Clinical trial NCT02188719
Conditions: Liver Transplantation
Keywords: Donor-Alloantigen-Reactive Regulatory T Cell Therapy Tregs Cell- and Tissue-Based Therapy Immunotherapy, Adoptive Liver Transplantation
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2014-12-17; Completion date: 2019-06-18
Enrollment: 15 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is look at the safety of:

* Taking a specific combination of immunosuppressant drugs after liver transplantation
* Receiving one of three different doses of donor-alloantigen-reactive regulatory T cells (darTregs) while taking this specific combination of drugs

Detailed description:
After liver transplantation, immunosuppressants must be taken every day to prevent the body from injuring the transplanted liver by a process called rejection. People who take these drugs may experience side effects.

Studies show that some of body's cells, called T regulatory cells (Tregs), may play a part in accepting the transplanted liver. The investigators are learning about whether scientists can take Tregs from the blood of a liver transplant recipient and teach them to protect the transplanted liver from rejection. In the laboratory, the recipient Tregs are exposed to cells from the liver donor. Research data suggests that giving these "donor reactive" Tregs back to the transplant recipient might allow a liver transplant recipient to take lower doses of immunosuppressants, or perhaps to stop them altogether, without rejecting the liver.

Arms: arm 1: 3 subjects (Cohort 1a) at site 1 (UCSF) and 3 subjects (Cohort 1b) from site 2 (Mayo Rochester) will receive Treg-Supportive immunosuppression (IS) regimen and will not receive Donor-Alloantigen-Reactive T Regulatory Cells (darTregs). Progression from one cohort to the next will depend on the cumulative incidence of sentinel adverse events. arm 2: At least 3 subjects will receive a single infusion of 50 million darTregs. Progression from one cohort to the next will depend on the cumulative incidence of sentinel adverse events. arm 3: At least 3 subjects will receive a single infusion dose of 200 million darTregs. Progression from one cohort to the next will depend on the cumulative incidence of sentinel adverse events. arm 4: Six subjects will receive a single infusion of 800 million darTregs.
Interventions: intervention 1: Anti-Thymocyte Globulin - Rabbit intervention 2: darTreg Infusion intervention 3: Everolimus intervention 4: Tacrolimus intervention 5: Mycophenolate mofetil intervention 6: Prednisone intervention 7: Acetaminophen intervention 8: Diphenhydramine intervention 9: Anti-Infective Prophylaxis intervention 10: Leukapheresis intervention 11: Blood draws intervention 12: Liver biopsies intervention 13: Liver transplantation
Intervention descriptions: intervention 1: Liver transplantation/Treg-supportive immunosuppression (IS) treatment. Subjects will be given a dose range of 3.0-4.5 mg/kg total, in divided doses of 1.5 mg/kg/day. Subjects who meet eligibility criteria for Thymoglobulin® administration will be given 1.5 mg/kg intravenously (IV) on post-operative day 3, within 72 hours of transplantation. Additional doses of 1.5 mg/kg IV will be administered until CD3 count is \<50/mm\^3 or when the maximal dose of 4.5/mg/kg has been given. intervention 2: A single dose darTreg infusion (Cohorts 2 - 4) will be received as per protocol. intervention 3: Liver transplantation/Treg-supportive immunosuppression (IS) treatment. Subjects meeting eligibility criteria for Treg-supportive IS regimen will begin EVR no sooner than 30 days after liver transplantation and no later than 44 days after transplantation; with target trough levels of 6-8 μg/L.EVR target trough levels will be further reduced to 4-6 μg/L 24 - 26 weeks after transplantation. intervention 4: Liver transplantation/Treg-supportive immunosuppression (IS) treatment. Between 30 and 44 days following transplant: Subjects who are not eliminated by Exclusion Criteria C1 (Protocol Section 14.3.1) will proceed in the study and receive either TAC-based or EVR- based IS, based on eligibility Criteria C2 (Section 4.3.4 )

* TAC-based IS: reduce TAC trough level to 3-8 μg/dL; continue MMF
* EVR-based IS: reduce TAC trough level to 3-8 μg/dL; EVR target trough level of 6-8 μg/dL; decrease then discontinue MMF intervention 5: Liver transplantation/Treg-supportive immunosuppression (IS) treatment. 1000 mg total daily dose. MMF will be initiated within 24 hours of transplantation. MMF must be discontinued as soon as target EVR trough levels have been achieved. intervention 6: Liver transplantation/Treg-supportive immunosuppression (IS) treatment. Solumedrol 500 mg will be given IV on the day of transplantation. Additional Solumedrol will be prescribed according to site-specific standard of care. Oral prednisone should be initiated once oral medication is tolerated. intervention 7: Pre-medication for single dose darTreg infusion (Cohorts 2 - 4). 650mg of acetaminophen will be administered intravenously or by mouth 30-60 minutes prior to the darTreg infusion. intervention 8: Pre-medication for single dose darTreg infusion (Cohorts 2 - 4). 25-50mg of diphenhydramine will be administered intravenously or by mouth 30-60 minutes prior to the darTreg infusion. intervention 9: Intravenous ganciclovir and/or oral Valcyte will be administered for the prophylaxis of cytomegalovirus (CMV) and Epstein-Barr Virus (EBV) for at least six months after liver transplantation. intervention 10: Leukapheresis is necessary to ensure collection of adequate numbers of autologous Tregs to support ex vivo expansion of darTregs for infusion after liver transplantation.

Participants enrolled in Cohorts 3 and 4 will undergo leukapheresis. Participants enrolled in Cohort 2 will have either whole blood collection or leukapheresis for the purpose of isolating autologous Tregs for later manufacture. If a cohort 2 subject has a hemoglobin level \>/=10.5 gm/dL, he or she will undergo phlebotomy. If the patient has a hemoglobin level \</=10.5 gm/dL and remains eligible for the study, the patient will undergo leukapheresis. intervention 11: Blood draws are necessary to carefully and frequently evaluate allograft function after liver transplantation and treatment with Treg-supportive IS as well as after darTreg infusion. Peripheral blood samples will be collected and analyzed per protocol throughout subject participation in this study. intervention 12: Subjects will have a liver biopsy for this study 12-14 weeks after transplantation. For subjects receiving darTregs, a second biopsy will be performed 7-10 days after darTregs infusion. intervention 13: Inclusion in this trial is in the setting of subjects defined as having end-stage liver disease and listed for primary solitary liver transplant.

Protocol documents:
Prot_000.pdf: 
 
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Tregs in Liver Transplantation (RTB-002) 
                                                           Version 7.0/ August 1, 2018 
Donor-Alloantigen-Reactive Regulatory T Cell Therapy in 
Liver Transplantation 
 Version 7.0/ August 1, 2018 
IND# 15479 
Study Sponsor(s): The National Institute of Allergy and Infectious Diseases (NIAID) 
Grant #:  R34AI095135 and U01AI110658-01 
Study Drug Manufacturer/Provider:  Sanofi US 
PRINCIPAL INVESTIGATOR 
SANDY FENG, MD, PHD 
Professor of Surgery 
Director, Abdominal Transplant 
Surgery Fellowship 
University of California, San 
Francisco 
505 Parnassus Ave. 
Box 0780, M-896 
San Francisco, CA  94143-0780 
Phone: (415) 353-8725 
Fax: (415) 353-8709 
E-mail: sandy.feng@ucsfmedctr.org  
CO- PRINCIPAL INVESTIGATOR  
JEFFREY BLUESTONE, PHD 
Executive Vice Chancellor and Provost 
A.W. Clausen Distinguished Professor 
in Medicine 
University of California, San Francisco 
513 Parnassus Ave. 
Box 0400, HSW-1128 
San Francisco, CA  94143-0780 
Phone: (415) 476-4451 
Fax: (415) 476-9634 
E-mail: jeffrey.bluestone@ucsf.edu 
CO- PRINCIPAL INVESTIGATOR  
SANG-MO KANG, MD, FACS 
Associate Professor 
University of California, San 
Francisco 
513 Parnassus Ave. 
Box 0780, HSE-520 
San Francisco, CA  94143-0780 
Phone: (415) 353-1888  
E-mail: Sang-
Mo.Kang@ucsfmedctr.org 
 
CO- PRINCIPAL INVESTIGATOR  
QIZHI TANG, PHD 
Associate Professor 
Director, UCSF Transplantation 
Research Lab 
University of California, San 
Francisco 
513 Parnassus Ave. 
Box 0780, HSE-520 
San Francisco, CA  94143-0780 
Phone: (415) 476-1739 
Fax: (415) 502-8326 
E-mail: qizhi.tang@ucsfmedctr.org 
BIOSTATISTICIAN  
DAVID IKLÉ, PHD 
Senior Statistical Scientist 
Rho, Inc. 
 
6330 Quadrangle Drive 
Chapel Hill, NC 27517 
Phone: (910) 558-6678 
Fax: (919) 408-0999  
E-mail: David_Ikle@rhoworld.com 
MEDICAL OFFICER 
NANCY BRIDGES, MD 
Chief, Transplantation Branch 
Division of Allergy, Immunology, 
and Transplantation 
NIAID 
5601 Fishers Ln, 6B31 
Bethesda, MD 20892 
Phone: (301) 480-6316 
E-mail: nbridges@niaid.nih.gov  
 
PROGRAM OFFICER 
DEBORAH HAYES, MS 
Transplantation Branch 
Division of Allergy, Immunology and 
Transplantation 
NIAID 
5601 Fishers Ln, 6B35 
Bethesda, Maryland 20892 
Phone: (240) 627-3496 
E-mail: dhayes@niaid.nih.gov 
REGULATORY OFFICER 
JULIA GOLDSTEIN, MD 
Office of Regulatory Affairs 
Division of Allergy, Immunology, and 
Transplantation 
NIAID 
5601 Fishers Ln, 7B29 
Bethesda, MD 20892  
Phone: (240) 627-3509 
Email: goldsteinj@niaid.nih.gov 
PROJECT MANAGER 
TINA SLEDGE, RN 
Transplantation Branch 
Division of Allergy, Immunology, 
and Transplantation  
NIAID 
5601 Fishers Ln, 6B28 
Bethesda, MD 20892  
Phone: (240) 627-3561   
Email: sledget@niaid.nih.gov 
Confidentiality Statement 
The information contained within this document is not to be disclosed in any way without the prior permission of the Protocol Chair, or the Division 
of Allergy, Immunology and Transplantation, NIAID of the National Institutes of Health. 
  
 
 


 
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Tregs in Liver Transplantation (RTB-002) 
                                                           Version 7.0/ August 1, 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
INVESTIGATOR SIGNATURE PAGE 
Protocol: 
Tregs in Liver Transplantation  
Version/Date:  
   7.0 / August 1, 2018 
Site Principal Investigator:   
  
Title:   Donor-Alloantigen-Reactive Regulatory T Cell Therapy in Liver Transplantation  
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. 
3900 Paramount Parkway 
Morrisville, NC 27560 
Phone: (919) 456-4229 
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 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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Tregs in Liver Transplantation (RTB-002) 
                                                           Version 7.0/ August 1, 2018 
Protocol Synopsis 
Title 
Donor-Alloantigen-Reactive Regulatory T Cell (darTreg) Therapy in Liver Transplantation 
Short Title 
darTregs in Liver Transplantation (deLTa) 
Protocol Number 
RTB-002 
ClinicalTrials.gov 
Identifier 
NCT02188719 
Clinical Phase 
Phase I 
Safety Objective 
This study will evaluate the safety, tolerability, and dose limiting toxicities (DLTs) of a Treg-supportive 
Immunosuppression (IS) regimen and darTreg infusion for adult, de novo, liver transplant recipients. 
Mechanistic 
Objectives 
This study will evaluate 1) the effects of a Treg-supportive IS regimen and Treg infusion on 
immunological profiles of liver transplant recipients, and 2) explore the hypothesis that ex vivo-
expanded darTregs will be biologically active. 
Study Design 
A multi-center, open-label, dose escalation, pilot trial with four cohorts (Treg-supportive IS only group 
+ three darTreg dosing groups) conducted in a phased schema. 
Safety Outcome 
The safety, tolerability, and dose-limiting toxicities (DLTs) of darTreg therapy given within the context 
of a Treg-supportive IS regimen will be evaluated with the rate of the following events within 40 
weeks of transplantation. 
 
Clinical outcomes that will be described are: 
1. Incidence and severity of biopsy proven acute rejection (AR) and/or chronic rejection (CR)  
2. Incidence of ≥ Grade 3 infections as defined in Section 14.3.1 
3. Incidence of wound complications (≥ CTCAE Version 4.0, Grade 3) 
4. Incidence of anemia, neutropenia, and/or thrombocytopenia (according to study defined 
reporting requirements, Section 14.3.1 Grading Criteria)  
5. Incidence of adverse events (AEs) attributable to the darTreg infusion including infusion 
reaction / cytokine release syndrome (CRS) (≥ CTCAE Grade 3), and malignant cellular 
transformation.    
Mechanistic 
Outcomes  
1. Treg frequency in peripheral blood using Treg TruCount assay 
2. Persistence of infused darTregs in blood and biopsies using deuterium labeling and T cell 
repertoire analysis  
3. darTreg and darTconv frequency in peripheral blood using an in vitro allofrequency assay 
4. Multicolor flow cytometry (MFC) to assess effect on immune profiles  
5. Functional assessment of Treg in peripheral blood using a mixed lymphocyte reaction (MLR) 
suppression assay 
6. Histology and immunophenotype of liver biopsies 
Accrual Objective 
18 - 24 subjects: 
• 
Cohort 1:  3 subjects from Center 1 (Cohort 1a) + 3 subjects from Center 2 (Cohort 1b)  
• 
Cohort 2:  At least 3, and up to 6 subjects (Dose A)   
• 
Cohort 3:  At least 3, and up to 6 subjects from either Center (Dose B) 
• 
Cohort 4:  6 subjects from either Center (Dose C). 
Study Duration 
4 year accrual period, 40 weeks of follow-up after transplantation 


 
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Treatment 
Description  
Treg-supportive IS regimen: 
• 
Rabbit anti-thymocyte globulin (Thymoglobulin®) induction (3-4.5 mg/kg started by day 3) 
• 
Corticosteroids (500 mg intra-operatively, taper to off by week 5) 
• 
Mycophenolate mofetil (1000 mg total daily dose) 
• 
TAC, with target trough level of 6-8 µg/dL 
 
Between 30 and 44 days following transplant:  Subjects who are not eliminated by Exclusion Criteria C1 
(Section 4.3.3) will proceed in the study and receive either TAC-based or EVR-based IS, based on 
eligibility Criteria C2 (Section 4.3.4) 
• 
TAC-based IS: reduce TAC trough level to 3-8 µg/dL; continue MMF  
• 
EVR-based IS:  reduce TAC trough level to 3-5 µg/dL; EVR target trough level of 6-8 µg/dL; 
decrease then discontinue MMF 
Escalating darTregs therapy:  
Eligible and consented patients in Cohorts 2, 3, or 4 will have peripheral blood mononuclear cells 
(PBMCs) collected, which will be cryopreserved for subsequent induction, purification, and expansion 
of darTregs.  At the time of transplant and after verification of the participant’s ongoing eligibility, donor 
cells will be collected and banked for production of stimulated donor B cells.  Donor B cells will be used 
to generate darTregs for infusion at 11-20 weeks after transplant.   
Cohort 1 – Treg-supportive IS only 
Cohort 2 – darTreg infusion, 50 million (range 25 to 60 million) 
Cohort 3 – darTreg infusion, 200 million (range 100-240 million) 
      Cohort 4 – darTreg infusion, 800 million (range 400-960 million) 
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. End-stage liver disease and listed for deceased-donor primary solitary liver transplant 
3. Between 21 and 70 years of age at the time of study entry/consent 
4. Have a calculated MELD score ≤ 25 at the time of study entry/consent  
5. Female and male subjects with reproductive potential must agree to use effective methods of 
birth control for the duration of the study 
6. If history of HCV, have completed or in current treatment for HCV AND have no detectable HCV 
RNA 
7. Subjects with HCC meeting Milan criteria   


 
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Exclusion Criteria 
Exclusion criteria A (assessed at study enrollment, prior to study procedures).  Subjects with any of the 
following are ineligible for study participation: 
1. End stage liver disease secondary to autoimmune etiology (autoimmune hepatitis, primary 
biliary cirrhosis, or primary sclerosing cholangitis) 
2. History of less than 5 years remission of malignancy, except for 1) HCC or 2) history of 
adequately treated in-situ cervical carcinoma, or adequately treated basal or squamous cell 
carcinoma of the skin  
3. History of previous organ, tissue or cell transplant 
4. Serologic evidence of human immunodeficiency 1 or 2 infection 
5. Epstein Barr Virus (EBV) sero-negativity (EBV naïve candidates) 
6. Cytomegalovirus (CMV) sero-negativity (CMV naïve candidates) if donor is positive 
7. Chronic use of systemic glucocorticoids or other IS, or biologic immunomodulators  
8. Chronic condition requiring anti-coagulation after liver transplantation 
9. Any chronic illness or prior treatment which, in the opinion of the investigator, precludes study 
participation 
10. Participation in any other studies that involved investigational drugs or regimens in the 
preceding year 
11. Received any vaccination within 28 days prior to leukapheresis or blood collection for Treg 
manufacture 
12. Hemoglobin <9.0 g/dL within 10 days prior to screening  
13. Neutrophils <1,500/µL within 10 days prior to screening 
14. Platelets <40,000/µL within 10 days prior to screening 
Exclusion Criteria 
(continued) 
Exclusion criteria B (assessed prior to administration of Thymoglobulin®): 
1. Calculated MELD score greater than 25 at the time of deceased donor liver transplant 
2. Last AFP obtained prior to liver transplantation >400 µg/L for candidates with hepatocellular 
carcinoma (HCC) 
3. Unacceptable PBMC product for participants enrolled in Cohorts 2, 3, or 4, per UCSF HICTF 
manufacturing specifications  
4. Absence of donor cells for manufacturing for any participant  
5. HLA-DR matched to donor at both loci 
6. Subject is <21 or > 70 years of age at the time of transplantation 
7. Located in the intensive care unit (ICU) 72 hours after transplantation 
8. Hemoglobin <8.0 g/dL  
9. Absolute neutrophil count <1,200/µL  
10. Platelets <40,000/µL  
11. Positive pregnancy test for females of child bearing potential 
12. Unexpected histopathology on back table liver biopsy that contraindicates the initiation of 
Treg-supportive IS regimen. 
13. Development of a condition requiring chronic anti-coagulation 
14. Hypersensitivity to rabbit proteins or any excipient in Thymoglobulin®  
15. Detectable HCV RNA (if history of HCV (e.g. HCV serology positive at screen A)) or less than six 
months after end of treatment for HCV at the time of transplantation (i.e., does not meet 
criteria for SVR)  


 
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Exclusion Criteria 
(continued) 
Exclusion criteria C1 (assessed at day 30-44 after transplantation for continuation in the trial) 
All subjects regardless of eligibility for EVR conversion, with any of the following will not receive 
darTregs and will move into safety follow up. 
1. Explanted liver with evidence of increased risk of recurrent cancer risk (HCC tumor burden 
exceeding the Milan criteria; presence of vascular invasion; cholangiocarcinoma morphology 
2. Insufficient depletion of recipient T cells, defined as a nadir CD3 count ≥50 cells/µL (50 
cells/mcL) or total lymphocyte count ≥ 0.10 x 109/L if CD3 count is unavailable 
3. Development of a condition requiring chronic anti-coagulation 
4. Clinical evidence of untreated biliary obstruction 
5. ALT >2.0 x upper limit of normal (ULN) 
6. Inability to taper off corticosteroids by 44 days (+/- 2 days)  after transplant 
7. Detectable circulating HCV RNA at any time since study entry 
Everolimus Conversion criteria C2 (assessed prior to conversion to EVR-based IS regimen; EVR cannot 
be initiated prior to 30 days after liver transplantation).  Subjects with any of the following will remain 
on TAC-based IS regimen.   
1. Evidence of hepatic artery stenosis or thrombosis by Doppler examination or angiography 
within 7 days prior to conversion 
2. Urine protein/creatinine ratio >1.0 mg/mg within 7 days prior to conversion 
3. Calculated GFR less than 30 ml/min per MDRD4 equation within 7 days prior to conversion 
4. Physical examination documentation of abnormal wound healing or uncontrolled wound 
infection 
5. Hemoglobin <8.0 g/dL within 7 days prior to conversion 
6. Absolute neutrophil count <1,200/µL within 7 days prior to conversion 
7. Platelets <50,000/µL within 7 days prior to conversion 
Exclusion Criteria 
(continued) 
Exclusion criteria D (assessed prior to darTreg-infusion): 
1. Inability or unwillingness of participant to give written informed consent 
2. Unacceptable darTreg product. 
3. Detectable circulating EBV or CMV DNA within 10 days prior to darTreg infusion 
4. Detectable circulating HBV DNA within 10 days prior to darTreg infusion 
5. Detectable circulating HCV RNA within 10 days prior to darTreg infusion 
6. ALT >1.5x ULN within 10 days prior to darTreg infusion 
7. Most recent, but not greater than 10 days prior to darTreg infusion, 12 hour TAC trough levels 
of > 8 µg/L for all subjects  
8. Most recent, but not greater than 10 days prior to darTreg infusion, 12 hour EVR trough levels 
of < 5 µg/L for subjects on EVR 
9. For subjects on EVR-based IS, received MMF within 10 days prior to Treg infusion 
10. Evidence of AR or CR according to Banff criteria on allograft biopsy on local assessment 
11. Received any vaccination within 14 days prior to darTreg infusion 
12. Positive pregnancy test for females of child bearing potential 
13. Inability or unwillingness to comply with study protocol or procedures 
14. Calculated eGFR less than 40 ml/min per MDRD4 equation within 10 days prior to infusion. 
 
 


 
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Tregs in Liver Transplantation (RTB-002) 
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Study Contacts: Participating Centers 
 
 
SITE INVESTIGATOR 
Sandy Feng, MD, PhD 
Director, Abdominal Transplant Surgery 
Fellowship 
Professor of Surgery 
University of California, San Francisco 
505 Parnassus Ave. 
San Francisco, CA 94143-0780 
Phone:  415-353-8725 
Fax:  415-353-8709 
Email: Sandy.Feng@ucsfmedctr.org 
 
SITE INVESTIGATOR 
Timucin Taner, MD PhD 
Mayo Clinic – Rochester 
Assistant Professor of Surgery 
E. Rolland Dickson Research Scholar 
in Transplantation 
Division of Transplantation Surgery 
Mayo Clinic 
Rochester, Minnesota 
Phone: 507-266-1580 
Email:  taner.timucin@mayo.edu 
SITE INVESTIGATOR 
Josh Levitsky, MD, MS 
Associate Professor 
Division of Hepatology and 
Comprehensive Transplant Center 
Feinberg School of Medicine 
Northwestern University 
676 North St. Clair Street Ste. 1900 
Phone: 312-695-4413 
Fax:  312-695-0036 
Email:  Josh.Levitsky@nm.org 
 


 
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Study Contacts: Core Laboratories 
 
 
ALLOFREQUENCY, MLR SUPPRESSION,  
TREG TRUCOUNT & CELLULAR ASSAYS 
Sang-Mo Kang, MD, FACS 
Associate Professor 
Surgical Director, Intestinal Rehabilitation 
and Transplantation 
University of California, San Francisco  
513 Parnassus Ave. 
Box 0780, HSE-520 
San Francisco, CA 94143-0780 
Phone:  415-353-1888 
Email:  Sang-Mo.Kang@ucsfmedctr.org 
 
 
HLA TYPING 
Victor Corpuz, Rajalingam Raja 
Immunogenetics and Transplantation 
Laboratory  
University of California, San Francisco 
Box 0508  
San Francisco, CA 94143-0508 
Phone: (415) 476-3886 
Fax: (415) 502-4926 
Email: Victor.Corpuz@ucsf.edu, 
ajalingam.Raja@ucsf.edu 
 
HISTOLOGY AND IMMUNOPHENOTYPE  
Anthony J. Demetris, MD 
Core Director 
University of Pittsburgh Medical 
Center 
Division of Transplantation 
Pathology  
Department of Pathology 
Montefiore University Hospital 
Room E-741 
Pittsburgh, PA 15261 
Phone: 412-647-2072 
Email: demetrisaj@upmc.edu 
 
ITN SAMPLE REPOSITORY 
BioStorage Technologies, Inc. 
2910 Fortune Circle West, Suite E 
Indianapolis, IN 46241 
Phone: (866) 697-2675 
Email: Operations@Biostorage.com 
MFC AND TCR REPERTOIRE 
Paul Wallace, PhD 
Flow Core Director 
Roswell Park Cancer Institute 
Bldg 10759 Room S631 
Elm & Carlton Streets 
Buffalo, NY 14263 
Phone: 716-845-8471 
Email: Paul.Wallace@RoswellPark.org 
TCR SEQUENCING  
Caterina Bertucci, MPA 
Lead Project Manager & Grants 
Administrater 
Adaptive Biotechnologies 
1551 Eastlake Avenue East, Suite 200 
Seattle, WA 98102 
Phone: 206-436-6357 
Email: 
cbertucci@adaptivebiotech.com 
TREG MANUFACTURING AND  
DEUTERIUM LABELING 
Qizhi Tang, PhD 
Associate  Professor  
Director, Transplantation Research Lab 
University of California, San Francisco 
San Francisco, CA 94143-0780 
Phone: 415-476-1739 
Email: Qizhi.Tang@ucsfmedctr.org 
DETECTION OF DEUTERIUM-
LABELED TREGS 
 
Marc Hellerstein, MD, PhD 
Professor  
Dept of Nutritional Science & 
Toxicology 
University of California, Berkeley 
54 Mulford Hall 
Berkeley, CA 94720-3104 
Phone: 510-643-3104 
EMAIL: MARCH@BERKELEY.EDU 
HTLV TESTING (DONORS) 
O’Dina Hurlburt 
Customer Service Manager  
Creative Testing Solutions  
Attention:  Special Testing 
2424 W. Erie Drive 
Tempe, AZ  85282 
Phone:  602-343-7021 
PBMC CENTRAL CELL PROCESSING  
 
Mike Sheldon  
Rutgers University Cell Repository 
604 Allison Road, Room C120 
Piscataway, NJ 08854 
Phone: 732-445-7190 
Email: ITNDNA@biology.rutgers.edu 
 
 


 
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Tregs in Liver Transplantation (RTB-002) 
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Table of Contents 
Table of Contents .................................................................................................................................................................... 9 
Glossary of Abbreviations ..................................................................................................................................................... 14 
Study Definitions Page .......................................................................................................................................................... 16 
 
Background and Rationale ............................................................................................................................................ 18 
 
Background and Scientific Rationale ..................................................................................................................... 18 
 
Rationale for Selection of Investigational Product or Intervention ...................................................................... 18 
 
Rationale for Treg Therapy ........................................................................................................................... 18 
 
Rationale for Lymphocyte Depletion using Thymoglobulin® ........................................................................ 19 
 
Rationale for Everolimus Conversion ............................................................................................................ 20 
 
Rationale for a TAC-based IS Regimen .......................................................................................................... 21 
 
Rationale for Timing of Treg Administration ................................................................................................ 21 
 
Preclinical Experience ........................................................................................................................................... 22 
 
Reactivity of darTregs ................................................................................................................................... 22 
 
Phenotype of darTregs .................................................................................................................................. 22 
 
In Vitro Function of darTregs ........................................................................................................................ 23 
 
In vivo Function of darTregs in a Humanized Mouse Model of Transplantation ......................................... 23 
 
Clinical Experience with Treg Therapy .................................................................................................................. 25 
 
Study Hypotheses/Objectives ....................................................................................................................................... 28 
 
Safety Objective .................................................................................................................................................... 28 
 
Mechanistic Objectives ......................................................................................................................................... 28 
 
Study Design .................................................................................................................................................................. 29 
 
Description of Study Design .................................................................................................................................. 29 
 
Safety Outcomes ................................................................................................................................................... 29 
 
Mechanistic Outcomes ......................................................................................................................................... 30 
 
Selection of Participants and Clinical Sites/Laboratories.............................................................................................. 31 
 
Rationale for Study Population ............................................................................................................................. 31 
 
Inclusion Criteria ................................................................................................................................................... 32 
 
Exclusion Criteria ................................................................................................................................................... 32 
 
Screening and Enrollment Exclusion Criteria A (Stage 1) .............................................................................. 32 
 
Thymoglobulin® Exclusion Criteria B (Stage 2) ............................................................................................. 32 
 
Exclusion Criteria C1 ...................................................................................................................................... 33 
 
Everolimus Conversion Criteria C2 (Stage 2)................................................................................................. 33 
 
darTreg Infusion Exclusion Criteria D (Stage 3) ............................................................................................. 33 
 
Selection of Clinical Sites ....................................................................................................................................... 34 
 
Investigational Agents ................................................................................................................................................... 35 
 
Deuterium-labelled darTreg Product .................................................................................................................... 35 
 
Manufacturing, Formulation, Packaging, and Labeling ................................................................................ 35 


 
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5.1.1.1 
darTreg Formulation, Packaging and Labeling .......................................................................................... 35 
5.1.2 
Dosage, Preparation, and Administration ..................................................................................................... 35 
5.1.3 
Repeated darTregs Manufacturing ............................................................................................................... 36 
 
Thymoglobulin® (Anti-Thymocyte Globulin – Rabbit) .......................................................................................... 36 
 
Formulation, Packaging, and Labeling .......................................................................................................... 36 
 
Dosage, Preparation, and Administration (Package Insert) .......................................................................... 37 
 
Thymoglobulin® Study Dosing and Administration ...................................................................................... 37 
 
Drug Accountability ............................................................................................................................................... 38 
 
Toxicity Prevention and Management (Dose Escalation Plan) ............................................................................. 38 
 
Overview ....................................................................................................................................................... 38 
 
Enrollment Pace and Dynamics ..................................................................................................................... 39 
5.4.2.1. 
Cohort 1................................................................................................................................................. 39 
5.4.2.2. 
Cohorts 2, 3, and 4 ................................................................................................................................ 39 
 
Rationale for Staggering Intervals ................................................................................................................. 39 
 
Management Plan for Out of Range darTreg Doses ..................................................................................... 40 
 
Other Medications ........................................................................................................................................................ 41 
 
Immunosuppressive Medications ......................................................................................................................... 41 
 
Zortress® (Everolimus) .................................................................................................................................. 41 
 
Tacrolimus ..................................................................................................................................................... 41 
 
MMF .............................................................................................................................................................. 41 
 
Prednisone .................................................................................................................................................... 41 
 
Other permitted concomitant medications .................................................................................................. 41 
 
Prophylactic Medications ...................................................................................................................................... 41 
6.2.1 
Recommended Pre-Medications for Thymoglobulin® .................................................................................. 41 
6.2.2 
Pre-Medications for darTreg Infusion ........................................................................................................... 42 
6.2.3 
Anti-Infective Prophylaxis Post-Transplantation .......................................................................................... 42 
 
Vaccinations .......................................................................................................................................................... 42 
 
Prohibited Medications ......................................................................................................................................... 42 
 
Premature Discontinuation of Investigational Therapy ................................................................................................ 43 
 
Premature Discontinuation of Thymoglobulin and/or Treg Supportive IS Regimen ............................................ 43 
 
Premature Discontinuation of darTreg Infusion ................................................................................................... 43 
 
Study Mandated Procedures ........................................................................................................................................ 44 
 
Leukapheresis or Whole Blood Draw for PBMC Collection .................................................................................. 44 
 
Blood draws .......................................................................................................................................................... 44 
 
Liver biopsies ......................................................................................................................................................... 44 
 
Known and Potential Risks and Benefits to Participants .............................................................................................. 45 
 
Risks of Thymoglobulin® (Anti-Thymocyte Globulin – Rabbit) ............................................................................. 45 


 
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Risks of Everolimus, TAC, MMF, and Prednisone.................................................................................................. 46 
 
Risks of darTreg Infusion ....................................................................................................................................... 46 
 
Risks of autologous ex vivo expanded polyclonal Treg infusion ................................................................... 46 
 
Risks of autologous ex vivo expanded darTregs infusion ............................................................................. 46 
 
Risks of Study Mandated Procedures ................................................................................................................... 48 
 
Risks of Leukapheresis .................................................................................................................................. 48 
 
Risks of Blood Draw ....................................................................................................................................... 48 
 
Risks of Liver Biopsy ...................................................................................................................................... 48 
 
Potential Benefits .................................................................................................................................................. 48 
 
Study Visits ................................................................................................................................................................ 49 
 
Stage 1: Screening, Enrollment and PBMC Collection .......................................................................................... 49 
 
Stage 2:  Thymoglobulin® and TAC- or EVR-based Maintenance IS ...................................................................... 49 
 
Follow-up for Cohorts 1a and 1b .......................................................................................................................... 49 
 
Stage 3: darTreg Infusion for Cohorts 2, 3, and 4 ................................................................................................. 50 
 
Unscheduled Visits ................................................................................................................................................ 50 
 
Safety Follow Up ................................................................................................................................................... 50 
 
Visit Windows ....................................................................................................................................................... 50 
 
Mechanistic Assays ................................................................................................................................................... 51 
 
HLA Typing ............................................................................................................................................................ 51 
 
T Cell Phenotype and Function ............................................................................................................................. 51 
 
Donor HLA Antibody ............................................................................................................................................. 52 
 
Histology and Multiplex Immunohistochemistry .................................................................................................. 52 
 
Deuterium Labeling and Detection ....................................................................................................................... 52 
 
TCR Sequencing ..................................................................................................................................................... 53 
 
Biospecimen Storage................................................................................................................................................. 54 
 
Criteria for Participant Completion and Premature Study Termination ................................................................... 55 
 
Participant Completion ......................................................................................................................................... 55 
 
Participant Withdrawal Criteria ............................................................................................................................ 55 
 
Participant Replacement ....................................................................................................................................... 55 
 
Follow-up after Early Study Withdrawal ............................................................................................................... 55 
 
Follow-up after Premature Discontinuation from Stage 1 ........................................................................... 55 
 
Follow-up after Premature Discontinuation from Stage 2 or Stage 3 .......................................................... 55 
 
Safety Monitoring and Reporting ............................................................................................................................. 56 
 
Overview ............................................................................................................................................................... 56 
 
Definitions ............................................................................................................................................................. 56 
 
Adverse Event ............................................................................................................................................... 56 
14.2.1.1 Suspected Adverse Reaction ....................................................................................................................... 56 


 
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14.2.1.2 Unexpected Adverse Event ......................................................................................................................... 56 
 
Serious Adverse Event ................................................................................................................................... 56 
 
Grading and Attribution of Adverse Events .......................................................................................................... 57 
14.3.1 Grading Criteria .................................................................................................................................................. 57 
14.3.2 Attribution Definitions ....................................................................................................................................... 57 
 
Collection and Recording of Adverse Events ........................................................................................................ 58 
14.4.1 Collection Period ................................................................................................................................................ 58 
14.4.2 Collecting Adverse Events .................................................................................................................................. 58 
14.4.3 Exceptions to Collection ..................................................................................................................................... 58 
14.4.4 Recording Adverse Events .................................................................................................................................. 58 
 
Reporting of Serious Adverse Events and Adverse Events ................................................................................... 59 
14.5.1 Reporting of Serious Adverse Events to Sponsor............................................................................................... 59 
14.5.2 Reporting to Health Authority ........................................................................................................................... 59 
14.5.2.1 Annual Reporting ........................................................................................................................................ 59 
14.5.2.2 Expedited Safety Reporting......................................................................................................................... 59 
14.5.3 Reporting of Adverse Events to IRBs/IECs ......................................................................................................... 60 
 
Pregnancy Reporting ............................................................................................................................................. 60 
 
Reporting of Other Safety Information ................................................................................................................. 60 
 
Review of Safety Information ............................................................................................................................... 60 
 
Study Stopping Rules ..................................................................................................................................... 60 
 
Medical Monitor Review ............................................................................................................................... 61 
 
DSMB Review ................................................................................................................................................ 61 
 
Statistical Considerations and Analytical Plan .......................................................................................................... 63 
 
Overview ............................................................................................................................................................... 63 
 
Outcomes .............................................................................................................................................................. 63 
 
Measures to Minimize Bias ................................................................................................................................... 63 
 
Analysis Plan .......................................................................................................................................................... 63 
 
Analysis Samples ........................................................................................................................................... 63 
 
Analysis of Safety Outcomes ......................................................................................................................... 63 
 
Supportive Analyses ...................................................................................................................................... 63 
 
Analyses of Exploratory Outcomes ............................................................................................................... 63 
 
Descriptive Analyses ..................................................................................................................................... 64 
 
Interim Analyses .................................................................................................................................................... 64 
 
Sample Size Considerations .................................................................................................................................. 64 
 
Identification and Access to Source Data ................................................................................................................. 66 
 
Source Data ........................................................................................................................................................... 66 
 
Access to Source Data ........................................................................................................................................... 66 


 
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Protocol Deviations ................................................................................................................................................... 67 
 
Protocol Deviation Definitions .............................................................................................................................. 67 
 
Reporting and Managing Protocol Deviations ...................................................................................................... 67 
 
Ethical Considerations and Compliance with Good Clinical Practice ........................................................................ 68 
 
Statement of Compliance ..................................................................................................................................... 68 
 
Informed Consent Process .................................................................................................................................... 68 
 
Privacy and Confidentiality ................................................................................................................................... 68 
 
Publication Policy ...................................................................................................................................................... 69 
 
References ................................................................................................................................................................ 70 
Appendix 1.  Recipient Schedule of Events (Stages 1 and 2) ................................................................................. 75 
Appendix 2.  Recipient Schedule of Events (Cohort 1a and 1b Follow Up) ............................................................ 76 
Appendix 3.  Recipient Schedule of Events (Stage 3:  Cohorts 2, 3, and 4) ........................................................... 77 
Appendix 4.  Recipient Safety Follow Up after Thymoglobulin (Early Termination from Stage 2) ........................ 78 
Appendix 5.  Recipient Safety Follow Up (Early Termination from Stage 3) ......................................................... 79 
Appendix 6.  Donor Schedule of Events ................................................................................................................. 80 
 


 
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Glossary of Abbreviations 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
AR 
Acute Rejection 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CMV 
Cytomegalovirus 
CNI 
Calcineurin Inhibitor 
CR 
Chronic Rejection 
CRF 
Case Report Form 
CRS 
Cytokine Release Syndrome 
CTCAE 
Common Terminology Criteria for Adverse Events 
DAIT 
Division of Allergy, Immunology, and Transplantation 
dar 
Donor Alloantigen Reactive 
darTregs 
Donor-Alloantigen-Reactive T Regulatory Cells 
DSMB 
Data Safety Monitoring Board 
EBV 
Epstein Barr Virus 
EVR 
Everolimus 
FACS 
Fluorescence Activated Cell Sorting 
FDA 
Food and Drug Administration 
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 
MELD 
Model for End Stage Liver Disease 
MFC 
Multiparameter Flow Cytometry 
MIHC 
Multiplex Immunohistochemistry 


 
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MLR 
Mixed Lymphocyte Reaction 
MMF 
Mycophenolate Mofetil 
mSAP 
Mechanistic Statistical Analysis Plan 
mToR 
Mammalian Target of Rapamycin 
NIAID 
National Institute of Allergy and Infectious Disease 
PI 
Principal Investigator 
 polyTregs 
 Polyclonally expanded Tregs 
PTLD 
Post-Transplant Lymphoproliferative Disorder 
PBMC 
Peripheral Blood Mononuclear Cell 
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 Response (HCV aviremia: 24 weeks after completion of antiviral 
therapy)  
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 (AR) 
 
(1997 Banff Criteria) 
(Demetris A. J., 1997)  
AR occurs as defined by Banff histopathologic criteria. 
Clinical Rejection 
Allograft dysfunction or elevated LTs where a biopsy is either not performed or is 
inconclusive for AR that results in an increase in IS dosing or reinstitution of IS 
Chronic Rejection (CR) 
 
(2000 Banff Criteria) 
(Demetris A. D., 2000) 
Early and late CR will be defined by the 2000 Banff update on histopathologic staging and 
reporting of CR.  Features of early and late chronic rejection 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 
• Luminal narrowing by 
subintimal foam cells 
• Fibrointimal 
proliferation 
Large perihilar 
bile ducts 
• Inflammation damage and focal foam 
cell deposition 
• Mural fibrosis 
 
 
Lost to Follow-up 
Subject who cannot complete study visits due to inability to reach the subject, subject 
relocation, etc. 
Medical Monitor 
NIAID official who is responsible for the safety aspects of this trial. The MM is responsible for 
making the final causal relationship assessment of each serious adverse eventAE. 
Milan Criteria 
The Milan criteria state that a patient with HCC is selected for transplantation when he or 
she has: 
• 
one lesion smaller than 5 cm. 
• 
up to 3 lesions smaller than 3 cm. 
• 
no extrahepatic manifestations 
• 
no vascular invasion 


 
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NIAID Project Manager 
NIAID assigned project manager who is responsible for all day to day protocol related issues, 
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 
standard of care). 
Regulatory Affairs 
Officer 
NIAID assigned officer responsible for regulatory aspects of study, including IND submissions 
and other communications with FDA, as applicable. 
Rejection Activity Index 
A numeric score that is part of the 1997 Banff schema for liver rejection.  The RAI is based on 
adding 3 scores related to portal inflammation, bile duct inflammation, and venous 
endothelial inflammation. 
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. 
Severe Rejection  
(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 that 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 AR, but 
these occur in association with none or mild portal 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". 
Steroid Refractory 
Rejection 
Rejection that has failed to resolve with corticosteroid treatment, necessitating treatment 
with an antibody preparation such as Thymoglobulin®. 
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 (darTregs and Thymoglobulin®) and all protocol required 
medications.  
Withdrawal from 
Therapy 
Subject who stops study therapy prior to protocol described duration. 
 
 


 
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 Background and Rationale  
 Background and Scientific Rationale 
Although ongoing refinement of immunosuppression (IS) regimens has substantially reduced the incidence of AR after 
solid organ transplant, long-term outcomes have stagnated partly due to morbidity and mortality associated with 
generalized, lifelong IS.  The traditional approach to IS has emphasized non-specific suppression of T cell responses.  The 
more recent elucidation of regulatory T cells (Tregs) and their importance in regulating immune responses has encouraged 
the reconfiguration of IS regimens to favor Treg development and function with the ultimate goal of inducing graft 
tolerance (Waldmann, 2008) (Kang, 2007) (Walsh, 2004) (Yeung, 2009) (Sanchez-Fueyo A. S., 2006) (Sagoo P. G., 2008) 
(Long, 2009).  Multiple animal models have shown that adoptive transfer of Tregs can mitigate graft rejection and, in 
combination with “Treg-supportive” IS regimens, can induce long-term tolerance (Kang, 2007) (Riley, 2009) (Issa, 2010) 
(Nadig, 2010).   Treg-supportive IS regimens have included the initial de-bulking of donor-specific T cells.  Thymoglobulin®, 
a commonly used T-cell depleting agent in transplantation, appears to relatively spare Tregs (Sewgobind, 2009), thereby 
increasing Treg: T conventional cell (Tconv) ratio.  Additionally, mammalian Target of Rapamycin (mTOR) inhibitors, a class 
of drug to which everolimus (EVR) belongs, suppress effector T-cells while fostering Treg development (Demirkiran A. T., 
2008) (Demirkiran A. V., 2009).  We aim to translate these basic and clinical findings into a viable clinical protocol.  We 
propose to test the use of donor alloantigen-reactive Tregs (darTregs) in the context of T cell depletion, followed by either 
an EVR-based or TAC-based maintenance IS regimen as an approach to induce liver transplant tolerance.  For several 
reasons, the liver transplant setting is ideal to evaluate the safety of Treg therapy as a strategy to either increase the 
likelihood of and/or accelerate the development of tolerance.  First, liver allografts appear to be more tolerogenic than 
other allografts.  Compared to recipients of other organs, liver transplant recipients require less IS and are relatively spared 
from both humoral and CR.  Second, AR, albeit common, is readily treated without long-term sequelae, a key advantage 
for testing novel immunomodulatory agents and tolerance induction strategies.  Third, emerging data from IS withdrawal 
trials in liver transplant recipients indicate that the rate of spontaneous tolerance increases over time after 
transplantation, from less than 10% within 3 years to as high as 80% after 10 years (Bohne, 2012) (Sanchez-Fueyo A. , 
2011) (Feng S. U., 2012).  As the first step toward a long-term goal of Treg immunotherapy, we propose to determine the 
safety of darTregs in combination with T cell depletion followed by EVR- or TAC-based Treg-supportive in adult, de novo 
liver transplant recipients.  We hypothesize that ex vivo-expanded darTregs administered to adult, de novo liver transplant 
recipients in combination with T cell depletion followed by Treg-supportive maintenance IS will be safe.  Our study also 
aims to describe for the first time the persistence of administered darTregs in humans, as well as to describe the effects of 
T cell depletion and Treg-supportive IS on darTreg and darTconv after transplantation.  If successful, our study will define 
an approach for the therapeutic administration of Tregs, establish a new paradigm for the design of IS regimens, and set 
the stage for subsequent efficacy trials. 
 Rationale for Selection of Investigational Product or Intervention 
 
Rationale for Treg Therapy 
Liver transplantation can be life-saving therapy for liver failure.  However, the maintenance of the transplanted liver 
requires continuous IS to prevent rejection by the host immune system.  Although IS regimens have been refined over the 
last 20 years, with significant improvement in the prevention of AR, long-term outcomes have remained stagnant and the 
side effects of non-specific IS continue to cause morbidity and mortality (Feng S. , 2008).  Therefore, a main focus of 
research has been to promote tolerance to transplanted livers so that IS can be minimized or completely withdrawn 
(Bohne, 2012) (Sanchez-Fueyo A. , 2011) (Feng S. U., 2012) (Londono, 2012). 
 
In the past decade, we have learned that tolerance in organ transplantation is linked to the development and persistence 
of Tregs (Waldmann, 2008) (Kang, 2007) (Walsh, 2004) (Yeung, 2009) (Sanchez-Fueyo A. S., 2006) (Sagoo P. G., 2008) 
(Long, 2009).  Therapeutic administration of Tregs has proven efficacy to control allograft rejection in multiple animal 
models through bystander suppression and infectious tolerance (Kang, 2007) (Riley, 2009) (Issa, 2010) (Nadig, 2010).  In 
the setting of transplantation, most studies have utilized darTregs.  These Tregs have been either isolated from tolerant 
animals that have higher frequencies of darTregs, or enriched or expanded in vitro using donor-type stimulator cells.  On 
a cell for cell basis, our laboratory has demonstrated that darTregs are 10 to 100 times more potent at inhibiting 
alloreactive Tconvs in vitro than polyclonal Tregs (Brennan, 2011), and are approximately 5-6 times more potent on a cell 


 
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for cell basis than polyclonal Tregs in preventing rejection in a murine islet transplant model (Lee K, 2014).  Moreover, 
darTregs are potentially safer than non-specific Tregs by offering targeted therapy instead of polyclonal, indiscriminate 
regulation that could theoretically lead to more general IS (Golshayan, 2007) (Raimondi, 2010).  A GMP-compliant protocol 
to expand polyclonal Tregs 320 to 1250 fold ex vivo has been developed by our group and is currently being utilized for a 
trial in Type I diabetic patients  (Putnam, 2009).  Our group has subsequently developed a modified protocol to expand 
darTregs, allowing 200 to 1500 fold expansion with >70% of the Treg product demonstrating donor reactivity (Putnam AL, 
2013).  This protocol presents the opportunity to test the utility of darTregs in clinical organ transplantation.  
 
Studies in mouse models of transplantation show that a high ratio of Tregs to conventional T (Tconv) cells such as 1:1 to 
1:2, i.e. 33 to 50% of Tregs, is needed to prevent transplant rejection (Hara, 2001) (Graca, 2002), and tolerogenic treatment 
leads to early accumulation of 30% Tregs in the grafts (Fan, 2010).  Tregs exist in equilibrium with Tconvs in the steady 
state and the percentages of Tregs remain constant in an individual for years.  To alter this equilibrium in favor of Tregs, a 
combination of lymphodepleting the host and infusing expanded Tregs is needed. 
 
A 70 Kg young adult human has 460 x 109 lymphocytes, 165.5 x 109 CD4+ T cells, and 13.1 x109 Tregs [4].  Using the 
immunosuppressive regimen described in this protocol, we estimate that at 10-12 week after transplant, CD4+ T cell count 
will be reduced to 25%, 41 x 109, and 10%, 4 x 109, will be Tregs.  Thus, infusion of 14.5 x 109 polyclonal Tregs will increase 
the overall Treg frequency to 33%.  darTregs are reported to be 10 to 100 times more effective at suppressing Tconv 
proliferation to alloantigens than polyclonal Tregs in in vitro suppression assays (Peters, 2008) (Chen, 2009) (Sagoo P. e., 
2011) (Veerapathran, 2011).  This means only 1/100th to 1/10th darTregs, 0.14 x 109to 1.4 x 109, are needed to achieve 
the same efficacy as polyclonal Tregs.  
 
It should be noted that the number of Tregs needed to prevent rejection in humans is unknown.  The human 
immunological makeup is far more complex than that of laboratory mice born and maintained in specific pathogen-free 
cages.  The presence of memory alloreactive T cells due to heterologous immunity in humans makes transplant rejection 
more difficult to control (Adams, 2003) (Selin, 2006).  Therefore, even higher presence of Tregs may be needed to achieve 
efficacy in humans.  On the other hand, most of mouse Treg studies do not use immunosuppressive drugs, which is 
certainly not an option in early clinical trials.  Effective Treg dose in humans will likely vary depending concurrent 
immunosuppressive drugs.  Thus, the numbers we estimate above is an approximation that can be used to guide the 
design of initial dose-seeking Treg therapy clinical trials, through which range of effective doses can be defined. 
 
Based on all above considerations and the current manufacturing capacity, we selected to test three doses of darTregs.  
The initial dose in Cohort 2, 50 x 106, is below the range of estimated effective dose and is designed mainly to obtain safety 
profile of this novel therapy.  The second dose in Cohort 3, 200 x 106, is in the lower range of estimated effective dose. 
The last Cohort 4 will receive the highest dose, 800 x 106, which is what we can currently reliably manufacture.  We believe 
that this dose will most likely have an impact on alloimmune responses in patients. 
 
Rationale for Lymphocyte Depletion using Thymoglobulin® 
darTreg therapy has the potential to induce tolerance to the transplanted organ without impeding conventional immune 
responses.  However, because of the exceptionally high frequency of donor-reactive T cells, “debulking” of the host 
alloreactive repertoire and adjunct IS are needed to create a more favorable setting for Tregs to control alloimmunity and 
to ensure long-term graft tolerance.   
 
In the majority of animal transplant models of Treg therapy, efficacy appears to be optimal in a lymphodepleted host.  This 
is likely due to the extremely high precursor frequency of alloreactive T cells, which can be a barrier to significantly altering 
the darTreg/darTconv ratio with darTreg administration.  Lymphodepletion may favor the engraftment of the 
administered Tregs.  Importantly, Thymoglobulin® has also been shown to favor the growth of Tregs in vitro (Boenisch, 
2012), and to durably increase Treg percentages in humans after transplantation (Tang Q. J., 2012). 
 
Tregs exist in equilibrium with Tconvs in the steady state. The most effective way of altering this equilibrium is by 
lymphodepleting the host and infusing expanded Tregs.  We have estimated that an average 70Kg human has 165.5 x109 


 
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CD4+ T cells and 13.1 x109 Tregs (~8%) (Tang Q. K., 2012).  Thymoglobulin® treatment can deplete 95-99% of T cells from 
peripheral blood within one week after the initiation of the therapy.  Assuming the depletion efficiency in lymphoid tissues 
is similar to that in the peripheral blood as seen in non-human primates (Preville, 2001), the total CD4+ Tconvs will be 
reduced to about 4.6 x 109.  Depletion of Tregs by Thymoglobulin® is slightly less efficient (Lopez, 2006) (Morelon, 2010), 
90-98%, therefore approximately 0.6 x 109 Tregs, 13% of CD4+ cells, will remain after Thymoglobulin® depletion.    T cell 
counts recover from the nadir at one week, reaching an average plateau of 33% pre-transplant level for Tconvs and 41% 
pre-transplant level for Tregs at one month post-therapy (Tang Q. J., 2012).  Thus, at the time of darTreg infusion between 
11-20 weeks post-transplant, we expect that each patient will have a total of 50x109 CD4+ T cells and 5.4 x 109 Tregs.  
Infusion of expanded darTregs at the higher doses of 0.2 x 109 and 0.8 x109 may allow us to see an increase in darTreg 
numbers in circulation, at least for a short period of time post infusion.  Moreover, because endogenous darTregs are 
likely to be approximately 1-10% of the overall Tregs, the darTreg infusions are expected to have a correspondingly larger 
impact on the frequency of darTregs.   
 
In this trial, participants who are medically stable and progressing well after liver transplantation, without the need for 
ICU-level care by 72 hours after transplantation, will receive a short course of Thymoglobulin®, to achieve lymphocyte 
depletion.  The use of induction IS in liver transplantation setting in general and polyclonal Abs such as Thymoglobulin® in 
specific has markedly increased over the past decade (http://optn.transplant.hrsa.gov/ar2009/906a_li.htm). This trend 
likely resulted from implementation of the Model for End Stage Liver Disease (MELD) (Kamath, 2001) allocation policy that 
has advantaged candidates with renal dysfunction, prompting interest in strategies that reduce early post-transplant 
exposure to calcineurin inhibitors (CNIs) that have known nephrotoxicity.  Thymoglobulin® administration has therefore 
been used to delay introduction of CNIs (NCT00117689; NCT00564538; NCT00970073) (Soliman, 2007).   As of 2008, nearly 
30% of liver transplant recipients received induction therapy with approximately 14% receiving a polyclonal, T cell 
depleting agent (Thymoglobulin® or ATGAM).  Thus Thymoglobulin® use in liver transplantation is common and appears 
to be safe (Nair, 2008) (Boillot, 2009) (Soliman, 2007).   
 
Rationale for Everolimus Conversion  
In subjects for whom it is considered safe, Treg-supportive IS will be implemented by reduction in TAC and conversion to 
mTOR inhibitor-based IS at least 30 days after liver transplantation.  We will utilize EVR in this study due to its favorable 
pharmacokinetic profile compared to sirolimus, allowing more rapid adjustment of drug levels.  In contrast to CNIs, mTOR 
inhibitors have been shown in animal and human models to foster the survival and proliferation of Tregs (Raimondi, 2010) 
(Battaglia M. A., 2005) (Battaglia M. A., 2006) (Pothoven, 2010) (Bocian, 2010) (Delgoffe, 2009).  Moreover, recently, in 
February, 2013, EVR has received an indication as a maintenance IS agent in combination with reduced dose TAC if initiated 
30 days or more after liver transplantation.  
 
From a clinical perspective, mTOR inhibitors, as a class of drugs, have had efficacy in several different arenas, including 
the prevention of in-stent stenosis, the treatment of various cancers, and the prevention of rejection in the setting of solid 
organ transplantation.  With respect to oncologic indications, EVR has been approved for the treatment of advanced renal 
cell carcinoma, subependymal glial cell astrocytomas in tuberous sclerosis patients, angiomyolipoma associated with 
tuberous sclerosis complex, breast cancer, and pancreatic neuroendocrine tumors and is being studied for efficacy against 
gastric cancer, hepatocellular carcinoma (HCC), and lymphoma.  In transplantation, EVR has been approved for the 
prophylaxis of AR in adult kidney transplant recipients.  A registration trial entitled “A 24 month, multicenter, open-label, 
randomized, controlled study to evaluate the efficacy and safety of concentration-controlled EVR to eliminate or to reduce 
TAC compared to TAC in de novo liver transplant recipients” has been conducted (Novartis Trial CRAD001H2304, 2007-
001821-85; NCT00622869) (Di Simone, 2012).  Based on the results of this worldwide clinical trial, the United States Food 
and Drug Administration has given EVR an indication as a maintenance IS agent for liver transplant recipients. 
  
In this protocol, eligible participants will be treated with a regimen of low dose TAC (target trough levels of 3 – 5µg/L) and 
EVR (target trough levels of 6 – 8 µg/L) four to six weeks (not prior to day 30) after liver transplantation.  This closely 
mirrors the dosing and conversion approach used in the trial referenced above which has had outstanding efficacy along 
with excellent safety and tolerability (Di Simone, 2012) compared to the control arm of TAC alone.  At one year after 
transplantation, patients in the EVR + reduced TAC arm (comparable to the proposed Treg supportive IS regimen) had a 


 
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combined incidence of 6.7% for the primary efficacy endpoint of death, graft loss, and treated biopsy-proven AR which 
was non-inferior to that observed for the control arm (9.7%) (Di Simone, 2012). The one year incidence of treated biopsy 
proven AR alone was 2.9% compared to 7.0% in the control arm.  The estimated GFR however, was superior in the EVR + 
reduced TAC arm with a difference of 8.50 mL/min/1.73m2, 97.5% CI 3.74, 13.27 mL/min/1.73 m2, p<0.001 for 
superiority).   With delayed introduction of EVR, there was no increase in the frequency of wound healing complications 
that has been a concern with the use of mTOR inhibitors compared to the control arm which did not include EVR 
(Zuckermann, 2011) (Pengel, 2011) (Patel, 2009) (Flechner, 2009) (Di Simone, 2012).  Therefore, Thymoglobulin® followed 
by delayed conversion to EVR, has been carefully designed to maintain adequate IS and support Tregs (both endogenous 
and infused) while avoiding excessive IS. 
 
Rationale for a TAC-based IS Regimen  
Although mTOR-based IS may be ideal for Treg survival, the compelling results of a recent study suggest  that Treg infusion 
to subjects maintained on a regimen of TAC/MMF/prednisone is not only safe but also efficacious  (Todo S, 2016) in adults 
who underwent living donor liver transplantation (with splenectomy).  Subjects received a single dose of 
cyclophosphamide (40 mg/kg) on post-transplant day 8 to cytoreduce conventional Tcells (analogous to thymoglobulin in 
this trial) followed by a single infusion of a variable number of Tregs.  Remarkably, 7 of 10 patients were successfully 
weaned off of IS, beginning at 6 months post-transplant and now are 16 to 33 months off of all IS.  The three patients who 
failed IS withdrawal were reported to have autoimmune etiologies of liver disease.  Thus, despite theoretical concerns of 
the survival and efficacy of Treg administration in the setting of TAC and MMF therapy, there is clinical evidence to suggest 
efficacy.  It should also be noted that the Treg infusions using a variety of Treg products in a multi-national renal transplant 
trial (ONEStudy) have been administered in the setting of TAC/MMF/prednisone IS. 
 
This trial importantly enrolls subjects undergoing deceased donor liver transplantation.  As a consequence of increased 
cold ischemia time and reduced donor quality that results in substantial ischemia / reperfusion injury, a significant 
proportion of deceased donor liver recipients incur severe acute kidney injury in the immediate peri-transplant timeframe.  
Since trajectory of renal recovery is unpredictable, use of mTOR might not be appropriate because of low GFR and/or 
proteinuria.  These subjects will be maintained on a regimen of TAC and MMF, albeit at TAC trough levels and MMF doses 
lower than standard of care to optimize renal function.  We believe that this will be sufficient IS to prevent rejection since 
they have received thymoglobulin, which depletes T cells for months.  Finally, it is also possible that subjects who 
successfully convert to EVR-based IS may experience toxicities that mandate EVR discontinuation concomitant with re-
initiation of MMF.  In light of the results of the recent Japanese trial, allowing darTreg administration in subjects 
maintained on either TAC-EVR or TAC-MMF IS is a justifiable approach.  Obtaining safety and mechanistic data from 
subjects on either IS regimen in the context of darTreg administration will be valuable in proceeding with subsequent 
efficacy trials. 
 
Rationale for Timing of Treg Administration 
Most animal studies of Treg administration have administered Treg at the time of transplantation, in contrast to our study 
which proposes to give Tregs approximately 12-20 weeks after liver transplant.  There are no published studies to our 
knowledge that have examined the optimal timing for Treg administration. In addition, modeling such studies in animals 
is difficult due to the variable responses of animals to conventional IS.  In addition, the administration of T cell depleting 
agents often results in long term graft acceptance without further manipulation.  Liver transplantation in particular is 
difficult to model due to the natural tolerogenicity of liver allografts which often require no IS for long-term survival. 
 
We have chosen to delay Treg administration for several reasons.  First, in the setting of liver transplantation, the potential 
for complications is very high, especially early after transplantation.  In the context of a safety trial, administering Tregs at 
the time of transplant would be prohibitively risky.  In addition, we expect that the window of T cell depletion after 
Thymoglobulin® will last at least until the time of Treg administration, 11-20 weeks after liver transplant.  Thymoglobulin 
induction in renal transplant patients induced CD4 depletion out to at least 28 weeks, with no increase in CD4 counts 
between weeks 12-28 (Tang Q. J., 2012)(See Figure 1).  This timing will also allow us to transition eligible subjects to an 
mTOR-based regimen, which is difficult immediately after liver transplantation for reasons discussed in Section 1.2.2.  
Waiting 11-20 weeks is also beneficial to a safety trial because the incidence of rejection is expected to be lower at that 


 
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time point, allowing a cleaner evaluation of the safety of Treg administration.  Delayed administration will also enable us 
to manufacture darTregs as donor cells are required. 
 
 
Figure 1.  Repopulation of CD4+ T cells after Thymoglobuin induction in renal transplant patients.  X axis denotes weeks 
after transplant and Y axis represents Total cell number/ml blood (x 106).  Open circles, control arm.  Filled circles, 
thymoglobulin induction arm.   
Finally, theoretically, delaying Treg administration may have some benefit because it is well established that Tregs work 
better in a relatively quiescent milieu, rather than a highly pro-inflammatory environment as would be expected after a 
liver transplant.   Overall the timing of Treg administration in this study is designed to maximize safety while potentially 
optimizing efficacy.     
 Preclinical Experience 
 Reactivity of darTregs 
To determine the reactivity of the expanded darTregs toward the allogeneic stimulated B cells (sBc) used for primary 
expansion, we restimulated darTregs harvested on day 16 with the same sBcs used for darTreg expansion.  The 
proliferation of the darTregs was monitored using CFSE dilution (Figure 2.  Reactivity of darTregs in vitro).  On average, 
87.5% (range 72.5 to 95.2%) of the expanded darTregs proliferated in response to sBc stimulation. This essentially matched 
the proliferation achieved using anti-CD3 and anti-CD28 bead stimulation (average 88.8%, range 73.6 to 96%), suggesting 
that vast majority of the Tregs are reactive to the sBc allotype (Figure 2.  Reactivity of darTregs in vitro). 
 
Figure 2.  Reactivity of darTregs in vitro 
 Phenotype of darTregs 
Expanded darTregs were found to be CD3+CD4+ with minimal CD8+ T cell and CD19+ and contamination (Table 1. Phenotype 
of expanded darTregs and Figure 2. Phenotype of darTregs).  The majority of the CD4+ T cells were FOXP3+HELIOS+.  In 
contrast, similarly expanded Tconvs were mostly FOXP3-HELIOS+ (data not shown).  Previous reports have shown that co-
expression of CD27 and CD62L correlated with suppressive activity of the expanded Tregs and could be used to distinguish 
between expanded Tregs and contaminating effector T cells.  We found that Tregs expanded using this protocol were 
mostly CD27+CD62L+.  Lastly, the expanded Tregs have > 80% demethylated TSDR (Table 1. Phenotype of expanded 
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Figure 3. Phenotype of darTregs).  Collectively, the phenotype of Tregs expanded using allogeneic sBcs suggests that they 
are stable and committed Tregs.  
 
Table 1. Phenotype of expanded darTregs 
 
 
CD3+ 
CD19+ 
CD4+ 
CD8+ 
FOXP3+ 
HELIOS+ 
CD62L+CD27+ 
TSDR 
Mean 
97.1 
0.2 
97.1 
0.5 
83.0 
88.2 
85.4 
94.0 
SD 
2.6 
0.2 
1.9 
0.2 
10.8 
6.6 
6.4 
15.5 
n 
14 
14 
14 
14 
14 
14 
10 
10 
 
Figure 3. Phenotype of darTregs 
 
 In Vitro Function of darTregs  
To determine the suppressive activity of darTregs in vitro, we measured the ability of darTregs to suppress one way MLRs.  
In this assay, PBMCs from the same person as the darTregs were stimulated with irradiated PBMCs from the same person 
from whom the allogeneic sBcs were generated.  darTregs and polyclonally expanded Tregs (polyTregs) were added to the 
MLR at a ratio of 1 Treg per 5 PBMCs to 1 Treg per 125 PBMCs. Both polyTregs and darTregs suppressed the MLR (Figure 
4.  In vitro suppression of MLR by polyclonally expanded Tregs and darTregs).  Side-by-side comparison show that the 
darTregs are 5 to 25 fold more potent at suppressing MLR than polyTregs.  
Figure 4.  In vitro suppression of MLR by polyclonally expanded Tregs and darTregs 
 
 In vivo Function of darTregs in a Humanized Mouse Model of Transplantation 
Using a model of alloimmune-mediated injury of human skin allografts (Sagoo P. N., 2011)  we compared the protective 
function of darTregs and PolyTregs. We transplanted BALB/c.Rag2-/-γc-/- mice with human skin from a HLA-DR0401+ donor 
and allow the grafts to heal for 6 weeks.  Then we isolated effector cells by depleting CD25+ cells from PBMC.  These 
effector cells were transferred to the mice with healed in skin grafts alone or in combination with different preparations 
of syngeneic Tregs at a ratio 5:1 effector cells:Treg cells.  PBMC donors were HLA-DR0401- and alloantigen-reactive Tregs 
from these donors were expanded using HLA-DR0401+ CD40L-sBc.  We then monitored the skin grafts in these mice until 
rejection or until up to a maximum of 6 weeks after PBMC reconstitution when the grafts were collected for histological 


 
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analysis.  Levels of human leukocyte engraftment in spleens were similar in the three groups of mice that received human 
PBMC alone or in combination with Tregs (Figure 5A).  No animal developed xenogeneic graft-versus-host disease 
symptoms confirmed by the maintenance of stable body weight (Figure 5B). 
 
Figure 5. Characteristics of Characteristics of the humanized mouse model of skin transplantation. A. PBMC 
reconstitution was determined at the end of the experiment, demonstrating that co-infusion of Tregs did not significantly 
alter the extent of PBMC reconstitution.  B. Body weight of the BALB/c.Rag2-/-γc-/- mice in four experimental groups was 
assessed to determine general health status, demonstrating that PBMC infusion did not induce graft-versus-host disease 
 
We then analyzed the skin grafts using immunofluorescent microscopy (Figure 6).  Compared to the skin grafts in control 
animals that did not receive PBMCs (Figure 6A), skin grafts in the PBMC alone group showed intense human CD45+ 
mononuclear cell infiltrates at the dermo-epidermal junctions with concomitant increase in keratinocyte proliferation, 
loss of involucrin, and decreased vascularization as indicated by the reduction in clustered CD31+ cells in the dermis (Figure 
6B).  These changes revealed active skin inflammation and loss of dermo-epidermal integrity mediated by the allogeneic 
human leukocytes.  All these inflammatory parameters in the grafts were reduced by co-injection of PolyTregs, correlating 
with an increase in FOXP3+ cells (Figure 6C).  Strikingly, skin grafts in mice that received alloantigen-reactive Tregs were 
nearly completely protected from histological features of graft injury and were indistinguishable from those in control 
grafts except for the infiltration of FOXP3+ cells at the dermo-epidermal junctions (Figure 6D).  Quantitative analysis of 
these histological findings demonstrated significant reduction in Ki67+ keratinocytes, increase in CD31+ vascular 
endothelial cells, correlating with significantly higher FOXP3+ to CD3+ cell ratios in grafts of mice injected with alloantigen-
reactive Tregs when compared to those in mice treated with polyTregs (Figure 6E). At a ratio of 5:1 effector:Tregs, 
darTregs, but not polyTregs, completely protected the skin grafts from pathological changes induced by the effectors cells. 


 
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Figure 6.  Suppression of skin allograft injury by PolyTregs and darTregs in vivo in a humanized mouse model. A to D. 
Skin graft integrity and human leukocyte infiltration were analyzed using immunofluorescent microscopy and 
representative results for each experimental group is shown. E. immunofluorescence micrograph images were analyzed by 
counting 4 to 6 high-powered visual fields per stain for each graft. Quantitative results from four experimental groups were 
then compared. One-way ANOVA with Bonferroni multiple comparison test was used to determine the statistical 
significance of the differences. 
 Clinical Experience with Treg Therapy 
The cellular product to be utilized in this clinical trial has not been previously used in humans.  Other relevant clinical 
experience with Treg infusion is in quite distinct settings. Three trials of Treg therapy for graft versus host disease (GvHD) 
in patients have been reported  (Trzonkowski, 2009)  (Brunstein, 2010) (Di lanni, 2011). The first-in-man cases by 
Trzonkowski et al involved two patients. The first patient had chronic GvHD two years after 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 an accumulative dose of 3 × 106/kg expanded donor Tregs.  A larger scale phase I 
trial by Brunstein et al. was reported in 2010 (Brunstein, 2010).  Twenty-three patients with advanced hematologic 
malignancy 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 blood samples that had 4 to 6 
HLA antigen match 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 detectable in circulation for up to 7 days.  During the one-year period after 
Treg infusion, the investigators observed no DLTs or increase in adverse events (AEs) when compared to historical controls. 
Incidences of severe acute GvHD were significantly reduced in patients who received Treg therapy.  The median time to 
development of GvHD in patients who received Treg infusion was longer compared to normal disease course although the 
delay was not significant.  The third 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 and Tconv transplants from the same donors.  The majority of 


 
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the patients received 2 × 106/kg Tregs with 1 × 106/kg Tconvs.  No adjunct IS was given after transplant. Patients 
demonstrated accelerated immune reconstitution, reduced CMV reactivation, and a lower incidence of tumor relapse and 
GvHD when compared to historical controls.  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), and CD4+CD127- Tregs sorted from CD4+CD25+ CliniMACS selected population (R. Negrin, Stanford).   
 
Recently, a clinical trial infusing Tregs in diabetic children was reported (Marek-Trzonkowska N., 2012).  The investigators 
administered Tregs in 10 type 1 diabetic children (aged 8-16 years) within 2 months after diagnosis.  In total, 4 patients 
received 10 × 106 Tregs/kg body wt, and the remaining 6 patients received 20 × 106 Tregs/kg body wt. The preparation 
consisted of sorted autologous CD3+CD4+CD127lo/-CD25+ Tregs expanded under good manufacturing practice conditions.  
No toxicity of the therapy was noted. A significant increase in the percentage of Tregs 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 
UI/kg body wt of insulin daily, with 2 patients out of insulin completely, whereas in the untreated group 6 out 10 required 
>0.5UI/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. 
   
The prior experience with polyclonal CD4+ T cells has not suggested significant safety concerns with the administration of 
ex vivo expanded autologous cells.  Over 400 infusions administered to subjects for treatment of HIV and myeloma have 
been performed by Bruce Levine and Carl June at the University of Pennsylvania (Levine B. , 2008).  The cellular products 
administered in these clinical trials were activated CD4+ and CD4+/CD8+ T cell polyclonal populations, which inherently 
have a high associated risk due the presence of T effector cells.  The proven safety of these potentially high risk populations 
in conjunction with the low risk of the presence of contaminating effector cells in the proposed product advocates a low 
probability of toxicity of ex vivo expanded autologous CD4+127lo/-25+ regulatory T cells.  Notably, the inclusion of CD127 
marker in our sorting procedure allows us to discriminate between T effector cells and true Tregs (Liu W., 2006).  
 
Our manufacturing group is producing polyclonal Tregs for a phase I trial of ex-vivo expanded, polyclonal Tregs in recent 
onset type I diabetics.  The trial completed enrollment in October 2013.  A total of 26 potential participants signed consent 
and underwent screening assessments.  Sixteen subjects were determined to be eligible and were enrolled.  Two of these 
sixteen subjects were withdrawn from the study due to failure of Treg product to meet release criteria.  Fourteen subjects 
(age range 18-43, 6 females and 8 males) were treated with a single dose of Ex Vivo Expanded Autologous CD4+CD127lo/-
CD25+ Polyclonal Tregs. Three subjects were treated in the first dosing cohort with 0.05x108 cells, three subjects were 
treated in the second dosing cohort with 0.4x108 cells, 4 subjects were treated in the third dosing cohort with a target 
dose of 3.2 x 108 cells, and 4 subjects were treated in the fourth dosing cohort with a target dose 26 x 108 cells. As of 
February 2014, all 14 subjects were active in the study.  The first treated subject has completed the scheduled 2.5-year 
follow-up visit, and the last treated subject has completed the scheduled 3 month follow-up visit. 
 
As of December 2013, 91 AEs have been reported in 15 subjects since the beginning of the trial.  All 14 treated subjects 
reported at least 1 AE. One subject who underwent phlebotomy but was withdrawn before treatment reported 1 AE 
before withdrawal from the trial. Fifty-eight events were judged as grade 1 in severity, 25 were judged as grade 2, and 8 
were judged as grade 3. There were no grade 4 or 5 AEs.  Thirty events were judged to be possibly related, 15 unlikely 
related, and 46 unrelated to study therapy. Three SAEs have been reported. One judged unlikely and two judged unrelated 
to study participation.  
 
The most common system organ class affected was Infections and Infestations followed by Gastrointestinal Disorders and 
General Disorders and Administration Site Conditions. Of 22 infections recorded, 13 were upper respiratory infections. Of 
those, nine were judged grade 1 and 4 were judge grade 2 in severity. One infection, reported as grade 2 pharyngitis, was 
subsequently demonstrated to reflect a new CMV infection that occurred prior to treatment with Tregs.  
 


 
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Two of the patients in cohorts 3 and one of the patients in cohort 4 are at Yale.  Whole blood collected from the Yale 
patients were shipped to UCSF for expansion.  After 14 days of expansion, the cellular products were shipped back to Yale 
for infusion, demonstrating the feasibility of the UCSF manufacturing team and facility to support multi-site studies.  In 
addition, three of the cohort 3 patients and all 4 cohort 4 patients received Tregs that were labeled with deuterium during 
expansion, which allowed longitudinal monitoring of the infused cells in circulation.  Six-month follow-up of cohort 3 and 
3-month follow-up of cohort 4 patients show deuterium among circulating Tregs shortly after infusion.  The signals peaked 
in the first two weeks and then decayed with a half-life of approximately 1 month (Figure 7).  The signals from cohort 3 of 
3.2x108 cells remained detectable at 6 months.  Together, these results demonstrate that the technology can detect 
3.2x108 cells infused into lymphoreplete hosts. 
 
Our expanded, darTreg product has a theoretical safety advantage compared to polyclonal Tregs in that darTregs will not 
have as many specificities as polyclonal Tregs, lessening the chance for non-specific IS. This first-in-class effort will generate 
some of the first clinical safety data in this area, provide information on kinetics of persistence, and possibly provide the 
first clues on how darTregs affect the alloimmune response in the setting of liver transplantation. 
 
 
Figure 7 Detection of infused deuterium-labeled Tregs.  
Peripheral blood Tregs were purified from study subjects in the T1D trial at indicated time after Treg infusion. Levels of 
deuterium in the purified Tregs were analyzed using gas chromatography-mass spectrometry. Blue symbols in the graph
represent the 3 subjects in cohort 3 that received 3.2 x 108 Tregs and red symbols represent the 4 subjects in cohort 4 that
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 Study Hypotheses/Objectives 
 Safety Objective 
This study will evaluate the safety, tolerability, and dose limiting toxicities (DLTs) of a darTreg infusion for adult, de novo, 
liver transplant recipients who have received a T cell depleting agent and are maintained on TAC- or EVR-based Treg-
supportive IS regimen. 
 Mechanistic Objectives 
This study will evaluate the effects of the Treg-supportive IS regimens on the immunological profiles of liver transplant 
patients.  This study will also describe for the first time the persistence of administered darTregs in humans. 
 
 


 
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 Study Design 
 Description of Study Design  
This is a multi- center, open-label, dose- escalation, pilot study in which adult subjects undergoing primary solitary liver 
transplant will receive a T cell depletion agent (Thymoglobulin®) followed by Treg-supportive IS regimen based on either 
EVR or reduced-dose TAC (Cohorts 1a and 1b).  Subjects in subsequent cohorts will also receive a single infusion of 
autologous, darTregs (Cohort 2: 50 million darTregs; Cohort 3: 200 million darTregs: or Cohort 4: 800 million darTregs).  
For individual participants, there are up to three successive stages in the study with specific eligibility criteria for each 
stage.  As shown in Figure 8, the three stages are:   
• 
Screening and Enrollment  
• 
Eligibility assessment for EVR conversion and proceeding to T-reg infusion 
• 
darTreg Infusion  
Subjects will be followed for 40 weeks after transplant, during which clinical data along with peripheral blood (PBMCs and 
serum) and liver biopsy samples will be collected and analyzed (Appendix 1, 2 3).  
 
Figure 8.  Three Stages of Trial 
The four cohorts are summarized below, with details about the dose escalation plan in Section 5.4.  
• 
Cohort 1:  3 subjects from Center 1 (Cohort 1a) + 3 subjects from Center 2 (Cohort 1b); these subjects will undergo 
T cell depletion followed by TAC- or EVR-based IS (Stage 1 and 2; they will not receive darTreg infusion (Stage 3)). 
• 
Cohort 2:  3 subjects meeting eligibility criteria for all 3 Stages will receive 50 million darTregs (Dose A range 25-
60 million). 
• 
Cohort 3:  3 subjects meeting eligibility criteria for all 3 Stages will receive 200 million darTregs (Dose B range 100-
240 million). 
• 
Cohort 4:  6 subjects meeting eligibility criteria for all 3 Stages will receive 800 million darTregs (Dose C range 400-
960 million). 
 Safety Outcomes 
The safety, tolerability, and ties of darTreg therapy given within the context of either a TAC- or EVR-based IS regimen will 
be evaluated with the rate of the following events within 40 weeks of transplantation: 
Clinical outcomes that will be described are: 
1. 
Incidence and severity of biopsy-proven AR and/or CR (Demetris A. J., 1997) (Demetris A. D., 2000) 
2. 
Incidence of ≥ Grade 3 infections as defined in Section 14.3.1 
3. 
Incidence of wound complications (≥ CTCAE Grade 3) 


 
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4. 
Incidence of anemia, neutropenia, and/or thrombocytopenia (according to study defined reporting 
requirements, Section 14.3.1)  
5. 
Incidence of AEs attributable to the darTreg infusion including infusion reaction / cytokine release syndrome 
(CRS) (≥ CTCAE Grade 3), and malignant cellular transformation. 
 Mechanistic Outcomes 
Blood and liver biopsy samples from all patients enrolled in the study will be analyzed using MFC, T cell functional assays, 
and/or immunohistochemical techniques to describe the effect of TAC- vs EVR-based IS with or without darTreg 
administration on immune cell profiles over time.  We will also describe the persistence of administered darTregs after 
infusion using TCR repertoire analysis as well as deuterium labeling of administered darTregs. 
 
Mechanistic outcomes that will be assessed are: 
1. 
Treg frequency in peripheral blood using Treg TruCount assay 
2. 
Persistence of infused darTregs in peripheral blood and biopsies using deuterium labeling and T cell repertoire 
analysis 
3. 
darTreg and darTconv frequency in peripheral blood using an in vitro allofrequency assay 
4. 
MFC to assess effect on immune profiles 
5. 
Functional assessment of Tregs in peripheral blood using a MLR suppression assay 
6. 
Histology and immunophenotype of liver biopsies 
 
 


 
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 Selection of Participants and Clinical Sites/Laboratories 
 Rationale for Study Population 
The liver transplant setting was chosen for multiple reasons.  First, the liver is considered an organ of immune privilege. 
As such, it is the only solid organ transplant associated with an appreciable rate of spontaneous operational tolerance 
(Demetris A. J., 2009) (Mazariegos G. , 2011). Although historical evidence was based on sporadic case reports and single 
center experiences (Assy, 2007) (Eason, 2001) (Girlanda, 2005) (Mazariegos G. , 2011) (Mazariegos G. J., 1997) (Oike, 2002) 
(Orlando, 2008) (Pons, 2003) (Ramos, 1995) (Takatsuki, 2001) (Tisone, 2006) (Tryphonopoulos, 2005) (Devlin, 1998), 
several ongoing or recently completed clinical trials have provided strong corroborating evidence (NCT00105235; 
NCT00135694; NCT00320606; NCT00436722; NCT00647283; NCT00668369; NCT01198314) (Martinez-Llordella, 2008) 
(Bohne, 2012) (Feng S. U., 2012)  (Benitez C, 2013).  Emerging data from these experiences and trials indicate that the 
likelihood of spontaneous tolerance increases with time after transplantation with estimated frequency of <10% for 
patients <3 years after transplant, 10% for patients between 3–6 years after transplant, 35% for those between 6–10 years 
after transplant, and >80% for patients >10 years after transplant (Martinez-Llordella, 2008) (Bohne, 2012) (Benitez C, 
2013).  This data suggests a substantial opportunity to increase the likelihood and/or accelerate the development of 
tolerance through novel approaches based on rigorous biological mechanisms.  Second, it is known that, among solid 
organ allografts, the liver is uniquely resilient to rejection processes.  AR, particularly for the non-hepatitis C virus (HCV), 
non-autoimmune population that we aim to enroll, has not exerted a detrimental event on patient or graft survival 
(Wiesner, 1998).  Similarly, in the modern IS era, CR not only occurs rarely (Jain, 2001) but is also amenable to treatment 
(Inomata, 2001) (Blakolmer K. A., 2000) (Blakolmer K. E., 1999). Third, as a corollary of the above, a basic premise 
underlying all of the IS withdrawal trials is that AR occurring in a highly supervised setting, such as a clinical trial, will be 
diagnosed and treated expeditiously, leading to rapid resolution without any long-term sequelae.  Therefore, determining 
an optimal dose of darTregs in this pilot dose escalation trial will set the stage for a Phase II safety and efficacy trial that 
includes attempted IS withdrawal. 
 
MELD Score 
We have chosen MELD score of ≤25 somewhat arbitrarily as the MELD score is a continuous metric of liver disease severity.  
As such, there is no obvious discrete threshold.  A MELD score of 25 corresponds to an approximate 20% risk of death 
from liver disease in 90 days.  This threshold will exclude severely / critically ill patients.   
 
Historically, the median laboratory MELD score of UCSF liver transplant recipients is 19.3 while that of Emory is significantly 
lower.  So a threshold of 25 will exclude less than one third of all deceased donor liver transplant candidates, ensuring the 
feasibility of our study design. 
 
Minimum Circulating Tregs 
We are requiring a minimum threshold of circulating Tregs at the time of recruitment for a practical reason.  In order to 
be able to manufacture a darTreg product, we will need to be able to isolate a sufficient starting population of Tregs from 
the liver transplant candidate through phlebotomy or leukapheresis.  Patients with very low concentrations of Tregs may 
not yield sufficient Treg numbers to support darTreg production. 
 
After transplantation, we do believe that we need to achieve a dominance of Tregs over Tconvs.  We aim to achieve this 
goal by the combination of depletion and administration of increasing doses of darTregs. 
 
HLA-DR Matching 
We are excluding liver transplant recipients who are matched to their donor at both HLA-DR as this will compromise the 
generation of the darTreg product. 
 
 
 


 
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 Inclusion Criteria 
Subjects who meet all of the following criteria are eligible for enrollment as study participants.  Inclusion criteria should 
be re-affirmed as appropriate at each stage of the study.   
1. 
Able to understand and provide informed consent 
2. 
End-stage liver disease and listed for deceased-donor primary solitary liver transplant 
3. 
Between 21 and 70 years of age at the time of study entry /consent 
4. 
Have a calculated MELD score ≤ 25 at the time of study entry /consent 
5. 
Female and male subjects with reproductive potential must agree to use FDA approved methods of birth control 
for the duration of the study. 
6. 
If history of HCV, have completed or are in current treatment for HCV AND have no detectable HCV RNA 
7. 
Subjects with HCC meeting Milan criteria 
 Exclusion Criteria 
 Screening and Enrollment Exclusion Criteria A (Stage 1) 
Below are exclusion criteria to be assessed at study enrollment, prior to Stage 1 study procedures.  Subjects who meet 
any of these criteria are ineligible for study participation.   
1. 
End stage liver disease secondary to autoimmune etiology (autoimmune hepatitis, primary biliary cirrhosis, or 
primary sclerosing cholangitis) 
2. 
History of less than 5 years remission of malignancy, except for 1) HCC or 2), history of adequately treated in-situ 
cervical carcinoma, or adequately treated basal or squamous cell carcinoma of the skin 
3. 
History of previous organ, tissue or cell transplant 
4. 
Serologic evidence of human immunodeficiency 1 or 2 infection 
5. 
Epstein Barr Virus (EBV) sero-negativity (EBV naïve candidates)  
6. 
Cytomegalovirus (CMV) sero-negativity (CMV naïve candidates) if donor is positive 
7. 
Chronic use of systemic glucocorticoids or other IS, or biologic immunomodulators  
8. 
Chronic condition requiring anti-coagulation after liver transplantation 
9. 
Any chronic illness or prior treatment which, in the opinion of the investigator, precludes study participation 
10. Participation in any other studies that involved investigational drugs or regimens in the preceding year 
11. Received any vaccination within 28 days prior to leukapheresis or blood collection for Treg manufacture 
12. Hemoglobin <9.0 g/dL within 10 days prior to screening 
13. Neutrophils <1,500/µL within 10 days prior to screening 
14. Platelets <40,000/µL within 10 days prior to screening 
 Thymoglobulin® Exclusion Criteria B (Stage 2) 
Below are exclusion criteria to be assessed prior to administration of Thymoglobulin®.  Subjects who meet any of these 
criteria should not receive Thymoglobulin®. 
1. 
Calculated MELD score greater than 25 at the time of deceased donor liver transplant 
2. 
Last AFP obtained prior to liver transplantation >400 µg/L for candidates with HCC 
3. 
Unacceptable PBMC product for participants enrolled in Cohorts 2, 3, or 4, per UCSF HICTF manufacturing 
specifications   
4. 
Absence of donor cells for manufacturing for any participant  
5. 
HLA-DR matched to donor at both loci 
6. 
Subject is < 21 or > 70 years of age at the time of transplantation 
7. 
Located in the ICU 72 hours after transplantation 
8. 
Hemoglobin <8.0 g/dL  
9. 
Absolute neutrophil count <1,200/µL  
10. Platelets <40,000/µL  
11. Positive pregnancy test for females of child bearing potential 
12. Unexpected histopathology on back table liver biopsy that contraindicates the initiation of Treg supportive IS 
regimen. 
13. Development of a condition requiring chronic anti-coagulation 


 
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14. Hypersensitivity to rabbit proteins or any excipient in Thymoglobulin® 
15. Detectable HCV RNA (if history of HCV (e.g. serology positive at screen A)) or less than six months after end of 
treatment for HCV at the time of transplantation (i.e. does not meet criteria for SVR)  
 Exclusion Criteria C1 
Below are exclusion criteria to be assessed at day 30-44 after transplantation for continuation in the trial.  All subjects 
regardless of eligibility for EVR conversion, with any of the following will not receive darTregs and will move into safety 
follow up. 
1. Explanted liver with evidence of increased risk of recurrent cancer (HCC tumor burden exceeding the Milan criteria; 
presence of vascular invasion; cholangiocarcinoma morphology) 
2. Insufficient depletion of recipient T cells, defined as a nadir CD3 count ≥50 cells/µL (50 cells/mcL) or total 
lymphocyte count ≥ 0.1 x 109/L if CD3 count is unavailable 
3. Development of a condition requiring chronic anti-coagulation 
4. Clinical evidence of untreated biliary obstruction 
5. ALT >2.0 x upper limit of normal (ULN) 
6. Inability to taper off corticosteroids by 44 days (+/- 2 days) after transplant 
7. Detectable circulating HCV  
 Everolimus Conversion Criteria C2 (Stage 2) 
Below are exclusion criteria to be assessed prior to conversion to EVR-based IS regimen.  Subjects who meet any of these 
criteria should not be converted to EVR-based IS regimen.  EVR cannot be initiated prior to 30 days after liver 
transplantation.  Subjects with any of the following will remain on TAC-based IS regimen. 
1. 
Evidence of hepatic artery stenosis or thrombosis by Doppler examination or angiography within 7 days prior to  
conversion 
2. 
Urine protein/creatinine ratio >1.0 mg/mg within 7 days prior to conversion 
3. 
Calculated GFR less than 30 ml/min per MDRD4 equation within 7 days prior to conversion 
4. 
Physical examination documentation of abnormal wound healing or uncontrolled wound infection 
5. 
Hemoglobin <8.0  g/dL within 7 days prior to conversion 
6. 
Absolute neutrophil count <1,200/µL within 7 days prior to conversion 
7. 
Platelets <50,000/µL. within  7 days prior to conversion 
 darTreg Infusion Exclusion Criteria D (Stage 3) 
Below are exclusion criteria to be assessed prior to darTreg infusion for subjects in Cohorts 2, 3, and 4 only.  Subjects in 
Cohort 2, 3, or 4 who meet any of these criteria should not receive a darTreg-infusion: 
1. 
Inability or unwillingness of participant to give additional written informed consent  
2. 
Unacceptable darTreg product 
3. 
Detectable circulating EBV or CMV DNA within 10 days prior to darTreg infusion 
4. 
Detectable HBV DNA within 10 days prior to darTreg infusion 
5. 
Detectable circulating HCV RNA within 10 days prior to darTreg infusion 
6. 
ALT >1.5x ULN within 10 days of darTreg infusion 
7. 
Most recent, but not greater than 10 days prior to darTreg infusion, 12 hour TAC trough levels of > 8 µg/L for all 
subjects  
8. 
Most recent, but not greater than 10 days prior to darTreg infusion, 24 hour EVR trough levels of < 5 µg/L for all 
subjects on EVR 
9. 
For subjects on EVR-based IS, received MMF within 10 days prior to darTreg infusion  
10. Evidence of AR or CR according to Banff criteria on protocol allograft biopsy based on local assessment 
11. Received any vaccination within 14 days prior to darTreg infusion 
12. Positive pregnancy test for females of child bearing potential  
13. Inability or unwillingness of participant to comply with study protocol or procedures 
14. Calculated GFR less than 40 ml/min per MDRD4 equation within 10 days prior to infusion 
 


 
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 Selection of Clinical Sites  
UCSF performs approximately 130-140 adult liver transplants for chronic liver disease each year and has had outstanding 
outcomes based on SRTR data. At the time of protocol design, the median calculated MELD score at transplant is 19.3 
(Washburn, 2009) such that approximately 65% would have been eligible based on a calculated MELD score of ≤25 at the 
time of transplant.  However, multiple changes in US liver allocation policy has substantially changed the landscape of liver 
transplantation. Moreover, throughout the country, the median MELD score at transplant has increased significantly. At 
UCSF, nearly all subjects who undergo deceased donor liver transplantation across all blood groups with a laboratory 
MELD score of ≤25 are those who have been granted MELD exception points.  The vast majority of these are candidates 
with HCC which has, historically, accounted for approximately 30-35% of the annual deceased donor liver transplant 
volume.  Exclusion of candidates with active HCV in conjunction with HCC leaves approximately 15% or 20-22 recipients 
eligible for trial participation annually.  Because this trial has additional eligibility assessment steps after enrollment, 
including donor eligibility for use of cells for a cellular product, we anticipate some attrition at each phase.  Therefore, we 
aim to enroll a minimum of 6-8 patients per year to ensure that 4-5 will progress to darTreg infusion.  The pace of 
enrollment was on par with this plan until October 2015, when the “Wait and Cap” allocation policy for HCC was 
implemented.  The aim of this policy change is to better equalize waitlist mortality of patients with and without HCC.  Since 
the HCC MELD exception score is now capped at 34 and donor livers are regionally shared for candidates with MELD scores 
≥35, HCC candidates in Region 5 (UCSF), a region notorious for transplanting at high MELD scores, have much reduced 
access to transplantation.  Our partnership with large transplant centers outside of our region is therefore critical to 
achieve the enrollment goals for this trial.  The UCSF investigators have selected to partner with Mayo Clinic Rochester, 
Rochester, Minnesota and Northwestern Memorial Hospital, Chicago, Illinois, for many reasons.  Mayo Clinic Rochester 
and Northwestern perform approximately 75-80 and 80-95, respectively, adult liver transplants for chronic liver disease 
on an annual basis.  Both centers are located in United Network for Organ Sharing Region 7 while UCSF is located in Region 
5.  These two regions differ significantly in the MELD score required to access a deceased donor organ.  The lower disease 
severity at Mayo Clinic Rochester will increase the proportion of transplant recipients eligible to participate in this trial.  
  
In addition to the complementary clinical profile, the most compelling reason for us to partner with both Mayo Clinic 
Rochester and Northwestern is the breadth and depth of its infrastructure and expertise to support this highly complex 
trial of a novel therapy.  Investigators at Northwestern produce a polyclonal regulatory T cell product which has been 
administered to living donor kidney recipients to prevent rejection in a Phase I clinical trial (NCT02145325).  Both centers 
are also participating in another clinical trial administering darTreg therapy to facilitate IS withdrawal among adult living 
donor liver transplant recipients (CTOTC-12/ NCT02474199).  Participation in two trials involving the same product in a 
similar patient population will substantially increase their familiarity with the study procedures and processes and 
enhance their ability to conduct the proposed trial with high fidelity.  The combined infrastructure supporting both trials 
will escalate the commitment to the proper collection, processing, and shipment of samples for both darTreg 
manufacturing and for the accompanying mechanistic assays.  
 
 


 
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 Investigational Agents  
 Deuterium-labelled darTreg Product 
The darTreg product will be manufactured at Human Islet and Cellular Transplantation Facility (HICTF) and GMP Facility:  
UCSF Mission Center Building  
Rooms M640-650 
1855 Folsom Street 
San Francisco, CA 94103 
 Manufacturing, Formulation, Packaging, and Labeling 
Recipient T cell Banking 
Peripheral blood leukocytes will be collected via leukapheresis from participants enrolled into Cohorts 3 and 4 after 
successful screening and prior to transplantation.  Cohort 2 subjects might have leukapheresis or whole blood collection 
for PBMC isolation.  The whole blood or leukapheresis product will be transported to the manufacturing facility. PBMCs 
will be isolated using Ficoll density gradient centrifugation and cryopreserved in liquid nitrogen until the patient is eligible 
for darTreg infusion.  If HBV DNA is greater than 1,000 IU/mL at the time of PBMC collection, then the resulting PBMC 
product will not be utilized to manufacture darTregs.  At least 2x109 PBMCs (leukapheresis) or 2x108 PBMC (whole blood) 
are expected to be collected and banked from each subject.  If the whole blood or leukapheresis product does not contain 
sufficient numbers of Tregs, phlebotomy or leukapheresis can be repeated to ensure an adequate cell number for product 
manufacture before transplant.  
 
Donor B cell (sBcs) Banking and Activation 
On the day of transplantation, donor spleen and/or whole blood will be collected and transported to the manufacturing 
facility for processing into a single cell suspension followed by stimulation and expansion with irradiated KT64-
CD40L.HLADR0401 cells and expanded for 10 days in the presence of recombinant human IL-4, cyclosporine A and 
ganciclovir.  The CD40L-stimulated donor sBcs will be sorted, irradiated, and cryopreserved until needed for darTreg 
expansion.  Failure to collect donor cells will disqualify a subject from further trial participation for participants in Cohorts 
2, 3, or 4.  Participants in Cohorts 1a and 1b will be allowed to continue in the trial since they are not scheduled to undergo 
darTreg infusion.   
 
Treg Expansion 
The banked recipient PBMCs will be thawed, washed, stained and sorted for CD4+127lo/-25+ Tregs.  Purified Tregs will be 
cultured with irradiated sBcs at a ratio of 4:1 in deuterated media.  On day 11, cells will be further stimulated with beads 
conjugated with anti-CD3 and anti-CD28 mAbs-conjugated beads at a 1:1 ratio.  Beads will be removed and the appropriate 
number of cells will be counted and re-suspended in 100 mL of infusion media (see below) at the appropriate 
concentration, and aseptically filled in infusion bags.  The final product will be quarantined at 4°C until release. 
 
Each lot will be released for infusion if it meets the pre-defined specifications criteria, including ≥95% CD4+, ≥60% FOXP3+, 
≥75% TSDR+, ≤5% CD8+, ≤1% CD19+.  
5.1.1.1 darTreg Formulation, Packaging and Labeling 
The deuterium-labelled darTreg product is fresh, non-cryopreserved, sterile cell suspension of ≥85% viable, ≥95% CD4+, 
≥60% FoxP3+-, cells for IV administration of a single dose containing 50 (range: 25-60 x 106 cells ), 200 (range: 100-240 x 
106 cells ) or 800 x 106 (range: 400-960 x 106 cells) darTregs and formulated in 100 mL of sterile infusion solution containing 
49.02% (v/v) PlasmaLyte-A USP, 49.02% (v/v) Dextrose 5%-USP, 0.45% NaCl-USP, and 1.96% (v/v) 25% human serum 
albumin.  The product is supplied in a sterile, PVC, 150-mL infusion bag (Charter Medical catalog #T3000).  The final product 
should be stored and maintained at 2-10°C until ready to use.    
 
The product will be released with an interim Certificate of Analysis (COA).  The final COA will be provided once the sterility 
results become available.   
5.1.2 
Dosage, Preparation, and Administration  


 
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The deuterium-labelled darTreg product will be transported within UCSF and between UCSF and remote clinical centers 
in a shipping container with a temperature monitor and a coolant pack(s) to maintain the temperature between 2°C and 
10°C.  The product should be maintained in the shipping container until its administration.  The infusion of the product 
must occur within 30 hours after final formulation.  The identity of the donor, recipient and the product will be verified by 
two individuals prior to infusion. 
 
Subjects meeting eligibility criteria for darTreg infusion will receive a single IV dose containing 50 (range 25-60), 200 (range 
100-240), or 800 (range 400-960) million darTregs administered in approximately 20-30 minutes by gravity.  The line will 
be primed with saline prior to infusion and the product bag, tubing, and peripheral IV line will be flushed with up to 250ml 
normal saline to ensure the complete dose is infused.   
 
Pre-medications will be administered prior to infusion (see Section 6.2.2).     
 
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 CRS.  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 which will allow ongoing monitoring for any infusion-related signs and symptoms. 
5.1.3 
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.  Except for liver biopsy, the laboratory components of Screen D must be 
repeated within 10 days of planned infusion to ensure eligibility.  In any case, infusion of darTregs must take place no later 
than 20 weeks after transplantation.  If the second attempt at manufacturing fails, the subject will go into safety follow 
up (Appendix 4).    
 Thymoglobulin® (Anti-Thymocyte Globulin – Rabbit) 
Thymoglobulin® (ATG) is manufactured by Genzyme Corporation (Cambridge, MA).  Generic forms of the medication are 
also available but, for this trial, generic formulations should not be used.  Thymoglobulin® is licensed for the treatment of 
renal transplant AR in conjunction with concomitant IS.  Currently, there is no indication for the use of Thymoglobulin® as 
an induction therapy in the context of liver transplantation.  Data from the Scientific Registry of Transplant Recipients, 
however, shows that there has been an increasing trend for liver transplant recipients to be treated with induction therapy 
over the past 10 – 15 years.  In 1999, approximately 13% of liver transplant recipients received induction therapy with less 
than 2% receiving a polyclonal agent (Thymoglobulin® or ATGAM).  By 2008, nearly 30% of liver transplant recipients 
received induction therapy with approximately 14% receiving a polyclonal, T cell depleting agent (Thymoglobulin® or 
ATGAM).  Interest in using Thymoglobulin® has been driven by multiple motivations including the desire to reduce or even 
eliminate exposure to corticosteroids and to delay and / or minimize exposure to CNIs in an attempt to reduce 
nephrotoxicity and optimize short and long-term renal function.  Hence, T cell depletion is already an accepted approach 
to IS in liver transplant patients. 
 
For this trial, we have chosen to administer a course of Thymoglobulin® within 72 hours after transplantation with the 
goal of achieving adequate lymphocyte depletion.  We have chosen a dose range, 3.0 – 4.5 mg/kg that balances the desire 
for depletion with hematologic side effects of Thymoglobulin® administration.  This dose range has been shown to be as 
effective at depleting T cells as compared to the more common 6 mg/kg dosing (Wong, 2008).  Moreover, immunologic 
outcomes appear to be indistinguishable between dose ranges (Wong, 2008).  Dose dependent leukopenia and 
thrombocytopenia will be minimized by using lower doses, which will be particularly important for liver transplant patients 
who have depressed leukocyte and platelet counts at baseline.    
 Formulation, Packaging, and Labeling 
Thymoglobulin® is a purified, preservative-free, pasteurized, gamma immune globulin, obtained by immunization of 
rabbits with human thymocytes.    Thymoglobulin® is available as sterile, lyophilized powder to be reconstituted with 
sterile water for Injection (SWFI).  Each package contains a 10 mL vial of freeze-dried Thymoglobulin® (25 mg) NDC# 58468-


 
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0080-1.  Each 10 mL vial contains 25 mg anti-thymocyte globulin (rabbit) as well as 50 mg glycine, 50 mg mannitol, and 10 
mg sodium chloride.  After reconstitution with 5 mL SWFI, each vial of reconstituted product contains approximately 5 
mg/mL of Thymoglobulin®, of which >90% is rabbit gamma immune globulin (IgG).  The reconstituted solution has a pH of 
6.5 - 7.2.   
 
Vials should be stored in a refrigerator at 2°C to 8°C (36°F to 46°F) and protected from light.  The vials should not be frozen 
and should not be used after the expiration date indicated on the label.  Reconstituted Thymoglobulin® is physically and 
chemically stable for up to 24 hours at room temperature; however, room temperature storage is not recommended.  As 
Thymoglobulin® contains no preservatives; the reconstituted product should be used immediately.  Any unused drug 
remaining after infusion must be discarded. 
 Dosage, Preparation, and Administration (Package Insert) 
Reconstitution  
After calculating the number of vials needed, using aseptic technique, reconstitute each vial of Thymoglobulin® with 5 mL 
of SWFI.   
1. Allow Thymoglobulin® vials to reach room temperature before reconstituting the lyophilized product. 
2. Aseptically remove caps to expose rubber stoppers. 
3. Clean stoppers with germicidal or alcohol swab. 
4. Aseptically reconstitute each vial of Thymoglobulin® lyophilized powder with the 5 mL of SWFI. 
5. Rotate vial gently until powder is completely dissolved. Each reconstituted vial contains 25 mg or 5 mg/mL of 
Thymoglobulin®. 
6. Inspect solution for particulate matter after reconstitution. Should some particulate matter remain, continue to 
gently rotate the vial until no particulate matter is visible. If particulate matter persists, discard this vial. 
Dilution 
1. Transfer the contents of the calculated number of Thymoglobulin® vials into the bag of infusion solution (saline 
or dextrose).  Recommended volume: per one vial of Thymoglobulin® use 50 mL of infusion solution (total volume 
usually between 50 to 500 mL). 
2. Mix the solution by inverting the bag gently only once or twice 
Dosage and Administration 
The recommended dosage of Thymoglobulin® for treatment of acute renal graft rejection is 1.5 mg/kg of body weight 
administered daily for 7 to 14 days.   
 
The recommended route of administration is intravenous infusion using a high-flow vein.  Thymoglobulin® should be 
infused over a minimum of 6 hours for the first infusion and over at least 4 hours on subsequent days of therapy.  
Thymoglobulin® should be administered through an in-line 0.22 micrometer filter. 
 Thymoglobulin® Study Dosing and Administration 
For this study, subjects will be given a dose range of 3.0-4.5 mg/kg total.  Subjects who meet eligibility criteria for 
Thymoglobulin® will be given the first dose no later than 72 hours after transplantation.  For transplants occurring during 
overnight hours, the first dose might begin on Day 2.   
 
The first and second doses of Thymoglobulin® must be 0.75 mg/kg administered over 12 hours.  Additional doses of 0.75 
-1.5 mg/kg IV will be administered over 6-12 hours, or at the site investigator’s discretion, until CD3 count is less than or 
equal to 50 cells/µL (50 cells/mcL) or total lymphocyte count is less than or equal to 0.10 x 109/L when CD3 count is 
unavailable; or when the maximal dose of 4.5/mg/kg has been given. 
  


 
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Figure 9. EVR-based Regimen after Thymoglobulin® 
 
Figure 10. TAC-based Regimen after Thymoglobulin® 
 Drug Accountability   
The investigator will maintain adequate records of the disposition of the darTreg products and Thymoglobulin®, including 
the date, lot ID, and dose of the product, dose received, name of the recipient (participant-by-participant accounting), and 
a detailed accounting of any product that is accidentally or deliberately destroyed.  Used vials of Thymoglobulin will be 
maintained on site until drug reconciliation.  No Thymoglobulin vial may be destroyed without the expressed approval of 
study sponsor. 
 
Records for receipt, storage, use, and disposition will be maintained by the study site.  A dispensing log will be kept current 
for each participant.  All records regarding the disposition of the investigational product will be available for inspection.   
 Toxicity Prevention and Management (Dose Escalation Plan) 
 Overview 
Eligible patients will receive either no darTreg infusion (a total of 6 subjects, 3 at each clinical site; Cohorts 1a and 1b) or 
a single infusion of darTregs at one of 3 dose levels: 50, 200, and 800 million (Cohorts 2, 3, and 4).  Three patients will be 
planned for each darTreg dose, with the possibility of expanding to 6 patients for Cohorts 2 and 3 as required; the last 
dosing group will have 6 patients.   
 
Progression from one group to the next will be based on the cumulative frequency and severity of specific AEs as described 
in 14.8. 
Note:  If Myfortic is used, dosing should be 360 mg BID. 
Wk 11-20 
Note:  If Myfortic is used, dosing should be 360 mg BID. 


 
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 Enrollment Pace and Dynamics  
5.4.2.1. Cohort 1 
Subject enrollment will take place in a staggered fashion (Figure 11.  Staggered Enrollment ).  Center 1 will enroll the first 
group of 3 participants (Cohort 1a) that will receive Thymoglobulin®+EVR IS but will not receive darTregs.  Upon 
completion of enrollment and at least 12 weeks of observation for the last participant in Cohort 1a, and in the absence of 
any safety signals, enrollment will proceed in Cohort 2 (darTreg Dose A).  Enrollment into Cohort 2 from participating 
centers will not begin until enrollment in cohort 1b is complete.   
5.4.2.2. Cohorts 2, 3, and 4 
For Cohorts 2, 3, and 4 that will receive escalating darTreg doses, the first subject at each dose will be monitored for 4 
weeks after darTreg infusion to ensure that there are no safety signals (Section 14.8).  Subsequently, a 4-week observation 
period between darTreg infusions for the second and third subjects in the same cohort and between the last subject in 
one cohort and the first darTreg infusion in the next cohort is recommended but not mandatory.  If the expected time 
interval between darTreg infusions is less than two weeks, the PI and NIAID medical monitor will review the status of the 
study and determine whether Thymoglobulin® should be initiated, Progression from one cohort to the next will depend 
on the cumulative incidence of sentinel AEs as described in Section 14.8. 
 
Figure 11.  Staggered Enrollment  
 Rationale for Staggering Intervals 
All participants in Cohort 1a – those who receive Treg supportive IS alone - must be monitored for 12 weeks prior to 
enrollment of Cohort 2 and Cohort 1B by the second clinical site.  The rationale for selecting 12 weeks is that this interval 
covers the time period that has the highest incidence of important post-liver transplant complications such as rejection 
and infection, particularly bacterial and fungal infections related to the transplant operation including those directly 
related to the transplant incision / wound.  
 
Within each darTreg dosing cohort, there is a mandatory four-week observation period after the administration of 
darTregs to the 1st patient of each cohort before the 2nd patient can receive darTregs.  We have selected a shorter 
observation time interval for several reasons.  First, darTregs will be administered sometime between Week 11-20 after 
transplant.  This is a period of relative clinical quiescence that will readily permit optimal identification of AEs that may be 
precipitated by the darTreg infusion.  Liver transplant recipients have largely recovered from the transplant operation as 
well as from the baseline liver disease.  They continue on anti-viral (until 6 months after transplantation) and anti-


 
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pneumocystis prophylaxis (until at least 12 months after transplantation) such that the frequency of these infections is 
very low. Second, we anticipate that the majority of complications associated with darTreg administration (infusion 
reaction, AR, or infection) are most likely to occur early, soon after administration while the beneficial effect of darTreg 
administration may well evolve and/or intensify over time.  Infection may be precipitated by contamination of the darTreg 
preparation with infectious agents or with non-donor-alloantigen-reactive Tregs that lead to generalized over-
immunosuppression.  Similarly, rejection may be precipitated by contamination of the darTreg preparation with T effector 
cells.  In these cases, the deleterious impact of darTreg infusion is expected to manifest soon – within 4 weeks – after 
infusion.  While malignant transformation of infused cells is a concern, the timeframe of this complication as well as its 
manner of presentation is certainly difficult if not impossible to predict and therefore, this safety concern cannot be 
mitigated by any sort of mandatory waiting period.  
 Management Plan for Out of Range darTreg Doses 
For subjects in cohort 2, any product with less than 25 million darTregs will not be infused and subject will be replaced.  In 
cohort 3, products falling short of defined dose range for the cohort will be infused up to maximum of range for cohort 2 
in up to 2 subjects, with the subject(s) contributing to cohort 2.  The UCSF HICTF, NIAID MM, and study PI will review 
available information after the first instance of the dose falling short of target range, and again in the event of a second 
product not meeting the specified dose.  One subject will be allowed to receive study directed Thymoglobulin under 
circumstances described.  Recruitment and enrollment activities can continue during this time.  If a third instance of lower-
than-expected dose is manufactured, the study will be paused and DSMB will review available data prior to the trial 
proceeding.   
 
If a higher dose of darTregs is produced than specified for a cohort, the maximum defined dose for that cohort will be 
packaged in the product and infused.  Cells not infused will be used for research.     
Table 2. RTB-002/delta Dose Ranges 
 
Target Dose for Cohort 
Dose Range 
Cohort 2 
50 million cells 
25-60 million cells 
Cohort 3 
200 million cells 
100-240 million cells 
Cohort 4 
800 million cells 
400-960 million cells 
     
Figure 12. Algorithm for Out of Range darTreg Doses 
Less than
specified dose range
Within 
specified dose range
Greater than 
specified dose range
Infuse within 
dose range for the 
previous cohort 
and analyze 
subject with 
previous cohort
Infuse dose 
within dose range
Infuse maximum 
of dose range
Algorithm for Out of Range darTreg Doses
^ Recruitment/enrollment activities 
can proceed, inclusive of 
leukapheresis
1st instance for 
this cohort?
Is there a subject in the 
study who has received 
Thymoglobulin?
Review 
available 
data^
If number of darTregs manufactured is:
Batch does not
contain enough 
cells for dose 
range in previous 
cohort OR subject 
is in cohort 2
Cells are not
infused, subject is 
terminated and 
replaced
Batch contains enough 
cells for dose range in 
previous cohort
Yes
No
Yes
No
Proceed with manufacture 
and infuse if enough cells for 
dose range in previous cohort
Stop all study procedures 
pending review
 


 
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 Other Medications 
 Immunosuppressive Medications 
 Zortress® (Everolimus) 
For this study, EVR will be used in combination with TAC following taper and cessation of corticosteroids (Figure 9. EVR-
based Regimen after Thymoglobulin®).  The suggested starting dose for this trial is 1.0–1.5 mg bid since our target trough 
level is 6 to 8 µg/mL.  This is based on data from the de novo liver transplant EVR trial. Participants who had a trough level 
of 6-8 µg/mL required a mean (median, standard deviation) daily dose of 4.4 (4.0, 1.75) mg.  Subjects meeting eligibility 
criteria for EVR conversion (Screen C2) will begin EVR no sooner than 30 days after liver transplantation and no later than 
44 days after transplantation; with target trough levels of 6-8 µg/L.  EVR target trough levels will be further reduced to 4-
6 µg/L 24 – 26 weeks after transplantation. 
 Tacrolimus  
Tacrolimus will be initiated within 72 hours of liver transplantation, targeting 12 hour trough levels of 6-8 µg/L.   At 5-6 
weeks after transplantation, after EVR is initiated in eligible subjects, the TAC target 12 hour trough levels will be decreased 
to 3-5 µg/L (Figure 9. EVR-based Regimen after Thymoglobulin®).  Subjects ineligible for EVR conversion will remain on 
TAC with target levels of 3-8 µg/L with MMF (Figure 10. TAC-based Regimen after Thymoglobulin®). 
 
Inability to tolerate TAC leading to discontinuation or conversion to cyclosporine will result in early termination from the 
clinical trial and reversion to standard of care IS.   
 MMF 
MMF will be initiated within 24 hours of transplantation.  If Cellcept is used, then the dosing will be 500 mg twice daily 
intravenously or by mouth.  If Myfortic is used, then the dosing should be 360 mg twice daily by mouth.  For subjects who 
convert to EVR, MMF must be discontinued as soon as target EVR trough levels have been achieved.  Subjects ineligible 
for EVR conversion will remain on Cellcept or Myfortic.   
 Prednisone 
Solumedrol 500 mg will be given intravenously on the day of transplantation.  Additional Solumedrol will be prescribed 
according to center standard of care.  Oral prednisone should be initiated once oral medication is tolerated.  Steroid doses 
may be further tapered on an individualized basis depending on the clinical status of patient and function of graft.   
Corticosteroids must be discontinued by day 44 after transplant.  For subjects eligible for EVR, corticosteroids must be 
discontinued prior to initiating EVR.  
 Other permitted concomitant medications 
Other non-immunosuppressive concomitant medications used as standard of care in the management of the liver 
transplant subject (e.g. cholesterol lowering medications, blood pressure lowering medications, etc.) not specifically 
described in Section 6.3. Prohibited Medications are acceptable in the study. 
 Prophylactic Medications 
6.2.1 
Recommended Pre-Medications for Thymoglobulin® 
Solumedrol 500 mg IV is recommended prior to first dose of Thymoglobulin®; and Solumedrol 250 mg IV prior to the 
second dose of Thymoglobulin®.  Corticosteroid dosing prior to subsequent doses of Thymoglobulin® will be at the site 
investigator’s discretion.  Acetaminophen 500 mg PO and diphenhydramine 50 mg IV 1 hour prior to first and second doses 
of Thymoglobulin® is also recommended.  At the site investigator’s discretion, acetaminophen and diphenhydramine can 
be continued every 6 hours around the clock to cover the timeframe of the first two infusions. 
 
 
 
 


 
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6.2.2 
Pre-Medications for darTreg Infusion  
Pre-medications will be administered 30-60 minutes prior to the darTreg infusion.  Pre-medications will include 650 mg 
acetaminophen and 25-50mg diphenhydramine intravenously or by mouth. 
6.2.3 
Anti-Infective Prophylaxis Post-Transplantation 
Intravenous gancyclovir and/or oral Valcyte will be administered for the prophylaxis of CMV and EBV for at least six months 
after liver transplantation.  Participants should receive center standard of care prophylaxis for candida infection until 
discontinuation of corticosteroids and pneumocystis infection for the duration of the study.   
 Vaccinations 
Subjects should receive seasonal influenza vaccinations as SOC.  However, subjects should not receive any vaccination 
within 28 days prior to blood collection for darTreg manufacturing and/or within 14 days prior to the actual infusion (see 
Schedule of Events and darTreg exclusion criteria).  In addition, the subjects should not have any vaccination for 28 days 
after the date of darTreg infusion.  These requirements are in place to minimize the chance of having enrichment of 
influenza-reactive Tregs in the product and ensure that Treg infusion does not negatively impact influenza immunity 
elicited by the vaccine. 
 Prohibited Medications 
Cyclosporine cannot be used as an alternative to tacrolimus in this trial. The indication for use of EVR in liver 
transplantation for the primary prophylaxis of AR is in combination with tacrolimus.  
 
IS medications other than those specified in the protocol are similarly disallowed.  These include, but are not limited to 
azathioprine, basiliximab, alemtuzumab, sirolimus, rituximab, intravenous immune globulin, and belatacept.   
 
The use of live vaccines will be proscribed during the follow up period, as per standard of care for liver transplant 
recipients.  Examples include (but are not limited to) the following: intranasal influenza, measles, mumps, rubella, oral 
polio, BCG, yellow fever, varicella, and TY21a typhoid vaccines. 
 
Grapefruit and grapefruit juice inhibit cytochrome P450 3A4 and P-gp activity and should therefore be avoided with 
concomitant use of EVR.  Patients with rare hereditary problems of galactose intolerance, the Lapp lactase deficiency or 
glucose-galactose mal absorption should not take EVR as this may result in diarrhea and malabsorption. 
 
Administration of medications known to interact with tacrolimus and/or EVR is allowed but tacrolimus and EVR levels 
should be carefully monitored and dosing titrated to maintain the target levels to minimize toxicity while maintaining 
efficacy. 
 
 
 


 
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 Premature Discontinuation of Investigational Therapy 
 Premature Discontinuation of Thymoglobulin and/or Treg Supportive IS Regimen 
Study IS regimen may be prematurely discontinued for any participant for any of the following reasons: 
1. Inability to tolerate at least 3 mg/kg Thymoglobulin® or inability to deplete CD3 count to less than or equal to 
50µ/L (50/mcL) or total lymphocyte count to less than equal to  0.1 x 109/L after study Thymoglobulin® 
administration 
2. Inability to tolerate TAC- or EVR-based maintenance IS  
3. The diagnosis of severe AR (Banff criteria) at any time after transplantation 
4. The diagnosis of early or late CR (Banff criteria) at any time after transplantation 
5. The occurrence of steroid refractory rejection at any time after transplantation 
6. Poor compliance with study procedures. 
7. Investigator decision that the study IS regimen and/or future study treatment is no longer in the best interest of 
the participant.   
8. Failure to manufacture and supply the cellular product two times for the same subject  
There are a myriad of diagnoses that might lead an investigator to determine that the study IS regimen and/or darTreg 
infusion is no longer in the best interest of the participant.  While there are likely an infinite number of specific reasons, 
general reasons include severe, life-threatening infections, malignancy (excluding skin cancer), GvHD, pneumonitis, severe 
renal dysfunction / proteinuria, serious medical co-morbidity such as myocardial infarction, and logistical issues including 
patient relocation and record of non-compliance.   
 
The (Data Safety Monitoring Board) DSMB will be provided with information about cases of premature discontinuation 
occurring in any stage.  Subjects who have prematurely discontinued the study regimen will enter into a schedule of events 
(SOE) of reduced monitoring intensity for purpose of safety (Please refer to Section 13.4 and Safety Follow-Up SOE) 
 Premature Discontinuation of darTreg Infusion 
darTreg infusion will be stopped and will not be restarted if there is a hypersensitivity reaction, a CTCAE Grade ≥3 infusion-
related reaction, a CTCAE Grade ≥3 CRS, or any other infusion related serious adverse event. 
 
 


 
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 Study Mandated Procedures 
 Leukapheresis or Whole Blood Draw for PBMC Collection 
Leukapheresis may be necessary to ensure collection of adequate numbers of autologous Tregs to support ex vivo 
expansion of darTregs for infusion after liver transplantation, particularly for those enrolled in Cohorts 3 and 4.  The study 
team will make a decision in the best interest of a particular subject based on the current clinical picture including assigned 
study cohort, peripheral Tregs in circulation, and hemogloblin level.  If a subject has a hemoglobin level greater than 10 
gm/dL, he or she will have the option to undergo phlebotomy.  If the patient has a hemoglobin level less than or equal to 
10 gm/dL and remains eligible for the study, the patient will undergo leukapheresis.  Number of Tregs in peripheral 
circulation will also guide decision for method of PBMC collection (leukapheresis or whole blood) where subjects should 
have at least 30/µL peripheral blood Tregs for whole blood collection.  For subjects to have whole blood draw, 450-500ml 
whole blood will be collected.  
 Blood draws 
Blood draws are necessary to carefully and frequently evaluate allograft function after liver transplantation as well as after 
darTreg infusion.  Liver tests are the most reliable marker for post-transplant complications including AR.  Finally, blood 
draws will also be done to enable the planned mechanistic studies aimed at delineating the presence and impact of infused 
darTregs. 
 Liver biopsies  
For all subjects, local pathology information will be obtained for assessment of eligibility criteria B (no central pathology 
assessment).  If a pathology report is unavailable from donor records, a liver biopsy will be obtained on the back table. 
 
For patients enrolled in Cohort 1 (either 1a or 1b), there will be a second protocol liver biopsy performed 12 weeks after 
transplantation for central laboratory assessments.   
 
For subjects enrolled in Cohorts 2, 3, or 4, there are two additional protocol biopsies after the initial, back table biopsy.  
The second protocol liver biopsy will be obtained approximately 10-14 weeks after transplantation, and approximately 7 
– 10 days prior to darTreg infusion to ensure that darTregs will only be infused in the context of a normal allograft with 
essentially normal allograft histology and the absence of any diagnostic criteria for AR or CR.  Eligibility criteria D will be 
assessed with local pathology information.  However, slides and additional tissue will be collected for a central reading 
and mechanistic assays.  The darTregs eligibility biopsy does not have to be repeated if a second preparation of darTregs 
is manufactured and infusion of the product is delayed.  The third and last protocol liver biopsy for Cohorts 2, 3, or 4 will 
be one week after darTreg infusion for central laboratory assessments.   
 
There will be up to two passes for each protocol biopsy.  Up to one additional pass might be required during clinically 
indicated (standard of care) biopsies to obtain adequate tissue for the planned mechanistic studies.  The same set of 
specimens will be collected for each 16g biopsy (protocol driven or clinically indicated/for cause) in the following priority: 
1) 1.5 cm fixed in formalin for histopathology; 2) 1.5-2.0 cm in buffered saline for detection of deuterium labeled cells; 3) 
0.5-1cm in RNALater for gene expression and other mechanistic studies. 
 
 


 
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 Known and Potential Risks and Benefits to Participants 
 Risks of Thymoglobulin® (Anti-Thymocyte Globulin – Rabbit) 
Risks associated with Thymoglobulin® include immune-mediated reactions, infections, reactivation of infection, malaise, 
dizziness, sepsis, thrombocytopenia, neutropenia, and increased incidence of malignancies.  Occasional reactions are 
observed at the infusion site including pain, swelling and erythema. 
 
Immune-mediated reactions 
In rare instances, serious immune-mediated reactions have been reported with the use of Thymoglobulin and consist of 
anaphylaxis or severe cytokine release syndrome (CRS). Very rarely, fatal anaphylaxis has been reported. If an anaphylactic 
reaction occurs, the infusion should be terminated immediately. Medical personnel should be available to treat patients 
who experience anaphylaxis. Emergency treatment such as 0.3 mL to 0.5 mL aqueous epinephrine (1:1000 dilution) 
subcutaneously and other resuscitative measures including oxygen, intravenous fluids, antihistamines, corticosteroids, 
pressor amines, and airway management, as clinically indicated, should be provided. Thymoglobulin or other rabbit 
immunoglobulins should not be administered again for such patients. Severe, acute infusion-associated reactions (IARs) 
are consistent with CRS attributed to the release of cytokines by activated monocytes and lymphocytes. In rare instances, 
these reported reactions are associated with serious cardiorespiratory events and/or death. Thymoglobulin contains a 
mixture primarily of antibodies to T cell antigens, but it is largely unknown which specificities mediate the alteration in 
immunoregulation. Thymoglobulin may potentially contain or promote undesired or harmful antibody specificities, but 
which may be difficult to predict, identify or to exclude. Live vaccines should not be administered to patients about to 
receive, receiving, or after treatment with Thymoglobulin. Concomitant administration of Thymoglobulin with live virus 
vaccines carries a potential of uncontrolled viral replication in the immunosuppressed patient. There is insufficient 
information to fully define the extent of the risk, or the period of time during which the risk exists. If administered, live 
viruses may interfere with Thymoglobulin treatment. Skin testing is not advised prior to Thymoglobulin administration. 
 
Infection 
Thymoglobulin® is routinely used in combination with other IS agents.  Infections (bacterial, fungal, viral and protozoal), 
reactivation of infection (particularly CMV) and sepsis have been reported after Thymoglobulin® administration in 
combination with multiple IS agents.  Severe acute infections can be fatal.   
 
Malignancy 
Use of IS agents, including Thymoglobulin®, may increase the incidence of malignancies, including lymphoma or post-
transplant lymphoproliferative disease (PTLD). 
 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
The carcinogenic and mutagenic potential of Thymoglobulin® and its potential to impair fertility have not been studied. 
 
Hematologic Effects 
Thrombocytopenia and/or leukopenia (including lymphopenia and neutropenia) have been identified and are reversible 
following dose adjustments.  When thrombocytopenia and/or leukopenia are not part of the underlying disease or 
associated with the condition for which Thymoglobulin is being administered, the following dose reductions are suggested: 
• 
A reduction in dosage must be considered if the platelet count is between 50,000 and 75,000 cells/mm3 or if the 
white blood cell count is between 2,000 and 3,000 cells/mm3; 
• 
Stopping Thymoglobulin treatment should be considered if persistent and severe thrombocytopenia (< 50,000 
cells/mm3) occurs or leukopenia (< 2,000 cells/mm3) develops. 
 
White blood cell and platelet counts should be monitored during and after Thymoglobulin therapy 
 
Pregnancy: Pregnancy Category C 
Animal reproduction studies have not been conducted with Thymoglobulin®.  It is also not known whether Thymoglobulin® 
can cause fetal harm or can affect reproduction capacity.  Thymoglobulin® should be given to a pregnant woman only if 


 
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clearly needed.  Pregnancy testing will be routinely performed in female patients pre-transplant.  Although extremely 
unlikely, any pregnant patient will be excluded from the study. 
 Risks of Everolimus, TAC, MMF, and Prednisone 
TAC, MMF, and prednisone are considered standard of care maintenance IS medications in liver transplantation.  EVR is 
not as widely used in liver transplantation but recently received an FDA-approved indication for liver transplantation.  The 
risks for dose related toxicities and side effects from these immunosuppressive medications are not different for subjects 
in this trial.  However, the reduced dosing or target trough levels in conjunction with other agents in the Treg-supportive 
IS regimen may expose subjects to an increased risk of rejection.  
 Risks of darTreg Infusion 
 Risks of autologous ex vivo expanded polyclonal Treg infusion  
Although polyclonal Treg infusion has been innocuous in animal models, there is currently scant experience in humans.  
Three Treg therapy trials in GvHD have been reported.  The first-in-man trial by Trzonkowski et al involved two patients 
(Trzonkowski, 2009).  The first patient had chronic GvHD two years after transplantation.  After receiving 0.1 x 106/Kg flow 
sorted ex vivo expanded Tregs from the donor, the symptoms subsided and the patient was successfully withdrawn from 
IS.  The second patient had acute GvHD disease that progressed despite three infusions with an accumulative dose of 3 x 
106/Kg expanded donor Tregs.  A larger scale phase I trial led by Brunstein et al (Brunstein, 2010) enrolled twenty three 
patients with advanced hematologic malignancy.  The patients were 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 blood samples that had 4 to 6 HLA match with the recipient.  Up to 6 x 106/Kg Tregs were infused after ex vivo 
expansion using anti-CD3 and anti-CD28 conjugated beads.  The infused Tregs were detectable in circulation for up to 7 
days.  During the one-year period after Treg infusion, the investigators observed no DLTs and AEs when compared to 
historical controls.  Incidences of acute and chronic GvHD were reduced in patients received Treg therapy.  The third 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 and Tconv transplants from the same donors.  A majority of the patients received 2 x 106/Kg Tregs 
with 1 x 106/Kg Tconvs and no adjunct IS was given after transplant.  Patients demonstrated accelerated immune 
reconstitution, reduced CMV reactivation, low incidence of tumor relapse and GvHD.  Collectively, these studies show that 
Treg therapy has minimal toxicity in the setting of GvHD. 
 
Recently, a phase I/II study applying polyclonally expanded FACS purified Tregs to type diabetic patients has been reported 
by the Trzonkowski group (Marek-Trzonkowska N, 2012).  The study enrolled 10 type 1 diabetic children (aged 8-16 years) 
within 2 months after diagnosis.  Four patients received 10 × 106 Tregs/kg body wt and the remaining 6 patients received 
20 × 106 Tregs/kg body wt.  The patients were followed for 4-5 months after Treg infusion and no toxicity of the therapy 
was noted.  The authors concluded that Treg therapy was safe and well tolerated in children. 
 
At UCSF, a phase I Treg dose-escalating trial evaluating safety of Treg therapy in type 1 diabetic patients is ongoing.  As of 
January 23, 2013, 10 enrolled subjects have received a single infusion of Tregs.  Three subjects were treated in the first 
dosing cohort with 0.05x108 cells, three subjects were treated in the second dosing cohort with 0.4x108 cells, and four 
subjects were treated in the third dosing cohort with 3.2x108 cells. Notably, two of these 4 subjects were enrolled at 
treated at Yale (New Haven, Connecticut). Thirty-four AEs have been reported in these subjects. All 6 subjects in Cohorts 
1 and 2 had at least one adverse event.   Twenty-eight events were judged as mild in severity, three were judged as 
moderate, and three were judged as severe.  Sixteen were judged to be possibly related, four unlikely related and 14 
unrelated to study therapy.   No SAEs have been reported.  No events that would disallow dose escalation have occurred.  
The Data Safety and Monitoring Board will be reviewing the data in accordance with the protocol plan to determine if the 
study can proceed to enroll the final cohort that will receive a cell dose of 26 x108 cells in four subjects. 
 Risks of autologous ex vivo expanded darTregs infusion 
There is limited experience in humans using the darTreg product.  Because infusion of ex vivo explanded effector T cells 
has been reported to be associated with infusion reactions, recipients of arTregs should be closely monitored after infusion 


 
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for possible infusion reactions including nausea, hypotension, fevers, chills, and pain.  Since the arTreg product is 
immunosuppressive, possible side effects include over-immunosuppression with increased risks of infections and difficulty 
clearing infections. 
   
A safety concern of Treg infusion is potential impairment of immune responses to pathogens and/or malignancies.  
darTregs utilized in this trial are unlikely to possess the full breadth of specificities offered by non-specific, polyclonal Treg 
pool.  Moreover, animal studies suggest that adoptive transfer of alloantigen-specific Tregs does not alter immune 
responses to viral pathogens (Bushell, 2005).  The infusion of Tregs could theoretically impair anti-tumor immunity, which 
could be of particular concern in transplant recipients at increased risk of PTLD and/or recurrent HCC. With the infusion 
of darTregs, there is a theoretical possibility that donor reactive T effector cells may be infused and contribute to rejection.   
 
Lastly, an ancillary reagent used in the manufacturing of darTreg is a CD40L-expressing cell line, KT64.CD40L.HLADR0401, 
which is derived from the human myelogenous leukemia cell line K562.  These cells have been irradiated 10,000 rads (5 
times the lethal dose); they are therefore not expected to persist in darTreg cultures. To further prevent the infusion of 
replication competent K562 cells along with the darTregs, the final darTreg product must meet a release criterion of a 
negative assay for the BCR-ABL translocation, a genetic marker identifying K562 cells. The risk of transmission of 
myelogenous leukemia in the Treg product is considered to be negligible. 
 
Possible adverse reactions include, but are not limited to, abdominal cramps, anaphylaxis, back pain, chest tightness/pain, 
and/or chills at the time of infusion, fever ≤38.5⁰C, flashing, heat/pain at the IV site, hemoglobinuria, hives, mild rigors, 
nausea, headache, oliguria, pruritus, sever rigors, syncope, tachycardia, vomiting, and wheezing. 
 
 


 
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 Risks of Study Mandated Procedures  
 Risks of Leukapheresis  
The common risks of leukapheresis include bruising and discomfort at the site of needle placement, typically in the 
antecubital 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.  Our inclusion/exclusion criteria specify a minimum acceptable platelet count for participants.  Thrombosis and 
bleeding could theoretically occur, although they are rarely if ever observed. 
 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.  The amount of blood that may be drawn from adult subjects for 
research purposes will not be more than 168 mL over an eight-week period.  The additional amount of blood could 
contribute to the development of anemia.  The subject’s clinical condition will be taken into consideration to determine if 
research blood tests can be performed. 
 Risks of Liver Biopsy 
Mild AEs resulting from 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 adverse event 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.   
 
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 
This study provides no direct or immediate benefit to the participants. However, the results of the study could influence 
future care of transplant patients. 
 
 


 
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 Study Visits 
 Stage 1: Screening, Enrollment and PBMC Collection 
 
The first stage of the study encompasses screening A and enrollment.  Participants will be selected from the liver transplant 
waiting lists according to the study eligibility criteria (Screen A).  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. The potential participant will sign an 
informed consent form before undergoing any screening or study procedures.  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 meets inclusion criteria and exclusion criteria A (Section 
4.2 and 4.3.1  ). 
 
Eligible and consented participants enrolled in Cohort 2, 3, or 4 will undergo leukapheresis (or blood draw option for 
Cohort 2) at any time prior to transplantation to isolate PBMCs, which will be cryopreserved for subsequent Treg 
purification and expansion.  Participants who have detectable HBV DNA at the time of PBMC collection will be terminated 
from the trial.   At the time of transplantation, donor material for these participants will be collected to make donor sBcs 
and banked to support future expansion of darTregs.   
 
At the time of transplantation, participants will receive Solumedrol, 500 mg intravenously. Within 24 hours after 
transplantation, participants will be started on MMF in the form of either Cellcept 500mg bid intravenously or orally or 
Myfortic 360 mg bid by mouth.  Within 72 hours after transplantation, participants will be prescribed tacrolimus to target 
12-hour trough levels of 6 – 8 µg/L.  Please refer to Appendix 1.  Recipient Schedule of Events (Stages 1 and 2).  Note that 
collection of AEs and SAEs in Stage 1 are limited to events occurring within 72 hours after leukapheresis. 
 Stage 2:  Thymoglobulin® and TAC- or EVR-based Maintenance IS 
The second stage of the study begins after liver transplantation with administration of Thymoglobulin®.  The first dose 
must be initiated within 72 hours of transplantation (Appendix 1.  Recipient Schedule of Events (Stages 1 and 2).  Prior to 
Thymoglobulin® administration, enrollment inclusion criteria (Section 4.2) should be re-reviewed to ensure eligibility.  In 
addition, exclusion criteria B (Section 4.3.2 ) should also be reviewed prior to administration of Thymoglobulin®.  Note that 
collection of AEs and SAEs in Stage 2 begins with Thymoglobulin® administration. 
 
Between 5-6 weeks after transplantation, participants who continue to meet study eligibility Screen C1 (Section 4.3.3) will 
be assessed by everolimus conversion criteria C2 (Section 4.3.4) will initiate conversion to an EVR-based IS regimen.  EVR 
should be initiated at a dose of 1.0-1.5 mg bid, targeting 12 hour trough levels of 6-8 µg/L no earlier than 30 days after 
liver transplantation.  As EVR is introduced, both MMF and tacrolimus dosing should be reduced.  MMF should be 
completely discontinued when target EVR levels are achieved.  Tacrolimus should be reduced to target 12-hour trough 
levels of 3-5 µg/L.    
 
During Stage 2, study assessments will take place daily during administration of Thymoglobulin®. Starting at week 2, study 
assessments will occur weekly until 12 weeks after transplantation.  Participants will have assessments at weeks 2, 3, 4, 
6, 8, and 10 after transplantation.  Participants enrolled in Cohorts 2, 3, and 4 must undergo an allograft biopsy between 
week 10-14 after transplantation as part of the eligibility determination for darTreg infusion (Appendix 1.  Recipient 
Schedule of Events (Stages 1 and 2)and Appendix 3). 
 
Participants in Cohort 1 (1a and 1b) should undergo an allograft biopsy 12 weeks after transplantation. 
 Follow-up for Cohorts 1a and 1b 
Participants enrolled in Cohorts 1a and 1b who will not receive darTreg infusion will follow Appendix 2. Recipient Schedule 
of Events (Cohort 1a and 1b) starting at week 12 after transplantation.  Visits to the transplant center will take place at 
week 12, 16, 24 and 40 after transplantation.  Laboratory assessments will take place every 4 weeks until the end of the 
study, 40 weeks after transplantation.  Please refer to Appendix 2.  Recipient Schedule of Events (Cohort 1a and 1b Follow 
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 Stage 3: darTreg Infusion for Cohorts 2, 3, and 4 
Participants enrolled in Cohorts 2, 3, and 4 must undergo the following study procedures and visits.  darTreg infusion will 
take place between weeks 11-20 after liver transplantation and will require an overnight stay.  Screen D eligibility must be 
met.  Blood for central laboratory assessments will be drawn prior to the infusion and 24 hours after the infusion is 
complete.  Visits to the transplant center will take place 1 week and 4 weeks after darTreg infusion as well as 12 and 28 
weeks after darTreg infusion (approximately 24 and 40 weeks after transplantation, respectively).  A liver biopsy will be 
done in conjunction with the visit 1 week after darTreg infusion.  Thereafter, laboratory assessments must take place every 
two weeks for 10 weeks, followed by every 4 weeks until the end of the study, 28 weeks after darTreg infusion, or 40 
weeks after transplantation.  Details regarding study procedures including monitoring, laboratory assessment, and blood 
draws for mechanistic studies are specified in Appendix 1.  Recipient Schedule of Events (Stage 3:  Cohorts 2, 3, and 4).      
 Unscheduled Visits  
Unscheduled or additional visits will typically occur secondary to standard of care management for liver transplant 
recipients or secondary to concern for allograft dysfunction when there are liver tests that are elevated above baseline.  
Abnormal liver tests should be assessed according to center standard of care protocols.  A liver biopsy should be performed 
to confirm any clinical suspicion of rejection prior to the administration of any treatment for rejection.  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 LFT’s).   Blood for mechanistic assays are not required unless the unscheduled visit coincides with a 
scheduled visit with central laboratory assessments. 
 Safety Follow Up 
Subjects who are in Stage 2 or 3 and discontinue the study prematurely will be followed according to the safety follow up 
SOE (See Section 13.4, Appendix 4.  Recipient Safety Follow Up (Early Termination from Stage 2) and Appendix 5.  Recipient 
Safety Follow Up (Early Termination from Stage 3). 
 Visit Windows 
Study visits should take place within the following time limits shown in Table 2. Study Visit Windows 
Table 3. Study Visit Windows 
Stage 1 and 2 Study Visits 
Visit Window 
Screen A 
-6 Months 
Visit 1:  PBMC Collection for darTreg manufacturing 
Prior to transplant 
Visit 2:  Transplantation 
-24 hours Central labs can be 
drawn up to 1 year before 
solumedrol dose in OR 
Screen B 
-24 hours before first dose 
Visits 3a, 3b, 4, 5a, 5b:  Days 3, 4, and 5 after transplantation 
None 
Screen C and Visits 6-15, 15a, 15b:  Weeks 2-13 after transplantation 
± 2 days 
Cohort 1 Follow Up Study Visits 
Visit Window 
Visits 16:  Week 12 after transplantation 
-14/+10 days 
Visits 17-23:  Months 4-9 after transplantation 
± 5 days 
Stage 3 Study Visits 
Visit Window 
Screen D 
-10 days 
Visit T0:  Day of darTreg Infusion  
None 
Visit T2:  Day 1 after darTreg Infusion 
± 1 hour 
Visit T3:  Day 7 after darTreg Infusion 
± 1 day 
Visit T4-T5:  Day 14 and 28 after darTreg Infusion 
± 2 days 
Visits T6-T13:  Weeks 6, 8, 10, 12, 16, 20, 24 and 28 after darTreg Infusion 
± 5 days 
Safety Follow Up (Early Termination from Stage 2 or 3) 
Visit Window 
All visits 
± 7 days 
 
 
 


 
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 Mechanistic Assays 
 HLA Typing 
DNA will be isolated from whole blood collected in a K2EDTA tube from both recipients and donors using the QIAmp Blood 
Mini Kit™ (Qiagen, Inc.). Automated DNA sequencing using both commercial (Abbott Molecular Inc., Illinois and QIAGEN 
Inc., CA) and in house reagents will be used to HLA type the samples.  Loci sequenced will included HLA Class I (HLA-A, -B 
and -C) and HLA Class II (HLA-DRB1/3/4/5, -DQA1 and - DQB1). For Class I, exons 2 and 3 containing the defining 
polymorphic regions for most alleles will be amplified and sequenced.    For Class II, typically only exon 2 will be sequenced.  
Any sequencing ambiguities will be resolved by sequencing additional exon(s). 
 T Cell Phenotype and Function 
We will use MFC to profile leukocyte subpopulations, determine frequencies of donor-reactive T cells, assess donor-
specific suppression by darTregs, and use deuterium labeling and TCR sequencing to track infused darTregs.  Together, 
these assays allow us to assess the impact of darTregs therapy and/or Treg-supportive IS on the alloimmune profile of the 
patients. 
 
Treg TruCount analysis:  One ml of whole blood will be collected at the time points indicated in the SOE. The samples will 
be used directly for analysis without cryopreservation.  Fifty microliters of the blood will be aliquoted into a TruCount 
tube, stained with fluorochrome-conjugated antibodies to CD4, 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 determine if the counts are altered by darTreg therapy. 
 
Frequency of donor-reactive T cells:  We will use the assay described in Figure 13 to 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, especially in the 200 million and 800 million dose cohorts. 
 
Figure 13.  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-
transplant 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+.  PBMC for this assay will be obtained from whole 
blood on the day of transplantation.  The cultures will be stimulated with irradiated donor splenocytes to assess donor-
specific suppression and with anti-CD3 and anti-CD28 to assess nonspecific suppression. 
  
MFC panels: We will use MFC to determine the percentages of leukocyte subsets in peripheral blood using panels of 
antibodies developed in our lab.  Panels and markers are summarized in Table 3. MFC Panel Markers.  Adjustments might 
be made if future data from other trials are able to better inform the panel selection. 


 
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Table 4.  MFC Panel Markers   
Panel 
Cell # 
Markers 
Leukocyte panel 
0.25m 
CD45 
CD14 
CD3 
CD19 
CD56 
CD16 
CD4 
CD8 
CD7 
Effector/Memory/ 
Naïve T Cells 
0.5m 
CD3 
CD4 
CD8 
CD45RA 
CD27 
CD28 
CCR7 
 
 
Treg Panel 
1m 
CD3 
CD4 
CD25 
CD127 
FOXP3 
HELIOS 
 
 
 
B cells 
0.5m 
DR 
CD19 
CD20 
CD27 
CD38 
IgD 
 
 
 
 Donor HLA Antibody 
We will assess for the development of donor-specific antibodies as they have been implicated as a risk factor for acute 
rejection, chronic rejection, and the development of fibrosis (Musat, 2011) (Paterno, 2012) (Kaneku, 2012).   
 
Serum samples collected from participants at time points after liver transplantation as specified in the SOE and will be 
evaluated for the presence or absence of donor-specific alloantibodies.  Assessments will include donor-specific cross-
matching with cryopreserved cells, testing against a panel of cryopreserved lymphocytes and/or HLA antigen-coated 
microparticles.  
  Histology and Multiplex Immunohistochemistry 
In collaboration with Dr. A. Jake Demetris, we will perform extensive histology and mIHC analysis of protocol biopsy 
samples obtained prior to darTreg infusion and 1 week after darTreg infusion.  The cohort that does not receive darTreg 
will get a single biopsy at 12 weeks after transplantation, the approximate time of darTreg infusion for the other groups.  
The design of these analyses will be guided by histology and mIHC data from tolerant patients in the Feng 
Immunosuppression Withdrawal trial and our current understanding of liver transplant tolerance.  Histological analyses 
will evaluate 40 histopathological features to determine tissue integrity and degree of inflammation.  mIHC analyses are 
summarized in Table 4. mIHC Markers. 
 
Table 5. mIHC Markers 
mIHC Panel 
Rationale 
C4d/CD31 
Decrease in C4d deposits on the hepatic microvasculature is associated with liver transplant tolerance.  
Determine if Treg therapy leads to decrease in C4d deposits 
CD3/ƴδ -1/ ƴδ -2 
Portal tract ratio of ƴδ -1/ ƴδ -2 >1.0 is associated with operational tolerance. Determine if Treg therapy 
promotes this signature 
CD3/CD45RO/CD45RA 
Monitor the relative ratio of naïve to memory T cells; test the hypothesis that Treg therapy leads to a 
reduction in portal-based CD3+/CD45RO+ (memory) T cells 
CD4/Tbet/GATA-3/IL-
17/FoxP3 
Monitor the polarization of CD4+ lymphocytes within the allograft to determine whether an increase of 
putative Tregs contributes to tolerance. 
IL10/TGFβ/HLADR 
Monitor expression of immunomodulatory cytokines by HLA-DR expressing cells in the liver such as Kupffer 
and B cells. 
CK19/CD31/HLADR 
Up-regulation of HLA-DR on biliary epithelium (CK19+) and vascular endothelium (CD31+) makes these cells 
targets of immune rejection. Determine if Treg therapy prevents DR induction. 
 Deuterium Labeling and Detection 
 
Our clinical trial relies on transferring darTregs that have been expanded hundreds-of-fold in vitro.  These cells will be 
indistinguishable from endogenous Tregs by standard surface markers; therefore we will not be able to distinguish 
administered darTregs from endogenous Tregs.  Because the cells are being expanded prior to infusion, it provides a 
unique opportunity to label the cells prior to infusion to monitor engraftment and persistence of the administered 
darTregs.  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 (Klein PD, 1986) (Koletzko B1, 1997), and they have been safely used as 
cellular, molecular, and metabolic markers in patients and healthy controls for more than 6 decades (Klein PD, 1986) 
(Koletzko B1, 1997).  


 
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In Protocol RTB-002, Tregs from transplant patients will be labeled with deuterium by including the 2H label in the culture 
medium during the entirety of the expansion phase in vitro (day 0 to day 14) prior to infusion in the patient. Based on data 
from in vitro labeling studies, we expect the cells to be ~60% enriched for 2H.  After infusion, PBMCs will be isolated and 
stored at specified time points as described in the SOE.  DNA prepared from these samples will be analyzed by gas 
chromatography-mass spectrometry.  Based on prior studies, we expect sensitivities in the range of approximately ±0.05 
to 0.10% enrichment (which refers to the fraction of labeled molecules) (Busch, 2007) (Macallan, 1998).   As detailed in 
Section 1.2.2, we expect the patients to have about 5 x 109 endogenous Tregs 10 to 13 weeks after Thymoglobulin® 
treatment.  Therefore, an infusion of 200 and 800 x 106 darTreg in the higher dose groups will likely result in a detectable 
increase in overall Treg numbers, detectable by TruCount (BD Biosciences) and MFC assays (Section 10.2) at early time 
points.  Deuterium labeling will allow investigators to distinguish between infused darTregs and endogenous Tregs, thus 
unequivocally demonstrate the presence of infused darTregs in the periphery.  Similarly, presence of deuterium-labeled 
Tregs in the graft will be determined by analyzing genomic DNA extracted from FACS-purified CD4+ cells isolated from 
biopsy tissues collected at 1 week after darTreg infusion and at the time of rejection.  Deuterium labeling will be detectable 
for at least 4 cell divisions of the administered darTregs.  We expect this method of detection to be especially 
advantageous at early time points (up to 28 days) post infusion, prior to significant division, exit into tissues, and/or loss 
of darTregs. 
 TCR Sequencing 
Recent advances in sequencing technologies have led to PCR based strategies to detect rare T cell clones based on a unique 
TCR sequence.  Sensitivities of up to 1 in 100,000 cells have been reported (Robins, 2012) with this technique.  Using this 
technology, we have analyzed TCR usage of expanded darTregs.  Our results demonstrate that the repertoire of darTregs 
is narrower than the repertoire of Tregs freshly isolated from peripheral blood, but remained highly diverse. The highest 
frequency of a single T cell clone in the darTreg product was around 1% and the top 20 clones collectively represented 5 
to 10% of the total population.  Interestingly, by comparing the repertoire before and after expansion, we have found that 
the top 20 clones mostly undetectable in the pre-expansion Treg samples.  This suggests that we will be able to identify 
darTregs after infusion using TCR analysis.  We will determine the top clones in darTregs using sequencing and detect the 
top clones after infusion using PCR.  Although this technology may allow us to track cells early after infusion, this technique 
has the potential to be complementary to the deuterium labeling approach.  Deuterium labeling may be advantageous 
early after infusion as the percentage of labeled cells will be high.  However, with cell division, signal will be difficult to 
detect after approximately 4 divisions. The deuterium labeling approach has already proved successful in the Type I 
diabetes trial with polyclonally expanded Treg. TCR sequencing may potentially be less sensitive early, depending on the 
degree of oligoclonality; however, if successful clones persist and divide over time in patients, these clones will give a 
stronger signal over time.  PBMCs will be sorted for Treg from frozen, banked specimens and cell pellets will be used for 
TCR sequencing by Adaptive Biotechnologies. 
 


 
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 Biospecimen Storage 
 
Whole blood or leukapheresis samples for Treg manufacturing will be collected and cryopreserved in aliquots according 
to established SOPs and stored in UCSF GMP cell manufacturing facility.  Some of the aliquots will be sent to ITN repository 
for mechanistic studies. 
 
Samples collected solely for mechanistic studies will be sent to the ITN repository and stored under specific conditions to 
maintain long-term sample integrity, as well as specimen tracking from receipt to shipment to alternate locations.  A 21 
CFR Part 11 validated database system can be used to track shipment date, location shipped to, carrier, items shipped, 
amount shipped, barcode numbers, protocol number, and associated comments about each individual specimen.  Storage 
temperature, location, processing and aliquoting, and freeze/thaw events may also be recorded. 
 
If the study subject allows storage, the subject’s specimens will be stored indefinitely.  The subject can change their mind 
at any time and have their stored specimens destroyed by notifying the study physician in writing.  In such cases, the site 
coordinator would send all requests for sample destruction to the ITN.  The site will receive confirmation that the specimen 
was destroyed as requested.  If the subject’s samples have already been analyzed, then the data will be used as part of 
the overall analysis.  The subject can only request to have samples destroyed if they still exist, i.e. have not already been 
used in an experiment. 
 
Specimens at the ITN core or repository can only be transferred to another destination with appropriate authorization per 
ITN standard procedures.  Purpose for accessing/transferring the specimen (within study assay as defined by the protocol 
or future studies), evaluation of subject consent for the purpose provided, verification of specimen identifiers, and quality 
and quantity of the specimen are some of the items checked prior to authorization. 
 
If the purpose is for future studies, and the subject consents for storage for future use, the subject’s sample may be made 
available to the scientific research community per the ITN Sample Sharing Policy (www.immunetolerancenetwork.org).   
Any research conducted using stored samples for future use may also need appropriate regulatory approval, such as 
Institutional Review Board (IRB) per the study consent. 
 
Exceptions to the above banking condition apply to the samples used for Treg TruCount, and deuterium detection in blood 
and biopsies because the assays need to be performed on fresh samples.  Donor specimens will be stored until the end of 
study analyses and then be destroyed or anonymized. 
 
 


 
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 Criteria for Participant Completion and Premature Study Termination 
 Participant Completion 
Subjects in Cohorts 1a and 1b who have completed assessments at all study visits outlined in the SOE after lymphocyte 
depletion with Thymoglobulin° and TAC- or EVR-based Treg supportive IS regimen will have completed the study.  Subjects 
in Cohorts 2, 3, and 4 who have completed assessments at all study visits outlined in the schedule of events after receiving 
Thymoglobulin°, Treg supportive IS, and darTreg infusion will have completed the study.  The site PI will determine 
whether these participants should continue with the Treg supportive IS regimen or whether these participants should be 
converted to the site’s standard of care IS regimen. 
 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, because the investigator no longer 
believes participation is in the best interest of the participant or because of non-compliance with follow-up and/or 
study procedures, follow-up should be according to the safety follow-up schedules of events (Section 13.4).   
 Participant Replacement 
Any participant who is deemed ineligible to initiate Thymoglobulin° (Stage 2, Screening B) will be replaced. Any participant 
in Cohorts 2, 3, or 4 in whom Thymoglobulin° has been administered but is then deemed ineligible to receive darTreg 
infusion (Stage 3, Screening D) will be replaced if deemed appropriate after review by the NIAID MM, PI, and two 
transplant physicians independent of this study.  This does not replace or change the stopping rules detailed in Section 
14.8.1  . All decisions made related to participant replacement in this stage will be reviewed during the standard DSMB 
review as described in Section 14.8.1    Any participant who has received any part of a darTreg infusion (Stage 3) and 
subsequently terminates from this study prematurely will not be replaced. 
 Follow-up after Early Study Withdrawal 
 Follow-up after Premature Discontinuation from Stage 1 
Subjects who are prematurely withdrawn from Stage 1 of the study will be terminated from the study and will not be 
followed.  Subjects who move to Stage 2 but discontinue from the trial prior to receiving study directed Thymoglobulin 
will not be followed.   
 Follow-up after Premature Discontinuation from Stage 2 or Stage 3 
All subjects who are prematurely withdrawn from Stage 2 and have received any study directed Thymoglobulin®(without 
receiving darTregs) will have a safety follow up visit 4 weeks after stopping study therapy (Appendix 4).  In addition, these 
subjects will continue the safety follow up schedule (Appendix 4) until 40 weeks after transplantation.  Subjects 
prematurely discontinuing study therapy from Stage 3 after receiving any part of a darTreg infusion will be followed 
according to Appendix 5 starting with a follow up visit within 4 weeks after the decision to stop study therapy.  
 
 


 
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 Safety Monitoring and Reporting 
 Overview 
This section defines the types of safety data that will be collected under this protocol and outlines the procedures for 
appropriately collecting, grading, recording, and reporting those data. AEs that are classified as serious according to the 
definition of health authorities must be reported promptly to the sponsor DAIT/NIAID or other Sponsor.  Appropriate 
notifications will also be made to site PIs, 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 Event 
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 Adverse Events (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 agents (Section 
5) caused the AE. For the purposes of safety reporting, ‘reasonable possibility’ means there is evidence to suggest a causal 
relationship between the drug and the AE. A 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)). 
14.2.1.2 Unexpected Adverse Event  
An AE or suspected adverse reaction is considered “unexpected” if it is not listed in the package insert 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 IND.  
 
“Unexpected” also refers to AEs or suspected adverse reactions that are mentioned in the package insert 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 (21 CFR 312.32(a). 
 Serious Adverse Event  
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.  
3. Inpatient hospitalization or prolongation of existing hospitalization (Please see Section 0 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. 
 
 
 


 
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 Grading and Attribution of Adverse Events 
14.3.1 Grading Criteria 
The study site will grade the severity of AEs experienced by the study subjects according to the criteria set forth in the 
NCI-CTCAE – Version 4.0 for all AEs with the exception of Infection.  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 with the exception of infection and hematologic events described below.  
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 
Other than those described below, any events grade 2 or higher (including study defined grading of infections) will be 
recorded on the appropriate AE CRF for this study. 
 
For hematologic events listed below, grade 3 or higher will be recorded on the appropriate AE case report form (CRF) for 
this study:   
• 
Neutrophil count decreased 
• 
Anemia (Hemoglobin decreased) 
• 
Platelet count decreased  
• 
Total lymphocyte count decreased  
• 
Lymphocyte subset count decreased (including CD4) 
 
Decreased CD4 lymphocytes and lymphocyte counts within 6 months after last study dose of Thymoglobulin® do not 
require reporting. 
 
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. Changes in grade from screening to baseline will also 
be recorded as AEs, but are not treatment-emergent. 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. 
14.3.2 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 5. Attribution of Adverse Events.  For additional 


 
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information 
and 
a 
printable 
version 
of 
the 
NCI-CTCAE 
manual, 
consult 
the 
NCI-CTCAE 
web 
site: 
http://ctep.cancer.gov/reporting/ctc.html. 
Table 6.  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 
Definite 
The AE is clearly related. 
Attribution assessment for the following study drugs and procedures will be made when a SAE is reported:      
• 
Lymphocyte depletion with Thymoglobulin° 
• 
Treg-supportive IS regimen – reduced dose tacrolimus, reduced dose MMF, reduced dose methylprednisolone, 
and EVR (if applicable) 
• 
darTreg  
• 
PBMC collection (Leukapheresis or 450-500 ml blood draw) 
• 
Liver biopsy 
Other than 450-500 ml blood draw for PBMC collection, AEs associated with blood draws do not have to be reported 
unless the investigator deems an event reportable. 
 Collection and Recording of Adverse Events 
14.4.1 Collection Period 
During Stage 1, AEs will be collected from the time of first study procedure (leukapheresis or 450-500 ml blood draw), 
until 72 hours after completion of the leukapheresis procedure or 450-500 ml blood draw.  AEs occurring outside the 72 
hour window should be reported if the investigator deems a possible association with the protocol mandated 
leukapheresis or blood draw. 
 
During Stages 2 and 3 (including Cohort 1 follow up), AEs will be collected from the time of first Thymoglobulin® 
administration, until a subject completes study participation or until 30 days after he/she prematurely withdraws (without 
withdrawing consent) or is withdrawn from the study. 
 
Additionally, elevated liver tests (i.e. ALT, AST, ALP, GGT, and bilirubin) must be reported starting 7 days after 
transplant.  Events involving unexpected elevations of liver tests in the first week should be reported according the site 
investigator’s judgment. 
14.4.2 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 14.3 Grading and Attribution of AEs. 
14.4.3 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. 
14.4.4 Recording Adverse Events 
Throughout the study, the investigator will record AEs and SAEs as described previously (Section 14.2, Definitions) 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, 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 
14.5.1 Reporting of Serious Adverse Events to Sponsor  
This section describes the responsibilities of the site investigator to report SAEs to the sponsor via the Statistical and 
Clinical Coordinating Center (SACCC) eCRF.  Timely reporting of AEs is required by 21 CFR and ICH E6 guidelines.  
 
Site investigators will report all SAEs to the sponsor, regardless of relationship or expectedness 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. 
14.5.2 Reporting to Health Authority 
After an AE requiring 24 hour reporting (13.5.1, Reporting of SAEs to Sponsor) is submitted by the site investigator and 
assessed by DAIT/NIAID, there are two options for DAIT/NIAID to report the AE to the health authority: 
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 (Section14.2.2, Suspected Adverse Reaction, and Unexpected AE) 
• 
Serious and not a suspected adverse reaction (Section 14.2.2, Suspected Adverse Reaction) 
• 
Pregnancies not reported as SAEs 
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  
This option, 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, Suspected Adverse Reaction 
and Section 14.2.2, Unexpected AE and 21 CFR 312.32(c)(1)i).  
The sponsor shall report any suspected adverse reaction that is both serious and unexpected.  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 protocols, 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 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.   
 


 
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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. 
 
14.5.3 Reporting of Adverse Events to IRBs/IECs 
All investigators shall report AEs, including expedited reports, in a timely fashion to their respective IRBs/IECs in 
accordance with applicable institutional 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/IEC 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 as expedited events 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 medical monitor, and the NIAID 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 that there are significant 
safety concerns.  
 
In addition to the dose escalation plan (Section 5.4), the SACCC will continuously monitor accumulating safety data to 
determine if any of the following criteria are satisfied. If a criterion is satisfied, the trial will be placed on hold pending 
DSMB review, during which event rates and other clinical data will be evaluated. During this holding period, screening, 
enrollment, and initiation of any new study treatment or procedure will cease.  Specifically, participants who are in Stage 
1 will not be allowed to initiate Stage 2 and participants in Stage 2 will not be allowed to initiate Stage 3.  However, 
participants who have received Thymoglobulin® will be allowed to proceed to EVR conversion unless specifically 
disallowed by the Medical Monitor.   
 Study Stopping Rules 
The decision to pause enrollment pending DSMB review will be based on the occurrence of selected SAEs described below: 
1. Death or graft loss 


 
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2. Infections of Grade ≥3 as defined in Section 14.3.1 
3. Severe AR  
a. Histological:  according to 1997 Banff criteria (Demetris A. D., 2000) 
b. Clinical:  steroid refractory rejection (see Study Definitions) 
4. CR (early or late) according to 2000 Banff criteria (Demetris A. J., 1997) 
5. PTLD 
6. Malignancy excluding recurrent HCC and skin cancer 
7. CTCAE ≥ 3 Infusion reaction 
8. Failure to manufacture and supply the cellular product for 2 subjects. 
 
Since the trial is small and these events are of particular concern, and the risks are expected to be different in the two 
stages of the trial, absolute thresholds for the numbers of such events have been established for each event in each stage 
that would satisfy the stopping rule. 
Table 7.  Minimum Numbers of Selected AEs in each Stage that would Satisfy the Stopping Rule 
Selected SAE 
Lymphocyte 
Depletion/Treg 
Supportive IS  
Stage 2 
darTreg Infusion 
Stage 3 
Death or graft loss 
1 
1 
Grade ≥3 Infection (Section 14.3.1) 
1 
1 
Severe AR (histological or steroid refractory rejection) 
2 
1 
CR 
1 
1 
PTLD 
1 
1 
Malignancy excluding recurrent HCC and in situ skin cancer 
1 
1 
CTCAE ≥ 4 Infusion reaction 
N/A 
1 
 
Every patient who initiates Stage 2, defined as receiving any amount of a Thymoglobulin® infusion, will contribute to the 
assessment of the safety of Treg supportive IS.  At the point in time when the patient initiates Stage 3, defined as receiving 
any amount of a darTreg infusion, he/she will contribute to the safety assessment for darTreg infusion.  Any death or 
Grade ≥3 infection (defined in Section 14.3.1) occurring in subjects prior to initiation of Thymoglobulin® infusion (prior to 
Stage 2) will not stop the trial.   
 
The UCSF HICTF, NIAID MM, and study PI will review available information after the first instance of the dose falling short 
of target range, and again in the event of a second product not meeting the specified dose.  If a pause is required due to 
manufacturing failures, one subject will be allowed to receive study directed Thymoglobulin during the review process.  
Recruitment and enrollment activities can continue during this time.  If a third instance of lower-than-expected dose is 
manufactured, the study will be paused and DSMB will review available data prior to the trial proceeding.   
 Medical Monitor Review 
The DAIT/NIAID Medical Monitor shall receive at least monthly reports from the SACCC compiling new and accumulating 
information on AEs, SAEs, and pregnancies recorded by the study site(s) on appropriate eCRFs. 
 
In addition, the Medical Monitor shall review and make decisions on the disposition of the SAE and pregnancy reports 
received from the site investigator via the SACCC (See Sections 14.5 and 14.6 ).   
 DSMB Review  
The DSMB shall review safety data at least yearly during planned DSMB Data Review Meetings.  Data for the planned 
safety reviews will include, at a minimum, a listing of all reported adverse 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 PI or DAIT/NIAID.  Please 
see Section 5.4 Dose Escalation Plan/Toxicity Management.  
 


 
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 Statistical Considerations and Analytical Plan 
 Overview  
The objectives of this multi-center, open-label, pilot dose-escalation study are described in Section 3. Study Design.  Briefly, 
they are to evaluate the safety, tolerability, DLTs and biological activity of ex vivo-expanded darTregs in combination with 
lymphocyte depletion using Thymoglobulin° and TAC- or EVR-based Treg-supportive IS in adult liver transplant recipients.   
 Outcomes  
The study design as well as the safety, clinical, and mechanistic outcomes are described in Section 3.  
 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.  Subjects who do not receive Thymoglobulin® or subjects 
who receive Thymoglobulin®, but do not subsequently receive darTregs will be replaced according section 13.3.   
  Analysis Plan 
Statistical analyses of the safety and clinical outcomes will be descriptive, 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 Samples 
The Safety Sample will be comprised of all study subjects who are consented and receive any study therapy, including 
leukapheresis, T cell depletion followed by TAC- or EVR-based Treg-supportive IS and darTreg infusion.  This excludes 
consented subjects who were terminated before receiving any study treatment. 
 
Safety and clinical outcomes will be analyzed within the Safety Sample as a whole and within the following two treatment-
related analysis periods during which: 
• 
Subjects receive any Thymoglobulin°, followed by TAC- or EVR-based darTreg supportive IS  
• 
Subjects receive darTreg infusions at any dose. 
 
Subjects will contribute AEs and weeks at risk to each analysis period for as long as they are on the corresponding study 
treatment in that analysis period. Thus, if they progress from T cell depletion to darTreg infusion at any dose, they will 
contribute subsequent AEs and weeks at risk to the darTreg infusion analysis period when they initiate any darTreg 
infusion and that will continue until the end of their follow-up time on study. 
 
In addition, a third subset of the safety sample will include any enrolled subject who has PBMCs collected for darTreg 
manufacturing (phlebotomy or leukapheresis) and may or may not receive subsequent Thymoglobulin®, darTreg 
supportive IS or darTreg infusions. 
 Analysis of Safety Outcomes   
As the safety outcomes are all person-week incidence rates of selected AEs as described in Section 3.2, they will be 
estimated using proportions and exact two-sided 95% confidence intervals in the Safety Sample as a whole and within the 
two analysis periods defined above.   
 Supportive Analyses  
Since this is a relatively small pilot study, no sub-group analyses, sensitivity analyses or covariate adjustments are planned. 
 Analyses of Exploratory Outcomes 
Mechanistic assays are designed to monitor various Treg cell subset levels over time before and immediately after the 
initiation of any darTreg infusion. These Treg cell subset measures 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. 


 
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 Descriptive Analyses  
Disposition of subjects will be summarized in the Safety Sample.  Reasons for termination of subjects who did not receive 
whole blood draw or leukapheresis for manufacturing, those who underwent whole blood draw or leukapheresis for 
manufacturing but did not receive liver transplantation, and those who underwent whole blood draw or leukapheresis for 
manufacturing and liver transplantation but did not receive any Thymoglobulin or Treg supportive IS will be tabulated.    
 
Standard descriptive statistics for continuous and categorical variables will be used to summarize the following on all 
subjects in the Safety Sample and for the two 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  
This pilot study is designed to evaluate 18 subjects through Stage 3 in a modified 3+3 dose escalation design. Therefore, 
no formal power and sample size analyses have been performed. However, with 18 evaluable subjects undergoing darTreg 
supportive IS for 12 weeks of follow-up (6 subjects for 40 weeks of follow-up) and up to 12 evaluable subjects undergoing 
darTreg infusions for 28 weeks of follow-up after infusion, exact 95% confidence intervals on person-week incidence rates 
will vary in width by analysis period.  For example, the following table (Table 8. Incidence Rates and Confidence Intervals 
for Selected Numbers of Events and Person-Weeks) shows incidence rates and confidence intervals for selected numbers 
of events and person-weeks of 216 (18 subjects x 12 weeks), 336 (12 subjects x 28 weeks), and 384 (6 subjects x 40 weeks 
+ 12 subjects x 12 weeks) in the two analysis periods. It is not known at this time how many subjects may be expected to 
comprise the entire Safety Sample, which includes subjects who only received leukapheresis. 
 
 


 
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Table 8. Incidence Rates and Confidence Intervals for Selected Numbers of Events and Person-Weeks 
Number 
of 
Events 
Total 
Person-
Weeks 
Person-Weeks 
Incidence Rate 
Lower 
95% 
Confidence Limit 
Upper 95% Confidence 
Limit 
2 
216 
0.0093 
0.0011 
0.0334 
4 
216 
0.0185 
0.0050 
0.0474 
6 
216 
0.0278 
0.0102 
0.0605 
8 
216 
0.0370 
0.0160 
0.0730 
10 
216 
0.0463 
0.0222 
0.0851 
12 
216 
0.0556 
0.0287 
0.0970 
14 
216 
0.0648 
0.0354 
0.1087 
16 
216 
0.0741 
0.0423 
0.1203 
18 
216 
0.0833 
0.0494 
0.1317 
20 
216 
0.0926 
0.0566 
0.1430 
 
2 
336 
0.0060 
0.0007 
0.0215 
4 
336 
0.0119 
0.0032 
0.0305 
6 
336 
0.0179 
0.0066 
0.0389 
8 
336 
0.0238 
0.0103 
0.0469 
10 
336 
0.0298 
0.0143 
0.0547 
12 
336 
0.0357 
0.0185 
0.0624 
14 
336 
0.0417 
0.0228 
0.0699 
16 
336 
0.0476 
0.0272 
0.0773 
18 
336 
0.0536 
0.0317 
0.0847 
20 
336 
0.0595 
0.0364 
0.0919 
 
2 
384 
0.0052 
0.0006 
0.0188 
4 
384 
0.0104 
0.0028 
0.0267 
6 
384 
0.0156 
0.0057 
0.0340 
8 
384 
0.0208 
0.0090 
0.0410 
10 
384 
0.0260 
0.0125 
0.0479 
12 
384 
0.0313 
0.0161 
0.0546 
14 
384 
0.0365 
0.0199 
0.0612 
16 
384 
0.0417 
0.0238 
0.0677 
18 
384 
0.0469 
0.0278 
0.0741 
20 
384 
0.0521 
0.0318 
0.0804 
 
 
 


 
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 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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 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 well-being 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 PI 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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 Ethical Considerations and Compliance with Good Clinical Practice 
 Statement of Compliance 
This clinical study will be conducted using good clinical practice, 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.  The PI or designee listed on the FDA 1572 will review the consent and 
answer questions.  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. A copy of the signed consent form will be given to the participant. 
 
The consent process will be ongoing; the consent form will be reviewed with each subject in Cohorts 2, 3, and 4 prior to 
darTreg infusion.  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) except for those 
lawfully required for manufacture and administration of cell therapies, and to ensure that the appropriate cell product is 
administered to the intended recipient. 
 
 


 
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 Publication Policy 
The publication guidelines and policies stipulated in the grant will apply to this trial. 
 
 


 
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Tregs in Liver Transplantation (RTB-002) 
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Tregs in Liver Transplantation (RTB-002) 
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                                                           Version 7.0/ August 1, 2018 
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36. 
 
 
 


 
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Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 1.  Recipient Schedule of Events (Stages 1 and 2) 
 
Visit Number
Screen A  
1
2
Screen B 3a10 3b
4
5a
5b10
6
7
8
Screen C
9
10
11
12
13
14
15 15a1315b13 15c1315d1315e13 15f13
CIB/ Rej.14
Visit Window 
- 6 Months
Prior to Tx
-3mons 
-24 hrs
N/A
Inclusion Criteria and Exclusion Criteria A
x
x
Inclusion Criteria and Exclusion Criteria B
x 
Inclusion Criteria and Exclusion Criteria C
x2
Informed Consent
x
Demographics (DOB, Gender, Ethnicity)
x
Medical History
x
Physical Examination
x
x
x
x
x
x12
x
HCC Assessment, Pathology, Treatment
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
x
Adverse Event/Serious Adverse Event Assessment 
x6
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
→
x
PBMC Collection (450ml Phlebotomy 1 or Leukapheresis for Cohort 2, Leukapheresis for Cohorts 3 and 4)
x
Solumedrol 500 mg 
x7
Prednisone Taper
→
→
→
→
→
→
→
→
→
→
MMF (Cellcept 500 mg BID or Myfortic 360 mg BID)
x
x 
x
x
x
x
x
x
x
Tacrolimus  
Thymoglobulin® Infusion (3-4.5 mg/kg) 
EVR Conversion
darTreg Expansion (Cohorts 2, 3, and 4 Only)
Pregnancy Test (Urine or Serum hCG)
2
HLA Typing 
x
Viral Serology: HIV Ab,  CMV IgG, EBV IgG, HBV (HBsAg, HBcAb, HBeAb, HBeAg, HDVAb), HCVAb3
x
Viral Nucleic Acid Testing: HBV DNA (for HBV positive subjects)
x
x
Viral Nucleic Acid Testing: HCV NAT (Screen A: most current test w ithin 6 months prior to txp, Screen C: all subjects)
x
x 
x
CBC (w ith differential and platelets)
x
x8
x 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
CD3 Count
x10
x
x
x
x10
CD4 Count
x
Basic Chemistry  (Na, K, Cl, CO2, Creatinine, BUN)
x
x8
x 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Liver Function Tests (ALT, AST, Alk Phos, GGT, T Bilirubin, D Bilirubin) 
x
x8
x 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
INR/PTT
x
x8
x 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
AFP4
x
x4
Tacrolimus Level
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
EVR Level5
x
x
x
x
x
x
x
x
x
x
x
x
x
Ultrasound and Doppler Examination
x
Urine Protein/Creatinine Ratio
x
Local Pathology Results - Graft Routine Histology
x9
x
Protocol Liver Biopsy 
x
Treg TruCount (2ml Green Heparin Tube)
2
Allofrequency Assay & MFC-Leukocyte, Treg, Tact/mem, B Cell Panels (10 ml Green Heparin Tube)
107
10
MLR Suppression Responder Cells (10-10 ml Green Heparin Tubes)
100
MLR Suppression (2-10ml Green Heparin Tubes)
207
20
Alloantibody Analysis-Donor HLA Ab (3ml Serum Separator Tube)
37
2
133
30
Stage 2
W20
Collect only when 
CIB visit 
coincides with 
scheduled visit 
with central labs.
Discontinue steroids by w eek 5
2 Screening for eligibility C1 and C2 can be assessed between 5-6 weeks (30-44 days) after transplantation.  Everolimus can be started on or after 30 days after liver transplantation if eligibility criteria are met (Screen C1).  
W11
Study Assessments
W5
Investigational Agents and Immunosuppressive Medications
PBMC 
Collection1
Initiate w ithin 72 hrs of tx, target trough 6-8 µg/L5
Clinically 
Indicated Bx/ 
Rejection
Screen A  
Day 0 
(Tx)11
W4
x13
Screen 
C1, C22
W9 W10
W16W18
TAC target trough 3-5 µg/L5
EVR target trough 6-8 µg/L 2, 5
+/-2 days
W12
W3
W7 W8
None
First dose within 72 hrs of tx
10
W6
W13W14
D2
6 Prior to Thymoglobulin, collect AE's/SAE's occurring within 72 hours after PBMC Collection only.  Collect all AE's/SAE's grade 2 and higher from the time of first Thymoglobulin infusion.
9 Liver allograft biopsy should be obtained prior to transplantation (back table) for local pathology reading only; no specimen for central pathology
7 Central laboratory assessments may be drawn up to one year BEFORE solumedrol dose in OR.  Once drawn, mechanistic labs do not need to be repeated.    
10 Screening should take place within 24 hours prior to Thymoglobulin®.  The first dose of Thymoglobulin® is to be given within 72 hours after transplantation.  Additional doses of 0.75-1.5 mg/kg IV will be administered until CD3 count is ≤50 cells/µL (50 cells/mcL) or total lymphocyte count is ≤ 
0.10 x 109/L; or when maximum dose of 4.5/mg/kg has been given.  Day 2 and Day 6 visits are only needed if Thymoglobulin is given on these days.
14 Local laboratory assessments collected (retrospective chart review) at the time of clinically indicated biopsy should reflect reason for biopsy (e.g. elevated LFT's).    
13Subjects in Cohorts 2, 3, and 4 should have protocol biopsy between week 10 and 14 to assess eligibility D for Stage 3 (See Stage 3 SOE).  Subjects in Cohort 1 should have a protocol biopsy at week 12 after transplant (See Stage 2 Follow Up).  Visits 15a-15f should be conducted to collect 
weekly labs if subject has not started Stage 3 SOE.
8 CBC, Basic Chemistry, Liver Function Tests, and PT/INR/PTT should be collected as close to, but prior to transplant as possible.
12 Visits to the transplant center are required for physical exams.  A transplant center visit and physical exam is not required for Cohort 1 at Week 10; physical exam and transplant center biopsy visit between Week 10-14 for Cohorts 2, 3, and 4 only.    
11 Treg expansion should be initiated 2-3 weeks prior to planned Treg infusion if subjects continue to meet eligibility criteria.  Please refer to Stage 3 Schedule of Events. .  
STAGE 1
5 First EVR level should be obtained within one week of first dose (+/- 2 days).  MMF discontinuation and TAC trough 3-5 µg/L for subjects converting to EVR only.  Subjects continuing on TAC-based IS should have troughs 6-8 µg/L.
W2
D3
D4
D5
3 HIV/HBV/HCV serology can be within one year of screening.
4 AFP is only required for subjects known to have HCC.  At Screen A, AFP should be drawn if unavailable within 3 preceding months; AFP should be redrawn at time of transplant.  After transplant, AFP should be recorded every 3 months.
Central Laboratory Assessments
1 PBMC Collection Visit does not apply to Cohort 1.  Cohort 2 can have whole blood collection for PBMC isolation if hemoglobin is ≥10.5 mg/dl; leukapheresis if <10.5 mg/dl.
Local Laboratory Assessments
D/c MMF once EVR target trough is reached.
5
Screen B 
x13
x11
D6


 
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Page 76 of 80 
Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 2.  Recipient Schedule of Events (Cohort 1a and 1b Follow Up) 
 
 
Time after Transplant
W12
W20
W24
W28
W32
W36
W40
Time after darTregs**
T0**
T56**
T84** Tw16** Tw20** Tw24** Tw28**
Visit Number
16
18
19
20
21
22
23
CIB/ Rej.
Visit Window 
-14/+10 
days
Within -14 days of study 
visit
Physical Examination
x
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
→
→
→
→
→
→
→
x
Concomitant Medications (inlcuding treatment for rejection and HBV)
→
→
→
→
→
→
→
x
Adverse Event/ Serious Adverse Assessment
→
→
→
→
→
→
→
x
Tacrolimus Maintenance  
EVR Maintenance
HBV DNA and HBsAb Quantitative
x
HCV RNA (for previously HCV positive subjects only)
x
CBC (w ith differential and platelets)
x
CD4 Count
x
x
x
Basic Chemistry  (Na, K, Cl, CO2, BUN)
x
x
x
x
x
x
x
x
Liver Function Tests (ALT, AST, Alk Phos, GGT, T Bilirubin; D Bilirubin) 
x
x
x
x
x
x
x
x
INR/PTT
x
AFP2
x
x
x
Tacrolimus Level
x
x
x
x
x
x
x
x
EVR Level
x
x
x
x
x
x
x
x
Local Pathology Results - Graft Routine Histology
x
Protocol Liver Biopsy1 
x
x
Allofrequency Assay & MFC-Leukocyte, Treg, Tact/mem, B Cell Panels (10 ml Green Heparin Tube)
10
10
10
MLR Suppression (2-10ml Green Heparin Tubes)
20
20
20
Treg Detection: TCR Sequencing (10ml Green Heparin Tube)
10
10
10
Alloantibody Analysis-Donor HLA Ab (3ml Serum Separator Tube)
3
3
3
43.0
43.0
43.0
Collect only when 
CIB visit coincides 
with scheduled 
visit with central 
** Corresponds w ith Cohorts 2, 3, and 4 time points (post drTreg infusion).
EVR target trough 4-6 µg/L
Local Laboratory Assessments
Investigational Agents and Immunosuppressive Medications
EVR target trough 6-8 µg/L
Clinically Indicated  
Biopsy/ Rejection
Central Laboratory Assessments
Study Assessments
TAC target trough 3-5 µg/L
1For each protocol (W12) or clinically indicated biopsy, one specimen w ill be in formalin for unstained slides, one specimen in PBS for deuterium tracking, and one specimen in RNALater.
2AFP is only required for subjects know n to have HCC.


 
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Page 77 of 80 
Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 3.  Recipient Schedule of Events (Stage 3:  Cohorts 2, 3, and 4) 
 
 
 
Time after Transplant
W13
W14
W16
W18
W20
W22
W24
W28
W32
W36
W40
Tw2
Tw4
Tw6
Tw8 Tw10 Tw12
Treg Infusion
T02
Visit Number
Screen D  
T0
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T13
CIB/ Rej.
Visit Window
-10 days 
before Tregs
None
±1 hr
±1 day
Within -14 days 
of study visit
Inclusion Criteria and Exclusion Criteria D
x
Treg Informed Consent 
x
Physical Examination
x
x
x
x
x
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
→
→
→
→
→
→
→
→
→
→
→
→
→
→
x
Concomitant Medications (including treatment for rejection and HBV)
→
→
→
→
→
→
→
→
→
→
→
→
→
→
x
Adverse Event/ Serious Adverse Assessment
→
→
→
→
→
→
→
→
→
→
→
→
→
→
x
darTreg Infusion (50, 200, or 800) (infuse between 11 and 20weeks  after txp) 
x
Tacrolimus Maintenance  
Everolimus Maintenance
Pregnancy Test (Urine or Serum hCG)
x
HBV DNA 
x
HCV RNA (All Subjects)
x
CMV and EBV DNA
x
x
x
x
x
CBC (w ith differential and platelets)
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
CD4 Count
x
x
x
x
x
x
x
Basic Chemistry  (Na, K, Cl, CO2, Creatinine, BUN)
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Liver Function Tests (ALT, AST, Alk Phos, GGT, T Bilirubin; D Bilirubin) 
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
AFP4
x
x
Tacrolimus Level
x
x
x
x
x
x
x
x
x
x
x
x
x
EVR Level
x
x
x
x
x
x
x
x
x
x
x
x
x
Local Pathology Results - Graft Routine Histology
x3
x
Protocol Liver Biopsy5
x3
x
x
Allofrequency Assay & MFC-Leukocyte, Treg, Tact/mem, B Cell Panels      
(10 ml Green Heparin Tube)
10
10
10
10
10
10
MLR Suppression (2-10ml Green Heparin Tubes)
20
20
20
20
20
Treg Detection: TCR Sequencing (10ml Green Tube)
10
10
10
10
10
10
Treg Detection: Heavy Glucose Labeling (10ml Green Tube)
10
10
10
10
10
Alloantibody Analysis-Donor HLA Ab (3 ml Serum Separator Tube)
3
3
43.0
30.0
50.0
50.0
50.0
53.0
Central Laboratory Assessments
TAC target trough 3-5 µg/L
Collect only 
w hen CIB visit 
coincides w ith 
scheduled visit 
w ith central 
labs.
Clinically 
Indicated 
Biopsy/Rej
STAGE 31
T28
T14
T7
T70
Tw16
darTreg Week 1 (Tw)2
darTreg Days (T)2
± 5 days
Local Laboratory Assessments
±2 days
1 Stage 3 begins w ith screen D for darTreg infusion.  If a second attempt at Treg infusion is planned, laboratory assessments (except for liver biopsy) for Screen D must be repeated.
5 For each protocol (Eligibility D and Day 7) or clinically indicated biopsy, one specimen w ill be in formalin for unstained slides, one specimen in PBS for deuterium tracking, and one specimen in RNALater.
3 Subjects in Cohorts 2, 3, and 4 should have biopsy betw een w eek 10 and 12 to assess eligibility D for Stage 3.  
4 AFP is only required for subjects know n to have HCC.  
2 T0 designates day of darTreg infusion.  h = hour, T = day, Tw  = w eek; w here (x) indicates number of hours, days, or w eeks after darTReg infusion. 
Tw24 Tw28
EVR target trough 4-6 µg/L
Investigational Agents and Immunosuppressive Medications
EVR target trough 6-8 µg/L
Study Assessments
T42
T84
h24
T56
Tw20
Screen D  
(-T1)
W12
Time Points (Time after darTregs)


 
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Page 78 of 80 
Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 4.  Recipient Safety Follow Up after Thymoglobulin (Early Termination from Stage 2) 
 
 
 
Time Points 
Wk 4 
after 
Term 1 W121 W241 W403
Clinically 
Indicated 
Biopsy/Rej
Visit Number
F1
F1a
F2
F3
CIB
Visit Window 
Within -14 days 
of study visit
Study Assessments
Physical Examination
x
x
Review/Collect Current Immunosuppressive & Anti-Infective Medications
x
x
x
x
x
Rejection Episodes
→
→
→
→
→
Adverse Event/ Serious Adverse Event Assessment
→
→
→
→
→
Local Laboratory Assessments
CBC (w ith differential and platelets) 
x
x
x
x
x
CD4 Count
x
x
x
x
Basic Chemistry  (Na, K, Cl, CO2, BUN, creatinine)
x
x
x
x
x
Liver Function Tests (ALT, AST, Alk Phos, T Bilirubin; D Bilirubin, GGT) 
x
x
x
x
x
Tacrolimus Level2
x
x
x
x
x
EVR Level2
x
x
x
x
x
Central Laboratory Assessments
Allofrequency Assay & MFC-Leukocyte, Treg, Tact/mem, B Cell Panels (10 ml Green Heparin Tube)
10
10
10
103
MLR Suppression (2-10ml Green Heparin Tubes)
20
20
20
203
TCR Sequencing (10ml Green Heparin Tube)
10
10
10
103
Alloantibody Analysis-Donor HLA Ab (3ml Serum Separator Tube)
3
3
3
33
* This schedule should be utilized for subjects who terminate from the study after receiving any study directed Thymoglobulin, but no 
darTregs.
± 7 days 
1 Subjects w ho prematurely terminate from Stage 2 of the study after receiving a partial dose of study Thymoglobulin should have the 
first safety follow  up visit 4 w eeks after stopping protocol therapy.  After the first follow  up visit, the subject w ill use the next 
applicable study visit and continue this schedule until 40 w eeks after transplant.  Follow  up visit F1 (4 w eeks after termination) 
should be replaced w ith W12 (F1a) or W24 (F2) if the subject terminates w ithin 4 w eeks of that visit. 
3 Central laboratory assessments do not need to be collected for CIB if collected less than 6 w eeks ago.
2 Tacrolimus and EVR levels should be collected from standard of care labs, if available.  


 
Confidential 
Page 79 of 80 
Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 5.  Recipient Safety Follow Up (Early Termination from Stage 3) 
 
 
 
Time Points (Weeks after darTregs)
Wk 121 Wk 28
Time Points (Weeks after Transplant)
W241
W402
Visit Number
F1
F2
F3
CIB
Visit Window 
Within -14 days of 
study visit
Physical Examination
x
x
Review/Collect Current Immunosuppressive and Anti-Infective Medications
x
x
x
Rejection Episodes
→
→
→
→
Adverse Event/ Serious Adverse Event Assessment
→
→
→
→
CBC (w ith differential and platelets)
x
x
x
x
CD4 Count
x
x
x
Basic Chemistry  (Na, K, Cl, CO2, BUN, creatinine)
x
x
x
x
Liver Function Tests (ALT, AST, Alk Phos, T Bilirubin; D Bilirubin, GGT) 
x
x
x
x
Tacrolimus Level3
x
x
x
x
Everolimus Level3
x
x
x
x
Local Pathology Results - Graft Routine Histology
x
Protocol Liver Biopsy
x
Allofrequency Assay & MFC-Leukocyte, Treg, Tact/mem, B Cell Panels (10 ml Green Heparin Tube)
10
10
MLR Suppression (2-10ml Green Heparin Tubes)11
20
20
TCR Sequencing (10ml Green Heparin Tube)
10
10
Heavy Glucose Labeling (10ml Green Heparin Tube)
10
10
Alloantibody Analysis-Donor HLA Ab (3ml Serum Separator Tube)
3
3
0.0
53.0
53.0
* This schedule should be utilized for subjects who terminate from the study after receiving any darTregs.
± 7 days 
1 Subjects w ho prematurely terminate from Stage 3 of the study should have the the first safety follow  up visit 4 w eeks after stopping protocol therapy.  Follow  up visit F1 (4 
w eeks after termination) should be replaced w ith visit F2 or visit F3 if the subject terminates w ithin 4 w eeks of that visit. 
4 For each clinically indicated biopsy, one specimen w ill be in formalin for unstained slides, one specimen in PBS for deuterium tracking, and one specimen in RNALater.
3 Tacrolimus and EVR levels should be collected from standard of care labs, if available.  
Study Assessments
2 The second follow -up visit takes place 10 months (w eek 40) after transplant.  
Central Laboratory Assessments
Local Laboratory Assessments
Collect only w hen 
CIB visit coincides 
w ith scheduled 
visit w ith central 
labs.
Wk 4 
after 
Term1 
Clinically 
Indicated 
Biopsy/ 
Rejection


 
Confidential 
Page 80 of 80 
Tregs in Liver Transplantation (RTB-002) 
                                                            Version 7.0/ August 1, 2018 
Appendix 6.  Donor Schedule of Events 
  
 
Study Assessments/Data Collection1
Demographics (DOB, Gender, Ethnicity)
Medical and Social History 
Physical Assessment
ABO Blood Type
HIV Ab, HCV Ab, HBsAg, HBcAb
EBV Ab
HBV, HCV, HIV, WNV1 NAT
RPR
T.Cruzi Ab1
Manufacturing and Central Laboratory Assessments
Donor Eligibility per 21CFR 1271 including CMV Total Antibody, HTLV 1 and 2) (6ml Red Top) (Cohort 2, 3, 4 only)
Donor Blood for Banking or HLA Typing2 (3ml Gold or Red Top Tube)(Cohort 2, 3, 4 only)
1cm3 Spleen (B Cells and HLA Typing)
Donor Blood for Manufacturing (70ml Green Top Tubes) (UCSF only)
 1Data should be collected from medical records or UNOS records retrospectively. Except for WNV and T. Cruzi, viral tests should be but 
ordered if not available.  WNV NAT and T. Cruzi Ab should be collected for study/manufacutring records if available.  
2An additional tube should be collected w henever available for subjects in Cohort 2, 3, and 4.  

SAP_001.pdf: 
 
 
 
 
 
 
 
 
darTreg Statistical Analysis Plan 
 
 
 
Donor-Reactive Regulatory T Cell Therapy in Liver Transplantation 
 
 
 
 
Version: 1.0 
 
Date: 06MAY2020 
 
Study Sponsor: The National Institute of Allergy and Infectious Diseases (NIAID) 
 
NCT02188719 
 
 
 
 
 
 
 
 
PREPARED BY 
 
Katharine Spain, MS  
 
 
 
 
Rho, Inc. 
2635 E NC Hwy 54 
Durham, NC 27713 
 
 
 
 
 
 
 
 
 
 
Tel: 919-408-8000 
 
 
 
 
katie_spain@rhoworld.com  
 
 
 
 
 
 
 
 
 
Confidential 
This document is confidential and proprietary to the National Institute of Allergy and Infectious Diseases. 
Acceptance of this document constitutes agreement by the recipient that no unpublished information 
contained herein will be reproduced, published, or otherwise disclosed without the prior written approval 
of the National Institute of Allergy and Infectious Diseases, except that this document may be disclosed 
to appropriate Institutional Review Boards under the condition that they keep the information confidential. 
 


 
 
APPROVALS 
Approved: 
 
Date: 
 
 
Sandy Feng, MD, PhD 
Treg Principal Investigator 
 
 
 
 
Date: 
 
 
Nancy Bridges, MD 
NIAID Medical Monitor 
 
 
 
 
Date: 
 
 
Michelle Sever, PhD 
Rho Senior Reviewer 
 
 
 
 
Date: 
 
 
Katharine Spain, MS  
Rho Senior Statistician 
 
 
 
 
 


TABLE OF CONTENTS 
1. 
INTRODUCTION ..................................................................................................... 7 
2. 
STUDY DESIGN ...................................................................................................... 7 
2.1 
Safety Outcomes .......................................................................................... 9 
2.2 
Safety Objective ........................................................................................... 9 
5. 
ANALYSIS SAMPLES ........................................................................................... 11 
5.1 
Screen Failure Analysis .............................................................................. 11 
6. 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS ........................ 11 
7. 
INTERIM ANALYSES AND DATA MONITORING ................................................. 12 
8. 
STATISTICAL METHODOLOGY AND STATISTICAL ANALYSES ...................... 12 
8.1 
Analysis Methods .............................................................................. 12 
9. 
ADDITIONAL SAFETY EVALUATIONS ................................................................ 13 
9.1 
Overview of Safety Analysis Methods ......................................................... 13 
9.2 
Adverse Events .......................................................................................... 13 
9.3 
Deaths/Graft Losses, Serious Adverse Events, and Other Significant Adverse Events
 ................................................................................................................... 13 
9.4 
Clinical Laboratory Evaluation .................................................................... 14 
9.5 
Vital Signs, Physical Findings, and Other Observations Related to Safety.. 14 
9.5.1 
Vital Signs ......................................................................................... 14 
9.5.2 
Physical Examinations ...................................................................... 14 
9.5.3 
Protocol/For-Cause Biopsies ............................................................. 14 
9.5.4 
Creatinine/GFR ................................................................................. 14 
 


 
List of Abbreviations 
 
AE  
Adverse Event  
ALT  
Alanine Aminotransferase  
CFR  
Code of Federal Regulations  
CI  
Confidence Interval  
CMV  
Cytomegalovirus  
CNI  
Calcineurin Inhibitor  
CRF  
Case Report Form  
CTCAE  
Common Terminology Criteria for Adverse Events  
DAIT  
Division of Allergy, Immunology, and Transplantation  
dr  
Donor Reactive  
darTregs  
Donor-Reactive T Regulatory Cells  
DSMB  
Data Safety Monitoring Board  
EBV  
Epstein Barr Virus  
EVR  
Everolimus  
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  
MELD  
Model for End Stage Liver Disease  
MFC  
Multiparameter Flow Cytometry  
MIHC  
Multiplex Immunohistochemistry  
MLR  
Mixed Lymphocyte Reaction  
AE  
Adverse Event  
ALT  
Alanine Aminotransferase  
CFR  
Code of Federal Regulations  
CI  
Confidence Interval  
CMV  
Cytomegalovirus  
CNI  
Calcineurin Inhibitor  
CRF  
Case Report Form  
CTCAE  
Common Terminology Criteria for Adverse Events  
DAIT  
Division of Allergy, Immunology, and Transplantation  
dr  
Donor Reactive  
darTregs  
Donor-Reactive T Regulatory Cells  


DSMB  
Data Safety Monitoring Board  
EBV  
Epstein Barr Virus  
EVR  
Everolimus  
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  
CTCAE  
Common Terminology Criteria for Adverse Events  
DAIT  
Division of Allergy, Immunology, and Transplantation  
dr  
Donor Reactive  
darTregs  
Donor-Reactive T Regulatory Cells  
DSMB  
Data Safety Monitoring Board  
EBV  
Epstein Barr Virus  
EVR  
Everolimus  
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  
MELD  
Model for End Stage Liver Disease  
MFC  
Multiparameter Flow Cytometry  
MIHC  
Multiplex Immunohistochemistry  
MLR  
Mixed Lymphocyte Reaction  
MMF  
Mycophenolate Mofetil  
mSAP  
Mechanistic Statistical Analysis Plan  


mToR  
Mammalian Target of Rapamycin  
NIAID  
National Institute of Allergy and Infectious Disease  
PI  
Principal Investigator  
PTLD  
Post -Transplant Lymphoproliferative Disorder  
PBMC  
Peripheral Blood Mononuclear Cell  
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  
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  
 


1. 
INTRODUCTION 
This statistical analysis plan (SAP) only includes analyses related to the clinical endpoints 
outlined in the protocol. Mechanistic analyses will be performed at the Immune Tolerance 
Network (ITN), and a separate mechanistic statistical analysis plan (MSAP) will be created to 
detail the planned analyses. Relevant clinical data from the study will be submitted to the ITN 
Biomarker and Discovery Research (BDR) and ITN Bioinformatics Groups (BiG) to permit the 
mechanistic data analyses. 
 
1.1 Background and Scientific Rationale 
Although ongoing refinement of immunosuppression (IS) regimens has substantially reduced the 
incidence of acute rejection after solid organ transplant, long-term outcomes have stagnated partly due 
to morbidity and mortality associated with generalized, lifelong IS. The traditional approach to IS has 
emphasized non-specific suppression of T cell responses. The more recent elucidation of regulatory T 
cells (Tregs) and their importance in regulating immune responses has encouraged the reconfiguration of 
IS regimens to favor Treg development and function with the ultimate goal of inducing graft tolerance 
(Waldmann, 2008) (Kang, 2007) (Walsh, 2004) (Yeung, 2009) (Sanchez-Fueyo, 2006) (Sagoo, 2008) 
(Long, 2009). Multiple animal models have shown that adoptive transfer of Tregs can mitigate graft 
rejection and, in combination with “Treg-supportive” IS regimens, can induce long-term tolerance (Kang, 
2007) (Riley, 2009) (Issa, 2010) (Nadig, 2010). Treg-supportive IS regimens have included the initial de-
bulking of donor-specific T cells. Thymoglobulin®, a commonly used T-cell depleting agent in 
transplantation, appears to relatively spare Tregs (Sewgobind, 2009), thereby increasing Treg: T 
conventional cell (Tconv) ratio. Additionally, mammalian Target of Rapamycin (mTOR) inhibitors, a class 
of drug to which everolimus (EVR) belongs, suppress effector T-cells while fostering Treg development 
(Demirkiran A. T., 2008) (Demirkiran A. V., 2009). We aim to translate these basic and clinical findings 
into a viable clinical protocol. We propose to test the use of donor reactive Tregs (darTregs) in the 
context of a Treg-supportive IS regimen as an approach to induce liver transplant tolerance. For several 
reasons, the liver transplant setting is ideal to evaluate the safety of Treg therapy as a strategy to either 
increase the likelihood of and/or accelerate the development of tolerance. First, liver allografts appear to 
be more tolerogenic than other allografts. Compared to recipients of other organs, liver transplant 
recipients require less IS and are relatively spared from both humoral and chronic rejection. Second, 
acute rejection, albeit common, is readily treated without long-term sequelae, a key advantage for testing 
novel immunomodulatory agents and tolerance induction strategies. Third, emerging data from IS 
withdrawal trials in liver transplant recipients indicate that the rate of spontaneous tolerance increases 
over time after transplantation, from less than 10% within 3 years to as high as 80% after 10 years 
(Bohne, 2012) (Sanchez-Fueyo A. , 2011) (Feng S. U., 2012). As the first step toward a long-term goal of 
Treg immunotherapy, we propose to determine the safety of darTregs in combination with a Treg-
supportive IS regimen in adult, de novo liver transplant recipients. We hypothesize that ex vivo-expanded 
darTregs administered to adult, de novo liver transplant recipients in combination with a Treg-supportive 
IS regimen will be safe. Our study also aims to describe for the first time the persistence of administered 
darTreg in human, as well as to describe the effects of Treg-supportive IS on darTreg and drTconv after 
transplantation. If successful, our study will define an approach for the therapeutic administration of 
Tregs, establish a new paradigm for the design of IS regimens, and set the stage for subsequent efficacy 
trials. 
2. 
STUDY DESIGN  
This is a three center, open-label, dose escalation, pilot study in which adult subjects undergoing primary 
solitary liver transplant will receive a Treg-supportive IS regimen alone (Cohorts 1a and 1b) or a Treg-
supportive IS regimen followed by a single infusion of autologous, darTregs (Cohort 2: 50 million 


darTregs; Cohort 3: 200 million darTregs: or Cohort 4: 800 million darTregs). There are three successive 
stages in the study with specific eligibility criteria that participants must meet prior to proceeding to the 
next stage. As shown in Figure 1, the three stages are:  
 
Screening and Enrollment  
 
Treg Supportive IS Regimen and,  
 
darTreg Infusion  
 
Subjects will be followed for 40 weeks after transplant, during which clinical data along with peripheral 
blood (PBMCs and serum) and liver biopsy samples will be collected and analyzed.  
 
 
 
 
 
Figure 1. Three Stages of Trial 
 
The four cohorts are summarized below: 
 
Cohort 1: 3 subjects from Center 1 (Cohort 1a) + 3 subjects from Center 2 (Cohort 1b); these 
subjects will only receive Treg supportive IS study therapy (Stage 1 and 2; they will not receive 
darTreg infusion (Stage 3)).  
 
Cohort 2: 3 subjects meeting eligibility criteria for all 3 Stages will receive 50 million darTregs 
(Dose A).  
 
Cohort 3: 3 subjects meeting eligibility criteria for all 3 Stages will receive 200 million darTregs 
(Dose B).  
 
Cohort 4: 6 subjects meeting eligibility criteria for all 3 Stages will receive 800 million darTregs 
(Dose C).  


2.1 
Safety Outcomes 
The safety, tolerability, and dose limiting toxicities of darTreg therapy given within the context of a Treg 
supportive IS regimen will be evaluated with the rate of the following events within 40 weeks of 
transplantation: 
Clinical outcomes that will be described are: 
1.  Incidence and severity of biopsy-proven acute and/or chronic rejection 
2.  Incidence of ≥ Grade 3 infections as defined in Section 13.3.1 
3.  Incidence of wound complications (≥ CTCAE Grade 3) 
4.  Incidence of anemia, neutropenia, and/or thrombocytopenia (≥ CTCAE Grade 3) 
5.  Incidence of adverse events attributable to the darTreg infusion including infusion 
reaction/cytokine releaser syndrome (≥ CTCAE Grade 3), and malignant cellular transformation. 
2.2 
Safety Objective 
This study will evaluate the safety, tolerability, and dose limiting toxicities (DLT’s) of a Treg-supportive IS 
regimen and darTreg infusion for adult, de novo, liver transplant recipients. 
3. 
Sample Size Considerations 
This pilot study is designed to evaluate 18 subjects through Stage 3 in a modified 3+3 dose escalation 
design. Therefore, no formal power and sample size analysis have been performed. However, with 18 
evaluable subjects undergoing darTreg supportive IS for 12 weeks of follow-up (6 subjects for 40 weeks 
of follow-up) and up to 12 evaluable subjects undergoing darTreg infusions for 28 weeks of follow-up, 
exact 95% confidence intervals on person-week incidence rates will vary in width by analysis period. For 
example, the following table (Table 1) shows incidence rates and confidence intervals for selected 
numbers of events and person-weeks of 216 (18 subjects x 12 weeks), 336 (12 subjects x 28 weeks), 
and 384 (6 subjects X 40 weeks and 12 subjects X 12 weeks) in the two analysis periods. It is not known 
at this time how many subjects may be expected to comprise the entire Safety Sample.  
 
Table 1. Incidence Rates and Confidence Intervals for Selected Numbers of Events and Person-Weeks 
 
Number of 
Events 
Total Person-
Weeks 
Person-Weeks 
Incidence Rate 
Lower 95% 
Confidence Limit 
Upper 95% 
Confidence Limit 
2 
216 
0.0093 
0.0011 
0.0334 
4 
216 
0.0185 
0.0050 
0.0474 
6 
216 
0.0278 
0.0102 
0.0605 
8 
216 
0.0370 
0.0160 
0.0730 
10 
216 
0.0463 
0.0222 
0.0851 
12 
216 
0.0556 
0.0287 
0.0970 
14 
216 
0.0648 
0.0354 
0.1087 
16 
216 
0.0741 
0.0423 
0.1203 
18 
216 
0.0833 
0.0494 
0.1317 
20 
216 
0.0926 
0.0566 
0.1430 
 
2 
336 
0.0060 
0.0007 
0.0215 
4 
336 
0.0119 
0.0032 
0.0305 
6 
336 
0.0179 
0.0066 
0.0389 
8 
336 
0.0238 
0.0103 
0.0469 


10 
336 
0.0298 
0.0143 
0.0547 
12 
336 
0.0357 
0.0185 
0.0624 
14 
336 
0.0417 
0.0228 
0.0699 
16 
336 
0.0476 
0.0272 
0.0773 
18 
336 
0.0536 
0.0317 
0.0847 
20 
336 
0.0595 
0.0364 
0.0919 
 
2 
384 
0.0052 
0.0006 
0.0188 
4 
384 
0.0104 
0.0028 
0.0267 
6 
384 
0.0156 
0.0057 
0.0340 
8 
384 
0.0208 
0.0090 
0.0410 
10 
384 
0.0260 
0.0125 
0.0479 
12 
384 
0.0313 
0.0161 
0.0546 
14 
384 
0.0365 
0.0199 
0.0612 
16 
384 
0.0417 
0.0238 
0.0677 
18 
384 
0.0469 
0.0278 
0.0741 
20 
384 
0.0521 
0.0318 
0.0804 
4.        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. 
 
In general, in frequency tables of variables with different categories (e.g., for discontinuation 
reason, race): if no subjects belong to a certain category, including “Missing”, across all treatment 
groups, then the printing of this category should be suppressed.  Otherwise, if the number of 
subjects in a category for a treatment group is zero, then a zero should be displayed for the 
number, and the percentage should be left blank. 
 
Numeric variables will be summarized using n, mean, standard deviation (SD), median, min, max.  
The min/max will be reported at 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 will be 
reported at two more significant digits than the precision of the data.  Descriptive statistics will be 
displayed in the order: n, mean, SD, median, min, max 
 
The median will be reported as the average of the two middle numbers if the dataset contains 
even numbers. 
 
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, then report “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. 
 


 
In general, columns with character values will have the header and column values left aligned. 
Numeric columns will be centered on their decimal place with headers also centered. 
 
In the first column, if text wraps onto another line indent one additional space. For subgroups, 
indent two spaces. 
 
Units of measurement - International units SI will be used for clinical laboratory data as a 
standard presentation.  The metric system will be used whenever possible. Thus, weight will be in 
kilograms and height in centimeters. Temperature will be presented in Celsius degrees. 
 
For general footnotes, ‘Note:’ will come before any bracketed footnotes. 
 
All listings will be sorted in order of treatment, subject, and time of assessment (e.g., visit, time, 
and/or event). 
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. 
5. 
ANALYSIS SAMPLES 
The Safety Sample will be comprised of all study subjects who are consented and receive any study 
therapy, including leukapheresis, Treg-supportive IS and darTreg infusion. This excludes consented 
subjects who were terminated before receiving any study treatment.  
 
Safety and clinical outcomes will be analyzed within the Safety Sample as a whole and within the 
following two treatment-related analysis periods during which:  
• Subjects receive any darTreg supportive IS  
• Subjects receive darTreg infusions at any dose.  
 
Subjects will contribute adverse events and weeks at risk to each analysis period for as long as they are 
on the corresponding study treatment in that analysis period. Thus, if they progress from darTreg 
supportive IS to darTreg infusions at any dose, they will contribute subsequent adverse events and 
weeks at risk to the darTreg infusion analysis period when they initiate any darTreg infusion and that will 
continue until the end of their follow-up time on study. 
5.1 
Screen Failure Analysis 
At each stage of the study, subject’s eligibility will be re-evaluated. Reasons for study discontinuation for 
screen failures will be summarized at each stage, both overall and within each cohort. 
6. 
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS 
Summary descriptive statistics for baseline and demographic characteristics will be provided for all 
enrolled participants. Demographic data will include age, race, sex, body weight, and height; these data 
will be presented in the following manner:  
 
 
Continuous data (i.e., age, body weight, and height) will be summarized descriptively by mean 
standard deviation, median, and range.  
 
Categorical data (i.e., sex and race) will be presented as enumerations and percentages.  
Both Recipient and Donor demographic and baseline characteristic data will also be presented in data 
listings by subject. 


7. 
INTERIM ANALYSES AND DATA MONITORING 
There are no interim analyses planned for this study. 
 
The protocol chair, the ITN clinical trial physician, the NIAID medical monitor, and the NIAID Transplant 
Data and Safety Monitoring Board (DSMB) will periodically review safety data. Enrollment of subjects in 
the trial and/or potentially progression from one stage of the trial to the subsequent stage for current trial 
subjects may be suspended at any time if any of these reviews concludes that there are significant safety 
concerns. Stopping rules are described in Section 18.3.1 of the protocol. 
 
The progress of the study will be monitored by the NIAID Transplant DSMB. The DSMB will be chartered 
to review safety data and to make recommendations regarding continuation, termination, or modification 
of the study.  The DSMB will 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 when an event occurs that is of sufficient concern 
to the NIAID medical monitor or protocol co-chairs to warrant review, or when an event occurs that could 
contribute to a pre-defined stopping rule as specified in the Protocol. 
 
Findings will be reported to IRBs and health authorities. 
8. 
STATISTICAL METHODOLOGY AND STATISTICAL ANALYSES 
8.1 
Analysis Methods 
All analyses will be run on the safety population with no sensitivity analyses, sub-group analyses, or 
covariate adjustments planned. 
8.1.1 Safety Outcome Analyses 
 
The safety outcomes will be considered in two ways:  the absolute number and the person-week 
incidence rates (Section 3.2) of selected adverse events following transplantation.  The person-week 
incidence rate will be estimated using proportions and exact binomial two-sided 95% confidence intervals 
for the Safety Sample as a whole and within the two analysis periods defined above.  The absolute 
number and the estimated incidence proportions of the following will be reported: 
 
 
Biopsy-proven acute and/or chronic rejection 
 
Grade ≥ 3 infections as defined in Section 13.3.1 of the Protocol 
 
Wound complications (≥ CTCAE Grade 3) 
 
Anemia, neutropenia, and/or thrombocytopenia (≥ CTCAE Grade 3) 
 
Adverse events attributable to the darTreg infusion including infusion reaction/cytokine releaser 
syndrome (≥ CTCAE Grade 3), and malignant cellular transformation. 
 
Descriptive statistics will also be presented for: 
 
 
The severity of biopsy-proven acute and/or chronic rejection 
 
Reasons for termination of subjects who did not receive leukapheresis 
 
Use of concomitant medications 


9. 
ADDITIONAL SAFETY EVALUATIONS 
9.1 
Overview of Safety Analysis Methods 
All safety analysis will be carried out using the safety sample defined in Section 5 above unless 
otherwise noted. Missing safety information will not be imputed. Safety will be analyzed through the 
reporting of adverse events, vital signs, physical examinations, and changes in routine laboratory values. 
9.2 
Adverse Events 
All adverse events will be classified by system organ class and preferred term, according to a 
standardized thesaurus (MedDRA version 17.0). The severity of AEs will be classified using the National 
Cancer Institute’s Common Toxicity Criteria for Adverse Events, with the exception of infections which 
will be graded according to protocol definitions. 
 
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 (SAEs) 
 
AEs that lead to study discontinuation 
 
AEs with an outcome of death 
 
AEs reported by maximum severity 
In addition, adverse events classified by MedDRA system organ class and preferred term will be 
summarized for each study cohort and overall for each of the following: 
 
All AEs 
 
AEs by maximum severity 
 
AEs by study stage 
The summary tables will present the total number of events as well as the number and percentage of 
subjects experiencing the events.  When reporting the number of AEs, if the same AE occurs for a 
subject on multiple occasions the event will be counted once for each occurrence.  When reporting the 
number of subjects experiencing the events, a subject will only be counted once if they ever experience 
an event within the particular system organ class or preferred term.  Percentages will be based on the 
number of subjects in the analysis population. 
A summary table of incidence rates adverse events classified by system organ class and MedDRA 
preferred term will be provided.  Incidence rate for a system organ class or preferred term will be defined 
as the number of adverse events divided by the person-time since the subject enrolled. 
Separate data listings will be provided for AEs leading to study discontinuation. 
9.3 
Deaths/Graft Losses, Serious Adverse Events, and Other Significant Adverse Events 
SAEs will be listed and summarized in the same manner described in section 9.2. Separate displays 
listing and summarizing death, graft loss, grade 3 or higher infections (per protocol definitions), severe 
acute cellular rejection (histological or clinical), chronic rejection, PTLD, and malignancies (excluding 
recurrent HCC and skin cancer), will also be created. 


9.4 
Clinical Laboratory Evaluation 
Data listings will be provided for clinical laboratory measurements including serum chemistry, 
hematology, and liver tests. They will be sorted by subject ID, laboratory parameter and time of 
assessment.  Results will be standardized to the international system of units (SI), where possible.  
Laboratory normal ranges for serum chemistry and hematology results will be included and out-of-range 
flags (H or L) will be used to denote abnormal values.  Standard reference ranges will be used instead of 
site specific normal ranges.  Liver tests will be compared to the subject’s baseline test values to 
determine abnormal values. 
Selected serum chemistry and hematology tests with numeric results will be plotted to show patterns 
over time.  Tests with qualitative results (such as ‘present’ or ‘positive’) will not be plotted. 
9.5 
Vital Signs, Physical Findings, and Other Observations Related to Safety 
9.5.1 Vital Signs 
Descriptive statistics of vital signs results and change from baseline of vital signs will be summarized for 
each study group and overall. Data listings will be provided for vital signs measurements. They will be 
sorted by treatment group, subject, vital sign parameter and time of assessment. 
9.5.2 Physical Examinations 
Physical examination results of normal, abnormal, and not done will be summarized as frequencies and 
percentages by body system and visit. Data listings will be provided for physical examination results and 
sorted by treatment group, subject, body system and time of assessment.  
9.5.3 Protocol/For-Cause Biopsies 
Protocol and for-cause biopsies will be summarized as frequencies for each cohort and overall at each 
timepoint.  Data listings for each individual subject will be provided with detailed biopsy information (i.e.-
fibrosis, inflammation, diagnosis, etc.).  
9.5.4 Creatinine/GFR 
Creatinine/GFR values will be plotted over time for each subject to assess kidney function.  Data listings 
of GFR values will be provided for all subjects with at least one abnormal GFR result.