resumption of IS Withdrawal 
after darTregs Infusion” below).  Only subjects who receive 300-500 x 106 darTregs and who meet the 
primary endpoint of 75% reduction in CNI from baseline after darTregs will be offered the opportunity to 
continue IS withdrawal until complete discontinuation of IS (secondary endpoint).  Those who receive 
100-300 x 106 darTregs will only be allowed to proceed to the primary endpoint but will not be eligible 
for complete IS withdrawal. 
Study Enrollment/ IS 
Withdrawal Inclusion 
Criteria 
Subjects who meet all of the following criteria are eligible for enrollment as study participants:  
1. Able to understand and provide informed consent  
2. Have received primary, solitary, living donor liver transplant more than 24 months but less than 84 
months ago  
3. Have a living donor who is willing to consent to one time phlebotomy of 100 mLs to enable 
manufacture of darTregs 
4. Between 18 and 70 years of age at the time of study entry/consent  
5. Have ALT consistently <60 U/L and either alkaline phosphatase consistently <150 U/L or GGT 
consistently <60 U/L  
6. Currently receiving a calcineurin inhibitor (CNI) with 12 hour trough levels consistently <6.0ng/mL for 
tacrolimus;  <150ng/mL for cyclosporine  
7. Currently receiving CNI monotherapy or CNI and ONE of the following:   
a. Prednisone: maximum dose of 5mg / day  
b. Mycophenolate mofetil (MMF):  maximum dose of 500mg bid for Cellcept or 360mg bid 
for Myfortic 
8. Female and male subjects with reproductive potential must agree to use effective methods of birth 
control for the duration of the study 
9. If history of HCC, LT recipients who have: 
a. AFP less than 100 µg/L at the time of transplant AND 
b. Explanted liver: 
i. 
with tumor burden within the Milan criteria and 
ii. 
without macro- or micro-vascular invasion and 
iii. 
without any lesions with poorly differentiated HCC and 
iv. 
without cholangiocarcinoma morphology  
c. Risk Estimation of Tumor Recurrence After Transplant (RETREAT) Score less than or equal to 3 
10. If history of HCC, at the time of enrollment, subjects must also: 
a. Be 36 months or more post-transplant AND 
b. Without evidence of recurrent HCC defined as  
i. AFP within normal limits for performing laboratory 
ii. Confirmatory chest CT and 
iii. Confirmatory CT or MRI of the abdomen and pelvis 
11. If history of HCV, recipients must be: 
a. Cured of HCV as defined by achieving SVR and be greater than or equal to six months after the 
end of treatment 
b. HCV RNA negative at time of study enrollment 
 
 


 
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Study Enrollment/ IS 
Withdrawal Exclusion 
Criteria  
Subjects who meet any of these criteria are not eligible for study enrollment.   
1. Transplant for liver disease secondary an autoimmune etiology (e.g. autoimmune hepatitis, primary 
sclerosing cholangitis, or primary biliary cirrhosis)  
2. Matched at both HLA-DR loci to the donor 
3. Organ, tissue or cell transplant prior to or after the primary solitary living donor liver transplant 
4. For subjects with hepatitis B, detectible HBV DNA  
5. History of malignancy within 5 years of enrollment. History of adequately treated in-situ cervical 
carcinoma and/or skin cancer (basal or squamous cell) will be permitted.  
6. Serologic evidence of human immunodeficiency 1 or 2 infection 
7. Epstein Barr Virus (EBV) sero-negativity (EBV naïve) if living donor is EBV sero-positive 
8. Cytomegalovirus (CMV) sero-negativity (CMV naïve) if living donor is CMV sero-positive 
9. Calculated GFR less than 50 mL/min/1.73m2 at the time of enrollment   
10. An episode of AR within one year of enrollment 
11. Systemic illness requiring or likely to require recurrent or chronic IS drug use   
12. Any chronic condition for which anti-coagulation cannot be safely interrupted for liver biopsy 
13. Positive pregnancy test  
14. Participation in any other studies that involved investigational drugs or regimens in the preceding 
year 
15. Any other condition, in the investigator’s judgment, that increases the risk of darTregs infusion or 
prevents safe trial participation 
16. Unwilling or unable to adhere to study requirements and procedures 
17. Screening liver biopsy with any of the following histological criteria, as determined by the reading of 
a central pathologist (Table 9) 
darTregs Infusion 
Inclusion Criteria 
Subjects will initiate IS withdrawal and, at the beginning of the 2nd step of the withdrawal algorithm 
(week 1- 2), undergo a final assessment to ensure eligibility for darTregs infusion.   Subjects must meet 
the following criteria to receive darTregs infusion: 
1. Stable liver tests, defined as ALT and either alkaline phosphatase or GGT either within normal limits 
OR <1.5 X baseline 
2. No detectible circulating EBV or CMV DNA prior to darTregs infusion, assessed at the time of PBMC 
collection for manufacture 
3. For subjects with HBV, no detectible circulating HBV DNA prior to darTreg infusion, assessed at the 
time of PBMC collection for manufacture 
4. Able to understand and provide informed consent  
darTregs Infusion 
Exclusion Criteria 
Subjects who meet any of these criteria are not eligible for darTregs infusion:   
1. Diagnosis of AR after initiation of IS withdrawal 
2. Any vaccination given within 28 days prior to Treg collection for Treg production 
3. Receipt of a vaccination within 14 days prior to Treg infusion 
4. Unacceptable darTregs product 
5. Positive pregnancy test  
6. Clinical evidence of viral syndrome less than 7 days prior to darTregs infusion 
Eligibility Criteria to 
Resume IS Withdrawal 
after darTregs infusion 
Subjects are eligible to resume IS withdrawal after darTregs infusion if all criteria below are met: 
1. Subject received at least 100 x 106 darTregs 
2. ALT and either alkaline phosphatase or GGT remain within normal limits or ≤ 1.5 x baseline after 
darTregs infusion 
3. For subjects with elevated liver tests as defined above, local pathology reading of liver biopsy 6-10 
days after darTregs infusion is without acute rejection according to Banff criteria 
4. IS withdrawal resumes no later than 14 days after darTregs infusion 
5. Site principal investigator determines it is acceptable for the study subject to resume IS withdrawal 
 
 


 
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Study Contacts: Participating Centers 
SITE INVESTIGATOR 
 
Professor of Surgery 
Director, Abdominal Transplant Surgery 
Fellowship 
Benioff Children’s Hospital 
University of California, San Francisco 
 
 
 
 
SITE INVESTIGATOR 
 
Mayo Clinic – Rochester 
Assistant Professor of Surgery 
E. Rolland Dickson Research Scholar 
in Transplantation 
Division of Transplantation Surgery 
Mayo Clinic 
 
 
SITE INVESTIGATOR 
 
Associate Professor 
Division of Hepatology and 
Comprehensive Transplant Center 
Feinberg School of Medicine 
Northwestern University 
 
 
 


 
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Study Contacts: Core Laboratories 
 
 
TREG MANUFACTURING  
 
 
Associate  Professor  
Director, Transplantation Research Lab 
University of California, San Francisco 
San Francisco, CA 94143-0780 
 
 
DETECTION OF DEUTERIUM-
LABELED TREGS  
  
Professor  
Dept of Nutritional Science & 
Toxicology  
University of California, Berkeley  
  
  
  
MFC PANEL 
 
Flow Core Director 
Roswell Park Cancer Institute 
 
 
 
 
 
 
HLA TYPING AND ALLOANTIBODIES 
 
Main Hospital Level B,  
CPMC Davies Campus 
University of California, San Francisco 
 
 
 
 
HTLV TESTING (DONORS) 
 
 
Customer Service Manager 
Creative Testing Solutions 
Attention: Special Testing 
 
 
HISTOPATHOLOGY  
 
Core Director 
University of Pittsburgh Medical 
Center 
Division of Transplantation 
Pathology  
Department of Pathology 
Montefiore University Hospital 
 
 
 
 
PRECISION FOR MEDICINE 
SAMPLE REPOSITORY 
 
 
 
 
 
PBMC CENTRAL CELL PROCESSING 
 
Rutgers University 
 
Rutgers University Cell Repository 
 
 
 
 


 
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Table of Contents 
Glossary of Abbreviations ..................................................................................................................................................... 15 
Study Definitions Page .......................................................................................................................................................... 17 
1. 
Background and Rationale ............................................................................................................................................ 20 
 
Mortality and morbidity of conventional IS medications ..................................................................................... 20 
 
Spontaneous / operational tolerance after LT ...................................................................................................... 21 
 
Previous experiences with IS withdrawal for adult and pediatric LT recipients ........................................... 21 
 
Recent trials of IS withdrawal for pediatric LT recipients ............................................................................. 21 
 
Recent trials of IS withdrawal for adult LT recipients ................................................................................... 22 
 
Rationale for Accelerating Successful IS Minimization / Withdrawal ................................................................... 23 
 
Induction of Transplantation Tolerance ............................................................................................................... 23 
 
Rationale for Selection of Investigational Product or Intervention ...................................................................... 24 
 
Rationale for darTregs Therapy .................................................................................................................... 24 
 
Rationale for darTregs Dosing ....................................................................................................................... 24 
 
Dosing precedents in humans ................................................................................................................... 24 
1.5.2.2 
Estimated efficacy dose for darTregs in organ transplantation ................................................................ 25 
1.5.2.3 
Dose limitations imposed by darTregs manufacturing capacity ............................................................... 26 
 
Rationale for Proposed IS Withdrawal Algorithm ......................................................................................... 27 
 
Rationale for Timing of Treg Administration during IS Minimization/Withdrawal ....................................... 28 
 
Rationale for Timing of IS Withdrawal Resumption after darTregs Administration ..................................... 29 
 
Clinical Experience with Treg Therapy .................................................................................................................. 30 
 
Treg Therapy for Treatment or Prevention of GvHD .................................................................................... 31 
 
Treg Therapy in Type 1 Diabetes ................................................................................................................... 32 
1.6.2.1 
Treg Therapy in Children with Type 1 Diabetes ........................................................................................ 32 
1.6.2.2 
Pharmacokinetics and Product Metabolism in Humans ........................................................................... 33 
1.6.2.3 
Treg Therapy in Adults with Type 1 Diabetes ........................................................................................... 34 
1.6.2.4 
Safety of Treg Therapy in Adults with Type 1 Diabetes ............................................................................ 34 
1.6.2.5 
Feasibility of Multi-Site Trials .................................................................................................................... 35 
2. 
Study Objectives............................................................................................................................................................ 36 
 
Primary Safety Objective:  darTregs Infusion ........................................................................................................ 36 
 
Secondary Safety Objective: IS Withdrawal .................................................................................................. 36 
 
Primary Efficacy Objective:  IS Minimization ........................................................................................................ 36 
 
Secondary Efficacy Objective:  Tolerance ............................................................................................................. 36 
 
Mechanistic Objectives ......................................................................................................................................... 36 
3. 
Study Design .................................................................................................................................................................. 37 
 
Description of Study Design .................................................................................................................................. 37 
 
Primary Safety Endpoint ....................................................................................................................................... 37 
 
Secondary Safety Endpoints.................................................................................................................................. 38 


 
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Primary Efficacy Endpoint ..................................................................................................................................... 38 
 
Secondary Efficacy Endpoint ................................................................................................................................. 38 
 
Primary Mechanistic Endpoint .............................................................................................................................. 38 
 
Secondary Mechanistic Endpoints ........................................................................................................................ 38 
 
Exploratory Mechanistic Endpoints ...................................................................................................................... 38 
4. 
Selection of Participants and Clinical Sites/Laboratories.............................................................................................. 39 
 
Rationale for Study Population ............................................................................................................................. 39 
 
Rationale for Adult Living Donor LT Recipients ............................................................................................. 39 
 
Rationale for Adult Living Donor LT Recipients 2-6 Years after Transplantation.......................................... 39 
 
Rationale for Inclusion of Subjects with History of HCC ............................................................................... 40 
 
Rationale for Enrollment of Liver Transplant Recipients with History of HCV .............................................. 40 
 
Study Enrollment/ darTregs Infusion Eligibility Criteria ........................................................................................ 40 
 
Study Enrollment / IS Withdrawal Inclusion Criteria .................................................................................... 40 
 
Study Enrollment / IS Withdrawal Exclusion Criteria .................................................................................... 41 
 
darTregs Infusion Eligibility Criteria ...................................................................................................................... 42 
 
darTregs Infusion Inclusion Criteria .............................................................................................................. 42 
 
darTregs Infusion Exclusion Criteria .............................................................................................................. 42 
 
Eligibility Criteria to Resume IS Withdrawal after darTregs Infusion .................................................................... 43 
 
Clinical Sites........................................................................................................................................................... 43 
 
Manufacturing Facility .................................................................................................................................. 43 
5. 
Investigational Intervention: IS Withdrawal ................................................................................................................. 44 
 
IS Withdrawal ........................................................................................................................................................ 44 
 
CNI taper algorithm ....................................................................................................................................... 44 
5.1.1.1. Pause in CNI taper ..................................................................................................................................... 44 
 
Prednisone taper algorithm .......................................................................................................................... 44 
 
MMF taper algorithm .................................................................................................................................... 45 
 
Windows During IS Withdrawal ............................................................................................................................ 45 
 
Logistical Pause during Step 2 of CNI Withdrawal Algorithm ............................................................................... 45 
 
Definition of Operational Tolerance ..................................................................................................................... 46 
 
Allograft Dysfunction ............................................................................................................................................ 46 
 
Acute Rejection (AR) ............................................................................................................................................. 46 
 
Treatment of AR ............................................................................................................................................ 46 
 
Resolution of AR ............................................................................................................................................ 47 
 
Chronic Rejection (CR) .................................................................................................................................. 47 
 
Premature Discontinuation of IS Withdrawal ....................................................................................................... 47 
6. 
Investigational Agent: darTregs Infusion ...................................................................................................................... 48 
 
Formulation, Packaging, and Labeling .................................................................................................................. 48 


 
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Dosage, Preparation, and Administration............................................................................................................. 48 
 
Drug Accountability ............................................................................................................................................... 49 
 
Intervals between darTregs infusions ................................................................................................................... 49 
 
Repeated darTregs Manufacturing ....................................................................................................................... 49 
 
Premature Discontinuation of darTregs Infusion ................................................................................................. 49 
7. 
Other Medications ........................................................................................................................................................ 50 
 
mTOR inhibitors .................................................................................................................................................... 50 
 
Prophylactic Medications ...................................................................................................................................... 50 
 
Pre-Medications for darTregs Infusion ......................................................................................................... 50 
 
Anti-Infective Prophylaxis after Corticosteroid or Antibody Treatment for Rejection ................................. 50 
 
Vaccinations .......................................................................................................................................................... 50 
 
Other permitted concomitant medications .......................................................................................................... 50 
8. 
Study Mandated Procedures ........................................................................................................................................ 51 
 
Blood Draws .......................................................................................................................................................... 51 
 
Leukapheresis or Blood Draw for PBMC Collection .............................................................................................. 51 
 
Liver biopsies ......................................................................................................................................................... 51 
 
Protocol Mandated Liver Biopsies ................................................................................................................ 51 
 
Clinically Indicated (For Cause) Biopsies ....................................................................................................... 51 
9. 
Known and Potential Risks and Benefits to Participants .............................................................................................. 52 
 
Risks of IS Withdrawal ........................................................................................................................................... 52 
 
Risk of Treatment for Rejection .................................................................................................................... 52 
 
Risks of darTregs infusion ..................................................................................................................................... 53 
 
Risks of Study Mandated Procedures ................................................................................................................... 54 
 
Risks of Blood Draw ....................................................................................................................................... 54 
 
Risks of Leukapheresis .................................................................................................................................. 54 
 
Risks of Liver Biopsy ...................................................................................................................................... 54 
 
Potential Benefits of darTregs Infusion to Facilitate IS Minimization and/or Complete Withdrawal .................. 54 
10. Study Visits .................................................................................................................................................................... 56 
 
Living Donor .......................................................................................................................................................... 56 
 
LT Recipient ........................................................................................................................................................... 56 
 
Screening, Enrollment, and Initiation of IS Withdrawal................................................................................ 56 
 
Assessments during IS Withdrawal (High Frequency Schedules) ................................................................. 56 
 
darTregs Infusion and Resumption of IS Withdrawal ................................................................................... 57 
10.2.3.1 Adjudication of Discrepant Pathology Readings for the Post-darTreg infusion (Day 7) Biopsy ................ 57 
 
Medium Frequency Schedule ....................................................................................................................... 57 
 
Unscheduled Visits ........................................................................................................................................ 58 
 
Visit Windows ....................................................................................................................................................... 58 


 
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11. Mechanistic Assays ....................................................................................................................................................... 59 
 
darTregs pharmacokinetics ................................................................................................................................... 59 
 
Treg TruCount Analysis ......................................................................................................................................... 59 
 
MFC Panels ............................................................................................................................................................ 59 
 
Donor Specific Assays ............................................................................................................................................ 60 
 
Frequency of donor-reactive T cells .............................................................................................................. 60 
 
In vitro suppression ....................................................................................................................................... 60 
 
Alloantibodies ....................................................................................................................................................... 60 
 
HLA Typing .................................................................................................................................................... 60 
 
Histology and Multiplex Immunohistochemistry (mIHC) ..................................................................................... 61 
 
Gene Expression Profiling ..................................................................................................................................... 61 
12. Biospecimen Storage..................................................................................................................................................... 63 
13. Criteria for Participant Completion and Premature Study Termination ....................................................................... 64 
 
Participant Completion ......................................................................................................................................... 64 
 
Study Completion .......................................................................................................................................... 64 
 
Participant Withdrawal Criteria ............................................................................................................................ 64 
 
Participant Replacement ....................................................................................................................................... 64 
14. Safety Monitoring and Reporting ................................................................................................................................. 65 
 
Overview ............................................................................................................................................................... 65 
 
Definitions ............................................................................................................................................................. 65 
 
Adverse Events (AEs) ..................................................................................................................................... 65 
14.2.1.1. Suspected Adverse Reaction ..................................................................................................................... 65 
 
Unexpected AEs ............................................................................................................................................ 65 
 
Serious Adverse Events ................................................................................................................................. 65 
 
Grading and Attribution of Adverse Events .......................................................................................................... 66 
 
Grading Criteria ............................................................................................................................................. 66 
 
Attribution Definitions .................................................................................................................................. 67 
 
Collection and Recording of Adverse Events ........................................................................................................ 67 
 
Collection Period ........................................................................................................................................... 67 
 
Collecting Adverse Events ............................................................................................................................. 67 
 
Exceptions to Collection ................................................................................................................................ 67 
 
Recording Adverse Events ............................................................................................................................. 67 
 
Reporting of Serious Adverse Events and Adverse Events ................................................................................... 68 
 
Reporting of SAEs to Sponsor ....................................................................................................................... 68 
 
Reporting to Health Authority ...................................................................................................................... 68 
14.5.2.1. Annual Reporting ...................................................................................................................................... 68 
14.5.2.2. Expedited Safety Reporting ....................................................................................................................... 68 


 
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Reporting of AEs to IRBs ................................................................................................................................ 69 
 
Pregnancy Reporting ............................................................................................................................................. 69 
 
Reporting of Other Safety Information ................................................................................................................. 69 
 
Review of Safety Information ............................................................................................................................... 69 
 
MM Review ................................................................................................................................................... 69 
 
DSMB Review ................................................................................................................................................ 69 
 
Study Stopping Rules ..................................................................................................................................... 70 
15. Statistical Considerations and Analytical Plan .............................................................................................................. 71 
 
Statistical Analyses ................................................................................................................................................ 71 
 
Analysis Populations ..................................................................................................................................... 71 
 
Endpoint Assessments .......................................................................................................................................... 71 
 
Safety Endpoints ........................................................................................................................................... 71 
 
Efficacy Endpoints ......................................................................................................................................... 72 
 
Measures to Minimize Bias ........................................................................................................................... 72 
 
Supportive Analyses ...................................................................................................................................... 72 
 
Analyses of Exploratory Mechanistic Outcomes ........................................................................................... 72 
 
Descriptive Analyses ..................................................................................................................................... 72 
 
Interim Analyses .................................................................................................................................................... 72 
 
Sample Size Considerations .................................................................................................................................. 72 
16. Identification and Access to Source Data ..................................................................................................................... 75 
 
Source Data ........................................................................................................................................................... 75 
 
Access to Source Data ........................................................................................................................................... 75 
17. Protocol Deviations ....................................................................................................................................................... 76 
 
Protocol Deviation Definitions .............................................................................................................................. 76 
 
Reporting and Managing Protocol Deviations ...................................................................................................... 76 
18. Ethical Considerations and Compliance with Good Clinical Practice ............................................................................ 77 
 
Statement of Compliance ..................................................................................................................................... 77 
 
Informed Consent Process .................................................................................................................................... 77 
 
Privacy and Confidentiality ................................................................................................................................... 77 
19. Publication Policy .......................................................................................................................................................... 78 
20. References .................................................................................................................................................................... 79 
Appendix 1. Living Donor Assessments ................................................................................................................. 82 
Appendix 2.  Study Entry and IS Withdrawal (High Frequency) ............................................................................ 82 
Appendix 3.  Logistical Pause in Step 2 SOE .......................................................................................................... 84 
Appendix 4.  darTregs Infusion SOE ...................................................................................................................... 85 
Appendix 5.  IS Withdrawal after darTregs Infusion to Step 5/75% CNI Reduction (High Frequency) .................. 86 
Appendix 6.  Complete Immunosuppression Withdrawal (High Frequency) ......................................................... 87 


 
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Appendix 7.  Medium Frequency Schedule after Rejection ................................................................................... 88 
Appendix 8.  Medium Frequency Schedule after Partial or Complete IS Withdrawal ........................................... 89 
 
 
Figure 1. Cause specific probability of death after LT over time .......................................................................................... 20 
Figure 2. Expansion and suppressive function of darTregs. ................................................................................................. 27 
Figure 3. Treg Tracking by Stable Isotope Labeling. .............................................................................................................. 33 
Figure 4. Survival of infused polyclonal Tregs in transplant patients. .................................................................................. 34 
Figure 5. Study Design ........................................................................................................................................................... 37 
Figure 6. Assay for measuring frequency of donor-reactive T cells ...................................................................................... 60 
 
Table 1. 12 Operationally Tolerant Pediatric LT Recipients from WISP-R (as of September 30, 2014) ................................ 21 
Table 2. Seven Non-Tolerant Participants from WISP-R ....................................................................................................... 22 
Table 3. Relationship of time after transplantation with proportion of tolerant subjects in NCT00647283 ....................... 23 
Table 4. Prevalence of tolerance among subjects enrolled in AWISH .................................................................................. 23 
Table 5. Treg infusions and IS withdrawal outcomes for 10 adult LT recipients: Hokkaido University. ............................... 26 
Table 6. Completed and ongoing IS withdrawal clinical trials .............................................................................................. 28 
Table 7. Stepwise Outcome of IS Withdrawal for AWISH Subjects Maintained on CNI Monotherapy ................................ 29 
Table 8. Summary of Treg cell therapy in humans ............................................................................................................... 31 
Table 9. Central Pathology Histological Inflammatory and/or Fibrosis Criteria for Screening Biopsy* ............................... 42 
Table 10. Number of Patients Eligible for ARTEMIS .............................................................................................................. 43 
Table 11. CNI Withdrawal Algorithm .................................................................................................................................... 44 
Table 12. MMF Withdrawal Algorithm ................................................................................................................................. 45 
Table 13.  Tolerance Biopsy Criteria*.................................................................................................................................... 46 
Table 14. SOE Visit Windows ................................................................................................................................................ 58 
Table 15. ARTEMIS MFC panels ............................................................................................................................................ 60 
Table 16. ARTEMIS Immunohistochemistry Panels .............................................................................................................. 61 
Table 17. Attribution of Adverse Events ............................................................................................................................... 67 
Table 18.  Selected AEs that Constitute a Study Stopping Rule ............................................................................................ 70 
Table 19. Analyses of Safety Endpoints ................................................................................................................................ 71 
Table 20. Analyses of Efficacy Endpoints .............................................................................................................................. 72 
Table 21. Sample Size Power Calculation ............................................................................................................................. 73 
Table 22. Incidence Rates and Confidence Intervals for 9 Subjects Attempting Complete IS Withdrawal after Treatment 
with darTregs ........................................................................................................................................................................ 74 
Table 23. Incidence Rates and Confidence Intervals for 11 Subjects Attempting Complete IS Withdrawal after Treatment 
with darTregs ........................................................................................................................................................................ 74 
 


 
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Glossary of Abbreviations 
AE 
Adverse Event 
AFP 
Alpha fetoprotein 
ALT 
Alanine Aminotransferase 
ACR 
Acute Cellular Rejection 
AR 
Acute Rejection 
CFR 
Code of Federal Regulations 
CR 
Chronic Rejection 
CI 
Confidence Interval 
CMV 
Cytomegalovirus 
CNI 
Calcineurin Inhibitor 
CRF 
Case Report Form 
CT 
X-ray computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DSMB 
Data Safety Monitoring Board 
EBV 
Epstein Barr Virus 
FACS 
Fluorescence Activated Cell Sorting 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GFR 
Glomerular Filtration Rate 
GMP 
Good Manufacturing Practice 
GvHD 
Graft Versus Host Disease 
HBV 
Hepatitis B Virus 
HCC 
Hepatocellular Carcinoma 
HCV 
Hepatitis C Virus 
HIV 
Human Immunodeficiency Virus 
ICH 
International Conference on Harmonization 
ICU 
Intensive Care Unit 
IgG 
Immunoglobulin G 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
IS 
Immunosuppression 
LT 
Liver Transplantation or liver transplant 
MELD 
Model for End Stage Liver Disease 
MFC 
Multiparameter Flow Cytometry 


 
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MIHC 
Multiplex Immunohistochemistry 
MLR 
Mixed Lymphocyte Reaction 
MM 
Medical Monitor 
MMF 
Mycophenolate Mofetil 
mSAP 
Mechanistic Statistical Analysis Plan 
NIAID 
National Institute of Allergy and Infectious Disease 
PI 
Principal Investigator 
PTLD 
Post -Transplant Lymphoproliferative Disorder 
PBMC 
Peripheral Blood Mononuclear Cell 
RAI 
Rejection Activity Index 
RETREAT 
Risk Estimation of Tumor Recurrence After Transplant 
SACCC 
Statistical and Clinical Coordinating Center 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Suspected Adverse Reaction 
sBc 
Stimulated B cell 
SOC 
Standard of Care 
SOP 
Standard Operating Procedure 
SVR 
Sustained Virological Responses  
SWFI 
Sterile Water for Injection 
Tac 
Tacrolimus 
Tconv 
Conventional T Cell 
Treg 
Regulatory T Cell 
TSDR 
Treg-Specific Demethylation Region 
ULN 
Upper Limit of Normal 
UNOS 
United Network for Organ Sharing 
 


 
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Study Definitions Page  
Acute Rejection, Severe 
(1997 Banff Criteria) 
(Demetris A. J., 1997) 
Histopathologically, severe AR shows brisk portal inflammation that expands most of the triads 
and often extends into the periportal hepatic parenchyma, similar to moderate AR. More 
importantly, there is inflammation in and around the connective tissue sheath surrounding the 
terminal hepatic venules, which extends out into the perivenular parenchyma and is associated 
with perivenular hepatocyte necrosis. In fact, the combination of perivenular inflammation and 
associated necrosis is the critical lesion used to recognize severe AR. The associated 
mononuclear perivenular inflammation helps to distinguish ischemia-induced perivenular 
necrosis from that seen with severe AR. In some of these cases, the accompanying bile duct 
damage is severe, with disruption of the ductular basement membrane and even partial 
destruction of the duct, recognized by the presence of a few biliary epithelial cells. This can 
quickly lead to bile duct loss, or CR.  
 
It should be stressed the perivenular necrosis and inflammation of severe AR must occur in 
combination with the typical portal inflammation and duct damage characteristic of AR. 
Perivenular necrosis and inflammation alone can be seen with more mild forms of rejection, 
but these occur in association with none or mild portal inflammation and none or mild bile 
duct damage.  
 
In the vast majority of cases, the total score will range from 7 to 9 and the venular 
inflammation score is by definition a "3". 
Acute Rejection, Steroid 
Refractory 
Rejection failing to resolve with corticosteroid treatment, necessitating treatment with an 
antibody preparation such as Thymoglobulin®. 
Allograft Dysfunction 
ALT >120 U/L, alkaline phosphatase >300 U/L, or GGT >120 U/L.   
Autoimmune hepatitis 
Autoimmune hepatitis is defined by histopathological features of autoimmune hepatitis on liver 
biopsy described as moderate to severe, interface and/or perivenular necro-inflammatory 
activity mediated by an infiltrate that is comprised of >30% plasma cells, in more than an 
occasional foci.  Histopathological features will be taken in context with one or more of the 
following clinical parameters:   
1. Liver tests  
2. ANA, ASMA, ALKMA, and quantitative IgG titers 
Baseline Liver Tests  
(ALT, alkaline 
phosphatase, and GGT) 
Baseline liver tests are defined as the average of two laboratory tests: those obtained just prior 
to and at the study entry screening visit. 


 
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Chronic Rejection (CR) 
 
(2000 Banff Criteria) 
(Demetris A. D., 2000) 
Early and late chronic allograft rejection will be defined by the 2000 Banff update on 
histopathologic staging and reporting of CR.  Features of early and late CR are listed below. 
Structure 
Early CR 
Late CR 
Small bile ducts 
(≤60 µm) 
 
• Degenerative changes involving a 
majority of ducts: eosinophilic 
transformation of the cytoplasm; 
increased N:C ratio; nuclear 
hyperchromasia; uneven nuclear 
spacing; ducts only partially lined by 
biliary epithelial cells 
• Bile duct loss in <50% of portal tracts 
• Degenerative changes in 
remaining bile ducts 
• Loss in >50% of portal tracts 
 
Terminal hepatic 
venules and zone 
3 hepatocytes 
 
• Intimal/luminal  inflammation 
• Lytic zone 3 necrosis and inflammation 
• Mild perivenular fibrosis 
• Focal obliteration  
• Variable inflammation  
• Severe (bridging) fibrosis 
Portal tract 
hepatic arterioles 
• Occasional loss involving <25% of 
portal tracts 
 
• Loss involving >25% of portal 
tracts 
 
Other 
• So-called ‘‘transition’’ hepatitis with 
spotty necrosis of hepatocytes 
• Sinusoidal foam cell accumulation; 
marked cholestasis 
Large perihilar 
hepatic artery 
branches 
 
• Intimal inflammation, focal foam cell 
deposition without luminal 
compromise 
• Lumenal narrowing by subintimal 
foam cells 
• Fibrointimal proliferation 
Large perihilar 
bile ducts 
• Inflammation damage and focal foam 
cell deposition 
• Mural fibrosis 
 
 
Investigational Agent 
darTregs (Please see Section 6) 
Liver pathology, 
explanted liver 
Explanted liver for subjects with history of HCC must meet the following criteria: 
• Tumor burden within Milan criteria AND 
• Without macro- or micro-vascular invasion AND 
• Without any lesions with poorly differentiated hepatocellular carcinoma AND 
• Without cholangiocarcinoma morphology  
Lost to Follow-up 
Subject who cannot complete study visits due to inability to reach the subject, subject 
relocation, etc. 
Medical Monitor (MM) 
NIAID official who is responsible for the safety aspects of this trial. The MM is responsible for 
making the final assessment of each serious adverse event (SAE). 
Milan Criteria 
The Milan criteria state that a patient with HCC is eligible for transplantation if the tumor 
burden is: 
• 
one lesion smaller than 5 cm or up to 3 lesions smaller than 3 cm with 
• 
no extrahepatic manifestations and 
• 
no vascular invasion 
NIAID Project Manager 
NIAID assigned project manager who is responsible for all day to day protocol-related 
operations, including version control, consent review, etc. 
Principal Investigator 
Investigator awarded NIH funding for the grant. 
Program Officer 
NIAID official who oversees the scientific and budgetary aspects of the grant. 
Protocol Mandated 
Procedures 
Any procedure performed solely for the purpose of this research study (not site-specific SOC). 
Regulatory Affairs 
Officer 
NIAID assigned officer responsible for regulatory aspects of study, communications with FDA, 
and GCP compliance, as applicable. 
Resolution of Acute 
Rejection 
ALT and either alkaline phosphatase or GGT are either ≤ 1.5 X ULN or ≤ 1.5 baseline or less than 
≤ 1.5x ULN 


 
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Site Principal 
Investigator 
Lead investigator listed on the FDA 1572 at a participating center who is responsible for the 
conduct of the study at that center. 
Stable liver tests 
ALT and alkaline phosphatase or GGT either ≤1.5 X upper limit of normal or ≤1.5 X baseline  
Study Termination 
Subjects who complete the study, are lost to follow up, withdraw consent, or die during the 
study.  Data and specimens will no longer be expected from subjects who are terminated from 
the study. 
Study Therapy 
The investigational agents and all protocol required interventions and medications.  
Sustained Virological 
Response 
SVR is defined as undetectable HCV RNA 12 weeks after end of treatment 
Tolerance 
Tolerance will be adjudicated 1 year after the last dose of IS. ALT and GGT must be less than or 
equal to 1.5X baseline. Baseline ALT and GGT are defined as the average of two laboratory 
tests: those obtained just prior to and at the initial screening visit. In addition, liver biopsy will 
be read by central pathology and adjudicated according to strict, predetermined criteria.  A 
tolerance adjudication committee will review and determine tolerance outcome for subjects 
who meet central pathology criteria but have elevated liver tests above 1.5X baseline.  
Withdrawal from 
Therapy 
Subject who stops study therapy prior to protocol described duration. 
 
 


 
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1. Background and Rationale  
 Mortality and morbidity of conventional IS medications 
It is well known that conventional immunosuppression (IS) imposes substantial mortality risk for adult liver transplant (LT) 
recipients over their lifetime.  The probability of death after LT has been reported to be trimodal: steepest in the first 6 
months after LT, fairly flat between 6 months and 8 years, and then rises again (Watt KD, 2010).  The late increase in the 
risk of death is thought to reflect the cumulative impact of IS exposure.  Examining the causes of death for LT recipients 
strongly supports the notion that IS contributes substantially to increased mortality risk over time. 
 
Standard IS medications suppress the immune system in a generalized, non-specific manner that leaves the transplant 
recipient vulnerable to infection and malignancy.  Within the first 6 months to 1 year after LT, infection is the dominant 
cause of death (Figure 1) (Watt KD, 2010).  While deaths secondary to infection continues to accumulate over time after 
LT, the slope is modest and steady over the decade plus follow-up period.  However, the probability of death from hepatic 
causes (inclusive of recurrent disease) and malignancy increases steeply over time, representing the 1st and 2nd most 
common etiologies of death in the medium- and long-term after LT (Watt KD, 2010), (Chandok N, 2012), (Schoening WN, 
2013), (Schoening WN, 2013), (Rodriguez-Peralvarez M, 2014).  Among the drugs that are commonly employed in current 
IS regimens, calcineurin inhibitors (CNIs), the pillar of modern IS regimens, are most strongly associated with a pro-
oncologic effect (Rodriguez-Peralvarez M, 2014).   Minimization of lifetime exposure to CNIs may significantly mitigate the 
escalating risk of de novo malignancy for stable, long-term LT recipients. 
 
Figure 1. Cause specific probability of death after LT over time 
In addition to increasing mortality risk secondary to immunologically-mediated mechanisms of weakening host defenses 
against infection and malignancy, CNIs are also well known to predispose to and/or exacerbate non-immunologically 
mediated medical co-morbidities that threaten the longevity of adult LT recipients. CNI exposure exerts a strong 
deleterious impact on renal function, particularly in the current climate of LT in the United States where deceased donor 
liver allocation is based on the Model for End Stage Liver Disease (MELD) score that has resulted in a steep increase in the 
proportion of LTs performed in candidates with acute kidney injury and/or chronic kidney disease (CKD) (Sharma P S. D., 
2011) (Sharma P G. N., 2013).  There is substantial literature that adult LT recipients are highly vulnerable to the 
development of severe chronic kidney disease (CKD) and even end stage renal disease (ESRD) over time (Ojo AO, 
2003)(Sharma P G. N., 2013).  Severe CKD in a LT recipient, defined as a GFR <30 ml/min/1.73m2, has been recently 
reported to increase the risk of death exponentially (Allen AM, 2014).  As a result, renal failure emerges as an increasingly 
common cause of death late after LT (Watt KD, 2010) (Allen AM, 2014).  There is interest in CNI minimization and/or 
withdrawal as a primary strategy to reduce CNI-mediated kidney injury (Farkas SA, 2009) (Saner FH, 2012)(Penninga L, 
2012) (McKenna G, 2010; Trotter J, 2012).  Although spontaneous operational tolerance may allow for IS/CNI withdrawal 
with reasonable success late after LT, the ability to induce tolerance with darTregs, and thereby minimize and/or 
completely discontinue CNIs early after LT is likely to better salvage and preserve renal function. 


 
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In addition to nephrotoxicity resulting in CKD/ESRD that predisposes to late mortality for LT recipients, CNIs are strongly 
associated with other potent atherosclerosis risk factors such hypertension, dyslipidemia (low high-density lipoproteins 
and high triglycerides), obesity, and diabetes / insulin resistance – the primary components of the metabolic syndrome 
(Berenson, 1992) (Davis, 2001) (Nair S. S., 2002) (Shalev, 2005) (Textor SC, 2000) (Varo E, 2002).   These considerations are 
particularly important because the indication for LT is anticipated to, over the 5-10 decades, shift dramatically away from 
viral hepatitis (both hepatitis B and hepatitis C) to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis 
(NASH). The epidemiology of metabolic syndrome is regarded as not only a national but also a worldwide public health 
care concern with the hepatic component manifest as NAFLD/NASH (AJ., 2011).  The influx of candidates undergoing LT 
with metabolic disease will undoubtedly translate into a heavy burden of metabolic syndrome post-transplant and focus 
intense attention on the negative impact of standard IS medications such as CNIs on these important medical co-
morbidities (Pagadala M, 2009) (Watt KD, 2010) (Oliveira CP, 2013).  Again, there is interest in reducing lifetime CNI 
exposure to avoid precipitating or exacerbating the components of metabolic syndrome that, alone or in combination 
with CKD/ESRD, predispose to cardiovascular morbidity and mortality for LT recipients.  
 Spontaneous / operational tolerance after LT 
 Previous experiences with IS withdrawal for adult and pediatric LT recipients 
Historically, several different single centers have reported their experiences with transplant recipients who were no longer 
taking IS medications.  Transplant recipients were electively, incidentally (non-adherence), or obligatorily (major 
contraindication to ongoing IS) weaned from IS.  In sum, these series suggest that approximately 19% of selected adult 
and/or pediatric LT recipients are operationally tolerant (Mazariegos GV, 1997) (Oike, 2002) (Ramos, 1995) (Takatsuki, 
2001) (Devlin, 1998) (Assy, 2007) (Tisone, 2006) (Eason JD, 2005) (Tryphonopoulos, 2005).  
 Recent trials of IS withdrawal for pediatric LT recipients 
A pilot trial of IS withdrawal in 20 pediatric recipients of parental living donor transplants 4 or more years after LT 
(ITN029ST; WISP-R; NCT900320606) identified 12 tolerant subjects who have been off drug for 70.5-91.0 months as of 
September 30, 2014 (Table 1) (Feng S, 2012).  Predictors of successful IS withdrawal were increased time after LT (tolerant 
versus non-tolerant subjects were 99.46±29.97 versus 69.70±13.67 months p=0.025), absent or minimal portal 
inflammation (p=0.03), and decreased C4d score (p=0.04) on screening liver biopsy were predictors of successful IS 
withdrawal. 
 
Table 1. 12 Operationally Tolerant Pediatric LT Recipients from 
WISP-R (as of September 30, 2014) 
Pt 
Liver 
Disease 
Age (years) 
CNI Dose at 
Study Start 
Months 
off IS 
Tx 
Study Start 
1 
BA 
0.32 
8.2 
Tac 1.0 mg bid 
107 
2 
BA 
0.57 
8.2 
Tac 0.35 mg bid 
107 
3 
BA 
0.56 
8.8 
Tac 0.25 mg bid 
106.5 
4 
BA 
0.58 
12.1 
Tac  0.5 mg bid 
91.5 
5 
BA 
0.42 
10.3 
CsA  45 mg bid 
91 
7 
BA 
0.42 
11.7 
CsA  38 mg bid 
45.3* 
9 
BA 
0.75 
5.2 
Tac  0.5 mg bid 
86.5 
11 
BA 
0.61 
6.4 
CsA  50 mg bid 
101 
12 
BA 
0.56 
11.2 
CsA  50 mg qd 
105 
14 
AIAT 
0.32 
10.2 
CsA  35 mg qd 
92 
16 
PFIC 
2.5 
8.9 
Tac  2.0 mg qd 
97 
17 
NSC 
1.5 
7.0 
Tac  1.0 mg qd 
91 
*Subject withdrew consent and was terminated from the study 45.3 
months after the last IS dose. 
 


 
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The data from the pilot IS withdrawal trial (WISP-R) for pediatric recipients of parental living donor LTs, led by Dr. Feng, 
suggest that IS withdrawal is safe and feasible. Twelve of the 20 enrollees (60%) have achieved the primary endpoint of 
one year off IS with normal graft function assessed by liver tests. Protocol biopsies performed more than two years and 
again more than four years after the last dose of IS have not shown significant histological change compared with the 
screening biopsies performed more than three or five years earlier, respectively. Eight patients failed to achieve the 
primary endpoint: one for violation of an exclusion criterion and seven for biopsy proven (n=2) or clinical (n=5) rejection. 
Five of the seven participants developed elevated liver tests during withdrawal, between 43 and 169 days after initiating 
IS withdrawal; 1 participant developed elevated liver tests after cessation of withdrawal, 333 days after initiating 
withdrawal. The seven participants were all treated with increased IS. Three received bolus corticosteroids (intravenous 
and/or oral) plus an increase in CNI dose beyond their dose at study start (escalation). Among the 4 that were not treated 
with corticosteroids, 1 was treated with escalation of IS while 3 were treated simply with return to their IS dose (baseline) 
at the start of the study (Table 2). 
  
Table 2. Seven Non-Tolerant Participants from WISP-R 
Pt 
Liver 
Disease 
Age (years) 
CNI Dose at 
Study Start 
Peak 
ALT/GGT 
Biopsy 
(Central Pathology) 
IS 
Management 
Elevated ALT/GGT 
Start 
(D)* 
End 
(D)* 
Duration 
(Days) 
Tx 
Study 
Start 
8 
OTC 
7.5 
15.3 
Tac4.5 mg bid 
121/244 
ACR indeterminate 
Baseline 
43 
162 
119 
15 
BA 
1.1 
6.2 
Tac1mg qam /  
0.5 mg qpm 
318/98 
ACR moderate 
IV+PO steroids 
Escalation 
88 
190 
102 
6 
BA 
0.75 
8.0 
Tac1.0 mg bid 
562/127 
ACR indeterminate 
Escalation 
155 
515 
360 
20 
BA 
0.63 
5.0 
CsA50 mg bid 
312/197 
ACR indeterminate 
/ biliary stricture 
PO steroids 
Escalation 
169 
222 
53 
10 
BA 
0.44 
6.6 
CsA25 mg bid 
134/25 
ACR indeterminate 
Baseline 
333 
402 
69 
19^ 
BA 
0.50 
5.2 
Tac0.4 mg bid 
NA 
ACR mild 
Baseline 
NA 
18^ 
BA 
0.48 
6.6 
Tac1.0 mg qd 
NA 
ACR indeterminate 
PO steroids 
Escalation 
NA 
 
Lastly, there is an ongoing, large prospective multicenter trial involving 12 pediatric transplant centers in North American 
(United States and Canada) (iWITH; RTB001; NCT01638559) that is fully enrolled.  Compared to the pilot trial just 
described, this trial includes both deceased and living donor LT recipients. The primary objective of the trial will be to 
determine the frequency of operational tolerance in a well-defined population of pediatric LT recipients.  Preliminarily, 
based on the observed incidence of AR, the prevalence of tolerance in this trial will be very comparable to the pilot study, 
in spite of including deceased donor LT recipients.  This is harmonious with the literature that suggests minimal 
immunologic benefit of HLA matching in the liver transplant setting (Balan V, 2008) (Lan X, 2010) (Yosry A, 2012). 
 Recent trials of IS withdrawal for adult LT recipients 
A European, multi-center, adult IS withdrawal trial enrolled 102 adult deceased donor LT recipients of which 41 (40%) 
were operationally tolerant (V-2005-CE512090-O; NCT00647283 (Benitez, 2013).  Clinical parameters that differentiated 
tolerant from non-tolerant patients included increased time after transplantation (p<0.0001), increased age (p<0.0005), 
and male gender (p=0.016). Specifically, with respect to time after LT, among subjects who were 3.0-5.7 years after LT, 
only 3 of 24 (12.5%) succeeded with IS withdrawal, compared to 19 of 24 (79.2%) among subjects who were >10.6 years 
after LT (Table 3).   
 


 
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Table 3. Relationship of time after 
transplantation with proportion of 
tolerant subjects in NCT00647283 
Years since 
transplant 
Tolerant 
N 
% 
3.0 - 5.7 
3 / 24 
12.5 
5.7 - 10 
19 / 50 
38.0 
>10.6 
19 / 24 
79.2 
 
Finally, a trial entitled “Gradual Withdrawal of Immune System Suppressing Drugs in Patients Receiving a Liver Transplant” 
(AWISH/ITN030ST/NCT00135694) that enrolled adult de novo LT recipients receiving standard IS who initiate IS 
withdrawal between one and two years after deceased donor LT.  In total, 76 subjects attempted IS withdrawal; 5/30 
(16.7%) subjects with HCV and 6/46 (13.0%) subjects with non-immune, non-viral liver disease were identified as tolerant 
(Table 4).   
 
Taken together, the historical experiences and the recent adult and pediatric withdrawal trials convincingly show that 
operational tolerance indeed occurs in both adult and pediatric LT recipients, and perhaps more often in the latter 
(Mazariegos GV, 1997) (Oike, 2002) (Ramos, 1995) (Takatsuki, 2001) (Feng, 2012). AR episodes occurring during controlled, 
highly supervised IS withdrawal were consistently mild to moderate in histologic severity, almost never requiring antibody 
treatment, and resolved without permanent allograft damage or dysfunction (Mazariegos GV, 1997) (Ramos, 1995) 
(Takatsuki, 2001). Graft loss related to IS withdrawal has not been observed. Presumably, close surveillance ensures 
expeditious detection, diagnosis, and treatment of allograft rejection. The literature supports the intuition that delayed 
diagnosis and treatment leads to the varied outcomes of rejection occurring outside of the close surveillance characteristic 
of IS withdrawal protocols (Neil, 2001).  Time after LT has emerged as a consistent factor relative to the prevalence of 
operational tolerance.  Among adults 2-6 years after LT, the prevalence of tolerance is low, approximating 13%.  
 Rationale for Accelerating Successful IS Minimization / Withdrawal 
One of the primary findings of the large, European, prospective multi-center trial of IS withdrawal for adult recipients of 
deceased donor LTs is that the frequency of operational tolerance is highly dependent on time after transplantation 
(Benitez, 2013)(Table 3).  The success rate of IS withdrawal increased from 12.5% (3 of 24) to 38.0% (19 of 50) to 79.2% 
(19 of 24) for patients who were 3.0-5.7, 5.7–10.6, and >10.6 years after LT (Table 3).  Moreover, in AWISH, where de novo 
adult deceased donor LT recipients who received standard IS underwent IS withdrawal that was initiated 1-2 years after 
LT, the frequency of operational tolerance among recipients with non-immune or non-viral liver disease was 13.0% (6/46).   
Finally, WISP-R, a multi-center prospective pilot trial of IS withdrawal for pediatric recipients of living parental donor LTs, 
increased time after transplantation was identified as significantly associated with operational tolerance (Feng, 2012).  
Therefore, spontaneous operational tolerance does occur among adult and pediatric LT recipients but only at a low 
frequency early after LT (<6 years).  Since the toxicities of conventional IS as delineated above (Section 1.1) are cumulative, 
LT recipients could derive substantial benefit if standard IS in general and CNIs in specific could be minimized and/or 
discontinued earlier, within the first 2-6 years after LT.  darTregs may therefore represent a strategy to actively control 
the allo-immune response and substantially increase the success rate of IS minimization or discontinuation in the early 
post-transplant timeframe.   
 Induction of Transplantation Tolerance 
Acquisition of immune tolerance to self-antigens or allo-antigens is an active process that requires antigen exposure. 
Tolerogenic antigen exposure leads to inactivation of antigen-reactive T cells through apoptosis, injury and induction of 
immune regulatory mechanisms that maintain tolerance. The current approach of IS for transplant recipients blindfolds 
the immune system to prevent rejection, but also impedes tolerance induction. This may explain why spontaneous 
transplant tolerance is rare and the best predictor of tolerance is time after transplant, likely through the cumulative effect 
Table 4. Prevalence of tolerance among 
subjects enrolled in AWISH 
Etiology of Liver 
Disease 
Tolerant 
N 
% 
HCV+ 
5 / 30 
16.7 
Non-immune, non-viral 
6 / 46 
13.0 


 
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of low level of donor antigen exposure over a long period of time. Ideally, transplant patients should receive 
immunoregulatory regimens blocking rejection while allowing donor antigen recognition and tolerance induction. Such 
immunoregulatory regimens, including Treg therapy, have promoted tolerance and IS-independent graft survival in animal 
models of transplantation. While most of these experimental regimens are given in the peri-transplant period, we think a 
similar approach can be rationally applied to patient undergoing IS withdrawal at a later time point after transplantation.  
In our previous pilot trial of IS withdrawal in pediatric LT recipients (Feng, 2012) many years after transplantation, along 
with AWISH, a trial of IS withdrawal in adults starting 1-2 years after LT, we observed de novo production of donor-specific 
antibodies, demonstrating new immune recognition of donor antigen during IS withdrawal.  Thus, IS withdrawal may 
present an important opportunity to induce tolerance using an immunoregulatory regimen. 
 Rationale for Selection of Investigational Product or Intervention 
 Rationale for darTregs Therapy 
The recent elucidation of Tregs and their importance in suppressing autoimmunity and alloimmunity has inspired new 
thinking in managing alloresponses. Emerging data suggests designing IS regimens with a “Treg-centric” approach to 
promote regulation may favor induction of graft tolerance and improve long-term graft outcomes (Wood, 2003) 
(Bluestone, 2004) (Walsh, 2004) (Kang, 2007) (Sagoo P. G., 2008) (Waldmann, 2008) (Long, 2009). Unlike generalized IS 
regimens, Tregs are long-lived and function in a dominant and antigen-specific manner. Thus, therapeutic infusion of Tregs 
has potential to induce long-term donor-specific tolerance without impeding desired immune responses to pathogens and 
tumors in transplant patients. Research in animal models has demonstrated Tregs can be used to treat many auto-
inflammatory diseases such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, and multiple 
sclerosis. In addition, Treg therapies are efficacious in controlling alloimmune responses in graft-versus-host disease 
(GvHD) as well as organ and cell transplantation in animal models.  
 Rationale for darTregs Dosing 
The Treg dose in protocol CTOTC-12 was selected based on three primary factors: prior human experience, estimated 
effective dose, and manufacturing capacity at the UCSF facility.  
 Dosing precedents in humans 
Currently, there are five published reports of Treg therapy in humans, four studies in adults for GvHD (Di lanni, 2011) 
(Trzonkowski, 2009) (Brunstein, 2010) (Martelli MF D. I., 2014) and one study in children with new onset type 1 diabetes 
(Marek-Trzonkowska N., 2012). All these studies administered polyclonal Tregs, and currently no prior experiences with 
darTregs have been reported.  Since polyclonal Tregs are most similar to darTregs when compared to other cellular 
therapies, it is nonetheless helpful to review the dosing and safety information from these studies.  The results of these 
protocols, particularly with respect to the safety of Treg administration, are detailed in Section 1.6.  The discussion below 
provides the rationale for selection of Treg doses for administration to human subjects participating in protocol CTOTC-
12. 
 
The highest dose used in the referenced GvHD studies was 2 infusions of 3 x 106 Tregs/kg each on day 1 and day 15 after 
cord blood transplantation. No infusion reactions or severe adverse events (AEs) was observed with this dose. In fact, the 
investigator reported that maximal tolerated dose could not be assessed due to limitation in manufacturing capacity 
(Brunstein, 2010).  
 
In the type 1 diabetes pediatric study (Marek-Trzonkowska N., 2012), children age 8 to 16 were given 10 x 106 Tregs/kg 
(n=4) or 20 x 106 Tregs/kg (n=6), corresponding to 700 – 1,400 x 106 Tregs for the average 70kg adult human.  It is worth 
noting that the Treg expansion protocol used for the Marek-Trzonkowska study is comparable to that currently used at 
UCSF for polyclonal Treg manufacture and many manufacturing processes are shared with the darTregs manufacturing 
process for CTOTC-12.   
 
In an adult, UCSF-sponsored ongoing type 1 diabetes trial at UCSF, subjects in four escalating dose cohorts have been 
infused with 5, 40, 320, and 2,600 x 106 Tregs total dose, corresponding to approximately 0.07, 0.6, 4.6, and 38 x 106 
Tregs/kg.  All 14 patients in four dosing levels have been administered. As of September 2014, all 14 subjects were active 


 
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in the study.  Cohorts 1-4 are 3, 2.5, 1.5, and 1 year after administration, respectively. No infusion reaction of any grade 
was observed in any patient.  Four serious adverse events (SAEs) deemed unrelated to Treg infusion were reported, as of 
August 2014. 
 
Thus, based on published data and UCSF’s clinical experience, infusions of up to 2,600 x 106 (38 x 106/kg) polyclonal Tregs 
were well tolerated.  
1.5.2.2 Estimated efficacy dose for darTregs in organ transplantation 
Results from preclinical studies in mouse models of transplantation in the past 30 years provide strong rationale for the 
use of Tregs to induce transplant tolerance (Tang Q, 2013).  In these models, a high percentage of Tregs, as many as 30-
50% of CD4+ cells, is needed to prevent transplant rejection (Hara, 2001) (Graca, 2002). To achieve this high percentage 
of Tregs in a 70kg adult human, an infusion of 4,500 x 106 (642 x 106 Tregs/kg) Tregs would be needed. This dose can be 
reduced by 80 to 90% (to 500-1,000 x 106 total dose) if darTregs are used (Lee K, 2014).  While these estimates provide 
useful guidelines for dose selection in humans, these should not be directly applied because of the many differences 
between mouse models and human trial design such as the use of concurrent IS and the timing of Treg infusion.  Typically, 
Tregs are given around the time of transplantation in mouse models without additional IS when strong activation signals 
and highly inflammatory conditions increase the resistance of effector T cells to Treg-mediated suppression.  Thus, it is 
very likely that a lower percentage/ fewer Tregs would be required to induce tolerance when IS is present to control T cell 
activation and when Tregs are given years after transplantation without acute tissue injury and inflammation of the 
surgery. 
Recently, an ongoing clinical trial of Treg therapy in LT conducted in Japan has been reported, providing valuable clues 
(Yamashita, 2013) (Yamashita, 2014) (Todo S, 2016).  This trial was conducted in de novo adult, living donor LT recipients 
on a standard triple IS regimen comprised of corticosteroids, tacrolimus, and MMF.  Recipient peripheral blood 
mononuclear cells (PBMCs) expanded with irradiated donor PBMCs in the presence of anti-CD80 and anti-CD86 
antibodies to block co-stimulation were administered 13 days after LT and 8 days after a single dose of cyclophosphamide 
(40mg/kg).   In total, 610 - 2,590 x 106 expanded autologous PBMCs expanded in the presence of donor cells and 
containing an average of 28% Foxp3+ cells (31 - 466 x 106 Foxp3+ Tregs) were infused (Table 5).  IS was withdrawn 
gradually starting 6 months after LT. As of July, 2014 (personal communication; presentation at the World Transplant 
Congress, July 2014, San Francisco, CA; manuscript in preparation), 7 of 10 treated patients have been weaned off IS for 
6-23 months with five recipients off IS for greater than 12 months (13 to 23 months), Three of 10 have failed IS 
withdrawal, two secondary to AR and one secondary to IS re-initiation for brachial plexus neuritis who later developed 
AR while on IS.  The cell manufacturing protocol has been shown to inactivate effector T cells and favor the outgrowth of 
Tregs (Davies JK, 2009). Similarly manufactured cells have also been used therapeutically to control GvHD (Guinan EC, 
1999) (Davies JK G. J., 2008), and kidney transplant rejection in non-human primates (Bashuda H, 2005). 
To determine appropriate Treg dosing, the outcome relative to the dose of Foxp3+ Tregs administered was considered.  
Three patients received only 590 - 630 x 106 PBMCs, corresponding to 31 to 43 x 106 Foxp3+ Tregs.  Two of these three 
subjects developed AR while 1 successfully discontinued IS and has remained off for 23 months.  The other 6 subjects 
who have successfully discontinued IS received 790 to 2,590 x 106 PBMCs, corresponding to 94 to 466 x 106 Foxp3+ Tregs.  
The other patient who failed IS withdrawal received 272 x 106 Foxp3+ Tregs failed IS withdrawal due initially to brachial 
plexus neuritis and later AR.  
This study suggests that Treg doses of <50 x 106 Tregs are not sufficient to induce operational tolerance (1 success out of 
3).  However, doses between 100 and 300 x 106 Tregs have some efficacy (2 successes out of 3) and doses >300 x 106 Tregs 
appear to be reliably adequate (4 successes out of 4) to support successful IS withdrawal.  
 
The product used in this Japanese trial is enriched for donor alloantigen specificity as is the darTregs product we plan to 
use.  However, there are several important differences regarding the context of Treg administration between their trial 
design and CTOTC-12.  The depletional therapy that they administered prior to Treg infusion (cyclophosphamide) is likely 
to support Treg expansion, engraftment, and function; depletion is perceived to lower the Treg dose needed for efficacy.  
However, they administer Tregs 13 days after LT, during the highly inflamed peri-transplant period to subjects who are 


 
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receiving a high IS burden in general and high CNI dosing in specific.  Both of these factors may hamper the survival and 
function of their infused Tregs.  CTOTC-12 study design administers darTregs to LT recipients with stable liver function on 
modest doses of CNIs long after transplantation, an immunologically quiescent timeframe but without depletion.  We 
believe that these are compensatory differences and therefore can reasonably extrapolate their dosing regimen in this 
clinical trial. 
Table 5. Treg infusions and IS withdrawal outcomes for 10 adult LT recipients: Hokkaido University. 
Pt # 
Total Cells 
Infused x106 
CD4+CD25+Foxp3+ 
Cells Infused  (x106) 
Days after 
LDLT* 
Current IS 
DSA 
Months off IS* 
1 
610 
31 
1620 
none 
Neg 
33 
2 
2,540 
466 
1543 
none 
Class II+ 
31 
3 
790 
94 
1515 
none 
Class II+ 
32 
4 
2,450 
441 
1410 
none 
Neg 
29 
7 
2,590 
318 
1263 
none 
Neg 
13 
8 
700 
304 
1186 
none 
Neg 
18 
10 
1,200 
289 
1018 
none 
Neg 
16 
5 
630 
43 
1326 
FK 4 mg/d 
ND 
ACR; POD 394 
9 
590 
33 
1284 
FK 4 mg/d 
ND 
ACR; POD 365 
6 
1,180 
272 
1123 
 MMF 500 mg/d 
PSL 5 mg/d 
ND 
Brachial plexus 
neuritis POD 206;  
ACR POD 311 
*As of April 30, 2015 
 
Although the Japanese study has many differences to our proposed study, this is currently the only Treg cell therapy trial 
in solid organ transplantation and the closest to our clinical scenario of adult LT. In sum total, we estimate that the 
requisite dose of darTregs to facilitate IS withdrawal in stable, long-term LT recipients may be similar to that reported in 
the Japanese trial.  We estimate that a dose of <100 x 106 darTregs will have low efficacy; that a dose of 100 – 300 x 106 
will have moderate efficacy; and that a dose of >300 x 106 will have high efficacy for successful IS minimization / 
discontinuation. 
1.5.2.3 Dose limitations imposed by darTregs manufacturing capacity 
The UCSF Treg manufacturing facility can routinely produce more than 600 x 106 darTregs from 1 unit of blood (450 ml) or 
500 x 106 leukapheresis products, a quantity that is approximately double the dose estimated to be effective based on the 
LT Treg trial conducted in Japan.  On average, 6.6 x 106 (range 3 to 11.9 x 106) Tregs can be purified from one unit of blood 
and expanded >200 fold (range 200 to 4000) during the 16-day ex vivo culture (Figure 2A) to produce more than 600 x 106 
Tregs (Figure 2B).  The expanded darTregs retain their phenotypes and are highly suppressive of donor antigen stimulated 
proliferation in vitro (Figure 2B).   
 
Taken together, we plan to infuse a targeted dose of 400 x 106 ± 100 darTregs in this trial.  A range of +/- 25% change from 
this targeted dose is allowed such that the range of darTregs to be infused is 300 - 500 x 106.  Subjects receiving this dose 
will have the option, if eligible, to proceed to complete IS withdrawal.  If less than 100 x 106 darTregs are produced for a 
patient, the product will not be infused because of the low likelihood of efficacy based on data from the Japanese LT Treg 
trial.   If 100 - 300 x 106 darTregs are produced, the product will be infused and the patient will be allowed to start IS 
withdrawal because a favorable probability of efficacy with this dose compared to no treatment (Table 7 and Table 8).  
Participants receiving 100 – 300 x 106 darTregs will only be allowed to progress to the primary endpoint: 75% reduction of 
CNI dosing along with Prednisone or MMF discontinuation (if they entered the trial on Prednisone or MMF). 
 


 
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Figure 2. Expansion and suppressive function of darTregs.   
 
 Rationale for Proposed IS Withdrawal Algorithm 
The IS withdrawal as proposed (Section 5.1; Table 11. CNI Withdrawal Algorithm, Table 12. MMF Withdrawal Algorithm) 
is based on recently completed or currently ongoing clinical trials for pediatric or adult liver transplant recipients (Table 6. 
Completed and ongoing IS withdrawal clinical trials).  For comparison purposes, the currently proposed trial is listed as 
the 
last 
two 
entries: 
“ARTEMIS 
Minimization” 
and 
“ARTEMIS 
Complete 
Withdrawal”. 
The 
AWISH 
(ITN030ST/NCT00135694) and the European multi-center (Benitez, 2013) trials which provide historical controls for the 
proposed trial are also listed.   
 
Essentially all trials withdraw IS completely over approximately 9 – 12 months, reducing IS doses in 6 – 10 steps, each of 
4 – 8 week duration.  The European multi-center trial (Benitez, 2013) has a less regimented withdrawal algorithm, with 
less clearly delineated dose reduction algorithm and a “target” withdrawal timeframe of 6 – 9 months.  We designed the 
ARTEMIS withdrawal algorithm to fit well within the parameters of these tested approaches.  There is no published data 
that speaks to the impact of either a significantly shorter or much longer weaning protocol on the safety or efficacy of 
withdrawal.  
 
The currently proposed trial seeks to enroll subjects who are “early” after liver transplantation (2 – 6 years) who have a 
low likelihood, in the absence of darTregs, of successful IS minimization or complete discontinuation.  Such subjects, when 
managed according to standard of care, are typically on a calcineurin-inhibitor (CNI) based regimen but often with an 
additional medication, either Prednisone or mycophenolate.  The other adult withdrawal trials (de la Garza RG, 2013) 
(Benitez, 2013) similarly allow subjects on one- or two-drug therapy to attempt withdrawal.   AWISH is unique, only 
allowing subjects on monotherapy even though withdrawal is initiated early after transplantation, between 1 – 2 years 
after transplant.  This trial, however, enrolled subjects prior to liver transplantation and the protocol, rather than standard 
of care, determined the IS regimen from the time of transplantation. 
 
We have decided to withdraw both CNI and either prednisone or MMF simultaneously rather than sequentially (Section 
5.1) to allow gradual reduction of drugs, while maintaining the overall timeframe of withdrawal at approximately one year 
(48-50 weeks).  We believe that gradual reduction of prednisone, from the maximal allowable entry dose of 5 mgs daily 
to none over 16 weeks (Section 5.1.2  ), is important for subjects who have been maintained on corticosteroids for as 
many as six years.  There is little precedent to suggest that there is a difference in efficacy or safety with sequential versus 
simultaneous withdrawal of two IS medications. 
 
 
 
 
 
A. Fold expansion of darTregs using 8 
distinct donor-recipient combinations.   
B. In vitro suppression of donor antigen 
stimulated proliferation comparing darTregs 
and polyclonal Tregs from the same 
individual.   The graph is a summary of 5 
different pairs of darTregs and polyclonal 
products. 


 
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Table 6. Completed and ongoing IS withdrawal clinical trials 
STUDY 
Adult / 
Pediatric 
Time after 
Transplantation at 
Initiation of IS 
Withdrawal: 
 
Inclusion 
Criterion; 
[Actual Data] 
# IS 
Medications 
at Baseline 
Number of 
Dose 
Reductions 
Duration 
at Each 
Dose 
Minimum 
Duration of IS 
Withdrawal* 
ITN029/WISP-R 
Pediatric 
> 4 yrs; 
 
[median (range): 
102.0 (60.0 – 183.8) 
mos] 
1 
8 
4 – 6 
weeks 
36 weeks 
iWITH 
> 4 yrs; 
 
[mean ± SD: 
104.4 ± 39.8 mos] 
1 
8 
4 – 6 
weeks 
36 weeks 
ITN030/AWISH 
Adult 
1 – 2 yrs 
1 
8 
8 weeks 
56 weeks 
Benitez et al., 
Hepatology 
20131 
> 3 yrs; 
 
[mean ± SD: 
103 ± 47 mos] 
1 or 2; 
 
Sequential 
withdrawal if 2 
drugs 
Manuscript 
states: “. . . 
doses were 
gradually 
reduced by ¼ 
to ½ until the 
minimum 
feasible daily 
dose” 
3 weeks 
Manuscript 
states: “Target 
completion time 
for drug 
discontinuation 
was 6 to 9 
months.” 
De la Garza et 
al., Liver 
Transplantation 
20132 
> 3 yrs; 
 
[median (range): 
112 (72-160) mos] 
1 or 2; 
 
Unspecified 
whether 
simultaneous 
or sequential 
withdrawal if 2 
drugs 
6 - 10 
one month 
6 - 10 months 
ARTEMIS  
Minimization 
2 – 6 yrs 
1 or 2; 
 
Simultaneous 
withdrawal if 2 
drugs 
5 
6 - 8 
weeks 
24 – 26 weeks 
ARTEMIS 
Complete 
Withdrawal 
8 
6 – 12 
weeks 
48 – 50 weeks 
*The last dose reduction is complete discontinuation of IS.  This “step” does not contribute to the total duration of IS withdrawal.  “Pauses” 
for additional monitoring of liver tests can lengthen the duration of withdrawal.  Hence, the minimum duration of withdrawal is shown.  
 
1Benitez C. (2013). Prospective multi-center clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients. 
Hepatology, 58(5), 1824-35. 
2de la Garza RG. (2013). Trial of Complete Weaning From Immunosuppression for Liver Transplant 
Recipients: Factors Predictive of Tolerance. Liver Transplantation, 19:937–44 
 Rationale for Timing of Treg Administration during IS Minimization/Withdrawal 
Protocol CTOTC-12 is designed to administer darTregs at a time that will maximize clinical efficacy and thereby increase 
the likelihood that participants will derive the benefit of successful IS minimization or discontinuation.   
 
To enter the trial, subjects must be between two and six years after living donor LT and stable on low to moderate dose 
CNI, as defined by 12 hour trough CNI levels.  As patients during this post-LT timeframe may still be maintained on a 2nd 
drug, subjects can enter the trial if they are similarly maintained on low to moderate dose of a 2nd IS medication.  There is 
precedent for allowing adult subjects on more than a single drug to enter IS withdrawal trial (Benitez, 2013). 


 
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We have imposed specific limits for 12 hour CNI trough levels to ensure that the infused darTregs are not exposed to high 
circulating levels of CNIs.  Moreover, the darTregs infusion will occur after a 25% reduction of the study entry CNI dose.  
Treg function depends on activation by the cognate interaction between T cell receptors and the antigen-presenting cells.  
High CNI doses are often used early after transplantation to block Treg activation and suppress IL-2 production by 
conventional T cells.  Tregs are thus deprived of this essential survival and growth factor in the early post-transplant period.  
However, reports suggest that low to moderate CNI levels, as would be expected for the population enrolled in this study 
who are not recently transplanted, may be less harmful for Tregs because 1) Tregs have more active Ca++ signaling 
machinery than conventional T cells requiring a higher CNI concentration to be blocked; 2) IL-2 receptor on Tregs have 
higher binding affinity than those expressed on conventional T cells and effectively compete when IL-2 production is 
restricted.  Therefore, the function or persistence of the infused Tregs should not be critically impacted by the ongoing, 
moderate CNI doses subjects will be taking in this protocol (Brandt C, 2009) (Calvo-Turrubiartes M, 2009). Tregs also 
function by conferring regulatory activities to conventional T cells, termed “infectious tolerance.”  This activity depends 
on antigen activation of conventional T cells and Tregs (Qin S., 1993), (Dieckmann, 2002), (Jonuleit, 2002), (Kendal, 2011)) 
that may also be inhibited by exposure to high levels of CNIs.  Finally, subjects may enter the trial if they are on a modest 
dose of a 2nd IS medication, either Prednisone or MMF.  At the doses typically given to patients 2-6 years after liver 
transplantation, neither of these two drugs have a substantial impact on Treg function or longevity (Lim DG1, 2010).   
 
We have deliberately selected to administer darTregs at the end of the 2nd IS withdrawal step for several reasons.  First, 
as mentioned above, darTregs will be infused after 25% reduction of CNI dosing.  Second, delaying darTregs infusion will 
eliminate subjects who develop abnormal liver tests/AR very early in the IS withdrawal algorithm, perhaps those who are 
the farthest from tolerance.  The 1st step (Table 7) stipulates that the morning and evening doses be combined into a 
single dose, taken in the morning.  As such, there is no dose reduction.  The 2nd step entails a 25% dose reduction.  In 
AWISH (ITN030ST/NCT00135694), out of 46 subjects on CNI monotherapy who initiated IS withdrawal, a single subject 
each failed at the 1st and at the 2nd step.  Third, administration at the end of the 2nd step will also deliver darTregs more 
proximal to the time when immune responses are escalating, as reflected by the timing of AR in AWISH 
(ITN030ST/NCT00135694).   
Table 7. Stepwise Outcome of IS Withdrawal for AWISH Subjects Maintained on CNI Monotherapy 
STEP  
IS REDUCTION: 
% OF STARTING DOSE 
# SUBJECTS 
ENTERING STEP 
# SUBJECTS 
FAILING DURING STEP 
% FAILURE  
PER STEP 
CUMULATIVE 
FAILURE RATE 
1 
0%* 
46 
1 
2.2% 
2.2% 
2 
25% 
45 
1 
2.2% 
4.3% 
3 
50% 
44 
11 
25.0% 
28.3% 
4 
75% 
33 
18 
54.5% 
67.4% 
5 
85.7% 
15 
4 
26.7% 
76.1% 
6 
92.9% 
11 
4 
36.4% 
84.8% 
7 
96.4% 
7 
1 
14.3% 
87.0% 
8 
100% 
6 
0 
0% 
87% 
*First step combines twice daily dosing into a single daily dose and therefore does not involve dose reduction. 
 Rationale for Timing of IS Withdrawal Resumption after darTregs Administration 
The protocol mandates that IS withdrawal be initiated soon, within 14 days after darTregs infusion to maximize the 
therapeutic effects and potential benefit of darTregs administration.  Initiation of IS withdrawal soon after darTregs 
infusion will favor tolerance induction for the following reasons.  First, CNI is known to be detrimental to darTregs function 
and homeostasis (Baan, 2005) (Rudensky, 2006) (Pascual J, 2008).  Although CTOTC-12 patients will receive lower doses 
of CNIs compared with typical transplant patients, further reduction in CNI exposure will be beneficial for the infused 
Tregs.  Second, it is well established that Treg induction of transplantation tolerance depends on a process called infectious 


 
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tolerance (Kendal AR, 2010).  The infused darTregs create a tolerogenic microenvironment in the tissue, allowing host T 
cells activated in this environment to acquire regulatory properties and thereby propagating tolerance.  Infectious 
tolerance requires therapeutic Tregs and host T cells to be activated by their T cell receptors, which is blocked by CNI.  
Therefore it is proposed that reduction of CNI doses will favor development of infectious tolerance.  Third, our experience 
with the ex-vivo expanded autologous CD4+CD127lo/-CD25+ Tregs thus far show that the infused Tregs peaked in the 
circulation within two weeks of infusion and persisted at a relatively low level for at least 3 months (data shown in UCSF 
IB).  We think it is best to reduce CNI exposure when the infused Tregs are at the highest circulating levels to maximize 
the effect of Treg therapy.   
 Clinical Experience with Treg Therapy 
There have been and continue to be multiple clinical experiences with Treg infusion in distinct settings that provide 
relevant information to this protocol with respect to both the safety and the efficacy of Tregs.  Tregs have been 
administered to adults and children, to patients on and off generalized IS, for wide-ranging indications including the 
prevention or treatment of GvHD, the stabilization and/or reversal of type I diabetes mellitus, and the induction of 
tolerance after LT (Table 8).  
 
 


 
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 Treg Therapy for Treatment or Prevention of GvHD 
Initial human experiences in Treg therapy were all for GvHD.  The first-in-man use of Tregs was by Trzonkowski et al. to 
treat established GvHD (Trzonkowski, 2009).  As such, these two patients were also receiving IS.  The first patient had 
chronic GvHD two years after bone marrow transplantation.  After receiving 0.1 × 106/kg fluorescence-activated cell 
sorting (FACS) purified ex vivo expanded Tregs from the donor, the symptoms subsided and the patient was successfully 
withdrawn from IS.  The second patient had acute disease that progressed despite three infusions with a cumulative dose 
of 3 × 106/kg expanded donor Tregs.   After the initial report in 2009, the investigators continued to enroll patients who 
are taking conventional IS with chronic and acute GvHD for Treg therapy.  Their recent review suggests that Treg therapy 
is effective at reversing chronic GvHD and allowing patients to be weaned off IS, but ineffective at controlling acute GvHD 
(Trzonkowski P, 2013) 
 
In all of the other GvHD trials reported thus far, Tregs were given at the time of stem cell transplant to prevent the 
development of GvHD.  A phase I trial by Brunstein et al. was reported in 2010 (Brunstein, 2010).  Twenty-three patients 
with advanced hematologic malignancies were enrolled and treated with two units of umbilical cord blood as source of 
stem cells and effector T cells.  Tregs were isolated using anti-CD25 immunomagnetic bead selection from third-party cord 
Table 8. Summary of Treg cell therapy in humans 
Study 
Patients 
Treg 
Adjunct 
IS 
Follow
-up 
Major findings 
Status 
Lead 
Dise
ase 
N 
Ag
e 
Type
* 
Dose 
Safety 
Efficacy 
Trzonkow
ski 
GvH
D 
2+ 
40, 
44, 
NR 
A 
0.1-3 x 106/kg 
2 - 4 x 106/kg 
Steroid, 
ATG, 
MMF, 
Tac 
2-3 
mos 
No relapse 
Reverse 
cGvHD, 
ineffective 
in aGvHD 
Published 
2009; 
ongoing  
Brunstein 
 
GvH
D 
23 
12-
70 
B 
0.1-6 x 106/kg 
MMF, 
CsA or 
Sirolimu
s 
2-24 
mos 
No increase 
in 
infections 
or relapse 
No cGvHD 
Published 
2010 
Di Ianni 
GvH
D 
28 
21-
60 
C 
2-4 x 106/kg 
none 
12 mos 
No increase 
in relapse, 
positive 
response to 
flu vaccine 
Reduced 
aGvHD 
Published 
2011 
Edinger 
GvH
D 
9 
NR 
C 
5 x 106/kg 
none 
NR 
No severe 
infections, 
no relapse 
No GvHD 
Reported 
2011 
Marek-
Trzonkow
ski 
T1D 
10 
8-
16 
A 
10-20 x 106/kg 
none 
4 mos 
No safety 
concerns 
C-peptide 
stabilizatio
n 
Published 
2012 
Yamashita 
LT 
10 
39-
59 
D 
0.6 - 2.6x109 
(8.6 – 37.1 x 
106/kg) 
CYA, 
Steroid, 
MMF, 
CNI 
16-36 
mos 
No infusion 
AE 
7 pts off IS 
Abstract, 
ongoing  
Bluestone 
T1D 
14 
18-
43 
A 
0.05 - 2.6x109 
(0.7 – 37.1 x 
106/kg) 
none 
3-27 
mos 
No  SAEs 
related to 
therapy 
Stable c-
peptide 
Published 
2015 
Treg type: 
A: FACS purified polyclonally expanded nTreg, most similar to UCSF protocol 
B: MACS-enriched polyclonally expanded nTregs  
C: MACS-enriched non-expanded nTregs  
D: PBMC expanded with irradiated allogeneic PBMC with costimulation 
blockade 
 
Abbreviations: 
GvHD: graft-versus-host disease; cGvHD: chronic GvHD; aGvHD: acute GvHD 
IS: immunosuppression  
T1D: type 1 diabetes CsA: cyclosporine 
Tac: Tacrolimus 
CNI: calcineurin inhibitor of either tacrolimus or cyclosporine 
MMF: mycophenolate mofetil 
ATG: anti-thymocyte globulin 
CYA: Cyclophosphamide 
NR: not reported 


 
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blood donors who were matched at least 4 of 6 HLA loci with the recipient.   Up to 6 × 106/kg Tregs, expanded ex vivo 
using anti-CD3 and anti-CD28 conjugated beads, were infused.  The infused Tregs were detectible in circulation for up to 
7 days.  During the one-year period after Treg infusion, the investigators observed no dose-limiting toxicities or increase 
in AEs when compared to historical controls. Incidences of severe acute GvHD were significantly reduced in patients who 
received Treg therapy.  For those who developed GvHD after Treg therapy, the median time to disease onset was longer.  
The investigator monitored opportunistic infections and tumor relapse rates to determine if Treg therapy led to over-IS in 
these severely immunocompromised individuals.  They reported that Treg treatment did not increase opportunistic 
infection or tumor relapse rates when compared to historical control patients who underwent the same regimen without 
Treg infusion.   
 
Another published GvHD prophylaxis trial enrolled 28 patients with high-risk hematological malignancies (Di lanni, 2011). 
Patients received anti-CD25 immunomagnetic bead-enriched donor Tregs without ex vivo expansion four days before 
receiving one haplo-mismatched hematopoietic stem cell transplant (average 9.4 x 106 stem cells/kg) and CD19-depleted 
donor PBMC as source of conventional T cells (Tconvs) from the same donors.  The majority of the patients received 2 × 
106/kg Tregs with 1 × 106/kg Tconvs.  No adjunct IS was given after transplant due to the lower risk of GvHD secondary to 
the infusion of a lower dose of donor T cells.  Patients demonstrated accelerated immune reconstitution, reduced 
cytomegalovirus (CMV) reactivation, a lower incidence of tumor relapse, and less GvHD when compared to historical 
controls.  More recently, the same group published their phase II trial using a similar protocol in 43 high-risk leukemia 
patients.  The results were consistent with their previous report that Tregs given concomitantly to stem cells and Tconv 
cells enabled infusion of higher dose of Tconv cells for the prevention of cancer relapse without increasing GvHD (Martelli 
MF D. I., 2014).   
 
Similar GvHD trials are being conducted with non-expanded Tregs (CD4+CD25hi) (P. Hoffmann and M. Edinger in 
Regensburg, Germany), Tregs grown in IL-10 and sirolimus (M. Grazia-Roncarolo, Milan), CD4+CD25+ Tregs 
immunoselected via CliniMACS (M.F. Martelli, Italy), and CD4+CD127- Tregs sorted from a CD34+CD25+ CliniMACS selected 
population (R. Negrin, Stanford).   
 Treg Therapy in Type 1 Diabetes 
1.6.2.1 Treg Therapy in Children with Type 1 Diabetes 
A clinical trial infusing Tregs in diabetic children has been reported (Marek-Trzonkowska N., 2012).  The investigators 
administered Tregs to 10 type 1 diabetic children (aged 8-16 years) within 2 months after diagnosis.  In total, 4 patients 
received 10 × 106 Tregs/kg, and the remaining 6 patients received 20 × 106 Tregs/kg. The preparation consisted of sorted 
autologous CD3+CD4+CD127lo/-CD25+ Tregs expanded under good manufacturing practice (GMP) conditions.  The children 
were not receiving IS.  For safety, the investigators monitored infusion reactions, episode of hyper- or hypo-glycemia 
episodes, and infections.  No toxicity of the Treg therapy was noted. To assess the efficacy of the treatment, the 
investigators monitored percentages of circulating Tregs in patients after infusion and metabolic parameters including 
fasting C-peptide levels, HgbA1c, and daily insulin requirements.  A significant increase in Treg percentages in the 
peripheral blood was observed on the day of infusion.  These patients were followed along with matched type 1 diabetic 
patients not treated with Tregs.  Half a year after type 1 diabetes onset (4-5 months after Tregs infusion), 8 patients treated 
with Tregs still required <0.5 IU/kg of insulin daily, with 2 patients no longer requiring insulin.  In the untreated group, 6 
out of 10 required > 0.5 IU/kg. In addition, plasma C-peptide levels were significantly higher in the treated group as 
compared with those not treated.  The investigators concluded that the administration of Tregs was safe and tolerable in 
children with recent-onset type 1 diabetes.   
 
Since the publication of this trial, the lead investigator has presented updates at international meetings that the metabolic 
improvements in Treg-treated children did not persist beyond 6 months after treatment.  Given the promising profile of 
the treatment, the team has reported treatment of additional children with repeated Treg infusions resulting in more 
sustained effects.      
 
 


 
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1.6.2.2 Pharmacokinetics and Product Metabolism in Humans 
Stable isotope labeling has been employed to track CD4+CD127lo/-CD25+ polyclonal Tregs in the peripheral vascular space 
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 and in the study “A Pilot Trial of CD4+CD127lo/-CD25+ Polyclonal Treg 
Adoptive Immunotherapy in Renal Transplant Recipients (TASKp)” conducted under IND 15711.  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. 
 
In the T1D Phase 1 study, three subjects in cohort 3 and four subjects in cohort 4 were treated with 2H-labeled Tregs. The 
2H2 (deuterium) label was incorporated into replicating DNA such that 59.8% +/- 1.03% (SD) of the deoxyribose in purine 
deoxyribonucleosides isolated from cellular DNA was labeled in the expanded Tregs (range 58.2% to 60.9%). The labeled 
cells were transferred into the patients and blood samples were harvested at various time points after administration. 
Tregs sorted from purified peripheral blood mononuclear cells (PBMCs), were analyzed for deuterium enrichment of in 
the DNA Figure 3. As seen in Figure 8 (inset), at day 1, there was a significant percentage of deuterium label in the DNA of 
circulating Tregs in each individual. The maximal percentage of the adoptively transferred PolyTregs occurred by 7-14 
days, after which point there was a decline in the percentage of labeled Tregs in the circulation. Thus, by ~ 90 days post 
infusion about 25% of the peak labeling in cells was still observed in the circulation. Interestingly, this percentage stabilized 
over the next 9 months resulting in the prolonged presence of labeled Tregs in the circulation at least one year after 
transfer. Our pharmacokinetic analysis of the survival of the Tregs indicated a two-phase decay curve, with the average 
half-life of the fast decay phase of about 19.6 days (range 4.7 to 32.5 days) and a second slow decay phase with a half-life 
of a year or more in 4 of 7 patients studied.     
 
Figure 3. Treg Tracking by Stable Isotope Labeling.  
Three subjects (002-015, 007-102, and 002-017) were treated with a single dose of 2H-labeled Tregs at a target dose of 3.2 x108 cells, 
and four subjects (002-018, 002-019, 007-103, and 002-022) were treated with a target dose of 26 x108 cells that were approximately 
60% enriched for the 2H-label. Peripheral blood was collected on days 1, 4, 7, 14, 28, 91, 182, and 364 days post infusion, and Tregs 
were sorted from the peripheral blood. Following isolation and hydrolysis of genomic DNA, the 2H isotopic enrichment of the purine 
deoxyribonucleosides in Tregs sorted from whole blood was assessed by gas chromatography/mass spectrometry. Background 
enrichment of unlabeled Tregs was ≤0.1% for each of the seven subjects. 


 
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Stable isotope labeling has also been utilized to track CD4+CD127lo/-CD25+ Polyclonal Tregs in the peripheral blood in the 
two subjects in the TASKp trial using the same procedure. Data has been obtained for the first 6 months for subject 1 and 
for the first month for subject 2. The pharmacokinetic profile of Tregs in these two patients was similar to that obtained 
from T1D patients who received the same dose of Tregs in spite of the fact that these patients were being treated with 
immunosuppressive drugs (Figure 4).  
Figure 4. Survival of infused polyclonal Tregs in transplant patients. 
Two kidney transplant recipients (TASKp1 and TASKp2) and three type 1 diabetes patients (002-015, 007-102, and 002-017) were 
treated with a single dose of 2H-labeled Tregs at a target dose of 3.2 x108 cells. Peripheral blood was collected on days 1, 4, 7, 14, 28, 
91, and 182 days post infusion, and Tregs were sorted. Following isolation and hydrolysis of genomic DNA, the 2H isotopic enrichment 
of the purine deoxyribonucleosides in Tregs sorted from whole blood was assessed by gas chromatography/mass spectrometry.  
1.6.2.3 Treg Therapy in Adults with Type 1 Diabetes  
The UCSF Diabetes Center group completed enrollment and infusion of expanded, polyclonal Tregs in recent onset adult 
type I diabetics in a phase 1 trial in October of 2013.  Tregs were manufactured at UCSF GMP facility by a team that will 
also be responsible for producing Tregs for this trial.  The UCSF T1D trial included 4 escalating dosing cohorts – 3 patients 
each at 5 x 106 and 40 x 106 Tregs and 4 patients each at 320 x 106 and 2,600 x 106 Tregs (Bluestone JA, 2015 ).   
1.6.2.4 Safety of Treg Therapy in Adults with Type 1 Diabetes 
As of May 2015, 145 AEs have been reported in 15 subjects since the beginning of the trial.  All 14 treated subjects have 
reported at least 1 AE.  One subject who underwent phlebotomy but was withdrawn before treated reported 1 AE before 
withdrawal from the trial. Ninety-two events were judged as mild in severity, 42 were judged as moderate, 9 were judged 
as severe, and 2 were judged as life-threatening.  Thirty-one events were judged to be possibly related, 33 unlikely related 
and 81 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 nine 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. 
 
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. 


 
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In the 24 hours following infusion, only 4 AEs in 4 subjects were reported. Two were mild headache, one was mild nausea 
and one was mild abdominal pain. Thus infusion reactions have been limited. 
 
Among the AEs, four were serious adverse events (SAE). 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.  
 
A review of laboratory parameters demonstrates a pattern of an approximately 0.5-1 g/dL drop in hemoglobin after 
protocol-specified phlebotomy performed day -16 to -14, which recovers by day 28 to 91 post-infusion. One subject was 
noted to have an elevated LDH, which was judged to be due to a CMV infection acquired prior to Treg infusion as discussed 
above. This same individual had detectable CMV on days 7, 14 and 21.  CMV was undetectable by day 28, and the infection 
resolved without antiviral treatment. Otherwise neither EBV nor CMV has been detected in the trial. 
1.6.2.5 Feasibility of Multi-Site Trials 
Two of the enrolled patients in cohort 3 and one of four patients in cohort 4 were at Yale University.  Whole blood collected 
from Yale was shipped to UCSF for expansion.  After 14 days of expansion, the cellular products were shipped back to Yale 
for infusion.  These experiences demonstrate that raw materials and Treg products are sufficiently stable for overnight 
shipment to the UCSF manufacturing team and that the manufacturing facility can support multi-site studies.   
 
 
 
 


 
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2. Study Objectives  
 Primary Safety Objective:  darTregs Infusion 
This study will evaluate the safety and tolerability of a single infusion of donor alloantigen reactive regulatory T cells 
(darTregs) in adult LT recipients. 
 Secondary Safety Objective: IS Withdrawal 
This study will evaluate the safety of IS 1) reduction and 2) discontinuation after one IV dose of darTregs. 
 Primary Efficacy Objective:  IS Minimization 
The study will evaluate the ability of a single IV dose of darTregs to reduce baseline, standard of care (SOC) CNI dose by 
75% along with discontinuation of either prednisone or mycophenolate mofetil (MMF), as applicable.   
 Secondary Efficacy Objective:  Tolerance 
This study will determine the number and proportion of LT recipients who become operationally tolerant with darTregs 
infusion.  We hypothesize that a single dose of darTregs in a well-defined cohort of LT recipients will induce operational 
tolerance as revealed by subsequent IS withdrawal. 
 Mechanistic Objectives 
We will assess the pharmacokinetic profile of darTregs by measuring the level of deuterium-labeled darTregs in circulation.  
Potential impact of darTregs therapy on immunological profiles will be assessed by comparing leukocyte phenotypes and 
tissue histology in protocol and for-cause biopsies, and alloantibody before and after darTregs infusion. 
 
 
 
 


 
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3. Study Design 
 Description of Study Design  
This is a multi-center, open-label clinical trial that aims to enroll at least 9 and up to 11 adult LT recipients to receive 300-
500 million darTregs 12–26 weeks after initiating IS withdrawal.  Up to 18 subjects will be enrolled to target 9-11 subjects 
eligible for IS withdrawal and darTregs infusion.  Subjects who have had a study eligibility biopsy alone and those who 
have begun IS withdrawal will receive darTregs if the subject continues to be eligible.  After darTregs infusion, subjects 
will resume IS withdrawal.  Subjects will be assessed for their ability to reduce IS by 75% while maintaining normal liver 
tests.  Subjects willing to proceed with IS withdrawal will then attempt to discontinue IS altogether. Those able to maintain 
normal liver tests and demonstrate stable allograft histology for one year in the complete absence of IS will be identified 
as operationally tolerant. 
 
Up to 18 subjects will be enrolled in the study to account for potential dropout prior to darTregs infusion.  Dropout can 
occur based on screening liver biopsy findings (estimated to occur at 20-33% frequency; 3–5 subjects) in Screen 1; or IS 
withdrawal failure during the early steps of IS withdrawal (10%; 1-2 subjects) in Screen 2.  Subject participation is 
anticipated to be 24 months.  Therefore, the total study duration is projected to be 42 months. 
 
 
Figure 5. Study Design 
 Primary Safety Endpoint 
The safety and tolerability of a single infusion of darTregs administered to adult living donor LT recipients will be assessed 
24 weeks after darTregs by describing:  
1. Occurrence of CTCAE Grade 3 or higher AEs attributable to the darTregs infusion including infusion reaction / 
cytokine release syndrome 
2.  Occurrence of study defined Grade 3 or higher infections  
3. Occurrence of any malignancy, including PTLD  


 
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 Secondary Safety Endpoints 
The trial will assess the safety of IS withdrawal in the context of darTregs therapy by describing the following secondary 
safety endpoints: 
1. Rate of composite outcome measure including refractory acute rejection, chronic rejection,  re-transplantation, 
and death 
2. Incidence of biopsy proven or clinical acute rejection and/or chronic rejection 
3. Timing of biopsy proven or clinical acute rejection and/or chronic rejection 
4. Severity of biopsy proven acute rejection and/or chronic rejection  
 Primary Efficacy Endpoint 
The efficacy of a single IV dose of darTregs will be assessed by the number and proportion of LT subjects who are able to 
reduce CNI dosing by 75% and discontinue a 2nd IS drug (if applicable) with stable liver tests for at least 12 weeks.   The 
frequency of successful CNI minimization will be compared to historical cohorts of comparable adult LT recipients 
undergoing IS withdrawal.    
 Secondary Efficacy Endpoint 
The efficacy of a single IV dose of darTregs infusion will be assessed by determining the number and percentage of subjects 
who have received darTregs and are identified as operationally tolerant, defined by maintaining stable allograft function 
(assessed by liver tests) and histology (determined by central pathologist reading in comparison to screening liver biopsy 
at study entry) in the absence of IS for one year.  The frequency of tolerance will be compared to historical cohorts of 
adult liver transplant recipients undergoing IS withdrawal.   
 Primary Mechanistic Endpoint 
The level and persistence of deuterium-labeled darTregs in the circulation will be determined by serial measurements of 
deuterium content in DNA from purified peripheral blood Tregs after darTregs infusion using gas chromatography-mass 
spectrometry (GC-MS) testing. 
 Secondary Mechanistic Endpoints 
The overall increase of darTregs in circulation will be assessed using the alloreactive T cell frequency (ATF) assay 
 Exploratory Mechanistic Endpoints 
The immunologic impact of infused darTregs will be determined by assessing the following: 
• 
Leukocyte phenotypes before and after darTregs infusion using multi-parameter flow cytometry (MFC). 
• 
Alloantibody responses before and after darTregs infusion during IS withdrawal. 
• 
Histology and multiplex immunohistochemistry of protocol and for cause biopsies    
• 
The composition of immune infiltrate in liver biopsies post Treg infusion and at the time of for-cause biopsies will 
be profiled using single-cell RNA+TCRseq 
 
 


 
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4. Selection of Participants and Clinical Sites/Laboratories 
 Rationale for Study Population 
The study aims to prospectively identify a cohort of stable, adult, living donor LT recipients who are 24 months but less 
than 84 months after transplantation to attempt IS withdrawal.  Subjects will initiate IS withdrawal, receive a single 
infusion of darTregs, and resume IS withdrawal, to reduce IS by 75% and possibly discontinue all IS while maintaining 
stable allograft function and stable allograft histology.   
 Rationale for Adult Living Donor LT Recipients 
Adult living donor LT recipients are selected for multiple reasons. First, manufacturing of the darTregs product requires 
access to donor lymphocytes, either from the peripheral blood or from the spleen.  Consent of living donors for HLA typing 
is required to ensure there is at least one mismatch at the DR locus and, pending full eligibility assessment of the recipient, 
70 mLs of blood will be drawn to make stimulated B cells (sBcs) for darTregs manufacturing.  Second, adult to adult living 
donor LT has matured over the past 15 years such that it is no longer perceived as an experimental or non-standard 
procedure.  Patients 24-84 months after living donor LT with a good profile of liver tests and liver histology do not pose 
any contraindication to enrollment in a trial of IS withdrawal supported by darTregs. 
 Rationale for Adult Living Donor LT Recipients 2-6 Years after Transplantation 
The decision to enroll subjects who are 24-84 months after LT is informed by the outcomes of previous IS withdrawal 
trials.  As the primary endpoint for this trial is IS minimization, defined as successful reduction of SOC CNI dosing by 75% 
and, if applicable, discontinuation of a 2nd IS medication, we aimed to select a population with expected low success 
rates.  Previous IS withdrawal trials in both adult and pediatric deceased and living donor LT recipients indicate that the 
success rate of tolerance increases with time after LT (Feng, 2012; Benitez, 2013). 
 
Prospective IS withdrawal trials also provide data regarding failure rates for subjects who are early after LT.  The 
prospective European multi-center trial (NCT00647283) allowed adult recipients more than 3 years after LT to enroll and 
withdraw IS.  In that trial, as shown in Table 3, subjects who were 3.0-5.7 years after LT had a 12.5% (3 of 24) prevalence 
of operational tolerance. This frequency is strikingly similar to the 13% frequency (6 of 46) of tolerance observed in AWISH 
(ITN030ST/NCT00135694) for participants who were 1-2 years post LT for non-viral, non-immune liver disease. Therefore, 
the study cohort is expected to have a 12-13% prevalence of operational tolerance.  The relatively low rate of success, as 
demonstrated by AWISH (ITN030ST/NCT00135694) and the European trial (Benitez, 2013), suggests that a 
contemporaneous control group for this safety study is not warranted as it would expose subjects to unnecessary risk. 
 
The low frequency of tolerance – complete discontinuation of IS for one year with stable liver tests and allograft histology 
- also reflects the low frequency of success with reducing CNI dosing by 75%.  As can be seen in Table 7, 31 of 46 (67.4%) 
AWISH (ITN030ST/NCT00135694) subjects did not tolerate a 75% CNI dose reduction, with 2 (4.3%) failing during steps 1 
or 2 and 29 (63%.0%) subjects failing during steps 3 or 4.    
 
Finally, it is important to note that these two trials enrolled adult deceased donor LT recipients while CTOTC-12 proposes 
to enroll adult living donor LT recipients.  There are two primary lines of evidence to suggest that the historical cohort is 
an appropriate comparator group in that the success rate of IS minimization and discontinuation is not substantially 
affected by whether the donor was living or deceased.  First, as presented in Section 1.2.2  , WISP-R, a pilot trial that 
enrolled pediatric parental living donor LT recipients, identified 12 (60%) tolerant subjects and 7 (35%) non-tolerant 
subjects; one subject has an indeterminate phenotype secondary to early study discontinuation.  Currently, the iWITH trial 
has enrolled 88 subjects, of whom 57 (64.8%) were recipients from deceased donors.  As of 10/31/2014, 10 subjects are 
still undergoing withdrawal.  Among the 78 other subjects, 31 (35.2%) have experienced AR while 46 (60.5%) have 
successfully discontinued all IS medications from less than one month to 16 months.   One subject returned to IS therapy 
after 13 months.  Although only a few subjects off of IS currently meet the definition of operational tolerance, we 
anticipate that the vast majority of these subjects will ultimately meet tolerance criteria based on the fact that previous 
adult and pediatric withdrawal trials have observed a negligible rate of developing AR after discontinuing IS. Moreover, 
the observed rate of AR thus far strongly also strongly supports our contention that donor type – living versus deceased – 
does not strongly impact success / failure rates of IS withdrawal.  Second, in clear contrast to kidney transplantation, the 


 
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degree of HLA matching has not exerted a significant influence either on the risk of rejection or on the outcomes after LT 
(Balan V, 2008) (Lan X, 2010) (Yosry A, 2012)(A Shaked, 2009).   We hypothesize that a single infusion of darTregs given 
towards the end of step 2 (25% CNI dose reduction) will enable subjects to reduce CNI dosing by 75% (and discontinue a 
2nd IS medication, if applicable) while maintaining stable liver tests. 
 Rationale for Inclusion of Subjects with History of HCC 
Eligibility criteria relative to hepatocellular carcinoma (HCC) in this trial has been strongly associated with low rates of 
recurrent HCC.  The requirement for ALL of the criteria to be satisfied results in a cohort of HCC recipients with extremely 
low HCC recurrence risk, particularly as subjects must be more than three years after LT.   
 Rationale for Enrollment of Liver Transplant Recipients with History of HCV 
Remarkable advances have recently developed in the treatment of chronic hepatitis C virus (HCV) infection before and 
after LT. What is unique about HCV is that the RNA replicon is cytoplasmic and does not establish latency or incorporate 
into the cellular genome, like HIV or other viruses (HSV, VZV). Thus, patients with HCV who achieve sustained virological 
responses (SVR = virus undetectable at least 3 months after antiviral therapy) are ‘cured’ and are not at risk of recurrence 
from the initial virus. It is well known that achievement of a SVR following HCV therapy is associated with significantly 
improved long term outcomes, such as reduced rates of fibrosis progression and liver decompensation (Morgan TR, 2010). 
This is also true in patients who are cured of HCV with antiviral therapy following LT; they have no risk of recurrence and 
have excellent long term graft function and outcomes, similar to LT recipients who never had HCV infection (Levitsky J, 
2013). In the past, HCV therapy, however, included the use of interferon which was both difficult to tolerate following LT 
and was associated with rejection and immune-mediated graft dysfunction (Levitsky J F. M., 2012). Fortunately, in the 
past 2 years, direct-acting antiviral agents (DAAs) have been approved to treat patients with chronic HCV infection, 
including LT recipients, resulting in 90%+ cure rates with minimal side effects or complications (Brown RS, 2015) (Kwo PY, 
2014). Liver transplant centers, including those in the ARTEMIS study, are actively treating recipients with HCV infection 
and have a growing population of patients achieving SVR. Given the virus does not relapse and is effectively cured with 
therapy, there is no reason non-viremic patients with excellent histology should be excluded from cellular therapy and 
tolerance trials, such as ARTEMIS, as they represent a similar immunological risk as non-HCV non-immune patients. In 
addition, immunosuppression withdrawal can be achieved in viremic patients, as HCV expands CD8+ Tcells that are less 
responsive (e.g. “exhausted), further supporting enrollment of non-viremic patients, an even lower risk group, into 
withdrawal studies (Bohne F, 2014). 
 Study Enrollment/ darTregs Infusion Eligibility Criteria 
 Study Enrollment / IS Withdrawal Inclusion Criteria 
Subjects who meet all of the following criteria are eligible for enrollment as study participants.   
1. Able to understand and provide informed consent 
2. Have received primary, solitary, living donor LT more than 24 months but less than 84 months ago 
3. Have a living donor who is willing to consent to one time phlebotomy of 100mLs to enable manufacture of 
darTregs 
4. Between 18 and 70 years of age at the time of study entry / consent 
5. Have ALT consistently <60 U/L and either alkaline phosphatase consistently <150 U/L or GGT consistently <60 U/L 
during the preceding 12 months 
6. Currently receiving a calcineurin inhibitor (CNI) with 12 hour trough levels consistently <6.0ng/mL for tacrolimus;  
<150ng/mL for cyclosporine during the preceding 6 months 
7. Currently receiving CNI monotherapy or CNI and ONE of the following:   
a. Prednisone:  maximum dose of 5 mg / day 
b. Mycophenolate mofetil (MMF):  maximum dose of 500 mg bid for Cellcept® or 360 mg bid for Myfortic® 
8. Female and male subjects with reproductive potential must agree to use effective methods of birth control for 
the duration of the study 
9. If history of HCC, recipients who have: 
a. AFP less than 100 µg/L at the time of transplant 
b. Explanted liver: 


 
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i. With tumor burden within the Milan criteria and 
ii. Without macro- or micro-vascular invasion and 
iii. Without any lesions with poorly differentiated HCC and 
iv. Without cholangiocarcinoma morphology  
c. Risk Estimation of Tumor Recurrence After Transplant (RETREAT) Score less than or equal to 3 
10. If history of HCC, at the time of enrollment, subjects must also: 
a. Be more than 36 months post-transplant AND 
b. Without evidence of recurrent HCC defined as  
i. AFP within normal limits for performing laboratory 
ii. Confirmatory chest CT and 
iii. Confirmatory CT or MRI of the abdomen and pelvis 
11. If history of HCV, recipients must be: 
a. Cured of HCV (greater than or equal to six months after the end of treatment 
b. HCV RNA negative at time of study enrollment 
 Study Enrollment / IS Withdrawal Exclusion Criteria  
Subjects who meet any of these criteria are not eligible for IS withdrawal.   
1. 
Transplant for liver disease secondary to an autoimmune etiology (e.g. autoimmune hepatitis, primary sclerosing 
cholangitis, primary biliary cirrhosis) 
2. 
Matched at both HLA-DR loci to the donor 
3. 
Organ, tissue, or cell transplant prior to or after the primary solitary living donor LT 
4. 
For subjects with HBV, detectible HBV DNA 
5. 
History of malignancy within 5 years of enrollment.  History of adequately treated in-situ cervical carcinoma 
and/or adequately treated skin cancer (basal or squamous cell) will be permitted 
6. 
Serologic evidence of human immunodeficiency 1 or 2 infection 
7. 
Epstein Barr Virus (EBV) sero-negativity (EBV naïve) if living donor is EBV sero-positive 
8. 
Cytomegalovirus (CMV) sero-negativity if living donor is CMV sero-positive 
9. 
Calculated glomerular filtration rate less than 50 mL /min/1.73m2 at time of enrollment 
10. AR within one year of enrollment 
11. Systemic illness requiring or likely to require recurrent or chronic IS drug use 
12. Any chronic condition for which anti-coagulation cannot be safely interrupted for liver biopsy 
13. Positive pregnancy test  
14. Participation in any other studies that involved investigational drugs or regimens in the preceding year 
15. Any other condition, in the investigator’s judgment, that increases the risk of darTregs infusion or prevents safe 
trial participation 
16. Unwilling or unable to adhere to study requirements and procedures 
17. Screening liver biopsy with acute rejection, early or late chronic rejection according to Banff criteria, or any 
inflammatory activity and/or fibrosis in excess of histological criteria, as determined by the reading of a central 
pathologist (Table 9) 
 
 
 


 
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Table 9. Central Pathology Histological Inflammatory and/or Fibrosis Criteria for Screening Biopsy* 
Compartment 
Findings 
Portal inflammation and 
interface activity 
This is preferably absent, but minimal to focal mild portal mononuclear 
inflammation may be present. Interface necro-inflammatory activity is absent or 
equivocal/minimal and, if present, involves a minority of portal tracts and not 
generally associated with fibrosis. 
Centrizonal/perivenular 
inflammation 
Negative for perivenular inflammation. 
Bile duct changes 
Lymphocytic bile duct damage, ductopenia, and biliary epithelial senescence 
changes are absent unless there is an alternative, non-immunological explanation 
(e.g. biliary strictures). 
Fibrosis** 
Fibrosis should be graded according to Venturi C, et al., AJT 2012.  Portal fibrosis: 
0-3 but portal-to-portal bridging (Ishak3), if present, must be not more than rare.  
Peri-sinusoidal and peri-venular fibrosis:0-1 only.  
Arteries 
Findings for obliterative or foam cell arteriopathy and isolated lymphocytic 
arteritis (isolated v lesions) are negative. 
*Patients with underlying AIH, PBC, or PSC are excluded (Demetris A. , 2012) 
Patients with severe architectural distortion for other reasons (e.g. nodular regression hyperplasia, outflow obstruction etc.) 
should also not be included. 
** Fibrosis for both non-HCV subjects and HCV subjects with SVR should be graded as follows (Venturi C, 2012): 
Portal/periportal: 0 – 3 
Peri-sinusoidal:  0 – 3. 
Perivenular:  0 – 3. 
 
 darTregs Infusion Eligibility Criteria 
 darTregs Infusion Inclusion Criteria 
Subjects will initiate IS withdrawal and, at the beginning of the 2nd step of the withdrawal algorithm (week 1-2), undergo 
a final assessment to ensure eligibility for darTregs infusion.  Subjects must have, since initiating IS withdrawal: 
1. Stable liver tests, defined as ALT and alkaline phosphatase or GGT either ≤1.5 X upper limit of normal or ≤1.5 X 
baseline.   
2. No detectible circulating EBV or CMV DNA prior to Treg Infusion, assessed at the time of PBMC collection for 
manufacture 
3. For subjects with HBV, no detectible circulating HBV DNA prior to Treg infusion, assessed at the time of PBMC 
collection for manufacture 
4. Able to understand and provide informed consent 
 
If liver tests are abnormal after time of initial darTregs Infusion screening and PBMC collection, final eligibility must be 
confirmed with the study team prior to darTregs infusion.  
 darTregs Infusion Exclusion Criteria 
1. Diagnosis of AR after initiation of IS withdrawal 
2. Any vaccination given within 28 days prior to Treg collection for darTregs production 
3. Receipt of a vaccination within 14 days prior to darTregs infusion 
4. Unacceptable darTregs product 


 
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5. Positive pregnancy test 
6. Clinical evidence of viral syndrome less than 7 days prior to darTregs infusion 
 Eligibility Criteria to Resume IS Withdrawal after darTregs Infusion 
Subjects are eligible to resume IS withdrawal after darTregs infusion if all criteria below are met: 
1. Subject received at least 100 x 106 darTregs 
2. ALT and either alkaline phosphatase or GGT remain within normal limits or ≤ 1.5 x baseline after darTregs infusion 
3. For subjects with elevated liver tests as defined above, local pathology reading of liver biopsy 6-10 days after 
darTregs infusion is without acute rejection according to Banff criteria 
4. IS withdrawal resumes no later than 14 days after darTregs infusion 
5. Site principal investigator determines it is acceptable for the study subject to resume IS withdrawal 
 Clinical Sites 
This trial will recruit adult living donor recipients 24-84 months after LT from three sites in the United States.  All 
participating centers are consistently high volume adult to adult living donor LT centers with annual volumes shown below.  
Assuming a clinical trial start date in 2016 and an enrollment period extending to the end of 2017 (12-18 months), Table 
10 shows that there were a total of 331 living donor LTs performed amongst the three sites during the period 1/1/2009 – 
12/31/2015.  This volume of adult living donor LTs should be adequate to support the estimated enrollment of 18 subjects, 
such that 12-15 subjects will be eligible to initiate IS withdrawal and 10-14 subjects will be eligible to receive darTregs and 
at least 9 (and up to 11) subjects will receive 300-500 X 106 darTregs.  All three institutions have a strong track record of 
clinical trial investigation and are therefore accustomed to recruiting and enrolling patients for clinical trials.    
 
Table 10. Number of Patients Eligible for ARTEMIS 
Center 
ADULT LIVING DONOR TRANSPLANT VOLUMES 
2016 
2015 
2014 
2013 
2012 
2011 
2010 
2009 
Mayo 
22 
20 
23 
19 
20 
22 
15 
16 
UCSF 
29 
31 
18 
13 
9 
9 
8 
5 
Northwestern 
10 
8 
19 
16 
8 
21 
17 
19 
TOTAL 
61 
54 
60 
48 
37 
52 
40 
40 
 Manufacturing Facility 
darTregs will be manufactured at the Human Islet and Cellular Transplantation Facility, a GMP Facility which is an FDA-
registered 4,500 sq. ft. laboratory at UCSF.  Quality Assurance is independent of cell manufacturing and over-sees 
operations.   
 
 


 
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5. Investigational Intervention: IS Withdrawal 
 IS Withdrawal 
Participants who fulfill all eligibility criteria will withdraw IS.  Subjects may enter on CNI monotherapy or a CNI-based 
regimen with either Prednisone or MMF as a second IS medication.  Subjects on two drugs will reduce dosing of both drugs 
simultaneously.  The algorithm for dose reduction for each agent is delineated below. 
 CNI taper algorithm 
Subjects on CNI inhibitor monotherapy with either tacrolimus or cyclosporine will change the CNI dosing according to the 
algorithm shown in Table 11.  Step 2 has a variable duration to allow for flexibility in timing of darTregs infusion which 
should be administered during the last week of the 2nd step.  Moreover, a ±7 day window is allowed for IS reduction at 
each step, including Step 2.    
 
Finally, there is the option of invoking a “logistical pause” of up to 26 weeks (see Section 5.4) during Step 2 of the CNI 
taper algorithm in the event of manufacturing and/or patient scheduling/logistical conflicts, including manufacturing 
failure of the first darTreg lot and the requirements to manufacture a second production run.  During such a logistical 
pause, tapering of Prednisone and/or MMF will also be suspended.   
 
Subjects who attain a 75% reduction of CNI dosing will also, if applicable, have discontinued Prednisone or MMF (refer to 
Table 11 and Table 12 below).  They will remain at this dose for a total of 12 weeks with ongoing monitoring of liver tests.  
If liver tests remain stable, they will have met the primary efficacy endpoint of the study.  Subjects will then be asked 
whether they wish to continue with IS withdrawal, attempting complete discontinuation.  Only subjects who receive 300 
– 500 x 106 darTregs will have the option of complete IS withdrawal.      
5.1.1.1. Pause in CNI taper 
At any step, additional monitoring at a specific dose level may be undertaken prior to continuing withdrawal.  Unless a 
logistical pause is implemented during Step 2, intentional “pauses” can last no longer than 4 weeks by which time 
withdrawal must resume or a biopsy must be performed.  Unless a study stopping rule is applied or a stipulation approved 
by the NIAID MM and/or Data Safety Monitoring Board (DSMB), failure to resume withdrawal will be considered a failure 
of IS withdrawal.  Aside from logistical pauses during Step 2, the total duration of pauses during withdrawal cannot exceed 
8 weeks during the initial IS withdrawal attempt.   
During Step 1 and Step 2 before darTregs is administered, a pause cannot exceed 4 weeks.    
 
Table 11. CNI Withdrawal Algorithm 
STEP 
DOSE (mg) 
FREQUENCY 
% REDUCTION 
DURATION 
ENTRY 
X 
BID 
n/a 
1 
2X 
QD 
0 
6 weeks 
2 
1.5X 
QD 
25 
6 – 8 weeks* 
Administer darTregs during the last 2 weeks of Step 2 
3 
X 
QD 
50 
6 weeks 
4 
X 
5 D / week 
63.5% 
6 weeks 
5 
X 
QOD 
75 
12 weeks 
Continuation of CNI Withdrawal pending 2nd consent 
6 
X 
2X/WK 
86 
6 weeks 
7 
X 
1X/WK 
93 
6 weeks 
8 
OFF CNIs 
 Prednisone taper algorithm 
If a subject enters the trial on prednisone, the maximal allowable dose is 5 mg/day.  The dose will be reduced by 1mg 
increments every 4 weeks until the subject has discontinued Prednisone.   As with CNI reduction, a decision may be made 
to undertake additional monitoring prior to continuing with dose reduction.  Such intentional “pauses” can last no longer 


 
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than 4 weeks each and the total duration of pauses during Prednisone withdrawal cannot exceed 8 weeks.  Prednisone 
tapering will also be stopped as part of a logistical pause during Step 2 during the CNI taper algorithm (see Section 5.4) for 
a maximum of 26 weeks. 
 MMF taper algorithm 
If a subject enters the trial on MMF, the maximal allowable dose is Cellcept® 500 mg bid or Myfortic® 360 mg bid.  The 
algorithm for dose reduction is shown in Table 12.  Depending on the dose at entry, MMF will be discontinued either 8 or 
16 weeks after initiation of IS withdrawal.  As with CNI reduction, a decision may be made to undertake additional 
monitoring prior to continuing with dose reduction.  Such intentional “pauses” can last no longer than 4 weeks each.  The 
total duration of pauses during MMF withdrawal cannot exceed 4 weeks if there is one step or 8 weeks if there are two 
steps.  MMF tapering will also be stopped as part of a logistical pause during Step 2 during the CNI taper algorithm (see 
Section 5.4) for a maximum of 26 weeks.   
 
Table 12. MMF Withdrawal Algorithm 
STEP 
STARTING DOSE:   
Cellcept 500 mg bid OR Myfortic 360 mg bid 
AM Dose 
PM Dose 
DURATION 
1 
250 or 180 
250 or 180 
8 weeks 
2 
250 or 180 
0 
8 weeks 
OFF MMF 
 
STEP 
STARTING DOSE: 
Cellcept 500 mg am / 250 mg pm OR 
Myfortic 360 mg am / 180 mg pm 
1 
250 or 180 
250 or 180 
8 weeks 
2 
250 or 180 
0 
8 weeks 
OFF MMF 
 
STEP 
STARTING DOSE: 
Cellcept 250 mg bid OR Myfortic 180 mg bid 
1 
250 or 180 
0 
8 weeks 
OFF MMF 
 Windows During IS Withdrawal 
Dose reductions for each drug can occur within a 7-day window at each dose level. The overall duration of the IS 
withdrawal algorithm will be determined by the duration of CNI withdrawal, varying with each subject’s need to “pause”.   
 Logistical Pause during Step 2 of CNI Withdrawal Algorithm 
During Step 2, an IS withdrawal pause of up to 26 weeks + 7 days (CNI and Prednisone / MMF, as appropriate) can be 
invoked to accommodate logistical challenges.  The logistical pause may be triggered by any number of constraints related 
to scheduling, collection, and shipping of donor or recipient manufacturing materials.  Similarly, a logistical pause might 
be utilized when faced with limitations imposed by the darTreg manufacturing facility, at the clinical site for the infusion, 
and/or with subject’s availability for an overnight hospital stay and post-infusion biopsy.  
 
During the logistical pause, liver tests must be monitored every two weeks until 12 weeks after the last IS dose change.  
Thereafter, liver tests must be monitored at least every 4 weeks until the pause is terminated.  If allograft dysfunction 
develops during the logistical pause, protocol guidelines must be followed (Section 5.5 Allograft Dysfunction). 
 
At the end of the logistical pause, the subject must continue to meet all eligibility requirements to proceed with darTreg 
infusion.   
 
 
 


 
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 Definition of Operational Tolerance 
Tolerance will be adjudicated 1 year after completing complete IS withdrawal.  Tolerance in this study is based on stable 
liver tests and central pathology liver biopsy reading. ALT and either alkaline phosphatase or GGT must be either ≤1.5X 
normal limits or ≤1.5X baseline.  Baseline ALT, alkaline phosphatase, and GGT are defined as the average of two laboratory 
tests: those obtained just prior to study entry and at the study entry screening visit.  A tolerance adjudication committee 
will convene to review cases, if warranted.  A liver biopsy will be obtained and compared to the baseline biopsy.  Pathologic 
criteria for tolerance are defined in Table 13 below.   
Table 13.  Tolerance Biopsy Criteria* 
Compartment 
Findings 
Portal inflammation and interface 
activity 
Increased portal inflammation (in comparison with a pre-weaning biopsy sample), 
especially in association with histopathological evidence of tissue damage manifest as:  
focally worsening or more prevalent lymphocytic bile duct damage, interface hepatitis, 
fibrosis, or the appearance of definite venous endotheliitis. 
Centrizonal/perivenular inflammation 
New onset perivenular inflammation (in comparison with a pre-weaning biopsy 
sample) associated with even mild perivenular necro-inflammatory activity.  Note:  
these changes might be present in the absence of typical portal changes of rejection. 
Bile duct changes 
New-onset biliary epithelial cell senescence changes or ductopenia when sampling 
problems and/or an alternative, non-immunological explanation (e.g. biliary strictures) 
can be reasonably excluded 
Fibrosis** 
Greater than 1 grade increase in fibrosis in any one compartment: (a) 
portal/periportal; (b) peri-sinusoidal; or (c) perivenular fibrosis; or new onset bridging 
fibrosis without an alternative explanation (e.g. biliary strictures) that is reasonably 
prevalent and not readily explained by a possible sampling error. 
Arteries 
Any evidence of foam cell or obliterative arteriopathy 
*Patients with underlying AIH, PBC, or PSC are excluded (Demetris A. , 2012). 
** Fibrosis should be graded as follows (Venturi C, 2012): 
Portal/periportal: 0 – 3 
Peri-sinusoidal:  0 – 3. 
Perivenular:  0 – 3. 
 Allograft Dysfunction 
Allograft dysfunction occurs when ALT is >120 U/L, alkaline phosphatase is >300 U/L, or GGT is >120 U/L.  If allograft 
dysfunction is unexplained, liver biopsy must be performed, although liver tests can be repeated once for confirmation 
prior to biopsy. At the discretion of the site principal investigator, allograft biopsy may be triggered by elevated liver tests 
below the threshold that defines allograft dysfunction but above the individual subject's baseline. 
 Acute Rejection (AR) 
Clinically indicated biopsies will be read locally according to the Banff global assessment criteria (Demetris A. J., 1997) to 
guide prompt clinical decision-making.  If the biopsy is non-diagnostic, other causes of liver dysfunction should be 
thoroughly considered.  Liver biopsies will then be reviewed centrally and data analysis will be based on central pathology 
readings.  In the case of a non-diagnostic biopsy performed for either allograft dysfunction or elevated liver tests (liver 
tests above baseline but not meeting criteria for allograft dysfunction), including biopsies read as “indeterminate” AR, a 
clinical management decision to increase or to reinitiate IS constitutes a clinical diagnosis (as opposed to a histologic 
diagnosis) of AR. 
 Treatment of AR 
Treatment for AR will be determined by the site investigator.  The therapeutic regimen can be comprised of one or more 
of the following treatment options: 


 
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• 
Steroid pulse:  a defined course of steroids, prescribed either intravenously or orally or both 
• 
Dose increase:  resumption (if completely discontinued) or dose increase of CNI, MMF, or prednisone 
o For CNI and MMF: this terminology can only be used for the drug was part of the subject’s maintenance 
IS regimen immediately prior to study entry 
o For Prednisone:  this terminology does not include the “pulse” of steroids used to treat acute rejection 
but can be utilized if a subject entered the trial on prednisone and returns to a maintenance IS regimen 
that includes prednisone 
• 
Initiation of an additional agent(s) 
o This terminology does not apply to a corticosteroid pulse (defined below) which represents a discrete 
course of treatment and does not apply if a new IS medication is initiated to substitute for another 
medication. 
• 
Substitution:  substituting one IS medication for another. Examples include: 
o Tacrolimus for cyclosporine, or vice versa 
o mTOR inhibitor for prednisone, CNI, or MMF 
o MMF for prednisone 
• 
Antibody treatment:  administration of a course of an antibody preparation, typically thymoglobulin 
 
Antibody treatment should be reserved for AR refractory to intravenous corticosteroid therapy and given ideally after a 
second biopsy demonstrating the absence of treatment response.   
 Resolution of AR 
ALT and alkaline phosphatase/GGT will be used to assess whether AR has resolved. AR is considered resolved when ALT 
and alkaline phosphatase or GGT are either ≤1.5 X ULN or ≤1.5 baseline. 
 Chronic Rejection (CR) 
A diagnosis of CR requires abnormal total and direct bilirubin and liver histology that fulfills Banff criteria. CR will be 
treated according to center SOC.  Any participant who develops CR will be considered to have failed IS withdrawal and 
will enter follow up according to Appendix 7.  Medium Frequency Schedule after Rejection. 
 Premature Discontinuation of IS Withdrawal 
Participants who fail IS withdrawal will be followed for 52 weeks from the date of rejection (Appendix 7.  Medium 
Frequency Schedule after Rejection). 
Subjects who require a pause beyond 26 weeks in Step 2 or otherwise do not receive darTregs will not continue IS 
withdrawal and will have 26 weeks of follow-up from the time of last dose change or rejection (Appendix 7).  Subjects 
who experience rejection will have 52 weeks of follow up as previously described.  Any further changes to IS will be 
determined by the site investigator. 
 
 


 
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6. Investigational Agent: darTregs Infusion 
 Formulation, Packaging, and Labeling 
Collection of Recipient T cells  
Peripheral blood leukocytes will be collected from eligible participants approximately 9-12 weeks after initiation of IS 
withdrawal.  Collection will be by phlebotomy (450 mls whole blood) or leukapheresis.  The whole blood or leukopheresed 
cells will be immediately transported to the manufacturing facility. PBMCs will be isolated by density gradient 
centrifugation using Ficoll. At least 1x109 PBMCs are expected to be collected from each subject.  If the whole blood or 
leukapheresis product does not contain sufficient numbers of Tregs, leukapheresis or phlebotomy can be repeated to 
ensure an adequate cell number for product manufacture.  
 
Blood collected at off-site location will be shipped via next-day service to UCSF and processed as described above. 
 
Donor B cell (sBcs) Production and Banking 
Peripheral blood leukocytes will be collected from living donors by phlebotomy (70 mls whole blood) and transported to 
the UCSF manufacturing facility for processing and production of sBcs.  sBcs will be generated by stimulation with 
irradiated KT64-CD40L.HLADR0401 cells and expanded for 10 days in the presence of commercially available recombinant 
human IL-4. The donor sBcs will be irradiated and cryopreserved until needed for darTregs expansion.   
 
darTregs Expansion 
Sorted CD4+CD25+CD127lo/- Treg obtained from the processed PBMC will be ex vivo expanded for a total of 16 days in 
medium supplemented with deuterated glucose in co-cultures with the donor sBcs followed by a second stimulation with 
commercially available anti-CD3 and anti-CD28 Mab-coated magnetic beads for secondary stimulation.  darTregs will be 
expanded in medium supplemented with deuterated glucose to label the product for tracking after infusion.  At the end 
of the 16-day expansion, the anti-CD3 and anti-CD28 beads will be removed by magnetic separation. 
 
darTregs packaging 
The darTregs product will be filled in sterile 150 ml PVC infusion bags.  The filled product will be maintained at 2-10oC at 
all times until administration.  A Certificate of Analysis documenting the lot release test results will accompany the final 
product during delivery to the bedside.  Infusion must occur within 30 hr of final product formulation.   
 
Prior to lot release, the product label will be affixed to the infusion bag and will include the product identifier, date of cell 
harvest, expiration date and time, and the name and unique identifier of the intended recipient.   
 
The darTregs products manufactured for subjects at an off-site location will be shipped from UCSF to the clinical site via 
next-day service using validated conditions and containers.  The remote site will test the received product according to 
UCSF established procedures.  Qualified staff at the remote site will receive the darTregs and verify the recorded shipment 
temperatures were maintained within the validated range, perform a cell count, viability determination, to ensure product 
quality characteristics have been retained during shipping.  The data collected will be reported to UCSF prior to infusion.   
 Dosage, Preparation, and Administration  
The product is a sterile cell suspension of 400 ± 100 x 106 darTregs (range 300-500 x 106) in 100 mL of 49.02% (v/v) 
PlasmaLyte-A, 49.02@ (v/v) Dextrose 5%, 0.45% NaCl, and 1.96% (v/v) 25% human serum albumin and filled in a sterile 
infusion bag.infused IV by gravity in approximately 20 to 30 minutes.  The IV line should be primed with saline prior to 
administration of the product.  Following administration of the product the bag, tubing and peripheral IV line are flushed 
with normal saline to ensure the complete dose is infused.  The IV line will be maintained after the infusion and the subject 
will be asked to remain in the clinical research unit for a minimum of 24 hours, to allow ongoing monitoring for any 
infusion-related signs and symptoms.   
 
Preparations less than 100 x 106 darTregs will not be infused.  Cells not infused will be used for research (See Section 
1.5.2.3 and 13.3).  
 


 
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Pre-medications (acetaminophen 650 mg and diphenhydramine 25-30 mg) will be administered prior to infusion (Section 
7.2.1  ).  Vitals signs will be monitored before, during, and after the infusion.  Emergency medical equipment will be 
available during the infusion in case the subject has an allergic response or an infusion reaction that can result in a cytokine 
release syndrome.   
 Drug Accountability   
Under Title 21 of the Code of Federal Regulations (21CFR §312.62), the investigator will maintain adequate records of the 
disposition of the darTregs products, including the date and quantity of the investigational product received, to whom the 
investigational product was dispensed (participant-by-participant accounting), and a detailed accounting of any 
investigational product that is accidentally or deliberately destroyed. 
 
Records for receipt, storage, use, and disposition will be maintained by the study site.  A dispensing log will be kept current 
for each participant.  This log will contain the identification of each participant and the date and quantity of biologic 
product dispensed. 
 
All records regarding the disposition of the investigational product will be available for inspection.  Unused product will 
be de-identified and destroyed.  They may be utilized for laboratory studies.   
 Intervals between darTregs infusions 
Eligible subjects will receive a single infusion of darTregs at the end of the 2nd step of IS withdrawal.   darTregs infusions 
will be staggered between subjects.  There will be a mandatory observation interval of 4 weeks after each darTregs 
infusion for the first 3 subjects to ensure that there are no acute safety signals (Section 14.8.3  ) related to either the 
infusion and/or the resumption of IS withdrawal.  Subsequently, for the remaining 6 subjects, a 4 week interval between 
darTregs infusions is preferred.  However, if the expected time interval between darTregs infusions should be less than 4 
weeks, the PI and NIAID medical monitor will determine together whether there are any safety concerns based on 
available study data to preclude administration of the darTregs infusion at a shorter interval than 4 weeks.   
 Repeated darTregs Manufacturing  
The manufacturing process can be repeated once for a particular subject if there or technical or logistical issues with the 
first preparation.  The study team will review the available manufacturing information to determine if a second 
manufacturing attempt is likely to be successful.  The laboratory components of darTregs infusion eligibility criteria must 
be repeated within 10 days of planned infusion to ensure eligibility.  In any case, infusion of darTregs must take place no 
later than 26 weeks during Step 2 of IS withdrawal.  If the second attempt at manufacturing fails, the subject will be 
followed for at least 26 weeks from the time of last dose change (Section 5.7).     
 Premature Discontinuation of darTregs Infusion 
A darTregs infusion will be stopped for an individual subject and will not be restarted if any of the following occur: 
• 
hypersensitivity reaction 
• 
CTCAE Grade 3 or higher infusion-related reaction, including cytokine release syndrome,  
• 
any other infusion-related SAE.   
A subject who receives any part of the Treg infusion will be followed for at least 52 weeks from the date of darTregs 
infusion. 
 
No more than two manufacturing attempts will be undertaken for a particular subject. 
 
 
 


 
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7. Other Medications 
 mTOR inhibitors 
Subjects cannot be on mTOR inhibitors (sirolimus or everolimus) at time of study entry.  However, an mTOR inhibitor may 
be used to treat AR or CR.   
 Prophylactic Medications 
 Pre-Medications for darTregs Infusion 
Pre-medications will be administered 30-60 minutes prior to the darTregs infusion.  Pre-medications will include 650 mg 
acetaminophen and 25-50 mg diphenhydramine intravenously or by mouth. 
 Anti-Infective Prophylaxis after Corticosteroid or Antibody Treatment for Rejection 
Participants should receive center SOC prophylaxis after oral or intravenous corticosteroids, with or without a course of 
rabbit thymoglobulin for treatment of rejection.   
 Vaccinations 
Subjects should receive seasonal influenza and other vaccinations as SOC.  However, subjects eligible for darTregs should 
not receive any vaccination within 28 days prior to blood collection or leukapheresis for darTregs manufacturing and/or 
within 14 days prior to the actual infusion (see Schedule of Events and Treg exclusion criteria).  In addition, the subjects 
should not have any vaccination for 28 days after the date of darTregs infusion.  These time parameters are in place to 
minimize the chance of having enrichment of influenza-reactive Tregs in the product and ensure that darTregs infusion 
does not negatively impact influenza immunity elicited by the vaccine. 
 Other permitted concomitant medications 
Other non-IS concomitant medications used as SOC in the management of the LT subject not specifically described in 
Section 5.6.1  are acceptable in the study. 
 
 


 
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8. Study Mandated Procedures 
 Blood Draws 
Laboratory blood draws are necessary to carefully and frequently evaluate allograft function during and after IS 
withdrawal.  In this trial, darTregs infusion will be administered during the time course of IS withdrawal.  Liver tests are 
the most reliable marker for all etiologies of allograft dysfunction, including AR and CR.  Blood draws will also be done to 
enable the planned mechanistic studies.  Please refer to Appendices 2-7. 
 Leukapheresis or Blood Draw for PBMC Collection 
PBMCs will be collected via leukapheresis or phlebotomy.  Leukapheresis will be performed for participants who are 
relatively anemic, defined as a hemoglobin ≤10.5 gm/dL.  For subjects who are not anemic, whole blood collection (450 
mls) is an option for PBMC collection.     
 
Leukapheresis will take place in an apheresis center with qualified staff.  The time requirement for the procedure is usually 
between 1 and 1.5 hours to process 6 liters of blood, which historically yields at least 1 x 109 PBMCs needed for 
manufacture.  Use of anti-coagulant and specific processing procedures at participating clinical sites will be reviewed.  
Subjects will be monitored throughout the procedure and replacement therapy (e.g. calcium) will be provided, if required. 
 
Donor and recipient PBMC collection for manufacturing can be repeated up to two times as long as blood volume limits 
for research are not exceeded for a given time period. 
 Liver biopsies  
Liver biopsies both protocol and for cause, will be done during the trial.  For each biopsy, a 16G needle will be used and a 
total of 4cm of tissue will be required.  Therefore, two passes may be required for each liver biopsy performed during the 
trial.  A separate consent that describes the procedure and its risks in detail, including the need for more than a single 
pass to obtain adequate tissue will be signed prior to each biopsy. 
 Protocol Mandated Liver Biopsies  
Subjects will have two to three protocol mandated liver biopsies during study participation.  All subjects will undergo a 
protocol biopsy to determine eligibility for study entry and IS withdrawal.  A second liver biopsy will be performed 6-10 
days after darTregs infusion.  If a subject succeeds at withdrawing completely from IS, they will undergo a third protocol 
biopsy to adjudicate operational tolerance one year after the last IS dose.   
 Clinically Indicated (For Cause) Biopsies 
Clinically indicated biopsies will be obtained when liver tests are elevated at the discretion of the site principal investigator 
or mandatorily, when allograft dysfunction thresholds as defined in Section 5.5, unless there is another explanation for 
allograft dysfunction.  Local pathology reading will direct treatment of the subject.  Both blood and tissue specimens, will 
be collected for central laboratory assessments at the time of a for cause biopsy. 
 
 


 
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9. Known and Potential Risks and Benefits to Participants 
 Risks of IS Withdrawal 
The main risks of IS withdrawal are (1) AR and/or CR (2) silent / subclinical chronic allograft injury (3) graft loss and (4) 
potential immune and non-immune complications associated with increased dosing or reinstitution of IS.  The data from 
recent pediatric as well as adult trials provides significant reassurance that these risks are minimal when IS withdrawal is 
conducted within the context of a clinical trial under highly supervised conditions (Benitez, 2013). In spite of low risk, for 
completeness of consideration, we will segment AR risks into mild, moderate and severe risk categories based on severity 
as determined by Banff criteria (Demetris A. A., 2006) (Demetris A. J., 1997) (Demetris A. D., 2000) and response to 
therapy.  CR will be assigned to the severe risk category.  We will also address the potential impact of AR on short and 
long-term graft function and on short and long term IS exposure. Although severe AR is a defined risk, we consider it to 
be unlikely given the (1) enrollment of carefully selected subjects who have undergone screening allograft biopsy (2) 
intense monitoring of allograft function during IS withdrawal and (3) preliminary results from multiple IS withdrawal trials 
in both adults and children.   
 
Mild 
IS withdrawal will trigger episodes of AR. AR episodes that occur under close monitoring inherent in IS withdrawal 
protocols have been mild to moderate in histological severity and easily reversed, almost never requiring treatment with 
potent antibodies. Allograft recovery from mild AR, diagnosed according to Banff criteria (Demetris A. A., 2006) (Demetris 
A. J., 1997) (Demetris A. D., 2000) is almost always complete, occurring without significant fibrosis, architectural distortion, 
or loss of function (Mazariegos GV, 1997) (Ramos, 1995) (Tisone, 2006) (Koshiba, 2007) (Feng, 2012).  Although treatment 
of mild AR requires increased IS dosing relative to the dose at which AR occurred, it is expected that overall IS exposure 
during and after the trial would be comparable (Benitez, 2013). 
 
Moderate 
Moderate risks associated with IS withdrawal include the risk of moderate AR, diagnosed according to Banff criteria 
(Demetris A. A., 2006) (Demetris A. J., 1997) (Demetris A. D., 2000). Although it is expected that moderate AR will be 
effectively treated by brief exposure to corticosteroids and/or dose increase, substitution, or addition of IS medications. 
Allograft recovery from moderate AR should be complete, occurring without significant fibrosis, architectural distortion, 
or loss of function. Therefore, long-term and indefinite increased IS exposure is not anticipated.  
 
A second moderate risk of IS withdrawal is related to progressive allograft changes that may occur silently without changes 
in liver tests. In the ITN029, a pilot trial of IS withdrawal for pediatric LT recipients, 11 of 12 participants have undergone 
late protocol biopsies more than 4 years after the last IS dose.  Compared to screening biopsies, there was no systematic 
change in allograft histology. Moreover, in NCT00647283, a multi-center prospective trial of IS withdrawal for adult LT 
recipients, protocol biopsy 3 years after IS withdrawal have not exhibited any evidence of progressive allograft 
histopathology after AR associated with IS withdrawal.  Therefore, while the limited nature of the currently available data 
urge caution, they do not robustly support the notion that IS withdrawal is inherently associated with substantial risk for 
histological deterioration.  
 
Severe 
Severe risks associated with IS withdrawal include steroid unresponsive or refractory AR or CR, as defined by Banff criteria 
(Demetris A. A., 2006) (Demetris A. J., 1997) (Demetris A. D., 2000).  In each of these scenarios, there is increased risk of 
graft loss leading to a requirement for re-transplant along with increased short- and long-term IS exposure. Consequently, 
each scenario is associated with increased susceptibility to infection, elevated blood pressure, renal dysfunction, and/or 
diabetes. Among adult and pediatric LT recipients who have undergone IS withdrawal, severe rejection has been 
exceedingly rare. 
 Risk of Treatment for Rejection 
All transplant recipients require IS as SOC and the well described risks of IS (Section 1.1) are no greater for study 
participants. However, rejection during the trial will require treatment with one or more of the following: corticosteroids, 


 
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increase in CNI dosing, conversion to a different IS agent, addition of a new IS medication, and/or thymoglobulin. The 
additional exposure to IS agents and possible concomitant medications (i.e. prophylaxis for opportunistic infections) as a 
result of rejection during this trial is a risk that study participants might not otherwise have.  Moreover, although the 
subjects will have had reduced IS exposure during IS withdrawal prior to failure, an episode of rejection will certainly 
necessitate increased IS exposure in the short-term but may also result in increased IS exposure cumulatively and/or in 
the long-term. 
 Risks of darTregs infusion  
Transfusion/Infusion Reaction  
Side effects reported from previous human trials involving T cell infusions include transient fever, chills, and/or nausea.  
Infusion reactions are, however, usually self-limited and resolve without any permanent sequelae.  Pre-medications will 
be administered to mitigate the risk and severity of infusion reactions. 
 
Immune Suppression 
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 in general or darTregs in particular will alter 
protective immunity. 
 
Infection 
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 darTregs is far below the resident Treg population.  
Moreover, the darTregs product is manufactured to be highly enriched for Tregs with donor-specificity.  We will, however, 
take additional precautions and exclude subjects with detectible EBV, CMV, or HBV DNA prior to infusion. 
 
Loss of Tumor Surveillance 
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 tumors in preclinical models.  The impact of Tregs on tumor 
surveillance in the organ transplant recipient is unknown.  Clearly, the long term follow up of all treated patients will 
determine whether there is evidence of an increase in the frequency of tumors.  The population under study, adult LT 
recipients who are two to six years after LT is a generally low-risk population for malignancies, with the exception of skin 
cancer.   
 
PTLD 
Treg IS has been shown to enhance tumor growth in some small animal model systems. Thus, complications such as PTLD 
are possible on a theoretical basis. Clinical experience in transplant recipients suggests that the risk of PTLD is highest in 
those who develop a primary EBV infection while immunosuppressed.  Therefore, we are excluding patients who are EBV 
naïve to minimize the risk.  We will also check EBV DNA shortly prior to planned darTregs infusion and exclude any subjects 
with detectible EBV DNA. 
 
Rejection 
It is possible that darTregs could have anti-allograft effect and thus precipitate AR. This may occur secondary to 
contamination of the darTregs product by T effector cells or secondary to instability of the regulatory phenotype such that 
darTregs change from a regulatory to an effector phenotype after in vivo adoptive transfer.  These risks may be mitigated 
by the IS that subjects will still be taking when darTregs are administered.       
 
 


 
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 Risks of Study Mandated Procedures  
 Risks of Blood Draw 
Risks of blood draw or venipuncture are typically minimal with temporary local discomfort.  More serious risks would 
include ecchymosis and, rarely, localized infection.  There is also the risk of anemia, particularly for the large volume blood 
draw (450 mls) for darTregs manufacturing.  However, subjects are screened for study eligibility and are not expected to 
have significant baseline anemia. 
 Risks of Leukapheresis 
The common risks of leukapheresis include bruising and discomfort at the site of needle placement, typically in the ante 
cubital fossae.  Calcium level in blood may fall due the citrate anticoagulant used to prevent clotting in the leukapheresis 
instrument.  Hypocalcemia can lead to perioral or digital numbness and tingling.  Calcium replacement may be used during 
the procedure and is routinely used at the conclusion of the procedure.  Platelet count may fall due to platelet loss during 
processing.  Hemorrhagic complications due to thrombocytopenia have not been reported in normal donors.  Thrombosis 
and bleeding could theoretically occur, although they are rarely if ever observed.  The study population consists of 
relatively healthy subjects with excellent liver tests and liver function.  In addition, subjects will have undergone rigorous 
assessment of general and cardiac health as part of the standard evaluation for liver transplant candidacy that is standard 
of care for both clinical sites.  As a result, subjects are expected to tolerate the procedure with a low side effect profile. 
 Risks of Liver Biopsy 
Participation in this clinical trial includes protocol biopsies and, potentially, additional for cause or clinically indicated 
biopsies. 
 
Mild risks of a liver biopsy include local pain during and for a short period of time (hours or at most days) after the 
procedure that will be experienced to some degree by every participant.  The second AE that is typically of mild to 
moderate severity is bleeding.  Although some bleeding likely occurs with every biopsy, it typically does not result in any 
symptoms; the only sign might be a small decrement in hemoglobin / hematocrit.  More serious bleeding after a liver 
biopsy is typically diagnosed by a significant drop in the hemoglobin / hematocrit that does not cause any symptoms.  The 
risk of requiring a transfusion secondary to excessive bleeding is 0.5 to 1% (Rockey, 2009).  Even rarer would be 
symptomatic hemorrhage and/or the requirement for operative or other procedural intervention to stop bleeding.   
 
Another potential complication of liver biopsy is a cholangitis, a clinical syndrome characterized by fever, abdominal pain, 
and abnormal liver tests.  The etiology of cholangitis occurring after liver biopsy is occult biliary stricture, a known long-
term complication after LT (Porrett PM, 2009).  Biliary stasis resulting from biliary stricture can lead to bacterial 
colonization of the biliary tree.  Liver biopsy may precipitate biliary leak and/or bacteremia producing the signs and 
symptoms delineated above. Treatment of cholangitis typically requires in-patient hospitalization for intravenous 
antibiotics.  While diagnostic procedures including radiographic imaging are often undertaken, intervention is typically 
undertaken electively after recovery from the acute episode unless necessitated by ongoing hemodynamic instability in 
spite of intravenous antibiotics and maximal supportive therapy.    
 
Other potentially serious risks associated with liver biopsy include pneumothorax or colonic perforation.  If either were to 
occur, hospitalization as well as procedural or operative intervention may be necessary.  Finally, there is a very small risk 
of death after liver biopsy, estimated at 0.1 to 0.01% (Rockey, 2009). 
 Potential Benefits of darTregs Infusion to Facilitate IS Minimization and/or Complete Withdrawal 
There might be no direct benefit to study participants undergoing IS withdrawal and/or receiving darTregs infusion. 
 
Autologous, ex vivo expanded darTregs are administered to a population of adult LT recipients 2-6 years after LT in an 
effort to facilitate IS minimization and induce tolerance earlier after transplantation.  It is anticipated that successful IS 
minimization and/or withdrawal may offer benefits by reducing the risk of both mortality and morbidity for adult LT 
recipients.   
 


 
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One of the primary findings of the prospective multi-center trial of IS withdrawal for adult and pediatric LT recipients is 
that the frequency of operational tolerance is highly dependent on time after transplantation (Feng, 2012) (Benitez, 2013).  
If autologous darTregs demonstrate efficacy, allowing subjects 2-6 years after LT to successfully minimize or completely 
discontinue IS, then study participants would reap the benefits of lower lifetime exposure to conventional, non-specific IS 
drugs which have been extensively described in the literature and summarized in Section 1.1.  
 
Although successful CNI dose reduction of 75% with discontinuation of a 2nd IS agent or complete IS discontinuation of IS 
would likely offer the greatest benefit, attempted but failed withdrawal can provide valuable information as to the 
subject’s required IS dosing to prevent AR.  For example, if AR occurs late in the withdrawal algorithm, when a subject is 
taking 14.3% (Step 6) or less, then the subject’s ultimate and long-term maintenance regimen, after treatment of AR and 
stabilization, may well be less than the dose at study entry.  As result, participation in this clinical trial may reduce the 
long-term cumulative IS burden for a subject. 
 
 


 
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10. Study Visits  
 Living Donor 
Prior to performing the screening liver biopsy in the liver recipient, the living donor will be consented and undergo a 
buccal swab for HLA typing.  This will enable determination of the degree of HLA DR matching between the donor and 
the recipient.  If the donor and recipient are not fully matched at DR, then the recipient can undergo screening liver 
biopsy.  Once the recipient is deemed fully eligible to initiate IS withdrawal, then 100 mls of blood will be collected from 
the living donor for the manufacturing of darTregs and mechanistic assays.  The blood must be collected within 6 weeks 
of the recipient initiating IS withdrawal.  Additional blood will be collected at the same study visit to for infectious 
disease screening, as required by CFR 1271 (Appendix 1. Living Donor Assessments).  This will complete study 
participation for the living donor.  
 LT Recipient 
 Screening, Enrollment, and Initiation of IS Withdrawal 
Participants will be identified by reviewing the LT recipients cared for by the UCSF and Mayo Clinic transplant centers 
according to the study eligibility criteria (Section 4.2).  The potential participant will sign an informed consent form 
before undergoing any screening or study procedures.  The LT recipient will be asked to perform a buccal swab for HLA 
typing as part of screening for the study.  If the subject continues to be eligible, the research study will be explained in 
lay terms to each potential research participant by the site PI or designee listed on FDA form 1572.  Surrogate consent 
will not be permitted.  Once the informed consent form has been signed, the participant will be assigned a unique 
participant number.  All screening procedures will be completed to ensure that the subject eligibility criteria.  Very 
importantly, the site PI or designee listed on the FDA form 1572 will need to determine if the living donor is willing to 
consent to study participation.   
 
Consented and otherwise eligible participants will undergo a protocol liver biopsy that will be read by the central 
pathologist.  If the central pathology reading is permissive of IS withdrawal, then IS withdrawal can be initiated.  IS 
withdrawal must begin within 8 weeks of the screening biopsy.    
 Assessments during IS Withdrawal (High Frequency Schedules) 
Participants undergoing withdrawal, both prior to and after darTregs infusion will be assessed with liver tests (ALT, alkaline 
phosphatase, GGT, and total and direct bilirubin) once every two weeks during withdrawal and for 12 weeks after 
achieving 75% CNI reduction and, finally, for 12 weeks after successful complete withdrawal, as applicable (Appendix 2.  
Study Entry and IS Withdrawal (High Frequency); Appendix 3.  Logistical Pause in Step 2 SOE, Appendix 4.  darTregs Infusion 
SOEAppendix 5.  IS Withdrawal after darTregs Infusion to Step 5/75% CNI Reduction (High Frequency) or Appendix 6.  
Complete Immunosuppression Withdrawal (High Frequency)). During high frequency follow up, telephone consultations 
will collect medical history, concomitant medications, and AEs between transplant center visits. Transplant center visits 
with phlebotomy for mechanistic studies will be required every 12 weeks.  
 
All subjects will have leukapheresis or 450 mls of blood drawn for darTregs manufacturing approximately 3-5 weeks into 
the 2nd step of CNI withdrawal.  Blood will be immediately processed to isolate circulating Tregs and placed into culture 
for darTregs manufacturing.    If a logistical pause is used, blood draw or leukapheresis will be repeated (see Appendix 3). 
 
The visit schedule will commence every two weeks from the start of IS withdrawal (Appendix 2.  Study Entry and IS 
Withdrawal (High Frequency) but will be interrupted by the darTregs infusion.  The high frequency visit schedule will 
resume 2 weeks after darTregs infusion and continue until 12 weeks after the subject starts the 5th CNI withdrawal step 
(75% CNI dose reduction).  
 
If the subject consents to continue IS withdrawal after darTregs, the subject will resume the high frequency visit schedule 
until 12 weeks after the last CNI dose, unless the subject fails IS withdrawal.    
 


 
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 darTregs Infusion and Resumption of IS Withdrawal 
Participants will receive one IV infusion of darTregs during an overnight stay.  darTregs will be infused through a peripheral 
intravenous catheter over 20-30 minutes.  All participants will be closely monitored for infusion reactions as well as other 
AEs.  Blood will be drawn for clinical and mechanistic assessments 1 day after infusion. Liver tests will be evaluated to 
determine if the subject is eligible to resume IS withdrawal according to the guidelines stipulated in Section 4.4.  IS 
withdrawal can resume as early as the next day, and no later than 14 days after darTregs infusion.  The final determination 
regarding the resumption of IS withdrawal after darTregs infusion should always be the clinical judgment of the site 
principal investigator.  
 
Eligible subjects will be instructed on how to reduce their IS prior to discharge from the hospital after the darTregs infusion.  
Subjects will be asked to return to the transplant center for a protocol biopsy 6-10 days after darTregs infusion (Appendix 
3.  Logistical Pause in Step 2 SOE, Appendix 4.  darTregs Infusion SOE).  
 
Subjects with elevated liver tests after darTregs infusion are ineligible to continue IS withdrawal until further evaluation.  
No changes in IS, including Prednisone or MMF, if applicable, should be made until liver tests are rechecked OR the 
protocol liver biopsy performed 6-10 days after darTregs infusion has been evaluated by the local pathologist.  If liver tests 
improve and meet eligibility criteria OR liver biopsy does not show rejection, IS withdrawal can resume.   
 
All participants who receive darTregs will be followed for a minimum of 52 weeks after the infusion.  The time on high or 
medium frequency assessment schedules will depend on whether IS withdrawal is continued (Step 6-8) and the duration 
of IS withdrawal (Appendix 5.  IS Withdrawal after darTregs Infusion to Step 5/75% CNI Reduction (High Frequency) and 
Appendix 6.  Complete Immunosuppression Withdrawal (High Frequency)). All subjects, irrespective of outcome, will 
continue to follow-up with their transplant center according to center SOC indefinitely.      
10.2.3.1 Adjudication of Discrepant Pathology Readings for the Post-darTreg infusion (Day 7) Biopsy 
As described previously (Section 5.5), the local pathology reading will guide clinical decision making.  However, discrepant 
reads where the central pathologist finds rejection on the day 7 biopsy after darTregs will require a review of clinical and 
pathology data by the PI, site investigator, and NIAID MM within 14 days of awareness. 
 Medium Frequency Schedule 
Medium frequency schedule entails laboratory assessments every 4 weeks, telephone consultations to collect ongoing 
changes in medical history and concomitant medications every 8 weeks.  A transplant center visit will be required at the 
end of medium frequency follow-up.  
The duration on medium frequency schedule of events will vary by subject and depend on the status of the subject 
entering the medium frequency schedule.  Reasons for leaving high frequency and entering medium frequency include 
failing IS withdrawal secondary to AR (biopsy-proven or clinical AR), elevated liver tests without diagnosis of AR, ineligibility 
to proceed with IS withdrawal after darTregs infusion, or prolonged duration of pauses during IS withdrawal.  Subjects 
who fail IS withdrawal (with or without receiving darTregs) will utilize the medium frequency follow up schedule.  These 
subjects will enter the medium frequency schedule four weeks after failing IS withdrawal and continue on the schedule 
for 52 weeks.  A transplant center visit is required at the end of medium frequency follow-up and marks the end of study 
participation.   
 
Subjects will also enter medium frequency after completing the requisite high frequency schedule required after 
successful 75% CNI dose reduction or successful discontinuation of IS altogether.  Subjects will continue with medium 
frequency visits (every 4 weeks) until 52 weeks after darTregs infusion or 52 weeks after the last dose of IS, whichever is 
longer.  The visit at the end of medium frequency follow up will be at the transplant center and marks the end of study 
participation.  For subjects who are completely off IS, a biopsy will be performed to adjudicate tolerance.     
 
 
 


 
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 Unscheduled Visits  
Additional visits for a liver biopsy will occur when there is allograft dysfunction or at the discretion of the site investigator 
(Section 5.5 and 8.3.2  ).  In addition to research specimens, local pathology results generated with unscheduled visits will 
be collected for the study.     
Local laboratory assessments recorded for the study at the time of clinically indicated biopsy should reflect reason for 
biopsy (e.g. elevated liver tests).    
 
If a participant needs a clinically indicated/for-cause biopsy within 4 weeks prior to a scheduled protocol visit, then all 
scheduled blood collections can be collected at the same time as the for-cause biopsy in lieu of the scheduled visit.  
Similarly, a telephone visit and local laboratory assessments are not required for the study if a subject has a transplant 
center visit.  
 Visit Windows 
Study visits should take place within the following time limits shown below:  
 
Table 14. SOE Visit Windows 
 
 
High Frequency SOE Visit Windows 
Study Visit 
Visit Window 
Study Entry Eligibility Screening Visit (Screen 1) 
Eligibility labs and biopsy should be within 8 weeks prior to 
initiation of IS withdrawal 
Remote Visits (Telephone and Local Liver Tests) 
± 3 days 
Transplant Center Visits  
± 14 days 
Recipient PBMC Collection for Manufacturing 
17 (UCSF) or 18 (Mayo) days before darTreg infusion 
Medium Frequency SOE Visit Windows 
Study Visit 
Visit Window 
Entry into Medium Frequency SOE 
Within 2 weeks after rejection (Appendix 7) 
Within 4 weeks after completing high frequency SOE (Appendix 8) 
Week 2 (Appendix 7) 
± 3 days 
Remote Visits (Telephone and Local Liver Tests) 
± 5 days 
End of Study 
± 14 days 
darTregs Infusion SOE Visit Windows 
Study Visit 
Visit Window 
darTregs Eligibility Screening Visit 
Between day 14-41 of the 2nd withdrawal step (10-13 weeks) 
Treg Blood Draw for darTregs manufacturing 
Between day 18-45 of the 2nd withdrawal step 
Day 0 = darTregs Infusion 
During the last 2 weeks of the 2nd withdrawal step  
Day 1 after darTregs infusion 
± 1 hour 
Protocol biopsy visit after darTregs infusion 
Between 6-10 days after darTregs infusion 
Resumption of IS withdrawal  
Between day 1-14 after darTregs infusion; corresponds to between 
day 36 – day 63 of the 2nd withdrawal step 
Day 14 after darTregs infusion 
± 2 days 
 
 
 


 
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11. Mechanistic Assays 
The goal of mechanistic studies in ARTEMIS is to determine the pharmacokinetics of darTregs and their impact on the 
alloimmune status of subjects.  We will define darTregs pharmacokinetics by measuring deuterium enrichment in 
circulation.  We will evaluate alloimmunity using leukocyte phenotyping, mixed lymphocyte reaction, serum donor-specific 
antibody levels, subclass, and C1q activity, and multi-parameter immunohistochemical analysis of graft biopsies.  Details 
on rationale, sample collection, processing, and storage, assay procedures, and data analysis for each of the assay can be 
found below.  Since there is no established biomarker for graft tolerance and the number of patients to be enrolled in this 
trial is low, most of the mechanistic assessments described below are exploratory in nature.   
 darTregs pharmacokinetics 
PK will be determined by assessment of deuterium labelled genomic DNA isolated from PBMC and biopsies of darTregs 
recipients.  Since darTregs are autologous, they will be indistinguishable from endogenous Tregs by standard surface 
markers and administered darTregs will not be identifiable using conventional means.  The T cells are expanded prior to 
infusion, providing a unique opportunity to label the cells during culture so that the presence and persistence of the 
administered darTregs can be traced.  For over a decade, the Hellerstein group, 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 6 decades.  
 
We plan to label darTregs with deuterium by including the 2H label in the culture medium during the entirety of the 
expansion phase in vitro (day 0 to day 16) prior to infusion in the patient. Based on data from in vitro labeling studies, we 
expect the cells to be ~60% enrichment for 2H.  After infusion of the deuterium-labelled investigational product, PBMCs 
will be isolated and cryopreserved at time points stipulated in the SOE.  Tregs will be sorted from the thawed PBMCs in 
batches and genomic DNA will be prepared from these samples for gas chromatography-mass spectrometry analysis of 2H 
enrichment as shown in Figure 2.  In addition, genomic DNA from subsets of peripheral blood CD4+ Tconv will be analyzed 
to determine if infused Tregs lose Treg markers after infusion.    Based on prior studies, we expect sensitivities in the range 
of 0.05 to 0.10% enrichment (which refers to the fraction of labeled molecules) (Busch, 2007) (Macallan, 1998).  Peripheral 
blood results from this analysis will be presented as number of infused darTregs per ml of blood and % of darTregs among 
all Tregs in circulation.   
 Treg TruCount Analysis 
Samples collected for this assay will be used directly for analysis without cryopreservation.  Blood will be aliquoted into a 
TruCount tube, stained with fluorochrome-conjugated antibodies to CD4, CD45, CD25 and CD127, and analyzed on a flow 
cytometer to enumerate the numbers of Tregs.  This assay would allow us to obtain the absolute Treg counts in one 
microliter of blood and establish the counts at baseline; and later determine if counts are altered by darTregs therapy. 
 MFC Panels 
The MFC analysis is focused on defining leukocyte subsets, T cell activation/exhaustion status, and CD4+ and CD8+ Treg 
frequencies.  Extensively validated MFC panels will be used to quantify changes in leukocyte populations in blood (Table 
15) collected prior to during and after IS withdrawal.  Blood will be collected at time points specified on the SOE.  The 
blood samples will be shipped to a central laboratory for processing into PBMC and then cryopreserved.  Absolute counts 
of each cell subsets, percentages and percent changed over baseline will be calculated. Changes in the values will be 
correlated with the level of IS, stage of IS withdrawal, Treg therapy, and the final outcome of IS withdrawal. 
 
 
 
 
 
 
 


 
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Table 15. ARTEMIS MFC panels 
Panel 
Names 
Markers 
Rationale 
Leuko 
CD3, CD4, CD14, CD16, CD19, CD56, HLA-DR  
To determine the numbers and percentages of T cells, B cells, 
subsets of monocytes, subsets of NK cells and dendritic cells 
Treg 
CD3, CD4, CD8, CD25, CD127, FOXP3, HELIOS  To determine the number of Tregs 
Tact 
CD3, CD4, CD8, CD27, CD28, CD45RA, CCR7 
To determine the activation status of T cells 
Texh 
CD3, CD4, CD8, CD57, PD1, Tim3 
To determine the percentage of T cells that express exhausted or 
inhibited phenotype that is implicated in transplant tolerance 
 Donor Specific Assays 
 Frequency of donor-reactive T cells  
We will use the assay described in Figure 6to determine the frequency of donor-reactive CD4+ Tconvs, CD8+ T cells, and 
Tregs at time points indicated in the SOE. We expect to see an increase in darTregs shortly after infusion. 
 
Figure 6. Assay for measuring frequency of donor-reactive T cells 
 In vitro suppression 
We will assess suppression by Tregs isolated from patients at time points indicated in the SOE. Pre- infusion PBMCs will 
be used as responders mixed with Tregs from various time points. Tregs will be sorted from frozen PBMCs based on the 
cell surface phenotype of CD4+CD127lo/-CD25+.  The cultures will be stimulated with irradiated donor PBMC to assess 
donor-specific suppression and with anti-CD3 and anti-CD28 to assess nonspecific suppression. 
 Alloantibodies 
HLA antibodies in general and DSAs in specific have not been strongly implicated as a common risk factor for AR, CR, or 
graft loss in LT (Kaneku H. , 2010). Nevertheless, their presence at baseline may preclude successful IS withdrawal. 
Moreover, increasing breadth and/or strength of DSAs during IS withdrawal is evidence of an anti-donor immune 
response. The presence and strength of HLA antibodies/DSA at baseline and longitudinally, will also be analyzed for 
correlations with histological findings (C4d and MHC class II staining patterns and intensities on protocol and indication 
liver biopsies) (Feng, 2012).  Finally, we will also plan to characterize the IgG subclass and the C1q activity of DSAs as these 
additional parameters are increasingly correlated to the functional importance of DSA in liver and/or kidney 
transplantation (Gao ZH, 2004) (Lobashevsky A, 2010) (Kaneku H, 2012) (Sutherland SM, 2012) (Freitas MC, 2013) (Loupy 
A, 2013 ). 
 HLA Typing 
HLA typing will be performed for all donor and recipient pairs from buccal cells collected at the time of study screening.  
HLA typing data will be used to conduct alloantibody studies described above.   


 
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 Histology and Multiplex Immunohistochemistry (mIHC) 
In collaboration with Dr. A. Jake Demetris, we will perform extensive histology and mIHC analysis of protocol biopsy 
samples obtained at screening, 1 week after Treg infusion, all for-cause biopsy samples, and 12 months after last IS dose 
(if applicable).  Banff criteria will be used to score histologic findings as described in Section 4 and 5.  The design of these 
analyses will be guided by histology and mIHC data from tolerant patients in iWITH and our current understanding of LT 
tolerance.  Histological analyses will evaluate 40 histopathological features to determine tissue integrity and degree of 
inflammation.  mIHC analyses are summarized in Table 16. 
 
Table 16. ARTEMIS Immunohistochemistry Panels 
Panel 
Rationale 
C4d/CD31  
Determine extent and intensity of C4d deposits on hepatic microvasculature as a barometer of anti-
donor reactivity; test hypothesis that total C4d score ≥ 6 is associated with withdrawal failure  
CD3/γδ-1/γδ-2  
Test hypothesis that portal tract ratio of γδ-1/γδ-2 > 1.0 is associated with operational tolerance. 
CK19/CD31/HLA-DR 
Test hypothesis that inappropriate expression of HLA-DR on bile ducts is associated with failed 
withdrawal; monitor rejection targets, CK19+ biliary epithelium); CD31+ endothelium, for immune 
activation via up regulation of HLA-DR, which is not normally expressed. 
CD3/CD45RO/CD45RA 
Monitor relative ratio of naïve to memory T cells; test the hypothesis that an increase in portal-based 
CD3+/CD45RO+ (memory) T cells is associated with failed ISW 
CD4/Tbet/GATA-3/IL-
17/FoxP3 
Monitor polarization of CD4+ lymphocytes within the allograft to determine whether an increase of 
putative regulatory T cells contributes to tolerance 
IL10/TGFβ/HLADR 
Monitor expression of immunomodulatory cytokines by HLA-DR expressing cells in the liver such as 
Kupffer’s cells and B cells. 
CD56/PD-1/CD3 
Determine the relative number/ratio of CD3+, CD56+, and PD-1+ lymphocytes and whether changes in 
NKT cells in the liver is associated with operational tolerance 
CD5/CD19/CD27/IgG 
Determine ratio of naïve to memory B cells and B1:B2 intra-hepatic B cells and whether memory B cells 
or B-regulatory cells residing in the liver might contribute to allograft acceptance or rejection 
  Gene Expression Profiling 
A piece of liver tissue from both protocol driven and clinically indicated liver biopsies will be preserved in RNALater and 
banked at -80oC.  Future studies might include examining how levels of RNA expression of immunoregulatory genes such 
as IL-10, Tim3, PD1, etc., changes in the setting of IS withdrawal and donor antigen exposure.  
 Single Cell RNA+TCRseq of Infiltrates 
Phenotype and specificity of graft–infiltrating T cells may provide important clues to their long-term impact on graft 
outcomes and the effect of the novel study regimen.  While cells from biopsy offers most biologically relevant data 
obtained from the target tissue, the challenge in analyzing graft-infiltrating immune cells is their low number. With single 
cell RNAseq combined with TCRseq, rich mechanistic data can be generated with limited samples.  Figure 7 shows that 
this approach can detect CD4 and CD8 T cells, CD3-NKG7+ NK cells, B cells, monocytes, and dendritic cells in kidney 
biopsies. From patients undergoing subclinical inflammation.  In addition, close to 2000 T cells with paired TCR were 
identified (Figure 7B) and global gene expression profile of the expanded top clones can be obtained (Figure 7C).  In the 
examples shown, the top clones in the first biopsy are CD8 cytotoxic cells (clone1, highly expressed XCL2, GZMB, and GNLY) 
and effector cytokine producing cells (clone 2 and 3 highly express HSPA1B, LTB, IL-7R, and MTE), whereas the #1 top 
clone  in second biopsy are likely regulatory T cells that highly expressed IL10, CTLA-4, LAG3, PDCD1, HAVCR2 and low 
expression of all granzymes and top clones 2 and 3 are cytotoxic memory cells with higher expression of granzymes, GNLY, 
LTB, and CCL5.  Thus, single-cell RNA+TCRseq from biopsy is feasible and provides high dimension data for dissecting graft 
infiltrating immune cells.  In addition, tracking the infiltrating T cell clones using single cell TCRseq also digitizes the TCR 
so that the sequence may be used in follow-up analysis for specificities of the TCR if needed.   To do this, paired TCRab 
chains can be expressed in reporter cell lines to test donor or common pathogen specificity using assay established in Tang 
lab (Spence, 2018). 
 


 
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Figure 7. Single-cell RNA+TCRseq analyses of kidney biopsies.  
 


 
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12. Biospecimen Storage 
Biological specimens obtained under this protocol may be used in future assays to reevaluate biological responses as 
additional research tests are developed over time. Blood for gene expression, PBMCs, and serum will be collected at time 
points already scheduled for the core mechanistic studies, in order to allow specimens to be stored for use in new assays 
that have yet to be optimized or conceived, or assays performed by other CTOT or CTOTC consortia members for cross-
validation studies.  Appropriate informed consent will be obtained for both the collection and storing of samples.  The 
specimens from these evaluations may be stored beyond the funding period.  
 
 
 
 
 


 
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13. Criteria for Participant Completion and Premature Study Termination 
 Participant Completion 
After study completion, all subjects, whether or not on IS, will revert to SOC follow-up at the site investigator's transplant 
center.   
 Study Completion 
Subjects who do not initiate IS withdrawal will discontinue study participation without further study follow up.  Subjects 
who fail IS withdrawal, whether prior to or after receiving darTregs, will be followed for a minimum of 52 weeks from the 
date of rejection for safety.  Subjects who receive darTregs will be followed for a minimum of 52 weeks after darTregs 
infusion, irrespective of any other outcome.  Subjects who consent to and succeed at complete IS withdrawal will be 
followed for 52 weeks after the last IS dose and, at the end of that follow-up, will be assessed for tolerance (blood and 
biopsy).   
 Participant Withdrawal Criteria  
Participants may be prematurely terminated from the study without any further follow-up for the following reasons: 
1. The participant elects to withdraw consent from all future study activities, including follow-up 
2. The participant is “lost to follow-up” (i.e., no further follow-up is possible because attempts to reestablish contact 
with the participant have failed)  
3. The participant dies 
4. If a participant prematurely terminates from the study because of graft loss or because of non-compliance with 
follow-up and/or study procedures   
5. If the principal investigator, site investigator, and/or medical monitor (MM) believe study intervention is no longer 
in the best interest of the participant. 
If a subject prematurely terminates from the trial, the subject’s IS regimen will be determined by the site investigator 
based on the best interest of the subject.  
 Participant Replacement 
Any subject who receives less than 100 x 106 darTregs will be replaced.   
 
 


 
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14. Safety Monitoring and Reporting 
 Overview 
This section defines the types of safety data that will be collected under this protocol and outlines procedures for 
appropriately collecting, grading, recording, and reporting those data. AEs that are classified as serious according to the 
definition of health authorities as well as other events of interest must be reported promptly (Section 14.5) to the sponsor 
DAIT/NIAID.  Appropriate notifications will also be made to site principal investigators, Institutional Review Boards (IRBs) 
and health authorities.  
 
Information in this section complies with ICH Guideline E2A: Clinical Safety Data Management: Definitions and Standards 
for Expedited Reporting, ICH Guideline E-6: Guideline for Good Clinical Practice, 21CFR Parts 312 and 320, and applies the 
standards set forth in the National Cancer Institute (NCI) CTCAE, Version 4.0: http://ctep.cancer.gov/reporting/ctc.html. 
 Definitions 
 Adverse Events (AEs) 
Any untoward or unfavorable medical occurrence 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 
International Conference on Harmonization E-6 Guidelines for Good Clinical Practice) (from OHRP "Guidance on Reviewing 
and 
Reporting 
Unanticipated 
Problems 
Involving 
Risks 
to 
Subjects 
or 
Others 
and 
AEs 
(1/15/07)" 
http://www.hhs.gov/ohrp/policy/advevntguid.html#Q2 )  
14.2.1.1. Suspected Adverse Reaction  
A suspected adverse reaction is any AE for which there is a reasonable possibility that the investigational agent (Section 
6) caused the AE. For the purposes of IND safety reporting, ‘reasonable possibility’ means there is evidence to suggest a 
causal relationship between the drug (i.e. darTregs) and the AE. A suspected adverse reaction implies a lesser degree of 
certainty about causality than adverse reaction, which means any AE caused by a drug (21 CFR 312.32(a)). 
 
Suspected adverse reactions associated with IS withdrawal, blood draw, or liver biopsy are collected and reported to the 
sponsor.  The sponsor will relay any suspected adverse reactions to the DSMB, as appropriate. 
 Unexpected AEs  
For events assessed in association with the investigational agent (darTregs), an AE or suspected adverse reaction is 
considered “unexpected” if it is not listed in the investigator’s brochure or is not listed at the specificity, severity or rate 
of occurrence that has been observed; or is not consistent with the risk information described in the general 
investigational plan or elsewhere in the study protocol.  
 
“Unexpected” also refers to AEs or suspected adverse reactions that are mentioned in the investigator’s brochure as 
occurring with a class of drugs or as anticipated from the pharmacological properties of the drug, but are not specifically 
mentioned as occurring with the particular drug under investigation (darTregs) (21 CFR 312.32(a). 
 
For events assessed in association with IS withdrawal or liver biopsy, an AE or suspected adverse reaction is considered 
“unexpected” if it is not listed in the protocol or is not listed at the specificity, severity or rate of occurrence that has been 
observed. 
 Serious Adverse Events 
An AE or suspected adverse reaction is considered “serious” if, in the view of either the investigator or Sponsor, it results 
in any of the following outcomes (21 CFR 312.32(a)): 
1. Death. 
2. A life-threatening event: An AE or suspected adverse reaction 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 suspected adverse reaction that, had it occurred in a more severe form, might have caused death.  


 
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3. Inpatient hospitalization or prolongation of existing hospitalization (Please see Section 13.4.3 for exceptions). 
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 events that might not result in death, be life threatening, or require hospitalization may be 
considered serious when, based upon medical judgment, they might jeopardize the subject and might require 
medical or surgical intervention to prevent one of the outcomes listed above. 
The events of interest below should be reported as a serious adverse event (within 24 hours of awareness) even if the 
event does not meet serious criteria:   
• 
Biopsy Proven or Clinical (Treated) AR  
• 
CR 
• 
Infusion Reactions CTCAE grade 2 or higher 
• 
Malignancy, PTLD 
• 
Infections study defined grade 3 or higher 
 
 Grading and Attribution of Adverse Events 
 Grading Criteria 
The study site will grade the severity of AEs experienced by the study subjects according to the criteria set forth in the 
National Cancer Institute’s CTCAE – Version 4.0 for all AEs.  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 study investigators and has been deemed appropriate for 
the subject population to be studied in this protocol for all AEs except infection and elevated liver tests.  
 
AEs will be graded on a scale from 1 to 5 according to the following standards in the NCI-CTCAE manual: 
• 
Grade 1 = mild AE. 
• 
Grade 2 = moderate AE. 
• 
Grade 3 = severe and undesirable AE. 
• 
Grade 4 = life-threatening or disabling AE. 
• 
Grade 5 = death. 
 
For any AE of Infection, the following grading system will be used for study participants: 
• 
Grade 1 =  asymptomatic; clinical or diagnostic observation only; intervention with oral antibiotic, antifungal, 
or antiviral agent only; no invasive intervention required 
• 
Grade 2 =  symptomatic; intervention with intravenous antibiotic, antifungal, or antiviral agent; invasive 
intervention may be required  
• 
Grade 3 =  any infection associated with hemodynamic compromise requiring pressors; any infection 
necessitating ICU level of care; any infection necessitating operative intervention; any infection involving the 
central nervous system; any infection with a positive fungal blood culture; any proven or probable aspergillus 
infection; any tissue invasive fungal infection; any pneumocystis jiroveci infection 
• 
Grade 4 =  life-threatening infection 
• 
Grade 5 =  death resulting from infection 
 
Events, grade 2 or higher will be recorded on the appropriate AE case report form for this study. 
 
For grading an abnormal value or result of a clinical or laboratory evaluation (including, but not limited to, a radiograph, 
an ultrasound, an electrocardiogram etc.), a treatment-emergent AE is defined as an increase in grade from baseline or 
from the last post-baseline value that doesn’t meet grading criteria.  If a specific event or result from a given clinical or 
laboratory evaluation is not included in the NCI-CTCAE manual, then an abnormal result would be considered an AE if 
changes in therapy or monitoring are implemented as a result of the event/result. 


 
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 Attribution Definitions 
The relationship, or attribution, of an AE to the study therapy regimen or study procedure(s) will initially be determined 
by the site investigator and recorded on the appropriate AE/SAE form.  Final determination of attribution for safety 
reporting will be determined by DAIT/NIAID.  The relationship of an AE to study therapy regimen or procedures will be 
determined using the descriptors and definitions provided in (Table 17).  For additional information and a printable version 
of the NCI-CTCAE manual, consult the NCI-CTCAE web site: http://ctep.cancer.gov/reporting/ctc.html. 
 
Table 17. Attribution of Adverse Events 
Code 
Descriptor 
Relationship (to primary investigational product and/or other concurrent mandated study therapy or 
study procedure) 
Unrelated Category 
1 
Unrelated 
The AE is clearly not related. 
Related Categories 
2 
Possible 
The AE has a reasonable possibility to be related; there is evidence to suggest a causal relationship. 
3 
Related 
The AE is clearly related. 
 
Attribution assessment for the following study interventions and procedures will be made when a SAE is reported:   
o IS withdrawal 
o darTregs infusion 
o Blood draw (Donor or Recipient) 
o Leukapheresis 
o Liver biopsy 
 Collection and Recording of Adverse Events 
 Collection Period 
AEs will be collected from the time of study mandated liver biopsy procedure until a subject completes study participation 
or until 30 days after he/she prematurely withdraws.   
 Collecting Adverse Events 
AEs (including SAEs) may be discovered through any of these methods: 
• 
Observing the subject 
• 
Interviewing the subject (e.g., using a checklist, structured questioning, diary, etc.) 
• 
Receiving an unsolicited complaint from the subject 
In addition, an abnormal value or result from a clinical or laboratory evaluation can also indicate an AE, as defined in 
Section 14.2.1  . 
 Exceptions to Collection 
Elective hospitalizations, hospitalization solely for a diagnostic procedure, or hospital admissions to conduct protocol 
mandated procedures are not to be collected as an AE unless hospitalization is prolonged due to complications. 
 
Only AEs associated with protocol mandated liver biopsy will be collected from the time of the study eligibility biopsy to 
the initiation of initial IS withdrawal.   
 
A pause in IS withdrawal alone is not reportable as an AE. 
 Recording Adverse Events 
Throughout the study, the investigator will record AEs and SAEs as described previously on the appropriate AE/SAE form 
regardless of the relationship to study therapy regimen or study procedure.   
 


 
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Once recorded, an AE/SAE will be followed until it resolves with or without sequelae.  AE/SAE will be followed until the 
end of study participation, or until 30 days after the subject prematurely withdraws (without withdrawing consent) /or is 
withdrawn from the study, whichever occurs first. 
 Reporting of Serious Adverse Events and Adverse Events 
 Reporting of SAEs to Sponsor  
This section describes the responsibilities of the site investigator to report serious AEs to the sponsor via the SACCC eCRF.  
Timely reporting of AEs is required by 21 CFR and ICH E6 guidelines.  
 
Site investigators will report all SAEs, regardless of relationship or expectedness to blood draw or liver biopsy with research 
specimen collection, IS withdrawal, or Treg infusion within 24 hours of discovering the event. 
 
For SAEs, all requested information on the AE/SAE form should be provided.  However, unavailable details of the event 
will not delay submission of the known information.  As additional details become available, the AE/SAE should be updated 
and submitted. 
 Reporting to Health Authority 
SAEs (Section 14.2.3  14.4.2  ) submitted by the site investigator are assessed by the DAIT/NIAID medical monitor.  
DAIT/NIAID will report the SAE to the appropriate health authorities as follows: 
14.5.2.1. Annual Reporting 
DAIT/NIAID will include in the annual study report to health authorities all AEs classified as: 
• 
Serious, expected, suspected adverse reactions  
• 
Serious and not a suspected adverse reaction  
• 
Serious, unexpected suspected adverse reactions occurring prior to Treg infusion. 
Note that all AEs (not just those requiring 24-hour reporting) will be reported in the Annual IND Report. 
14.5.2.2. Expedited Safety Reporting  
DAIT/NIAID shall notify the FDA and all participating site investigators of expedited Safety Reports within 15 calendar days; 
unexpected fatal or immediately life-threatening suspected adverse reaction(s) shall be reported as soon as possible or 
within 7 calendar days. 
 
Expedited reporting, with 2 possible categories, applies if the AE is classified as one of the following: 
 
Category 1:  Serious and unexpected suspected adverse reaction [SUSAR] (Section 14.2.1.1, 14.2.3  and 21 CFR 
312.32(c)(1)i).  The sponsor shall report any suspected adverse reaction that is both serious, unexpected and occurs after 
Treg infusion.  The sponsor shall report an AE as a suspected adverse reaction only if there is evidence to suggest a causal 
relationship between the study drug and the AE, such as: 
1. A single occurrence of an event that is uncommon and known to be strongly associated with drug exposure (e.g., 
angioedema, hepatic injury, or Stevens-Johnson Syndrome); 
2. One or more occurrences of an event that is not commonly associated with drug exposure, but is otherwise 
uncommon in the population exposed to the drug (e.g., tendon rupture); 
3. An aggregate analysis of specific events observed in a clinical trial (such as known consequences of the underlying 
disease or condition under investigation or other events that commonly occur in the study population 
independent of drug therapy) that indicates those events occur more frequently in the drug treatment group than 
in a concurrent or historical control group. 
 
Category 2: Any findings from studies that suggests a significant human risk  
The sponsor shall report any findings from other epidemiological studies, analyses of AEs within the current study or 
pooled analysis across clinical studies or animal or in vitro testing (e.g. mutagenicity, teratogenicity, carcinogenicity) that 


 
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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 package insert or other aspects of the overall conduct of the study.   
 Reporting of AEs to IRBs 
All investigators shall report AEs, including expedited reports, in a timely fashion to their respective IRBs/IECs in 
accordance with applicable regulations and guidelines.  All IND Safety Reports to the FDA shall be distributed by 
DAIT/NIAID or designee to all participating institutions for site IRB submission. 
 Pregnancy Reporting 
The investigator shall be informed immediately of any pregnancy in a study subject or a partner of a study subject.  A 
pregnant subject shall be instructed to not stop taking IS study medication unless directed by his/her study physician.  The 
investigator shall counsel the subject and discuss the risks of continuing with the pregnancy and the possible effects on 
the fetus.  Monitoring of the pregnant subject shall continue until the conclusion of the pregnancy.    
 
The investigator shall report to the SACCC all pregnancies within 1 business day of becoming aware of the event using the 
Pregnancy form.  All pregnancies identified during the study shall be followed to conclusion and the outcome of each must 
be reported.  The Pregnancy form shall be updated and submitted to the SACCC when details about the outcome are 
available.  When possible, similar information shall be obtained for a pregnancy occurring in a partner of a study subject. 
 
Information requested about the delivery shall include: 
o Gestational age at delivery 
o Birth weight, length, and head circumference 
o Gender 
o Appearance, pulse, grimace, activity, and respiration (APGAR) score at 1 minute, 5 minutes, and 24 hours after 
birth, if available 
o Any abnormalities. 
 
Should the pregnancy result in a congenital abnormality or birth defect, an SAE shall be submitted to the SACCC using the 
SAE reporting procedures described above.  Pregnancies reported as SAE’s will be reported to the FDA as described above. 
 Reporting of Other Safety Information 
An investigator shall promptly notify the site IRB as well as the SACCC using the AE/SAE form when an “unanticipated 
problem involving risks to subjects or others” is identified, which is not otherwise reportable as an AE. 
 Review of Safety Information 
The PI, the NIAID MM, and the NIAID/DAIT Transplant DSMB will review safety data on an ongoing basis. Enrollment and 
initiation of study treatment may be suspended at any time if any of these reviews conclude there are significant safety 
concerns.  Study treatment includes first withdrawal attempt, darTregs infusion, and continuation of IS withdrawal.  
 MM Review 
The NIAID MM shall receive monthly reports from the SACCC compiling new and accumulating information on AEs and 
SAEs recorded by the study site(s) on appropriate eCRFs. 
 
In addition, the MM shall review and make decisions on the disposition of the SAE reports (including any infusion related 
events) received from the site investigator via the SACCC in a real time manner.   
 DSMB Review  
The DSMB shall review safety data at least biannually 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.   
 
The DSMB will be informed of an Expedited Safety Report in a timely manner.  
 


 
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In addition to the pre-scheduled data reviews and planned safety monitoring, the DSMB may be called upon for ad hoc 
reviews. The DSMB will  review any event that potentially impacts safety at the request of the principal investigator or 
DAIT/NIAID.  
 Study Stopping Rules 
NIAID and the SACCC will continuously monitor accumulating safety data to determine if any stopping rule criterion is 
satisfied. The criteria for pausing enrollment pending DSMB review is based on any occurrence of selected AEs delineated 
in Table 18.  Since the trial is small and the above events are of particular concern, a single occurrence of any event listed 
will require DSMB and/or MM review within 48 hours.   
 
In addition to AE’s described, failure to manufacture and supply the cellular product for 2 subjects will also require a pause 
in the trial. 
 
If a study stopping rule is met, study enrollment will be paused, darTregs infusions will be held, and darTregs 
manufacturing should not begin.  If the study is stopped due to darTregs infusion reactions, subjects who have already 
received darTregs will continue with the IS withdrawal algorithm.  If any other stopping rule is met, subjects will remain 
at their current IS dose without further dose reduction until the DSMB and/or NIAID MM authorizes the study to continue.  
Any subject who has successfully completed IS withdrawal will remain off drug unless clinical condition mandates 
otherwise.  In the event that darTregs manufacturing for an eligible subject was already initiated every attempt will be 
made to expedite review such that a prompt decision is made regarding the appropriate management for these subjects.   
 
Table 18.  Selected AEs that Constitute a Study Stopping Rule 
Any time during the study 
After darTregs Infusion 
Death or graft loss 
CTCAE Grade 3 or higher AEs attributable to the darTregs infusion 
including infusion reaction/cytokine release syndrome in 2 subjects 
CR* 
Any infections of Grade 3 or higher as defined in Section 14.3.1   
Severe AR (histological or steroid 
refractory rejection)* 
Any malignancy, including PTLD  
* Banff criteria 
 
Cessation of IS withdrawal mandated by the NIAID DSMB or MM review will not be considered a “pause”. 
 
 
 


 
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15. Statistical Considerations and Analytical Plan 
 Statistical Analyses 
Statistical analyses of the safety and clinical outcomes will be descriptive for the analysis samples defined below in section 
15.1.1., employing standard methods for the estimation of person-week incidence rates and their exact two-sided 95% 
confidence intervals. Statistical analyses of the mechanistic outcomes will be exploratory in nature. The plans for statistical 
analyses of study data will be described in more detail in a Statistical Analysis Plan (SAP). 
 Analysis Populations 
The summary descriptive analyses will be performed on the following subject populations: 
1. All subjects who give informed consent and undergo the screening liver biopsy. 
2. The Intent-to-treat I sample (ITT1) will be defined as all subjects who initiate IS withdrawal 
3. The Intent-to-treat II sample (ITT2) will be defined as all subjects who receive darTregs. 
4. The Per-Protocol sample (PP) will be defined as all subjects who receive at least 300 x 106 cells of darTregs. 
 Endpoint Assessments 
 Safety Endpoints  
Table 19 describes the safety endpoints and their corresponding parameters to be estimated in the study.  Safety 
endpoints will be listed or summarized, as appropriate, using standard descriptive statistics for continuous and categorical 
data. 
 
Table 19. Analyses of Safety Endpoints  
ENDPOINT 
ANALYSIS PARAMETER 
ANALYSIS 
POPULATION 
Primary Endpoints - darTregs Infusion 
 
Incidence of AEs (CTCAE grade ≥3) 
attributable to darTregs infusion 
(including infusion reaction/cytokine 
release) 
Number of AEs of CTCAE grade ≥ 3 
Possibly or Definitely related to darTregs 
infusion 
ITT2, PP 
Incidence of infections ≥grade 3  
Number of infectious AEs of grade ≥ 3 
(defined in Section 14.3.1) 
ITT2, PP 
Incidence of any malignancy including 
PTLD  
Number of participants with any malignancy 
including PTLD  
ITT2, PP 
Secondary Endpoints - IS withdrawal 
 
Rate of composite outcome measure 
including refractory AR, CR, re-
transplantation, and death 
Proportion of participants with at least one of 
the events with exact binomial 95% 
confidence limits 
ITT1, PP 
Incidence of biopsy proven or clinical 
AR and/or CR  
Number of biopsy proven or clinical AR or 
CR AEs 
ITT1, PP 
Timing of biopsy proven or clinical AR 
and/or CR 
Time from darTregs infusion to AR and/or 
CR 
ITT2, PP 
Severity of biopsy proven AR and/or CR 
Banff criteria 
ITT1, PP 


 
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 Efficacy Endpoints 
Table 20 describes efficacy endpoints and their corresponding parameters to be estimated in the study.  Efficacy endpoints 
will be listed or summarized, as appropriate, using standard descriptive statistics for continuous and categorical data.  
 
Table 20. Analyses of Efficacy Endpoints 
ENDPOINT 
ANALYSIS PARAMETER 
ANALYSIS 
SAMPLE 
Primary Endpoint 
 
Number and proportion of subjects who reduce CNI by 75% and 
discontinue a 2nd IS drug, if applicable, with stable liver tests 
Proportion with exact binomial 95% 
confidence limits 
ITT1, ITT2, PP 
Secondary Endpoint 
 
Number and proportion of subjects who successfully withdraw 
from all IS after darTregs infusion  
Proportion with exact binomial 95% 
confidence limits 
ITT2, PP 
Time duration that subjects tolerate complete IS discontinuation  
Time interval between the last IS dose 
and reinstitution of IS 
ITT2, PP 
Number and proportion of subjects who successfully withdraw 
from all IS after darTregs infusion and are operationally tolerant 
Proportion with exact binomial 95% 
confidence limits 
ITT2, PP 
 Measures to Minimize Bias  
 Although the study is open-label, the mechanistic analyses of recipient specimens at the central laboratories will be 
blinded with respect to the status of the recipient in the study. 
 Supportive Analyses  
Since this is a small pilot study, no sub-group analyses, sensitivity analyses or covariate adjustments are planned. 
 Analyses of Exploratory Mechanistic Outcomes 
Mechanistic assays are designed to monitor the impact of darTregs therapy on the immune system and the liver graft.  
These objectives are accomplished by performing measurement on the peripheral blood and the liver biopsy samples 
collected before and after darTregs infusion.  The results will be summarized using standard statistical methods for 
continuous variables and will be displayed graphically by subject over time.  The exploratory analyses of the mechanistic 
outcomes will be described in more detail in the SAP. 
 Descriptive Analyses  
Disposition of subjects will be summarized in the all subjects receiving Tregs (ITT2).  Standard descriptive statistics for 
continuous and categorical variables will be used to summarize the following on all subjects for the analysis periods 
defined above: 
• 
baseline and demographic characteristics of the subjects 
• 
use of concomitant medications 
• 
reasons for early termination 
• 
all reported AEs  
 Interim Analyses 
 No formal interim analyses of this study are planned.  
 Sample Size Considerations  
The proposed trial is designed as a pilot trial to assess the safety of administering autologous, darTregs to stable adult 
living donor LT recipients. Currently, as of 11/1/2014, ex vivo expanded, autologous, darTregs have not yet been 
administered to solid organ transplant recipients taking IS medications. There are however two trials open to enrollment 
that plan to administer darTregs to adult, de novo, living donor kidney transplant recipients (NCT02244801) and adult, de 
novo, deceased donor liver transplant recipients (NCT02188719).  As a pilot trial designed to assess safety, this trial design 
cannot be driven by specific sample size calculations based on previous data related to the safety of darTregs 
administration as none exists.  
 


 
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We however propose to use available data regarding the success, the failure, and the timing failure of IS withdrawal among 
stable adult liver transplant patients to demonstrate the prospect that darTregs will offer a benefit in terms of increasing 
the success rate of IS minimization, complete withdrawal, and/or operational tolerance.   
 
Previous IS withdrawal trials in adult liver transplant recipients together demonstrate that patients who are 1 – 6 years 
after LT have an approximate operationally tolerant success rate of 13%.  Moreover, in AWISH (NCT00135694), among the 
44 subjects who tolerated a 25% decrease in their CNI dosing, 66% failed in that they were unable to tolerate 75% dose 
reduction.  We propose that darTregs infusion will increase the proportion of subjects able to tolerate a 75% CNI dose 
reduction along with, if applicable, complete discontinuation of either Prednisone or MMF for 12 weeks while maintaining 
stable liver tests.  We will examine the power to assess this as the primary endpoint in the following table.  We have 
selected to administer darTregs to 9-11 subjects, who will comprise the ITT2 analysis sample.  If the actual failure rate is 
22% (2/9 subjects), then we will have 89% power at an alpha level of 0.05 to conclude that this failure rate differs from 
the baseline failure rate of 66% in the absence of darTregs using an exact one-sided binomial test.  Similarly, with eleven 
subjects, if the actual failure rate is 27% (3/11 subjects), then we will have 85% power at an alpha level of 0.05 to conclude 
that this failure rate differs from the baseline failure rate of 66% in the absence of darTregs using an exact one-sided 
binomial test. 
Table 21. Sample Size Power Calculation  
Sample 
Size 
Failure 
Rate 
Power 
Alpha 
8 
1/8 
0.988 
0.047 
2/8 
0.886 
3/8 
0.658 
4/8 
0.385 
 
10 
1/10 
0.987 
0.022 
2/10 
0.879 
3/10 
0.650 
4/10 
0.382 
 
11 
1/11 
0.998 
0.043 
2/11 
0.965 
3/11 
0.845 
4/11 
0.631 
5/11 
0.384 
Subjects who are able to maintain stable liver tests for 12 weeks after 75% CNI reduction and discontinuation of 
Prednisone or MMF will be offered the opportunity to continue CNI withdrawal aiming to stop CNIs.  Subjects will be 
considered to have successfully discontinue all IS medications if they maintain stable liver tests for 12 weeks after the last 
IS / CNI dose.  Subjects will be considered to be operationally tolerant if they maintain stable liver tests and stable allograft 
histology 52 weeks after the last IS / CNI dose.  While not the primary endpoint of the study, this is an important secondary 
endpoint and thus warrants discussion.  Tables 22Error! Reference source not found. and 23 show that if 4 or more 
subjects either succeed at complete discontinuation or become tolerant after darTregs treatment, the one-sided 95% 
Confidence Limit is >13% which is the historical rate of complete IS discontinuation and operational tolerance. 
 
 


 
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Table 22. Incidence Rates and Confidence Intervals for 9 Subjects Attempting Complete IS Withdrawal 
after Treatment with darTregs 
Number of Subjects Able to Discontinue All 
IS or Achieve Operational Tolerance 
Incidence Rate (%) 
One-Sided 95% Lower 
Confidence Limit (%) 
1 
11.11 
0.57 
2 
22.22 
4.10 
3 
33.33 
9.77 
4 
44.44 
16.88 
5 
55.56 
25.14 
6 
66.67 
34.49 
7 
77.78 
45.04 
8 
88.89 
57.09 
9 
100.00 
71.69 
 
Table 23. Incidence Rates and Confidence Intervals for 11 Subjects Attempting Complete IS Withdrawal 
after Treatment with darTregs 
Number of Subjects Able to Discontinue All 
IS or Achieve Operational Tolerance 
Incidence Rate (%) 
One-Sided 95% Lower 
Confidence Limit (%) 
1 
9.09 
0.47 
2 
18.18 
3.33 
3 
27.27 
7.88 
4 
36.36 
13.51 
5 
45.45 
19.96 
6 
54.55 
27.12 
7 
63.64 
34.98 
8 
72.73 
43.56 
9 
81.82 
52.99 
10 
90.91 
63.56 
11 
100.00 
76.16 
 
 
 
 


 
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16. Identification and Access to Source Data 
 Source Data 
Source documents and source data are considered to be the original documentation where subject information, visits 
consultations, examinations and other information are recorded.   Documentation of source data is necessary for the 
reconstruction, evaluation and validation of clinical findings, observations and other activities during a clinical trial.  
 Access to Source Data 
The site investigators and site staff will make all source data available to the DAIT/NIAID, as well as to relevant health 
authorities.  Authorized representatives as noted above are bound to maintain the strict confidentiality of medical and 
research information that may be linked to identified individuals. 
 
 


 
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17. Protocol Deviations 
 Protocol Deviation Definitions 
Protocol Deviation – The investigators and site staff will conduct the study in accordance to the protocol; no deviations 
from the protocol are permitted.  Any change, divergence, or departure from the study design or procedures constitutes 
a protocol deviation.  As a result of any deviation, corrective actions will be developed by the site and implemented 
promptly. 
 
Major Protocol Deviation (Protocol Violation) - A Protocol Violation is a deviation from the IRB approved protocol that 
may affect the subject's rights, safety, or wellbeing and/or the completeness, accuracy and reliability of the study data.  In 
addition, protocol violations include willful or knowing breaches of human subject protection regulations, or policies, any 
action that is inconsistent with the NIH Human Research Protection Program’s research, medical, and ethical principles, 
and a serious or continuing noncompliance with federal, state, local or institutional human subject protection regulations, 
policies, or procedures.  
 
Non-Major Protocol Deviation - A non-major protocol deviation is any change, divergence, or departure from the study 
design or procedures of a research protocol that does not have a major impact on the subject's rights, safety or well-being, 
or the completeness, accuracy and reliability of the study data. 
 Reporting and Managing Protocol Deviations 
The study site principal investigator has the responsibility to identify, document and report protocol deviations as directed 
by the study Sponsor.  However, protocol deviations may also be identified during site monitoring visits or during other 
forms of study conduct review.  
 
Deviations that impact the ability of the Sponsor and PI to assess the study outcomes will be collected for this study. When 
a deviation is identified, the site will record the deviation on the eCRF.  In addition, the site will be responsible to report 
deviations to the applicable IRB, according to local guidelines.   
 
The SACCC will compile monthly listings of deviations.  NIAID, the study PI and DSMB will review deviations on a regular 
basis.  Protocol deviations will also be included in the annual report to FDA. 
 
 
 


 
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18. Ethical Considerations and Compliance with Good Clinical Practice 
 Statement of Compliance 
This clinical study will be conducted using good clinical practice (GCP), as delineated in Guidance for Industry: E6 Good 
Clinical Practice Consolidated Guidance, and according to the criteria specified in this study protocol.  Before study 
initiation, the protocol and the informed consent documents will be reviewed and approved by each site’s IRB, NIAID, and 
FDA.  Any amendments to the protocol or to the consent materials will also be approved by the same entities before they 
are implemented. 
 Informed Consent Process 
The consent process will provide information about the study to a prospective participant and will allow adequate time 
for review and discussion prior to his/her decision.  Donor and recipient consents for buccal swab/HLA typing will be 
mailed.  Once the LT recipient is determined to be eligible for the study, the principal investigator or designee listed on 
the FDA 1572 will review the consent and answer questions with both the donor and recipient prior to further study 
procedures.  A physician listed on the 1572 must participate in the consent process.  The prospective participant will be 
told that being in the trial is voluntary and that he or she may withdraw from the study at any time, for any reason.  All 
participants will read, sign, and date a consent form before undergoing any study procedures.  Consent materials will be 
presented in participants’ primary language.  
 
The consent process will be ongoing; the consent form will be reviewed with each subject again prior to proceeding with 
complete IS withdrawal.  The consent forms will be revised when important new safety information is available, the 
protocol is amended, and/or new information becomes available that may affect participation in the study.  
 
The consent process must be appropriately documented in the subject’s records.  A copy of the signed consent forms 
should be given to each subject. 
 Privacy and Confidentiality 
A participant’s privacy and confidentiality will be respected throughout the study.  Each participant will be assigned a 
unique identification number and these numbers rather than names will be used to collect, store, and report participant 
information.  Site personnel will not transmit documents containing personal health identifiers (PHI) to the study sponsor 
or their representatives. 
 
 


 
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19. Publication Policy 
The CTOTC publication guidelines and policies will apply to this trial. 
 
 


 
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20. References 
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Bashuda H, K. M. (2005). Renal allograft rejection is prevented by adoptive transfer of anergic T cells in nonhuman 
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of parental living donor liver transplants. Journal of the American Medical Association, 307(3), 283-93. 
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Hara, M. e. (2001). IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. Journal of 
Immunology, 166(6), 3789-96. 
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Appendix 1. Living Donor Assessments 
 
 
 
Visit Number
D1
D2
Visit Window
After 
Recipient 
Screen 1*
Donor Consent
x
Demographics (Age at Donation, Gender, Ethnicity)
x
Medical and Social History (Conduct prior to scheduling PBMC Collection)1
x
Physical Examination
x
Donor HLA Typing (Buccal Swab)3
HLA Typing Class I and II
CMV IgG4
EBV IgG4
Creative Testing Solutions Profile H without ABO/Rh (HBsAg, HBc, HCV, HIV/HCV/HBV 
NAT IDS, WNV NAT IDS, HIV 1/2, HTLV 1/2, Syphilis Screen MHA-TP, CMV, T. cruzi) 
(2 x 6 ml EDTA Lavender Top and 1 x 6 ml Serum Red Top)
x
Donor HLA Typing (Buccal Swab)2
x
HLA Typing Class I and II
x3
CMV IgG4
x
EBV IgG4
x
Mayo Test ID CTBMT (Secondary ID 89562) (HBsAg, HBc, HCV, HIV/HCV/HBV NAT 
IDS, WNV NAT IDS, HIV 1/2, HTLV 1/2, Syphilis Screen MHA-TP, CMV, T. cruzi)
x
PBMC Collection for darTreg Manufacturing (70ml in Green Heparin Tubes)
x
Donor Reactive T Cell Frequency & In Vitro Suppression (30 ml in Green Heparin Tubes) 
x
4 CMV IgG and EBV IgG should be obtained only if donor tested negative at the time of donation.  EBV IgG should be obtained if not 
previously available.
Study Assessments
Screening
PBMC 
Collection
Mayo Donor Screening Tests2
UCSF and NW Donor Screening Tests2
PBMC Collection for darTregs Manufacture
Central Laboratory Assessment
2 UCSF w ill send all donor testing to CTS.  Mayo w ill have donor testing performed locally.  Both sites w ill send results to manufacturing 
facility and enter results in study database.  Please order specific tests/ item numbers for FDA approved tests.
3 HLA typing must be repeated if testing performed previously did not include DR, DP and DQ.
* Donor blood collection can be draw n after recipient is deemed eligible for w ithdraw al, preferably after Step 1. Donor blood draw  can 
be earlier but not less than 18 days prior to planned recipient PBMC collection.
1 Donor medical and social history questionnaire can be conducted remotely and repeated, as needed, on the day of PBMC Collection.
x3x
x
x


 
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Appendix 2.  Study Entry and IS Withdrawal (High Frequency) 
 
Week 16 Week 18
Visit Number
Screen 1
ISW 1
1
2
3
4
5
PBMC
6
7
8
9
CIB
Visit Window
Start ISW
17 or 18 days 
prior to 
infusion
± 14 days
Replaces closest 
study visit
x
Study/ IS Withdrawal Eligibility
x
Informed Consent
x
Telephone Consultation
x2
x
x
x
Transplant Center Visit
x2
x
x
Physical Examination/ Vital Signs
x
x
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
→
→
→
→
HBV (HBsAg, HBcAb), HCV (HCVAb) serology (retrospective data from pre-transplant w ork up)
x
Recipient HLA Typing (Buccal Swab)
x
Pregnancy Test
x
CMV, EBV IgG3
x3
HBV by PCR (for subjects with HBV)
x
x
HCV RNA (for subjects with history of HCV)
x
HIV 1 & 2 serology
x
Hemoglobin A1C
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x
x
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
x
x
x
x
CBC (with differential and platelets) 
x
x
CD4 Count
x
Tacrolimus Level
x
AFP8
x
x
Chest CT8 
x
Abdominal CT or MRI8
x
Local Pathology Results - Graft Routine Histology
x
Leukapheresis or 450 ml Blood Collection for PBMC Isolation/ Treg Manufacturing
x5
Protocol Biopsy
x7
x7
Treg TruCount (2ml Green Heparin Tube)
x
MFC Panels -Leukocyte, Treg, Tact, Texh (2 x 10ml Green Heparin Tube)
x
x
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x
x
Alloantibodies (3ml Red Top Tube)
x
x
PBMCs for Banking (4 x 10ml Green Heparin Tube)
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x6
x
Serum for Banking (7ml Red Top Tube)
x
x6
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x
x6
x
8 AFP, CT, MRI  is only required for subjects known to have HCC.  At Screen A, AFP, Chest CT and abdominal CT or MRI should be done if unavailable within 3 preceding months.  After enrollment AFP should be collected every transplant center visit.
This schedule of events should be used at the time of screening for study entry and initial IS withdrawal.  IS withdrawal must begin no later than 8 weeks after date of screening biopsy.  The duration on this SOE will vary by subject and depend 
on pauses during IS withdrawal.  However, the maximum time allowed on this SOE is 18 weeks.  At step 2 of IS withdrawal, subjects should be screened for darTregs eligibility (Appendix 4, darTreg SOE).  Subjects who do not meet eligibility 
for darTregs should switch to Appendix 6, Medium Frequency Follow Up.
Step 1 (6 weeks)
± 3 days
Local Laboratory Assessments
Study Assessments
Immunosuppression Withdrawal Algorithm
Step 2 (6-8 weeks)
IS Withdrawal 
Study/ISW 
Eligibility1
Start ISW
Week 2
Week 4
Week 6
Possible visits if 
ISW was paused
Week 8 Week5 10
± 3 days
PBMC
Week5 12Week5 14
PBMC Collection for darTregs Manufacture
x4
← Any single pause during Step 1 or 2 must be less than 4 weeks.  The maximum time allowed on this SOE is18 weeks 5 →
Clinically 
Indicated 
Biopsy
7 For Screen 1 biopsy and clinically indicated biopsies prior to darTreg, biopsy specimen should be prioritized: 1) 2.0cm formalin, 2) 1.0-1.5cm PBS for flow/deuterium tracking, and 3) 0.5-1.0cm RNALater.
2 Transplant center visit not required to start ISW.  
4 CBC with differential can be obtained once between week 10-12.  This test can be performed locally prior to transplant center visit.
Central Laboratory Assessments
3 CMV and EBV IgG testing should be done only if recipient CMV and EBV IgG was negative at the time of transplant.  EBV IgG should be obtained if not previously available. Donor EBV and CMV IgG must be also be negative for EBV and CMV negative 
6 Specimens for banking should be collected at the time of PBMC collection OR just prior to darTreg infusion, not both. 
1 Laboratory tests for Screen 1 should be dated within 8 weeks prior to start of IS withdrawal.
5 Leukapheresis or blood collection for Treg manufacturing should occur 17 (UCSF) to 18 (Mayo or NU) days prior to planned date of infusion, at the transplant center visit.  HBV by PCR should be obtained on the day of PBMC collection for darTregs 
manufacture. Specimens for this visit will likely be collected at the week 10 or week 12 visit.This is also the time of Treg eligibility screening (Appendix 4).  Subjects should continue with liver tests every 2 weeks until darTreg infusion.  If logistical pause is 
used, please complete visits 8 and 9 on this SOE and move to Appendix 3 (Logistical Pause SOE).  Both a safety pause and logistical pause can be used in Step 2.  However, each pause cannot exceed the specified weeks. 


 
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Page 84 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
 
Appendix 3.  Logistical Pause in Step 2 SOE 
 
 
 


 
Confidential 
Page 85 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
Appendix 4.  darTregs Infusion SOE 
Treg 
Eligibility1
Treg Infusion
Day 1
Resume 
ISW4
Day 7
Visit Number
Screen 2
T0
T1
ISW 2
T2
Visit Window
During ISW 
Step 2 
During last 2 
w eeks of Step 2
± 1 hour
≤ 14 days after 
Tregs
-1/+3 days
Treg Infusion Eligibility Criteria
x
Physical Examination/ Vital Signs
Review/Collect Concomitant Medications
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
400 x 106 darTreg Infusion 
x
Resume IS withdrawal (Step 3) no later than day 14 after darTreg infusion
Pregnancy Test
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x2
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x2
x
x
x
CBC (w ith differential and platelets)
x
CD4 Count
x
x
x
Tacrolimus Level
Local Pathology Results - Graft Routine Histology
x
Protocol Biopsy
x5
MFC Panels -Leukocyte, Treg, Tact, Texh (2x10ml Green Heparin Tube)
x3
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x3
Alloantibodies (3ml Red Top Tube)
x3
Treg PK: Heavy Glucose Labeling (10ml Green Tube)
x
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x3
x
x
Serum for Banking (7ml Red Top Tube)
x3
x
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x3
x
x
1 Treg Eligibility screening visit does not require a transplant center visit.  High frequency visits should continue until darTreg infusion.
4 IS w ithdraw al instructions should be given in person.  This can be done at the infusion visit if eligibility is met.  IS w ithdraw al can start as early as one day after Tregs but no later than 14 days after Treg 
infusion.  
5For Visit T2/Day 7 after darTreg or clinically indicated biopsy after darTreg, biopsy specimen should be prioritized: 1) 2.0cm PBS for flow /deuterium tracking, 2) 1.0-1.5cm formalin, and 3) 0.5-1.0cm RNALate
3 Blood for central laboratory assessments should be collected prior to darTreg infusion at Visit T0.  Specimens for banking should not be repeated at T0 if already collected at time of PBMC collection.
2 Laboratory tests for Screen 2 (Treg eligibility) should be dated w ithin 14 days prior to Treg infusion.  Fresh draw  is not required.
Screening (Screen 2) for Treg infusion should start at Step 2 of initial ISW.  Tregs must be infused during the last 2 weeks of Step 2.  ISW must resume within 14 days 
after darTreg infusion. Once ISW is resumed, Appendix 4, ISW after darTregs SOE should be followed two weeks after date of IS dose reduction (1 week after Day 7 visit).  
Study Interventions 
Study Assessments
Local Laboratory Assessments
Central Laboratory Assessments
→
x
 


 
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Page 86 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
Appendix 5.  IS Withdrawal after darTregs Infusion to Step 5/75% CNI Reduction (High Frequency) 
 
 
Weeks after Resuming IS Withdrawal (darTreg Infusion)
W2
W4
W6
W8
W10 W121 W14
W16
W18
W20
W22 W241 W26
W28
W30
W32
Visit Number
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
T14
T15
T16
T17
T18
E1
CIB
Visit Window
± 3 
days
± 14 
days
± 14 
days
Replaces 
closest study 
visit
Telephone Consultation
x
x
x
x
x
Complete Withdrawal Informed Consent
x
Transplant Center Visit
x
x
x
x
Physical Examination/ Vital Signs
x
x
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
CMV, EBV by PCR
x
x
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x
x
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
CBC (with differential and platelets) 
x
x
x
CD4 Count
x
x
x
x
x
AFP
x
x
Local Pathology Results - Graft Routine Histology
x
x
Protocol Biopsy
x2
MFC Panels -Leukocyte, Treg, Tact, Texh (2x10ml Green Heparin Tube)
x
x
x
x
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x
x
x
x
Alloantibodies (3 ml Red Top Tube)
x
x
x
x
Treg PK: Heavy Glucose Labeling (10ml Green Tube)
x
x
x
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x
x
x
x
Serum for Banking (7ml Red Top Tube)
x
x
x
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x
x
x
x
± 3 days
Central Laboratory Assessments
2 For Visit T2/Day 7 after darTreg or clinically indicated biopsy after darTreg, biopsy specimen should be prioritized: 1) 2.0cm PBS for flow/deuterium tracking, 2) 1.0-1.5cm formalin, and 3) 0.5-1.0cm RNALater.
1 A study visit should be done in person at the transplant center 12 weeks after resuming IS Withdrawal and at the completion of Step 5.  The Step 5 End Visit should take place at week 24 or later (if there were pauses 
during ISW).  If the subject consents to continue withdrawal, instructions for next dose change should be given and Appendix 5 should be followed with assessments every 2 weeks.  If the subject is maintaining at 75% IS, 
medium frequency visits (Appendix 7) should start 4 weeks after W24.
IS Withdrawal: Maximum time allowed for withdrawal to Step 5 is 32 weeks
Step 3 (6 weeks)
Local Laboratory Assessments
± 3 days
■ This schedule of events should be used after darTreg infusion, starting 2 weeks after resuming IS withdrawal.  There will be a day 7 transplant center visit (shown on the Treg SOE) before starting this SOE.  
The duration on this SOE will vary by subject and depend on pauses during IS withdrawal.  However, the maximum time allowed for withdrawal to Step 5 is 32 weeks, less if pauses were made previously.  
■ If a subject fails IS withdrawal at any time, the medium frequency schedule should be used (Appendix 7) until 52 weeks after date of darTregs infusion or date of rejection. 
■ When CNI has been reduced by 75% (step 5) for 12 weeks, subjects will either 1) continue IS withdrawal on high frequency visits using Appendix 6, or 2) switch to medium frequency (Appendix 8) until 52 
weeks after darTregs.
Study Assessments
Immunosuppression Withdrawal
Clinically 
Indicated 
Biopsy
Step 4 (6 weeks)
Step 5 (12 weeks)
Biweekly visits after darTregs
← Any single pause during must be less than 4 weeks, 8 weeks cummulative.  →
Possible visits if              
ISW was paused
± 3 days
Additional visits if ISW was 
paused.
Step 
5 
End


 
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Page 87 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
Appendix 6.  Complete Immunosuppression Withdrawal (High Frequency) 
 
W2
W4 
W6 
W8
W10
W12
Weeks after Resuming IS Withdrawal (darTreg Infusion)
W26
W28
W30
W32
W34
W36
W38
W40
W42
W44
Visit Number
T19
T20
T21
T22
T23
T24
T25
T26
T27
T28
E2
CIB
Visit Window
± 14 
days
± 14 
days
Replaces closest 
study visit
Telephone Consultation
x
x
x
x
x
x
Transplant Center Visit
x
x
x
Physical Examination/ Vital Signs
x
x
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
→
→
→
→
→
CMV, EBV by PCR
x
x
x
HCV RNA (for subjects with history of HCV)
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x
x
x
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
CBC (with differential and platelets) 
x
x
x
x
CD4 Count
x
x
x
x
AFP2
x
x
x
Chest CT2 
x
Abdominal CT or MRI2
x
Local Pathology Results - Graft Routine Histology
x
Protocol Biopsy
x3
x4
MFC Panels -Leukocyte, Treg, Tact, Texh (2x10ml Green Heparin Tube)
x
x
x
x
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x
x
x
x
Alloantibodies (3ml Red Top Tube)
x
x
x
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x
x
x
x
Serum for Banking (7ml Red Top Tube)
x
x
x
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x
x
x
x
2AFP, CT, MRI  is only required for subjects know n to have HCC.
3 Only subjects w ho are completely off IS should have protocol biopsy to assess for tolerance.  For tolerance biopsy (off IS after darTreg), biopsy specimen should be prioritized: 1) 2.0cm formalin, 2) 1.0-1.5cm PBS, and 3) 0.5-1.0cm RNALater.
1For some subjects, 52 w eeks of follow  up can be completed using this schedule, w ithout moving to medium frequency.  These subjects should have the End of Study visit after completing 12 w eeks of high frequency follow  up after last dose of IS, even 
if longer than 52 w eeks
Local Laboratory Assessments
Central Laboratory Assessments
4 For Visit T2/Day 7 after darTreg or clinically indicated biopsy after darTreg, biopsy specimen should be prioritized: 1) 2.0cm PBS for flow /deuterium tracking, 2) 1.0-1.5cm formalin, and 3) 0.5-1.0cm RNALater.
■ This schedule of events should be used if subject consents to continue IS withdrawal.  The duration on this SOE will vary by subject and depend on pauses during IS withdrawal.  However, the maximum time 
allowed for Steps 6 & 7 is 20 weeks; less if pauses were made previously.  
± 3 days
Study Assessments
Immunosuppression Withdrawal
IS Withdrawal 
Step 6 (6 weeks)
■ If a subject successfully completes withdrawal, a transplant center visit with protocol biopsy should take place 12 weeks after starting Step 7.  The subject will continue high frequency visits on this SOE for 12 
weeks after the biopsy.  If the subject has reached 52 weeks of follow up after receiving darTregs and resuming IS withdrawal, the last study visit will be after 12 weeks of follow up medium frequency schedule is 
not needed.  If the subject has not reached 52 weeks, Appendix 8, Medium Frequency should be followed until 52 weeks after darTreg.
■ If a subject fails IS withdrawal at any time, the medium frequency schedule should be used (Appendix 7) until 52 weeks after rejection. 
12 week follow up after IS 
discontinuation (Step 8)
± 3 days
Extension if ISW paused
Biweekly visits Step 6 & 7
Possible visits if             
ISW is paused
Clinically 
Indicated 
Biopsy
Step 7 (6 weeks)
 
± 3 days
←Any single pause during must be less than 4 weeks, 8 weeks cummulative.→
End of 
Study1 
C1
C2
C3
C4
C5
C6


 
Confidential 
Page 88 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
Appendix 7.  Medium Frequency Schedule after Rejection 
 
Visit Number 
R1 
M1R M2R M3R M4R M5R M6R M7R M8R M9R M10RM11RM12R
E3
CIB
Visit Window 
± 3 days
± 14 days
- 6 w eeks
Physical Examination/ Vital Signs
x
Telephone Visit
x
x
x
x
x
x
Transplant Center Visit
x
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
CMV, EBV by PCR
x
x
HCV RNA (for subjects with history of HCV)
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x
x
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
CBC (with differential and platelets) 
x
x
x
C4D Count
x
x
x
AFP2
x
x
Chest CT2 
x
Pelvic CT or MRI2
x
Local Pathology Results - Graft Routine Histology
x
Protocol Biopsy
x3
MFC Panels -Leukocyte, Treg, Tact, Texh (2x10ml Green Heparin Tube)
x
x
x
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x
x
x
Alloantibodies (3ml Red Top Tube)
x
x
x
Treg PK: Heavy Glucose Labeling (10ml Green Tube)
x
x
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x
x
x
Serum for Banking (7ml Red Top Tube)
x
x
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x
x
x
2AFP, CT, MRI  is only required for subjects known to have HCC.
± 5 days
Central Laboratory Assessments
W21
1Subjects with rejection should have a follow up visit 2 weeks after CIB, then continue with medium frequency schedule.
■ Subjects who experience rejection on medium frequency follow up (partial or complete ISW after darTregs) will restart this medium frequency SOE until 52 weeks.
3 For Visit T2/Day 7 after darTreg or clinically indicated biopsy after darTreg, biopsy specimen should be prioritized: 1) 2.0cm PBS for flow/deuterium tracking, 2) 1.0-1.5cm formalin, and 3) 0.5-1.0cm 
RNALater.
W16 W20
Study Assessments
Local Laboratory Assessments
W4
W8
■ Subjects failing IS withdrawal will enter into medium frequency follow up 2 weeks after rejection.  Note the study visit weeks restart at the time the subject starts medium frequency 
follow up (e.g. two weeks after completing final 12 weeks of high frequency follow up).  Subjects should be followed for 52 weeks total follow up after rejection.  
■ All subjects must have at least 52 weeks of follow up after receiving darTregs and resuming IS withdrawal; this will be a combination of high and medium frequency visits. 
W24
Clinically 
Indicated 
Biopsy
W32 W36 W40 W44 W48
W28
Weeks on Medium Frequency Follow Up
End of 
Study 
W12


 
Confidential 
Page 89 of 89 
darTregs for Tolerance in Liver Transplantation (CTOTC-12) 
                                                        Version 7.0/ October 15, 2018 
Appendix 8.  Medium Frequency Schedule after Partial or Complete IS Withdrawal 
 
 
Weeks Continuing at Reduced CNI (from Appendix 4)
W28 W32 W36 W40 W44 W48 W52
Weeks after Resuming IS Withdrawal (darTreg Infusion)
W52 W56 W60 W64 W68 W72 W76
Visit Number for Follow Up (Partial ISW: a, Completed ISW: b)
M1
M2
M3
M4
M5
M6
M7
E4
CIB
Visit Window 
± 14 days
- 6 w eeks
Physical Examination/ Vital Signs
Telephone Visit
x
x
x
Transplant Center Visit
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
→
→
→
→
→
→
→
→
→
Review/Collect Concomitant Medications
→
→
→
→
→
→
→
→
→
Adverse Event/Serious Adverse Event Assessment 
→
→
→
→
→
→
→
→
→
CMV, EBV by PCR
x
HCV RNA (for subjects with history of HCV)
x
Basic Chemistry  (Glucose, Na, K, Cl, CO2, Creatinine, BUN)
x
x
Liver Tests (ALT, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x
x
x
x
x
x
x
x
CBC (with differential and platelets) 
x
x
C4D Count
x
x
AFP2
x
Chest CT2 
x
Pelvic CT or MRI2
x
Local Pathology Results - Graft Routine Histology
x
Protocol Biopsy
x3
x4
MFC Panels -Leukocyte, Treg, Tact, Texh (2x10ml Green Heparin Tube)
x
x
Donor-reactive Tcell Frequency/In Vitro Suppression (10ml Green Heparin Tube)
x
x
Alloantibodies (3ml Red Top Tube)
x
x
Treg PK: Heavy Glucose Labeling (10ml Green Tube)
x
x
PBMCs for Banking (2 x 10ml Green Heparin Tube)
x
x
Serum for Banking (7ml Red Top Tube)
x
x
mRNA for Banking (2 x 2.5 Paxgene Tube)
x
x
■ Subjects who do not receive darTregs after starting IS withdrawal will have at least 26 weeks of follow up after last IS dose change.  52 weeks of follow 
up is required if rejection occurs.  
■ All subjects must have at least 52 weeks of follow up after receiving darTregs and resuming IS withdrawal; this might be accomplished without using 
the medium frequency schedule or will be a combination of high and medium frequency visits. 
■ Subjects successful with partial or complete withdrawal should use this SOE after completing 12 weeks of high frequency follow up (Appendix 5 or 6). 
The duration on this SOE will vary by subject and depend on pauses during IS withdrawal.  
Clinically 
Indicated 
Biopsy
Study Assessments
Local Laboratory Assessments
Central Laboratory Assessments
± 5 days
Weeks on Medium Frequency Follow Up
W4
W8 W12 W16 W20 W24 W28
End of 
Study1 
1The last study visit should take place when 52 weeks of follow up (after darTregs and resuming IS withdrawal) have been completed.  The timing will vary by 
subject.
3 Only subjects who are completely off IS should have protocol biopsy to assess for tolerance.  For tolerance biopsy (off IS after darTreg), biopsy specimen should 
be prioritized: 1) 2.0cm formalin, 2) 1.0-1.5cm PBS, and 3) 0.5-1.0cm RNALater.
4 For Visit T2/Day 7 after darTreg or clinically indicated biopsy after darTreg, biopsy specimen should be prioritized: 1) 2.0cm PBS for flow/deuterium tracking, 2) 
2AFP, CT, MRI  is only required for subjects known to have HCC.

SAP_001.pdf: 
 
 
DAIT/Rho STATISTICAL ANALYSIS PLAN 
 FINAL  
 
 
 
 CTOTC-12: Safety of Donor Alloantigen Reactive Tregs to Facilitate 
Minimization and/or Discontinuation of Immunosuppression in Adult 
Liver Transplant Recipients 
VERSION: 
1.0 
DATE: 
04MAR2020 
SPONSOR: 
Division of Allergy, Immunology, and Transplantation 
National Institute of Allergy and Infectious Diseases – NIH 
 
 
 
 
 
 
PREPARED BY: 
Rho 
 
 
 
 
 
 
 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  2 of 24 
 
DAIT/Rho STATISTICAL ANALYSIS PLAN 
ACKNOWLEDGMENT AND SIGNATURE SHEET 
 
Safety of Donor Alloantigen Reactive Tregs to Facilitate Minimization 
and/or Discontinuation of Immunosuppression in Adult Liver 
Transplant Recipients  
 
Approved: 
__________________________________ Date: 
_______________ 
 
 
 
 
Protocol Chair/Principal Investigator 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
 
 
 
NIAID Medical Monitor 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
 
 
 
 
Rho Scientist 
 
 
 
Approved: 
__________________________________ Date: 
_______________ 
 
 
 
 
 
Rho Statistician 
 
 
 
 
 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  3 of 24 
 
Document History 
 
Version 
 
Date 
 
Change(s) 
 
Author 
0.1 
 
Initial Draft 
 
0.2 
 
 
 
1.0 
04MAR2020 
Final Version 
Katharine Spain 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  4 of 24 
 
Table of Contents 
 
1. 
PROTOCOL SYNOPSIS ........................................................................................... 6 
2. 
INTRODUCTION ..................................................................................................... 11 
3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS .................................... 12 
4. 
ANALYSIS SAMPLES ............................................................................................ 13 
5. 
STUDY SUBJECTS ................................................................................................ 14 
5.1. 
Disposition of Subjects ................................................................................. 14 
5.2. 
Demographic and Other Baseline Characteristics ......................................... 14 
6. 
STUDY OPERATIONS ............................................................................................ 15 
6.1. 
Protocol Deviations ...................................................................................... 15 
6.2. 
Treatment Adherence................................................................................... 15 
7. 
ENDPOINT EVALUATION ...................................................................................... 16 
7.1. 
Overview of Analysis Methods ...................................................................... 16 
7.1.1. Multicenter Studies .................................................................................. 16 
7.1.2. Assessment Time Windows ..................................................................... 16 
7.2. 
Primary Efficacy Endpoint............................................................................. 17 
7.2.1. Computation of the Primary Efficacy Endpoint .......................................... 17 
7.2.2. Primary Analysis of the Primary Efficacy Endpoint .................................... 17 
7.2.3. Sensitivity Analyses of the Primary Efficacy Endpoint ............................... 17 
7.3. 
Primary Safety Endpoint ............................................................................... 17 
7.3.1. Computation of the Primary Safety Endpoint ............................................ 17 
7.3.2. Primary Analysis of the Primary Safety Endpoint ...................................... 17 
7.3.3. Additional Analyses of the Primary Safety Endpoint .................................. 17 
7.4. 
Secondary Efficacy Endpoints ...................................................................... 17 
7.4.1. Number and proportion of subjects who successfully withdrawal from all IS after 
darTregs infusion ..................................................................................... 18 
7.4.2. Duration that subjects tolerate complete IS discontinuation....................... 18 
7.4.3. Number and proportion of subjects who successfully withdrawal from all IS after 
darTregs infusion and are operationally tolerant ....................................... 18 
7.5. 
Secondary Safety Endpoints ........................................................................ 18 
7.5.1. Rate of composite outcome measure including refractory AR, CR, re- 
transplantation, and death........................................................................ 18 
7.5.2. Incidence of biopsy proven or clinical AR and/or CR ................................. 18 
7.5.3. Timing of biopsy proven or clinical AR and/or CR in ITT2 population ......... 18 
7.5.4. Severity of biopsy proven or clinical AR and/or CR in ITT2 population....... 19 
8. 
SAFETY EVALUATION .......................................................................................... 20 
8.1. 
Overview of Safety Analysis Methods ........................................................... 20 
8.2. 
Adverse Events ............................................................................................ 20 
8.3. 
Deaths and Serious Adverse Events............................................................. 21 
8.4. 
Clinical Laboratory Evaluation ...................................................................... 21 
8.5. 
Vital Signs, Physical Findings, and Other Observations Related to Safety ..... 21 
8.5.1. Vital Signs ............................................................................................... 21 
8.5.2. Physical Examinations ............................................................................. 21 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  5 of 24 
 
9. 
INTERIM ANALYSES AND DATA MONITORING ................................................... 22 
10. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL .......................... 23 
 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  6 of 24 
 
 
 


CTOTC 
Statistical Analysis Plan 
Protocol No. CTOTC-12 
<04 March 2020> 
 
 
Version: <1.0> 
Page  7 of 24 
 
1. 
PROTOCOL SYNOPSIS 
Title 
Safety of Donor Alloantigen Reactive Tregs (darTregs) to Facilitate Minimization and/or 
Discontinuation of Immunosuppression in Adult Liver Transplant Recipients (ARTEMIS) 
Short Title 
darTregs for CNI Reduction 
Protocol Number 
CTOTC-12 
ClinicalTrials.gov 
Identifier 
NCT02474199 
Clinical Phase 
Phase I/II 
Primary Safety 
Objective 
This study will evaluate the safety and tolerability of a single IV dose of the darTregs product. 
Secondary Safety 
Objective 
This study will evaluate the safety of immunosuppression (IS) 1) minimization and 2) 
discontinuation after one IV dose of darTregs. 
Primary Efficacy 
Objective 
The study will evaluate the ability of a single IV dose of darTregs to reduce baseline, 
standard of care (SOC) calcineurin inhibitor (CNI) dosing by 75% along with discontinuation 
of either prednisone or mycophenolate mofetil (MMF), as applicable. 
Secondary Efficacy 
Objective 
This study will determine whether a single IV dose of darTregs can induce operational tolerance. 
Mechanistic Objectives 
We will assess the pharmacokinetic profile of darTregs by measuring the level of deuterium-
labeled Tregs in circulation. Potential impact of darTregs therapy on immunological profiles will 
be assessed by comparing leukocyte phenotypes, tissue histology and gene expression in protocol 
and for-cause biopsies, and alloantibody before and after darTregs infusion. 
Study Design 
A multi-center, open-label clinical trial in adult liver transplant recipients with two 
primary interventions: 
 
administration of a single IV dose of darTregs and 
 
reduction of IS dosing with possible complete IS discontinuation. 
Adult living donor liver recipients two to six years after LT will initiate IS withdrawal. Twelve to 
14 weeks later, they will receive a single dose of darTregs and continue IS withdrawal. 
Primary Safety 
Endpoint 
The safety and tolerability of a single infusion of darTregs administered to LT recipients will be 
assessed 24 weeks after darTregs by describing: 
1. Occurrence of CTCAE Grade 3 or higher adverse events (AEs) attributable to the 
darTregs infusion including infusion reaction / cytokine release syndrome 
2. Occurrence of study defined Grade 3 or higher infections 
3. Occurrence of any malignancy, including PTLD 
Secondary Safety 
Endpoint 
The trial will assess the safety of IS withdrawal in the context of darTregs therapy by 
describing the following secondary safety endpoints: 
1. Rate of composite outcome measure including refractory acute rejection, chronic 
rejection,  re- transplantation, and death 
2. Incidence of biopsy proven or clinical acute rejection and/or chronic rejection 
3. Timing of biopsy proven or clinical acute rejection and/or chronic rejection 
4. Severity of biopsy proven acute rejection and/or chronic rejection 


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Primary Efficacy 
Endpoint 
The efficacy of a single IV dose of darTregs will be assessed by the number and proportion of LT 
subjects who are able to reduce CNI dosing by 75% and discontinue a 2nd IS drug (if applicable) 
with stable liver tests for at least 12 weeks. The frequency of successful CNI minimization will 
be compared to historical cohorts of comparable adult LT recipients undergoing IS withdrawal. 
Secondary Efficacy 
Endpoints 
The efficacy of a single IV dose of darTregs infusion will be assessed by determining the 
number and percentage of subjects who have received darTregs and are identified as 
operationally tolerant, defined by maintaining stable allograft function (assessed by liver tests) 
and histology (determined by central pathologist reading in comparison to screening liver biopsy 
at study entry) in the absence of IS for one year. The frequency of tolerance will be compared 
to historical cohorts of adult liver transplant recipients undergoing IS withdrawal. 
Primary Mechanistic 
Endpoints 
The level and persistence of deuterium-labeled darTregs in the circulation will be determined 
by serial measurements of deuterium content in DNA from purified peripheral blood Tregs 
after darTregs infusion using gas chromatography-mass spectrometry (GC-MS) testing. 
Secondary Mechanistic 
Endpoints 
The overall increase of darTregs in circulation will be assessed using the alloreactive T cell 
frequency (ATF) assay 
Exploratory 
Mechanistic Endpoints 
The immunologic impact of infused darTregs will be determined by assessing the following: 
 
Leukocyte phenotypes before and after darTregs infusion using multi-parameter flow 
cytometry (MFC). 
 
Alloantibody responses before and after darTregs infusion during IS withdrawal. 
 
Histology and multiplex immunohistochemistry of protocol and for cause biopsies 
 
The composition of immune infiltrate in liver biopsies post Treg infusion and at 
the time of for-cause biopsies will be profiled using single-cell RNA+TCRseq 
Accrual Objective 
Up to 18 participants will be screened and enrolled to target up to 11 participants eligible for 
both IS withdrawal and darTregs infusion 
Study Duration 
The maximal length of trial participation for an individual subject is anticipated to be 2 years. 
The total trial duration will be 3 years. 
 
One year period to accrue nine patients eligible for both IS withdrawal and darTregs 
infusion that will be given approximately 10-11 weeks after initiating IS withdrawal 
 
Minimum of 52 weeks follow-up after any AR episode or or darTregs infusion 
 
For all subjects able to discontinue IS, 52 weeks of follow-up after the last IS dose to 
assess for operational tolerance 
The duration of study participation will vary by subject depending on his/her duration of IS 
withdrawal. Accounting for a one year enrollment period, the primary endpoint for all 
participants will be assessed approximately 1.5 years after trial initiation. 
Treatment Description 
All eligible participants will initiate IS withdrawal. Approximately 10-12 weeks after 
initiating IS withdrawal, subjects will have autologous Tregs collected for darTregs 
manufacturing. 
During the last 2 weeks of IS withdrawal Step 2 (CNI reduced by 25%), a single total dose of 400 
x 106 ± 100 deuterium-labeled darTregs will be infused intravenously. The subject will then, if 
eligible, resume IS withdrawal within 2 weeks after darTregs infusion (see Section entitled 
“Resumption of IS Withdrawal after darTregs Infusion” below).  Only subjects who receive 300-
500 x 106 darTregs and who meet the primary endpoint of 75% reduction in CNI from baseline 
after darTregs will be offered the opportunity to continue IS withdrawal until complete 
discontinuation of IS (secondary endpoint). Those who receive 100-300 x 106 darTregs will only 
be allowed to proceed to the primary endpoint but will not be eligible for complete IS 
withdrawal. 


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Study Enrollment/ IS 
Withdrawal Inclusion 
Criteria 
Subjects who meet all of the following criteria are eligible for enrollment as study participants: 
1. Able to understand and provide informed consent 
2. Have received primary, solitary, living donor liver transplant more than 24 months but less 
than 84 months ago 
3. Have a living donor who is willing to consent to one time phlebotomy of 100 mLs 
to enable manufacture of darTregs 
4. Between 18 and 70 years of age at the time of study entry/consent 
5. Have ALT consistently <60 U/L and either alkaline phosphatase consistently <150 
U/L or GGT consistently <60 U/L 
6. Currently receiving a calcineurin inhibitor (CNI) with 12 hour trough levels consistently 
<6.0ng/mL for tacrolimus;  <150ng/mL for cyclosporine 
7. Currently receiving CNI monotherapy or CNI and ONE of the following: 
a. 
Prednisone: maximum dose of 5mg / day 
b. Mycophenolate mofetil (MMF): maximum dose of 500mg bid for Cellcept or 
360mg bid for Myfortic 
8. Female and male subjects with reproductive potential must agree to use effective 
methods of birth control for the duration of the study 
9. If history of HCC, LT recipients who have: 
a. AFP less than 100 µg/L at the time of transplant AND 
b. Explanted liver: 
i. 
with tumor burden within the Milan criteria and 
ii. 
without macro- or micro-vascular invasion and 
iii. 
without any lesions with poorly differentiated HCC and 
iv. 
without cholangiocarcinoma morphology 
c. Risk Estimation of Tumor Recurrence After Transplant (RETREAT) Score less than or equal 
to 3 
10. If history of HCC, at the time of enrollment, subjects must also: 
a. Be 36 months or more post-transplant AND 
b. Without evidence of recurrent HCC defined as 
i. AFP within normal limits for performing laboratory 
ii. Confirmatory chest CT and 
iii. Confirmatory CT or MRI of the abdomen and pelvis 
11. If history of HCV, recipients must be: 
a. Cured of HCV as defined by achieving SVR and be greater than or equal to six months 
after the end of treatment 
b. HCV RNA negative at time of study enrollment 


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Study Enrollment/ IS 
Withdrawal Exclusion 
Criteria 
Subjects who meet any of these criteria are not eligible for study enrollment. 
1. Transplant for liver disease secondary an autoimmune etiology (e.g. autoimmune hepatitis, 
primary sclerosing cholangitis, or primary biliary cirrhosis) 
2. Matched at both HLA-DR loci to the donor 
3. Organ, tissue or cell transplant prior to or after the primary solitary living donor liver 
transplant 
4. For subjects with hepatitis B, detectible HBV DNA 
5. History of malignancy within 5 years of enrollment. History of adequately treated in-situ 
cervical carcinoma and/or skin cancer (basal or squamous cell) will be permitted. 
6. Serologic evidence of human immunodeficiency 1 or 2 infection 
7. Epstein Barr Virus (EBV) sero-negativity (EBV naïve) if living donor is EBV sero-positive 
8. Cytomegalovirus (CMV) sero-negativity (CMV naïve) if living donor is CMV sero-positive 
9. Calculated GFR less than 50 mL/min/1.73m2 at the time of enrollment 
10. An episode of AR within one year of enrollment 
11. Systemic illness requiring or likely to require recurrent or chronic IS drug use 
12. Any chronic condition for which anti-coagulation cannot be safely interrupted for liver biopsy 
13. Positive pregnancy test 
14. Participation in any other studies that involved investigational drugs or regimens in the 
preceding year 
15. Any other condition, in the investigator’s judgment, that increases the risk of darTregs 
infusion or prevents safe trial participation 
16. Unwilling or unable to adhere to study requirements and procedures 
17. Screening liver biopsy with any of the following histological criteria, as determined by the 
reading of a central pathologist (Table 9) 
darTregs Infusion 
Inclusion Criteria 
Subjects will initiate IS withdrawal and, at the beginning of the 2nd step of the withdrawal 
algorithm (week 1- 2), undergo a final assessment to ensure eligibility for darTregs infusion.  
Subjects must meet the following criteria to receive darTregs infusion: 
1. Stable liver tests, defined as ALT and either alkaline phosphatase or GGT either within 
normal limits OR <1.5 X baseline 
2. No detectible circulating EBV or CMV DNA prior to darTregs infusion, assessed at the time 
of PBMC collection for manufacture 
3. For subjects with HBV, no detectible circulating HBV DNA prior to darTreg infusion, 
assessed at the time of PBMC collection for manufacture 
4. Able to understand and provide informed consent 
darTregs Infusion 
Exclusion Criteria 
Subjects who meet any of these criteria are not eligible for darTregs infusion: 
1. Diagnosis of AR after initiation of IS withdrawal 
2. Any vaccination given within 28 days prior to Treg collection for Treg production 
3. Receipt of a vaccination within 14 days prior to Treg infusion 
4. Unacceptable darTregs product 
5. Positive pregnancy test 
6. Clinical evidence of viral syndrome less than 7 days prior to darTregs infusion 
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Eligibility Criteria to 
Resume IS Withdrawal 
after darTregs infusion 
Subjects are eligible to resume IS withdrawal after darTregs infusion if all criteria below are met: 
1. Subject received at least 100 x 106 darTregs 
2. ALT and either alkaline phosphatase or GGT remain within normal limits or ≤ 1.5 x 
baseline after darTregs infusion 
3. For subjects with elevated liver tests as defined above, local pathology reading of liver 
biopsy 6-10 days after darTregs infusion is without acute rejection according to Banff 
criteria 
4. IS withdrawal resumes no later than 14 days after darTregs infusion 
5. Site principal investigator determines it is acceptable for the study subject to resume IS 
withdrawal 
Study Stopping Rules 
If one of the following events occurs any time during the study, the study will be paused for 
DSMB review: 
 
Death or graft loss 
 
Chronic Rejection 
 
Severe AR (histological or steroid refractory rejection) 
If any of the following events occurs after darTregs Infusion, the study will be paused for 
DSMB review: 
 
CTCAE Grade 3 or higher AEs attributable to the darTregs infusion including infusion 
reaction/cytokine release syndrome in 2 subjects 
 
Any infections of Grade 3 or higher as defined in Section 14.3.1 
 
Any malignancy, including PTLD 
Failure to manufacture and supply the cellular product for 2 subjects will also require a pause 
in the trial. 
 


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2. 
INTRODUCTION 
This statistical analysis plan includes pre-planned analyses related to the study objectives 
outlined in the protocol. 
 
 


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3. 
GENERAL ANALYSIS AND REPORTING CONVENTIONS 
The following analyses and reporting conventions will be used: 
 
Categorical variables will be summarized using counts (n) and percentages (%) and will 
be presented in the form “n (%).”  Percentages will be rounded to one decimal place. 
 
Numeric variables will be summarized using n, mean, standard deviation (SD), median, 
minimum (min), maximum (max).  The min/max will be reported at the same level of 
significance as original data.  The mean and median will be reported at one more 
significant digit than the precision of the data, and SD w ill be reported at two more 
significant digits than the precision of the data. 
 
The median will be reported as the average of the two middle numbers if the dataset 
contains an even number of observations. 
 
Test statistics including t and z test statistics will be reported to two decimal places.  
 
P-values will be reported to three decimal places if greater than or equal to 0.001.  If less 
than 0.001, the value will be reported as “<0.001.”  A p-value can be reported as “1.000” 
only if it is exactly 1.000 without rounding.  A p-value can be reported as “0.000” only if it 
is exactly 0.000 without rounding. 
If departures from these general conventions are present in the specific evaluations section of 
this SAP, then those conventions will take precedence over these general conventions.  
 


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4. 
ANALYSIS SAMPLES 
The summary descriptive analyses will be performed on the following subject populations: 
1. All subjects who give informed consent and undergo the screening liver biopsy (Safety 
Sample). 
2. The Intent-to-treat I sample (ITT1) will be defined as all subjects who initiate IS 
w ithdrawal 
3. The Intent-to-treat II sample (ITT2) will be defined as all subjects who receive darTregs. 
4. The Per-Protocol sample (PP) will be defined as all subjects who receive at least 300 x 
106 cells of darTregs. 
 
 
 
 


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5. 
STUDY SUBJECTS 
5.1. 
Disposition of Subjects  
The disposition of all enrolled subjects will be summarized in tables and listed. 
The numbers and percentages of subjects in each analysis sample, and completing each 
tapering level, as well as reasons for early termination from the study will be presented.  For 
subjects discontinuing study treatment early, the reasons for discontinuing study treatment 
early will also be presented.   
 
5.2. 
Demographic and Other Baseline Characteristics  
Summary descriptive statistics for baseline and demographic characteristics will be reported for 
the ITT1, ITT2, PP samples.  Characteristics to be summarized include age, race, ethnicity, 
sex, body weight at screening, height, time from transplant and primary reason for transplant. 
 
 
 
 
 


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6. 
STUDY OPERATIONS 
6.1. 
Protocol Deviations 
Protocol deviations will be listed by site with information such as type of deviation, severity of 
the deviation (major or non-major), date of occurrence, and the reason for the deviation.  
Protocol deviations will be summarized in tabular format by type of deviation.   
 
6.2. 
Treatment Adherence 
A summary of each subject’s adherence to the tapering schedule will be presented in graphical 
form, with indications for pauses at each level and any deviations from the recommended dose 
at each taper level. 
A summary of the number of cells infused will be presented for the darTregs as well as a 
categorical summary of the number of subjects that received between 100 and 300 x 106 cells of 
darTregs, and the number of subjects that received at least 300 x 106 cells of darTregs. 
 
 


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7. 
ENDPOINT EVALUATION 
 
7.1. 
Overview of Analysis Methods 
 
7.1.1. 
Multicenter Studies 
 
Study subjects will be recruited from three study sites.  Due to the small number of subjects in 
the study, study data will be analyzed as a whole, and no formal accommodation for site-to-site 
variation will be made.  
7.1.2. 
Assessment Time Windows 
 
Allowable visit windows for all scheduled visits are provided in Table 7-1. 
Table 7-1 
Visit Windows  
 
High Frequency SOE Visit Windows 
Study Visit 
Visit Window 
Study Entry Eligibility Screening Visit (Screen 1) Eligibility labs and biopsy should be within 8 weeks 
prior to initiation of IS withdrawal 
Remote Visits (Telephone and Local Liver Tests) ± 3 days 
Transplant Center Visits 
± 14 days 
Recipient PBMC Collection for Manufacturing 
17 (UCSF) or 18 (Mayo) days before darTreg infusion 
Medium Frequency SOE Visit Windows 
Study Visit 
Visit Window 
Entry into Medium Frequency SOE 
Within 2 weeks after rejection (Appendix 7) 
Within 4 weeks after completing high frequency SOE 
(Appendix 8) 
Week 2 (Appendix 7) 
± 3 days 
Remote Visits (Telephone and Local Liver Tests) ± 5 days 
End of Study 
± 14 days 
darTregs Infusion SOE Visit Windows 
Study Visit 
Visit Window 
darTregs Eligibility Screening Visit 
Between day 14-41 of the 2nd withdrawal step (10-13 weeks) 
Treg Blood Draw for darTregs manufacturing 
Between day 18-45 of the 2nd withdrawal step 
Day 0 = darTregs Infusion 
During the last 2 weeks of the 2nd withdrawal step 
Day 1 after darTregs infusion 
± 1 hour 
Protocol biopsy visit after darTregs infusion 
Between 6-10 days after darTregs infusion 
Resumption of IS withdrawal 
Between day 1-14 after darTregs infusion; corresponds to 
between day 36 – day 63 of the 2nd withdrawal step 
Day 14 after darTregs infusion 
± 2 days 
 
Unscheduled visits may also occur throughout the study.  All data will be included in analyses, 
regardless of time of assessment. 
 


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7.2. 
Primary Efficacy Endpoint 
The primary efficacy endpoint is the number and proportion of subjects who reduce CNI by 75% 
and discontinue a 2nd IS drug, if applicable, with stable liver tests for at least 12 weeks.   
7.2.1. 
Computation of the Primary Efficacy Endpoint 
All subjects that successfully complete Step 5 of the tapering algorithm and have stable liver 
tests (ALT and alkaline phosphatase or GGT either ≤1.5 X upper limit of normal or ≤1.5 X 
baseline) for at least 12 weeks will be counted as meeting the primary efficacy endpoint 
7.2.2. 
Primary Analysis of the Primary Efficacy Endpoint 
The primary efficacy endpoint will be descriptively summarized with an exact binomial 
proportion and an exact 95% confidence interval using the ITT1 population. 
 
7.2.3. 
Sensitivity Analyses of the Primary Efficacy Endpoint 
 
A sensitivity analysis using both the ITT2 and PP populations will also be performed. 
7.3. 
Primary Safety Endpoint 
The primary safety endpoint is a descriptive analysis of three outcomes assessed 24 weeks 
after darTregs: 
 
CTCAE Grade 3 or higher AEs attributable to the darTregs infusion including infusion 
reaction / cytokine release syndrome 
 
study defined Grade 3 or higher infections (Protocol section 14.3.1) 
 
any malignancy, including PTLD 
 
7.3.1. 
Computation of the Primary Safety Endpoint 
All subjects who have any of the listed outcomes above will be counted as meeting the primary 
safety endpoint. 
7.3.2. 
Primary Analysis of the Primary Safety Endpoint 
The primary safety endpoint will be descriptively summarized with an exact binomial proportion 
and an exact 95% confidence interval using the ITT1 population. 
7.3.3. 
Additional Analyses of the Primary Safety Endpoint 
The outcomes in the primary safety endpoint will also be analyzed individually.   A sensitivity 
analysis using the PP population will also be performed. 
 
7.4. 
Secondary Efficacy Endpoints 
All secondary efficacy endpoint analyses will use the ITT2 population, with a sensitivity analysis 
using the PP population.  


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7.4.1. 
Number and proportion of subjects who successfully withdrawal 
from all IS after darTregs infusion 
Variable: Complete withdrawal from all IS 
Analysis: Proportion with exact binomial 95% confidence limits 
7.4.2. 
Duration that subjects tolerate complete IS discontinuation 
Variable: Time off all IS 
Analysis: Kaplan-Meier estimates of time to re-initiation of IS after completion of ISW 
(or censoring if no re-initiation occurs by the end of study). Duration will be displayed 
graphically with a Kaplan-Meier curve and the corresponding median and two-sided 
95% confidence interval. 
7.4.3. 
Number and proportion of subjects who successfully withdrawal 
from all IS after darTregs infusion and are operationally tolerant 
Variable: Complete withdrawal from all IS and meets definition of operational 
tolerance (Protocol section 5.4)  
Analysis: Proportion with exact binomial 95% confidence limits 
 
7.5. 
Secondary Safety Endpoints 
All secondary safety endpoint analyses will use the ITT1 population, with a sensitivity analysis 
using the PP population unless otherwise stated.  
7.5.1. 
Rate of composite outcome measure including refractory AR, CR, re- 
transplantation, and death 
Variable: Occurrence of refractory AR, CR, re-transplantation, or death 
Analysis: Proportion with exact binomial 95% confidence limits 
7.5.2. 
Incidence of biopsy proven or clinical AR and/or CR 
Variable: Biopsy proven or clinical AR or CR 
Analysis: Proportion with exact binomial 95% confidence limits; Also number of 
biopsy proven or clinical AR and/or CR AEs 
7.5.3. 
Timing of biopsy proven or clinical AR and/or CR in ITT2 population 
Variable: Time to biopsy proven or clinical AR and/or CR   
Analysis: Kaplan-Meier estimates of time to re-initiation of IS after completion of ISW 
(or censoring if no rejection occurs by the end of study). Duration will be displayed 


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graphically with a Kaplan-Meier curve and the corresponding median and two-sided 
95% confidence interval. 
7.5.4. 
Severity of biopsy proven or clinical AR and/or CR in ITT2 population 
Variable: Biopsy proven or clinical AR or CR  
Analysis: Proportion of subjects in each observed BANFF severity category 
 
 
 
 
 


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8. 
SAFETY EVALUATION 
8.1. 
Overview of Safety Analysis Methods 
All safety analyses will be carried out using the safety sample defined in Section 4 unless 
otherwise noted.  Missing safety information will not be imputed.  These analyses will not be 
stratified by site.   
 
8.2. 
Adverse Events 
 
All AEs will be classified by system organ class (SOC) and preferred term, according to a 
standardized thesaurus (Medical Dictionary for Regulatory Activities [MedDRA] version 18.0).  
The severity of AEs will be classified using the National Cancer Institute’s (NCI’s) Common 
Toxicity Criteria for Adverse Events (CTCAE) version 4.0.  Each AE is entered on the electronic 
case report form (eCRF) once at the highest severity. As such, no additional data manipulation 
is needed to identify events. 
 
An overall summary table will be developed to report the number of events and the number and 
percentage of subjects having at least one event in the following categories:  
 
AEs 
 
AEs indicated as serious 
 
AEs of interest (Acute Rejection, Clinical Rejection, Chronic Rejection, Malignancy, Study 
defined Grade 3 or higher infections, and Grade 3 or higher Infusion Reactions)AEs that lead 
to study drug discontinuation 
 
AEs with an outcome of death 
 
SAEs that were reported as being related to ISW or darTregs 
 
AEs reported by severity 
In addition, AEs classified by MedDRA SOC and preferred term will be summarized for each 
treatment group and overall for each of the following: 
 
All AEs 
 
AEs by maximum severity 
 
AEs by relationship to ISW or darTregs (site investigator determination) 
Summary tables will present the total number of events as well as the number and percentage 
of subjects experiencing the events.  If a subject experiences the same AE on multiple 
occasions, the event will be counted once for each occurrence when reporting the number of 
AEs.  When reporting the number of subjects experiencing the events, a subject will only be 


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counted once if they experience an event within the particular SOC or preferred term.  
Percentages will be based on the number of subjects in the safety population. 
8.3. 
Deaths and Serious Adverse Events 
 
Serious adverse events (SAEs) will be listed and summarized in the same manner described in 
Section 8.28.3.  As applicable, separate displays listing and summarizing death, including time 
to death and cause of death, will also be created. 
8.4. 
Clinical Laboratory Evaluation 
 
Clinical laboratory measurements include serum chemistry, urinalysis, and hematology.  Results 
w ill be converted to standardized units where possible.  For numeric laboratory results, 
descriptive statistics of laboratory values and the change from baseline of laboratory values will 
be presented for each analysis sample and overall.  For categorical laboratory results, the 
number and percentage of subjects reporting each result will be presented for each analysis 
sample and overall.   
 
Liver test data will be plotted to show patterns over time.  For each test with a numeric result, 
data w ill be plotted as a spaghetti plot where each subject’s values will be plotted and 
connected by line segments, forming one line per subject.  For each test with a numeric result, 
quantile plots with treatment group means (or medians) as well as 25th and 75th percentiles 
plotted over time will be created.  Tests with qualitative results (such as “present” or “positive”) 
w ill not be plotted.  Normal ranges will also be plotted using Harrison’s normal range. 
 
 
8.5. 
Vital Signs, Physical Findings, and Other Observations Related to Safety 
8.5.1. 
Vital Signs 
Vital sign measures will be plotted over time for each subject.  If needed to fulfill regulatory 
reporting obligations, listings of vital sign data over time may also be created. 
 
8.5.2. 
Physical Examinations 
If needed to fulfill regulatory reporting obligations, listings of physical examination data will be 
produced.  These listings may include, but are not limited to hepatic body system results.   
 
 
 


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9. 
INTERIM ANALYSES AND DATA MONITORING 
The progress of the study will be monitored by the Data and Safety Monitoring Board (DSMB).  
The NIAID Transplant DSMB w ill be chartered to review safety data and to make 
recommendations regarding continuation, termination, or modification of the study.  The DSMB 
w ill formally review the safety data at least yearly.  The discontinuation of study treatment will 
also be periodically reported to the DSMB.  
In addition, safety data will be reviewed by the DSMB w hen an event occurs that is of sufficient 
concern to the National Institute of Allergy and Infectious Diseases (NIAID) medical monitor or 
protocol chair to warrant review, or when an event occurs that could contribute to a predefined 
stopping rule specified in the protocol.  
Findings will be reported to Institutional Review Boards (IRBs) and health authorities. 
 
 


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10. 
CHANGES TO THE ANALYSES PLANNED IN THE PROTOCOL