Conditions: Liver Transplant
Keywords: alloantigen-reactive Tregs (arTreg) immunosuppression operational tolerance
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 42
Healthy volunteers: no

Brief summary:
This is a single-center, prospective, open-label, non-randomized clinical trial exploring cellular therapy to facilitate immunosuppression withdrawal in liver transplant recipients.

Detailed description:
The researchers in this study plan to enroll 9 participants. Eligible participants will receive a single dose of Treg product (arTreg). The target dose is at least 90 to 500 x 10\^6 total cells.

Participants who successfully withdraw from all immunosuppression (IS) will undergo a research biopsy at 52 weeks following IS discontinuation to determine whether they meet the primary efficacy outcome of operational tolerance. Participants determined to be operationally tolerant will be followed until 104 weeks following IS discontinuation. Participants who fail drug withdrawal after 52 weeks but before 104 weeks will be followed until week 104 or 12 weeks after resuming immunosuppression, whichever is longer.

Participants who do not successfully withdraw from all IS will complete 104 weeks of High Intensity Safety Follow-up after failing immunosuppression withdrawal.

\*\*\* IMPORTANT NOTICE: \*\*\* The National Institute of Allergy and Infectious Diseases and the Immune Tolerance Network do not recommend the discontinuation of immunosuppressive therapy for recipients of cell, organ, or tissue transplants outside of physician-directed, controlled clinical studies. Discontinuation of prescribed immunosuppressive therapy can result in serious health consequences and should only be performed in certain rare circumstances, upon the recommendation and with the guidance of your health care provider.

Arms:
arm 1: arTreg: alloantigen-reactive T regulatory cells

The investigational product is donor alloantigen-reactive regulatory T cells (arTreg). Supportive regimen for receipt of arTregs includes everolimus, leukapheresis, cyclophosphamide, and mesna.

Note: Participants who receive at least the minimum Treg product (arTreg) dose of 30 to \<90 x10\^6 total cells will be included in intent-to-treat analysis.

Interventions: intervention 1: arTreg intervention 2: leukapheresis intervention 3: cyclophosphamide intervention 4: mesna intervention 5: everolimus
Intervention descriptions: intervention 1: Eligible participants will receive a single dose of Treg product (arTreg). The target dose is at least 90 x 10\^6 total cells.

Method of receipt: peripheral intravenous (IV) infusion, administered over 20 to 30 minutes. intervention 2: Leukapheresis will be the method employed to recover peripheral blood mononuclear cells (PBMCs) from the allograft recipient. The recipient will undergo the procedure prior to initiating the cyclophosphamide conditioning regimen.

Procedure on Day -3 (-1 day) prior to Treg product (arTreg) IV infusion. intervention 3: 40 mg/kg administered intravenously (IV) following leukapheresis and between 1 to 3 days prior to Treg product (arTreg) infusion, per institutional standard of care. intervention 4: Mesna is administered:

* Intravenously to inhibit hemorrhagic cystitis induced by cyclophosphamide, and
* In conjunction with the cyclophosphamide, per institutional practice with CTX. intervention 5: EVR is approved for prophylaxis of allograft rejection in adults receiving a liver transplant. Per protocol: Post transplantation, subject will initially receive standard IS with tacrolimus (TAC),plus a mycophenolate product and/or steroids.Subsequently, evaluation for eligibility to be converted to EVR-based IS regimen will occur and, when applicable, proceed. Once the optimal EVR trough level is achieved,TAC dose will be reduced. When target EVR and TAC levels are maintained over two consecutive measurements, ALT liver function test (LFT) is ≤50 U/L, GGT LFT is ≤ the upper limit of normal or ≤ 1.5 times the baseline GGT, subject will be considered successfully converted to EVR-based IS regimen. EVR doses will be administered/monitored/adjusted over time.

Protocol document:
Prot_SAP_000.pdf: 
Immune Tolerance Network 
CONFIDENTIAL 
Page 1 
Protocol ITN074ST 
Version 4.0 
January 18, 2022 
LITTMUS: Liver Transplantation with Tregs at UCSF  
A Phase I/II Drug Withdrawal Study of Alloantigen-Specific 
Tregs in Liver Transplantation 
 Protocol ITN074ST 
Version 4.0 (January 18, 2022) 
IND # 18329 
IND Sponsor: DAIT NIAID 
Funded by: DAIT NIAID 
Protocol Chair 
Sandy Feng, MD, PhD 
Professor, Department of Surgery 
University of California, San Francisco 
505 Parnassus Ave., Box 0780, M-896 
San Francisco, CA 94143 
Tel: 415- 353-8725  Fax: 415- 353-8709 
Email: Sandy.Feng@ucsfmedctr.org 
 
Protocol Manufacturing Chair 
Qizhi Tang, PhD 
Professor, Department of Surgery 
Director, UCSF Transplantation Research Lab 
University of California, San Francisco 
513 Parnassus Ave., Box 0780, HSE-520 
San Francisco, CA 94143-0780  
Tel: 415-476-1739  Fax: 415-502-8326 
Email: qizhi.tang@ucsfmedctr.org 
ITN Clinical Trial Physician 
Sindhu Chandran, MBBS 
Associate Director, Clinical and Translational 
Medicine 
Clinical Trials Group,  
Immune Tolerance Network 
University of California, San Francisco 
Diabetes Center 
513 Parnassus Avenue, HSW 11, Box 0534 
San Francisco, CA 94143-0534 
Tel: 415-610-4284   
Email: schandran@immunetolerance.org 
 
NIAID Medical Monitor 
Nancy D. Bridges, MD 
Chief, Transplantation Branch, Division of 
Allergy, Immunology and Transplantation, NIAID 
5601 Fisher Lane, Room 6B31 
Rockville, MD 20852-6601  
Tel: 240-627-3535  
Email: nbridges@niaid.nih.gov 
NIAID Regulatory Affairs 
Julia Goldstein, MD 
Senior Regulatory Affairs Officer 
Division of Allergy, Immunology and 
Transplantation, NIAID 
5601 Fisher Lane, Room 7B29 
Rockville, MD 20852-6601  
Tel: 240-627-3509 
Email: goldsteinj@niaid.nih.gov 
 
ITN Lead Biologist 
Cynthia Breeden, MPH 
Emory University School of Medicine 
Department of Surgery, Division of 
Transplantation 
Larsen Lab 
Woodruff Memorial Research Building 
101 Woodruff Circle, Room 5314 
Atlanta, GA 30322 
Tel: 404-379-5811 
Email: cbreeden@immunetolerance.org 


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LITTMUS: Liver Transplantation with Tregs at UCSF  
ITN Manager of Clinical Operations 
Geo Gaile 
Manager, Clinical Operations 
Clinical Trials Group 
Immune Tolerance Network 
University of California, San Francisco 
Diabetes Center 
513 Parnassus Avenue, HSW 11, Box 0534 
San Francisco, CA 94143-0534 
Phone: 415-854-7485    Fax: 415-353-4404 
Email: ggaile@immunetolerance.org 
NIAID Project Manager 
Natasha Watson, BSN 
Division of Allergy, Immunology, and 
Transplantation 
National Institute of Allergy and Infectious 
Diseases 
5601 Fishers Lane 
Rockville, MD 20852 
Tel: 301-335-8476  Fax: 240-627-3115 
Email: nwatson@niaid.nih.gov 
Rho Scientist 
Karen Kesler, PhD 
Senior Statistical Scientist 
Rho Inc. 
2635 East NC Highway 54  
Durham, NC 27713  
Tel: 919-595-6244  Fax: 919-408-0999 
Email: karen.kesler@rhoworld.com  
 
This clinical study is supported and conducted by the Immune Tolerance Network, which is 
sponsored by the National Institute of Allergy and Infectious Diseases. 
This document is confidential. It is provided for review only to investigators, potential investigators, 
consultants, study staff, and applicable independent ethics committees or institutional review boards. It 
is understood that the contents of this document will not be disclosed to others without written 
authorization from ITN and NIAID unless it is necessary to obtain informed consent from potential study 
participants. 
 
 


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LITTMUS: Liver Transplantation with Tregs at UCSF  
 
 
SITE INVESTIGATOR SIGNATURE PAGE 
Protocol Number:  
  
Version Number/Date: 4.0 / January 18, 2022 
  
Protocol Title:   A Phase I/II Drug Withdrawal Study of Alloantigen-Specific Tregs in Live
Transplantation 
  
IND/IDE Sponsor:  The National Institute of Allergy and Infectious Diseases (NIAID)      
[Amend if another IC. Change “IND/IDE Sponsor” to “Study Sponsor” if no IND/IDE]             
Return Signed Form to: 
The original signature page must be kept for your records. Return an electronic PDF copy of the signed 
signature page (*as described below) to the DAIT Regulatory Management Center via the applicable DAIT 
RMC email address for the protocol/network. 
  
If PPD is not the Regulatory Management Center for this protocol, e.g., for an Investigator Initiated clinical 
trial, contact the DAIT Regulatory Officer for instructions on where to send the completed signature page. 
I confirm that I have read the above protocol in the latest version. I understand it, and I will work 
according to the principles of Good Clinical Practice (GCP) as described in the United States Code of 
Federal Regulations (CFR) – 45 CFR part 46 and 21 CFR parts 50, 56, and 312, 812 and in the 
International Conference for Harmonisation (ICH) document entitled Integrated Addendum to ICH 
E6(R1): Guideline for Good Clinical Practice E6(R2). 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. 
 
[*The site Principal Investigator should sign and date at the indicated location below. A written 
signature/date is acceptable (e.g., scanned and sent via email as a PDF version). An electronic signature is 
also acceptable (e.g., sent via email as a PDF version).] 
 
____________________________________     
Site Principal Investigator  (Print) 
 
____________________________________                                      _________________ 
Site Principal Investigator (Signature)                                                          Date 


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TABLE OF CONTENTS 
TABLE OF CONTENTS ............................................................................................................. 4 
SYNOPSIS ................................................................................................................................ 10 
ABBREVIATIONS.................................................................................................................... 15 
STUDY DEFINITIONS ............................................................................................................. 18 
1. 
BACKGROUND AND RATIONALE ................................................................................ 20 
1.1 
BACKGROUND INFORMATION .................................................................................. 20 
 
Study Summary ......................................................................................................................... 20 
 
IS Withdrawal in Liver Transplantation .................................................................................... 21 
 
Current Trial .............................................................................................................................. 21 
1.2 
RATIONALE ................................................................................................................. 22 
 
Rationale for Treg Therapy in Allogeneic Transplantation ....................................................... 22 
 
Why Explore Treg Efficacy in Liver Transplantation? ............................................................. 22 
 
Antigen-Specific vs. Polyclonal Tregs ...................................................................................... 23 
 
T-regulatory Cell Product.......................................................................................................... 23 
 
Rationale for Conditioning with CTX Prior to Treg Infusion ................................................... 27 
1.3 
POTENTIAL RISKS AND BENEFITS ............................................................................ 28 
 
Potential Risks ........................................................................................................................... 28 
 
Potential Benefits ...................................................................................................................... 36 
2. 
OBJECTIVES ................................................................................................................... 36 
2.1 
PRIMARY OBJECTIVE ................................................................................................. 36 
2.2 
SECONDARY OBJECTIVES.......................................................................................... 36 
2.3 
MECHANISTIC OBJECTIVES ....................................................................................... 36 
3. 
STUDY DESIGN ............................................................................................................... 36 
3.1 
DESCRIPTION .............................................................................................................. 36 
3.2 
STUDY DURATION ...................................................................................................... 37 
3.3 
STUDY ENDPOINTS ..................................................................................................... 38 
 
Primary Endpoints ..................................................................................................................... 38 
 
Secondary Endpoints ................................................................................................................. 39 
 
Exploratory Mechanistic Endpoints .......................................................................................... 39 
3.4 
PREMATURE TERMINATION OR SUSPENSION OF THE TRIAL / STOPPING RULES 39 
 
Ongoing Review........................................................................................................................ 39 


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Study Stopping Rules Guidance ................................................................................................ 39 
4. 
ELIGIBILITY ................................................................................................................... 41 
4.1 
RECIPIENT (ELIGIBILITY 1 – PRE-TRANSPLANT) ..................................................... 41 
 
Inclusion Criteria ....................................................................................................................... 41 
 
Exclusion Criteria...................................................................................................................... 41 
4.2 
LIVING DONOR ............................................................................................................ 42 
 
Inclusion Criteria ....................................................................................................................... 42 
 
Exclusion Criteria...................................................................................................................... 42 
4.3 
DECEASED DONOR ..................................................................................................... 42 
 
Inclusion Criteria ....................................................................................................................... 42 
 
Exclusion Criteria...................................................................................................................... 42 
4.4 
PREMATURE DISCONTINUATION OF STUDY THERAPY ......................................... 42 
4.5 
PREMATURE TERMINATION OF A PARTICIPANT FROM THE STUDY .................... 43 
5. 
STUDY THERAPIES, MEDICATIONS AND PROCEDURES ......................................... 44 
5.1 
OVERVIEW ................................................................................................................... 44 
5.2 
STUDY MEDICATIONS ................................................................................................ 45 
 
Investigational Agent: Donor Alloantigen-Reactive Regulatory T Cells (arTreg) .................... 45 
 
arTreg Preparative Agents ......................................................................................................... 47 
 
Maintenance Immunosuppressive Agents ................................................................................. 50 
 
Concomitant Medications ......................................................................................................... 52 
 
Drug Accountability .................................................................................................................. 53 
 
Assessment of Compliance with Study Medications ................................................................ 53 
5.3 
POST-TRANSPLANT STUDY PROCEDURES ............................................................... 53 
 
Study Stage 1: Initial Post-transplant IS .................................................................................... 54 
 
Study Stage 2: EVR Conversion ............................................................................................... 56 
 
Study Stage 3: arTreg Manufacturing and Investigational Treatment ....................................... 57 
 
Study Stage 4: TAC Withdrawal ............................................................................................... 61 
 
Study Stage 5: EVR/Prednisone Withdrawal ............................................................................ 62 
 
Study Stage 6: Post-IS Withdrawal Surveillance and Evaluation ............................................. 64 
5.4 
OTHER STANDARD STUDY PROCEDURES ................................................................ 64 
 
Procedure for Temporary Pause in IS Withdrawal .................................................................... 64 
 
Procedure for IS Resumption .................................................................................................... 65 
 
Assessment and Treatment of Allograft Dysfunction ............................................................... 65 
6. 
STUDY ASSESSMENTS AND WINDOWS ...................................................................... 68 


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6.1 
OVERVIEW ................................................................................................................... 68 
6.2 
VISIT WINDOWS .......................................................................................................... 68 
6.3 
GENERAL ASSESSMENTS ........................................................................................... 71 
 
Recipient ................................................................................................................................... 71 
 
Living Donor ............................................................................................................................. 72 
 
Deceased Donor ........................................................................................................................ 72 
6.4 
CLINICAL ASSESSMENTS AND PROCEDURES .......................................................... 72 
 
Recipient ................................................................................................................................... 72 
6.5 
CLINICAL LABORATORY ASSESSMENTS ................................................................. 72 
 
Recipient ................................................................................................................................... 72 
 
Living Donor ............................................................................................................................. 73 
 
Deceased Donor ........................................................................................................................ 73 
6.6 
ARTREG MANUFACTURE ........................................................................................... 74 
 
Recipient ................................................................................................................................... 74 
 
Living Donor ............................................................................................................................. 74 
 
Deceased Donor ........................................................................................................................ 74 
6.7 
LIVER BIOPSY ............................................................................................................. 74 
 
Types of Biopsies ...................................................................................................................... 74 
 
Central and Local Biopsy Assessments ..................................................................................... 74 
 
Biopsy Technique and Handling ............................................................................................... 75 
 
Recording of Biopsy Assessments ............................................................................................ 75 
6.8 
MECHANISTIC ASSESSMENTS ................................................................................... 75 
7. 
MECHANISTIC ASSAYS ................................................................................................. 75 
7.1 
RATIONALE FOR IMMUNE STUDIES .................................................................. 75 
7.2 
MECHANISTIC HYPOTHESES ..................................................................................... 76 
7.3 
PROPOSED MECHANISTIC ASSAYS ........................................................................... 76 
 
HLA Typing .............................................................................................................................. 76 
 
Functional Assays ..................................................................................................................... 76 
 
Graft-Related Assays ................................................................................................................ 78 
 
Treg Cell Product Characterization ........................................................................................... 79 
7.4 
QUALITY CHECK/PRELIMINARY STUDIES ............................................................... 79 
7.5 
FUTURE / UNPLANNED STUDIES ............................................................................... 79 
8. 
ADVERSE EVENTS ......................................................................................................... 80 
8.1 
OVERVIEW ................................................................................................................... 80 


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8.2 
DEFINITIONS ............................................................................................................... 80 
 
Adverse Event ........................................................................................................................... 80 
 
Suspected Adverse Reaction and Adverse Reaction ................................................................. 80 
 
Unexpected AE ......................................................................................................................... 80 
 
Serious Adverse Event .............................................................................................................. 81 
8.3 
COLLECTING AND RECORDING ADVERSE EVENTS ................................................ 81 
 
Collection Period ....................................................................................................................... 81 
 
Collecting Adverse Events ........................................................................................................ 81 
 
Recording AEs .......................................................................................................................... 82 
 
Severity of Adverse Events to be Recorded .............................................................................. 82 
 
Methods of Recording ............................................................................................................... 82 
8.4 
GRADING AND ATTRIBUTION OF ADVERSE EVENTS ............................................. 82 
 
Grading...................................................................................................................................... 82 
 
Attribution ................................................................................................................................. 84 
8.5 
REPORTING SERIOUS ADVERSE EVENTS ................................................................. 84 
 
Reporting SAEs to the IND Sponsor ......................................................................................... 84 
 
Reporting SAEs to Health Authorities ...................................................................................... 85 
 
Reporting SAEs to the DSMB .................................................................................................. 86 
 
Reporting of AEs to IRBs/IECs ................................................................................................ 86 
8.6 
REPORTING PREGNANCY........................................................................................... 86 
8.7 
REPORTING OF OTHER SAFETY INFORMATION ...................................................... 87 
8.8 
REVIEW OF SAFETY INFORMATION ......................................................................... 87 
 
Medical Monitor Review .......................................................................................................... 87 
 
DSMB Review .......................................................................................................................... 87 
9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ................................... 87 
9.1 
ANALYSIS SAMPLES ................................................................................................... 87 
9.2 
ANALYSIS OF ENDPOINTS ......................................................................................... 88 
 
Overview ................................................................................................................................... 88 
 
Primary Endpoints ..................................................................................................................... 88 
 
Secondary Endpoints ................................................................................................................. 88 
 
Safety Analysis.......................................................................................................................... 89 
 
Exploratory Analysis ................................................................................................................. 89 
 
Medical History ......................................................................................................................... 89 
 
Use of Medications ................................................................................................................... 89 


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9.3 
SAMPLE SIZE ............................................................................................................... 89 
9.4 
MISSING DATA ............................................................................................................ 90 
9.5 
POOLED ANALYSIS WITH OTHER LITTMUS TRIALS ............................................... 90 
9.6 
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL PLAN ................... 90 
10. ACCESS TO SOURCE DATA/DOCUMENTS .................................................................. 90 
11. QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 91 
12. ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE .................................................................................................................. 91 
12.1 
STATEMENT OF COMPLIANCE .................................................................................. 91 
12.2 
INFORMED CONSENT ................................................................................................. 91 
12.3 
PRIVACY AND CONFIDENTIALITY ............................................................................ 92 
13. PUBLICATION POLICY ................................................................................................. 92 
14. REFERENCES ................................................................................................................. 93 
 
LIST OF APPENDICES 
Appendix 1A. Study Stage 1, 2, and 3A: Schedule of Events – Recipient Enrollment and EVR 
Conversion ................................................................................................................................ 97 
Appendix 1B. Schedule of Events – Donor Enrollment and Participation ............................................ 100 
Appendix 2. Study Stage 3 (A, B, C): Schedule of Events – Investigational Treatment ....................... 101 
Appendix 3. Study Stage 4: Schedule of Events – TAC Withdrawal .................................................... 104 
Appendix 4. Study Stage 5: Schedule of Events – EVR/Prednisone Withdrawal ................................. 105 
Appendix 5. Study Stage 6: Schedule of Events – Post-withdrawal Surveillance and Evaluation ........ 107 
Appendix 6. Schedule of Events – High Intensity Safety Follow-up .................................................... 108 
Appendix 7. Schedule of Events – Low Intensity Safety Follow-up ..................................................... 109 
Appendix 8. Schedule of Events – For-Cause Biopsy ........................................................................... 110 
 
LIST OF TABLES 
Table 1. arTreg Products at MGH-DFCI and UCSF ............................................................................... 24 
Table 2. Summary of Rejection Events following arTreg Infusion in ARTEMIS/CTOCc012 ............... 26 
Table 3. Comparison of Liver Transplant Trials at UCSF Evaluating arTreg ......................................... 27 
Table 4. Reported or Registered Clinical Trials of Treg Cell Therapy .................................................... 33 
Table 5. Biopsy Criteria for Operational Tolerance: Comparison of Biopsy Obtained 52 Weeks after 
the Last Dose of Immunosuppression to the Pre-Weaning (Visit 300) Biopsy57 ...................... 38 
Table 6. Stopping Rule Table for a 20% Manufacturing Failure Threshold ........................................... 40 
Table 7. Reconstitution in Preparation for IV Infusion ........................................................................... 48 
Table 8. Post-Transplant Immunosuppression ........................................................................................ 55 
Table 9. Study Stage 5: EVR/Prednisone Withdrawal. Eligibility 5 Biopsy Findings for IS 
Minimization57 .......................................................................................................................... 64 
Table 10. Banff Schema: Grading of Acute Liver Allograft Rejection ................................................... 66 
Table 11. Recipient Visit Windows ......................................................................................................... 68 
Table 12. Living/Deceased Donor Visit Windows .................................................................................. 71 
Table 13. Attribution of Adverse Events ................................................................................................. 84 


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Table 14. Analysis of Secondary Endpoints ............................................................................................ 88 
Table 15. Incidence Rates and Confidence Intervals for 9 Participants Attempting Complete IS 
Withdrawal following an Infusion of Treg Product (arTreg) .................................................... 89 
 
LIST OF FIGURES 
Figure 1. Study Design ............................................................................................................................ 37 
Figure 2. Overview of an Individual’s Participation (Study Stage 1 to 6) ............................................... 45 
Figure 3. Post-Transplant IS and EVR Conversion Regimen (Study Stage 1, 2, and 3A) ...................... 55 
Figure 4. Investigational Treatment Study Regimen (Study Stage 3) ..................................................... 58 
Figure 5. TAC Withdrawal and EVR/Prednisone Withdrawal Regimen (Study Stage 4, 5 and 6) ......... 62 
 
 


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SYNOPSIS 
Title 
A Phase I/II Drug Withdrawal Study of Alloantigen-Specific Tregs in Liver 
Transplantation 
Short Title 
Liver Transplantation with Tregs at UCSF 
IND Sponsor 
DAIT NIAID 
Conducted by 
Immune Tolerance Network 
Protocol Chair(s)  
Sandy Feng, MD, PhD 
Accrual Objective 
9 participants who receive the target Treg product (arTreg) dose 
Study Treatment 
The investigational product is donor alloantigen-reactive T regulatory cells 
(arTregs). The supportive regimen for this product includes everolimus 
(EVR), leukapheresis, cyclophosphamide (CTX), and mesna. 
Study Design 
This is a single-center, prospective, open-label, non-randomized clinical 
trial exploring cellular therapy to facilitate immunosuppression (IS) 
withdrawal in liver transplant recipients. The primary investigational 
product is arTreg. Study interventions include: 1) EVR conversion; 2) 
administration of CTX and mesna prior to a single intravenous (IV) dose of 
the arTregs, and 3) tacrolimus (TAC) discontinuation followed by 4) 
complete IS withdrawal.  
We plan to accrue 9 participants who will receive the target arTreg dose of 
at least 90 x 106 cells (section 5.2.1.4). This will comprise the PP sample. 
Participants who receive ≥30 x 106 cells but <90 x 106 cells as a result of 
low cell yield will not count towards the planned accrual, and no more than 
4 participants will be allowed in this group. This group will be included in 
the ITT sample. See section 9.1 for additional details on the analysis 
samples. 
The plan for IS, cellular therapy and IS withdrawal is described in sections 
5.1 to 5.3.5.  
Participants who successfully withdraw from all IS will undergo research 
biopsies as outlined in section 1.3.1.9. The research biopsy at 52 weeks 
following IS discontinuation will be used to determine whether they meet 
the primary efficacy endpoint of operational tolerance. Participants 
determined to be operationally tolerant will be followed until 104 weeks 
following IS discontinuation. Participants who fail IS withdrawal after 52 
weeks but before 104 weeks will be followed until week 104 or 12 weeks 
after resuming IS, whichever is longer (Appendix 5). 
Participants who do not successfully withdraw from all IS will complete 
104 weeks of High Intensity Safety Follow-up (Appendix 6) after failing IS 
withdrawal.  
All other participants will be followed per section 4.4.  


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Study Duration 
Accrual duration (section 3.1) will be 113 weeks (2.2 years) from first 
enrollment. 
Total duration of study participation will be influenced by the participant’s 
course during the study.  
• 
Participants who participate in all phases of the study, i.e. who receive 
arTregs, achieve complete IS withdrawal, and are operationally tolerant 
(Study Definitions) at 52 weeks off IS will be followed for 2 years after 
the last IS dose (Appendix 5).  
• 
Participants who receive arTregs, achieve complete IS withdrawal, and 
then resume IS (sections 5.4.2 and 5.4.3.2) will be followed for 2 years 
after first completing IS withdrawal or 12 weeks after resuming IS, 
whichever is longer (Appendix 5).  
• 
Participants who receive arTregs, but are not eligible to initiate or do 
not complete IS withdrawal will be followed for 2 years from the date 
they were deemed ineligible or failed IS withdrawal (Appendix 6).  
• 
Participants who receive CTX but do not receive arTregs will be 
followed for 1 year post CTX administration (Appendix 7). 
• 
Participants who initiate EVR conversion but do not receive CTX will 
be followed for up to 30 days as outlined in 4.4 (RFU visit in Appendix 
2).  
Primary Objective 
The primary objectives are to evaluate the safety and efficacy of a single IV 
dose of autologous, donor alloantigen-specific T regulatory cell product, 
arTreg, to induce operational tolerance early after adult living or deceased 
donor liver transplantation. 
Primary Endpoints 
The primary safety endpoints are defined as: 
1. Number and severity of adverse events (AEs) attributed to arTreg. 
2. Number and severity of AEs attributed to a) leukapheresis, b) CTX, or 
c) mesna. 
The primary efficacy endpoint is operational tolerance. Operational 
tolerance is defined as: 
1. Discontinuation of all IS for 52 weeks; 
2. ALT ≤ 50 U/L* for ≥ 2 measurements separated by ≥ 1 week in the 6 
weeks prior to the liver biopsy at 52 weeks after the last IS dose; and 
3. Liver biopsy at 52 weeks (± 4 weeks) after the last IS dose that meets 
the biopsy criteria for operational tolerance (Table 5) as assessed by 
central pathology. 
*If the investigator determines that abnormal laboratory results are related 
to an intercurrent illness, the investigator may request that the participant’s 
data be reviewed by an adjudication committee. If the adjudication 


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committee agrees, they may grant an extension of the primary endpoint visit 
window (+ 4 weeks). This adjudication committee will consist of the NIAID 
Medical Monitor, the ITN Clinical Trial Physician, and a Protocol Chair 
from another LITTMUS protocol (section 1.1.1). 
Secondary 
Endpoints 
1. Incidence of grade 3 or greater infections following arTreg infusion. 
2. Number and severity of biopsy-proven acute rejection (AR) and/or 
clinical rejection events at any time after arTreg infusion. 
3. Number of chronic rejection (CR) events at any time after arTreg 
infusion. 
4. Number of participants who develop any malignancy. 
5. The proportion of participants who discontinue TAC ≥ 12 weeks with 
ALT ≤ 50 U/L and a liver biopsy 12 - 26 weeks after the last TAC dose 
that meets biopsy criteria for IS minimization (Table 9) as assessed by 
central pathology. 
6. Durability of operational tolerance defined as the time from achieving 
the primary endpoint to IS reinitiation or to the end of trial 
participation. 
Exploratory 
Mechanistic 
Endpoints 
Mechanistic studies to evaluate the effects of the arTreg on the 
immunological profiles of liver transplant recipients. These endpoints 
include the number of participants with:  
• 
evidence of a tolerance-associated gene profile1 as described by A. 
Sanchez-Fueyo et al; 
• 
a loss or change of alloresponses following arTreg infusion; 
• 
an increase in peripheral blood Treg numbers after arTreg infusion; 
• 
an increase in intragraft Treg numbers after arTreg infusion. 
The hypotheses and associated assays for these endpoints are described in 
sections 7.2 and 7.3. 
Inclusion Criteria 
RECIPIENT 
Individuals must meet all of the following criteria to be eligible: 
1. Able to understand and provide informed consent. 
2. Male or female, 18 to 70 years of age at the time of enrollment. 
3. End-stage liver disease and listed for a living or deceased-donor 
primary solitary liver transplant. 
4. Agreement to use contraception; section 5.2.4.4. 
5. Removed in protocol version 3.0. 
6. Removed in protocol version 4.0. 
7. Positive EBV antibody test. 


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8. In the absence of contraindication, vaccinations must be up to date per 
the DAIT Guidance for Patients in Transplant Trials (Refer to the 
Manual of Procedures). 
LIVING DONOR 
Living donors must meet all of the following criteria to be eligible: 
1. Able to understand and provide informed consent. 
2. Male or female, 18 to 70 years of age at the time of enrollment. 
3. Meets site-specific clinical donor eligibility requirements. 
4. Meets donor eligibility manufacturing requirements within 7 days 
before or after the blood collection for manufacturing. 
5. Willingness to donate appropriate biologic samples. 
DECEASED DONOR 
Deceased donors must meet the following criteria for their recipients to 
remain eligible: 
1. Meets site-specific clinical donor eligibility requirements. 
2. Meets donor eligibility manufacturing requirements. 
Exclusion Criteria 
RECIPIENT 
Individuals who meet any of the following criteria will not be eligible: 
1. History of previous organ, tissue, or cell transplant. 
2. For CMV antibody negative recipients, a CMV antibody positive 
donor. 
3. Known contraindication to CTX or mesna. 
4. Serologic evidence of HIV-1/2 infection. 
5. The need for chronic anti-coagulation or anti-platelet agents other than 
aspirin that cannot be safely discontinued for a minimum of 1 week to 
safely perform a liver biopsy. 
6. End stage liver disease secondary to autoimmune etiology (such as 
autoimmune hepatitis, primary biliary cirrhosis, or primary sclerosing 
cholangitis), or other contraindications to drug withdrawal. 
7. Psychological, familial, sociological, or geographical factors potentially 
hampering compliance with the study protocol and follow-up visit 
schedule. 
8. Any condition that, in the opinion of the investigator, may interfere 
with study compliance. 
9. History of cardiac disease (ischemic heart disease requiring 
revascularization, history of or current treatment for dysrhythmia, or 
evidence of congestive heart failure) unless cleared by a cardiologist. 


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10. Any past or current medical problems, treatments or findings which, in 
the opinion of the investigator, may pose additional risks from 
participation in the study, may interfere with the candidate’s ability to 
comply with study requirements or may impact the quality or 
interpretation of the data obtained from the study. This includes past, 
present, or future enrollment in studies that affect eligibility at the time 
of EVR conversion. 
11. History of malignancy or any concomitant malignancy, except HCC, 
completely treated in-situ cervical carcinoma, or completely treated 
basal cell carcinoma (Study Definitions).  
12. Chronic use of systemic glucocorticoids or other immunosuppressives, 
or biologic immunomodulators. 
LIVING DONOR  
There are no exclusion criteria for living donors. 
DECEASED DONOR 
There are no exclusion criteria for deceased donors.  
 
 
 


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ABBREVIATIONS 
ADL 
activities of daily living 
AE 
adverse event 
AFP 
A-fetoprotein 
ALT 
alanine aminotransferase 
APCs 
antigen presenting cells  
AR 
acute rejection 
arTreg 
alloantigen-reactive Tregs 
CBC 
complete blood count 
CFR 
Code of Federal Regulations 
CKD 
chronic kidney disease 
CMV 
Cytomegalovirus 
CNI 
calcineurin inhibitor  
COVID-19 
coronavirus disease 2019 
CR 
Chronic rejection 
CRF 
chronic renal failure 
CRS 
cytokine release syndrome 
CTX 
cyclophosphamide 
DAIT 
Division of Allergy, Immunology, and Transplantation 
DFCI 
Dana-Farber Cancer Institute 
DLTs 
dose limiting toxicities 
DMF 
drug master file 
DSA 
donor specific antibodies  
DSMB 
Data and Safety Monitoring Board 
EBV 
Epstein-Barr virus 
eCRF 
electronic case report form 
ES 
enrolled sample 
EVR 
everolimus 
FACS 
Fluorescence-activated cell sorting 
FDA 
US Food and Drug Administration  
GCP 
good clinical practice 
GGT 
gamma-glutamyl transpeptidase 
GMP 
good manufacturing practice 


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GVHD 
graft verse host disease 
HBV 
hepatitis B virus 
HCC 
hepatocellular carcinoma 
HCV 
hepatitis C virus  
HICTF 
Human Islet and Cellular Transplantation Facility  
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
HSCT 
hematopoietic stem cell transplantation  
HSV 
herpes simplex virus 
IBW 
Ideal body weight 
ICH 
International Council for Harmonisation 
IRB 
institutional review board 
IS 
immunosuppression 
ISW 
immunosuppression withdrawal 
ITN 
Immune Tolerance Network 
ITT 
intent to treat  
IV 
intravenous 
MELD 
Model for End-Stage Liver Disease  
MGH 
Massachusetts General Hospital 
MMF 
mycophenolate mofetil 
NCI-
CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse Events 
(version 5.0, November 27, 2017) 
nCoV 
novel coronavirus 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NINV 
non-immune, non-viral 
PBMC  
peripheral blood mononuclear cells  
PCP 
pneumocystis pneumonia  
PP 
per protocol 
Pred 
prednisone 
PTLD 
post-transplant lymphoproliferative disorder 
RFU 
reduced follow-up 
RT-PCR 
reverse transcriptase- polymerase chain reaction 
SAE 
serious adverse event 
SAP 
statistical analysis plan 


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SAR 
suspected adverse reaction 
SARS-
CoV-2 
severe acute respiratory syndrome coronavirus 2 
sBc 
stimulator B cell 
SDCC 
Statistical and Data Coordinating Center 
SOC 
standard of care 
SS 
safety sample 
T1D 
type 1 diabetes 
TAC 
tacrolimus 
Tmem 
T memory cells  
Tr1 
Type 1 regulatory T cells 
Tregs 
regulatory T cells 
UCSF 
University of California, San Francisco 
ULN 
upper limit of normal 
UTI 
urinary tract infection  
VOD 
Veno-occlusive liver disease 
WOCBP 
women of childbearing potential  
 
 
 


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STUDY DEFINITIONS 
Acute rejection  
Diagnosed in accordance with Banff global assessment criteria 
(section 5.4.3.2.1). 
Allograft dysfunction 
When ALT ≥ 100 U/L and/or GGT is elevated relative to baseline. 
Elevation of GGT sufficient to meet allograft dysfunction is defined as 
≥ 2-fold the ULN if the baseline value was < than the ULN; or, ≥ 2-
fold the baseline value if the baseline value was ≥ than the ULN 
(section 5.4.3.1). 
Baseline GGT 
The average of two GGTs taken at the following times: GGT taken at 
EVR Conversion Eligibility (V101[R]); and GGT taken at the 
initiation of EVR conversion (V101S) (section 5.3.4.1). 
Chronic rejection 
Diagnosed in accordance with Banff global assessment criteria 
(section 5.4.3.2.2). 
Clinical rejection 
Participants who are treated empirically based on investigator clinical 
suspicion in cases where a biopsy is indeterminate or in rare cases 
where a biopsy cannot be performed (section 5.4.3.2.3).  
Completely treated 
in-situ cervical or 
basal cell carcinoma 
Cleared by an appropriate provider. 
Enrolled sample 
All participants who sign consent (section 9.1).  
Failed IS withdrawal 
All participants who do not complete IS withdrawal, or resume IS 
unless determined otherwise by an adjudication committee (sections 
4.4, 5.4.2, and 5.4.3.2.3).  
Failure to initiate 
study therapy 
Participants who do not initiate study therapy for any of the following 
reasons will be terminated: 
• Failure to meet study continuation parameters post-transplant 
(section 5.3.1.1) 
• 
Inability to tolerate TAC during Study Stage 1, leading to 
discontinuation of TAC or conversion to cyclosporine (section 
5.3.1.2) 
• Failure to meet criteria for Eligibility 2: EVR Conversion (section 
5.3.2) 
• Failure to initiate EVR conversion (section 5.3.2.4).  
See section 4.5. 
Initiation of EVR 
conversion 
EVR conversion is initiated once the participant receives their first 
dose of EVR (section 5.3.2.4). 
Intent to treat sample 
All participants who receive at least the minimum arTreg dose 
(section 9.1).  


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Manufacturing 
failure 
Onset of arTreg manufacture which does not result in a product 
meeting release criteria for infusion.  
Minimum arTreg 
dose 
30 to <90 x 106 cells (section 5.2.1.4). 
Onset of arTreg 
manufacture 
Initiation of first leukapheresis procedure 
Operational 
tolerance 
Discontinuation of IS for 52 weeks; ALT ≤ 50 U/L* for ≥ 2 
measurements separated by ≥ 1 week in the 6 weeks prior to the liver 
biopsy at 52 weeks after the last IS dose; and Liver biopsy at 52 weeks 
(± 4 weeks) after the last IS dose that meets the biopsy criteria for 
tolerance. 
*If the investigator determines that abnormal laboratory results are 
related to an intercurrent illness, the investigator may request that the 
participant’s data be reviewed by an adjudication committee. If the 
adjudication committee agrees, they may grant an extension of the 
primary endpoint visit window (+4 weeks). This adjudication 
committee will consist of the NIAID Medical Monitor, the ITN 
Clinical Trial Physician, and the Protocol Chair from another 
LITTMUS protocol (Table 5; sections 1.1.1 and 3.3.1). 
Per protocol sample 
All participants who receive at least the target arTreg dose (section 
9.1).  
Resolution of 
rejection episode 
An episode of biopsy-confirmed or clinical rejection will be 
considered resolved when ALT (with or without elevated GGT) ≤ 50 
U/L. For these cases, it is recognized that GGT levels decline very 
slowly following rejection and therefore will not be used to define 
resolution. If an episode of biopsy-confirmed or clinical rejection 
involves elevated GGT alone, it will be considered resolved when 
GGT is ≤ 1.5 x baseline levels. For participants with biopsy-confirmed 
rejection, a repeat biopsy to further document resolution of rejection 
may be performed at the discretion of the investigator (section 
5.4.3.3). 
Safety sample 
All participants who initiate EVR conversion (section 9.1).  
Screen failure 
Participants who do not meet criteria for Eligibility 1: Pre-transplant 
(section 4.1) are failures and will be terminated (section 4.5). 
Steroid refractory 
acute rejection 
AR that has failed to resolve with steroid treatment (section 5.4.3.2.1). 
Study therapy 
The investigational product is donor alloantigen-reactive T regulatory 
cells (arTregs). The supportive regimen for this product includes EVR, 
leukapheresis, CTX, and mesna (section 4.4).  
Successful EVR 
conversion 
Target EVR and TAC levels are maintained over two consecutive 
measurements with ALT ≤ 50 U/L and GGT ≤ the ULN or ≤ 1.5 x the 
baseline GGT (section 5.3.2.4). 
Target arTreg dose 
90 to 500 x 106 cells (section 5.2.1.4). 


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1. BACKGROUND AND RATIONALE  
1.1 
BACKGROUND INFORMATION 
 
Study Summary 
The success of liver transplantation is limited by the complications of chronic IS, including 
medication-related organ toxicities and the risks of cancer, infection, and cardiovascular 
disease. Transplantation tolerance, defined as the ongoing acceptance of the transplanted graft 
in the absence of IS, but with otherwise normal immunologic function, would be of great 
potential benefit. One proposed approach to tolerance is to modify the host immune response 
through infusion of autologous, functionally immunosuppressive regulatory cells. As a 
demonstration that exogenous cells can favorably modify the host immune response, animal 
models show that immunological tolerance can be adoptively transferred to organ recipients 
using cells from another recipient tolerized to the same donor.2 Transfer of tolerance has been 
achieved in a rodent model with purified preparations of regulatory immune cells.3 Such 
adoptively transferred T cells can also lead to infectious tolerance by generating an ongoing 
regulatory response among the recipient’s T cells, which have not been manipulated ex vivo.4 
Infectious tolerance remains even after the disappearance of the original tolerance-inducing 
cells. In humans, T cells with regulatory and immunosuppressive characteristics can be purified 
and expanded from peripheral blood mononuclear cells (PBMCs) or other cell sources. Several 
groups have explored the use of such cells for their potential utility to suppress allogeneic 
responses. Thus, controlling an organ recipient’s alloimmune response against a donor organ 
using cellular therapy is seen as a promising approach that might ultimately supplement current 
pharmacologic approaches, or allow patients to minimize IS. Several clinically relevant 
approaches using Tregs, Type 1 regulatory T cells (Tr1), mesenchymal stromal cells, tolerance-
inducing dendritic cells and macrophages with regulatory potential have been proposed and are 
being studied.5-9 Among these, the use of Tregs is among the most promising.10,11 
To advance our understanding of the use of different Treg products in organ transplantation, the 
Immune Tolerance Network and NIAID have created a consortium, called LITTMUS (Liver 
Transplantation with Tregs at MGH and UCSF) to carry out independent studies of regulatory 
cell therapy to induce immunologic tolerance in liver transplantation. This consortium is 
modeled on the precepts guiding other cooperative efforts exploring cellular therapy in solid 
organ transplantation such as the ONE Study consortium (http://www.onestudy.org/). Initial 
studies in this consortium will be carried out by investigators at the University of San Francisco 
(UCSF) and the Massachusetts General Hospital (MGH) who will conduct single-center, 
single-arm, phase I/II drug withdrawal studies of alloreactive Treg products in living- or 
deceased-donor liver transplant recipients. The studies will use a common clinical protocol 
(identical inclusion/ exclusion criteria, study design, endpoints, etc.), and site-specific, locally 
manufactured autologous Treg products, each of which has been used in previous clinical 
studies. Early after liver transplantation, infusion of the Treg product will be preceded by 
lymphodepletion using CTX, and followed by IS minimization and complete IS withdrawal 
within 30 months after liver transplantation. This study, protocol ITN074ST, will be conducted 
at UCSF and will use an alloantigen-reactive Treg cellular product called arTregs, 
manufactured at UCSF. arTregs are produced by culturing purified recipient Tregs with 
activated and irradiated donor stimulated B cells (sBc). 
The consortium is open to future participation by other institutions with appropriate regulatory 
cell manufacturing and clinical trial infrastructure.  


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IS Withdrawal in Liver Transplantation 
Liver transplantation is the only effective treatment for end stage liver disease.12 However, as 
with any organ transplant, most recipients require lifetime IS; this carries with it a significant 
burden of associated costs as well as on-target and off-target side effects. The long-term goal of 
this research is to develop novel approaches that will facilitate reduction, and potentially 
elimination, of IS.  
Liver allografts have long been thought to have natural tolerogenic potential. In small animal 
models, liver allografts are often accepted spontaneously and in large animals, “prope” 
tolerance was reported by Calne.13 There is also evidence from older clinical studies that the 
likelihood of spontaneous tolerance post liver transplant may exceed 50% in appropriately 
selected patients, compared with only 5% with other organs such as the kidney.14-18 These 
observations provided the foundation for recent clinical trials of IS withdrawal in stable 
pediatric and adult liver transplant recipients. Previous experiences with IS withdrawal in liver 
transplantation patients not only provide evidence for the safety of drug withdrawal in this 
population, but also establish the baseline of spontaneous tolerance for the proposed trial. 
That IS withdrawal in adult liver transplant recipients can be performed with an acceptably low 
risk of permanent graft injury or graft loss is supported by a number of recent trials. For 
example, Benitez et al. found that of 98 patients who attempted withdrawal, 57 (58%) had AR, 
which in all cases was readily reversed with treatment.16 In 21 of 57 cases, rejection was 
reversed merely by increasing calcineurin inhibitor (CNI) doses and levels. In the remaining 
patients, escalation of CNIs was combined with low-dose steroids (20 mg over 4-6 weeks; 30 
patients), moderate-dose steroids (40-60 mg over 4-6 weeks; 4 patients), and steroid boluses (1 
patient). Ultimately, normal graft function (based on liver function tests) was restored in all 
cases. The likelihood of successful drug withdrawal was strongly correlated with time after 
transplant: operational tolerance was achieved in only 13% (3 of 24) of those 3-5.7 years post-
transplant, compared with 38% (19 of 50) if 5.7-10.6 years and 80% (19 of 24) if >10.6 years 
post-transplant. 
Similar results were observed in DAIT-sponsored Protocol ITN030ST (NCT00135694), an 
ITN-supported withdrawal trial in liver transplant recipients that enrolled both a hepatitis C 
virus (HCV) arm and a non-immune, non-viral (NINV) arm.19 Of 30 HCV participants, five 
were found to be tolerant (17%). In the NINV arm, 5 of 47 were tolerant (11%). Of note, in this 
trial, IS withdrawal began as early as one year post-transplant, which may in part explain the 
comparatively lower rate of tolerance observed versus that seen in the European trials, although 
differences in cohorts may also be contributory.16 All rejections were successfully treated with 
reinstitution of maintenance IS and in only 5 cases was a steroid bolus required.19 Together, 
these two studies suggest that IS withdrawal can be attempted with a low likelihood of 
irreversible graft injury or graft loss if rejection occurs. 
 
Current Trial 
The objectives of the current trial are to: (1) Perform an open label phase I/II pilot study to 
assess the safety and preliminary efficacy of arTreg administration followed by IS withdrawal 
in recipients of deceased or living donor liver transplants, and (2) Conduct detailed 
immunological monitoring of the arTreg recipients.  
A trial in which Treg infusion is delayed until several weeks to months post-transplant has a 
number of advantages compared to earlier administration of Tregs, including: 1) avoiding the 


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immediate perioperative period in which surgery-associated inflammation may induce a milieu 
hostile to Treg survival and function; 2) allowing transition to a Treg-supportive IS regimen 
(EVR) after the initial perioperative period, once that drug’s risks, such as impaired wound 
healing, have decreased; 3) allowing the proposed use of leukapheresis and CTX after the 
patient has recovered from the transplant surgery; and 4) avoiding enrollment of the subset of 
patients who have poor outcomes post-liver transplant. In the proposed study, patients will be 
treated with a lymphodepleting agent, CTX, prior to Treg infusion to maximize the ability of 
the delivered cells to undergo expansion post-infusion and to shift the balance of Treg to 
Teffector cells in favor of Tregs. 
1.2 
RATIONALE 
 
Rationale for Treg Therapy in Allogeneic Transplantation 
Based on numerous animal and clinical studies, our working hypothesis is that long-term drug-
free allograft acceptance requires a combination of deletion of a large fraction of alloreactive 
cells and the development or delivery of regulatory cells capable of inhibiting the specific 
immune response to the allograft. Within this framework, the focus of the current protocol is 
the clinical development of donor alloantigen-specific Tregs as a cellular therapeutic that can 
be used in conjunction with conventional IS drugs that spare Tregs; i.e., mTOR inhibitors. 
It is clearly established from mouse models (scurfy) and patients with IPEX syndrome that 
Tregs (as defined by their expression of the transcription factor Foxp3) are required for the 
maintenance of self-tolerance.1-3 Increasing data from human and animal models suggests that 
maintaining tolerance to autologous antigens is not the sole role of Tregs, and that Tregs may 
also play a crucial role in fostering tolerance to alloantigens.4-10 Stable graft function on low 
dose IS is closely correlated with high numbers of Tregs, both in blood and in the grafts 
themselves.20 Moreover, many experimental protocols to induce tolerance or to allow IS 
minimization are based on strategies that promote Treg induction or function, or, more recently, 
that use Treg-based cellular therapies.8 Even the induction of mixed chimerism, initially 
thought to induce tolerance solely via deletion, is very likely to depend on regulation as well.11-
13 While cellular therapies have not been demonstrated to lead to tolerance to date, further 
investigation, as described in the current protocol, is warranted. 
We hypothesize that the function of Tregs in maintaining immune tolerance in transplant 
patients can be harnessed through administration of ex-vivo expanded autologous Treg 
products. In solid organ and hematopoietic stem cell transplantation (HSCT), Tregs are 
necessary and, under defined experimental conditions in animal models,21 sufficient to establish 
transplantation tolerance. Tregs have a unique and robust therapeutic profile that is distinct 
from small-molecule and biological IS and anti-inflammatory drugs. Tregs can migrate to sites 
of inflammation and have multiple distinct immune regulatory mechanisms to respond to a 
spectrum of inflammatory conditions at different sites.22,23 Tregs are long-lived and, in addition, 
can exhibit linked suppression and infectious tolerance by inducing new Tregs.24 Therefore, 
tolerance induced by a single infusion of Tregs may persist even beyond the survival of the 
original therapeutic cells.  
 
Why Explore Treg Efficacy in Liver Transplantation? 
As noted elsewhere in this background section, several groups, including at UCSF and MGH, 
have embarked on safety studies of Treg products in renal transplantation; those experiences 
lead us to believe that exploring the potential efficacy of arTregs to induce transplantation 


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tolerance after liver transplantation might be medically important to improve current practices 
in clinical immunotherapy. 
Liver transplantation is an ideal platform for testing the safety and efficacy of Treg for four 
reasons. First, because of its inherent relatively low immunogenicity, liver transplant recipients 
are usually maintained on lower IS doses than recipients of other organs. Second, surveillance 
for rejection uses simple blood tests, where transaminase levels can give early indications of 
cellular injury representing a potential rejection event. Third, possibly in part as a result of the 
ability to detect rejection early, rejection is relatively easier to reverse in the liver recipient 
compared to other organ transplants. Finally, because of the inherent regenerative capacity of 
the liver, the long-term sequelae of rejection, when rapidly detected and treated, are 
minimal.15,16 
Developing safe approaches to minimize or eliminate the need for long-term IS in liver 
transplant recipients could have a significant impact on the health of thousands of patients. 
Furthermore, it is possible that such approaches might be extended to the very large numbers of 
recipients of other organ and tissue transplants, making a significant impact on many lives. 
 
Antigen-Specific vs. Polyclonal Tregs 
Activation of the immune suppressive function of Tregs requires engagement of the cells’ 
antigen-specific T-cell receptor (TCR), and thus Treg-mediated suppression is antigen specific. 
Experimental models of Treg therapy in transplantation demonstrate that donor-reactive Tregs 
are more effective in inducing tolerance than polyclonal Tregs, because anti-donor, allo-
reactive Tregs preferentially migrate and accumulate in graft tissues, and suppress the 
activation of anti-graft immune responses, locally and in graft-draining lymph nodes.21,25 
 
T-regulatory Cell Product 
 
Summary of Clinical Experience for arTregs: MGH-DFCI and UCSF  
Several approaches have been developed to utilize ex vivo selected or expanded Tregs for 
clinical use. Additional sections will discuss these approaches in more detail. Features of Treg 
products developed at different centers are illustrated in the following table. 


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Table 1. arTreg Products at MGH-DFCI and UCSF 
 
Trial/Protocol 
Responder 
Stimulator 
IND 
(DMF) 
Indication 
N 
Costim 
blockade 
MGH-DFCI 
Product 1 
Guinan, NEJM 199926 Donor bone 
marrow 
Irradiated 
recipient PBMC 
 
Haplo-
identical 
BMT 
NA 
CTLA4-Ig 
(Repligen) 
Product 1 
Davies Blood 200827 
Donor bone 
marrow 
Irradiated 
recipient PBMC 
 
Haplo-
identical 
BMT 
19 
CTLA4-Ig 
(Repligen) 
Product 1a 
Davies Blood 200827 
Donor bone 
marrow 
Irradiated 
recipient PBMC 
 
Haplo-
identical 
BMT 
5 
Anti-B7.1 and 
anti-B7.2 
Product 2 
Multi-center PBSCT 
(NCT00376480 or 
NCT00475384). 12 are 
described ASH abstract 
2008.28 
Donor 
PBMC 
Second 
haploidentical 
donor or 
irradiated 
recipient PBMC 
12085 
Haplo-
identical 
PBSCT 
16 
Anti-B7.1 and 
anti-B7.2 
Product 3 
ONE Study 
(NCT02091232)29 
Recipient 
PBMC 
Irradiated living 
organ donor 
PBMC 
15907 
Kidney 
3 
belatacept 
Product 3  
LITTMUS/ITN073ST∞ 
Recipient 
PBMC 
Irradiated living 
or deceased 
organ donor 
PBMC 
18123 
Liver 
Proposed 
belatacept 
UCSF 
Product 1 
TASK/CTOT-21∞ 
(NCT02711826)25  
Recipient 
PBMC 
Irradiated 
activated living 
donor B cells 
16626 
(15431) 
Kidney 
1 
Not applicable 
Product 1 
DART (ONE Study) 
(NCT02244801) 
Recipient 
PBMC 
Irradiated 
activated living 
donor B cells 
16043 
(15431) 
Kidney 
2 
Not applicable 
Product 1 
DELTA/RTB-002∞ 
(NCT02188719) 
Recipient 
PBMC 
Irradiated 
activated 
deceased donor 
splenocytes 
15479 
(15431) 
Liver 
1 
Not applicable 
Product 1 
ARTEMIS/CTOTc-12∞ 
 (NCT02474199) 
Recipient 
PBMC 
Irradiated 
activated living 
donor B cells 
16345 
(15431) 
Liver 
5 
Not applicable 
Product 1*  
LITTMUS/ITN074ST∞ 
Recipient 
PBMC 
Irradiated 
activated living 
donor B cells or 
deceased donor 
splenocytes 
18329 
(15431) 
Liver 
Proposed Not applicable 
* See section 5.2.1 for a summary of arTreg manufacture. 
∞ On-going, DAIT NIAID-sponsored clinical trials. 
 
UCSF Treg Product 
1.2.4.2.1 
Overview 
The Treg product (arTreg) that will be used in this trial, is being used in several DAIT NIAID-
sponsored solid organ transplantation clinical trials (Table 1). 


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1.2.4.2.2 
Rationale for the Treg Product Proposed Dose 
The arTreg dose selection is based on three factors: prior human experience, estimated effective 
dose, and current manufacturing capacity.  
Prior Human Experience The safety and efficacy of Treg therapy in humans have been 
evaluated in several clinical studies in HSCT and autoimmune diabetes using polyclonal 
Tregs.30-35 Data obtained in these studies show promising safety and efficacy profiles. 
There are 8 published reports of Treg therapy in humans, five in GVHD,30,31,33,35,36 two in type 1 
diabetes (T1D),32,34 and one in liver transplantation.10 All these studies administered polyclonal 
Tregs, except the liver transplantation study in which a non-selected preparation of allo-reactive 
Tregs was infused. As of August 2020, 9 patients have received UCSF manufactured arTregs, 3 
in kidney transplantation and 6 in liver transplantation. The highest dose of polyTreg infused 
into GVHD patients was ~7 x 109 cells (100 x 106/kg) and the highest dose in T1D patients was 
2.6 x 109 cells (~38 x 106/kg). The highest dose of arTregs infused into a liver transplant 
recipient was 400 x 106. All studies reported that all Treg infusions have been well tolerated 
with no infusion reactions and no increase infection or cancer relapse in GVHD patients. 
Estimated Effective Dose 
In the liver transplant trial most similar to protocol ITN074ST10, 610 - 2,590 x 106 autologous 
PBMCs stimulated for 13 days with irradiated donor PBMCs in the presence of antibodies to 
CD80 and CD86 were infused.10 The product lots were administered after CTX for 
lymphodepletion and contained an average of 28% FOXP3+ cells: the lowest dose infused 
contained 31 x 106 and the highest dose infused contained 466 x 106 Foxp3+ Tregs. The results 
showed that Treg doses of 30 to <90 x 106 Tregs have suboptimal efficacy to induce operational 
tolerance (2 successes out of 4). However, doses between 90 and 500 x 106 Tregs appeared to 
be more efficacious (5 successes out of 6) to support successful IS withdrawal. Based on these 
published studies, we estimate that a dose of 90 - 500 x 106 will likely be well tolerated by 
patients and support successful IS discontinuation. 
Manufacturing Capacity 
Current experiences of the UCSF manufacturing facility support the feasibility of producing the 
selected dose of arTregs proposed in this trial. Manufacturing yield for the arTreg lots vary 
depending on the frequency of Tregs in peripheral blood and the ability of the arTregs to 
expand using the established processes. Some end-stage liver disease patients have less 
peripheral Tregs due to fewer CD4+ T cells in the circulation compared to normal control 
subjects. This deficit can be partially corrected using leukapheresis to ensure isolation of 5 x 
106 Tregs in the starting material. In the UCSF experience (unpublished data), clinical arTreg 
expansion ranges between 7 to 390 fold (average: 101 fold). The referenced UCSF data support 
the feasibility of manufacturing a minimum dose of 30 to <90 x 106 arTreg cells and the 
planned target dose of 90 to 500 x 106 arTreg cells proposed in protocol ITN074ST.  
1.2.4.2.3 
Clinical Studies with the arTreg UCSF Product 
Overview 
The UCSF group has developed a GMP-compliant process to manufacture autologous donor 
arTregs using activated and irradiated sBc for selective expansion of alloantigen-reactive 
Tregs.25 There are currently several DAIT-sponsored INDs using the arTreg product 


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manufactured at UCSF. The manufacturing process is described in detail in UCSF-sponsored 
DMF 15431 (Table 1). 
Kidney Trials 
As of April 2018, a total of seven subjects have been enrolled in the TASK/CTOT-21 study 
(IND #15431, NCT02711826). Three subjects were randomized to receive arTregs. Of these 
three subjects, 2 did not receive arTregs due to 1) possible EBV reactivation in the donor B 
cells 2) failure to meet the target arTreg dose. One kidney transplant recipient received 432.5 x 
106 arTregs at approximately 14 months post-transplant. There were no infusion reactions. The 
subject experienced 2 SAEs, which included CMV infection and antibody mediated rejection, 
which were both deemed unrelated to the arTreg infusion (unpublished results). 
As of the end of 2017, 2 kidney transplant recipients have received 300 x 106 arTregs on day 3 
after transplant in the DART study (IND #16043, NCT02244801). One subject experienced a 
urinary tract infection (UTI) SAE was a pre-existing condition in this patient and deemed not 
related to arTreg infusion. The other subject experienced a SAE of elevated serum creatinine 3 
days after arTreg infusion that was treated and responded to steroid pulse. The biopsy did not 
meet the criteria of rejection. It was deemed possibly related to arTreg infusion (unpublished 
results). 
Liver Trials 
As of April 2018, a total of six subjects have thus far been enrolled in the ARTEMIS/CTOTc-
12 study (IND #16345, NCT02474199); all subjects were eligible by biopsy to proceed with IS 
withdrawal and all subjects tolerated the first two steps of IS withdrawal and were eligible to 
receive arTreg infusion. For two of the six, an infusible dose of arTregs could not be made so 
no further IS withdrawal was undertaken and the subjects were moved to safety follow-up. The 
other four subjects received arTregs: 2 received the full dose (300 – 500 million) while 2 
received a “suboptimal” dose (100 – 300 million). One subject who received the full dose has 
achieved the primary endpoint of 75% CNI reduction. The other three subjects experienced 
rejection as detailed in Table 2. The only other reported adverse event was “bronchitis” (AE 
Grade 2) that occurred prior to arTreg infusion (unpublished results). 
Table 2. Summary of Rejection Events following arTreg Infusion in ARTEMIS/CTOCc012 
Subject 
Days 
after 
arTreg 
infusion 
Central 
pathology 
AE Grade 
SAE 
Comments 
8001 
83 
Mild to 
moderate 
2 = Moderate 
No 
Subject discontinued all IS 
the day after arTreg 
infusion 
6001 
167 
Indeterminate 
to mild 
3 = Severe 
Yes – 
hospitalization 
 
6003 
33 
Mild 
2 = Moderate 
No 
Subject had recent 
diarrheal illness with mild 
liver tests abnormalities at 
time of arTreg infusion 
 


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Comparison with ongoing trials  
Table 1 notes ongoing DAIT NIAID-sponsored clinical trials testing the UCSF manufactured 
product, arTreg, in adult solid organ transplantation recipients. The deLTa and ARTEMIS trials 
evaluate arTregs in liver transplantation. As shown in Table 3, protocol ITN074ST differs from 
the above referenced protocols in many key aspects, including patient population (living versus 
deceased donor recipients), time after transplantation, lymphocyte depletion agent, backbone 
IS, and IS management/withdrawal. These differences may ultimately provide insights as to the 
optimal context for arTreg administration with respect to both safety and efficacy.  
Table 3. Comparison of Liver Transplant Trials at UCSF Evaluating arTreg 
 
LITTMUS (proposed) 
deLTa 
ARTEMIS 
Lead site(s) 
UCSF and MGH 
UCSF 
UCSF 
Living vs deceased donor 
Living or Deceased 
Deceased 
Living 
Time after liver transplantation 
Early / de novo 
Early / de novo 
2 - 7 yrs post-transplant 
Lymphodepletion 
Cytoxan 
ATG 
None 
Backbone IS 
Full dose mTOR 
Full dose 
mTOR or CNI 
Low dose CNI 
Treg product 
Allo-reactive 
Allo-reactive 
Allo-reactive 
Immunosuppression withdrawal 
Yes 
No 
Yes 
 
Rationale for Conditioning with CTX Prior to Treg Infusion 
 
Role of in vivo homeostatic expansion 
Use of Treg therapy in organ transplantation is limited by multiple practical constraints on cell 
collection and timely and effective ex vivo manipulation. An additional consideration is the 
capacity for in vivo expansion. In general, all lymphocyte subclasses expand under conditions 
of lymphopenia (“homeostatic expansion”), although both memory T cells and Tregs are 
favored (i.e., proliferate more vigorously) compared with naïve T cells.37  
Bolton HA et al. have demonstrated in an allogeneic murine HSCT model that co-
administration of Treg and conventional CD4+ T cells was not an effective means of GVHD 
prophylaxis, whereas administration of Treg to lymphopenic mice followed by slower 
homeostatic expansion of all subsets resulted in better Treg reconstitution and decreased 
evidence of alloreactivity, emphasizing a probable role of Tregs in constraining T memory cell 
expansion.38 In a non-human primate model of kidney transplantation in which ex vivo 
costimulatory blockade was used to generate anergic and allospecific Treg, administration of 
CTX to the non-human primates prior to anergic Treg infusion was essential to enable tolerance 
induction.39 
In HSCT studies, significant expansion of Treg, generated ex vivo using costimulatory 
blockade and administered as part of the allograft to highly lymphopenic patients after 
lymphomyeloablation, was evident from 60 days to at least 1 year post-transplant.40 
 
Approaches to achieve lymphopenia 
The optimal mechanism by which to achieve lymphopenia in the solid organ transplant setting 
is not known. Anti-lymphocyte or anti-T cell antibodies have relatively long half-lives, which 
may make them inappropriate to be administered prior to a T cell-based therapy. CTX, a 
nitrogen mustard-derived DNA alkylating agent, has a long history of use in autoimmune and 


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malignant lymphoid disease as a lymphodepleting and immunosuppressive agent. In addition, 
dose-related specific effects on T-cell subsets, and perhaps on innate immune cell populations, 
have been postulated.41  
 
Best timing for CTX 
CTX has been administered in the first week after allogeneic HSCT in large numbers of 
published animal and human settings.42 In these studies, the goal has not been lymphodepletion 
(as all such recipients are already profoundly lymphoablated using both myeloablative and 
reduced conditioning regimens), but rather selective eradication of alloreactive donor cells 
proliferating in response to the hematopoietic recipient/stimulator. This early CTX treatment 
window, which capitalizes on both lymphodepletion and selective allodepletion, would raise 
significant concerns and likely not be appropriate in the proposed liver transplant setting. CTX 
requires activation in the liver and rapid renal clearance to achieve efficacy with minimal 
toxicity. The high Model for End-Stage Liver Disease (MELD) score and the related medical 
issues observed during the pre-transplantation period in liver transplant candidates, as well as 
the complex status of liver transplant patients shortly after the surgery, make potential 
morbidity from CTX administration more likely. This is the case because the hepatic CTX 
metabolic pathway required to activate CTX prodrug to the active metabolites is frequently 
compromised. 
The goal of delayed CTX administration will be to capitalize on the clinical improvement and 
improved hepatic function of patients later after transplant. Rather than focusing on 
allodepletion, we would be harnessing the capacity of CTX to produce generalized 
lymphodepletion.43  
 
Effect of CTX on non-proliferating Teffector cells in vivo 
Although not well studied, a theoretical concern for the use of CTX is its potential lack of 
efficacy in depleting non-proliferating Tmemory cells. However, the use of standard induction 
and maintenance IS in the patients prior to CTX is designed to minimize the number of such 
cells. Persistent and proliferating alloreactive cells should be susceptible to CTX. Indeed, in 
mouse HSCT models, CTX administration has ameliorated ongoing graft versus host disease.44 
Such relative alloselectivity may be abrogated by CNI, perhaps by inhibition of IL-2-mediated 
proliferation.45  
 
Effect of CTX on Tregs in vivo 
CTX has also been shown, in a dose-dependent manner, to reduce the number of Tregs in vivo. 
In protocol ITN074ST, arTregs will be generated exogenously, rendering the effects of CTX on 
in vivo Tregs relatively unimportant. This has been confirmed in mouse studies of combining 
CTX preconditioning with donor-specific Treg infusion to induce islet transplant tolerance.46 
1.3 
POTENTIAL RISKS AND BENEFITS 
 
Potential Risks 
 
CTX (Cytoxan®) Risks 
Myelosuppression, IS, Bone Marrow Failure and Infections 


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CTX can cause myelosuppression (leukopenia, neutropenia, thrombocytopenia and anemia), 
bone marrow failure, and severe IS which may lead to serious and sometimes fatal infections, 
including sepsis and septic shock. Latent infections can be reactivated. 
Urinary Tract and Renal Toxicity 
Hemorrhagic cystitis, pyelitis, ureteritis, and hematuria have been reported with CTX. Medical 
and/or surgical supportive treatment may be required to treat protracted cases of severe 
hemorrhagic cystitis. Urotoxicity (bladder ulceration, necrosis, fibrosis, contracture and 
secondary cancer) may require interruption of CTX or cystectomy. Urotoxicity can be fatal. 
Urotoxicity can occur with short-term or long-term use of CTX. 
Cardiotoxicity 
Myocarditis, myopericarditis, pericardial effusion including cardiac tamponade, and congestive 
heart failure, which may be fatal, have been reported with CTX therapy. Supraventricular 
arrhythmias (including atrial fibrillation and flutter) and ventricular arrhythmias (including 
severe QT prolongation associated with ventricular tachyarrhythmia) have also been reported 
after treatment with regimens that included CTX. 
The risk of cardiotoxicity may be increased with high doses of CTX (in the range planned in 
the current trial), in patients with advanced age, and in patients with previous radiation 
treatment to the cardiac region and/or previous or concomitant treatment with other cardiotoxic 
agents. Particular caution is necessary in patients with risk factors for cardiotoxicity and in 
patients with pre-existing cardiac disease.  
In post-marketing reporting, the following have been reported in patients taking CTX: cardiac 
arrest, ventricular fibrillation, ventricular tachycardia, cardiogenic shock, pericardial effusion, 
myocardial hemorrhage, myocardial infarction, cardiac failure (including fatal outcomes), 
cardiomyopathy, myocarditis, pericarditis, carditis, atrial fibrillation, supraventricular 
arrhythmia, ventricular arrhythmia, bradycardia, tachycardia, palpitations, QT prolongation. 
Pulmonary Toxicity 
Pneumonitis, pulmonary fibrosis, pulmonary veno-occlusive disease and other forms of 
pulmonary toxicity leading to respiratory failure have been reported during and following CTX 
treatment. Late onset (greater than 6 months after start of CTX) pneumonitis appears to be 
associated with increased mortality. Pneumonitis may develop years after treatment with CTX. 
Secondary Malignancies 
CTX is genotoxic. Secondary malignancies (urinary tract cancer, myelodysplasia, acute 
leukemias, lymphomas, thyroid cancer, and sarcomas) have been reported in patients treated 
with CTX-containing regimens. The risk of bladder cancer may be reduced by prevention of 
hemorrhagic cystitis. 
Veno-occlusive Liver Disease 
Veno-occlusive liver disease (VOD) including fatal outcome has been reported in patients 
receiving CTX-containing regimens. A cytoreductive regimen in preparation for bone marrow 
transplantation that consists of CTX in combination with whole-body irradiation, busulfan, or 


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other agents has been identified as a major risk factor. VOD has also been reported to develop 
gradually in patients receiving long-term low-dose immunosuppressive doses of CTX. Other 
risk factors predisposing to the development of VOD include preexisting disturbances of 
hepatic function, previous radiation therapy of the abdomen, and a low performance status. 
Infertility 
Male and female reproductive function and fertility may be impaired in patients being treated 
with CTX. CTX interferes with oogenesis and spermatogenesis. It may cause sterility in both 
sexes. Development of sterility appears to depend on the dose of CTX, duration of therapy, and 
the state of gonadal function at the time of treatment. CTX-induced sterility may be 
irreversible. 
Embryo-Fetal Toxicity 
CTX can cause fetal harm when administered to a pregnant woman. Exposure to CTX during 
pregnancy may cause birth defects, miscarriage, fetal growth retardation, and fetotoxic effects 
in the newborn. CTX is teratogenic and embryo-fetal toxic in mice, rats, rabbits and monkeys. 
Female patients of reproductive potential should avoid becoming pregnant and should use 
highly effective contraception during treatment and for 1 year after completion of therapy. 
Pregnancy Category D Risk Summary 
CTX can cause fetal harm when administered to a pregnant woman based on its mechanism of 
action and published reports of effects in pregnant patients or animals. Exposure to CTX during 
pregnancy may cause fetal malformations, miscarriage, fetal growth retardation, and toxic 
effects in the newborn. 
Malformations of the skeleton, palate, limbs and eyes as well as miscarriage have been reported 
in humans after exposure to CTX in the first trimester. Fetal growth retardation and toxic 
effects manifesting in the newborn, including leukopenia, anemia, pancytopenia, severe bone 
marrow hypoplasia, and gastroenteritis have been reported after exposure to CTX. 
Nursing Mothers 
CTX crosses into breast milk. Neutropenia, thrombocytopenia, low hemoglobin, and diarrhea 
have been reported in infants breast fed by women treated with CTX.  
Common Adverse Reactions  
More common side effects include; neutropenia, fever or chills, nausea, vomiting, anorexia, 
abdominal discomfort, gastrointestinal pain and diarrhea, alopecia, skin rash, and pigmentation 
of the skin and changes in nails. 
 
Mesna (MESNEX®) Risks 
Hypersensitivity Reactions 
MESNEX may cause systemic hypersensitivity reactions, including anaphylaxis. These 
reactions may include fever, cardiovascular symptoms (hypotension, tachycardia), acute renal 
impairment, hypoxia, respiratory distress, urticaria, angioedema, laboratory signs of 
disseminated intravascular coagulation, hematological abnormalities, increased liver enzymes, 


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nausea, vomiting, arthralgia, and myalgia. These reactions may occur with the first exposure or 
after several months of exposure.  
Dermatologic Toxicity 
Drug rash with eosinophilia and systemic symptoms and bullous and ulcerative skin and 
mucosal reactions, consistent with Stevens-Johnson syndrome or toxic epidermal necrolysis 
have occurred. MESNEX may cause skin and mucosal reactions characterized by urticaria, 
rash, erythema, pruritus, burning sensation, angioedema, periorbital edema, flushing and 
stomatitis. These reactions may occur with the first exposure or after several months of 
exposure.  
The most frequently reported side effects of Mesna when given without chemotherapy include 
headache, injection site reactions, flushing, dizziness, nausea, vomiting, abdominal pain/colic, 
pain, constipation, decreased white blood cell count, decreased platelet count, decreased red 
blood cell count, somnolence, diarrhea, anorexia, fever, pharyngitis, hyperesthesia, influenza-
like symptoms, hair loss, and coughing. A false positive test for urinary ketones may occur in 
patients treated with Mesna. 
An uncommon side effect of Mesna is allergic reaction. Usually it is mild in the form of a skin 
rash or itching. This reaction rarely becomes severe. Mesna is contraindicated in patients 
with a known hypersensitivity to Mesna or other thiol compounds such as captopril and 
penicillamine. 
Pregnancy Category B 
There are no studies of Mesna in pregnant women. Reproduction studies performed in rats and 
rabbits at oral doses approximately 10 times the maximum recommended total daily human 
dose on a body surface area basis (1000 mg/kg in rabbits and 2000 mg/kg in rats) revealed no 
evidence of harm to the fetus due to Mesna. The incidence of malformations in human 
pregnancies has not been established for Mesna. No long-term studies in animals have been 
performed to evaluate the carcinogenic potential of Mesna. 
Nursing Mothers 
It is not known whether mesna or dimesna is excreted in human milk. Benzyl alcohol present in 
maternal serum is likely to cross into human milk and may be orally absorbed by a nursing 
infants. 
 
UCSF Treg Product Infusion Risks 
Although Treg infusion has been innocuous in animal models, there is relatively 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.36 The first patient had chronic GVHD two 
years after transplantation. After receiving 0.1 x 106/Kg of 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 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 al47 enrolled twenty-three patients with advanced hematologic malignancy. The 
patients were treated with two units of umbilical cord blood as a source of stem cells and 
effector T cells. Tregs were isolated using anti-CD25 immunomagnetic bead selection from 


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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 dose-limiting toxicities (DLTs) or 
increase in AEs compared to historical controls. Incidences of acute and chronic GVHD were 
reduced in patients received Treg therapy. This group later reported a third trial that enrolled 28 
patients with high-risk hematological malignancies.31 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, and 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 fluorescence-activated cell sorting 
(FACS) purified Tregs to type 1 diabetic patients has been reported by the Trzonkowski 
group.32 The study enrolled 10 type 1 diabetic children (aged 8-16 years) within 2 months after 
diagnosis. Four patients received 10 x 106 Tregs/kg body wt. and the remaining 6 patients 
received 20 x 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 the safety of Treg therapy in type 1 
diabetic patients has been performed.34 This was a dose escalation study in 14 subject in 4 
cohorts receiving doses of polyclonal autologous Tregs from 0.05 to 26 x 108 cells. This study 
demonstrated that infused Tregs were phenotypically stable. A proportion of cells were long-
lived and detectable in vivo up to 1 year after infusion. There were no infusion reactions 
reported and no grade 3 or higher events attributed to the cell therapy.  
In transplantation, one Treg therapy trial has been published.10 Another 12 trials are active and 
registered on ClinicalTrials.gov (Table 4) in liver and kidney transplantation.11 


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Table 4. Reported or Registered Clinical Trials of Treg Cell Therapy 
Targeted 
Enrollment 
Type of Tregs 
Type of 
Trials 
Trial ID 
Liver Trials 
10 
Autologous Tregs stimulated with irradiated 
donor PBMCs with costimulation blockade  
Phase 2 
Japan10 
24 
Autologous donor antigen–expanded Tregs 
Phase 1 
deLTa-UCSF, 
NCT02188719 
10 
Autologous polyclonally expanded Tregs 
Phase 1 & 2 
Nanjing, China, 
NCT01624077 
18 
Autologous donor antigen–expanded Tregs 
Phase 1 & 2  
ARTEMIS-UCSF, 
NCT02474199 
26 
Autologous polyclonally expanded Tregs 
Phase 1 & 2 
ThRIL-KCL, 
NCT02166177 
Kidney Trials 
3 
Autologous polyclonally expanded Tregs 
Phase 1 
TASKp-UCSF, 
NCT02088931 
45 
Autologous polyclonally or donor antigen– 
expanded Tregs 
Phase 1 & 2 
TASK-UCSF, 
NCT02711826 
10 
Autologous polyclonally expanded Tregs 
Phase 1 
TRACT-Northwestern, 
NCT02145325 
8 
Autologous Tregs stimulated with irradiated 
donor PBMCs with costimulation blockade 
Phase 1 
One Study–MGH, 
NCT02091232 
12 
Autologous polyclonally expanded Tregs 
Phase 1 
One Study–UK, 
NCT02129881 
9 
Autologous polyclonally expanded Tregs 
Phase 1 & 2 
One Study–Charité, 
NCT02371434 
8 
Autologous donor antigen–expanded Tregs 
Phase 1 
One Study–UCSF, 
NCT02244801 
 
Liver Allograft Rejection Due to IS Withdrawal Risks 
The primary risk of IS withdrawal is that of graft rejection. Graft rejection carries three 
attendant risks: risk of irreversible graft injury or failure, the need for liver biopsy to verify the 
diagnosis, and the risk accompanying treatment(s) needed to reverse the rejection once the 
diagnosis has been secured.  
Rejection-associated graft injury or graft loss 
Given the risk of rejection, the trial will be designed to allow early detection of rejection 
through frequent monitoring during the period of IS withdrawal and in the period early after IS 
cessation. Based on the experience of other withdrawal trials, with this approach it is expected 
that rejection episodes will be detected early and readily reversed. While theoretically possible, 
severe rejection leading to graft loss, re-transplantation or patient death is very unlikely and has 
been only rarely observed with IS withdrawal in liver allograft recipients. In 4 separate studies 
of IS withdrawal on a total of 267 adult liver allograft recipients published between 1997 and 
201316,18,48,49 only a single graft loss was reported.48,49  


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For-cause liver biopsy risks 
Liver biopsy to permit histopathological assessment remains the gold standard in diagnosis of 
rejection and will be employed in all suspected cases of rejection in this trial unless clinically 
contraindicated or logistically infeasible. The procedure is usually performed percutaneously 
under ultrasound guidance and local anesthetic. It is usually associated with mild pain lasting 
only a few hours (rarely a few days). The risk of significant bleeding requiring transfusion is 
0.5-1% and the risk of bile leak or injury to adjacent organs (pneumothorax, bowel perforation 
etc.) is <0.5%; the risk of one of these complications leading to death is estimated at 0.1-
0.01%.50 
Risk associated with treatment of rejection 
The experience reported to date in liver IS withdrawal trials suggests that rejection episodes 
that occur during staged weaning and serial monitoring every 2-3 weeks are readily reversed by 
reinstitution of CNI-based IS alone, or CNI in combination with low dose steroids. This may be 
associated with transient worsening of certain co-morbidities (diabetes, hypertension, 
hyperlipidemia, etc.), but is very unlikely to result in irreversible damage. The need to use 
strong IS regimens to reverse rejection (e.g. repeated steroid boluses, T cell depleting 
antibodies) may lead to increased risk of infection (e.g. CMV reactivation), malignancy and 
renal dysfunction. Within a carefully monitored clinical trial, the development of rejection 
episodes of such severity is extremely unlikely. In 4 separate studies of IS withdrawal on a total 
of 267 adult liver allograft recipients published between 1997 and 201316,18,48,49 only 2 episodes 
of severe rejection were reported.48,49 One episode resulted in graft loss and the second reversed 
with steroids alone. In these 267 patients, all other rejection episodes were graded as mild or 
moderate and were treated successfully with reinstitution of maintenance IS with or without 
steroids. 
 
Maintenance IS Risks 
Administration of all IS therapies used presently to prevent rejection of transplanted tissues 
carry general risks of opportunistic infection and malignancy, including lymphoma, and skin 
cancers. These agents are not recommended for nursing mothers, and it is recommended (and 
mandated in the current protocol) that women of childbearing potential (WOCBP) use effective 
contraception before, during and for at least 4 months following administration of these agents. 
The use of mycophenolate acid, TAC, and steroid IS is well established in liver transplantation. 
EVR is FDA approved for use in liver transplantation. The risks for dose-related toxicities and 
side effects from these IS medications are not different for participants in this trial. The reduced 
dosing in conjunction with other agents in the Treg-supportive IS regimen may expose the 
participant to an increased risk of rejection. 
 
Over Immunosuppression Risks 
Chronic IS is associated with a variety of side effects including infection, malignancy, 
hypertension, diabetes and increased risk of cardiovascular disease. Chronic CNI use is also 
associated with nephrotoxicity, which is responsible for a significant rate of chronic renal 
failure (CRF) at 5 years post orthotopic liver transplantation (OLT). CRF confers a significant 
mortality risk and many patients ultimately require renal replacement therapy.51-56 Patients 
demonstrating progressive loss in functional nephron mass are at greatest risk of progressing to 


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chronic kidney disease (CKD) which is known to confer a more than 4 fold increase in 
mortality risk.  
 
Venipuncture Risks 
Frequent blood draws to allow close monitoring of liver function and to assess for safety is 
essential for the trials safe conduct. Risks of blood draw or venipuncture are typically minimal 
with temporary local discomfort. Rare, but more serious risks would include ecchymosis, 
thrombophlebitis, and infection.  
The amount of blood that may be drawn from adult participants for research purposes will not 
be more than 550 mL over an eight-week period. The additional amount of blood could 
contribute to the development of anemia. The participant’s clinical condition will be taken into 
consideration to determine if research blood tests can be performed. 
 
Leukapheresis Risks 
Leukapheresis will be performed on the recipient prior to initiating the CTX conditioning 
regimen. Complications that may occur during leukapheresis include changes (increase or 
decrease) in blood pressure or pulse, bleeding, or hypocalcemia. Bleeding, fever, and infection 
from leukapheresis can also occasionally occur. On very rare occasions, these complications 
may be life-threatening. 
 
Liver Biopsy Risks 
Participants who meet eligibility for all phases of the study and successfully complete IS 
withdrawal will undergo a minimum of 3 biopsies per protocol. This is in addition to a pre-
implant biopsy performed on the liver prior to transplantation into the recipient. These biopsies 
will occur at the following time points: 
1. 5-15 days after the arTreg infusion, to evaluate the effect of arTreg infusion and eligibility 
to proceed in the study;  
2. Prior to withdrawing prednisone and EVR, to determine eligibility; and 
3. 52 weeks after successful IS withdrawal, to assess the primary endpoint of the study.  
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%.50 Symptomatic hemorrhage and/or the requirement for 
operative or other procedural intervention to stop bleeding occurs in <0.5% of procedures. 
Other potentially serious risks associated with liver biopsy include pneumothorax or colonic 
perforation. If either were to occur, hospitalization, procedural or operative intervention may be 
necessary. Finally, there is a very small risk of death, estimated at 0.1 to 0.01%.50 


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Potential Benefits 
This study provides no direct or immediate benefit to the participants. However, the results of 
the study could improve future care of transplant patients.  
2. 
OBJECTIVES 
2.1 
PRIMARY OBJECTIVE 
The primary objectives are to evaluate the safety and efficacy of a single IV dose of autologous, 
donor alloantigen-specific T regulatory cell product, arTreg, to induce operational tolerance 
early after adult living or deceased donor liver transplantation. 
2.2 
SECONDARY OBJECTIVES 
The secondary objective is to evaluate the safety and tolerability of cytoreduction in 
combination with the arTregs to facilitate IS withdrawal in liver transplant recipients. 
2.3 
MECHANISTIC OBJECTIVES 
The mechanistic objectives are to evaluate the effects of the infused arTregs on the 
immunological profiles of liver transplant recipients.  
3. 
STUDY DESIGN 
3.1 
DESCRIPTION 
This is a single-center, prospective, open-label, non-randomized clinical trial exploring cellular 
therapy to facilitate IS withdrawal in liver transplant recipients. The primary investigational 
product is arTreg. Study interventions include: 1) EVR conversion; 2) administration of CTX 
and mesna prior to a single IV dose of the arTregs; and 3) TAC discontinuation followed by 4) 
complete IS withdrawal.  
We plan to accrue 9 participants who will receive the target arTreg dose of at least 90 x 106 
cells (section 5.2.1.4). This will comprise the PP sample. Participants who receive ≥ 30 x 106 
cells but <90 x 106 cells as a result of low cell yield will not count towards the planned accrual, 
and no more than 4 participants will be allowed in this group. This group will be included in the 
ITT sample. See section 9.1 for additional details on the analysis samples. 
The plan for IS, cellular therapy and IS withdrawal is described in sections 5.1 to 5.3.5. 
Participants who successfully withdraw from all IS will undergo research biopsies as outlined 
in section 1.3.1.9. The research biopsy at 52 weeks following IS discontinuation will be used to 
determine whether they meet the primary efficacy endpoint of operational tolerance. 
Participants determined to be operationally tolerant will be followed until 104 weeks following 
IS discontinuation. Participants who fail IS withdrawal after 52 weeks but before 104 weeks 
will be followed until week 104 or 12 weeks after resuming IS, whichever is longer (Appendix 
5). 
Participants who do not successfully withdraw from all IS will complete 104 weeks of High 
Intensity Safety Follow-up (Appendix 6) after failing IS withdrawal.  


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All other participants will be followed per section 4.4.  
Eligible by Criteria 1?
Consented
Transplanted, Receiving SOC
Eligible by Criteria 2?
(Initiate AE Collection)
EVR Conversion
Successful Conversion?
Eligible by Criteria 3?
Leukapheresis?
Eligible by Criteria 4?
TAC Withdrawal
Complete TAC Withdrawal?
Yes
Eligible by Criteria 5?
EVR/Pred Withdrawal
Post-withdrawal Surveillance 
and Evaluation 
(2 years [+12 weeks if applicable])
Complete 
EVR/Pred Withdrawal?
Yes
Screen Failure
High Intensity 
Safety Follow-up
(2 years)
Yes
Yes
Yes
Yes
Yes
No
Reduced Safety Follow-up
(30 days)
No
No
No
No
CTX?
Treg Product
(arTreg)?
Low Intensity 
Safety Follow-up
(1 year)
Yes
No
No
No
No
Yes - target dose 
ACCRUED
Terminated Early 
(failure to initiate study therapy)
Meets Study Continuation 
Parameters Post-Tx?
Yes
No
No
Yes - minimum dose 
Eligible by Criteria 4?
Eligible by Criteria 5?
High Intensity 
Safety Follow-up
(2 years)
Yes
No
No
Yes – evaluated at investigator discretion
Yes – not evaluated at investigator discretion
Yes
No
No
Yes - < minimum dose
No
Yes
Yes
 
Figure 1. Study Design 
3.2 
STUDY DURATION 
Accrual duration (section 3.1) will be 113 weeks (2.2 years) from first enrollment. 
Total duration of study participation will be influenced by the participant’s course during the 
study.  
• 
Participants who participate in all phases of the study, i.e. who receive arTregs, achieve 
complete IS withdrawal, and are operationally tolerant (Study Definitions) at 52 weeks off 
IS will be followed for 2 years after the last IS dose (Appendix 5). 


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• 
Participants who receive arTregs, achieve complete IS withdrawal, and then resume IS 
(sections 5.4.2 and 5.4.3.2) will be followed for 2 years after first completing IS withdrawal 
or 12 weeks after resuming IS, whichever is longer (Appendix 5). 
• 
Participants who receive arTregs, but are not eligible to initiate or do not complete IS 
withdrawal will be followed for 2 years from the date they were deemed ineligible or failed 
IS withdrawal (Appendix 6). 
• 
Participants who receive CTX but do not receive arTregs will be followed for 1 year post 
CTX administration (Appendix 7). 
• 
Participants who initiate EVR conversion but do not receive CTX will be followed for up to 
30 days as outlined in 4.4 (RFU visit in Appendix 2). 
3.3 
STUDY ENDPOINTS 
 
Primary Endpoints 
 
Primary Safety Endpoints 
The primary safety endpoints are defined as: 
1. Number and severity of AEs attributed to arTregs. 
2. Number and severity of AEs attributed to a) leukapheresis, b) CTX, or c) mesna.  
 
Primary Efficacy Endpoint 
The primary efficacy endpoint is operational tolerance. Operational tolerance is defined as: 
1. Discontinuation of all IS for 52 weeks; 
2. ALT ≤ 50 U/L* for ≥ 2 measurements separated by ≥ 1 week in the 6 weeks prior to the 
liver biopsy at 52 weeks after the last IS dose; and 
3. Liver biopsy at 52 weeks (± 4 weeks) after the last IS dose that meets the biopsy criteria for 
operational tolerance (Table 5) as assessed by central pathology. 
*If the investigator determines that abnormal laboratory results are related to an intercurrent 
illness, the investigator may request that the participant’s data be reviewed by an adjudication 
committee. If the adjudication committee agrees, they may grant an extension of the primary 
endpoint visit window (+ 4 weeks). This adjudication committee will consist of the NIAID 
Medical Monitor, the ITN Clinical Trial Physician, and the Protocol Chair from another 
LITTMUS protocol (section 1.1.1). 
Table 5. Biopsy Criteria for Operational Tolerance: Comparison of Biopsy Obtained 52 
Weeks after the Last Dose of Immunosuppression to the Pre-Weaning (Visit 300) 
Biopsy57 
Compartment 
Findings 
Portal inflammation 
and interface activity 
Similar to pre-weaning (Visit 300) biopsy with no more than minimal or focal 
mild, portal mononuclear inflammation. No more than minimal interface 
necro-inflammatory activity, limited to a minority of tracts. 
Centrizonal/peri-
venular inflammation 
Similar to pre-weaning (Visit 300) biopsy with no more than minimal 
perivenular necro-inflammatory activity. 


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Bile duct changes 
Similar to pre-weaning (Visit 300) biopsy, without new onset biliary 
epithelial cell senescence changes or ductopenia involving > 10% of portal 
tracts. 
Fibrosis 
Similar to pre-weaning (Visit 300) biopsy, without progressive 
portal/periportal or perivenular fibrosis or architectural distortion (≤1 point 
increase on the Ishak scale). 
Arteries 
Negative for obliterative or foam cell arteriopathy. 
 
Secondary Endpoints 
1. Incidence of grade 3 or greater infections following arTreg infusion. 
2. Number and severity of biopsy-proven AR and/or clinical rejection events at any time after 
arTreg infusion. 
3. Number of CR events at any time after arTreg infusion. 
4. Number of participants who develop any malignancy. 
5. The proportion of participants who discontinue TAC ≥ 12 weeks with ALT ≤ 50 U/L and a 
liver biopsy 12 - 26 weeks after the last TAC dose that meets the biopsy criteria for IS 
minimization (Table 9) as assessed by central pathology. 
6. Durability of operational tolerance defined as the time from achieving the primary endpoint 
to IS reinitiation or to the end of trial participation. 
 
Exploratory Mechanistic Endpoints 
Mechanistic studies to evaluate the effects of arTregs on the immunological profiles of liver 
transplant recipients. These endpoints include the number of participants with: 
• 
evidence of a tolerance associated gene profile1 as described by A. Sanchez-Fueyo et al; 
• 
a loss or change of alloresponses following arTreg infusion; 
• 
an increase in peripheral blood Treg numbers following arTreg infusion; 
• 
an increase in intragraft Treg numbers following arTreg infusion.  
The hypotheses and associated assays for these endpoints are described in sections 7.2 and 7.3. 
3.4 
PREMATURE TERMINATION OR SUSPENSION OF THE TRIAL / 
STOPPING RULES 
 
Ongoing Review 
The Data Safety Monitoring Board (DSMB) will review safety data not less often than yearly. 
 
Study Stopping Rules Guidance 
 
Study-Related AEs Contributing to Stopping Rules  
If any one of the criteria listed below are met, study enrollment will be suspended and arTreg 
infusion will be held pending review of all pertinent data by the NIAID Transplant DSMB, 
DAIT NIAID, and the ITN. In the event that, at the time that the study is paused, there is a 
subject who has received CTX conditioning, the study PI and Medical Monitor will determine 
whether it is in the patient’s best interest to receive arTreg infusion, and the subject will be 
informed of the circumstances and the decision. 


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• 
Any CTCAE grade 4 or higher infusion reaction 
• 
Any study-defined grade 4 or higher infection (section 8.4.1) after initiation of EVR 
conversion 
• 
Any diagnosis of malignancy, except recurrent HCC and skin cancer after initiation of EVR 
conversion 
• 
Any death or graft loss after initiation of EVR conversion 
• 
Any grade 4 or higher hemorrhagic cystitis event related to CTX 
• 
Rejection after arTreg infusion: 
- 
Severe AR in two or more participants (Study Definitions) 
- 
Steroid resistant AR (section 5.4.3.3) 
- 
CR (Study Definitions) 
• 
AR or clinical rejection (Study Definitions) in ≥ 3 participants within 6 weeks after arTreg 
infusion 
 
Manufacturing Failure(s) Contributing to Stopping Rules 
• 
Four patients receive less than the target dose of arTregs (90 to 500 x 106 cells) 
• 
 ≥20% failure rate to manufacture a minimum dose of arTregs (see Study definition for 
manufacturing failure and minimum dose). The lower limit of the 95% confidence interval 
for the rate of manufacturing failure will be used for this calculation (see Table 6 below). 
This manufacturing stopping rule will not go into effect till after the 4th patient undergoes 
an attempt to manufacture arTregs.  
 
Table 6. Stopping Rule Table for a 20% Manufacturing Failure Threshold  
Number of 
subjects with 
manufacturing 
failure 
Number of 
subjects in 
whom 
manufacturing 
was attempted 
Cumulative 
incidence rate 
(%) 
Lower 95% 
exact 
confidence 
limit (%) 
3 
4 
75 
24.86 
4 
5 
80 
34.26 
4 
6 
66.67 
27.13 
4 
7 
57.14 
22.53 
5 
8 
62.5 
28.92 
5 
9 
55.56 
25.14 
 


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4. 
ELIGIBILITY 
4.1 
RECIPIENT (ELIGIBILITY 1 – PRE-TRANSPLANT) 
 
Inclusion Criteria 
Individuals must meet all of the following criteria to be eligible: 
1. Able to understand and provide informed consent. 
2. Male or female, 18 to 70 years of age at the time of enrollment. 
3. End-stage liver disease and listed for a living or deceased-donor primary solitary liver 
transplant. 
4. Agreement to use contraception; section 5.2.4.4. 
5. Removed in protocol version 3.0. 
6. Removed in protocol version 4.0. 
7. Positive EBV antibody test. 
8. In the absence of contraindication, vaccinations must be up to date per the DAIT Guidance 
for Patients in Transplant Trials (refer to the Manual of Procedures). 
 
Exclusion Criteria 
Individuals who meet any of the following criteria will not be eligible: 
1. History of previous organ, tissue, or cell transplant. 
2. For CMV antibody negative recipients, a CMV antibody positive donor. 
3. Known contraindication to CTX or mesna. 
4. Serologic evidence of HIV-1/2 infection. 
5. The need for chronic anti-coagulation or anti-platelet agents other than aspirin that cannot 
be safely discontinued for a minimum of 1 week to safely perform a liver biopsy. 
6. End stage liver disease secondary to autoimmune etiology (such as autoimmune hepatitis, 
primary biliary cirrhosis, or primary sclerosing cholangitis), or other contraindications to 
drug withdrawal. 
7. Psychological, familial, sociological, or geographical factors potentially hampering 
compliance with the study protocol and follow-up visit schedule. 
8. Any condition that, in the opinion of the investigator, may interfere with study compliance. 
9. History of cardiac disease (ischemic heart disease requiring revascularization, history of or 
current treatment for dysrhythmia, or evidence of congestive heart failure) unless cleared 
by a cardiologist. 
10. Any past or current medical problems, treatments or findings which, in the opinion of the 
investigator, may pose additional risks from participation in the study, may interfere with 
the candidate’s ability to comply with study requirements or may impact the quality or 
interpretation of the data obtained from the study. This includes past, present, or future 
enrollment in studies that affect eligibility at the time of EVR conversion. 


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11. History of malignancy or any concomitant malignancy, except HCC, completely treated in-
situ cervical carcinoma, or completely treated basal cell carcinoma (Study Definitions). 
12. Chronic use of systemic glucocorticoids or other immunosuppressives, or biologic 
immunomodulators. 
4.2 
LIVING DONOR 
 
Inclusion Criteria 
Living donors must meet all of the following criteria to be eligible: 
1. Able to understand and provide informed consent. 
2. Male or female, 18 to 70 years of age at the time of enrollment. 
3. Meets site-specific clinical donor eligibility requirements. 
4. Meets donor eligibility manufacturing requirements within 7 days before or after the blood 
collection for manufacturing. 
5. Willingness to donate appropriate biologic samples. 
 
Exclusion Criteria 
There are no exclusion criteria for living donors. 
4.3 
DECEASED DONOR 
 
Inclusion Criteria 
Deceased donors must meet the following criteria for their recipients to remain eligible: 
1. Meets site-specific clinical donor eligibility requirements. 
2. Meets donor eligibility manufacturing requirements. 
 
Exclusion Criteria 
There are no exclusion criteria for deceased donors. 
4.4 PREMATURE DISCONTINUATION OF STUDY THERAPY 
Study therapy: The investigational product is arTreg. The supportive regimen for this product 
includes EVR, leukapheresis, CTX, and mesna. 
Study therapy will be prematurely discontinued for the following events: 
• 
Hypersensitivity: A participant develops a persistent, life threatening hypersensitivity 
reaction to mesna or CTX, despite the measures described in sections 5.2.2.1 and 5.2.2.2. 
• 
The arTreg infusion will be stopped and will not be restarted if there is a hypersensitivity 
reaction, CTCAE grade ≥3 infusion related reaction, or any infusion-related serious AE 
(SAE).  
• 
AE: A participant experiences an AE attributable to the study therapy that, in the judgment 
of the principal investigator or the medical monitor, presents an unacceptable consequence 
or risk to the participant. 


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• 
Intercurrent illness or infection: A participant develops an illness or infection that requires 
treatment not consistent with protocol requirements; or, a participant develops an 
intercurrent illness that, in the judgment of the principal investigator, makes further 
participation in the study not in the patient’s best interest.  
• 
Protocol violation. If a participant cannot comply with the study protocol and the protocol 
deviation is sufficient to jeopardize his or her well-being or the integrity of the study. 
• 
Pregnancy. 
• 
The principal investigator or medical monitor determines that the study therapy is no longer 
in the best interests of the participant. 
• 
The participant is unwilling or unable to continue on study therapy. 
Participants who do not initiate any study therapy will be prematurely terminated.  
Participants who do not complete EVR conversion will complete 30 days of Reduced Safety 
Follow-up (RFU) Visit in Appendix 2. 
Participants who complete EVR conversion but do not receive any CTX will complete 30 days 
of RFU Visit in Appendix 2. This includes participants who initiated leukapheresis. 
Participants who receive any CTX or mesna but do not receive the arTreg infusion will 
complete 104 weeks of Low Intensity Safety Follow-up per Appendix 7. 
Participants who receive any infusion of arTregs but either do not attempt or do not complete IS 
withdrawal will complete 104 weeks of High Intensity Safety Follow-up per Appendix 6. 
Participants who do not complete IS withdrawal will have failed IS withdrawal. 
The DSMB will be provided reports on all cases of premature termination and discontinuation 
of study therapy. 
4.5 
PREMATURE TERMINATION OF A PARTICIPANT FROM THE 
STUDY 
Withdrawal of consent. Participants who withdraw consent for further treatment will be 
terminated from the study. Participants who initiate EVR conversion will be asked if they 
would be willing to complete an end-of-study visit to include the assessments in Visit 400 
(Appendix 5). 
Investigator decision. The principal investigator and/or Medical Monitor may choose to 
terminate a participant from further participation if they deem it is not in the interest of the 
participant.  
Screen failure. Participants who do not meet criteria for Eligibility 1: Pre-transplant (section 
4.1) are failures and will be terminated. 
Failure to initiate study therapy. Participants who do not initiate study therapy (Study 
Definitions) for any of the following reasons will be terminated: 
• 
Failure to meet study continuation parameters post-transplant (section 5.3.1.1) 


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• 
Inability to tolerate TAC during Study Stage 1, leading to discontinuation of TAC or 
conversion to cyclosporine (section 5.3.1.2) 
• 
Failure to meet criteria for Eligibility 2: EVR Conversion (section 5.3.2) 
• 
Failure to initiate EVR conversion (section 5.3.2.4). 
There is no upper limit on the number of participants who are prematurely terminated due to 
being screen failures or failing to initiate study therapy. 
Failure to return. Participants who do not return for visits and who do not respond to repeated 
attempts by the site staff to return, will be considered lost to follow-up. 
5. 
STUDY THERAPIES, MEDICATIONS AND PROCEDURES 
5.1 
OVERVIEW 
Figure 2 provides a visual guide to the sequential stages in the trial and an overview of an 
individual’s participation.  
This study proceeds through a defined sequence of stages before and after transplantation. 
Study recruitment and informed consent occurs prior to transplantation, with samples of blood, 
serum, and PBMC samples collected at any time after informed consent, but prior to the 
administration of any IS. 
After transplantation, participants will initially receive standard IS with TAC, along with a 
mycophenolate product and/or steroids, as described in section 5.3.1 (Study Stage 1: Initial 
post-transplant IS). 
After at least 30 days, participants will then be evaluated for eligibility to initiate Study Stage 
2: EVR conversion, as described in section 5.3.2. 
Participants completing this stage will be evaluated for Study Stage 3: arTreg Manufacturing 
and Investigational Treatment which includes leukapheresis, cyclophosphamide and arTreg 
infusion (section 5.3.3). In the case of participants who have received a liver transplant from a 
living donor, donor PBMCs used in arTreg manufacture will be obtained from the living donor, 
if not previously collected (section 5.3.3.1); the donor phlebotomy will be performed at least 14 
days prior to the recipient leukapheresis. 
Following arTreg infusion, participants will be evaluated for eligibility to initiate Study Stage 
4: TAC withdrawal (section 5.3.4). 
After completion of Study Stage 4, participants will be evaluated for eligibility to withdraw 
from the remaining maintenance IS (Study Stage 5: EVR/Prednisone withdrawal, section 
5.3.5). Participants who discontinue all IS according to protocol schedule will then enter Study 
Stage 6: Post-IS Withdrawal Surveillance and Evaluation. The primary endpoint of 
operational tolerance will be evaluated 52 weeks after scheduled complete IS withdrawal, and 
final study clinical evaluation will be evaluated 104 weeks after scheduled complete IS 
withdrawal. 


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Figure 2. Overview of an Individual’s Participation (Study Stage 1 to 6) 
Participation comprises pre-transplant, transplant and EVR conversion (upper left); investigational treatment (middle 
left and section 5.3.3); and IS withdrawal (middle right and sections 5.3.4 and 5.3.5). 
5.2 
STUDY MEDICATIONS 
 
Investigational Agent: Donor Alloantigen-Reactive Regulatory T Cells 
(arTreg) 
 
Overview 
The arTreg product is manufactured at the Human Islet and Cellular Transplantation Facility 
(HICTF) and GMP Facility, an FDA-registered facility located at UCSF. arTregs are 
autologous cells that have been collected from the transplant recipient by leukapheresis, 
purified to obtain the CD4+CD127lo/-CD25+ population and cultured for 16 days to selectively 
expand those that are reactive to alloantigens. The expanded cells are then concentrated and 
resuspended in a defined sterile solution at the required cell concentration for infusion. 
 
Manufacturing 
5.2.1.2.1 
Recipient Material 
Leukapheresis from the liver transplant recipient per section 5.3.3.2.4 will be transported to the 
UCSF manufacturing facility for isolating PBMCs and further processing. Onset of arTreg 
manufacture for a participant is defined as the initiation of the first leukapheresis procedure. 


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5.2.1.2.2 
Donor Material (Manufacture of sBcs) 
Deceased Donor 
Donor spleen and/or blood are recovered at the time of liver procurement and transported to the 
manufacturing facility for processing and manufacturing of sBcs. If donor tissue cannot be 
collected or cannot be used, the liver transplant recipient will not be able to participate further 
in this clinical trial, and will undergo applicable follow-up as detailed in section 4.4.  
Living Donor 
PBMC for manufacture of sBcs from living donors will be collected via phlebotomy at least 14 
days prior to the recipient leukapheresis. If the living donor becomes unable or unwilling to 
donate the required cells, the liver transplant recipient will not be able to participate further in 
this clinical trial, and will complete safety follow-up as detailed in section 4.4.  
Expanded sBcs are irradiated (25-35 Gy), banked and cryopreserved at 1-10 x 106 cells/mL 
prior to use in manufacture of the arTregs.  
5.2.1.2.3 
Treg Expansion 
arTregs are manufactured at UCSF’s HICTF by expansion in co-culture with donor sBc. First, 
CD4+127lo/-25+ Tregs are purified from recipient source cell material. This population is 
cultured at 1:4 ratio with irradiated donor sBcs in the presence of IL-2 for 11 days in medium 
supplemented with deuterated glucose to label the expanding cells. Cells are then restimulated 
with anti-CD3/anti-CD28 magnetic beads for 5 additional days. After restimulation, arTreg 
cells are harvested, washed and the beads removed by magnetic separation. Cells are then re-
suspended in 100 ml infusion solution, as described below. 
 
Formulation, Packaging, and Labeling 
arTreg is a sterile, single-cell suspension containing 30 to 500 x 106 donor allo-antigen-reactive 
regulatory T cells in 100 ml of a defined infusion solution containing 49.02% (v/v) 
PlasmaLyte-A, 49.02% (v/v) Dextrose 5% (v/v), 0.45% NaCl (v/v), and 1.96% (v/v) 25% 
human serum albumin (HSA), USP for IV administration. The arTreg product does not contain 
antibiotics or preservatives.  
After product labelling, the lot is quarantined at 4°C until release. Each lot is released for 
clinical use with an interim Certificate of Analysis (CoA), pending completion of the sterility 
test. A copy of the CoA will be included with each product lot delivered to the clinical site. The 
final CoA is issued when the sterility test result is available and a copy is also provided to the 
clinical center. Released product is transported to the clinical site refrigerated at 2-15°C in 
containers validated to maintain the set temperature for >30 hours after lot release. 
 
Dosage, Preparation, and Administration 
Eligible participants will receive a single dose of arTregs containing 30 to 500 x 106 total cells. 
The target dose is 90 to 500 x 106 total cells. However, if a minimum arTreg dose of 30 to <90 
x 106 cells is manufactured (sections 3.1 and 9.1), the product will be infused. If the dose 
obtained after product manufacture is < 30 x 106 cells, the product will not be infused (section 
1.2.4.2.2). If the dose obtained after product manufacture is > 500 x 106 cells, a dose aliquot 
will be prepared so that the administered dose will be ≤ 500 x 106 cells, and ≥ 30 to 500 x 106 


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total cells. Manufacturing failure for a participant is defined as the onset of arTreg manufacture 
which does not result in a product that meets release criteria for infusion. 
arTregs will be administered as a single infusion via a peripheral IV primed with saline by 
gravity over approximately 20 to 30 minutes. The product will be infused only after verification 
of the product label and the recipient ID by clinical study personnel to ensure the lot is 
administered to the correct recipient. Following administration of the arTreg, the infusion bag, 
tubing and peripheral IV line is flushed with up to 250 ml normal saline to ensure that the 
complete dose is infused.  
Pre-medications will be administered 30 - 60 minutes prior to arTreg infusion. Pre-medications 
are 650 mg acetaminophen and 25 - 50 mg diphenhydramine IV or by mouth. 
Vital signs will be taken just prior to, 5 minutes into, and at the end of the infusion, as well as 
30 minutes, 1 hour, and 2 hours after the infusion. Emergency medical equipment will be 
available during the infusion in case the participant has an allergic response or an infusion 
reaction that can result in a cytokine release syndrome (CRS).  
The IV line will be maintained after the infusion and the participant will remain in the clinical 
research unit for a minimum of 24 hours, which will allow ongoing monitoring for any 
infusion-related signs and symptoms. 
 
Product Accountability 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62), the investigator will 
maintain adequate records of the disposition of the products, including the date and quantity of 
the biologic received, to whom the biologic was dispensed (participant-by-participant 
accounting), and a detailed accounting of any biologic that is accidentally or deliberately 
destroyed. 
Records for receipt, storage, use, and disposition will be maintained by the study site. A log 
will be kept current for each participant. This log will contain the identification of each 
participant and the date and quantity dispensed. 
All records regarding the disposition of the investigational product will be available for 
inspection. 
 
arTreg Preparative Agents 
 
Cyclophosphamide (Cytoxan®) 
5.2.2.1.1 
Formulation, Packaging and Labeling 
CTX for Injection, USP, is a sterile, white cake available as 500 mg, 1 g, and 2 g strength vials. 
• 
500 mg vial contains 534.5 mg CTX monohydrate equivalent to 500 mg CTX and 375 mg 
mannitol 
• 
1 g vial contains 1069.0 mg CTX monohydrate equivalent to 1 g CTX and 750 mg 
mannitol 
• 
2 g vial contains 2138.0 mg CTX monohydrate equivalent to 2 g CTX and 1500 mg 
mannitol 


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Store vials at or below 25°C (77°F). During transport or storage of CTX vials, temperature 
influences can lead to melting of the active ingredient, CTX. 
CTX will be supplied by the hospital pharmacy. 
5.2.2.1.2 
Dosing 
CTX is administered prior to arTreg.  
CTX 40 mg/kg will be administered as described in section 5.3.3. It is intended as a strong 
lymphodepleting agent to allow homeostatic expansion of the infused arTregs (section 1.2.5). 
5.2.2.1.3 
Preparation and Handling 
Handle and dispose of CTX in a manner consistent with other cytotoxic drugs. Caution should 
be exercised when handling and preparing CTX for Injection, USP (lyophilized powder), or 
bottles containing CTX tablets. To minimize the risk of dermal exposure, always wear gloves 
when handling vials containing CTX for Injection, USP (lyophilized powder). 
Parenteral drug products should be inspected visually for particulate matter and discoloration 
prior to administration, whenever solution and container permit. Do not use CTX vials if there 
are signs of melting. Melted CTX is a clear or yellowish viscous liquid usually found as a 
connected phase or in droplets in the affected vials. Do not use CTX if it contains particles, is 
cloudy or discolored, or if the vial is cracked or damaged. 
CTX does not contain any antimicrobial preservative and thus care must be taken to assure the 
sterility of prepared solutions. Use aseptic technique. 
Reconstitution of CTX: reconstitute CTX using 0.9% Sodium Chloride Injection, USP or 
Sterile Water for Injection, USP with the volume of diluent listed below in Table 7. Add the 
diluent to the vial and gently swirl to dissolve the drug completely. 
Table 7. Reconstitution in Preparation for IV Infusion 
Strength 
Volume of Diluent 
CTX Concentration 
500 mg 
25 mL 
20 mg per mL 
1 G 
50 mL 
2 G 
100 mL 
 
Dilution of reconstituted CTX: Further dilute the reconstituted CTX solution to a minimum 
concentration of 2 mg per mL with any of the following diluents: 
• 
5% Dextrose Injection, USP 
• 
5% Dextrose and 0.9% Sodium Chloride Injection, USP 
• 
0.45% Sodium Chloride Injection, USP 
5.2.2.1.4 
Administration 
Participants will receive CTX, 40 mg/kg (based on lesser of ideal or actual body weight); CTX 
will be infused per institutional practice. 
Adults (18 years and older)58: 


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• 
IBW (male) = 50 kg + (2.3 kg x height in inches over 5 ft.) 
• 
IBW (female) = 45.5 kg + (2.3 kg x height in inches over 5 ft.) 
CTX should not be administered to patients who are pregnant or breast-feeding, or patients 
taking a tumor necrosis factor blocking medicine (e.g., etanercept). 
To reduce the likelihood of adverse reactions that appear to be administration rate-dependent 
(e.g., facial swelling, headache, nasal congestion, scalp burning), CTX should be infused very 
slowly. Duration of the infusion should be appropriate for the volume and type of carrier fluid. 
5.2.2.1.5 
Pretreatment 
Approximately 60 minutes prior to administration of CTX, the participant should be pre-
medicated with the following or managed per institutional standards to prevent nausea: 
• 
Decadron® (dexamethasone), 20 mg IV dose 
• 
Benadryl® (Diphenhydramine), 25 mg IV dose 
• 
Ativan® (Lorazepam) Injection, 1 mg IV dose 
• 
Zofran® (Ondansetron), 24 mg IV dose 
 
Mesna (MESNEX®) Injection 
5.2.2.2.1 
Formulation, Packaging and Labeling 
Mesna is an agent to inhibit hemorrhagic cystitis induced by CTX. The active ingredient is a 
synthetic sulfhydryl compound designated as sodium-2-mercaptoethane sulfonate. MESNEX® 
Injection is a sterile, nonpyrogenic, aqueous solution of clear and colorless appearance in clear 
glass multidose vials for IV administration. MESNEX® Injection contains 100 mg/mL mesna, 
0.25 mg/mL edetate disodium and sodium hydroxide for pH adjustment. MESNEX® Injection 
multidose vials also contain 10.4 mg of benzyl alcohol as a preservative. The solution has a pH 
range of 7.5-8.5. 
Mesna is converted to an inactive form in the blood. Then, as it is circulated through the 
kidneys, it is reactivated. The reactivated mesna works by interacting with metabolites 
(substances produced by breakdown of a drug in the body). Two specific chemotherapies, 
ifosfamide and CTX, when broken down, produce a metabolite acrolein. This metabolite is 
toxic to the bladder. Mesna binds to and inactivates acrolein thereby preventing or reducing 
bladder problems. 
Mesna will be supplied by the hospital pharmacy in the form of MESNEX® for injection. 
5.2.2.2.2 
Dosing 
Mesna is administered in conjunction with CTX. 
MESNEX® is dosed and given per institutional practice. 
5.2.2.2.3 
Preparation and Handling 
Determine the volume of MESNEX® injection for the intended dose. 
Dilute the volume of MESNEX® injection for the dose in any of the following fluids to obtain 
a final concentration of 20 mg/mL: 


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5% Dextrose Injection, USP 
• 
5% Dextrose and 0.2% Sodium Chloride Injection, USP 
• 
5% Dextrose and 0.33% Sodium Chloride Injection, USP 
• 
5% Dextrose and 0.45% Sodium Chloride Injection, USP 
• 
0.9% Sodium Chloride Injection, USP 
• 
Lactated Ringer’s Injection, USP 
The MESNEX® injection multidose vials may be stored and used for up to 8 days after initial 
puncture. 
Store diluted solutions at 25°C (77°F). Use diluted solutions within 24 hours. Do not mix 
MESNEX® injection with epirubicin, CTX, cisplatin, carboplatin, and nitrogen mustard. 
Parenteral drug products should be inspected visually for particulate matter and discoloration 
prior to administration whenever solution and container permit. Any solutions which are 
discolored, hazy, or contain visible particulate matter should not be used. 
5.2.2.2.4 
Administration 
MESNEX® is administered per institutional practice with CTX. 
5.2.2.2.5 
Pretreatment 
Participants will receive pre-hydration in order to establish good urine flow with dilute urine 
specific gravity. They must meet standards of their institutional protocol for CTX 
administration at the prescribed dose and frequency. 
 
Maintenance Immunosuppressive Agents 
 
Overview 
 
Tacrolimus (Prograf®) 
TAC is approved in the U.S for the prophylaxis of organ rejection in liver, kidney and heart 
transplantation. Generic TAC may be substituted at the discretion of the site investigator. 
For further information on TAC, please refer to the package insert for Prograf® at: 
http://www.astellas.us/docs/prograf.pdf. 
TAC will be administered post-transplant based on the center standard of care. Standard of care 
will be continued until participants are evaluated for EVR conversion and administered as 
described in section 5.3.1.2. TAC will later be withdrawn as described in section 5.3.4.4. 
TAC will be supplied by the hospital or local pharmacy. 
 
Prednisone 
For further information on prednisone, please refer to the drug label at: 
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=3115aef0-fd50-4ec8-a064-
3effb695f3f2#druglabelcontent.  


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Prednisone will be administered post-transplant based on the center standard of care. Standard 
of care will be continued until participants are evaluated for EVR conversion and administered 
as described in section 5.3.1.2.  
Prednisone may be discontinued at any time at the discretion of the site investigator.  
Participants receiving > 5 mg/day of prednisone at the time of TAC withdrawal will taper 
prednisone as described in section 5.3.4.4. Participants receiving 5mg/day of prednisone at the 
time of EVR withdrawal will withdraw prednisone as described in section 5.3.5.4. 
Prednisone will be supplied by the hospital or local pharmacy. 
 
Everolimus (Zortress®) 
EVR is indicated for the prophylaxis of organ rejection in liver and kidney transplantation. 
EVR is to be administered no earlier than 30 days post-transplant concurrently in combination 
with reduced doses of TAC and corticosteroids. 
For further information on EVR, please refer to the package insert for Zortress® at: 
https://www.pharma.us.novartis.com/sites/www.pharma.us.novartis.com/files/zortress.pdf. 
EVR will be administered as described in section 5.3.2.4 and later withdrawn as described in 
section 5.3.5.4. 
EVR will be supplied by the hospital or local pharmacy. 
 
Mycophenolate products 
MMF and mycophenolic acid are Inosine Monophosphate Dehydrogenase Inhibitors. MMF is 
the 2-morpholinoethyl ester of the active metabolite mycophenolic acid. MMF is approved in 
the U.S. for use in the prophylaxis of organ rejection in patients receiving allogeneic renal, 
cardiac or hepatic transplants. Mycophenolic acid is approved in the U.S. for use in the 
prophylaxis of organ rejection in patients receiving allogeneic renal transplants. 
For further information on MMF, please refer to the package insert for Cellcept® at: 
http://www.gene.com/gene/products/information/cellcept/pdf/pi.pdf. 
For further information on mycophenolic acid please refer to the package insert for Myfortic® 
at: http://www.pharma.us.novartis.com/product/pi/pdf/myfortic.pdf. 
Azathioprine may be used if participants are unable to tolerate MMF. 
A mycophenolate compound may be administered post-transplant based on the center standard 
of care. Standard of care will be continued until participants are evaluated for EVR conversion 
(section 5.3.1.2). 
Mycophenolate products will be supplied by the hospital or local pharmacy. 


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Concomitant Medications 
 
Prophylactic medications 
5.2.4.1.1 
Viral Prophylaxis 
IV ganciclovir (Cytovene®) and/or oral valganciclovir (Valcyte®) will be administered for 
prophylaxis of CMV and Epstein-Barr virus (EBV) post-transplant and following CTX and 
arTreg infusion according to the center’s standard of care. 
In addition, if not already receiving viral prophylaxis, five days following CTX administration, 
participants will receive a 7-day course of famciclovir (Famvir®) 500 mg PO daily for 
prophylaxis against herpes simplex virus (HSV).  
5.2.4.1.2 
Fungal and Yeast Prophylaxis 
Participants will receive oral fluconazole for fungal prophylaxis post-transplant according to 
the center’s standard of care. 
Participants will receive center standard of care prophylaxis for candida infection post-
transplant. 
5.2.4.1.3 
Pneumocystis Pneumonia (PCP) and Bacterial Infection Prophylaxis 
Participants will receive standard of care prophylaxis for PCP post-transplant until at least 52 
weeks after CTX and arTreg infusion. 
Five days following CTX administration, participants will receive prophylaxis against bacterial 
infections in the setting of neutropenia per institutional practice. 
5.2.4.1.4 
Other Prophylaxis 
Participants will receive Granulocyte-Colony Stimulating Factor (G-CSF) after CTX per 
institutional standard of care. 
 
Vaccinations 
Participants should receive seasonal influenza vaccination at some point during the influenza 
vaccination season as standard of care. However, participants must not receive any vaccination 
within 28 days prior to leukapheresis for arTreg manufacture, and for a period of 28 days after 
the arTreg infusion.  
Live vaccines are prohibited for the duration of the study. 
 
Prohibited medications 
Use of any IS medications other than those specified in this protocol, including oral courses of 
corticosteroids for >7 days for any reason, necessitates withdrawal from receiving further study 
therapy (Study Definitions) or protocol directed IS withdrawal. Topical or inhaled 
corticosteroids or steroid mouthwashes will not be considered IS medications. 
Other medications that in the opinion of the investigator may interfere or adversely interact 
with the study therapy specified in this protocol are prohibited. 


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Contraception 
WOCBP must use contraception with more than 80% effectiveness according to the FDA’s 
Office of Women’s Health (http://fda.gov/birthcontrol) from the time of enrollment until 52 
weeks after completion of the last study therapy due to the unknown effects of this protocol on 
the developing fetus. These participants must consult with their physician and determine the 
most suitable method(s) from this list to be used during this time. Abstinence is also an option.  
 
Reporting Concomitant Medications 
All prophylactic medications, vaccines, IS medications, anticoagulants and any antibiotics, 
antimicrobials, and antifungals taken by or administered to liver transplant recipients starting 
from transplant and continuing throughout their study participation will be reported. 
 
Drug Accountability 
Under federal regulations (21CFR 312.62) an investigator is required to maintain adequate 
records of the disposition of the investigational product, including the date and quantity of drug 
that was received, the participants to whom drug was dispensed (participant by participant 
accounting), and an account of any drug accidentally or deliberately destroyed. The investigator 
will ensure that the investigational product supplies are stored as specified in the protocol and 
pharmacy manual in a secured area, with access limited to authorized study personnel as 
described in the clinical study agreement. 
Records for receipt, storage, use, and disposition of the study drug will be maintained by the 
study sites. A drug-dispensing log will be kept current for each participant and will contain the 
identification of each participant and the date and quantity of drug dispensed. All remaining 
unused investigational product will be returned to the sponsor or sponsor’s representative after 
study termination, or destroyed with the permission of the sponsor in accordance with 
applicable law and study site procedures. If investigational product is to be destroyed locally, 
the investigator will provide documentation in accordance with sponsor’s specifications. 
All records regarding disposition of the investigational product will be available for inspection 
by the clinical trial monitor. 
 
Assessment of Compliance with Study Medications 
All study medications will be administered at sites by trained medical staff; compliance, 
therefore, will be monitored by the site and documented on the electronic case report forms 
(eCRFs). 
5.3 
POST-TRANSPLANT STUDY PROCEDURES 
As noted in Figure 2, this study proceeds through a defined sequence of stages post-
transplantation. Study recruitment and informed consent occurs prior to transplantation, with 
samples of blood, serum, and PBMC samples collected at any time after informed consent, but 
prior to the administration of any IS. 


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Study Stage 1: Initial Post-transplant IS 
 
Study Stage 1: Initial Post-Transplant IS – Study Continuation 
Parameters 
In the interim between transplant and Eligibility 2: EVR Conversion (section 5.3.2), 
confirmation of the following (tested at the time of transplant, with results available after 
transplant) must occur. If any of these requirements are not confirmed, the participant will be 
prematurely terminated from the study (section 4.5). 
1. Removed in protocol version 4.0. 
2. Participants must have a negative HCV PCR RNA test at transplantation. 
3. For participants with HCC, the last AFP obtained within 3 months prior to liver 
transplantation must be < 400 µg/L. 
4. The recipients’ explanted liver must not have evidence of increased risk of recurrent 
cancer: explant must be within the Milan criteria, with no vascular invasion, and with no 
cholangiocarcinoma morphology. 
5. For deceased donor recipients, there must be sufficient donor material according to arTreg 
manufacturing specifications. 
6. The transplanted donor liver must not have any unexpected histopathology discovered upon 
the pre-implant liver biopsy that contraindicates the initiation of EVR conversion. 
7. For CMV antibody-negative recipients, the donor must be CMV antibody-negative.  
8. The donor and recipient must be ABO blood type compatible. 
9. The donor and recipient must not be HLA-DR matched at both loci. 
10. For deceased donor recipients, the donor must be HCV antibody and NAT negative. 
11. Participants must not have received any investigational (unlicensed) drugs within 8 weeks 
prior to pre-transplant mechanistic sample collections or any time post-transplant.  
12. Participants must not have any new diagnosis of malignancy since transplant. 
 
Study Stage 1: Initial Post-Transplant Immunosuppression 
All participants will be placed on an initial post-transplant IS regimen based on the center’s 
standard of care (sections 5.2.3.2, 5.2.3.3, and 5.2.3.5). 
For this study, TAC is a required medication during the initial stages; inability to tolerate TAC 
during Study Stage 1, leading to discontinuation of TAC or conversion to cyclosporine, will 
result in early termination from the clinical trial. Please see Table 8 for additional details. 


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Table 8. Post-Transplant Immunosuppression 
Medication 
Parameter 
Prograf® (or 
generic 
equivalent) 
Participants must enter the EVR conversion with a Prograf® trough level of 6-10 ng/mL. 
Target trough levels will decrease to 3-5 ng/mL once the participant achieves a 
therapeutic EVR trough between 5-8 ng/mL. 
Mycophenolate 
Compound 
(MMF or 
equivalent)  
Participants may be taking mycophenolate mofetil (MMF, Cellcept®) or mycophenolic 
acid (Myfortic®). Azathioprine may be used if participants are unable to tolerate MMF. 
This medication should be discontinued approximately 7-14 days after starting EVR. 
If MMF, Myfortic, or azathioprine is not part of the participant’s immunosuppression 
regimen, the participant may continue with EVR conversion.  
Prednisone  
Participants must enter EVR conversion with daily dose ≤ 10 mg. 
In the interest of patient safety, clinical judgment should be exercised when tapering 
steroid doses, particularly if graft rejection has occurred.  
 
 
Figure 3. Post-Transplant IS and EVR Conversion Regimen (Study Stage 1, 2, and 3A) 


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Study Stage 2: EVR Conversion 
 
Study Stage 2: Timing to Apply Eligibility Criteria 2 for EVR 
Conversion 
Between day 30 and week 48 post liver transplant, participants will be evaluated for eligibility 
to be converted to an EVR-based IS regimen. Participants not meeting eligibility on initial 
evaluation may be re-evaluated throughout this period for eligibility, but must meet all 
eligibility requirements and begin EVR conversion prior to week 48 post-liver transplant. 
Laboratory assessments required to confirm eligibility must be completed within 7 days prior to 
initiating EVR conversion. 
Participants ineligible for conversion to an EVR-based IS regimen will be terminated from the 
study (section 4.5). 
 
Study Stage 2: Inclusion Criteria for EVR Conversion 
Participants must meet the following criteria to be eligible for conversion to EVR-based IS: 
1. Able to understand and provide informed consent. 
2. Negative result for 2019-nCoV by RT-PCR. Participants with a history of positive test 
result since enrollment will need two consecutively negative results to be eligible for EVR 
conversion. 
3. For participants with a history of HCV, completion of treatment for HCV and maintenance 
of a sustained viral response of ≥ 24 weeks duration at the time that eligibility for EVR 
conversion is assessed. 
 
Study Stage 2: Exclusion Criteria for EVR Conversion 
Participants who meet any of the following criteria will not be eligible for conversion to EVR-
based IS: 
1. ALT > 50 U/L or alkaline phosphatase > 200 U/L. 
2. Evidence of hepatic artery stenosis or thrombosis by Doppler or angiography. 
3. Urine protein/creatinine ratio > 1.0. 
4. Estimated GFR < 40 ml/min per 1.73m² as calculated by CKD-EPI method. 
5. Abnormal wound healing or uncontrolled wound infection per physical examination. 
6. Hemoglobin < 8.0 g/dL. 
7. Absolute neutrophil count < 1,200/µL. 
8. Platelets < 50,000/µL. 
9. AR episode according to Banff criteria or clinical rejection (Study Definitions) based on 
local assessment. Participants may be re-evaluated for eligibility ≥ 4 weeks following 
resolution of the AR episode (Study Definitions). 
10. Any new diagnosis of malignancy since transplant. 
 
Study Stage 2: EVR Conversion Schedule and Dosing  
EVR conversion must be initiated (Visit 101S) within 7 days of the eligibility visit (Visit 
101/101R).  


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At the start of conversion from TAC to EVR, the latter will be started at 1.5 mg BID with the 
dose adjusted to achieve a trough concentration of 5-8 ng/mL (Figure 3). EVR conversion is 
initiated once the participant receives their first dose of EVR. 
Seven to 14 days after initiation of EVR, the mycophenolate compound will be discontinued, if 
used. 
Once EVR trough concentration is ≥ 5 ng/mL, the baseline TAC dose will be cut approximately 
in half to achieve a trough concentration of 3-5 ng/mL. 
Once the target EVR and TAC levels are achieved and maintained over two consecutive 
measurements with ALT ≤ 50 U/L and GGT ≤ the upper limit of normal (ULN) or ≤ 1.5 x the 
baseline GGT (Study Definitions), participants will be considered successfully converted to 
EVR-based IS. 
 
Study Stage 3: arTreg Manufacturing and Investigational Treatment 
 
Overview of Study Stage 3 
Study Stage 3 involves a sequence of steps required for collection, manufacturing and 
administration of the investigational product. First, source cell material is collected from the 
transplant recipient in Stage 3A: Recipient Leukapheresis (section 5.3.3.2) for use in arTreg 
manufacturing. Next, in Stage 3B: pre-arTreg Conditioning (section 5.3.3.3) the conditioning 
regimen is administered, consisting of CTX with mesna. Stage 3C: arTreg infusion (section 
5.3.3.4) is the final step in stage 3. 
Donor phlebotomy (only if the recipient received a liver graft from a living donor) 
If the transplant recipient has received a liver from a live donor, the transplant donor will 
undergo phlebotomy at least 14 days prior to the recipient leukapheresis. The donor 
phlebotomy is performed in order to provide donor-derived stimulator cells to be used in the 
arTreg manufacturing process, as noted in 5.2.1.2.3. The donor must have negative result for 
2019-nCoV by RT-PCR within 7 days prior to blood collection for manufacturing. If the donor 
is unable or unwilling to successfully complete phlebotomy, the recipient will not be able to 
participate further in this clinical trial, and the transplant recipient will complete safety follow-
up as detailed in section 4.4. 
 


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Figure 4. Investigational Treatment Study Regimen (Study Stage 3) 
Recipient leukapheresis (Study Stage 3A) will be performed 16-17 days before arTreg infusion (Study Stage 3C). 
CTX conditioning (Study Stage 3B) will be administered after leukapheresis. CTX will be administered 24-72 hours 
before arTreg infusion (Study Stage 3C). 
 
Study Stage 3A: Recipient Leukapheresis for arTreg Product 
Manufacturing 
5.3.3.2.1 
Study Stage 3A: Timing to Apply Eligibility Criteria 3A – Recipient 
Leukapheresis for arTreg Product Manufacturing 
Following EVR conversion (Study Stage 2), between day 60 and week 52 post liver transplant, 
participants will be evaluated for eligibility for leukapheresis for arTreg manufacturing. 
Participants determined not eligible due to any of the criteria below may be re-evaluated, up to 
52 weeks post liver transplant. 
Leukapheresis must be performed no more than 28 days after establishing eligibility. 
Assessments requiring results within a shorter period prior to leukapheresis, as described in 
sections 5.3.3.2.2 and 5.3.3.2.3, may need to be repeated within this timeframe to confirm 
eligibility.  
If the liver allograft was from a living donor, donor phlebotomy will be performed at least 14 
days prior to the recipient’s leukapheresis. If the liver allograft was from a deceased donor, 
donor spleen and/or blood recovered at the time of liver procurement are used. 
5.3.3.2.2 
Study Stage 3A: Inclusion Criteria for Recipient Leukapheresis for 
arTreg Product Manufacturing 
Participants must meet all of the following criteria to be eligible for leukapheresis: 
1. Negative test for hepatitis C infection by HCV PCR RNA. 
2. Negative test for hepatitis B infection by HBV PCR DNA.  
3. Most recent 12-hour TAC trough levels < 8 µg/L measured within 10 days prior to 
leukapheresis. 
4. Most recent 24-hour EVR trough levels > 5 µg/L measured within 10 days prior to 
leukapheresis. 


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5. Negative test for 2019-nCoV by RT-PCR within 7 days prior to leukapheresis (may be 
repeated on the day of leukapheresis at the discretion of the site investigator). Participants 
with a history of positive test result since EVR conversion will need two consecutively 
negative results to be eligible for leukapheresis. 
6. Meets vaccination requirements per section 5.2.4.2. 
5.3.3.2.3 
Study Stage 3A: Exclusion Criteria for Recipient Leukapheresis for 
arTreg Product Manufacturing 
Participants who meet any of the following criteria will not be eligible for leukapheresis: 
1. Detectable circulating CMV DNA. 
2. Detectable circulating EBV DNA. 
3. Active bacterial infection, including but not limited to pneumonia, urinary tract infection 
(UTI), or sepsis. 
4. Estimated GFR < 40 ml/min per 1.73m² as calculated by CKD-EPI equation. 
5. Development of a condition requiring chronic anti-coagulation or anti-platelet agents other 
than aspirin that cannot be safely discontinued for a minimum of one week to safely 
perform a liver biopsy. 
6. History of rejection since initiating EVR conversion; evidence of AR or CR according to 
Banff criteria based on local assessment or clinical rejection (Study Definitions). 
7. ALT > 50 U/L. 
8. For living donor recipients, insufficient donor material according to arTreg manufacturing 
specifications. 
9. Hemoglobin < 8.0 g/dL. 
10. Absolute neutrophil count < 1,500/µL. 
11. Platelets < 50,000/µL. 
12. Any condition that, in the opinion of the investigator, may pose additional risks from 
continued participation in the study, may interfere with the participant’s ability to comply 
with study requirements or may impact the quality or interpretation of the data obtained 
from the study. 
13. MMF or equivalent administered within 10 days prior to leukapheresis. 
14. For females of child bearing potential, a positive pregnancy test. 
15. GGT > the ULN or > 1.5 x the baseline GGT (Study Definitions). 
5.3.3.2.4 
Study Stage 3A: Recipient Leukapheresis 
Leukapheresis will take place in an apheresis center with qualified staff. The time requirement 
for the procedure is usually between 1 and 1.5 hours to process 6 liters of blood, which will 
provide a minimum of 1 x 109 mononuclear cells. Participants will be monitored throughout the 
procedure and supportive measures such as oral calcium supplementation will be provided. 
If the leukapheresis product does not contain sufficient cell numbers for arTreg manufacturing, 
a 2nd leukapheresis can be performed if the participant continues to meet eligibility (section 
5.3.3.2). Eligibility for a 2nd leukapheresis must be established by week 52 post liver transplant. 


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Leukapheresis will be performed exactly 16-17 days prior to arTreg infusion. 
 
Study Stage 3B: pre-arTreg Conditioning (CTX) 
5.3.3.3.1 
Study Stage 3B: Timing to Apply Eligibility Criteria 3B – Pre-arTreg 
Conditioning 
Following the leukapheresis procedure, participants will be evaluated for eligibility to receive 
CTX. Blood counts collected prior to leukapheresis will be used to determine whether a 
participant may receive CTX, however, physician review prior to CTX administration is 
required to confirm eligibility. 
Participants who receive CTX cannot receive a second dose of CTX. 
5.3.3.3.2 
Study Stage 3B: Inclusion Criteria for Pre-arTreg Conditioning 
Participants must meet the following criterion to receive CTX: 
1. Confirmation from the manufacturing facility that arTreg product is expanding 
appropriately to yield an infusible product. 
5.3.3.3.3 
Study Stage 3B: Exclusion Criteria for Pre-arTreg Conditioning 
Participants who meet the following criteria will be ineligible to receive CTX: 
1. Active and untreated bacterial infection, including but not limited to pneumonia, UTI, or 
sepsis. 
2. Development of ischemic heart disease requiring revascularization, development of 
dysrhythmia requiring treatment, or clinical evidence of congestive heart failure. 
3. Any condition that, in the opinion of the investigator, may pose additional risks from 
continued participation in the study, may interfere with the participant’s ability to comply 
with study requirements or may impact the quality or interpretation of the data obtained 
from the study. 
5.3.3.3.4 
Study Stage 3B: CTX Conditioning Administration 
CTX will be administered after leukapheresis; the timing may vary from approximately 13-16 
days after leukapheresis. Information about dosing, preparation/handing, and administration of 
CTX, mesna, and pretreatment medications are described in sections 5.2.2.1 and 5.2.2.2.  
 
Study Stage 3C: arTreg Infusion 
5.3.3.4.1 
Study Stage 3C: Timing for Eligibility Criteria 3C – arTreg Infusion 
At least 24 hours after CTX administration and no more than 72 hours after CTX 
administration, participants will be evaluated for eligibility to receive arTreg infusion. 
5.3.3.4.2 
Study Stage 3C: Inclusion Criteria for arTreg Infusion 
Participants must meet the following criterion to be eligible to receive arTreg infusion: 
1. arTreg product which meets release criteria for infusion. 
5.3.3.4.3 
Study Stage 3C: Exclusion Criteria for arTreg Infusion 
There are no exclusion criteria at this step. 


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5.3.3.4.4 
Study Stage 3C: arTreg Infusion 
The arTreg product will be infused as described in section 5.2.1.4.  
5.3.3.4.5 
Study Stage 3C: IS Management and Treatment of Elevated Liver Tests 
after arTreg Infusion and Prior to TAC Withdrawal 
TAC dosing may be adjusted to maintain trough concentration of 3-7 ng/mL per investigator 
discretion for the treatment of elevated LFTs that occur after arTreg infusion and prior to 
initiating TAC withdrawal. EVR dosing should be continued to maintain a trough concentration 
of 5-8 ng/mL. 
 
Study Stage 4: TAC Withdrawal 
 
Study Stage 4: Timing to Apply Eligibility Criteria 4 – TAC Withdrawal 
Within 15 days after receiving the minimum or target arTreg dose (Study Definitions), 
participants will be evaluated for eligibility for TAC withdrawal. 
 
Study Stage 4: Inclusion Criteria for TAC Withdrawal 
Participants must meet the following criteria to be eligible for TAC withdrawal: 
1A. ALT < 75 U/L and GGT < 75 or < 1.5 x the baseline GGT (Study Definitions) 
*OR* 
1B. ALT ≥ 75 U/L or GGT ≥ 75 or ≥ 1.5 x the baseline GGT (Study Definitions): participants 
may not begin TAC withdrawal until the liver biopsy 5 to 10 days after receiving the 
arTregs is performed and assessed to have a Rejection Activity Index (RAI) of 0-3 per 
Banff Global Criteria based on Local Pathology50,59-61. 
 
Study Stage 4: Exclusion Criteria for TAC Withdrawal 
Participants who meet any of the following criteria will not be eligible for TAC withdrawal: 
1. History of rejection since leukapheresis; evidence of AR or CR according to Banff criteria 
on protocol allograft biopsy based on local assessment or clinical rejection (Study 
Definitions). 
2. Estimated GFR < 40 ml/min per 1.73m² as calculated by CKD-EPI equation. 
3. Development of a condition requiring chronic anti-coagulation or anti-platelet agents other 
than aspirin that cannot be safely discontinued for a minimum of 1 week to safely perform 
a liver biopsy. 
4. Any condition that, in the opinion of the investigator, may pose additional risks from 
continued participation in the study, may interfere with the participant’s ability to comply 
with study requirements or may impact the quality or interpretation of the data obtained 
from the study. 
 
Study Stage 4: IS Management during TAC withdrawal 
Withdrawal may be initiated within 14 days of TAC withdrawal eligibility confirmation. 
Participants who meet eligibility by inclusion criteria #1A will undergo a liver biopsy 5 to 15 
days after receiving the arTregs and may continue with TAC withdrawal if the biopsy is 
assessed to have a RAI of 0-3 per Banff Global Criteria based on Local Pathology.50,59-61 


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Withdrawal will occur over two 6-week intervals, for a total of 12 weeks in the absence of a 
pause. Withdrawal will proceed as indicated in Figure 5. Dose reductions can occur within a +7 
day window for each taper level. 
If the participant is receiving prednisone, the prednisone dose will be tapered to a maximum of 
5 mg/day by the completion of TAC withdrawal. Prednisone may have been discontinued at an 
earlier time point at the discretion of the investigator. The exact schedule for prednisone 
withdrawal may be varied at the discretion of the investigator based on the clinical and 
immunologic status of the participant. 
 
Figure 5. TAC Withdrawal and EVR/Prednisone Withdrawal Regimen (Study Stage 4, 5 and 6) 
 
Study Stage 5: EVR/Prednisone Withdrawal 
Participants who successfully complete Study Stage 4 will remain on EVR. In addition, these 
participants may optionally remain on ≤ 5 mg/day of prednisone; prednisone may have been 
discontinued at an earlier time point at the discretion of the investigator.  
 
Study Stage 5: Timing to Apply Eligibility Criteria 5 – EVR/Prednisone 
Withdrawal 
Between 12 to 26 weeks of receiving EVR ± prednisone alone, participants who received ≥ 90 
x 106 arTregs will be evaluated for eligibility for EVR/prednisone withdrawal. 


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Participants who received a dose of arTregs ≥ 30 x 106 cells but < 90 x 106 cells may be 
considered for EVR/prednisone withdrawal at the discretion of the investigator.  
 
Study Stage 5: Inclusion Criteria for EVR/Prednisone Withdrawal 
Participants must meet all of the following criteria to be eligible for EVR/prednisone 
withdrawal: 
1. Able to provide reaffirmation of informed consent. 
2. Received a minimum of 12 weeks of EVR following complete TAC withdrawal. 
3. Had a liver biopsy that fulfills the criteria in Table 9 based on central pathology. 
 
Study Stage 5: Exclusion Criteria for EVR/Prednisone Withdrawal 
Participants who meet any of the following criteria will not be eligible for EVR/prednisone 
withdrawal: 
1. History of rejection since initiating EVR conversion (Study Stage 2); evidence of AR or 
CR according to Banff criteria on protocol allograft biopsy based on local assessment or 
clinical rejection (Study Definitions). 
2. ALT > 50 U/L.      
3. Estimated GFR < 40 ml/min per 1.73m² as calculated by CKD-EPI method. 
4. Development of a condition requiring chronic anti-coagulation or anti-platelet agents other 
than aspirin that cannot be safely discontinued for a minimum of 1 week to safely perform 
a liver biopsy. 
5. Any condition that, in the opinion of the investigator, may pose additional risks from 
continued participation in the study, may interfere with the participant’s ability to comply 
with study requirements or may impact the quality or interpretation of the data obtained 
from the study.  
6. GGT > the ULN or > 1.5 x the baseline GGT (Study Definitions) 


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Table 9. Study Stage 5: EVR/Prednisone Withdrawal. Eligibility 5 Biopsy Findings for IS 
Minimization57 
Compartment 
Finding(s) 
Portal 
inflammation and 
interface activity 
No more than minimal or focal mild portal mononuclear inflammation. No more 
than minimal interface necro-inflammatory activity, limited to a minority of portal 
tracts. 
Centrizonal / 
perivenular 
inflammation† 
Negative for perivenular inflammation. 
Bile duct changes 
Absence of lymphocytic bile duct damage, ductopenia, and biliary epithelial 
senescence changes, unless there is an alternative, non-immunological explanation 
(e.g. biliary strictures). 
Fibrosis 
No more than mild fibrosis, with rare or no portal-to-portal bridging.  
Arteries+ 
Negative for isolated “v” lesions (lymphocytic arteritis), or obliterative or foam 
arteriopathy. 
† Modified from Banff 2012 recommendations62 because of widespread recognition that the lesion represents a 
rejection reaction and has the potential to progress and cause perivenular fibrosis after weaning. 
+ Isolated “v” lesions (lymphocytic arteritis) was added to the Banff 2012 because of evidence of similar lesions in 
renal allografts leading to a suboptimal outcome even in patients maintained on immunosuppression.63 
 
Study Stage 5: EVR/Prednisone Withdrawal 
The Schedule of Events (Appendix 4) specifies the study visits for Study Stage 5. 
EVR/Prednisone withdrawal will be initiated within 8 weeks of the eligibility biopsy. 
For participants receiving prednisone, prednisone withdrawal will occur over four 4-week 
steps, for a total of 16 weeks in the absence of a pause. EVR withdrawal will occur over four 4-
week steps followed by three 6-week steps, for a total of 34 weeks in the absence of a pause. 
Withdrawal will proceed as indicated in Figure 5. Dose reductions can occur within a +7 day 
window for each taper level. 
 
Study Stage 6: Post-IS Withdrawal Surveillance and Evaluation 
See section 5.4.2 for study procedures for the resumption of IS, and section 5.4.3 for the study 
procedures for assessment and treatment of allograft dysfunction. All participants who achieve 
complete IS withdrawal according to the withdrawal schedule will complete 104 weeks of Post-
withdrawal Surveillance and Evaluation per Appendix 5, Schedule of Events. 
If a participant has biopsy-proven rejection or resumes IS for clinical rejection during Study 
Stage 6, they will complete follow-up per section 5.4.2 and subsequent study-mandated 
biopsies are optional.  
5.4 
OTHER STANDARD STUDY PROCEDURES 
 
Procedure for Temporary Pause in IS Withdrawal 
IS withdrawal may be temporarily paused for up to 4 weeks. During this time interval, the 
participant will remain at the current dose. If the participant is withdrawing from two 
medications at the time of a pause, both medications will remain at the current dose. If the 
participant cannot proceed to the next dose reduction after 4 weeks, they will move to High 
Intensity Safety Follow-up (Appendix 6) and will considered to have failed IS withdrawal. 
Participants are allowed up to 3 non-consecutive pauses or a cumulative total of 12 weeks. 


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Cases in which extenuating circumstances lead to longer pauses will be considered by a 
committee consisting of the NIAID Medical Monitor, the ITN Clinical Trial Physician, and the 
Protocol Chair. This committee will adjudicate whether the participant may proceed with IS 
withdrawal even if the 12-week total has been exceeded. 
 
Procedure for IS Resumption  
Participants who meet the following criteria will receive treatment or be restarted on IS per 
section 5.4.3.3. 
1. Participants undergoing IS withdrawal who have biopsy-proven rejection or clinical 
rejection will discontinue further withdrawal and receive treatment. 
2. Participants who have completed IS withdrawal who have biopsy-proven rejection or 
clinical rejection will be restarted on IS. 
All participants who resume IS will have failed IS withdrawal unless determined otherwise by 
an adjudication committee (section 5.4.3.2.3). Participants who fail IS withdrawal will not be 
allowed a second attempt. 
Participants who fail IS withdrawal while undergoing IS withdrawal will complete 104 weeks 
of High Intensity Safety Follow-up per Appendix 6. If the participant fails IS withdrawal after 
completing IS withdrawal, the participant will continue on Study Stage 6: Post-withdrawal 
Surveillance and Evaluation per Appendix 5. These participants will be followed for 104 
weeks after completing Study Stage 5 (section 5.3.5) or 12 weeks after resuming IS, whichever 
is longer. 
 
Assessment and Treatment of Allograft Dysfunction 
 
Definition of Allograft Dysfunction and Indication for Allograft Biopsy 
Allograft dysfunction occurs when ALT ≥ 100 U/L and/or GGT is elevated relative to baseline.  
The baseline GGT for each participant is defined as the average of two GGT values taken at the 
following time points: 
• 
The GGT taken at EVR Conversion Eligibility (V101[R]) 
• 
The GGT taken at the initiation of EVR conversion (V101S) 
Elevation of GGT sufficient to meet the definition of allograft dysfunction is measured in 
relation to the baseline value as noted below: 
• 
If the baseline GGT was < the ULN and the current value is ≥ 2-fold the ULN  
Or,  
• 
If the baseline GGT was ≥ the ULN and the current value is ≥ 2-fold the baseline 
value. 
When allograft dysfunction occurs, participants should be thoroughly assessed for any 
concurrent illness or alternative diagnosis according to standard of care. Liver tests may be 
repeated for both verification and re-assessment. 


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If allograft dysfunction persists for > 7 days and no etiology is identified, a for-cause biopsy 
must be performed, unless medically contraindicated. If a for-cause biopsy has been performed 
and is non-diagnostic, further biopsies may be performed at the discretion of the investigator. 
At the discretion of the investigator, a for-cause biopsy may be triggered by elevated liver tests 
below the threshold that defines allograft dysfunction but above the individual participant’s 
baseline. 
 
Diagnosis of Rejection 
5.4.3.2.1 
Acute Rejection 
AR will be diagnosed in accordance with Banff global assessment criteria.50,59-61 Severity will 
be determined by Table 10.64 
Steroid refractory AR refers to AR that has failed to resolve with steroid treatment. 
Table 10. Banff Schema: Grading of Acute Liver Allograft Rejection 
Global 
Assessment 
Criteria 
Indeterminate 
Portal inflammatory infiltrate that fails to meet the criteria for the diagnosis of AR 
Mild 
Rejection infiltrate in a minority of triads that is generally mild and confined within 
the portal spaces with RAI ≤4. 
Moderate 
Rejection infiltrate expanding most of all of the triads. 
Severe 
As above for moderate, with spillover into periportal areas and moderate to severe 
perivenular inflammation that extends into the hepatic parenchyma and is associated 
with perivenular hepatocyte necrosis. 
NOTE: Global assessment of rejection grade made on a review of the biopsy specimen and after the 
diagnosis has been established. 
 
5.4.3.2.2 
Chronic Rejection 
CR will be diagnosed in accordance with Banff global assessment criteria.59-61 
5.4.3.2.3 
Clinical Rejection 
For cases where a biopsy is indeterminate or in rare cases where a biopsy cannot be performed, 
participants may be treated empirically based on the investigator’s judgment as to the 
likelihood of rejection based on clinical parameters. In these cases, participants will be 
considered to have clinical rejection. If a participant is treated for clinical rejection at the study 
site without undergoing a biopsy, all other visit assessments done at a for-cause biopsy visit 
(Appendix 8) will be done prior to initiating treatment. 
Participants with clinical rejection and no biopsy must undergo a biopsy within a week of 
initiating treatment for allograft dysfunction, unless medically contraindicated. 
In the event an investigator determines that a participant did not experience rejection, but was 
treated for rejection or received increased IS, the investigator may request that the participant’s 
data is reviewed by an adjudication committee. This adjudication committee will consist of the 
NIAID Medical Monitor, the ITN Clinical Trial Physician, and a Protocol Chair from an 
alternate consortium protocol (section 1.1.1). The central pathologist may also be consulted as 
needed. This committee will adjudicate whether the participant has experienced clinical 


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rejection. If the committee is unanimous in determining that the participant has not experienced 
clinical rejection, then the participant can continue with IS withdrawal, or if already off all IS, 
can remain off. If it is the opinion of any or all committee members that the participant has 
experienced clinical rejection, the participant has failed IS withdrawal, or if already off all IS, 
will have failed the primary endpoint. 
 
Guidelines for Management of Allograft Dysfunction or Rejection 
IS will be escalated or reinstituted for all participants who experience biopsy-confirmed or 
clinical rejection. Investigators may treat rejection according to center standard of care. 
The following, however, are recommended guidelines based on previous experience in liver 
allograft IS withdrawal trials for participant’s experiencing allograft dysfunction following 
initiation or completion of IS withdrawal: 
• 
Allograft dysfunction with no AR or indeterminate for AR, and without other explanatory 
diagnosis should be treated at the discretion of the site investigator with increased or 
reinstitution of IS. If liver tests do not improve within 4 weeks, repeat biopsy should be 
considered prior to further escalation of treatment. 
• 
Mild AR should be treated initially with increased or reinstitution of IS. If liver tests do not 
improve within 2 weeks, conversion or addition of another agent should be considered prior 
to administration of corticosteroids. If corticosteroids are administered, a dose of 20 mg 
oral prednisone (or equivalent) should be considered. Corticosteroids will be tapered down 
over a 4-week period. A second biopsy can be performed at any time at the investigator’s 
discretion. 
• 
Moderate AR with mild biochemical abnormalities and no jaundice should be treated with 
increased or reinstitution of IS and 20 mg oral prednisone (or equivalent) with taper down 
of steroid doses over a 4-week period. If liver tests do not improve within 2 weeks, 
conversion or addition of another agent should be considered prior to additional 
corticosteroids. A second biopsy can be performed at any time at the investigator’s 
discretion. 
• 
Moderate AR with marked biochemical abnormalities, severe AR, or CR, should be treated 
according to center standard of care. 
• 
Antibody treatment should be reserved for steroid-resistant rejection proven by repeat liver 
biopsy. 
An episode of biopsy-confirmed or clinical rejection will be considered resolved when ALT 
(with or without elevated GGT) is ≤ 50 U/L. For these cases, it is recognized that GGT levels 
decline very slowly following rejection and therefore will not be used to define resolution. If an 
episode of biopsy-confirmed or clinical rejection involves elevated GGT alone, it will be 
considered resolved when GGT is ≤ 1.5 x baseline levels. 
For participants with biopsy-confirmed rejection, a repeat biopsy to further document resolution 
of rejection may be performed at the discretion of the investigator.  


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6. 
STUDY ASSESSMENTS AND WINDOWS 
6.1 
OVERVIEW 
Appendices 1-8 present the schedule of events for this trial. The visit windows are outlined in 
section 6.2, while sections 6.3-6.7 detail the study assessments that are performed. The 
frequency of the study assessments are noted in the schedule of events. 
6.2 
VISIT WINDOWS 
 
Recipient  
All scheduled study visits and assessments must occur within the time limits specified below: 
• 
Visits as noted in Table 11. 
• 
Laboratory evaluations (LFTs and IS levels) done locally, should be completed within ±5 
days of the scheduled time points. 
• 
Up to 3 non-consecutive pauses (up to 4 weeks each) may occur during Visits 201-203 and 
Visits 301-307 combined (if needed) as outlined in section 5.4.1. 
• 
If a participant needs a for-cause biopsy within 8 weeks prior to an Appendix 1A to 
Appendix 4 visit, the mechanistic assessments collected during the For-cause Biopsy Visit 
will take the place of any mechanistic assessments to be collected during that upcoming 
Appendix 1A to Appendix 4 visit.  
• 
If a participant needs a for-cause biopsy within 8 weeks prior to an Appendix 5 to 
Appendix 7 visit, the For-cause Biopsy Visit will take the place of that upcoming Appendix 
5 to Appendix 7 visit. 
Table 11. Recipient Visit Windows 
 
Step 
Visit 
Time Point 
Visit 
Window 
Appendix 1A 
Eligibility 1: Pre-Transplant 
-1 
Within 26 weeks prior to Visit 0 
Transplant 
0 
Day 0 
Eligibility 2: EVR 
Conversion 
101/101R 
Day 30 to Week 48 post-transplant 
EVR Conversion Initiation 
101S 
Within 7 days of V101[R] 
Eligibility 3A: Recipient 
Leukapheresis for arTreg 
Manufacturing 
102/102R 
Day 60 to Week 52 post-transplant 


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Step 
Visit 
Time Point 
Visit 
Window 
Appendix 2 
Recipient Leukapheresis 
T-17/T-17R Within 28 days of V102[R] 
Eligibility 3B: pre-arTreg 
Conditioning (CTX) 
T-3 
CTX administration must occur no more than 72 
hours and no less than 24 hours before Treg infusion 
Eligibility 3C: arTreg 
Infusion 
T0 
Treg Day 0 
 
Post arTreg Infusion Visits 
T1 
Treg Day 1 
+24 hours 
T7 
Treg Day 7 
-2/+8 days 
T14 
Treg Day 14 
-2/+3 days 
T28 
Treg Day 28 
±5 days 
T84 
Treg Day 84 
±5 days 
Reduced Safety Follow-up 
RFU 
Within 30 days after unsuccessful EVR conversion, 
Visit T-17/T-17R, or Visit T-3 
Appendix 3 
TAC Taper Level 1 
201 
Within 14 days after confirming TAC withdrawal 
eligibility (Visit T1-T7) (section 5.3.4.4) 
TAC Taper Level 2 - Off 
202-203 
Every 6 weeks 
+7 days 
TAC Withdrawal Pause 
Visits 
201P 
During Taper Level 1 – up to 4 weeks (section 5.4.1) 
 
202P 
During Taper Level 2 – up to 4 weeks (section 5.4.1) 
 
Post TAC Withdrawal Week 
24 
204 
24 weeks after Visit 201 
±7 days 
Appendix 4 
Eligibility 5: EVR/Pred 
Withdrawal 
300 
Week 12 to 26 post TAC Withdrawal (section 5.3.5.1 
for participants who receive the minimum/target 
arTreg dose [Study Definitions])  
EVR/Pred Taper Level 1 
301 
Within 8 weeks after Visit 300 Biopsy 
EVR/Pred Taper Levels 2-4 
302-304 
Every 4 weeks 
+7 days 
EVR Taper Levels 5-7 
305-307 
Every 6 weeks 
+7 days 
EVR/Pred Withdrawal Pause 
Visits 
301P-307P 
During Taper Level 1 to 7 – up to 4 weeks each 
(section 5.4.1) 
Appendix 5 
Post-IS Withdrawal 
Surveillance and Evaluation 
Follow-up Week 0 
400 
Within 7 days after the last IS dose 
Post-IS Withdrawal 
Surveillance and Evaluation 
Follow-up Week 26 
401 
26 weeks after the last IS dose 
±2 weeks 
Post-IS Withdrawal 
Surveillance and Evaluation 
Follow-up Week 52 (Primary 
Endpoint Visit) 
402 
52 weeks after the last IS dose 
±4 weeks 
Post-IS Withdrawal 
Surveillance and Evaluation 
Follow-up Week 104 
403 
104 weeks after the last IS dose 
±4 weeks 
Post-IS Withdrawal 
Surveillance and Evaluation 
Follow-up Week 105-116 
404 
12 weeks after restarting IS (participants 
for who resume IS at the end of study 
follow-up to ensure they are followed 
for ≥12 weeks after restarting IS [section 
5.4.2]) 
±2 weeks 


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Step 
Visit 
Time Point 
Visit 
Window 
Appendix 6 
High Intensity Follow-up 
Week 0 
500 
Within 4 weeks after Visit T0 (participants who don’t 
receive the minimum/target arTreg dose [Study 
Definitions] or attempt TAC withdrawal), Visit 300 
(participants who do not attempt EVR/Pred 
withdrawal), or ISW failure 
High Intensity Follow-up 
Week 26 
501 
26 weeks after Visit T0 (participants 
who don’t receive the minimum/target 
arTreg dose [Study Definitions] or 
attempt TAC withdrawal), Visit 300 
(participants who do not attempt 
EVR/Pred withdrawal), or ISW failure 
±2 weeks 
High Intensity Follow-up 
Week 52 
502 
52 weeks after Visit T0 (participants 
who don’t receive the minimum/target 
arTreg dose [Study Definitions]) or 
attempt TAC withdrawal), Visit 300 
(participants who do not attempt 
EVR/Pred withdrawal), or ISW failure 
±4 weeks 
High Intensity Follow-up 
Week 104 
503 
104 weeks after Visit T0 (participants 
who don’t receive the minimum/target 
arTreg dose [Study Definitions] or 
attempt TAC withdrawal), Visit 300 
(participants who do not attempt 
EVR/Pred withdrawal), or ISW failure 
±8 weeks 
Appendix 7 
Low Intensity Follow-up 
Week 0 
600 
7 to 10 days after Visit T-3 
Low Intensity Follow-up 
Week 4 
601 
4 weeks after Visit T-3 
±5 days 
Low Intensity Follow-up 
Week 52 
602 
52 weeks after Visit T-3 
±8 weeks 
Appendix 8 
For-cause Biopsy 
FCB 
As needed 
 
 
Living/Deceased Donor 
All scheduled study visits and assessments must occur within the time limits specified below: 
• 
Visits as noted in Table 12. 


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Table 12. Living/Deceased Donor Visit Windows 
 
Step 
Visit 
Time Point 
Visit 
Window 
Appendix 1B 
Eligibility: Living Donor 
Transplant 
LD-1 
Within 26 weeks prior to Visit 0 in Appendix 1A 
LD0 
Day 0 to Week 26 post-transplant, but prior to Visit 
102/102R in Appendix 1A 
Eligibility: Deceased Donor 
Transplant 
DD0 
Day 0 
±7 days 
6.3 
GENERAL ASSESSMENTS 
 
Recipient 
Recipients will undergo the following general assessments during the course of their study 
participation: 
• 
Informed consent  
• 
Eligibility criteria 
• 
Study parameters for continuation (section 5.3.1.1)  
• 
Demographics – date of birth, sex, ethnicity, and race  
• 
Medical history – (within the last 12 months) includes liver disease history to determine 
time of diagnosis and ABO typing 
• 
Complete physical exam – includes height only at Visit 101 
• 
Limited physical exam – includes body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary 
• 
Vital signs – weight, temperature, blood pressure, respiratory rate, pulse 
• 
Adverse Events – participants will be assessed for AEs and recorded per section 8.3 
• 
Concomitant medications – all concomitant medications will be recorded per section 
5.2.4.5 
• 
Remote consultation – e.g. by telephone, email, or a combination of methods 
• 
Telemedicine consultation – requires investigator assessment of the participant by 
telephone or video conference 
• 
Events of interest – includes the following: 
- 
Rejection (section 5.4.3.2) 
- 
Infections: includes wound infection, bacteremia, cholangitis, pneumonia, meningitis, 
CMV viremia, EBV/PTLD, fungal infection, Cryptococcus, and others 
- 
Surgical complications or procedures, including biliary obstruction 
- 
Cytopenia 
- 
Evidence of CKD 


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- 
Recurrent HCC or any cancer 
• 
HCV treatment history – includes last date of treatment for participants with a history of 
HCV only (section 5.3.1.1)  
• 
Milan criteria, with no vascular invasion, and with no cholangiocarcinoma morphology – 
criteria apply to the recipient’s explanted liver (section 5.3.1.1) for participants with HCC 
only 
 
Living Donor 
Living donors will undergo the following general assessments during the course of their study 
participation: 
• 
Informed consent 
• 
Eligibility criteria 
• 
Demographics – date of birth, sex, ethnicity, and race 
• 
ABO typing (from the medical record) 
• 
AEs – participants will be assessed for AEs and recorded per section 8.3 
 
Deceased Donor 
Data to be collected from medical or UNOS records. 
• 
Eligibility criteria 
• 
Demographics – date of birth, sex, ethnicity, and race 
• 
ABO typing (from the medical record) 
6.4 
CLINICAL ASSESSMENTS AND PROCEDURES 
 
Recipient 
Recipients will undergo the following clinical assessments and procedures during the course of 
their study participation: 
• 
Doppler or angiography of the liver 
• 
Liver transplant – standard of care 
• 
Liver biopsy (section 6.7) 
6.5 
CLINICAL LABORATORY ASSESSMENTS 
 
Recipient 
These laboratory assessments may be performed at the study site or other qualified local 
laboratories: 
• 
Hematology – CBC with differential and platelets 
• 
Basic chemistry – BUN, creatinine, total bilirubin, direct bilirubin, GGT, AST, ALT, 
alkaline phosphatase 


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• 
Urine or blood hCG – pregnancy test for WOCBP 
• 
Liver Function Tests – ALT, GGT 
• 
CD4 count – in peripheral blood 
• 
HIV-1/2 –antibody immunoassay 
• 
HCV RNA – hepatitis C viral copy number by quantitative PCR 
• 
HBV DNA – hepatitis B viral copy number by quantitative PCR 
• 
EBV Antibody – EBV IgG (the assessment does not need to be repeated if the participant 
has a documented positive result) 
• 
EBV DNA – quantitative by PCR 
• 
CMV antibody – CMV IgG (the assessment does not need to be repeated if the participant 
has a documented positive result) 
• 
CMV DNA – quantitative by PCR 
• 
Estimated GFR – as determined by calculated CKD-EPI65 
• 
HLA typing – includes HLA-DR (retrospective data collection from the medical record – 
repeat testing is not required if already completed as standard of care) 
• 
AFP – section 5.3.1.1 
• 
Urinalysis – urine protein/creatinine ratio 
• 
TAC trough levels 
• 
EVR trough levels 
• 
2019-nCoV RT-PCR – see sections 5.3.2.2 and 5.3.3.2.2  
 
Living Donor 
These laboratory assessments may be performed at the study site or other qualified local 
laboratories: 
• 
HLA typing – includes HLA-DR (retrospective data collection from the medical record – 
repeat testing is not required if already completed as standard of care) 
• 
CMV antibody – CMV IgG (the assessment does not need to be repeated if the living donor 
has a documented positive result) 
• 
EBV antibody – EBV IgG (the assessment does not need to be repeated if the living donor 
has a documented positive result) 
• 
2019-nCoV RT-PCR within 7 days prior to blood collection for manufacturing 
 
Deceased Donor 
For all assessments, data to be collected from medical or UNOS records. If tests are not 
available, they should be ordered. 
• 
HLA typing – includes HLA-DR 


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• 
CMV antibody – CMV IgG 
• 
EBV antibody – EBV IgG 
• 
HCV testing – antibody and NAT  
6.6 
ARTREG MANUFACTURE 
 
Recipient 
• 
Leukapheresis (section 5.3.3.2.4) 
• 
2019-nCoV RT-PCR within 7 days prior to leukapheresis (may be repeated on the day of 
leukapheresis at the discretion of the site investigator) 
 
Living Donor 
• 
Blood collection for infectious diseases testing – compliant with the current local and 
federal regulations (21CFR 1271) and requirements  
• 
Blood collection by phlebotomy for manufacture of sBc using KT64.CD40L stimulation 
 
Deceased Donor 
• 
Blood collection for infectious diseases testing – compliant with the current local and 
federal regulations (21CFR 1271) and requirements  
• 
Donor spleen and/or blood collection for manufacture of sBc using KT64.CD40L 
stimulation 
6.7 
LIVER BIOPSY 
 
Types of Biopsies 
Research Biopsies  
Participants will undergo liver biopsies at times specified in the schedule of events (Appendices 
1-5). These biopsies will be used to monitor liver histology, to screen for subclinical rejection, 
and for mechanistic analyses. 
For–cause Biopsies  
For-cause biopsies (FCB) will be obtained to confirm suspected rejection and all mechanistic 
assessments listed in Appendix 8 should be performed. 
 
Central and Local Biopsy Assessments 
The guidelines for the use of local and central liver biopsy pathology assessments are outlined 
below. All will be analyzed per the Banff global assessment criteria.50,59-61,64 
For the purpose of determining Study Stage 4: TAC withdrawal eligibility, the local 
assessment will be used (section 5.3.4). 
For the purpose of determining Study Stage 5: EVR/Prednisone withdrawal eligibility 
(section 5.3.5), and the primary and secondary endpoint evaluations (sections 3.3.1 and 3.3.2), 
the central assessment will be used. 


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For the purpose of clinical management, the local pathology assessment will be used. The 
central pathology assessment will be available to the investigators for consideration at their 
discretion. 
For the purpose of evaluating the study stopping rules (section 3.4.2.1), the local pathology 
assessment will be used.  
 
Biopsy Technique and Handling 
The day 0 pre-implant research biopsy will be performed prior to transplantation of the liver 
into the recipient. Otherwise, research and for-cause biopsies will be performed per institutional 
practice guidance. Use of a 16- gauge needle is preferred. A minimum of two core biopsies 
should be obtained with a minimum of 1 cm of tissue per core biopsy. A consent specific for 
this procedure will be obtained according to the guidelines at the study site. 
 
Recording of Biopsy Assessments 
The local pathology assessment will be recorded on the eCRF by the site. The central pathology 
assessment will be recorded by the ITN central pathology core laboratory and the data will be 
transferred at regular intervals to the clinical database. 
6.8 
MECHANISTIC ASSESSMENTS 
• 
HLA typing – molecular typing may include both Class I (HLA-A, B, C) and Class II 
(HLA-DRB1, DRB3, DRB4, DRB5, DQA, DQB1, DPB1) 
• 
arTreg collection – archived cell product prior to infusion 
• 
PBMC collection – includes arTreg detection and characterization and PBMC assays. 
arTreg detection and characterization assessments performed on days 14, 28, and 84 
following arTreg infusion may be performed at the study site or remotely.  
• 
Serum collection – includes donor-specific HLA antibody testing and serum assays 
• 
Whole blood collection – gene expression 
• 
Liver biopsy collection – graft histology, including H&E, Trichrome, and 
immunohistochemistry 
See section 7 for detailed discussion of additional mechanistic assays. 
7. 
MECHANISTIC ASSAYS 
Although potential markers and technologies are listed, utilized markers and technologies will 
be selected and finalized based on literature and the state-of-the-art at the time of 
experimentation. Experiments may be performed in real-time, and/or in batches at (an) agreed 
upon point(s) in the trial to decrease the potential for variability between experiments. 
7.1 
RATIONALE FOR IMMUNE STUDIES 
A key hypothesis for this study is that adoptive transfer of alloantigen specific Tregs will allow 
for successful IS withdrawal and allow for operational tolerance. We hypothesize that these 
cells will dampen the anti-donor response, potentially by a variety of mediators. The current 
ITN073ST and ITN074ST trials and potential future alloantigen specific Treg trials, are/will be 


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similar in their design, with the major difference being the generation of and intervention 
surrounding the infusion of different Treg products. By limiting the differences between these 
trials we will be able to compare the phenotype, stability, and function of alloantigen specific 
Tregs produced by at least two different methods. Further, these studies may identify or further 
confirm characteristics that describe operational tolerance, or lack of rejection, regardless of 
method of induction. We propose the following immune studies to address the mechanistic 
hypotheses outlined in section 7.2.  
7.2 
MECHANISTIC HYPOTHESES 
Overall, we hypothesize that successful IS withdrawal will correlate with: 
1. Increased arTreg cells in the circulation and graft, 
2. Suppression of donor-reactive Teffector cells, 
3. Increased intragraft expression and/or presence of tolerance associated genes, and 
4. Decreased intragraft expression and/or presence of rejection associated genes.  
Functional assay hypotheses are: 
1. Tolerant participants will have a loss or altered direct and indirect alloresponses following 
arTreg infusion. 
2. Increased presence of arTreg cells will suppress the expansion of donor-reactive effector T 
cells (and potentially lead to their deletion or deviation from a pathogenic phenotype), and 
promote T cell exhaustion and senescence. 
Graft-related hypotheses are: 
1. Following arTreg infusion, transferred cells will be detectable within the graft. 
2. The change of effector and regulatory T cell balance will be reflected within the graft, 
along with increased intra-graft expression of tolerance-associated genes and decreased 
intra-graft expression of rejection-associated genes.  
Regulatory T cell product characterization hypotheses are: 
1. arTreg infusion will lead to a persistent increase of alloantigen-reactive Tregs in both the 
circulation and graft.  
7.3 
PROPOSED MECHANISTIC ASSAYS 
 
HLA Typing 
HLA typing for both the donor and recipient may be performed, in addition to the standard of 
care HLA typing performed for enrollment eligibility. Samples provided by the donors and 
recipients may be used to perform molecular typing for both Class I (HLA-A, B, C) and Class 
II (HLA-DRB1, DRB3, DRB4, DRB5, DQA, DQB1, DPB1).  
 
Functional Assays 
 
Control of Anti-Donor Responses: DSA 
Following liver transplant, development of DSA by the recipient may portend damage to the 
liver allograft. In addition to possibly being evident by clinical measures, this damage may also 
be evident upon histological investigation of the allograft. We will first test liver transplant 


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recipient serum for PRA (panel reactive antibody) to determine if the recipient has generated 
anti-HLA reactive antibodies. If this test is positive, we will then test these sera for DSA. 
Results from donor and recipient HLA typing (section 7.3.1) will be used in conjunction with 
solid phase, single antigen bead assays to determine if the recipients’ anti-HLA antibody is 
reactive with a donor-type HLA. To further describe and attribute function to these DSA, the 
IgG subclass (IgG1/2/3/4) and/or ability to bind C1q may be assessed.  
 
Detection of Donor Reactivity 
7.3.2.2.1 
Serum Assays 
We propose to collect serum samples to test for the presence of regulatory proteins and/or the 
absence of proteins indicative of graft rejection. This testing may use multiplex technology to 
assay cytokine and/or other protein amounts.  
7.3.2.2.2 
Allospecific T Cells 
We propose to test liver recipients for allospecific responses to their donor. We hypothesize 
that tolerant recipients will have a loss of both direct and indirect alloresponses following 
arTreg infusion. Donor reactive cells may be deleted, become exhausted, and/or become 
senescent.  
To this end, we will collect PBMCs from both donor and recipients. Splenic or lymph node 
cells may be substituted in the case of deceased donors. To detect direct responses, recipient 
antigen presenting cells may be cultured in the presence of donor PBMCs. To detect indirect 
responses, recipient dendritic cells may be “fed” donor antigen before culture with recipient 
PBMCs. Recipient anti-donor reactivity may be assessed by flow cytometry (cellular activation, 
proliferation, and/or cytokine production), cytokine secretion, and/or other methods. Suitable 
controls would be included.  
We may also identify donor-reactive T cell clones in recipients pre-transplant, and investigate 
their persistence/presence post-transplant. This experiment may include a direct recognition 
mixed lymphocyte reaction to identify recipient cells that proliferate in the presence of donor 
antigen. These cells may be sorted into CD8+, CD4+ Treg and CD4+ Tconv populations. 
Doing so would allow us to determine if the donor-reactive TCRs present were in the CD8+, 
CD4+ or Treg lymphocyte subsets. The TCR sequences of these cells would then be 
determined by deep sequencing. At specified times following transplant, recipient cells would 
again be sorted into subsets and their TCR sequences assessed by deep sequencing. By 
comparing pre-transplant donor-reactive TCRs to TCRs present post-transplant, we may be able 
to assess their persistence. This method has been previously published by Morris, et al.66 
Detection of exhausted, senescent, and/or anergic cells may include addition of antibodies 
and/or cytokines to cell cultures before assaying for anti-donor responses.  
7.3.2.2.3 
Immunophenotyping 
We have hypothesized that development of tolerance in liver transplant recipients is a dynamic 
process that can in part be monitored by changes in cell phenotype and populations as assessed 
by immunophenotyping.  
To this end, we may perform flow cytometry/immunophenotyping on both fresh and/or frozen 
PBMC to determine PBMC phenotype and proliferative status. Potential cell populations 
include, but are not limited to, T and B cells, and their subsets, co-stained with phenotypic 


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markers suggestive of anergy, senescence, exhaustion, activation, regulation, and recent thymic 
emigration. Such markers may include CD3, CD4, CD8, CD19, CD45RA, CD45RO, CCR7, 
CD57, PD-1, KLRG-1, CD28, CD80/86, HLA-DR, Foxp3, CD25, and CD31.  
 
Graft-Related Assays 
 
Histological Assessment 
Changes that occur within the graft, both in terms of structure and cellular content, may help 
detail interactions between the recipient’s immune system and the liver allograft, and be 
informative as to whether these interactions are beneficial or detrimental to the graft. To this 
end, we may use both paraffin-embedded and/or OCT-mounted frozen liver biopsy tissue to 
capture a snapshot of the state of the allograft. Stains such as hematoxylin and eosin (H&E) and 
Trichrome staining may be used to capture structural and gross changes within the graft. To 
further describe and/or detect the presence of a recipient anti-donor reaction, staining for bound 
Class II antibodies and/or C4d may be performed. To characterize the presence/identity of any 
cellular infiltrate within the graft, tissue may be examined for the presence/location of Tregs 
(Foxp3), Th1/2/17 cells (Tbet/GATA-3/IL-17), T cells (CD3/4/8), NK cells (CD56), activated 
cells (HLA-DR), and/or B cells (CD19/20). Other markers of interest may identify endothelial 
cells (CD31) and smooth muscle actin. Markers in addition to the examples listed above may be 
included, and their presence may be detected by fluorescence, enzyme reaction, mass 
spectrometry, or another method as appropriate.  
 
Gene Expression  
Gene expression of PBMCs and within the liver allograft itself may be informative as to the 
status of the recipient’s immune response to the allograft. If the recipient is experiencing, or 
about to experience, a rejection event, a signature of inflammatory mediators and/or of non-
tolerance may be present. In contrast, if the recipient is tolerant, or to become tolerant, of the 
allograft a signature of tolerance may be present.  
A gene signature of operational liver tolerance has been described in the blood and within the 
graft.1,67 Potential genes associated with tolerance include genes indicative of an increase in γδ 
T cells, and genes involved with iron homeostasis. We hypothesize that potential genes 
associated with rejection, as described by Bonaccorsi-Riani et al.68 will not be upregulated in 
tissue from tolerant participants. 
The methylation status of different genes may also indicate whether these genes are turned “on” 
or “off”, and how permanent the on/off may be. We may examine gene expression, miRNAs, 
and/or gene methylation in peripheral blood (whole blood, PBMC, and/or sorted PBMC cell 
subsets), and/or liver allograft biopsy tissue. DNA, total RNA, and/or miRNA would be 
extracted from these samples. cDNA would be reverse transcribed from RNA. Methods for 
testing may include microarray and/or RT-PCR technology to identify and/or confirm the 
presence/absence of genes of interest.  
 
Treg Product Detection within the Graft 
We hypothesize that we will be able to detect the infused arTreg cells within the graft using 
deuterium labelling of the cell product. While this labeling will detect the persistence of 
transferred cells, it may not necessarily detect the proliferation of these cells. Interrogation of 
biopsy specimens for these cells may occur at 5-15 days post arTreg infusion, eligibility for 
EVR/prednisone withdrawal, and 52 weeks after the last IS dose (Appendices 2-5). 


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Additionally, TCR sequences of arTreg cells may be determined, and biopsy samples 
interrogated for the presence of these clones. 
 
Treg Cell Product Characterization 
 
Detection of Transferred Cells Post-Transfer 
We hypothesize that we will be able to detect arTreg post transfer, and that the total number of 
Tregs detected in the blood will increase relative to pre-transfer numbers. To facilitate specific 
detection of the arTreg, deuterium labelling may be used. While this labeling will detect the 
persistence of transferred cells, it may not detect the proliferation of these cells. Historically, 
the circulating arTreg cell concentration increases immediately post-transfusion, peaks in the 
first two weeks, and gradually declines, remaining detectible for even 1 year after infusion. 
Interrogation of blood samples will occur prior to arTreg infusion, 1 day, and 1, 2, 4, and 12 
weeks after arTreg infusion.  
Additionally, TCR sequences of arTregs may be determined, and blood and/or biopsy samples 
interrogated for the presence of these sequences. 
 
Comparison of Treg products 
We propose to characterize and compare the different allo-reactive Tregs tested within the 
consortium. For example, RNA-seq and/or flow cytometry may be used to identify and describe 
their phenotype and potential regulatory mechanisms. We also may characterize the stability of 
the regulatory phenotype by epigenetic analysis of whole genome methylation.  
7.4 
QUALITY CHECK/PRELIMINARY STUDIES 
Samples acquired may be used for either quality checks (if deemed necessary) or preliminary 
studies that could potentially be applied to the entire cohort of participants. Examples of when a 
quality check may be requested include issues that may arise from shipping/storage, to ensure 
sites are collecting samples correctly, or to compare data generated longitudinally within a core 
lab. Preliminary studies may include a pilot experiment when changing to a new core or 
piloting an assay/technique that may be of interest to this, and/or other, studies.  
7.5 
FUTURE / UNPLANNED STUDIES 
Specimens stored during the trial may be used in future assays to reevaluate biological 
responses as research tests are developed over time. Additionally, samples may be used for 
assays/ experiments outside the scope of this proposal, such investigation of miRNA 
expression, differences in the TCR repertoire as evaluated by sequencing, proteomics or other 
explorations that may emerge and be compelling during and/or after the trial period. Re-
evaluations or new assays will only be performed on samples of participants who have 
consented for future research. Blood, tissue, and other biologic samples will be collected and 
saved to allow for the possible use in later gene association studies. Specific consent will be 
obtained for the storage and use of participant DNA. The ITN sample sharing policy will apply 
for the provision of samples to study or outside investigators. Investigators may request use of 
specimens for tolerance related and unrelated studies. Only specimens from participants that 
have consented to future and/or future genetic studies, as appropriate, will be shared. 


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8. 
ADVERSE EVENTS 
8.1 
OVERVIEW 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an AE or SAE as described in section 8.2. All AEs and SAEs will be 
recorded in the source documents and on the appropriate eCRF(s). All data will be reviewed 
periodically by the NIAID Transplant DSMB, which may provide recommendations to DAIT 
NIAID about withdrawing any participant and/or terminating the study because of safety 
concerns. 
AEs that are classified as serious according to the definition of health authorities must be 
reported promptly and appropriately to the NIAID, ITN, principal investigators in the trial, 
institutional review boards (IRBs), and health authorities. This section defines the types of AEs 
and outlines the procedures for appropriately collecting, grading, recording, and reporting them. 
Information in this section complies with 21CFR 312; ICH Guideline E2A: Clinical Safety 
Data Management: Definitions and Standards for Expedited Reporting; and ICH Guideline E-
6: Guidelines for Good Clinical Practice; and applies the standards set forth in the National 
Cancer Institute (NCI)s Common Terminology Criteria for Adverse Events Version 5.0 
(published November 27, 2017). This document is referred to herein as the “NCI-CTCAE 
manual.” 
8.2 
DEFINITIONS 
 
Adverse Event 
An AE is 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 (ICH E-6 Guidelines for GCP). 
 
Suspected Adverse Reaction and Adverse Reaction 
Suspected adverse reaction (SAR) means any AE for which there is a reasonable possibility that 
the study drug caused the AE. For the purposes of safety reporting, ‘reasonable possibility’ 
means there is evidence to suggest a causal relationship between the drug and the adverse 
reaction. 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)). 
 
Unexpected AE 
An AE or suspected adverse reaction is considered “unexpected” if it is not listed in the 
Investigator Brochure or 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 
Investigator Brochure or 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). 


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Serious Adverse Event 
An AE or SAR is considered “serious” if, in the view of either the investigator or DAIT/NIAID 
it results in any of the following outcomes (21 CFR 312.32(a)): 
1. Death 
2. A life-threatening event: An AE or SAR is considered “life-threatening” if, in the view of 
either the investigator or DAIT/NIAID, its occurrence places the subject at immediate risk 
of death. It does not include an AE or SAR that, had it occurred in a more severe form, 
might have caused death. 
3. Inpatient hospitalization or prolongation of existing hospitalization. 
4. Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions. 
5. Congenital anomaly or birth defect. 
6. Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the subject and may require medical or surgical intervention to prevent 
one of the outcomes listed above. This would also include confirmed cases of COVID-19. 
Elective hospitalizations or hospital admissions for the purpose of conduct of protocol-
mandated procedures are not to be reported as an SAE unless hospitalization is prolonged due 
to complications. 
8.3 
COLLECTING AND RECORDING ADVERSE EVENTS 
 
Collection Period 
Recipient 
AEs will be collected from the time informed consent is obtained during Study Stage 2: EVR 
Conversion (section 5.3.2) until the participant completes study participation or until 30 days 
after he/she prematurely withdraws (without withdrawing consent) or is withdrawn from the 
study. 
Living Donor 
AEs grade 3 and higher that occur within 48 hours of phlebotomy for manufacturing will be 
collected for the study unless the blood for manufacturing is collected within 24 hours of the 
donor hepatectomy.  
 
Collecting Adverse Events 
AEs (including SAEs) may be discovered through any of these methods: 
• 
Observing the participant. 
• 
Questioning the participant in an objective manner. 
• 
Receiving an unsolicited complaint from the participant. 


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• 
In addition, an abnormal value or result from a clinical or laboratory evaluation can also 
indicate an AE, as defined in section 8.3.4.1. 
 
Recording AEs 
Throughout the study, the investigator will record AEs and SAEs as described previously 
(section 8.2) on the appropriate eCRF regardless of the relationship to study therapy or study 
procedure. 
 
Severity of Adverse Events to be Recorded 
 
 Adverse Events 
All AEs, regardless of seriousness or severity, will be collected during the collection period 
specified in section 8.3.1.  
AE grades will be defined per CTCAE criteria. Where the CTCAE relies on site normal ranges 
to assess grades, the normal ranges listed in Harrison’s Principles of Internal Medicine, 18th 
edition (McGraw-Hill, 2011) will be used. 
Once recorded, an AE/SAE will be followed until it resolves with or without sequelae, or 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. 
 
Methods of Recording 
 
Recording AEs 
Throughout the study, the investigator will record all AEs per section 8.3.4.1 on the appropriate 
eCRF. The investigator will treat participants experiencing AEs appropriately and observe them 
at suitable intervals until their symptoms resolve or their status stabilizes. 
 
Recording SAEs 
SAEs will be recorded on the SAE eCRF and health authorities will be notified as outlined in 
section 8.5. 
8.4 
GRADING AND ATTRIBUTION OF ADVERSE EVENTS 
 
Grading 
The study site will grade the severity of AEs experienced by study participants according to the 
criteria set forth in the NCI-CTCAE National Cancer Institute‘s Common Terminology Criteria 
for Adverse Events 5.0 (published November 27, 2017). This manual provides a common 
language to describe level of severity, analyze and interpret data, and articulate the clinical 
significance of all AEs. 
AEs will be graded on a scale from 1 to 5 according to the following standards in the NCI-
CTCAE manual: 
• 
Grade 1: Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
• 
Grade 2: Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental ADL*.  


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• 
Grade 3: Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self care 
ADL**. 
• 
Grade 4: Life-threatening consequences; urgent intervention indicated.  
• 
Grade 5: Death related to AE. 
*Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, 
managing money, etc. 
**Self care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking 
medications, and not bedridden 
For additional information and a printable version of the NCI-CTCAE manual, go to 
http://ctep.cancer.gov/reporting/ctc.html. 
For grading an abnormal value or result of a clinical or laboratory evaluation (including, but not 
limited to, a radiograph, an ultrasound, an electrocardiogram etc.), a treatment-emergent AE is 
defined as an increase in grade from baseline or from the last post-baseline value that doesn’t 
meet grading criteria. If a specific event or result from a given clinical or laboratory evaluation 
is not included in the NCI-CTCAE manual, then an abnormal result would be considered an AE 
if changes in therapy or monitoring are implemented as a result of the event/result. 
It is acceptable for a protocol to use additional and/or alternative grading scale(s) for all AEs or 
only for drug or procedure-specific AEs; such scales need to be clearly described herein. 
 
AEs Related to the IP/Study Medications 
Recipient 
Attribution assessment for the following study drugs will be made when an AE is reported:  
• 
CTX and Mesna 
• 
Treg supportive IS regimen (TAC, Mycophenolate product, EVR, and Prednisone)  
• 
IS withdrawal (TAC, EVR, and Prednisone) 
 
AEs Related to Study Procedures 
Recipient 
Attribution assessment for the following study procedures will be made when an AE is 
reported: 
• 
Leukapheresis 
• 
arTreg Infusion 
• 
Liver biopsies 
• 
Research blood draws  
Living Donor 
Attribution assessment for the following study procedures will be made when an AE is 
reported: 


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• 
Phlebotomy for manufacturing 
 
Attribution 
AEs will be categorized for their relation to the study therapy or procedure. The principal 
investigator will make the initial determination of the relation, or attribution, of an AE to the 
study and will record the initial determination on the appropriate eCRF and/or SAE reporting 
form. The relation of an AE to the study will be determined using definitions in Table 13 
below. Final determination of attribution for safety reporting will be decided by DAIT/NIAID. 
Table 13. 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 adverse event is clearly not related: there is insufficient evidence to suggest 
a causal relationship. 
Related Categories 
2 
Possible 
The adverse event has a reasonable possibility to be related; there is evidence to 
suggest a causal relationship. 
3 
Related 
The adverse event is clearly related. 
8.5 
REPORTING SERIOUS ADVERSE EVENTS 
 
Reporting SAEs to the IND Sponsor 
This section describes the responsibilities of the site investigator to report SAEs to the sponsor 
via the SDCC eCRF. Timely reporting of AEs is required by 21CFR 312 and ICH guidelines. 
Site investigators will report all SAEs (section 8.2), regardless of relationship or expectedness 
to study procedures within 24 hours of discovering the event. All confirmed cases of COVID-
19 will also be reported as SAEs, regardless of severity. 
For SAEs, all requested information on the AE/SAE eCRF will be provided. However, 
unavailable details of the event will not delay submission of the known information. As 
additional details become available, the AE/SAE eCRF will be updated and submitted. 
As additional details become available, the SAE eCRF should be updated and submitted. Every 
time the SAE eCRF is submitted, it should be electronically signed by the investigator or sub 
investigator. 
For additional information regarding SAE reporting, contact Rho Product Safety: 
Rho Product Safety 
2635 East NC Highway 54  
Durham, NC 27713   Toll-free - (888) 746-7231 
SAE Fax Line: 1-888-746-3293 
Email: rho_productsafety@rhoworld.com 


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Reporting AEs of Study Specific Relevance (Non-Serious AE) to the 
IND Sponsor 
Allograft dysfunction, clinical rejection, and biopsy proven rejection NOT meeting serious 
criteria will be reported as AEs of Study Specific Relevance and will be recorded on the 
AE/SAE eCRF within 5 business days of discovering the event. 
 
Reporting SAEs to Health Authorities 
After an AE requiring 24 hour reporting (per section 8.2) is submitted by the site investigator 
and assessed by DAIT/NIAID, there are two options for DAIT/NIAID to report the AE to the 
appropriate health authorities: 
 
Annual Reporting 
DAIT/NIAID will include in the annual study report to the health authorities all AEs classified 
as:  
• 
Serious, expected, suspected adverse reactions described in section 8.2. 
• 
Serious and not a suspected adverse reaction described in section 8.2. 
• 
Pregnancies 
Note that all AEs (not just those requiring 24-hour reporting) will be reported to the health 
authorities in the Annual IND report. 
 
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 8.2.2 
and section 8.2.3 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 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 


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consent, investigator brochure or package insert or other aspects of the overall conduct of the 
study. 
The sponsor shall notify the appropriate health authorities and all participating 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. 
All principal investigators must report SAEs to their respective IRBs as mandated by them. 
 
Reporting SAEs to the DSMB 
The DAIT NIAID and ITN will provide the NIAID Transplant DSMB with data of all SAEs on 
an ongoing basis, including quarterly reports of all SAEs. 
 
Reporting of AEs to IRBs/IECs 
All investigators shall report AEs, including expedited reports, in a timely fashion to their 
respective IRBs/IECs in accordance with applicable regulations and guidelines. All Safety 
Reports to the FDA shall be distributed by DAIT/NIAID or designee to all participating 
institutions for site IRB/IEC submission. 
8.6 
REPORTING PREGNANCY 
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 stop taking study medication. 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 Statistical and Data Coordinating Center (SDCC) all 
pregnancies within 1 business day of becoming aware of the event using the Pregnancy eCRF. 
All pregnancies identified during the study shall be followed to conclusion and the outcome of 
each must be reported. The Pregnancy eCRF shall be updated and submitted to the SDCC 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: 
• 
Gestational age at delivery 
• 
Birth weight, length, and head circumference 
• 
Gender 
• 
Appearance, pulse, grimace, activity, and respiration (APGAR) score at 1 minute, 5 
minutes, and 24 hours after birth, if available 
• 
Any abnormalities. 
All pregnancy complications that result in a congenital abnormality, birth defect, miscarriage, 
and medically indicated abortion - an SAE shall be submitted to SDCC using the SAE reporting 
procedures described above. 


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8.7 
REPORTING OF OTHER SAFETY INFORMATION 
An investigator shall promptly notify the site IRB as well as the SDCC when an “unanticipated 
problem involving risks to subjects or others” is identified, which is not otherwise reportable as 
an AE. 
8.8 
REVIEW OF SAFETY INFORMATION 
 
Medical Monitor Review 
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, the Medical Monitor shall review and make decisions on the disposition of the SAE 
and pregnancy reports received by the SDCC. 
 
DSMB Review 
 
 Planned DSMB Reviews 
The DSMB shall review safety data at least yearly during planned Data Review Meetings. Data 
for the planned safety reviews will include, at a minimum, a listing of all reported AEs and 
SAEs. 
The DSMB will be informed of an Expedited Safety Report in a timely manner. 
 
Ad hoc DSMB Reviews 
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 protocol chair or DAIT/NIAID. In addition, an ad hoc 
comprehensive DSMB Safety Review will convene if any of the stopping guidelines listed in 
section 3.4.2 are met. After review of the data, the DSMB will make recommendations 
regarding study conduct and/or continuation. 
9. 
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN 
9.1 
ANALYSIS SAMPLES 
The analysis samples are described beginning with the most inclusive. 
Enrolled sample (ES): All participants who sign consent. 
Safety sample (SS): All participants who initiate EVR conversion.  
Intent to treat (ITT) sample: All participants who receive at least the minimum arTreg dose.  
Per protocol (PP) sample: All participants who receive at least the target arTreg dose. 
The minimum and target doses are described in section 5.2.1.4. 


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9.2 
ANALYSIS OF ENDPOINTS 
 
Overview 
The principal features of the plan for statistical data analysis are outlined in this protocol and 
will be described in greater detail in the statistical analysis plan (SAP). Analysis of study data 
will be conducted to address all objectives of the trial and other interrelationships among all 
data elements of interest to the Investigators and of relevance to the objectives of the study.  
 
Primary Endpoints 
 
Primary Safety Endpoints 
The primary safety endpoints, defined in section 3.3.1.1, will be descriptively summarized with 
counts and percentages. Frequency tables by the investigational product and investigational 
regimen and by CTCAE grade will be presented. 
AEs will be considered attributed to arTregs if the AE is reported with possible or related 
attribution to arTreg. AEs will be considered attributed to the investigational regimen if the AE 
is reported with possible or related attribution to a) leukapheresis, b) CTX, or c) mesna. The 
severity will be determined by the CTCAE grade.  
The primary safety endpoints will be analyzed using the safety, ITT and PP samples.  
 
Primary Efficacy Endpoint 
The primary efficacy endpoint, defined in section 3.3.1.2, will be descriptively summarized 
with a point estimate and two-sided, 95% exact binomial confidence interval. The numerator 
will be the number of participants that meet the primary efficacy endpoint.  
The primary efficacy endpoint will be analyzed using the ITT and PP samples.  
 
Secondary Endpoints 
The secondary endpoints are defined in 3.3.2. Table 14 describes the secondary endpoints and 
their corresponding analysis parameters to be estimated in the study. The table also lists the 
analysis samples in the order of importance. Secondary endpoints will be listed or summarized, 
as appropriate, using standard descriptive statistics for continuous and categorical data. 
Table 14. Analysis of Secondary Endpoints 
Endpoint 
Analysis Parameter 
Analysis 
Sample 
Incidence of infections grade 3 or 
greater following arTreg infusion 
Proportion of participants with an infectious AEs of grade 
≥3 (defined in 8.4.1) after arTreg infusion with a two-
sided, 95% exact binomial confidence interval 
ITT, PP, 
SS 
Number and severity of biopsy-
proven AR or clinical rejection 
events any time after arTreg 
infusion 
Number and the categorization of severity (per Banff) of 
biopsy-proven AR or clinical rejection events after 
arTreg infusion 
ITT, PP, 
SS 
Number of CR events any time after 
arTreg infusion 
Number of CR events after arTreg infusion 
ITT, PP, 
SS 
Number of participants who 
develop any malignancy 
Number of participants with any malignancy 
ITT, PP, 
SS 


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Endpoint 
Analysis Parameter 
Analysis 
Sample 
Proportion of participants who 
discontinue TAC ≥12 weeks with 
ALT ≤ 50 U/L and a liver biopsy 12 
- 26 weeks after the last TAC dose 
that meets the biopsy criteria for IS 
minimization (Table 9) as assessed 
by central pathology 
Proportion of participants who successfully discontinue 
TAC for ≥12 weeks (regardless of other IS), have ALT 
≤50 U/L, and liver biopsy 12 - 26 weeks after the last 
TAC dose that meets the biopsy criteria for IS 
minimization (Table 9), with a two-sided, 95% exact 
binomial confidence interval 
ITT, PP 
Time from achievement of the 
primary endpoint to IS reinitiation 
or to the end of trial participation 
The number of days from the week 52 primary endpoint 
biopsy to the earliest of either IS reinitiation or end of 
trial participation (only calculated for those deemed 
operationally tolerant for the primary endpoint)  
ITT, PP 
 
 
Safety Analysis 
All analyses will be repeated using the SS as labeled in Table 14. All AEs will be classified by 
body system and preferred term according to the MedDRA dictionary. Frequency tables by 
category of event (e.g. serious, related) and by NCI-CTCAE v.5.0 grade will be presented. 
Selected laboratory values will be summarized and displayed graphically.  
 
Exploratory Analysis 
Participants who are in the ES but are not in the SS will be utilized to explore relationships 
between their baseline characteristics and events of interest (section 6.3.1) that differentiate 
them from participants who go on to the SS.  
 
Medical History 
Medical history within the past 12 months—including the existence of current signs and 
symptoms—will be collected for each body system. 
 
Use of Medications 
All medications defined in section 55 taken by or administered to liver transplant recipients 
starting from transplant and continuing throughout their study participation will be collected. 
The number and percentage of participants receiving prior and concomitant 
medications/therapies will be presented overall and by medication class. 
9.3 
SAMPLE SIZE 
This pilot study is designed to evaluate 9 participants. Therefore, no formal power and sample 
size analyses have been performed. However, with 9 evaluable participants receiving at least 
the target arTreg dose (section 5.2.1.4), incidence rates of operational tolerance and 
corresponding two-sided, 95% exact binomial confidence interval are shown in Table 15.  
Table 15. Incidence Rates and Confidence Intervals for 9 Participants Attempting 
Complete IS Withdrawal following an Infusion of Treg Product (arTreg) 
Number of Participants able to 
Achieve Operational Tolerance 
Incidence Rate (%) 
95% Confidence Interval (%) 
0 
0 
(0, 33.63) 
1 
11.1 
(0.28, 48.25) 
2 
22.2 
(2.81, 60.01) 


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Number of Participants able to 
Achieve Operational Tolerance 
Incidence Rate (%) 
95% Confidence Interval (%) 
3 
33.3 
(7.49, 70.07) 
4 
44.4 
(13.70, 78.80) 
5 
55.6 
(21.20, 86.30) 
6 
66.7 
(29.93, 92.51) 
7 
77.8 
(39.99, 97.19) 
8 
88.9 
(51.75, 99.72) 
9 
100 
(66.37, 100) 
9.4 
 MISSING DATA 
Steps will be taken to avoid missing data. For all secondary analyses, no imputations for 
missing data will be performed. 
9.5 
POOLED ANALYSIS WITH OTHER LITTMUS TRIALS 
LITTMUS protocols were developed collectively for the explicit purpose of harmonizing study 
design, data collection, and analysis endpoints.  
The analyses will be repeated as specified above with all available data from other LITTMUS 
trials.  
9.6 
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL 
PLAN 
The principal features of both the study design and the plan for statistical data analysis are 
outlined in this protocol and in the SAP. Any change in these features requires either a protocol 
or an SAP amendment, which is subject to review by the DSMB, the study sponsor(s), and the 
health authorities. These changes will be described in the final study report as appropriate. 
10. 
ACCESS TO SOURCE DATA/DOCUMENTS 
The investigational sites participating in this study will maintain the highest degree of 
confidentiality permitted for the clinical and research information obtained from participants in 
this clinical trial. Medical and research records should be maintained at each site in the strictest 
confidence. However, as a part of the quality assurance and legal responsibilities of an 
investigation, the investigational sites must permit authorized representatives of the ITN, 
sponsor, and health authorities to examine (and to copy when required by applicable law) 
clinical records for the purposes of quality assurance reviews, audits, and evaluation of the 
study safety and progress. Unless required by the laws permitting copying of records, only the 
coded identity associated with documents or other participant data may be copied (and any 
personally identifying information must be obscured). Authorized representatives as noted 
above are bound to maintain the strict confidentiality of medical and research information that 
may be linked to identified individuals. The investigational sites will normally be notified in 
advance of auditing visits. 


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11. 
QUALITY CONTROL AND QUALITY ASSURANCE 
The principal investigator is required to keep accurate records to ensure that the conduct of the 
study is fully documented. The principal investigator is required to ensure that all eCRFs are 
completed for every participant entered in the trial. 
The sponsor is responsible for regular inspection of the conduct of the trial, for verifying 
adherence to the protocol, and for confirming the completeness, consistency, and accuracy of 
all documented data. 
The eCRFs will be completed online via a web-based electronic data capture (EDC) system that 
has been validated and is compliant with Part 11 Title 21 of the Code of Federal Regulations. 
Some data requirements will be addressed outside the EDC using SAS® software. Data queries 
will be issued and resolved within the EDC system or SAS®. 
Study staff at the site will enter information into the eCRFs, and the data will be stored 
remotely at a central database. Data quality will be ensured through the EDC system’s 
continuous monitoring of data and real-time detection and correction of errors. All elements of 
data entry (i.e., time, date, verbatim text, and the name of the person performing the data entry) 
will be recorded in an electronic audit trail to allow all changes in the database to be monitored 
and maintained in accordance with federal regulations. 
Study staff will enter data from a study visit on the relevant eCRFs within 3 days following the 
visit or the time when data become available. 
12. 
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE 
12.1 
STATEMENT OF COMPLIANCE 
This trial will be conducted in compliance with the protocol, current Good Clinical Practice 
(GCP) guidelines—adopting the principles of the Declaration of Helsinki—and all applicable 
regulatory requirements. 
Prior to study initiation, the protocol and the informed consent documents will be reviewed and 
approved by the sponsor and an appropriate ethics review committee or IRB. Any amendments 
to the protocol or consent materials must also be approved by the Sponsor, the IRB and 
submitted to FDA before they are implemented. 
12.2 
INFORMED CONSENT 
The informed consent form is a means of providing information about the trial to a prospective 
participant and allows for an informed decision about participation in the study. All participants 
(or their legally acceptable representative) must read, sign, and date a consent form before 
participating in the study, taking the study drug, and/or undergoing any study-specific 
procedures. If a participant does not speak and read English, the consent materials must be 
translated into the appropriate language. 
The informed consent form must be updated or revised whenever important new safety 
information is available, whenever the protocol is amended, and/or whenever any new 
information becomes available that may affect participation in the trial. 


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A copy of the informed consent will be given to a prospective participant for review. The study 
investigator, in the presence of a witness, will review the consent and answer questions. The 
participant will be informed that participation is voluntary and that he/she may withdraw from 
the study at any time, for any reason. 
12.3 
PRIVACY AND CONFIDENTIALITY 
A participant’s privacy and confidentiality will be respected throughout the study. Each 
participant will be assigned a sequential identification number. This number, rather than the 
participant’s name, will be used to collect, store, and report participant information. 
13. 
PUBLICATION POLICY 
The ITN policy on publication of study results will apply to this study. Authorized participants 
may find details regarding the policy statement on the ITN internet website at 
http://www.immunetolerance.org. 
 


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63. 
Wu KY, Budde K, Schmidt D, Neumayer HH, Rudolph B. Acute cellular rejection with isolated 
v-lesions is not associated with more favorable outcomes than vascular rejection with more 
tubulointerstitial inflammations. Clin Transplant 2014;28:410-8. 
64. 
Ramos RL, Teixeira LA, Ormonde LR, et al. Emergence of mupirocin resistance in 
multiresistant Staphylococcus aureus clinical isolates belonging to Brazilian epidemic clone 
III::B:A. J Med Microbiol 1999;48:303-7. 


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LITTMUS: Liver Transplantation with Tregs at UCSF  
65. 
Glomerular Filtration Rate (GFR) Calculators. 2018, at https://www.niddk.nih.gov/health-
information/communication-programs/nkdep/laboratory-evaluation/glomerular-filtration-rate-
calculators.) 
66. 
Morris H, DeWolf S, Robins H, et al. Tracking donor-reactive T cells: Evidence for clonal 
deletion in tolerant kidney transplant patients. Sci Transl Med 2015;7:272ra10. 
67. 
Martinez-Llordella M, Puig-Pey I, Orlando G, et al. Multiparameter immune profiling of 
operational tolerance in liver transplantation. Am J Transplant 2007;7:309-19. 
68. 
Bonaccorsi-Riani E, Pennycuick A, Londono MC, et al. Molecular Characterization of Acute 
Cellular Rejection Occurring During Intentional Immunosuppression Withdrawal in Liver 
Transplantation. Am J Transplant 2016;16:484-96. 


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Appendix 1A. Study Stage 1, 2, and 3A: Schedule of Events – Recipient Enrollment and 
EVR Conversion 
 
- 
Study Stage 1 
Study Stage 2 
Study Stage 3A 
 
Eligibility 1:  
Pre-Transplant 
Transplant 
Eligibility 2:  
EVR 
Conversion 
EVR 
Conversion 
Initiation 
Eligibility 3A: Recipient 
Leukapheresis for 
arTreg Manufacturing 
Window 
-26 Wks 
- 
D30 - Wk48 
Within 7 days 
of V101[R] 
D60 - Wk52 
Visit Number 
-11 
02 
101/101R2,3,4 
101S 
102/102R5.6 
General Assessments7  
Informed consent 
X8 
 
X 
 
 
Eligibility 
X 
 
X 
 
X 
Study parameters for continuation 
 
X 
 
 
Demographics 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Complete physical exam 
 
 
X9 
 
 
Limited physical exam 
 
 
 
 
X 
Vital signs 
 
 
X 
 
X 
Adverse Events 
 
 
X10 
 
X 
Concomitant medications 
 
X 
X 
 
X 
Events of interest 
 
X11 
 
 
HCV treatment history, if applicable 
 
 
X 
 
 
Milan criteria, with no vascular invasion, 
and with no cholangiocarcinoma 
morphology, if applicable 
 
X 
 
 
 
Clinical Assessments and Procedures 
Doppler or angiography 
 
 
X 
 
 
Liver transplant 
 
X12 
 
 
 
 
1 The local laboratory and mechanistic assessments do not need to be repeated if Visit -1 becomes out of window of Visit 0, as 
participants will undergo additional eligibility steps prior to initiating any study therapy (Study Definitions).  
2 Participants who fail to initiate study therapy (section 4.5) will be terminated from further study participation. 
3 Participants experiencing an AR episode may be re-evaluated for Eligibility 2: EVR Conversion at least 4 weeks following resolution 
of AR (section 5.3.2.3) (Visit 101R). If Visit 101R occurs within 8 weeks of Visit 101, do not collect repeat mechanistic 
assessments. 
4 Laboratory assessments needed to verify eligibility for EVR conversion must occur within 30 days after Visit 101/101R. EVR 
conversion must be initiated within 7 days after these laboratory assessments. 
5 Participants ineligible due to any of the Eligibility 3A: Recipient Leukapheresis for arTreg Product Manufacturing criteria may be 
re-evaluated up to 52 weeks post liver transplant (Visit 102R). If Visit 102R occurs within 8 weeks of Visit 102, do not collect 
repeat mechanistic assessments. 
6 For participants who either failed EVR conversion or successfully completed EVR conversion but did not meet Eligibility 3A: 
Recipient Leukapheresis for arTreg Product Manufacturing, complete the Reduced Follow-up Visit (RFU) in Appendix 2. 
7 Reference section 6.3.1 for specific assessment details. 
8 Consent will be obtained prior to notification of organ offer by transplant center to the participant and separately from obtaining 
consent for transplant. 
9 Includes height. 
10 Adverse events will be collected once informed consent is obtained during Study Stage 2: EVR conversion. 
11 Collected from time of transplant until Eligibility 2: EVR Conversion (Visit 101). 
12 See section 5.2.4.1 for prophylactic medications per the center’s standard of care post-transplant. 


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- 
Study Stage 1 
Study Stage 2 
Study Stage 3A 
 
Eligibility 1:  
Pre-Transplant 
Transplant 
Eligibility 2:  
EVR 
Conversion 
EVR 
Conversion 
Initiation 
Eligibility 3A: Recipient 
Leukapheresis for 
arTreg Manufacturing 
Window 
-26 Wks 
- 
D30 - Wk48 
Within 7 days 
of V101[R] 
D60 - Wk52 
Visit Number 
-11 
02 
101/101R2,3,4 
101S 
102/102R5.6 
Liver biopsy 
 
X13 
 
 
 
Local Laboratory Assessments14  
HLA typing15 
X 
 
 
 
 
Urine or blood hCG 
 
 
 
 
X  
Hematology 
X16 
X 
 
X  
Basic chemistry 
X16 
X 
 
X  
Liver function tests 
 
 
 
X 
 
CD4 count 
 
 
 
 
X 
Urinalysis 
 
 
X 
 
X 
Estimated GFR 
 
 
X 
 
X  
HIV-1/2 
X 
 
 
 
 
2019-nCoV RT-PCR 
 
 
X 
 
X 
HCV RNA 
 
X 
 
 
X  
HBV DNA 
 
 
 
 
X  
EBV Antibody 
X17 
 
 
 
 
EBV DNA 
 
 
 
 
X 
CMV Antibody 
X17 
 
 
 
 
CMV DNA 
 
 
 
 
X 
AFP18 
 
X 
 
 
 
TAC trough levels 
 
 
X 
 
X  
EVR trough levels 
 
 
 
 
X  
 
13 Liver allograft biopsy should be obtained on the pre-implant liver prior to transplantation into the recipient. Local pathology 
assessment must be obtained. 
14 Reference section 6.5.1 for specific assessment details. 
15 Retrospective data collection from the medical record. Repeat testing not required if already completed as standard of care. 
16 Must be performed within 24 hours of the mechanistic blood collections (PBMC, serum, and whole blood collections) and can occur 
any time after consent until transplant. If the PBMC collection occurs on two separate dates, complete this assessment on each 
collection date. 
17 The assessment does not need to be repeated if the participant has a documented positive result. 
18 Last assessment within 3 months prior to transplant for participants with HCC only.  


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- 
Study Stage 1 
Study Stage 2 
Study Stage 3A 
 
Eligibility 1:  
Pre-Transplant 
Transplant 
Eligibility 2:  
EVR 
Conversion 
EVR 
Conversion 
Initiation 
Eligibility 3A: Recipient 
Leukapheresis for 
arTreg Manufacturing 
Window 
-26 Wks 
- 
D30 - Wk48 
Within 7 days 
of V101[R] 
D60 - Wk52 
Visit Number 
-11 
02 
101/101R2,3,4 
101S 
102/102R5.6 
Study Medications 
Mycophenolate Compound (MMF or 
equivalent)19 
 
SOC 
X20 
 
Prednisone21 
 
SOC 
X (≤10 mg 
daily) 
X 
Tacrolimus 
 
SOC 
X (trough 6-
10 ng/mL) 
X (target trough 3-5 
ng/mL) 
Everolimus 
 
 
 
X 
X (target trough 5-8 
ng/mL) 
Mechanistic Laboratory Assessments 
HLA Typing22 
X23 
 
 
 
PBMC collection (for UCSF lab) 
 
 
 
 
X 
PBMC collection 
X23,24 
X 
 
 
Serum collection 
X23 
X 
 
 
Whole blood collection 
X23 
X 
 
 
Liver biopsy collection  
 
X 
 
 
 
 
19 If MMF, Myfortic, or azathioprine is not part of the participant’s IS regimen, the participant may continue with EVR conversion 
(Table 8). 
20 Discontinued approximately 7-14 days after starting EVR (Table 8). 
21 Prednisone may be discontinued at any time at the discretion of the investigator (section 5.2.3.3). 
22 Involves a buccal swab collection, however, blood/PBMCs will be used if needed. 
23 Mechanistic assessments can be collected any time after informed consent on Visit -1 but prior to the administration of any IS or 
blood products at the time of the transplant. 
24 Specimen will be collected using two mechanistic specimen collections kits. Both kits can be collected on the same day or on 
different dates. 


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Appendix 1B. Schedule of Events – Donor Enrollment and Participation 
 
Living Donor 
Deceased Donor 
Window 
Eligibility: Living Donor 
Eligibility: Deceased 
Donor 
Window 
-26 Wks 
D0 - Wk26 
±7 days 
Visit Number 
LD-1 
LD0 
DD01 
General Assessments 
 
Informed consent 
X 
 
 
Eligibility 
X 
X2 
X 
Demographics 
X 
 
X 
ABO Typing (from medical record) 
X 
 
X 
Adverse Events 
 
X3 
 
Local Laboratory Assessments4 
 
HLA typing5 
X 
 
X 
CMV Antibody 
X6 
 
X 
EBV Antibody 
X6 
 
X 
HCV testing 
 
 
X 
arTreg Manufacture 
 
Blood collection for infectious diseases donor 
testing 
 
X2 
X 
Blood collection by phlebotomy for 
manufacture of stimulated irradiated donor B 
cells (KT64.CD40L stimulation)  
 
X7 
 
Spleen and/or blood collection for 
manufacture of stimulated irradiated donor B 
cells (KT64.CD40L stimulation)  
 
 
X 
Mechanistic Laboratory Assessments8 
 
HLA Typing9 
 
X10 
X11 
PBMC collection 
 
X10 
 
Blood and/or spleen collection 
 
 
X 
 
1 For all assessments other than peripheral blood and/or spleen collection for arTreg and mechanistic collections, data to be collected 
from medical and/or UNOS records. If tests are not available, they should be ordered.  
2 Living donor must meet donor eligibility manufacturing requirements within 7 days prior to the blood collection for manufacturing. 
3 Adverse events ≥ grade 3 that occur within 48 hours of research blood draw for arTreg manufacturing will be collected for the study 
unless the blood for manufacturing is collected within 24 hours of the donor hepatectomy. 
4 For specific assessment details, reference section 6.5.2 for living donors and section 6.5.3 for deceased donors. 
5 Retrospective data collection from the medical record. Repeat testing not required if already completed as standard of care. 
6 The assessment does not need to be repeated if the living donor has a documented positive result. 
7 Must occur at least 14 days prior to the Recipient’s Leukapheresis Visit (Visit T-17/T-17R) in Appendix 2 (section 5.3.3.1). 
8 For deceased donors, mechanistic collections will occur if material is available. 
9 Involves a buccal swab collection, however, blood/PBMCs will be used if needed. 
10 May be collected prior to transplant if the donor has consented and is eligible, the donor’s recipient is found eligible per Eligibility 
1: Pre-Transplant, and the transplant date has been confirmed. 
11 Can be performed on any appropriate sample type collected for mechanistic purposes.  


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Appendix 2. Study Stage 3 (A, B, C): Schedule of Events – Investigational Treatment 
 
Study  
Stage 3A 
Study  
Stage 3B 
Study Stage 3C 
- 
 
Recipient 
Leukapheresis 
Eligibility 3B:  
pre-arTreg 
Conditioning (CTX) 
Eligibility 3C: 
arTreg 
Infusion 
Eligibility 4: 
TAC 
Withdrawal 
- 
Reduced Safety  
Follow-up 
Post arTreg Infusion Visits1 
arTreg Infusion Day 
-17 
-3 
0 
1 
7 
14 
28 
84 
Window 
≤28 days of 
V102[R] 
≤72 hours &  
≥24 hours before 
arTreg infusion (VT0) 
- 
+24  
hours 
-2/+8 
days 
-2/+3 
days 
±5  
days 
±5  
days 
≤30 days after 
unsuccessful EVR 
conversion,  
VT-17[R], or VT-3 
Visit Number 
T-17/T-17R2,3 
T-33,4 
T0 
T1 
T75 
T145,6 
T285,6 
T845, 6 
RFU 
General Assessments7 
 
Eligibility 
 
X 
X 
X8 
 
 
 
 
Complete physical 
exam 
 
 
X 
 
 
 
 
 
 
Limited physical exam 
 
X 
 
X 
X 
 
 
 
X 
Vital signs 
 
X 
X9 
X 
X 
 
 
 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Remote consultation 
 
 
 
 
 
X 
X 
X 
 
Clinical Assessments and Procedures 
 
Liver biopsy 
 
 
 
 
X10 
 
 
 
 
Local Laboratory Assessments11 
 
Hematology  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Basic chemistry 
X 
X 
X 
X 
X 
 
 
 
X 
CD4 count 
X 
 
X 
X 
X 
X 
X 
X 
 
Estimated GFR 
 
 
 
X 
 
 
 
 
 
 
1 For participants who receive any arTregs but are ineligible for or do not attempt TAC withdrawal, move to Appendix 6 (section 4.4) 
while completing these visits concurrently. 
2 If the leukapheresis product does not contain a sufficient number of cells for Treg manufacturing, a second leukapheresis can be 
performed (Visit T-17R) provided the participant continues to meet Eligibility 3A: Recipient Leukapheresis for arTreg Product 
Manufacturing (section 5.3.3.2). Visit 102R must be completed if more than 28 days have elapsed since the prior Visit 102 
assessments. Visit 102R to confirm eligibility for a second leukapheresis must be established by week 26 post liver transplant. 
3 For participants who do not receive any CTX or mesna (VT-3), complete the Reduced Follow-up Visit (RFU) (section 4.4). 
4 For participants who receive any CTX or mesna but do not receive any arTregs (VT0), move to Appendix 7 (section 4.4). 
5 This visit may occur concurrently with Appendix 3 for participants who initiate TAC withdrawal (V201) or Appendix 6 for 
participants who fail ISW (section 4.4). 
6 Participants who are unable or unwilling to travel to the study site will have the option of completing this study visit remotely. 
7 Reference section 6.3.1 for specific assessment details. 
8 For participants who receive the minimum or target arTreg dose, confirm Eligibility 4: TAC Withdrawal (section 5.3.4). For 
participants who meet eligibility, initiate TAC withdrawal (V201) in Appendix 3 within 14 days of eligibility determination. For 
participants who do not meet eligibility, complete visits in Appendix 6 (section 4.4). Complete remaining post arTreg Infusion 
Visits (T7-T84) concurrently with either Appendix 3 or Appendix 6 visits. 
9 Vital signs will be taken prior to, 5 minutes into, and at the end of the infusion, as well as 30 minutes, 1 hour, and 2 hours after the 
infusion (section 5.2.1.4). 
10 Local pathology assessment must be obtained to evaluate participants either for Eligibility 4: TAC Withdrawal or to continue TAC 
withdrawal (section 5.3.4). Assessment to be done 5-15 days after receiving the arTreg. 
11 Reference section 6.5.1 for specific assessment details. 


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Study  
Stage 3A 
Study  
Stage 3B 
Study Stage 3C 
- 
 
Recipient 
Leukapheresis 
Eligibility 3B:  
pre-arTreg 
Conditioning (CTX) 
Eligibility 3C: 
arTreg 
Infusion 
Eligibility 4: 
TAC 
Withdrawal 
- 
Reduced Safety  
Follow-up 
Post arTreg Infusion Visits1 
arTreg Infusion Day 
-17 
-3 
0 
1 
7 
14 
28 
84 
Window 
≤28 days of 
V102[R] 
≤72 hours &  
≥24 hours before 
arTreg infusion (VT0) 
- 
+24  
hours 
-2/+8 
days 
-2/+3 
days 
±5  
days 
±5  
days 
≤30 days after 
unsuccessful EVR 
conversion,  
VT-17[R], or VT-3 
Visit Number 
T-17/T-17R2,3 
T-33,4 
T0 
T1 
T75 
T145,6 
T285,6 
T845, 6 
RFU 
TAC trough levels 
 
X 
X 
X 
X12 
 
 
 
X13 
EVR trough levels 
 
X 
X 
X 
X 
 
 
 
X13 
arTreg Manufacture 
 
Leukapheresis 
X14,15 
 
 
 
 
 
 
 
 
Study Medications 
 
Prednisone 
X16,17 
 
X18 
SOC 
Tacrolimus 
X (target trough 3-5 ng/mL)17 
 
X18 
SOC 
Everolimus 
X (target trough 5-8 ng/mL) 
SOC 
Mesna19 
 
X 
 
 
 
 
 
 
 
Cyclophosphamide20 
 
X21 
 
 
 
 
 
 
 
Famciclovir 
 
X22 
 
 
 
 
 
Bacterial Prophylaxis 
 
X23 
 
 
 
 
 
 
 
G-CSF 
 
X24 
 
 
 
 
 
 
 
arTreg Infusion25 
 
 
X21 
 
 
 
 
 
 
Mechanistic Laboratory Assessments 
 
arTreg archiving 
 
 
X26 
 
 
 
 
 
 
PBMC collection (for 
UCSF lab) 
X 
 
X27 
X 
X 
X 
X 
X 
 
 
12 Complete only if the participant hasn’t initiated TAC withdrawal.  
13 Complete if the participant is receiving the IS medication that is relevant for the trough level. 
14 Performed exactly 16-17 days prior to arTreg infusion. See section 5.3.3.2.4 for leukapheresis details. 
15 PBMCs will be collected for mechanistic purposes from any leftover material. 
16 Prednisone may be discontinued at any time at the discretion of the investigator (section 5.2.3.3). 
17 For participants who have initiated TAC withdrawal (following Visit T1/T7), please see Appendix 3 for dosing instructions. 
18 See Appendix 3 for dosing instructions for participants who are undergoing TAC withdrawal, or Appendix 6 for participants who 
did not attempt TAC withdrawal or failed ISW. 
19 See section 5.2.2.2 for mesna dosing and administration details. 
20 Administered approximately 13-16 days after leukapheresis (VT-17/T-17R) (section 5.3.3.3.4). See section 5.2.2.1 for CTX dosing 
and administration details.5.2.2 
21 See section 5.2.4.1 for prophylactic medications per the center’s standard of care following CTX and arTreg infusion. 
22 If not already receiving viral prophylaxis, five days following CTX administration, participants will receive a 7-day course of 
famciclovir (Famvir®) 500 mg PO daily for prophylaxis against herpes simplex virus (HSV) (section 5.2.4.1.1). 
23 Five days following CTX administration, participants will receive prophylaxis against bacterial infections in the setting of 
neutropenia per institutional practice (section 5.2.4.1.3). 
24 After cyclophosphamide per section 5.2.4.1.4. 
25 See section 5.2.1.4 for arTreg dosing and administration details. 
26 May be collected <1 day prior to arTreg infusion. 
27 Mechanistic blood samples to be collected prior (-24 hours) to arTreg infusion. 


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Study  
Stage 3A 
Study  
Stage 3B 
Study Stage 3C 
- 
 
Recipient 
Leukapheresis 
Eligibility 3B:  
pre-arTreg 
Conditioning (CTX) 
Eligibility 3C: 
arTreg 
Infusion 
Eligibility 4: 
TAC 
Withdrawal 
- 
Reduced Safety  
Follow-up 
Post arTreg Infusion Visits1 
arTreg Infusion Day 
-17 
-3 
0 
1 
7 
14 
28 
84 
Window 
≤28 days of 
V102[R] 
≤72 hours &  
≥24 hours before 
arTreg infusion (VT0) 
- 
+24  
hours 
-2/+8 
days 
-2/+3 
days 
±5  
days 
±5  
days 
≤30 days after 
unsuccessful EVR 
conversion,  
VT-17[R], or VT-3 
Visit Number 
T-17/T-17R2,3 
T-33,4 
T0 
T1 
T75 
T145,6 
T285,6 
T845, 6 
RFU 
PBMC collection 
X28 
 
 
 
 
 
 
 
X29 
Serum collection 
X 
 
X27 
X 
 
 
 
 
X29 
Whole blood collection 
X 
 
X27 
X 
 
 
 
 
X29 
Liver biopsy collection 
 
 
 
 
X 
 
 
 
 
 
 
28 Collected from any leftover material from the leukapheresis bag used for arTreg manufacturing. 
29 Only for participants who successfully completed EVR conversion but do not receive any CTX, and/or mesna. Participants may 
have undergone leukapheresis. 


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Appendix 3. Study Stage 4: Schedule of Events – TAC Withdrawal 
 
Study Stage 4 
 
TAC Taper Level 
TAC Withdrawal  
Pause Visits1 
Post TAC 
Withdrawal  
1 
2 
Off 
 
 
After Initiating TAC Withdrawal 
Week 
 
0 
6  
(up to 102) 
12  
(up to 202) 
During Taper 
Level 1 
During Taper 
Level 2 
24 
Window 
 
≤14 days 
after TAC 
Withdrawal 
Eligibility 
Confirmation 
(Visit T1/T7) 
+7 days 
- 
±7 days 
Visit Number3 
 
201 
202 
2034 
201P 
202P 
2045 
General Assessments6 
 
 
Limited physical exam 
 
 
 
 
 
 
 
Vital signs 
 
 
 
 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
Concomitant medications 
 
X 
X 
X 
X 
X 
X 
Telemedicine consultation 
 
 
 
 
 
 
X 
Remote consultation 
 
X 
X 
X 
X 
X 
 
Local Laboratory Assessments7 
 
 
Hematology  
 
 
 
X 
 
 
X 
Basic chemistry 
 
 
 
X 
 
 
X 
LFTs (±5 day window) 
 
Every 2 Weeks8 
Every 2 Weeks8 
X9 
Estimated GFR 
 
 
 
 
 
 
 
TAC trough levels 
 
 
 
 
 
 
 
EVR trough levels 
 
 
 
X 
 
 
X 
EVR trough levels (±5 day window) 
 
Every 2 Weeks8 
Every 2 Weeks8 
X9 
Study Medications 
 
 
Prednisone 
 
tapered to 5 mg/day10,11 
tapered to 5 mg/day10,11 
X10 
Tacrolimus 
 
tapered per section 5.3.4.4 
tapered per section 5.3.4.4 
 
Everolimus 
 
X (target trough 5-8 ng/mL) 
X (target trough 5-8 ng/mL) 
X12 
 
1 Only complete additional visits as needed if TAC withdrawal is paused (section 5.4.1). 
2 In the event TAC withdrawal is paused (section 5.4.1). 
3 For participants who receive the minimum or target arTreg dose but either do not attempt TAC withdrawal (V201) or fail ISW, move 
to Appendix 6 (section 4.4). 
4 Complete 12 weeks after initiating TAC withdrawal. 
5 Complete 24 weeks after initiating TAC withdrawal unless the participant has moved to Appendix 4. 
6 Reference section 6.3.1 for specific assessment details. 
7 Reference section 6.5.1 for specific assessment details. 
8 Until V204 or until the participant is evaluated for Eligibility 5: EVR/Pred Withdrawal (V300 in Appendix 4), whichever is sooner. 
9 Complete every 2 weeks until the participant has been off TAC for 12 weeks. Once the participant has been off TAC for 12 weeks, 
complete every 4 weeks until the participant is evaluated for Eligibility 5: EVR/Pred Withdrawal (V300 in Appendix 4). 
10 Prednisone may be discontinued at any time at the discretion of the investigator (section 5.2.3.3). 
11 For participants receiving > 5 mg/day of prednisone at the time of TAC withdrawal, prednisone will be tapered as described in 
section 5.3.4.4. 
12 Target trough 5-8 ng/mL. 


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Appendix 4. Study Stage 5: Schedule of Events – EVR/Prednisone Withdrawal 
 
Study Stage 5 
  
  
Eligibility 5: 
EVR/Pred 
Withdrawal 
EVR/Pred Taper Level 
EVR Taper Level 
EVR/Pred 
Withdrawal 
Pause Visits1 
 
1 
2 
3 
4 
5 
6 
7 
After Initiating EVR/Pred Withdrawal 
Week2 
- 
0 
4 
8 
12 
16 
22 
28 
Up to 3  
4-week 
Pauses3 
Window 
Wk 12-26 
Post TAC 
Withdrawal 
≤8 Wks after 
V300 Biopsy 
+7 days 
- 
Visit Number4 
3005 
301 
302 
303 
304 
305 
306 
3076 
301P-307P 
General Assessments7 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
Eligibility 
X 
 
 
 
 
 
 
 
 
Limited physical exam 
X 
 
 
 
 
 
 
 
 
Vital signs 
X 
 
 
 
 
 
 
 
 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Telemedicine consultation 
 
 
 
 
 
X 
 
 
 
Remote consultation  
 
X 
X 
X 
X 
 
X 
X 
X 
Clinical Assessments and Procedures 
 
Liver biopsy 
X 
 
 
 
 
 
 
 
 
Local Laboratory Assessments8 
 
Hematology 
X 
 
 
 
 
X 
 
 
 
Basic chemistry 
X 
 
 
 
 
X 
 
 
 
LFTs (±5 day window) 
Every 4 
Weeks 
Every 2 Weeks 
Every 2 
Weeks 
Estimated GFR 
X 
 
 
 
 
 
 
 
 
EVR trough levels 
X 
 
 
 
 
 
 
 
 
Study Medications 
 
Prednisone9 
X 
tapered per section 5.3.5.4 
 
 
 
 
Everolimus 
X 
tapered per section 5.3.5.4 
X10 
Mechanistic Laboratory Assessments 
 
PBMC collection 
X 
 
 
 
 
 
 
 
 
Serum collection 
X 
 
 
 
 
 
 
 
 
 
1 Only complete additional visits as needed if EVR/Pred withdrawal was paused (section 5.4.1). Complete visit number that 
corresponds to the taper level that is paused, i.e. taper level 4 is paused, complete Visit 304P. 
2 Weeks reflect time after initiating EVR/Pred Withdrawal in the absence of any pauses (section 5.4.1). 
3 Three non-consecutive pauses (up to 4 weeks each) during IS (TAC [Appendix 3] and EVR/Pred) withdrawal (section 5.4.1). 
4 For participants who fail ISW or do not attempt EVR/Pred withdrawal, move to Appendix 6. 
5 Complete for all participants who successfully complete TAC withdrawal. For participants who received the minimum arTreg dose 
(Study Definitions), withdrawal will be at the discretion of the investigator (section 5.3.5.1). If the participant remains off TAC but 
does not initiate EVR/Pred withdrawal, move to Appendix 6. 
6 For participants who successfully complete ISW, move to Appendix 5. 
7 Reference section 6.3.1 for specific assessment details. 
8 Reference section 6.5.1 for specific assessment details. 
9 Prednisone may be discontinued at any time at the discretion of the investigator (section 5.2.3.3). For participants receiving 5 mg/day 
of prednisone at the time of EVR withdrawal, prednisone will be withdrawn as described in section 5.3.5.4. 
10 Tapered per section 5.3.5.4. 


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Study Stage 5 
  
  
Eligibility 5: 
EVR/Pred 
Withdrawal 
EVR/Pred Taper Level 
EVR Taper Level 
EVR/Pred 
Withdrawal 
Pause Visits1 
 
1 
2 
3 
4 
5 
6 
7 
After Initiating EVR/Pred Withdrawal 
Week2 
- 
0 
4 
8 
12 
16 
22 
28 
Up to 3  
4-week 
Pauses3 
Window 
Wk 12-26 
Post TAC 
Withdrawal 
≤8 Wks after 
V300 Biopsy 
+7 days 
- 
Visit Number4 
3005 
301 
302 
303 
304 
305 
306 
3076 
301P-307P 
Whole blood collection 
X 
 
 
 
 
 
 
 
 
Liver biopsy collection 
X 
 
 
 
 
 
 
 
 


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Appendix 5. Study Stage 6: Schedule of Events – Post-withdrawal Surveillance and 
Evaluation 
For participants who successfully complete ISW. 
 
Study Stage 6 
Follow-Up Week 
0 
26 
52 
104 
105-116 
Window 
≤7 days after 
last IS dose 
±2 wks 
±4 wks1 
±4 wks 
±2 wks 
Visit Number 
400 
401 
402 
403 
4042 
General Assessments3 
 
Limited physical exam 
X 
X 
X 
X 
 
Vital signs 
X 
X 
X 
X 
 
Adverse Events 
X 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
Remote consultation 
 
 
 
 
X 
Clinical Assessments and Procedures 
 
Liver biopsy 
 
 
X 
 
 
Local Laboratory Assessments4 
 
Hematology 
X 
X 
X 
X 
X 
Basic chemistry 
X 
X 
X5 
X 
X 
LFTs (±5 day window)6 
Every 2 
Weeks7 
Every 4 Weeks 
Every 8 
Weeks 
Per standard 
of care8 
Mechanistic Laboratory Assessments 
 
PBMC collection 
X 
X 
X 
X 
 
Serum collection 
X 
X 
X 
X 
 
Whole blood collection 
X 
X 
X 
X 
 
Liver biopsy collection 
 
 
X 
 
 
 
1 If the investigator determines that abnormal laboratory results prior to the Visit 402 liver biopsy are related to an intercurrent illness, 
the investigator may request that the participant’s data be reviewed by an adjudication committee, who may grant an extension of 
the primary endpoint visit window (3.3.1.2) 
2 Complete for participants who resume IS at the end of study follow-up to ensure they are followed for at least 12 weeks after 
restarting IS (section 5.4.2). Complete V404 12 weeks after restarting IS. 
3 Reference section 6.3.1 for specific assessment details. 
4 Reference section 6.5.1 for specific assessment details. 
5 Must be done within the two weeks prior to the biopsy. 
6 For participants who experience rejection (section 5.4.3.2), record results of all assessments collected from standard of care into the 
clinical database. 
7 Continue LFT schedule every 2 weeks from EVR Taper Level 7 Visit in Appendix 4. Once the participant has been off all IS for 14 
weeks, complete LFTs every 4 weeks until the participant’s next visit. 
8 Record results of all assessments collected from standard of care into the clinical database. 


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Appendix 6. Schedule of Events – High Intensity Safety Follow-up 
For participants who receive any arTreg but either do not attempt or fail ISW. 
Follow-Up Week 
0 
26 
52 
104 
Window 
≤4 wks after VT0, 
V300, or ISW 
Failure1 
±2 wks 
±4 wks 
±8 wks  
Visit Number 
500 
501 
502 
503 
General Assessments2 
Limited physical exam 
X 
X 
X 
X 
Vital signs 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
Local Laboratory Assessments3 
Hematology 
X 
X 
X 
X 
Basic chemistry 
X 
X 
X 
X 
LFTs (±5 day window)4 
Every 8 Weeks 
 
 
TAC trough levels5 
X6 
X6 
X6 
X6 
TAC trough levels (±5 day window)5 
Every 8 Weeks 
 
 
EVR trough levels5 
X6 
X6 
X6 
X6 
EVR trough levels (±5 day window)5 
Every 8 Weeks 
 
 
Mechanistic Laboratory Assessments 
PBMC collection 
X7 
X 
X 
 
Serum collection 
X7 
X 
X 
 
Whole blood collection 
X7 
X 
X 
 
 
1 Visit T0 for participants who don’t receive at least the minimum arTreg dose (Study Definitions) or attempt TAC withdrawal, Visit 
300 for participants who do not attempt EVR/Pred withdrawal, or ISW failure. 
2 Reference section 6.3.1 for specific assessment details. 
3 Reference section 6.5.1 for specific assessment details. 
4 Record results of all assessments collected from standard of care into the clinical database. 
5 If the participant is receiving the IS medication that is relevant for the trough level, record results of all assessments collected from 
standard of care into the clinical database. 
6 Complete if the participant is receiving the IS medication that is relevant for the trough level. 
7 If Visit 500 occurs within 8 weeks of an Appendix 2, Appendix 3 or Appendix 4 visit that included collection of this mechanistic 
assessment, do not repeat this mechanistic assessment during V500. 


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Appendix 7. Schedule of Events – Low Intensity Safety Follow-up 
For participants who receive any CTX and/or mesna but do not receive any arTreg 
Follow-Up Week 
0 
4 
52 
Window 
7-10 days after VT-3 
±5 days 
±8 wks 
Visit Number 
600 
601 
602 
General Assessments 1 
Limited physical exam 
X 
 
X 
Vital signs 
X 
 
X 
Adverse Events 
X 
X 
X 
Concomitant medications 
X 
X 
X 
Remote consultation 
 
X 
 
Local Laboratory Assessments2 
Hematology 
X 
X3 
X 
Basic chemistry 
X 
X 
X 
 
1 Reference section 6.3.1 for specific assessment details. 
2 Reference section 6.5.1 for specific assessment details. 
3 Complete assessment every 4 weeks until absolute neutrophil count ≥ 1,500/µL. 


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Appendix 8. Schedule of Events – For-Cause Biopsy 
Visit Number 
FCB1,2,3 
General Assessments4 
Limited physical exam 
X 
Vital signs 
X 
Adverse Events 
X 
Concomitant medications 
X 
Clinical Assessments and Procedures 
Liver biopsy 
X 
Local Laboratory Assessments5 
Hematology 
X 
Basic chemistry 
X 
Mechanistic Laboratory Assessments 
PBMC collection 
X 
Serum collection 
X 
Whole blood collection 
X 
Liver biopsy collection 
X 
 
 
1 If a participant needs a for-cause biopsy within 8 weeks prior to an Appendix 1A-Appendix 4 visit, the mechanistic assessments 
collected during the FCB visit will take the place of any mechanistic assessments to be collected during that upcoming Appendix 
1A-Appendix 4 visit. 
2 If a participant needs a for-cause biopsy within 8 weeks prior to an Appendix 5-Appendix 7 visit, the FCB visit will take the place of 
that upcoming Appendix 5-Appendix 7 visit. 
3 If a participant is treated for clinical rejection at the study site without a biopsy (section 5.4.3.2.3), complete all other visit 
assessments done at a for-cause biopsy visit prior to initiating treatment. 
4 Reference section 6.3.1 for specific assessment details. 
5 Reference section 6.5.1 for specific assessment details.