Clinical trial NCT02062944
Conditions: Acute Rejection of Liver Transplant Effects of Immunosuppressant Therapy
Keywords: Liver Transplant Sirolimus Rapamune Immunosuppressant Therapy Withdrawal
Sponsor: Northwestern University
Start date: 2013-03-01; Completion date: 2020-07-01
Enrollment: 21 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The significance of this clinical trial lies in its potential to increase the success of immunosuppression (IS) therapy withdrawal in liver transplant (LT) recipients, thus decreasing the negative impact of IS on their long-term outcomes. Lifetime immunosuppression (IS) with standard agents, the calcineurin inhibitors (CNI) cyclosporine and tacrolimus (TAC), is currently required at clinically recommended doses and trough levels to prevent allograft rejection. However, this occurs at the significant expense of long-term CNI toxicity, i.e. chronic kidney disease (CKD), hypertension, hyperlipidemia, diabetes, infections and malignancy. With improvements in early graft and patient survival, long term adverse IS effects have become increasingly important in this rapidly expanding patient population. The strategies to reduce long term CNI toxicity include dose minimization that still leaves patients on CNI therapy, conversion to non-CNI therapy, or even complete IS withdrawal. The second approach, conversion to non-CNI IS therapy, is attractive in the potential to stabilize or improve renal function and other CNI toxicities. One such non-nephrotoxic IS agent, the mammalian target of rapamycin inhibitor (mTOR-I) SRL, has a different mechanism of IS action and studies have shown that CNI to SRL conversion can stabilize renal dysfunction with a low risk of rejection. Yet even with these possible benefits, patients on SRL are still subject to lifetime IS therapy with side effects and costs, highlighting the need to investigate the strategies that promote full IS withdrawal without rejection (3rd approach), also known as 'operational tolerance'.

Detailed description:
Study Overview The study proposed is a prospective trial of controlled SRL monotherapy minimization and withdrawal in up to 25 stable non-immune non-viremic LT recipients. Given the sample size calculations (see Statistical section), we plan to enroll up to 25 SRL monotherapy patients for this study (Figure 1). All patients will be consented to undergo laboratory evaluation as well as an enrollment liver biopsy and blood tests. If the patient meets inclusion/exclusion (I/E) criteria (see below), SRL will be minimized over 6 months until once a week dosing is achieved. Repeat clinical and immunological blood tests as above will be performed, and if no biochemical signs of rejection, SRL will be discontinued with blood tests and liver biopsy 12 months later for similar biochemical, histological and immunological measures. At any concern for rejection, liver biopsy and assays for equivalent biochemical, histological and immunological measures will be performed and if rejection is diagnosed on biopsy, a second attempt at withdrawal will not be performed. Patients will be monitored as standard of care with clinic visits every 6 months and laboratory tests every 2 weeks. The total study length will be 18 months: 6 month minimization phase and 1 year follow-up after withdrawal success/failure. The primary outcome will be the proportion of tolerant patients off SRL therapy with normal liver biochemistry and graft histology at 18 months. Secondary outcomes will include the incidence, severity and reversibility of rejection, patient/graft survival, resolution of SRL and other non-specific IS effects, and the assessment of clinical/immunological biomarkers of tolerance. All continuous/categorical clinical variables will be compared with the appropriate statistical analyses.The goal is that the primary and secondary aims will provide valuable preliminary data to further elucidate the mechanisms of mTOR-I immunoregulation and for determining the initial clinical success/feasibility of the mTOR-I approach (vs. historical 20% CNI withdrawal success seen in studies from the Immune Tolerance Network \[ITN\] and other groups). This is all so as to guide a submission to the Immune Tolerance Network for larger, more adequately powered prospective trials comparing SRL vs. CNI withdrawal and accompanying biomarker predictors of tolerance. If this pilot study fails to show a correlation between our biomarkers and the success/failure of SRL withdrawal, or is associated with an unacceptable low (e.g. \<20%) rate of operational tolerance, then this would avoid the necessity for such large, expensive trials and support the continued development of alternative approaches to tolerance.

Consent and Initial Phase of Enrollment (see Study Protocol Figure- Appendix A) These liver transplant candidates (up to 25 SRL) will be approached for consideration and informed consent into the study. The consent form will include discussion of the risks/benefits of their current therapy (SRL) and the planned minimization/withdrawal. Specifically, the risks of minimization/withdrawal (i.e. developing acute or chronic rejection, alloimmune hepatitis during any portion of the study), although unlikely, will be a major emphasis of the consent process. The consent form will also include the strict requirement for patients to follow all instructions from the PI in regard to the close laboratory follow-up occurring throughout the trial, to diagnose any episode of rejection or concern as early as possible to be able to respond appropriately. All of the study procedures will be discussed with the patient during clinic visits. They will be informed that neither participation nor refusal will influence their clinical care. All laboratory tests or costs related to their care in the study, with the exception of non-standard of care items (i.e. liver biopsies, blood assays), will be the responsibility of the patient and/or his/her insurance company. They will be asked questions afterwards to verify comprehension and then sign the consent form documenting their agreement to participate. A signed copy of the consent form will be given to them. Participation is completely voluntary and they may discontinue participation the study at any time without affecting their care or participation in any other study. No financial compensation will be given.

Screening Evaluation at Enrollment (see Study Protocol Figure) After long term (\> 3 years post-LT, \> 3 months on SRL monotherapy) on SRL monotherapy, inclusion/exclusion criteria will be reviewed and if appropriate, consent as above will be obtained. If the patient agrees and signs consent, baseline standard of care screening laboratories complete blood count, comprehensive metabolic panel, sirolimus (CBC, CMP, SRL trough level, fasting lipid profile, hemoglobin A1C \[HbA1C\], urine protein/creatinine ratio) and non-standard of care biomarker assays (blood immunophenotyping, proteogenomics) and liver biopsy (histology and graft immunohistochemistry) will be performed as dictated by the protocol. In addition, the liver biopsy will be used to determine stability in graft function (i.e. no evidence of rejection or immune-mediated hepatitis) before considering minimization/withdrawal. SRL minimization/withdrawal will only be performed if clinically, biochemically and histologically \[by biopsy; liver transplant pathology read (Yang, Rao)\] stable. Throughout the entire study, liver function tests will be monitored every 2 weeks (monthly is standard of care, so the interim non-standard of care 2 week blood tests will be covered by the study funds). Patients entering the minimization/withdrawal phases will be reduced by a total dose of 50% of their baseline dose every month until patients are on 0.5 mg SRL daily. At this point, every other day dosing will begin x 1 month. If no LFT abnormalities, twice weekly dosing x 1 month, then once a week dosing x 1 month. Prior to complete discontinuation, repeat clinical (screening labs above) and blood biomarker assays (blood immunophenotyping, proteogenomics) will be performed. Liver biopsy will not be performed at this juncture unless there are biochemical signs of liver injury. If complete withdrawal is deemed safe (no evidence of biochemical abnormalities) patients will be withdrawn completely (i.e. the once/week SRL dose discontinued) and followed off IS therapy for one year. In this time period, liver function tests will be monitored every 2 weeks, as usual, and repeat clinical (screening labs above), liver biopsy and blood (blood immunophenotyping, proteogenomics) assays performed at the end of this year or at any concern for rejection.

Blood and Tissue Samples Standard of care laboratories (CBC, CMP) will be performed every month on all patients, and only LFTs performed in the interim 2 weeks between each month (non-standard of care; covered by the study funds) until complete withdrawal. 3 green top 5 mL and 3 red top 5 mL tubes of blood for biomarker assays will be taken on all patients prior to study enrollment, prior to complete withdrawal, and one year after withdrawal or at the time of suspected rejection. Liver biopsy (one 3 cm biopsy- 2 cm for histology and 1 cm for genomic assays) will be performed at baseline (pre-minimization) and 1 year post withdrawal. A separate clinical informed consent will be obtained each time a liver biopsy is performed. If the patient develops elevation in liver transaminases requiring a liver biopsy at any stage of the study protocol, blood (all for immune monitoring (IM) assays) will be requested from the patient at the time of the biopsy. If rejection occurs, the patient will be followed in the study until completion but not be further withdrawn from SRL or have a liver biopsy at the end of the study

Follow-up Laboratory follow-up is described above- every 2 weeks throughout the trial until complete withdrawal. Any biochemical (or clinical) signs or significant liver function test abnormalities will be acted on immediately either by liver biopsy or a pause in IS withdrawal per investigator discretion. All patients will be seen in the clinic every 3 months to assess for any new signs or symptoms or resolution of drug side effects in the minimization or withdrawal arm. Quality of life questionnaires; Post Liver Transplant Quality of Life Instrument (pLTQ) and the Promis-29 profile v1.0 will be administered at the study onset and after successful vs. unsuccessful withdrawal (end of study)

Outcome Measures The primary outcome will be the proportion of patients off SRL therapy with normal liver biochemistry and graft histology at 12 months (i.e. tolerant). Thus, the incidence of graft dysfunction (acute rejection, immune mediated or autoimmune hepatitis, chronic rejection) or non-tolerance will be assessed in this SRL withdrawal group and compared to the historical CNI group (20% tolerant; 80% failure) as the primary endpoint. This rate will be a composite of the cumulative number of biopsy-proven graft dysfunctions requiring conversion back to SRL or additional IS therapy or discontinuation of minimization/withdrawal that occur during the course of the study. Major secondary measures compared will be the predictive capacity of the blood and graft biomarker assays (immunophenotyping, genomic/proteomics) before and after minimization/withdrawal in the success vs. failure groups. Clinical secondary outcomes will be compared: the number of infectious complications, liver biopsy complications, cardiovascular outcomes (i.e. blood pressure, diabetes control, lipid levels), renal function, hematopoietic parameters, gastrointestinal effects, or other side effects of SRL that may or may not improve or develop with minimization/withdrawal. These will all be documented by a study database during patient visits, electronic charts and/or by phone communication. Finally, quality of life (Post Liver Transplant Quality of Life Instrument (pLTQ) and the Promis-29 profile v1.0) will be analyzed at the end of the study to determine the effect of IS minimization/withdrawal on other health benefits.

Arms: arm 1: SRL minimization will be performed if clinically, biochemically and histologically stable. Patients entering the minimization phases will be reduced every month by 50% of total dose of Sirolimus until they reach .5mg daily for one month. Then .5 mg every other day, then twice weekly, the once weekly dosing. This should take approximately 6 month to complete minimization. Liver function tests will be monitored every 2 weeks. For any patient developing liver dysfunction, liver biopsy will be performed. Patients will then be completely withdrawn and followed post-withdrawal for 12 months.
Interventions: intervention 1: Sirolimus
Intervention descriptions: intervention 1: SRL minimization will be performed if clinically, biochemically and histologically stable. Patients entering the minimization phases will be reduced every month by 50% of total dose of Sirolimus until they reach .5mg daily for one month. Then .5 mg every other day, then twice weekly, the once weekly dosing. This should take approximately 6 month to complete minimization. Liver function tests will be monitored every 2 weeks. For any patient developing liver dysfunction, liver biopsy will be performed. Patients will then be completely withdrawn and followed post-withdrawal for 12 months.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Northwestern University 
Comprehensive Transplant Center 
Feinberg School of Medicine 
 
 
 
Liver Transplant Tolerance Enhanced By Sirolimus Therapy 
 
 
 
 
 
Principal Investigator: 
Josh Levitsky, MD, MS 
Northwestern University 
Comprehensive Transplant Center 
676 N. St. Clair Street, Suite 1900 
Chicago, IL 60611 
(312) 695-4413 
 
Sub-Investigators: 
Joshua Miller, MD; James Mathew, PhD; Michael Abecassis, 
MD, MBA, Joseph Leventhal, MD, PhD, Daniel Ganger, MD, 
Sean Koppe, MD, Laura Kulik, MD, MS, Talia Baker, MD, 
John Fryer, MD, Juan Carlos Caicedo, MD, Anton Skaro, 
MD, PhD, Daniela Ladner, MD, MPH, Sam Rao, MD, 
Guang-Yu Yang, MD and Brittany Lapin, MPH 
 
 
 
IRB Protocol Number: 
STU00072766 
 
 
 
Date: 10-04-2017 
 
 
 
 
 
 
 
 
                                                              CONFIDENTIAL 
 
This document is confidential and the property of the Department of Surgery/Division of Organ 
Transplantation, Northwestern University-Feinberg School of Medicine.  No part of it may be transmitted, 
reproduced, published, or used by other persons without prior written authorization from the study sponsor. 


 
 
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Table of Contents 
STUDY SUMMARY ................................................................................................................................................... 1 
1 
INTRODUCTION .............................................................................................................................................. 3 
1.1 
BACKGROUND ............................................................................................................................................. 3 
1.2 
PRECLINICAL DATA ..................................................................................................................................... 4 
1.3 
CLINICAL DATA TO DATE............................................................................................................................ 4 
1.4 
DOSE RATIONALE AND RISK/BENEFITS ....................................................................................................... 5 
2 
STUDY OBJECTIVES .................................................................................................................................... 56 
3 
STUDY DESIGN ................................................................................................................................................ 6 
3.1 
GENERAL DESIGN ....................................................................................................................................... 6 
3.2 
PRIMARY STUDY ENDPOINTS ...................................................................................................................... 8 
3.3 
SECONDARY STUDY ENDPOINTS ................................................................................................................. 8 
3.4 
PRIMARY SAFETY ENDPOINTS ................................................................................................................... 89 
4 
SUBJECT SELECTION AND WITHDRAWAL.......................................................................................... 89 
4.1 
INCLUSION CRITERIA ................................................................................................................................. 89 
4.2 
EXCLUSION CRITERIA ............................................................................................................................... 89 
4.3 
SUBJECT RECRUITMENT AND SCREENING ................................................................................................... 9 
4.4 
EARLY WITHDRAWAL OF SUBJECTS ...................................................................................................... 1011 
4.4.1 
When and How to Withdraw Subjects .............................................................................................. 1011 
4.4.2 
Data Collection and Follow-up for Withdrawn Subjects ................................................................. 1011 
5 
STUDY DRUG ................................................................................................................................................. 11 
5.1 
SUBJECT COMPLIANCE MONITORING ........................................................................................................ 11 
5.2 
PRIOR AND CONCOMITANT THERAPY ........................................................................................................ 11 
6 
STUDY PROCEDURES .............................................................................................................................. 1112 
6.1 
VISIT 1 .................................................................................................................................................. 1112 
6.2 
VISIT 2 .................................................................................................................................................. 1112 
6.3 
MONITORING DURING MINIMIZATION. ....................................................................................................... 12 
6.4 
VISIT 3. ..................................................................................................................................................... 12 
6.5 
VISIT 4 ...................................................................................................................................................... 12 
6.6 
VISIT 5. ..................................................................................................................................................... 12 
 
7 
STATISTICAL PLAN ................................................................................................................................. 1213 
7.1 
SAMPLE SIZE DETERMINATION ............................................................................................................. 1213 
7.2 
STATISTICAL METHODS......................................................................................................................... 1213 
7.3 
SUBJECT POPULATION(S) FOR ANALYSIS .................................................................................................. 13 
8 
SAFETY AND ADVERSE EVENTS ............................................................................................................. 13 
8.1 
DEFINITIONS .............................................................................................................................................. 13 
8.2 
RECORDING OF ADVERSE EVENTS ............................................................................................................. 15 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS ........................................................................................... 1516 
8.3.1 
EC/IRB Notification by Investigator ................................................................................................ 1516 
8.4 
STOPPING RULES ................................................................................................................................... 1516 
8.5 
MEDICAL MONITORING ......................................................................................................................... 1516 
9 
DATA HANDLING AND RECORD KEEPING .......................................................................................... 16 
9.1 
CONFIDENTIALITY ..................................................................................................................................... 16 
9.2 
SOURCE DOCUMENTS ............................................................................................................................ 1617 
9.3 
CASE REPORT FORMS ............................................................................................................................ 1617 
9.4 
RECORDS RETENTION ............................................................................................................................ 1617 


 
 
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9.5 
AUDITITNG AND INSPECTING ................................................................................................................. 1617 
10 
ETHICAL CONSIDERATIONS .................................................................................................................... 17  
11 
STUDY FINANCES ..................................................................................................................................... 1718 
11.1 
FUNDING SOURCE ................................................................................................................................. 1718 
11.2 
CONFLICT OF INTEREST ......................................................................................................................... 1718 
11.3 
SUBJECT STIPENDS OR PAYMENTS ........................................................................................................ 1718 
13 
PUBLICATION PLAN ................................................................................................................................ 1718 
14 
REFERENCES ............................................................................................................................................. 1819 
 


 
 
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                                                  List of Abbreviations 
 
AE 
 
 
Adverse Event 
 
ALK Phos 
 
Alkaline Phosphatase 
 
ALT 
 
 
Alanine Aminotrasferase 
 
AST 
 
 
Aspartate Transaminase  
 
AUROC 
 
Area Under Receiver Operating Characteristic (Curve) 
 
CBC  
 
Complete Blood Count 
 
CKD  
 
Chronic Kidney Disease 
 
CMP  
 
Comprehensive Metabolic Panel 
 
CNI 
 
 
Calcineurin Inhibitor 
 
CRF 
 
 
Case Report Form 
 
Cr 
 
 
Creatinine 
 
CTC 
 
 
Comprehensive Transplant Center 
 
eCRF  
 
Electronic Case Report Form 
 
eGFR  
 
Estimated Glomerular Filtration Rate 
 
FDA  
 
Food and Drug Administration 
 
GCP 
 
 
Good Clinical Practice 
 
GFR 
 
 
Glomerular Filtration Rate 
 
GGT  
 
Gamma-Glutamyl Transpeptidase 
 
HBV  
 
Hepatitis B Virus 
 
HCV  
 
Hepatitis C Virus 
 
HbA1C 
 
Hemoglobin 
 
HIPAA 
          Health Insurance Portability and Accountability Act of 1996  
 
HIV 
 
 
Human Immune Deficiency Virus 
 
 
 
 


 
 
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I/E 
 
 
Inclusion/Exclusion 
 
ICH 
 
 
International Conference on Harmonization 
 
IHC 
 
 
Immunohistochemical  
 
IL-2 
 
 
Interleukin 2 
 
IM 
 
 
Immune Monitoring 
 
 
IRB 
 
 
Institutional Review Board 
 
IS 
 
 
Immunosuppression 
 
ITN 
 
 
Immune Tolerance Network 
 
LFT 
 
 
Liver Function Tests 
 
LT 
 
 
Liver Transplant 
 
MDRD-4 
 
Modification of Diet in Renal Disease-4 (version four) 
 
MLR  
 
Mixed Lymphocyte Reaction 
 
NIAID  
 
National Institute of Allergy and Infectious Disease 
 
NIH 
 
 
National Institutes of Health 
 
NUIRB 
 
Northwestern University Institutional Review Board 
 
OLT 
 
 
Orthotopic Liver Transplant 
 
PBMC  
 
Peripheral Blood Mononuclear Cell 
 
PI 
 
 
Principal Investigator 
 
pLTQ  
 
Post Liver Transplantation Quality of Life Instrument 
 
PTLD  
 
Post Transplant Lymphoproliferative Disorder 
 
QOL  
 
Quality of Life 
 
RM-ANOVA  
Repeated Measures o fAnalysis of Variance 
 
SAE 
 
 
Serious Adverse Event 
 
SRL 
 
 
Sirolimus 
 


 
 
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TAC  
 
Tacrolimus  
 
ULN  
 
Upper Limit of Normal 
 
 
 


Clinical Research Protocol Template 
 
page 1 
Version 1.0 
Study Summary 
Title 
Liver Transplant Tolerance Enhanced By Sirolimus Therapy 
Short Title 
Sirolimus-Enhanced Tolerance 
NU  
Protocol Number 
STU00071766 
Phase 
Phase 4, pilot study 
Methodology 
Open label prospective study of Sirolimus withdrawal 
Study Duration 
18 months 
Study Center(s) 
Single-center  
Objectives 
Objectives: 1. To determine if tolerance can be achieved successfully in a 
reasonable percentage (>20%) of liver transplant  (LT) recipients withdrawn 
from SRL monotherapy. 2. To investigate if blood/allograft metrics of 
immunoregulation correlate with successful SRL withdrawal in LT recipients.   
Number of Subjects 
25 Subjects meeting inclusion/exclusion criteria 
Diagnosis and Main 
Inclusion Criteria 
1) Adult LT recipients > 3 years post-LT and > 6 months on SRL 
monotherapy; 2) Primary living or deceased donor LT; 3) Consent to undergo 
full immunosuppression withdrawal (sirolimus [SRL] withdrawal) 
Study Product, Dose, 
Route, Regimen 
Sirolimus (patients on varying doses of daily oral tablet) 
Statistical 
Methodology 
The primary outcome will be the proportion of tolerant patients off SRL 
therapy with normal liver biochemistry and graft histology at 12 months. As a 
pilot study focused on acquiring preliminary data, there will be no control 
group maintaining SRL therapy or a control group withdrawing from different 
immunosuppressive therapy (i.e. calcineurin inhibitor [CNI] agents) as 
comparators. Immune assay changes with successful or failed withdrawal will 
also be compared with appropriate statistical tests. 
 
This document is a protocol for a human research study.  This study is to be conducted according 
to United States (US) and international standards of Good Clinical Practice (GCP); (Food and 
Drug Administration [FDA] Title 21, parts 312, 50, 56 and International Conference on 
Harmonization guidelines [ICH]), applicable government regulations and institutional research 
policies and procedures. 
By signing below, I agree to conduct the research by US and International Standards of GCP 
FDA Title 21, parts 312, 50, 56 and ICH guidelines.  Being the principal investigator (PI), I also 
agree to oversee that the subinvestigators and all research staff abide by the same standards and 
principles. 
 


 
 
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Name                                    Date 


 
 
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Introduction 
This document is a protocol for a human research study. This study is to be conducted according to US 
and international standards of Good Clinical Practice (FDA Title 21 parts 312, 50, 56 and International 
Conference on Harmonization guidelines), applicable government regulations and Institutional research 
policies and procedures.  
1.1 Background 
The significance of this clinical trial lies in its potential to increase the success of immunosuppression 
(IS) therapy withdrawal in liver transplant (LT) recipients, thus decreasing the negative impact of IS on 
their long-term outcomes. Lifetime immunosuppression (IS) with standard agents, the calcineurin 
inhibitors (CNI) cyclosporine and tacrolimus (TAC), is currently required at clinically recommended 
doses and trough levels to prevent allograft rejection. However, this occurs at the significant expense of 
long-term CNI toxicity, i.e. chronic kidney disease (CKD), hypertension, hyperlipidemia, diabetes, 
infections and malignancy (1, 2). With improvements in early graft and patient survival, long term 
adverse IS effects have become increasingly important in this rapidly expanding patient population. The 
strategies to reduce long term CNI toxicity include dose minimization that still leaves patients on CNI 
therapy, conversion to non-CNI therapy, or even complete IS withdrawal. The second approach, 
conversion to non-CNI IS therapy, is attractive in the potential to stabilize or improve renal function and 
other CNI toxicities. One such non-nephrotoxic IS agent, the mammalian target of rapamycin inhibitor 
(mTOR-I) SRL, has a different mechanism of IS action and studies have shown that CNI to SRL 
conversion can stabilize renal dysfunction with a low risk of rejection (3, 4). Yet even with these possible 
benefits, patients on SRL are still subject to lifetime IS therapy with side effects and costs, highlighting 
the need to investigate the strategies that promote full IS withdrawal without rejection (3rd approach), also 
known as ‘operational tolerance’.  
 
Therefore, specific to this proposal and supported by our preliminary data, an additional advantage of 
mTOR-I therapy lies in its potential to promote such a clinical immunoregulatory state that could 
facilitate IS minimization and complete withdrawal. As the most immunoregulatory solid organ 
transplanted, the liver houses numerous extramedullary hematopoietic and non-immunogenic cells and 
secretes several immunoregulatory proteins (5). As a result, the percentage of LT recipients able to 
undergo IS withdrawal is the highest of all solid organ transplant recipients, although still only ~ 20% 
successful to date and primarily only performed in CNI-treated patients in withdrawal studies (6). This 
clinically unacceptable percentage is likely due to known CNI mechanisms inhibiting immunoregulation 
and the lack of available, well-defined immune monitoring to detect immunoregulation or unresponsive 
states. A key difference between mTOR-I and CNI is their effect on regulatory T cells (Tregs) and 
regulatory ‘immature’ dendritic cells (DCregs) important in the suppression of auto- and allo-immune 
responses. As an inhibitor of IL-2 signaling after T cell activation, SRL blocks proliferation of 
alloreactive T cells but promotes Tregs (CD4+CD25highFOXP3+), regulatory cytokines (i.e. TGF-β1), and 
tolerogenic dendritic cells (DCreg) (7, 8). In contrast, CNIs inhibit interleukin-2 (IL-2) transcription and 
thus negatively affect Treg generation (9-11). Importantly and most relevant to this proposal, we have 
recently demonstrated systemic (peripheral blood mononuclear cell [PBMC], bone marrow, allograft) 
Treg/DCreg increases and enhanced tolerogenic proteogenomic markers in LT recipients converted from 
CNI to SRL (12, 13), as well as augmented allo-specific Treg function by SRL vs. CNIs in vitro (14).  
These data provide the clinical and mechanistic background that support SRL facilitating operational 
tolerance in this pilot study. 
 
The identification of biomarkers of tolerance is as important as the chosen IS regimen. Various reports 
demonstrate a high percentage of Tregs, DCregs, γδ Tcells (Vδ1/Vδ2 ratio), and specific gene signatures 
in tolerant LT recipients (6, 15, 16). We have utilized a number of in vitro assays testing donor-specific 


 
 
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Treg inhibition/recruitment (Treg MLR) and regulation of cytotoxic T cells (micro-cell mediated 
lympholysis, m-CML) induced by IS therapies and in clinical tolerance trials (14, 17-20). Also, we have 
utilized 
peripheral 
blood/allograft 
immunophenotyping 
for 
regulatory 
cell 
populations 
and 
immunoregulatory proteogenomic assays to look for signatures of tolerance and CKD in LT recipients (13, 
21). Yet the presence of Treg populations and differential gene array snapshots in ‘already-withdrawn’ 
patients or patients currently on IS therapy, without a clear understanding of their utility in monitoring 
during withdrawal, only provides circumstantial evidence for their role in tolerance. An array of assays 
performed throughout the tapering and withdrawal period, as described herein, would provide a more 
accurate measure of the effects of SRL on Treg function and immunoregulatory gene/protein expression, 
supporting their use as predictors of tolerance in future protocols. 
With the above background and rationale, we expect to develop two strategies to promote the ability to 
achieve and monitor LT tolerance. The first is initial CNI to SRL conversion to promote systemic 
immunoregulation (already performed in our preliminary work (13)), followed by SRL minimization and 
withdrawal (this Institutional Review Board [IRB] submission). This is to be compared to the established 
literature success rate (≈20%) of CNI withdrawal, as we do not feel it is clinically acceptable to include 
this as a control group (6). We hypothesize that the clinical use of SRL promotes immunoregulatory 
pathways and provides higher IS withdrawal success compared to CNI therapy. The second strategy aims 
to identify immunologic and proteogenomic assessments of immunoregulation, potentially enhanced by 
SRL conversion and predicting/correlating with successful IS withdrawal.  
 
In summary, even though this is a small clinical/translational pilot study, it has a potential for 
significance in the field of transplant medicine. If successful, it could serve as a model approach for 
further studies and national grant funding in LT tolerance. Specifically, the Immune Tolerance Network 
(National Institutes of Health/National Institute of Allergy and Infectious Disease [NIH/NIAID]) requests 
these pilot data from our center prior to conducting a large multicenter controlled study of SRL (or other 
mTOR-I) withdrawal in LT recipients. The downstream impact could be far-reaching, not only from a 
clinical standpoint in improving patient outcomes, but also lead to amplified research in mechanisms of 
mTOR-I therapy, other tolerogenic approaches and biomarkers of tolerance. The immunoregulatory 
signatures determined preliminarily in this study could eventually, if validated, facilitate ‘point-of-
service’ tests in clinical settings, either routinely or during modifications of therapy. Knowledge of such 
specific clinical and immunological characteristics and using an individualized approach would allow 
transplant clinicians to more accurately define and select appropriate candidates for withdrawal. This 
would satisfy our long term goal of developing a refined approach to more successfully withdraw IS 
therapy and improve outcomes for LT recipients.  
 
1.2 Preclinical Data 
Preliminary Data: We demonstrated that liver transplant (LT) recipients on SRL monotherapy had higher 
percentages 
of 
peripheral 
blood 
mononuclear 
cell 
(PBMC) 
immunophenotypic 
Tregs 
(CD4+CD25Hi+Foxp3+ cells) compared to recipients on CNIs (12).This led to a recently published CNI 
to SRL conversion trial demonstrating increased percentages of PBMC/bone marrow Tregs and PBMC 
DCregs (ILT3/4+) in LT recipients converted from tacrolimus to SRL monotherapy(13). Liver biopsy 
immunohistochemistry FOXP3:CD3 and CD4:CD8 ratios were significantly higher after conversion. 
Patient sera on TAC but not SRL suppressed Treg generation in mixed lymphocyte reactions. Finally, 
consistent with prior reports of genomic expression patterns and cellular assays as signatures of tolerance 
(16), we detected the expression of 289 novel genes and 22 proteins, several important in 
immunoregulatory pathways, after SRL conversion. In a separate in vitro study, we also demonstrated 
robust allospecific regulatory effects of SRL in Treg mixed lymphocyte reactions (14). Our preliminary 
data, in conjunction with other clinical and laboratory reports, provide the mechanistic rationale for the 


 
 
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concept that SRL may facilitate IS withdrawal in LT and that such assays should be tested in parallel as 
biomarkers of tolerance. We hypothesize that SRL will promote regulatory gene/protein expression 
patterns in the peripheral blood AND in the allograft, which can then be utilized as predictive assays in 
tolerance trials. To robustly test this hypothesis, we will collect serial blood and allograft samples 
before/after withdrawal and at the time of biopsy for possible rejection, all to be tested as markers of 
immunoregulation. This will consist of assays similar to those performed in our recent work (12, 13, 21). 
1.3 Clinical Data to Date 
We and others have shown the safety of CNI to SRL conversion (3, 4, 12, 13), but there are no current 
studies testing full IS withdrawal in patients on SRL monotherapy. There are numerous studies of CNI 
withdrawal that have shown together an approximate 20% success rate (6). Fortunately, the risk of graft 
failure due to rejection in all of these prior withdrawal studies was extremely low (<1%) (6), making 
withdrawal a reasonably safe option. However, even so, 20% success is not considered clinically high 
enough to be acceptable as a standard of care approach and few centers, if any, are withdrawal patients 
routinely from IS therapy because of this low rate, except for in the setting of aggressive malignancy, 
infection or post-transplant lymphoproliferative disorder (PTLD). Thus, our goal is that SRL will be 
associated with higher than 20% success, allowing further study of this approach to make IS withdrawal 
clinically more feasible and acceptable to clinicians. 
1.4 Dose Rationale and Risk/Benefits 
All our LT patients are on different doses of SRL therapy currently (on average 1-2 mg daily) and will be 
tapered off of SRL according to the study procedures. There is no new drug therapy introduced in this 
study. It is hypothesized that 50% (vs. 80% historical CNI withdrawal) will develop reversible rejection 
during either minimization or withdrawal of SRL, requiring reinstitution of immunosuppressive therapy 
(i.e. failure of withdrawal). Fortunately, previous studies involving withdrawal of IS in LT recipients 
(mainly from CNI therapy) have shown that patients who fail to withdraw and develop rejection have a 
minimal (<1%) risk of graft failure when IS reinstituted (6). Only one patient in the major withdrawal 
studies developed chronic rejection and required retransplantation. Thus it is, in the vast majority, 
reversible. This may not be the case with SRL therapy although none of our previously SRL converted 
patients developed rejection or lost their grafts. Therefore, we feel the risk of withdrawal, while not 
insignificant, is fairly low in comparison to the high potential for benefit of being completely withdrawn 
from IS therapy altogether, satisfying the concept of clinical equipoise. All of these risks and benefits will 
be discussed with the patient at the time of informed consent. For IS withdrawn patients in our study, we 
expect at minimum stability or improvement in all of the secondary outcomes related to chronic IS 
therapy. For liver biopsies on all patients, the risk includes the following: bleeding, infection, perforation 
of an organ surrounding the liver (all < 1%). The patient will undergo a separate informed consent (for the 
procedure and is different than the actual study consent) prior to all liver biopsies. We feel that liver 
biopsy is necessary so that the patient will not proceed into the trial if any histological graft dysfunction 
or rejection is detected on biopsy. While liver biopsy is invasive and has associated risks, they are 
outweighed by the benefit of knowledge of liver histology prior to the intervention and follow-up. 
Decisions on whether to undergo the intervention can therefore be more accurately determined and 
provide more safety than risk to the patient. All of these risks will be clearly discussed with the patient 
during the study process and documented by appropriate consents.  
2 Study Objectives 
Primary Objective: The goal is to determine if operational tolerance (normal graft function without IS 
therapy x 12 months) can be achieved successfully in LT recipients withdrawn from SRL.   
 
Secondary Objectives 


 
 
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1. To investigate if cellular, allograft and proteogenomic metrics of immune regulation are predictive of 
and persist following successful SRL withdrawal in LT recipients.  
 
2. To analyze any initial clinical benefit of minimization and withdrawal of SRL therapy.   
3 Study Design 
3.1 General Design 
Study Overview 
The study proposed is a prospective trial of controlled SRL monotherapy minimization and withdrawal in 
up to 25 stable non-immune non-viremic LT recipients. Given the sample size calculations (see Statistical 
section), we plan to enroll up to 25 SRL monotherapy patients for this study (Figure 1). All patients will 
be consented to undergo laboratory evaluation as well as an enrollment liver biopsy and blood tests. If the 
patient meets inclusion/exclusion (I/E) criteria (see below), SRL will be minimized over 6 months until 
once a week dosing is achieved. Repeat clinical and immunological blood tests as above will be 
performed, and if no biochemical signs of rejection, SRL will be discontinued with blood tests and liver 
biopsy 12 months later for similar biochemical, histological and immunological measures. At any concern 
for rejection, liver biopsy and assays for equivalent biochemical, histological and immunological 
measures will be performed and if rejection is diagnosed on biopsy, a second attempt at withdrawal will 
not be performed. Patients will be monitored as standard of care with clinic visits every 6 months and 
laboratory tests every 2 weeks. The total study length will be 18 months: 6 month minimization phase and  
1 year follow-up after withdrawal success/failure. The primary outcome will be the proportion of tolerant 
patients off SRL therapy with normal liver biochemistry and graft histology at 18 months. Secondary 
outcomes will include the incidence, severity and reversibility of rejection, patient/graft survival, 
resolution of SRL and other non-specific IS effects, and the assessment of clinical/immunological 
biomarkers of tolerance. All continuous/categorical clinical variables will be compared with the 
appropriate statistical analyses.The goal is that the primary and secondary aims will provide valuable 
preliminary data to further elucidate the mechanisms of mTOR-I immunoregulation  and for determining 
the initial clinical success/feasibility of the mTOR-I approach (vs. historical 20% CNI withdrawal success 
seen in studies from the Immune Tolerance Network [ITN] and other groups). This is all so as to guide a 
submission to the Immune Tolerance Network for larger, more adequately powered prospective trials 
comparing SRL vs. CNI withdrawal and accompanying biomarker predictors of tolerance. If this pilot 
study fails to show a correlation between our biomarkers and the success/failure of SRL withdrawal, or is 
associated with an unacceptable low (e.g. <20%) rate of operational tolerance, then this would avoid the 
necessity for such large, expensive trials and support the continued development of alternative approaches 
to tolerance. 
 
Consent and Initial Phase of Enrollment (see Study Protocol Figure- Appendix A) 
These liver transplant candidates (up to 25 SRL) will be approached for consideration and informed 
consent into the study. The consent form will include discussion of the risks/benefits of their current 
therapy 
(SRL) 
and 
the 
planned 
minimization/withdrawal. 
Specifically, 
the 
risks 
of 
minimization/withdrawal (i.e. developing acute or chronic rejection, alloimmune hepatitis during any 
portion of the study), although unlikely, will be a major emphasis of the consent process. The consent 
form will also include the strict requirement for patients to follow all instructions from the PI in regard to 
the close laboratory follow-up occurring throughout the trial, to diagnose any episode of rejection or 
concern as early as possible to be able to respond appropriately. All of the study procedures will be 
discussed with the patient during clinic visits. They will be informed that neither participation nor refusal 
will influence their clinical care. All laboratory tests or costs related to their care in the study, with the 
exception of non-standard of care items (i.e. liver biopsies, blood assays), will be the responsibility of the 


 
 
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patient and/or his/her insurance company. They will be asked questions afterwards to verify 
comprehension and then sign the consent form documenting their agreement to participate.  A signed 
copy of the consent form will be given to them.  Participation is completely voluntary and they may 
discontinue participation the study at any time without affecting their care or participation in any other 
study. No financial compensation will be given. 
 
Screening Evaluation at Enrollment (see Study Protocol Figure) 
After long term (> 3 years post-LT, > 3 months on SRL monotherapy) on SRL monotherapy, 
inclusion/exclusion criteria will be reviewed and if appropriate, consent as above will be obtained. If the 
patient agrees and signs consent, baseline standard of care screening laboratories complete blood count, 
comprehensive metabolic panel, sirolimus (CBC, CMP, SRL trough level, fasting lipid profile, 
hemoglobin A1C [HbA1C], urine protein/creatinine ratio) and non-standard of care biomarker assays 
(blood 
immunophenotyping, 
proteogenomics) 
and 
liver 
biopsy 
(histology 
and 
graft 
immunohistochemistry) will be performed as dictated by the protocol. In addition, the liver biopsy will be 
used to determine stability in graft function (i.e. no evidence of rejection or immune-mediated hepatitis) 
before considering minimization/withdrawal. SRL minimization/withdrawal will only be performed if 
clinically, biochemically and histologically [by biopsy; liver transplant pathology read (Yang, Rao)] 
stable. Throughout the entire study, liver function tests will be monitored every 2 weeks (monthly is 
standard of care, so the interim non-standard of care 2 week blood tests will be covered by the study 
funds). Patients entering the minimization/withdrawal phases will be reduced by a total dose of 50% of 
their baseline dose every month until patients are on 0.5 mg SRL daily. At this point, every other day 
dosing will begin x 1 month. If no LFT abnormalities, twice weekly dosing x 1 month, then once a week 
dosing x 1 month. Prior to complete discontinuation, repeat clinical (screening labs above) and blood 
biomarker assays (blood immunophenotyping, proteogenomics) will be performed. Liver biopsy will not 
be performed at this juncture unless there are biochemical signs of liver injury. If complete withdrawal is 
deemed safe (no evidence of biochemical abnormalities) patients will be withdrawn completely (i.e. the 
once/week SRL dose discontinued) and followed off IS therapy for one year. In this time period, liver 
function tests will be monitored every 2 weeks, as usual, and repeat clinical (screening labs above), liver 
biopsy and blood (blood immunophenotyping, proteogenomics) assays performed at the end of this year 
or at any concern for rejection.  
 
Blood and Tissue Samples 
Standard of care laboratories (CBC, CMP) will be performed every month on all patients, and only LFTs 
performed in the interim 2 weeks between each month (non-standard of care; covered by the study funds) 
until complete withdrawal.  3 green top 5 mL and 3 red top 5 mL tubes of blood for biomarker assays will 
be taken on all patients prior to study enrollment, prior to complete withdrawal, and one year after 
withdrawal or at the time of suspected rejection.  Liver biopsy (one 3 cm biopsy- 2 cm for histology and 1 
cm for genomic assays) will be performed at baseline (pre-minimization) and 1 year post withdrawal. A 
separate clinical informed consent will be obtained each time a liver biopsy is performed. If the patient 
develops elevation in liver transaminases requiring a liver biopsy at any stage of the study protocol, blood 
(all for immune monitoring (IM) assays) will be requested from the patient at the time of the biopsy. If 
rejection occurs, the patient will be followed in the study until completion but not be further withdrawn 
from SRL or have a liver biopsy at the end of the study 
 
Follow-up 
Laboratory follow-up is described above- every 2 weeks throughout the trial until complete withdrawal. 
Any biochemical (or clinical) signs or significant liver function test abnormalities will be acted on 
immediately either by liver biopsy or a pause in IS withdrawal per investigator discretion. All patients 
will be seen in the clinic every 3 months to assess for any new signs or symptoms or resolution of drug 
side effects in the minimization or withdrawal arm. Quality of life questionnaires; Post Liver Transplant 


 
 
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Quality of Life Instrument (pLTQ) and the Promis-29 profile v1.0 will be administered at the study onset 
and after successful vs. unsuccessful withdrawal (end of study) 
 
Outcome Measures 
The primary outcome will be the proportion of patients off SRL therapy with normal liver biochemistry 
and graft histology at 12 months (i.e. tolerant). Thus, the incidence of graft dysfunction (acute rejection, 
immune mediated or autoimmune hepatitis, chronic rejection) or non-tolerance will be assessed in this 
SRL withdrawal group and compared to the historical CNI group (20% tolerant; 80% failure) as the 
primary endpoint. This rate will be a composite of the cumulative number of biopsy-proven graft 
dysfunctions requiring conversion back to SRL or additional IS therapy or discontinuation of 
minimization/withdrawal that occur during the course of the study. Major secondary measures compared 
will be the predictive capacity of the blood and graft biomarker assays (immunophenotyping, 
genomic/proteomics) before and after minimization/withdrawal in the success vs. failure groups. Clinical 
secondary outcomes will be compared: the number of infectious complications, liver biopsy 
complications, cardiovascular outcomes (i.e. blood pressure, diabetes control, lipid levels), renal function, 
hematopoietic parameters, gastrointestinal effects, or other side effects of SRL that may or may not 
improve or develop with minimization/withdrawal. These will all be documented by a study database 
during patient visits, electronic charts and/or by phone communication. Finally, quality of life (Post Liver 
Transplant Quality of Life Instrument (pLTQ) and the Promis-29 profile v1.0) will be analyzed at the end 
of the study to determine the effect of IS minimization/withdrawal on other health benefits. 
3.2 Primary Study Endpoints 
The primary outcome will be the proportion of tolerant patients off SRL therapy with normal liver 
biochemistry and graft histology at 12 months. 
3.3 Secondary Study Endpoints 
Secondary outcomes will include the incidence, severity and reversibility of rejection, patient/graft 
survival, resolution of SRL and other non-specific IS effects, and the assessment of 
clinical/immunological biomarkers of tolerance. 
3.4 Primary Safety Endpoints 
Safety of the study will be monitored closely and determined by the the incidence, timing, severity and 
reversibility of rejection with withdrawal, graft function (biochemical and histological) throughout and at 
the end of the study, resumption of other IS therapies due to rejection and their toxicities (SRL,CNIs, 
prednisone, mycophenolic acid, other), complications from phlebotomies and liver biopsies, and 
patient/graft survival (patient/graft loss very unlikely to occur based on prior studies) 
4 Subject Selection and Withdrawal 
4.1 Inclusion Criteria 
1) Adult male and female recipients of all races, ≥ 18-75 years of age 
2) Patients who underwent primary living or deceased donor liver transplantation ≥ 3 years (previous to 
screening ) and on ≥ 3 months of stable SRL monotherapy 
3) Recipient of single organ transplant only 
4) Liver transplant for non-immune, non-viral (no hepatitis B or hepatitis C virus unless currently non-
viremic) causes 
5) Ability to provide informed consent and to comply with the study protocol of IS withdrawal. 


 
 
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4.2 Exclusion Criteria 
1) Inability or unwillingness to provide informed consent 
2) Acute cellular rejection within 12 months prior to enrollment 
3) Viral (viremic hepatitis B virus [HBV] or hepatitis C virus [HCV]) or immune-mediated liver 
disease (Autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis) history 
4) Abnormal liver function tests: Direct bilirubin ≥ 1 mg/dL;  ([ALT, AST, GGT] or alkaline phosphatase 
[AlkPhos] ≥ 2x [ULN]); 5) Abnormal graft histology at enrollment: a) ≥ Grade 2 inflammation or stage 2 
fibrosis;  b) Acute or Chronic Rejection;  c)  De-novo Autoimmune Hepatitis; d) inflammation of >50% 
of portal tracts; e) Other pathology not-specified but deemed high risk per the PI and pathologist; 6) 
Ongoing or recurrent substance abuse 
7) Retransplantation or combined liver-other organ  
8) Human Immunodeficiency Virus(HIV) co-infection 
9) Glomerular Filtration Rate (GFR)<30 ml/min by estimated glomerular filtration rate ([eGFR]-
[MDRD-4]) 
4.3 Subject Recruitment and Screening 
Recruitment and Informed Consent 
 
Nonimmune, nonviremic liver transplant patients on SRL therapy meeting I/E criteria (except for liver 
biopsy which will be performed only if all other I/E criteria met) will be approached consideration and 
informed consent into the study. The consent form will include discussion of the risks/benefits of their 
current therapy (SRL) and the planned minimization/withdrawal. Specifically, the risks of 
minimization/withdrawal (i.e. developing acute or chronic rejection, alloimmune hepatitis during any 
portion of the study) and procedural risks will be a major emphasis of the consent process. The consent 
form will also include the strict requirement for patients to follow all instructions from the PI in regard to 
the close laboratory follow-up occurring throughout the trial, to diagnose any episode of rejection or 
concern (elevation of liver transaminases) as early as possible to be able to respond appropriately. All of 
the study procedures will be discussed with the patient during clinic visits. They will be informed that 
neither participation nor refusal will influence their clinical care. All laboratory tests or costs related to 
their care in the study, with the exception of non-standard of care items (i.e. liver biopsy,  IM assays), will 
be the responsibility of the patient and/or his/her insurance company. They will be asked a few questions 
afterwards to verify comprehension and then sign the consent form documenting their agreement to 
participate.  A signed copy of the consent form will be given to them.  Participation is completely 
voluntary and they may discontinue participation in either study at any time without affecting their care.  
 
Screening Evaluation at the time of Minimization/Withdrawal 
 
If the patient agrees and signs consent, history and physical, QOL (quality of life) survey, and screening 
laboratories (CBC, CMP, Trough IS level, HBA1C, Lipid profile, Urine prot/cr ratio) will be performed. 
Following this, single pass liver biopsy (histology and graft immunohistochemical [IHC] assays) and 
blood assays will be performed as part of the immunological monitoring protocol. In addition, the liver 
biopsy will be used to determine stability in graft function (i.e. no evidence of rejection or immune-
mediated hepatitis) before considering minimization. SRL minimization/withdrawal will only be 
performed if clinically, biochemically and histologically (by biopsy) stable, following the I/E criteria. 
Patients entering the minimization/withdrawal phases will be reduced every month by 50% of total dose 
until they reach 0.5 mg daily for one month. They will then be reduced further by to 0.5 mg every other 
day, then twice weekly, then once weekly (all separated by one month at a time) dosing. This should take 
approximately 6 months to complete the minimization phase as most patients are currently on 1-3 mg of 
SRL daily. Throughout the study time period, liver function tests will be monitored every 2 weeks. For 


 
 
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any patient throughout the study developing liver dysfunction (direct bilirubin >1 mg/dL; ALT, AST, or 
AlkPhos > 2xULN), this will be confirmed on a repeat blood test within 1-2 weeks and if persistent, liver 
biopsy will be performed. Immediately before complete withdrawal, i.e. repeat clinical (screening labs) 
and blood assays will be performed. If complete withdrawal is deemed safe (no evidence of biochemical 
abnormalities, i.e. direct bilirubin < 1 mg/dL; ALT, AST, or AlkPhos < 2xULN), patients will be 
withdrawn completely (i.e. the once/week SRL dose discontinued) and followed off  SRL therapy for one 
year. In this time period, liver function tests will be monitored every 2 weeks. At 12 months following 
withdrawal, the same history and physical, QOL survey, screening labs and blood IM assays will be 
performed as in the study initiation, as well as the liver biopsy- all for the primary endpoint. If rejection or 
immune-mediated hepatitis is seen on this post-withdrawal biopsy or at any time during the study period, 
the patient will be reinitiated on IS therapy appropriately per the PI and not have another withdrawal 
attempt. All patients will be followed until 1 year post-attempted withdrawal, even if unsuccessful, 
although patients developing rejection or resumption of IS prior to this period will not have a final liver 
biopsy performed (i.e. only those presumed tolerant or who did not have rejection). 
 
Blood and Tissue Samples 
Standard of care laboratories complete blood count, liver function tests (CBC, LFTs) will be performed 
every 2 weeks on all patients.  Please see Figure 1 above for sample collections. A separate clinical 
informed consent will be obtained each time a liver biopsy is performed. If the patient develops elevation 
in liver transaminases requiring a liver biopsy at any stage of the minimization/withdrawal phase, blood 
assays will be requested from the patient at the time of the biopsy.  These immunological data in rejecting 
patients will be important to compare to patients who successfully are minimized and withdrawn. 
 
Follow-up 
Laboratory follow-up (CBC, CMP) is described above- every 2 weeks throughout the trial. Any 
biochemical (or clinical) signs or significant liver function test abnormalities will be acted on 
immediately either by liver biopsy or resumption of the recent IS dose.  All patients will be seen in the 
clinic every 3 months to assess for any new signs or symptoms or resolution of drug side effects in the 
minimization or withdrawal arm. The Post Liver Transplantation Quality of Life and Promis-29 quality of 
life surveys and IS cost tally will be performed prior to the study onset and after successful vs. 
unsuccessful withdrawal (end of study). 
4.4 Early Withdrawal of Subjects 
4.4.1 When and How to Withdraw Subjects 
Subjects may be withdrawn from the trial at any time for any reason, either by personal choice or due to 
medical indications. Subjects who are non-compliant with study guidelines, medications and clinic 
examinations will be withdrawn from the study. The NU IRB may also discontinue the study at any point. 
The subjects will be fully informed of these actions.  
4.4.2 Data Collection and Follow-up for Withdrawn Subjects 
If patients are withdrawn prematurely, they will still be followed through the follow-up time period of the 
study and beyond as per standard medical care. These data are important to collect to determine 
differences in this group compared to those who completed the study. This will include patient and graft 
survival data, as well as laboratory values and immunosuppressive therapy. If the subject is lost to follow-
up, we will make every attempt to contact the patient (i.e. phone calls, certified letters, attempts at 
contacting next-of-kin, etc). 


 
 
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5 Subject Compliance Monitoring  
5.1 Subject Compliance Monitoring 
 
The investigator and research coordinator will have full contact with the patient throughout the course of 
the study. Subjects will be seen in the clinic at baseline and every three months through the study. 
Laboratory tests will be monitored by the investigator and research coordinator, and lapses in compliance 
with laboratory tests will be determined by monitoring a schedule of events and follow-up for each 
patient. In addition, the patient will be called on a monthly basis prior to each tapering of SRL therapy to 
document compliance with the dosing and for any new symptoms. Persistent non-compliance will be an 
indication to remove the patient from the study, per investigator discretion.  
 
5.2  Prior and Concomitant Therapy 
 
All concomitant medicines will be collected at each study visit (ie; every 3 months).  There are no 
restrictions on concomitant medicines/therapies except for additional immunosuppression (IS) therapy or 
other therapies deemed unsafe per the investigator. 
 
6 Study Procedures 
6.1 
Visit 1: Screening/Pre-Withdrawal: (occurs at a Standard of Care Visit): If the patient agrees 
after reading through the consent and discussing the study with the study physician and/or research 
coordinator/nurse they sign the consent, then a history and physical will be performed. The Post 
Liver Transplantation Quality of Life and Promis-29 quality of life surveys will be administered 
and, laboratory tests (CBC, CMP, Trough SRL level, HBa1C, Lipid profile, Urine prot/cr ratio, and 
tolerance/immunophenotyping/genomic/proteomic assays) will be performed. 
6.2 
Visit 2: Liver Biopsy (+2 weeks of Screening/Pre-Withdrawal Visit): Single pass liver biopsy 
(histology and graft immunohistochemistry assays) and blood immune/biomarker 
(tolerance/immunophenotyping/genomic/proteomic)  assays will be performed as part of the 
immunological monitoring protocol.  
6.3 
Monitoring during Minimization (No visits): SRL minimization will only be performed if 
clinically, biochemically and histologically (by biopsy) stable, following the I/E criteria. Patients 
entering the minimization phases will be reduced every month by 50% of total dose until they 
reach 0.5 mg daily for one month. They will then be reduced further by to 0.5 mg every other day, 
then twice weekly, then once weekly (all separated by one month at a time) dosing. This should 
take approximately 6 months to complete the minimization phase as most patients are currently on 
1-3 mg of SRL daily. Throughout the study time period, liver function tests will be monitored 
every 2 weeks. For any patient throughout the study developing liver dysfunction (direct bilirubin 
>1 mg/dL; ALT, AST, or AlkPhos > 2xULN), this will be confirmed on a repeat blood test within 
1-2 weeks and if persistent, liver biopsy will be performed.  
6.4 
Visit 3: End of Minimization and Pre-Withdrawal (6 month clinic visit): Immediately before 
complete withdrawal, i.e. repeat clinical (screening labs as SOC, except SRL level not needed) and 
blood (tolerance/immunophenotyping/genomic/proteomic- research) assays will be performed. If 


 
 
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complete withdrawal is deemed safe (no evidence of biochemical abnormalities, i.e. direct bilirubin 
< 1 mg/dL; ALT, AST, or AlkPhos < 2xULN), patients will be withdrawn completely (i.e. the 
once/week SRL dose discontinued) and followed off SRL therapy for one year. In this time period, 
liver function tests will be monitored every 2 weeks as per 6.3 above.  A history and physical will 
be performed as SOC (every six months visits are SOC at our transplant center). 
6.5 
Visit 4: Post-Withdrawal (12 month clinic visit- SOC): history and physical, laboratories (CBC, 
CMP) will be performed. In this time period, liver function tests will be monitored every 2 weeks 
as per 6.3 above. 
6.6 
Visit 5: Post-Withdrawal (18 month clinic visit- study end): At 12 months following withdrawal, 
the same history and physical, the Post Liver Transplantation Quality of Life and Promis-29 quality 
of life surveys, labs (CBC, CMP, HBa1C, Lipid profile, Urine prot/cr ratio- SOC; and 
tolerance/immunophenotyping/genomic/proteomic assays- research) will be performed as in the 
study initiation, as well as the research liver biopsy- all for the primary endpoint. If rejection or 
immune-mediated hepatitis is seen on this post-withdrawal biopsy or at any time during the study 
period, the patient will be reinitiated on IS therapy appropriately per the PI and not have another 
withdrawal attempt. All patients will be followed until 1 year post-attempted withdrawal, even if 
unsuccessful, although patients developing rejection or resumption of IS prior to this period will 
not have a final liver biopsy performed (i.e. only those presumed tolerant or who did not have 
rejection). 
 
7 Statistical Plan 
7.1 Sample Size Determination 
We do not plan to utilize a control/non-withdrawn or CNI comparator group as 1) this is an initial pilot 
mechanistic study; 2) patients can serve as their own control (rejection vs. success) for this initial effort; 
3) our prior studies have shown that statistically significant changes in the blood/graft biomarkers can be 
detected with just 20 patients. These results will be critical in guiding power calculations for larger, 
prospective, controlled studies testing the specific biomarker predictors that are identified herein and 
whether SRL vs. CNI withdrawal is associated with a higher % of tolerance. In addition, our sample size 
is limited by our available funds and the numbers of patients on SRL monotherapy in our center 
(estimated around 40) 
7.2 Statistical Methods 
We have performed within subject comparisons and measurements on PBMC and biopsy using 
appropriate paired analysis (paired t-test or equivalent nonparametric test (Wilcoxon Signed Rank test) 
(12, 13). Repeated measures of analysis of variance (RM-ANOVA) were used to test the trajectory 
variations over time.  Two-sided F-test statistics were applied using α=0.05. For proteogenomic 
bioinformatics analysis, we have reported for each gene/protein comparison the T-test p-value, its non-
parametric analog (Kruskal Wallis test), the fold change, and for multi-analyte signatures, the AUROC 
curve (C-index) and the q-values by member genes/proteins. We were then able to calculate the predictive 
accuracy, sensitivity and specificity of each signature. For clinical endpoints, categorical and continuous 
variables will be analyzed with appropriate tests (Fisher’s exact, T-test). P<0.05 will be considered 
significant 


 
 
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7.3 Subject Population(s) for Analysis 
All subjects (successful vs. failed, withdrawn from the study vs. maintained) will be followed and 
analyzed at study end. We will also document the number of patients screened who did not meet I/E 
criteria at the study initiation. 
8 Safety and Adverse Events  
8.1 Definitions 
Adverse Event 
By definition an adverse event (AE) is defined as:  any symptom, sign, illness or 
experience that develops or worsens in severity during the course of the study.  Intercurrent 
illnesses or injuries should be regarded as adverse events.  Abnormal results of diagnostic 
procedures are considered to be adverse events if the abnormality: 
 results in study withdrawal 
 is associated with a serious adverse event 
 is associated with clinical signs or symptoms 
 leads to additional treatment or to further diagnostic tests 
 is considered by the investigator to be of clinical significance 
Adverse events are classified as serious or non-serious. 
Serious Adverse Event 
.  A serious adverse event is any AE that is:  
 fatal 
 life-threatening 
 requires or prolongs hospital stay 
 results in persistent or significant disability or incapacity 
 a congenital anomaly or birth defect 
 an important medical event 
 
Events that are expected, anticipated, related to the standard of care treatment or 
known adverse events/serious adverse events from standard of care medications or 
pre-existing conditions will not be reported. 
 
Important medical events are those that may not be immediately life threatening, but are 
clearly of major clinical significance.   They may jeopardize the subject, and may require 
intervention to prevent one of the other serious outcomes noted above.  For example, drug 
overdose or abuse, a seizure that did not result in in-patient hospitalization, or intensive 
treatment of bronchospasm in an emergency department would typically be considered 
serious.  
 
All adverse events that do not meet any of the criteria for serious will be regarded as non-
serious adverse events.  
Adverse Event Reporting Period 
For the purposes of this study, Adverse events and Serious Adverse events will be defined 
as those that are related to the procedures performed specifically for the study which are: 


 
 
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liver biopsy (regardless if performed percutaneously, transjugularly or open) and 
phlebotomy which takes place only to draw study-related blood.  The study period during 
which adverse events must be reported is normally defined as the period from the initiation 
of any study procedures to the end of the study treatment follow-up.  For this study, the 
study treatment follow-up the last visit made for the study.   
Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  The pre-existing 
conditions will be recorded prior to the start of the study.  A preexisting condition should 
be recorded as an adverse event if the frequency, intensity, or the character of the condition 
worsens during the study period. 
General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting 
condition.  At the end of the study, any new clinically significant findings/abnormalities 
that meet the definition of an adverse event must also be recorded and documented as an 
adverse event.  
Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are 
resolved, the subject is lost to follow-up, or the adverse event is otherwise explained.  At 
the last scheduled visit, the investigator should instruct each subject to report any 
subsequent event(s) that the subject, or the subject’s personal physician, believes might 
reasonably be related to participation in this study.  The investigator should notify the study 
sponsor of any death or adverse event occurring at any time after a subject has discontinued 
or terminated study participation that may reasonably be related to this study.  The sponsor 
should also be notified if the investigator should become aware of the development of 
cancer or of a congenital anomaly in a subsequently conceived offspring of a subject that 
has participated in this study.  
Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of 
the following conditions is met:  
 The laboratory abnormality is not otherwise refuted by a repeat test to confirm the 
abnormality 
 The abnormality suggests a disease and/or organ toxicity 
 The abnormality is of a degree that requires active management; e.g. change of 
dose, discontinuation of the drug, more frequent follow-up assessments, further 
diagnostic investigation, etc. 
Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be 
documented and reported as a serious adverse event unless specifically instructed otherwise 
in this protocol.  Any condition responsible for surgery should be documented as an 
adverse event if the condition meets the criteria for and adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported 
as an adverse event in the following circumstances: 


 
 
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 Hospitalization or prolonged hospitalization for diagnostic or elective surgical 
procedures for a preexisting condition.  Surgery should not be reported as an 
outcome of an adverse event if the purpose of the surgery was elective or diagnostic 
and the outcome was uneventful. 
 Hospitalization or prolonged hospitalization required to allow efficacy 
measurement for the study. 
 Hospitalization or prolonged hospitalization for therapy of the target disease of the 
study, unless it is a worsening or increase in frequency of hospital admissions as 
judged by the clinical investigator. 
8.2 Recording of Adverse Events 
At each contact with the subject, the investigator/designee must seek information on adverse 
events by specific questioning and, as appropriate, by examination.  All clearly related signs, 
symptoms, and abnormal diagnostic procedures results should recorded in the source document, 
though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded.  The clinical course of 
each event should be followed until resolution, stabilization, or until it has been determined that 
the study treatment or participation is not the cause.  Serious adverse events that are still ongoing 
at the end of the study period must be followed up to determine the final outcome.  Any serious 
adverse event that occurs after the study period and is considered to be possibly related to the 
study treatment or study participation should be recorded and reported immediately. 
8.3 Reporting of Serious Adverse Events 
8.3.1 EC/IRB Notification by Investigator 
Reports of all serious adverse events (including follow-up information) must be 
submitted to the Northwestern University Institutional Review Board (NUIRB0 within 10 
working days.  Copies of each report and documentation of NUIRB notification and 
receipt will be kept in the Clinical Investigator’s binder. 
8.4 Stopping Rules  
If any of the following criteria are met, study enrollment will be suspended and participants will 
be maintained on their current IS therapy or may be required to restart IS therapy: 1) Rejection 
induced by IS weaning resulting in death, retransplantation or listing for retransplantation in any 
one subject; 2) Either steroid-resistant rejection or chronic rejection occurring during weaning in 
>10% of subjects (2 patients) 
8.5 Medical Monitoring 
The Principal Investigator (Dr. Levitsky) will oversee the safety of the study.  This safety 
monitoring will include careful assessment and appropriate reporting of adverse events as noted 
above.  Subjects will be assessed at their visits and during phone calls for Aes/SAEs. 


 
 
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9 Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the following:  
 What protected health information (PHI) will be collected from subjects in this study 
 Who will have access to that information and why 
 Who will use or disclose that information 
 The rights of a research subject to revoke their authorization for use of their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts will 
be made to obtain permission to collect at least vital status (i.e. that the subject is alive) at the 
end of their scheduled study period. 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source 
data are contained in source documents   Examples of these original documents, and data records 
include; but are not limited to: hospital records, clinical and office charts, laboratory notes, 
memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded 
data from automated instruments, copies or transcriptions certified after verification as being 
accurate and complete, microfiches, photographic negatives, microfilm or magnetic media, x-
rays, subject files, and records kept at the pharmacy, at the laboratories, and at medico-technical 
departments involved in the clinical trial. 
9.3 Case Report Forms  
An electronic case report form (eCRF) is the primary data collection instrument for the study.   
9.4 Records Retention 
  
It is the investigator’s responsibility to retain study essential documents for at least 2 years on 
site after the study has been terminated with the NUIRB.   These documents should be retained 
off site up to 13 more years at the PIs discretion.  If kept off site, the records will be stored at 
O’Hare Records, in Rosemont, IL. 
 
9.5 Auditing and Inspecting 
The investigator will permit study-related audits, and inspections by the NU IRB, government 
regulatory bodies, and University compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data 
etc.).  The investigator will ensure the capability for inspections of applicable study-related 


 
 
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facilities (e.g. clinic, diagnostic laboratory, etc.).  As an investigator in this study acceptance of 
potential inspection by government regulatory authorities and applicable University compliance 
and quality assurance offices is expected. 
10 Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 parts 312, 50, 56 and International Conference on Harmonization 
guidelines), applicable government regulations and Institutional research policies and 
procedures. 
 
This protocol and any amendments will be submitted to the NU IRB, in agreement with local 
legal prescriptions, for formal approval of the study conduct.  The decision of the NU IRB 
concerning the conduct of the study will be made in writing to the investigator.   
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  A copy of the Subject Informed Consent Form follows this protocol.  This consent form 
will be submitted with the protocol for review and approval by the NU IRB for the study.  The 
formal consent of a subject, using the NU IRB-approved consent form, must be obtained before 
that subject is submitted to any study procedure.  This consent form must be signed by the 
subject or legally acceptable surrogate, and the investigator or investigator-designated research 
professional obtaining the consent.  
11 Study Finances 
11.1 Funding Source 
Funding will be requested through a specific fund allocated to the transplant Hepatology group at 
Northwestern. Additional funding provided through an NIH grant. 
11.2 Conflict of Interest 
Any investigator who has a conflict of interest with this study (patent ownership, royalties, or 
financial gain greater than the minimum allowable by their institution, etc.) will have the conflict 
reviewed by a properly constituted Conflict of Interest Committee. All Northwestern University 
investigators will follow the University conflict of interest policy. 
11.3 Subject Stipends or Payments 
None 
12 Publication Plan 
 
Any and all scientific, commercial and technical information disclosed by the Principal 
Investigator (sponsor) in this protocol or elsewhere should be considered the confidential 
proprietary property of the PI and the Comprehensive Transplant Center (CTC). 
 


 
 
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The results of this study may also be used for teaching, publications, and/or presentations at 
scientific meetings.  If individual results are discussed, the identity of the subjects will be 
protected by use of a study code number. 
 
13 References 
 
1. 
Gonwa TA, Mai ML, Melton LB, Hays SR, Goldstein RM, Levy MF, et al. End-stage 
renal disease (ESRD) after orthotopic liver transplantation (OLTX) using calcineurin-based 
immunotherapy: risk of development and treatment. Transplantation. 2001;72(12):1934-9. 
2. 
Ojo AO, Held PJ, Port FK, Wolfe RA, Leichtman AB, Young EW, et al. Chronic renal 
failure after transplantation of a nonrenal organ. N Engl J Med. 2003;349(10):931-40. 
3. 
Morard I, Dumortier J, Spahr L, Hadengue A, Majno P, Morel P, et al. Conversion to 
sirolimus-based immunosuppression in maintenance liver transplantation patients. Liver Transpl. 
2007;13(5):658-64. 
4. 
Fairbanks KD, Eustace JA, Fine D, Thuluvath PJ. Renal function improves in liver 
transplant recipients when switched from a calcineurin inhibitor to sirolimus. Liver Transpl. 
2003;9(10):1079-85. 
5. 
Starzl TE. The "privileged" liver and hepatic tolerogenicity. Liver Transpl. 
2001;7(10):918-20. 
6. 
Levitsky J. Operational tolerance: past lessons and future prospects. Liver Transpl. 
2011;17(3):222-32. 
7. 
Battaglia 
M, 
Stabilini 
A, 
Roncarolo 
MG. 
Rapamycin 
selectively 
expands 
CD4+CD25+FoxP3+ regulatory T cells. Blood. 2005;105(12):4743-8. 
8. 
Turnquist HR, Raimondi G, Zahorchak AF, Fischer RT, Wang Z, Thomson AW. 
Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but 
enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance. J 
Immunol. 2007;178(11):7018-31. 
9. 
Baan CC, van der Mast BJ, Klepper M, Mol WM, Peeters AM, Korevaar SS, et al. 
Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the 
induction of FOXP3 in human T cells. Transplantation. 2005;80(1):110-7. 
10. 
Szabo G, Gavala C, Mandrekar P. Tacrolimus and cyclosporine A inhibit allostimulatory 
capacity and cytokine production of human myeloid dendritic cells. J Investig Med. 
2001;49(5):442-9. 
11. 
Gao W, Lu Y, El Essawy B, Oukka M, Kuchroo VK, Strom TB. Contrasting effects of 
cyclosporine and rapamycin in de novo generation of alloantigen-specific regulatory T cells. Am 
J Transplant. 2007;7(7):1722-32. 
12. 
Levitsky J, Miller J, Wang E, Rosen A, Flaa C, Abecassis M, et al. Immunoregulatory 
profiles in liver transplant recipients on different immunosuppressive agents. Human 
Immunology. 2009;70(3):146-50. 
13. 
Levitsky J, Mathew JM, Abecassis M, Tambur A, Leventhal J, Chandresekaran D, 
Herrera N, Al-Saden P, Gallon L, Abdul-Nabi A, Yang GY, Kurian S, Salomon D, Miller J. 
Systemic Immunoregulatory and Proteogenomic Effects of Tacrolimus to Sirolimus Conversion 
in Liver Transplant Recipients. Hepatology 2012;(in press). 


 
 
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14. 
Levitsky J, Gallon L, Miller J, Tambur AR, Leventhal J, Flaa C, et al. Allospecific 
regulatory effects of sirolimus and tacrolimus in the human mixed lymphocyte reaction. 
Transplantation. 2011;91(2):199-206. 
15. 
Mazariegos GV, Zahorchak AF, Reyes J, Ostrowski L, Flynn B, Zeevi A, et al. Dendritic 
cell subset ratio in peripheral blood correlates with successful withdrawal of immunosuppression 
in liver transplant patients. Am J Transplant. 2003;3(6):689-96. 
16. 
Martinez-Llordella M, Lozano JJ, Puig-Pey I, Orlando G, Tisone G, Lerut J, et al. Using 
transcriptional profiling to develop a diagnostic test of operational tolerance in liver transplant 
recipients. J Clin Invest. 2008;118(8):2845-57. 
17. 
Levitsky J, Mathew JM, CW Flaa, A Rosen, AR Tambur, J Miller. The human "Treg 
MLR": Immune monitoring for FOXP3+ T Regulatory Cell Generation. Transplantation. 2009 
Dec;88(11):1303-11. 
18. 
Mathew JM, Blomberg B, Fuller L, Burke GW, Ciancio G, Kenyon N, et al. A novel 
micro-cell-mediated lympholytic assay for the evaluation of regulatory cells in human 
alloreactive CTL responses. Journal of Immunological Methods. 2003;272(1-2):67-80. 
19. 
Levitsky J, Leventhal JR, Miller J, Huang X, Chen L, Chandrasekaran D, et al. Favorable 
effects of alemtuzumab on allospecific regulatory T-cell generation. Hum Immunol. 2011. 
20. 
Yu Y, Miller J, Leventhal JR, Tambur AR, Chandrasekaran D, Levitsky J, et al. 
Requirement of cognate CD4+ T-cell recognition for the regulation of allospecific CTL by 
human CD4+ CD127- CD25+ FOXP3+ cells generated in MLR. PLoS One. 2011;6(7):e22450. 
21. 
Levitsky J, Salomon DR, Abecassis M, Langfelder P, Horvath S, Friedewald J, et al. 
Clinical and plasma proteomic markers correlating with chronic kidney disease after liver 
transplantation. Am J Transplant. 2011;11(9):1972-8.