Biomarkers Predicting Successful Tacrolimus Withdrawal and Everolimus (Zortress) Monotherapy Early After Liver Transplantation
Phase: NA
Conditions: Liver Transplantation
Sponsor: Northwestern University

 Everolimus
Clinical Study Protocol- CRAD001HUS190T
Biomarkers Predicting Successful Tacrolimus Withdrawal 
and Everolimus (Zortress®) Monotherapy Early After Liver 
Transplantation
Authors:
Josh Levitsky, MD, MS
Principle or Coordinating 
Investigator
Levitsky, J.
Sub-investigators: Daniel Ganger, MD, Christopher Moore, 
MD, Laura Kulik, MD, Steven Flamm, MD, Lisa 
VanWagner, MD, and Mary Rinella, MD (Transplant 
Hepatology).
Michael Abecassis, MD, MBA, Jonathan Fryer, MD, Talia 
Baker, MD, Anton Skaro, MD, Juan Carlos Caicedo, MD, 
and Daniela Ladner, MD (Transplant Surgery)
Dan Salomon, PhD- Scripps Clinic, La Jolla, California
Document type:
Clinical Study Protocol
Version number:
03.1
Release date:
20-MAR-2017
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Clinical Study Protocol (Version No. 3.1)
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Table of contents
Table of contents..................................................................................................................2
List of tables.........................................................................................................................4
List of figures.......................................................................................................................4
List of abbreviations ............................................................................................................5
Glossary of terms.................................................................................................................8
Protocol synopsis .................................................................................................................9
1
Background........................................................................................................................12
2
Purpose and rationale.........................................................................................................15
3
Objectives ..........................................................................................................................15
3.1
Primary endpoints ..................................................................................................15
3.2
Secondary endpoints ..............................................................................................15
3.3
Exploratory objectives ...........................................................................................16
4
Study design.......................................................................................................................18
5
Population ..........................................................................................................................19
5.1
Inclusion criteria ....................................................................................................19
5.2
Exclusion criteria ...................................................................................................20
6
Treatment...........................................................................................................................21
6.1
Investigational and control drugs...........................................................................21
6.2
Treatment arms ......................................................................................................21
6.3
Treating the patient ................................................................................................21
6.3.1
Patient numbering..................................................................................21
6.3.2
Dispensing the study drug .....................................................................21
6.3.3
Study drug supply, storage and tracking ...............................................22
6.3.4
Instructions for prescribing and taking the study drug..........................22
6.3.5
Permitted study drug dose adjustments and interruptions.....................22
6.3.6
Other concomitant treatment .................................................................22
6.3.7
Study drug discontinuation and premature patient withdrawal.............23
6.3.8
Study completion and post-study treatment ..........................................23
7
Visit schedule and assessments..........................................................................................23
7.1
Patient demographics/other baseline characteristics..............................................24
7.2
Treatment exposure and compliance......................................................................24
7.3
Efficacy..................................................................................................................24
7.4
Safety .....................................................................................................................25
7.4.1
Physical examination.............................................................................26
7.4.2
Vital signs..............................................................................................26
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Clinical Study Protocol (Version No. 3.1)
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7.4.3
Height and weight..................................................................................26
7.4.4
Laboratory evaluations ..........................................................................26
7.4.5
Pregnancy and assessments of fertility..................................................27
7.4.6
Appropriateness of safety measurements ..............................................27
7.4.7
Other biomarkers...................................................................................27
8
Safety monitoring ..............................................................................................................27
8.1
Adverse events.......................................................................................................28
8.2
Serious adverse event reporting .............................................................................28
8.3
Pregnancies ............................................................................................................29
9
Data review and database management.............................................................................29
9.1
Data collection .......................................................................................................29
9.2
Database management and quality control ............................................................30
10 Data analysis (Any or all sections may be included based on type of study)....................31
10.1
Populations for analysis .........................................................................................31
10.2
Analysis of the primary objective(s)......................................................................31
10.2.1
Variable .................................................................................................32
10.2.2
Statistical hypothesis, model, and method of analysis ..........................32
10.2.3
Handling of missing values/censoring/discontinuations .......................33
10.2.4
Supportive analyses...............................................................................33
10.3
Analysis of secondary objectives...........................................................................33
10.3.1
Efficacy (secondary)..............................................................................33
10.3.2
Safety.....................................................................................................33
10.3.3
Biomarkers ............................................................................................34
10.4
Sample size calculation..........................................................................................34
10.5
Power for analysis of critical secondary variables.................................................35
10.6
Interim analysis......................................................................................................35
11 Ethical considerations........................................................................................................35
11.1
Regulatory and ethical compliance........................................................................35
11.2
Informed consent procedures.................................................................................35
11.3
Responsibilities of the investigator and IRB/IEC/REB .........................................35
11.4
Publication of study protocol and results...............................................................36
12 Protocol adherence.............................................................................................................37
12.1
Protocol Amendments............................................................................................37
13 Figures and References......................................................................................................37
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List of tables
Table 7-1
Assessment schedule .............................................................................23
List of figures
Figure 1: New CD4+CD25HighFOXP3+Treg "Recruitment" by Allo-specific Tregs Generated in 
the Presence of Media, TAC or SRL in MLR……………………………………………...38
Figure 2: Increase in Phenotypic Regulatory T Cells and Regulatory Dendritic Cells with 
Tacrolimus to Sirolimus Conversion………………………………………………………….39
Figure 3: Increased FOXP3+ cells in Liver Biopsies after Tacrolimus to Sirolimus 
Conversion……………………………………………………………………………………40
Figure 4: Heat Map of Immune/Inflammatory Gene Transcripts (n=93) Before and After 
Tacrolimus to Sirolimus Conversion…………………………………………………………41 
Figure 5: Blood and Biopsy mRNA differences in LT recipients with acute rejection, HCV 
recurrence and mixed AR/HCV-recurrence………………………………………………….42
Figure 6: Time Table for Sample Collection and Assessment……………………………….43
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List of abbreviations
AASLD
American Association for the Study of Liver Diseases
AE
Adverse event
ALT
Alanine aminotransferase
ANC
Absoulte neutrophil count
AR
Acute rejection
AST
Aspartate aminotransferase
AUROC
Area Under Receiver Operating Characteristic (Curve)
CFSE
Carboxyfluorescein succinimidyl ester
CKD 
Chronic kidney disease
CNI
Calcineurin inhibitor
CBC
Complete blood count
Cr
Creatinine
CRF
Case Report/Record Form
CRD
Clinical Research and Development
CPO
Country Pharma Organization
CRO
Contract Research Organization
CSR
Clinical Study Report
CTC
Comprehensive Transplant Center
DR
Donor
DSA
Donor specific antibodies
DSMB
Data Safety Monitoring Board
ECG
Electrocardiogram
eCRF
Electronic case report form
eGFR
Estimated glomerular filtration rate
EVL
Everolimus
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
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HCV-R
Hepatitis C virus recurrence
HgbA1C
Hemoglobin A1C
HIPAA
Health Insurance Portability and Accountability Act
HIV
Human Immunodeficiency Virus
HLA
Human leukocyte antigen
HLA-DR
Human leukocyte antigen-donor
ICH
International Conference on Harmonization of Technical Requirements for 
Registration of Pharmaceuticals for Human Use
ICH
International Conference on Harmonization
I/E
Inclusion/exclusion
ICH
Immunohistochemical
IL-2
Interleukin 2
IM
Immune Monitoring
IMS
Immune Monitoring Signals
IRB
Institutional Review Board
IS
Immunosuppression
IVRS
Interactive Voice Response System 
LFT
Liver function tests
LT
Liver transplantation
MAP
Multi-Analyte Profiling
MFI
Mean Fluorescence Intensity
miRNA
Micro ribonucleic acid
MLR
Mixed lymphocyte reaction
MPA
Mycophenolic acid
mRNA
Messenger ribonucleic acid
NK cells
Natural killer cells
NU 
Northwestern University
NU IRB
Northwestern University Institutional Review Board
OLT
Orthotopic Liver Transplant
PBMC
Peripheral blood mononuclear cell
PHI
Protected health information
PI
Principal Investigator
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PTLD
Post-transplant lymphoproliferative disorder
PRED
Prednisone
RM-ANOVA
Repeated Measures of Analysis of Variance
RN
Registered nurse
SAE
serious adverse event
SOC
Standard of Care
SRL
Sirolimus
SI
Stimulation indices
TAC
Tacrolimus
TAC-WD
Tacrolimus withdrawal
Tregs
T-regulatory cells
WBC 
White blood cell
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Glossary of terms
Assessment
A procedure used to generate data required by the study
Control drug
A study drug used as a comparator to reduce assessment bias, 
preserve blinding of investigational drug, assess internal study 
validity, and/or evaluate comparative effects of the investigational 
drug
Enrollment
Point/time of patient entry into the study; the point at which 
informed consent must be obtained (i.e. prior to starting any of the 
procedures described in the protocol)
Investigational drug
The study drug whose properties are being tested in the study; this 
definition is consistent with US CFR 21 Section 312.3 and is 
synonymous with “investigational new drug.”
Medication number
A unique identifier on the label of each medication package in 
studies that dispense medication using an IVR system
Patient number
A number assigned to each patient who enrolls in the study. When 
combined with the center number, a unique identifier is created for 
each patient in the study.
Stage
A major subdivision of the study timeline; begins and ends with 
major study milestones such as enrollment, randomization, 
completion of treatment, etc.
Period
A minor subdivision of the study timeline; divides phases into 
smaller functional segments such as screening, baseline, titration, 
washout, etc.
Premature patient withdrawal
Point/time when the patient exits from the study prior to the 
planned completion of all study drug administration and 
assessments; at this time all study drug administration is 
discontinued and no further assessments are planned
Randomization number
A unique identifier assigned to each randomized patient, 
corresponding to a specific treatment arm assignment
Stop study participation
Point/time at which the patient came in for a final evaluation visit 
or when study drug was discontinued whichever is later
Study drug
Any drug administered to the patient as part of the required study 
procedures; includes investigational drug and any control drugs
Study drug discontinuation
Point/time when patient permanently stops taking study drug for 
any reason; may or may not also be the point/time of premature 
patient withdrawal
Variable
Information used in the data analysis; derived directly or indirectly 
from data collected using specified assessments at specified time 
points
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Protocol synopsis
Title of study: Biomarkers Predicting Successful Tacrolimus Withdrawal and Everolimus 
(Zortress®) Monotherapy Early After Liver Transplantation
Purpose and rationale:  
The purpose of this study is to develop a biomarker strategy testing transplant 
immunoregulation to guide safe elimination of calcineurin inhibitor (CNI) therapy in favor of 
everolimus (EVL) early after liver transplantation (LT). We plan to analyze a set of biomarkers 
testing genomic and immunophenotypic measures of immune reactivity and regulation, 
expected to be enhanced by EVL and predictive of successful conversion. 
Objectives: 
To identify cellular and genomic metrics of immune regulation that may predict 
successful TAC withdrawal in favor of EVL early after LT. 
Our overall objective is to utilize biomarkers that will clarify the decision to consider tacrolimus 
withdrawal/EVL (TAC-WD/EVL) and maintain EVL without TAC. Proving the hypothesis 
that EVL promotes early immunoregulation in those patients not developing rejection with 
TAC-WD would provide:  
 1) rationale for early CNI to EVL conversion when the benefit and risk are highest
 2) bioassays to guide selection of appropriate candidates for TAC-WD/EVL vs. continued 
TAC in patients likely to develop rejection with CNI withdrawal.
Primary endpoint: 
The primary endpoint is the peripheral blood Treg% (or other immunoregulatory cells) and 
messenger ribonucleic acid (mRNA) acute rejection (AR) signatures seen prior to and during 
either successful (no rejection) vs. failed (rejection) TAC-WD/EVL. 
Secondary endpoints: 
Clinical endpoints as correlates for the success and failure of TAC-WD/EVL will serve as 
secondary endpoints. This will include but not be limited to the following at 6 months (± 1 
month) post-LT: liver allograft and renal function, EVL & TAC side effects, 
infectious/malignant complications, and patient/graft survival. At study end, all secondary 
endpoints will be statistically compared between those who succeed or fail TAC-WD/EVL.
Population: 
We will select adult (≥ 18 yrs. of age) LT recipients without immune disease, active viral 
disease, and previous or combined organ transplantation to avoid the confounding of coexistent 
inflammatory/infectious processes and prior transplants on the biomarker analysis. 
Inclusion/Exclusion criteria: 
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Inclusion Criteria: 

Adult LT candidates ≥ 18 years of age

Listed for or recent (within 2 months) recipient of deceased or living donor 
liver transplantation
Exclusion Criteria: 

Combined or previous organ transplantation

Human immunodeficiency virus (HIV) infection

Inability to provide informed consent or comply with the protocol.

Transplantation for autoimmune liver disease (Autoimmune hepatitis, 
Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis)
Investigational and reference therapy: 
Zortress® (everolimus) at advised doses and trough levels. 
Study design: 
Overall: Prospective, open-label pilot study comparing immunoregulatory assays in non-
viremic LT patients undergoing TAC-WD/EVL. The patients will serve as their own control 
for assays, clinical, biochemical, and histological measures, and the groups will be divided and 
compared by the presence or absence of rejection before, during and after the TAC-WD/EVL 
phase (months 0-6 (± 1 month)). As this is a pilot study, we hope to generate these preliminary 
data to power a larger trial testing the specific biomarker(s) at more specific time-points 
identified here in more definitively determining who may or may not be successful with 
undergoing CNI withdrawal in favor of EVL. 
Efficacy assessments: 

The proportion of patients who are successfully withdrawn from TAC and continued on 
EVL without rejection or other complications at six months (± 1 month)  post-LT.

The following assessments following addition of EVL at one to two months and 
attempted withdrawal of TAC between 3-6 months (± 1 month) in favor of EVL 
maintenance: liver allograft and renal function, EVL & TAC side effects and 
discontinuations, infectious/malignant complications, and patient/graft survival. 
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
Biomarker assessments: Peripheral blood Treg% (or other immunoregulatory cells) and 
messenger ribonucleic acid (mRNA) AR signatures seen prior to and during either 
successful (no rejection) vs. failed (rejection) TAC-WD/EVL. 
Other assessments: 

Other samples collected for future studies (see exploratory objectives below)
Data analysis: 
The primary objective is to test the working hypothesis that, in a prospective study, 
cellular and genomic markers of immunoregulation become more pronounced after early 
rejection-free TAC-WD/EVL compared to failed (rejection) TAC-WD. Conversely, if these 
markers do not signify immunoregulation, we hypothesize that TAC-WD/EVL will not be 
successful (i.e. rejection will ensue requiring reinstatement of TAC). Our hypothesis is based 
on our previous observations that late mTOR-I conversion increased % of Treg/DCreg in 
blood/tissue sites and generated immunoregulatory proteogenomic signatures and that AR can 
be diagnosed in the peripheral blood by genomic microarrays. Therefore, the plan is to perform 
sequential flow cytometry immunophenotyping and genomic signature analysis in the blood 
before, during and after attempted TAC-WD/EVL. 
Studies Planned and Methods:
i) Blood Immunophenotyping. The approach will be to conduct flow cytometry 
immunophenotyping of peripheral blood before TAC-WD initiation (3 months (± 1 month)), 
right at total TAC-WD (4 months (± 1 month)), 2 weeks after full TAC-WD (4.5 months (± 1 
month)) and study end (6 months (± 1 month)) post-LT to analyze cellular changes. Methods: 
The Immune Monitoring Laboratory at NU will perform the PBMC immunophenotyping. For 
statistical analysis, we perform within subject comparisons on PBMC for both successful and 
failed TAC-WD/EVL groups using appropriate paired analysis (paired t-test or Wilcoxon 
Signed Rank test). For the main hypothesis testing, repeated measures of analysis of variance 
(RM-ANOVA) are used to test the trajectory variations between the groups on PBMC over 
time. Two-sided F-test statistics are applied; α=0.05 (stats by NU Transplant Statistician).
ii) Gene Expression Microarrays:  The approach will be to conduct parallel serial 
genomic bioassays of peripheral blood before and after attempted TAC-WD/EVL (same time 
points as immunophenotyping). Methods: The Genomics Core at Scripps will perform the 
microarray analysis. Blood will be collected in PaxGene tubes and sent to Scripps for the gene 
microarrays. For statistical analysis, we report for each mRNA and microRNA comparison the 
T-test p-value, its non-parametric analog (Kruskal Wallis test), the fold change, and for multi-
analyte signatures, the area under the receiver operating curve (concordance index/C-index) and 
q-values by member genes/proteins. We then calculate the predictive accuracy, sensitivity and 
specificity of each signature (statistics by Dr. Daniel Salomon’s informatics group at Scripps 
Clinic)
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1
Background
It has been assumed that standard IS agents, the calcineurin inhibitors (CNI) cyclosporine and 
tacrolimus (TAC), are indefinitely required to prevent allograft rejection following liver 
transplantation (LT). However, this has occurred at the expense of chronic CNI toxicity, e.g. 
chronic kidney disease (CKD), metabolic complications, infections and malignancy1-3. The 
mortality related to CKD is only remediated by a curative kidney transplant, usurping costs and 
valuable organ resources3. While CNI therapy is universally given immediately after LT, 
subsequent conversion from CNI to non-CNI therapy could reduce these adverse effects. 
Agents such as mammalian target of rapamycin inhibitors (mTOR-I) sirolimus (SRL) and 
everolimus (EVL) have a different mechanism of action and may stabilize or improve renal 
function in patients with CKD related to CNI therapy4-13. However, most of the studies have 
involved late CNI to mTOR-I conversion when CKD is already “set in” and likely irreversible. 
Thus, early full conversion (<1 year post-op), i.e. CNI discontinuation in favor of mTOR-I 
therapy) before the onset of irreversible CKD, would be the most optimal approach. However, 
higher SRL doses and troughs (≥8 ng/mL) are required in this time period given the lower 
immunosuppressive potency of SRL vs. CNI, but this can result in intolerable SRL side effects 
and discontinuation in ~30% and still lead to rejection 14-16. In addition, SRL has known risks 
of hepatic artery thrombosis in the early post-operative period and cause mortality in liver 
recipients, thus leading to a “black box warning” and lack of indication in this population. 
Taking these considerations into account, safer, more tolerable mTOR-I therapy (such as EVL) 
would be preferable for use in the LT population.
Nevertheless, faced with CKD progression, clinicians are commonly confronted with 
difficult decisions, none of which are clearly considered the most optimal: 1) maintain CNI 
therapy and accept the nephrotoxicity; 2) reduce CNI to sub-therapeutic levels and risk acute 
rejection; 3) reduce CNI doses and add other IS (mycophenolic acid or mTOR-I) to decrease 
the rejection risk, but accept the potential for CKD progression given the continued CNI therapy 
and the combined toxicity/immunosuppression of the second agent; 4) stop CNI and convert to 
a non-nephrotoxic agent (mTOR-I like EVL) to minimize CNI effects and over-
immunosuppression. While some studies have not shown an increased risk of rejection with the 
last approach12,13, a recent major randomized trial did demonstrate a higher risk compared to 
either TAC monotherapy or combination low dose TAC and EVL17. Despite this arm (TAC-
withdrawal and continued EVL; TAC-WD/EVL) being stopped early because of this, the 
remaining patients had the most preserved glomerular filtration rate (GFR) at one-year post-
LT. Thus, it would represent a major advance to be able to determine more specifically which 
patients would fare well (improved GFR without AR) or poorly (rejection) with early TAC-
WD/EVL. However, the absence of serial, specific biomarkers of immunoregulation available 
to guide this approach is a significant roadblock to achieving this endpoint. 
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Therefore, specific to this proposal and supported by our unique data in LT recipients18-20, an 
unexplored advantage of mTOR-I therapy lies in its potential to promote an immunoregulatory 
state. This could facilitate success with early CNI withdrawal in favor of EVL under current 
target doses/trough levels. As the most immunoregulatory organ transplanted, the liver houses 
numerous extramedullary immunoregulatory hematopoietic cells and secretes several 
immunoregulatory proteins21-24. The percentage of LT recipients able to undergo IS withdrawal 
directly from CNI therapy appears to be the highest of all organ recipients, although still only 
~20% successful 24. This clinically unacceptable rate may be due to CNI mechanisms inhibiting 
immunoregulation and the lack of predictive assays to detect immunoregulation. A key 
difference between mTOR-I and CNIs is their effect on regulatory T cells (Tregs) important in 
the suppression of alloimmunity. As an inhibitor of downstream IL-2 signaling after T cell 
activation, mTOR-I blocks alloreactive T cell proliferation but appears to promote Tregs 
(CD4+CD25highFOXP3+), regulatory cytokines (TGF-β1), and tolerogenic dendritic cells (DC2, 
ILT3+/4+) 25-31. In contrast, CNI therapy inhibits IL-2 transcription and has been thought to 
negatively impact Treg generation32-35. 
We have also uniquely demonstrated robust donor-specific regulatory effects of SRL in 
MLR cultures18. In these studies, TAC, SRL or media without agents were added separately to 
MLRs using HLA 2 DR matched and mismatched volunteers. Stimulation indices (SI) of 3H-
TDR uptake, cell proliferation, and the generation of carboxyfluoroscein succinimidyl ester 
(CFSE) labeled CD4+CD25HighFOXP3+ cells by flow cytometry were initially compared. Each 
group of MLR-generated cells were then added as third components to CFSE labeled 
responding cells in freshly prepared primary MLRs, to determine allo-specific vs. non-specific 
inhibitory and Treg “recruitment” effects (in vitro “infectious tolerance”). Tacrolimus inhibited 
SI and CD4+CD25HighFOXP3+ cell generation in both HLA-DR matched and mismatched pairs, 
particularly at therapeutic levels (≥ 5 ng/ml). SRL had an equivalent lymphoproliferative 
inhibitory effect in matched pairs but was associated with a significantly higher % generation 
of CD4+CD25HighFOXP3+ cells. SRL-MLR-generated Tregs added as third components allo-
specifically inhibited MLR proliferation and “recruited” additional autologous Tregs compared 
with addition of TAC- or media-MLR-generated cell preps [Fig. 1, ref18]. These results can 
explain the differing in vitro effects of these agents, now with donor-specific regulatory 
implications for mTOR-I. The immunoregulatory effects of EVL appear to be even more 
profound than SRL. In a Novartis supported laboratory study (submitted to the American 
Transplant Congress; manuscript in preparation), we analyzed and compared the in vitro 
immunoregulatory effects of EVL vs. SRL vs. TAC and found the greatest augmentation of 
Tregs with EVL in culture, particularly in combination with mycophenolate. 
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Clinically, we demonstrated in another study that LT recipients previously converted to 
SRL had higher percentages of PBMC Tregs compared to recipients on other IS therapies 19. 
This led to a prospective TAC to SRL conversion trial supported by the American Association 
for the Study of Liver Disease Career Development Award and Wyeth/Pfizer (Levitsky) 20. Our 
aim was to determine if systemic phenotypic Tregs (CD4+CD25highFOXP3+), regulatory 
dendritic cells (DCregs: ILT3+/4+), and immunoregulatory proteogenomic signatures increase 
in LT recipients converted directly from TAC to SRL monotherapy. Twenty non-immune non-
viremic LT recipients were successfully converted for renal dysfunction. The mean eGFR 
increased from 45.9±7.9 to 49.6±10.8 (p=0.003). Tregs increased significantly in PBMC, 
marrow [Fig. 2, from ref 20] and LT biopsy cultures. Biopsy immunohistology FOXP3:CD3 
and CD4:CD8 ratios became significantly higher after conversion [Fig. 3, from ref 20]. 
Peripheral blood DCregs increased significantly (Fig. 2, p<0.01). Both pre- (TAC) and post- 
(SRL) conversion sera inhibited MLR proliferation, although only TAC sera suppressed Treg 
generation. 289 genes and 22 proteins, many of which are important in immunoregulatory 
pathways, were newly expressed after conversion [Fig. 4: from ref 20]. In conclusion, these 
studies demonstrated that TAC to SRL conversion increases systemic (blood, allograft, 
marrow) Tregs, blood DCregs and regulatory proteogenomic signatures in LT recipients. 
However, it is not known if this profile correlates with and predicts successful early mTOR-I 
use (i.e. TAC-WD/EVL) as in this proposal.
Equally important as the chosen IS regimen is identifying predictive biomarkers, such 
as those we have tested above. Various reports demonstrate a higher percentage of 
CD4+CD25highFOXP3+ cells, DC2, γδ T cells and specific genomic signatures in tolerant LT 
recipients versus those on maintenance IS 36-45. Our collaborative group has previously 
performed proteogenomic assays (mRNA profiles, proteomic analytes) to identify markers of 
acute rejection (AR) and chronic allograft nephropathy in kidney recipients, as well as CKD 
post-LT46-48. In addition, we recently conducted a study profiling (mRNA gene expression) 114 
blood and 57 liver biopsy samples of patients with different clinical phenotypes, and have 
identified both blood and tissue mRNA signatures that can distinguish AR from other major 
causes of graft injury (hepatitis C recurrence, mixed HCV-R and AR, other causes) in LT 
recipients with high predictive accuracy (AUC 0.89 – 0.9; Figure 5)49. These biomarkers have 
the potential to be useful to enhance the specificity of diagnosis, particularly in managing 
patients 
with 
contrasting 
etiologies 
(e.g., 
AR 
vs. 
HCV-R). 
In 
addition, 
the 
immune/inflammatory nature of these AR profiles may help inform decisions to perform liver 
biopsies and in immune monitoring during immunosuppression reduction, as proposed herein.  
Yet, the presence of ‘snap-shot’ non-specific cell populations and differential gene/protein 
arrays only provides circumstantial evidence for their role in immunoregulation50,51. 
Prospective sequential assays would provide a more accurate measure of the effects of mTOR-I 
on immunoregulation. We anticipate that these expression patterns will be useful as regulatory 
signatures that may predict successful CNI withdrawal, as suggested by our recent trial and 
other reports20,39,42. 
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2
Purpose and rationale
We propose to develop a biomarker strategy testing transplant immunoregulation to guide safe 
elimination of CNI therapy in favor of EVL early after LT. We plan to analyze a set of 
biomarkers testing genomic and immunophenotypic measures of immune reactivity and 
regulation, expected to be enhanced by EVL and predictive of successful conversion. These 
bioassays will be compared in this initial pilot study prospectively between non-viremic LT 
recipients on TAC + EVL combination therapy (EVL added at 1 to 2 months) who are either 
successfully (no rejection) or unsuccessfully (rejection) withdrawn from TAC at month 3-4 (± 
1 month) from LT. Our overall objective is to utilize biomarkers that will clarify the decision 
to consider TAC-WD/EVL and maintain EVL without TAC. Proving the hypothesis that EVL 
promotes early immunoregulation in those patients not developing rejection with TAC-WD 
would provide 1) rationale for early CNI to EVL conversion when the benefit and risk are 
highest and 2) bioassays to guide selection of appropriate candidates for TAC-WD/EVL vs. 
continued TAC in patients likely to develop rejection with CNI withdrawal.
3
Objectives
Primary Objective: To identify cellular and genomic metrics of immune regulation that 
may predict successful TAC withdrawal in favor of EVL early after LT. 
It is our hypothesis that these markers of immunoregulation are more pronounced with 
successful (no rejection) vs. unsuccessful (rejection) TAC-WD/EVL. The approach will be to 
analyze the peripheral blood for phenotypic Tregs correlating with mRNA footprints of 
regulation. These analyses will be performed in a prospective pilot clinical trial of LT recipients 
on standard combination TAC and EVL at 1 to 2 months post-LT selected to undergo TAC-
WD/continued EVL at 3-4 months (± 1 month). Thus, based on the clinical trials, we expect 
rejection to occur in 20% in the 1-2 month period following TAC-WD/EVL and that this will 
be predicted by bioassays analyzed prior to and/or immediately after full TAC-WD. 
Alternatively, we expect 80% to not experience rejection with TAC-WD/EVL and that this will 
be predicted by bioassays analyzed prior to and/or right after full TAC-WD. Therefore, the use 
of these assays would eventually serve to detect and predict appropriate candidates for 
successful early TAC-WD/EVL, enhancing the use of EVL in LT and allowing patients to be 
spared of CNI toxicity past the first 3-4 months following LT. 
3.1
Primary endpoints
The primary endpoint is the peripheral blood Treg% (or other immunoregulatory cells) and 
mRNA AR signatures seen prior to and during either successful (no rejection) vs. failed 
(rejection) TAC-WD/EVL.
3.2
Secondary endpoints
Clinical endpoints as correlates for the success and failure of TAC-WD/EVL will serve as 
clinical secondary endpoints. This will include but not be limited to the following at 6 months 
(± 1 month) post-LT: liver allograft and renal function, EVL & TAC side effects, 
infectious/malignant complications, and patient/graft survival. At study end, all secondary 
endpoints will be statistically compared between those who succeed or fail TAC-WD/EVL.
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The other mechanistic biomarker endpoints tested potentially in the future (unfunded 
exploratory objectives) will focus on comparing biopsy and peripheral blood microarray 
signatures at the time of a rejection biopsy, comparing these biopsy signatures in rejections that 
occur before and after TAC-WD, and other predictive biomarkers (miRNA, DSA, proteomic, 
donor specific assays- see exploratory objectives below). 
The primary safety endpoints are the development of rejection with TAC-WD and any graft 
failure due to rejection, the latter of which is not expected to happen based on the registration 
studies. We expect that 20% of patients will experience rejection that is reversible with 
resumption of TAC +/- corticosteroids, without any graft loss, all based on the registration 
studies. However, if any patient experiences a graft loss due to the protocol (TAC-WD) or ≥30% 
rejection is seen, the study will be terminated due to safety concerns.
3.3
Exploratory objectives
As this is a pilot exploratory study, a number of possibile directions may emanate from these 
initial results: 
1) If the immunophenotypic and/or genomic biomarkers are preliminarily predictive (primary 
endpoint met) of AR, we would then imminently request funding to adequately power a larger 
study focusing in on those specific time points and biomarkers identified from the initial pilot 
study. This could include expansion of our single center study or perhaps a multicenter trial 
utilizing the AR biomarkers identified to guide patient management - e.g. continued TAC 
WD/EVL in patients without serial AR signals vs. no TAC WD or TAC resumption in patients 
with these predictive AR signals to avoid AR. This type of personalized approach would be 
transformational if it could be instituted in clinical practice - to avoid CNI toxicity in those 
predicted not reject and to avoid rejection in those predicted to reject with CNI withdrawal. 
2) If the immunophenotypic and genomic biomarkers are not predictive (primary endpoint not 
met) of AR, we would then request funding for other biomarkers testing from the blood samples 
we collected and stored pre/during/post-TAC WD. There may be alternative biomarkers that 
are more predictive of AR that will need to be explored, and we will have these samples 
available. These would include, but not limited to, the following assays which we have 
experience in performing:
i)
MicroRNA: miRNA measures can be performed from whole blood collected at each time 
point using the Illumina Truseq protocol.  In brief, Truseq small RNA library preparations 
are done on RNA from clinical samples that have been extracted using the Trizol protocol 
and purified using the RNEasy RNA purification method that has been standardized for 
small RNA preservation. 
ii) Donor Specific Antibodies (DSA): The adverse effects of DSA in clinical LT have been 
increasing recognized as shown by recent studies linking the development of DSA to 
rejection episodes 52,53. Therefore, we can test for the development of DSA in the current 
cohort of patients at the same time points. Since estimation of DSA is expensive, we can 
monitor for the presence of panel reactive antibodies in the sera serially at the various time 
points, and only test those with high PRA for DSA. The NU Comprehensive Transplant 
Center Lab can perform these assays. 
iii) Cellular and Plasma Proteomics: Our recent studies of TAC to SRL conversion in LT had 
revealed protein profiling of the serum/plasma can yield important information on the 
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immunoregulatory status of the patients20. Therefore, we will collect and store sera for 
future serial protein analysis (same time points), either as an antibody approach contracted 
out to Myriad RBM®, Austin, TX or performed at NU via a collaboration with Dr. Neil 
Kelleher whose laboratory performs top down/bottom up proteomics (both cells and 
plasma) by mass spectrometry.
iv) Donor-specific Immunoregulation. Regulatory T cells with donor-specificity are critical in 
maintaining transplant immunoregulation and may be even more sensitive than 
immunophenotyping or proteogenomic arrays. In future studies, pending funding, the 
approach will be to perform the Treg-MLR, assessing both donor-specific 
immunoregulation and hyporesponsiveness serially before and after EVL addition and 
TAC-WD/EVL or at rejection as a comparator - all compared with non-specific measures 
collected at the same time points 18,54,55. For this, donor spleen/lymph nodes at organ 
retrieval and recipient lymph nodes (collected from the native explant liver hilum) are 
cryopreserved at the time of transplantation. Recipient pre-transplant cryopreserved CFSE-
labeled lymph node cells or PBMC (1-5x105) are stimulated with cryopreserved donor or 
third party irradiated cells (1-5x105) in the presence of recipient PKH26 labeled pre- vs. 
post- transplant PBMC as third component modulators (as low as 5x102 cell dilutions). After 
7 days, standard 18-hour 3H-TdR incorporation assays and CD4+ CFSE quantitation 
(lymphoproliferation) are performed and the percentage inhibition calculated. The %Treg 
(CD4+CD25highFoxP3+ cells) are calculated in the CFSE-labeled responder population by 
flow cytometric analyses after gating out the PKH26 labeled modulators. RM-ANOVA is 
performed to test the hypothesis that recipients’ EVL-influenced post-operative cells 
“recruit” Tregs and inhibit proliferation in vitro in a donor-specific manner vs. TAC-
influenced cells, particularly in non-rejecting vs. rejecting subjects.
3) Finally, in either scenario, there may be interest from our group or Novartis in comparing 
diagnostic (not predictive) peripheral blood and biopsy genomic biomarkers at the time of AR 
occuring before and after TAC WD/EVL. We have already identified genomic AR signatures 
in blood and biopsy from our prior work (all TAC-treated recipients), which can serve as 
comparative data for this trial49. There may be differences in AR signals in TAC-treated patients 
vs. those who have TAC withdrawn and are on EVL monotherapy. With additional future 
funding, we can perform the same mRNA as above at the time of any AR and compare 
rejections that occur pre- and post-TAC withdrawal. This will help identify signals that may be 
more specific of EVL (CNI-free) rejection to be able to clinically utilize these signals to guide 
diagnosis and patient management. Also, we can determine if biopsy vs. peripheral blood 
biomarker AR signals are similar or different, so as to ascertain if blood instead of biopsies 
(which are risky & costly) can be utilized for rejection diagnosis specific to EVL (CNI-free) 
therapy. For now, we will store blood (sera/plasma, PBMC, RNA) and biopsy tissue (RNAlater) 
at the time of AR if these analyses are of future interest.
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4
Study design
Overall: Prospective, open-label pilot study comparing immunoregulatory assays in non-
viremic LT patients undergoing TAC-WD/EVL. The patients will serve as their own control 
for assays, clinical, biochemical, and histological measures, and the groups will be divided and 
compared by the presence or absence of rejection before, during and after the TAC-WD/EVL 
phase (months 0-6 (± 1 month)). We expect to find assay measures (primary objective) 
consistent with immunoregulation in patients who are successfully withdrawn from TAC 
compared to signs of immunoreactivity/lack of immunoregulation in those who fail TAC-WD 
due to rejection and require TAC reinstitution. 
Clinical Trial & Timelines (Figure 6 in Appendices):  Patients will be approached for study 
enrollment within six months before LT but can be enrolled up until 60 days post-LT. At 
transplant if possible, patients consented pre-LT will have donor spleen/lymph node cells and 
recipient lymph node/blood cells/sera cryopreserved. This will be collected for potential future 
studies of donor-specific immunoregulation and donor specific antibodies (funds not requested 
– see Exploratory Objectives). Post-transplant, patients will be treated as standard of care with 
the following protocol (no antibody induction): TAC therapy (target trough 7-10 ng/mL) +/- 
MPA between 1-3 g/d (Cellcept®, Myfortic® or generic alternate), corticosteroids (intravenous 
solumedrol 500 mg daily day 2, prednisone taper from 200 mg down to 20 mg daily at week 1, 
then slow taper to 5 mg daily by month 3). Patients not consent pre-LT will be approached 
within the first 60 days post-LT. At month 1 to 2 post-LT , study continuation criteria will be 
determined (see inclusion criteria). If met, EVL will be started (MPA discontinued if patient 
treated with this) and the EVL goal troughs will be 5-8 ng/ml and TAC 3-5 ng/ml 17. At 3 
months (± 1 month) post-LT, patients on stable TAC + EVL meeting continuation criteria will 
undergo the TAC-WD/EVL phase. TAC will be reduced by 50% of daily dose. After two 
weeks, if liver enzymes are stable, TAC will be reduced 50% each week and discontinued at 
month 4 (± 1 month) post transplant, if criteria are met. From month 4-6 (± 1 month), the 
patients will be on EVL therapy alone (trough 5-8 ng/ml) with stable prednisone 5 mg/day until 
study end (6 months (± 1 month)) or rejection (expected in ~20%). 
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Peripheral blood bioassays will be performed pre-TAC-WD (1 to 2 months and 3 months (± 1 
month)), immediately pre-TAC-WD (4 months (± 1 month)), 2 weeks post-TAC-WD (4.5 
months (± 1 month) post-LT), at biopsy-proven rejection in any phase of the study (before or 
after TAC-WD phase), and at 6 months (± 1 month) post-LT (Timeline Figure 6). We will also 
store extra blood samples (funds not requested – See Exploratory Objectives). Liver biopsies 
for standard histology will be performed only for cause at the time of rejection and not required 
by the protocol – extra tissue placed in RNAlater will be collected (funds not requested). The 
blood biomarkers will be compared between the groups (AR vs. no AR). Additionally, the 
patients will be monitored as standard of care with clinical laboratory tests (CBC, liver/renal 
function tests, IS trough levels) for other respective secondary endpoints (hematologic, 
liver/renal function) every 1-2 weeks. Clinical laboratory tests for other endpoints 
(hyperlipidemia- lipid profiles, diabetes- HbA1C, proteinuria- urine protein/creatinine) will be 
performed pre-EVL addition and at 6 months (± 1 month) post-LT, all per our standard of care. 
Routine clinic visits will occur per standard of care (1, 2, 3, 4 & 6 months) to monitor for side 
effects and document any adverse events, in addition to our research coordinators maintaining 
close communication with the patients. The study will be completed at 6 months (± 1 month) 
post-LT, which will include final assessments of clinical and laboratory outcomes. Case report 
forms and electronic databases documenting all immune assay results and clinical 
data/outcomes will be utilized for all endpoint comparisons.
5
Population
This is a single-center (Northwestern University) study.  We intend to enroll 25 subjects of 
which 20 are expected to undergo the TAC-WD/EVL phase of the study.
We will select adult LT recipients without immune disease, active viral disease, and previous 
or 
combined 
organ 
transplantation 
to 
avoid 
the 
confounding 
of 
coexistent 
inflammatory/infectious processes and prior transplants on the biomarker analysis. We will also 
have two ‘checks’ for study continuation after initial enrollment. The first is at one to two 
months post-LT to ensure there is no contraindication to starting EVL, that renal/liver function 
is reasonably preserved and stable, and that there is a low predicted risk of future TAC WD. 
The second check is to determine if TAC-WD at 3 months (± 1 month) is safe - similar criteria 
(no rejections, stable liver/renal function, etc) will be used.  
5.1
Inclusion criteria
Patients must give written informed consent 90 days prior to transplant or up to 60 days after 
transplant) before any assessment is performed.
Patients eligible for inclusion in this study have to fulfill all of the following criteria:
Inclusion/Exclusion criteria: 
Inclusion Criteria: 

Adult LT candidates ≥ 18 years of age
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
Listed for or recent (within 2 months) recipient of deceased or living donor 
liver transplantation
5.2
Exclusion criteria
Exclusion Criteria: 

Combined or previous organ transplantation

HIV infection

Inability to provide informed consent or comply with the protocol

Transplantation for autoimmune liver disease (Autoimmune hepatitis, 
Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis)
Criteria for Study Continuation and EVL addition (1 to 2 months post-LT):
1. Currently on TAC + prednisone +/- MPA therapy with no contraindication to starting EVL 
(i.e. wound infection; significant proteinuria (urine prot/cr ratio ≥1.0), hepatic artery 
thrombosis/stenosis, cytopenias (WBC <1.5, ANC<1.0, Platelet< 30, Hemoglobin <7), other 
deemed by investigator) 
2. No more than 2 rejection episodes or 1 steroid-resistant rejection requiring lymphodepletion 
therapy
3. No active biliary, infectious, or malignant process
4. eGFR ≥30 ml/min/1.73m2
5. No significant liver function abnormalities (Direct bilirubin, AST/ALT, Alkphos ≥ 2x 
elevated)
6. If on current or prior antiviral therapy, HBV and HCV viral load should be undetectable 
7. No combined transplantation (e.g. should be primary liver transplant alone)
Criteria for Study Continuation and TAC-WD initiation (3 months (± 1 month) post-LT):
1. Tolerating TAC + EVL combination
2. Same other criteria as 1 to 2 months criteria above 
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For all time points above, a ±1 month window is allowable except that EVL must not be started 
until ≥1 month post-LT and Visit 6 must occur ≥2 weeks after Visit 5
6
Treatment
6.1
Investigational and reference therapy
Zortress (Everolimus) is a macrolide immunosuppressant.  Zortress® (Everolimus) is a 
macrolide immunosuppressant.  The chemical name of everolimus is (1R, 9S, 12S, 15R, 16E, 
18R, 19R, 21R, 23S, 24E, 26E, 28E, 30S, 32S, 35R)-1, 18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-
4-(2hydroxyethoxy)-3-methoxycyclohexyl-1-methylethyl}-19,30-dimethoxy-15, 17, 21,23, 
29, 
35-hexamethyl-11, 
36-dioxa-4-aza-tricyclo[30.3.1.04,9] 
hexatriaconta-16,24,26,28-
tetraene-2, 3, 10,14,20-pentaone.
Everolimus inhibits antigenic and interleukin (IL-2 and IL-15) stimulated activation and 
proliferation of T and B lymphocytes.  In cells, everolimus binds to a cytoplasmic protein, the 
FK506 Binding Protein-12 (FKBP-12), to form an immunosuppressive complex (everolimus: 
FKBP-12) that binds to and inhibits the mammalian Target Of Rapamycin (mTOR), a key 
regulatory kinase.  In the presence of everolimus phosphorylation of p70 S6 ribosomal protein 
kinase (p70S6K), a substrate of mTOR, is inhibited.  Consequently, phosphorylation of the 
ribosomal S6 protein and subsequent protein synthesis and cell proliferation are inhibited.  The 
everolimus: FKBP-12 complex has no effect on calcineurin activity.  
In rats and nonhuman primate models, everolimus effectively reduces kidney allograft rejection 
resulting in prolonged graft survival.  
Everolimus pharmacokinetics has been characterized after oral administration of single and 
multiple doses to adult kidney transplant patients, hepatically-impaired patients, and healthy 
subjects.  
6.2
Treatment arms
All patients will be assigned to Zortress® (everolimus) at advised doses and trough levels. After 
enrollment criteria met, EVL will be added (goal trough level 5-8 ng/ml) and TAC reduced 
(goal trough level 3-5 ng/ml). When TAC is discontinued, EVL will be maintained at goal 
trough of 5-8 ng/ml and prednisone kept for at least the study time period (6 months (± 1 
month)) at 5 mg/day. See study design (Figure 6 in Appendices).
6.3
Treating the patient 
6.3.1
Patient numbering
Subjects entering the study will be given sequential numbers in the order they are screened, 
e.g.; 01, 02, 03….
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6.3.2
Dispensing the study drug
“Study drug” is obtained via a prescription from the physician and filled at any pharmacy (local 
walk-in/mail-order).  
6.3.3
Study drug supply, storage and tracking
Pharmacies should be storing the drug at room temperature: 25°C (77°C); excursions permitted 
to 15-30°C (59-86°F).  Medication should be protected from light and moisture.
6.3.4
Instructions for prescribing and taking the study drug
 “Study” drug will be obtained via a prescription.   Everolimus is taken orally twice daily with 
doses approximately 12 hours apart.  Everolimus can be taken with/without food.  
This is an approved drug for the following:
Kidney transplant in low-moderate immunologic risk.  It is to be used in combination with 
basiliximab, cyclosporine (reduced dose) and corticosteroids.
It is also approved for use in liver transplantation.   It is not to be administered earlier than 30 
days post-transplant.  It is to be used in combination with tacrolimus (reduced doses) and 
corticosteroids.
6.3.5
Permitted study drug dose adjustments and interruptions
Per protocol to maintain the required trough levels of tacrolimus and everolimus therapy (Figure 
6)
6.3.6
Other concomitant treatment
Any medication (prescribed or over-the-counter), alternative/complimentary medication, any 
medical or surgical procedure that the subject takes or undergoes during the study must be 
reported to the study team and will be reported in the CRF.  This is to ensure that any elevation 
in liver function tests or rejection episode is most likely related to immunosuppression 
withdrawal and does not have another cause outside of the study.
It is important to note that medications that are normally used in post-transplant patients 
(antivirals, antibacterials, and other prophylactic meds, along with other prescribed meds) are 
allowed in this protocol.
Medications with strong-moderate CYP3A4 inhibitors (cyclosporine, detoconazole, 
erythromycin, verapamil) and CYP3A4 inducers (e.g., rifampin) may affect everolimus 
concentrations.  In this case Zortress dose adjustments should be considered.
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6.3.7
Study drug discontinuation and premature patient withdrawal
Subjects may be withdrawn from the trial at any time for any reason, either by 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. If patients are withdrawn 
prematurely, they will still be followed through the follow-up time period of the study as per 
standard medical care. These data are important to collect to determine differences in this group 
compared to those who completed the study. 
6.3.8
Study completion and post-study treatment
Study will be completed at the six month (± 1 month) time point post-liver transplantation
7
Visit schedule and assessments
Table 7-1
Assessment schedule
Visit**
1
2
3
4
5
6
7
At 
rejection***
Early 
Term./Study 
Comp.
Day
-90 to 
+30
0
(Tx)
30
90
120
135
180
Inc./Exc. Criteria
X
X
X
X
Obtain informed consent X
Physical Exam
X
X
X
X
Donor/Recipient 
Collections
X*
Blood 
(Flow, 
PBMC, 
RNA, sera)
X*
X
X
X
X
X
Tissue RNA
X
Study Completion Form
X
X
X
*unfunded and not mandatory for this protocol
** ± 1 monthwindow allowable
Visit 1 = Patient enrollment SOC visit (I/E criteria, informed consent)
Visit 2= Transplant SOC- if possible, donor spleen/lymph nodes and recipient lymph node/blood
Visit 3= Day 30 SOC visit- if Inc./Exc. criteria met, add everolimus and stop MPA if being used.  Collect blood
if possible (unfunded for this study).
Visit 4= Day 90 SOC visit- if Inc./Exc. criteria met, start tacrolimus withdrawal.  Collect blood.
Visit 5= Day 120 SOC visit- if Inc./Exc. criteria met, stop tacrolimus.  Collect blood.
Visit 6 = Day 135 visit- collect blood.
Visit 7= Day 180 SOC visit- Study termination.  Collect blood.  
***At any biopsy concerning for rejection: Collect blood and tissue in RNAlater.  If rejection, study termination.
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7.1
Patient demographics/other baseline characteristics
Basic demographics (age, sex, race, cause of liver disease, past medical history) and laboratory 
values will be determined prior to enrollment. To determine continuation criteria at one to two 
months  and three months (± 1 month), the medical charts will be reviewed to determine 
laboratory values and ensure inclusion/exclusion criteria are met. Throughout the trial, history 
and physicals per standard of care will be performed at most of the study time points (see 
schedule above) as well as laboratory values. 
7.2
Treatment exposure and compliance
All immunosuppressant drug levels will be documented at the study time points, and 
compliance with the dosing will be assessed at each study time point by the PI and coordinator. 
Side effects of tacrolimus (renal, neurologic, metabolic, etc) and everolimus (hyperlipidemia, 
edema, proteinuria, etc) will be documented in the case report forms
7.3
Efficacy 
The major efficacy assessment is the percentage of patients successfully withdrawn from TAC 
therapy in favor of EVL therapy without rejection or major complications. Rejection will be 
evaluated by liver biopsy when indicated (typically 2-3 fold increase in liver 
aminotransferases). If rejection occurs during any point of the trial following EVL addition, the 
study will be terminated for that patient. 
Other efficacy endpoints will be the improvement in TAC side effects (renal, neurologic, 
metabolic, etc.) and development of EVL side effects (hyperlipidemia, edema, proteinuria, etc) 
in those successfully withdrawn from TAC without rejection. For those unable to withdraw 
from TAC due to rejection, we will assess for TAC side effects and EVL side effects if this 
drug is continued following rejection. Liver and renal function, and hematological parameters 
will be followed per SOC throughout this biomarker study
The PI and research coordinator will perform these assessments at the study time points at 
Northwestern and at any intervening time point if the patient needs a liver biopsy to exclude 
rejection or other complication (hospitalization, infection, etc.). Patients enrolled also will be 
contacted by phone for any changes in their immunosuppression dosing or other medications in 
between visits.
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7.4
Safety/Potential Adverse Events/Protection Against Risk
The risks to the patient are limited to the minor risk of performing venipuncture and the risk of 
rejection with TAC-WD. The National Cancer Institute Common Terminology Criteria for 
Adverse Events v3.0 (CTCAE), dated December 12, 2003 will be used to grade all adverse 
(either serious or non-serious) events. It is the responsibility of the Principal Investigator to 
oversee the safety of the study. This safety monitoring will include careful assessment and 
appropriate reporting of adverse events as noted above. Medical monitoring will include a 
regular assessment of the number and type of serious adverse events. Information on all adverse 
events (signs, symptoms, abnormal diagnostic procedures, treatments) will be recorded in the 
appropriate adverse event module of the case report form (CRF).  The clinical course of each 
event will be followed until resolution, stabilization, or until it has been determined that the 
study participation is not the cause.  Serious adverse events that are still ongoing at the end of 
the study period will be followed to determine the final outcome.  
Reports of all serious adverse events (including follow-up) will be submitted to the IRB within 
24 hours of the investigator’s knowledge of the event.  Follow-up reports will be submitted to 
the IRB within 10 working days.  The IRB/Office for the Protection of Research Subjects at 
NU is under the direction of Lewis Smith, MD- (312) 503-9338. As this is a prospective study 
using primary data collection with a drug of interest all adverse events (AEs) – including serious 
adverse events (SAEs) and safety endpoints (where relevant) – will be collected and recorded 
in the study and safety database, irrespective of causal association. All adverse reactions 
identified will be reported to the local Health Authority in accordance with national regulatory 
requirements.
All of the risks and benefits will be discussed with the patient at the time of informed consent. 
The patient will undergo informed consent prior to all liver biopsies and suspected rejection 
will be managed as per our standard of care (SOC). The biopsies will be performed using a 16-
18 Gauge BioPince® biopsy instrument, which utilizes a Tri-axial "Cut and Capture" cannula 
system. We will remove a 0.5-1 cm biopsy piece from the main 3 cm sample for genomic 
analysis. Thus, only one pass liver biopsy is required per SOC and not extra for the study. The 
patient will be monitored for 2 hours after each liver biopsy in the Procedure Unit at 
Northwestern. Vital signs will be performed before and every 15 minutes x 1 hour, every 30 
minutes x 1 hour per standard protocol. Any atypical symptom after biopsy, i.e. shortness of 
breath, hypotension, tachycardia, fever, severe abdominal pain, will be investigated 
immediately by the physician performing the biopsy. 
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The patient will be monitored for any complication of this study as he/she would as part of our 
standard management, i.e. monitoring of graft function, kidney function, trough IS levels, 
cholesterol levels, urine protein/creatinine ratio. This follows the clinical practice that has been 
occurring at our center over the last 20 years. There is a risk of rejection with TAC-WD (~20%) 
which will be discussed with the patient. If a graft loss occurs due to the study protocol or ≥30% 
rejection occurs with TAC-WD, the study will be terminated for safety reasons. This is 
counterbalanced by the equipoise of the potential for preservation of renal function and other 
TAC side effects by TAC-WD. The blood collection will occur at the time of the standard of 
care blood tests after transplantation and thus will not add additional venipuncture risk. The 
allowed amount of blood per venipuncture/day will not be exceeded according to our IRB rules. 
Overall, the patient will be well informed of our standard of care approaches and research 
protocols, with appropriate discussion and consent.
Another risk involves breach of patient confidentiality during the study. To avoid this, all 
informed consents, medical records, and electronic databases will be stored in a locked location 
at Northwestern, with access limited to study personnel. Patient samples utilized for research 
will be de-identified by a coding system only accessed by the Co-PIs. Special procedures for 
ensuring patient confidentiality will be implemented in accordance with the HIPAA. Data 
transmission and the distributed data systems will have multiple layers of security. Each study 
subject will be assigned a unique identification number. Only this number will be used to 
identify subjects in any individual tabulation. It is expected that only group data will be 
published. If individual subject data are to be published, no identifying information will be 
included. The study files will be maintained in a secure location. Access to computerized data 
will be restricted to study personnel. Password authentication will be enforced. These 
passwords will be changed on a quarterly basis and whenever the Database Administrator 
makes a determination for a security change. 
7.4.1
Physical examination
On visits 3, 4, 5, 7 and early termination an exam will be performed. 
7.4.2
Vital signs
Vital signs including blood pressure, pulse, respiratory rate and temperature will be taken at 
every SOC visit and study-related visit when the subjects come to clinic.
7.4.3
Height and weight
Subjects will be weighed at every visit and their height will be taken at the first study visit.
7.4.4
Laboratory evaluations
At liver transplant donor/recipient collection of lymph nodes and liver biopsy tissue will take 
place if the patient is consented prior to transplantation.  Liver biopsy tissue will also be 
collected at the time of a suspected rejection.
Blood samples for flow cytometry, PBMCs, RNA and sera will be collected at visits 3, 4, 5, 6, 
7 and rejection.
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Standard SOC laboratory tests will be performed per our transplant center post-LT protocol: 
CBC, CMP, trough IS levels three times weekly (0-1 month post-LT), two times weekly (1-3 
months post-LT), one time weekly (3-6 months post-LT).
7.4.5
Pregnancy and assessments of fertility
Pregnancy
There are no adequate and well-controlled studies of Zortress in pregnant women.  In rats and 
rabbits, everolimus crossed the placenta and was toxic to the conceptus.  The potential risk for 
humans in unknown.  Zortress should be given to pregnant women only if the potential benefit 
to the mother justifies the potential risk to the fetus.  Women of childbearing potential should 
be advised to use effective contraception methods while they are receiving Zortress and up to 8 
weeks after treatment has been stopped. 
Nursing Mothers
It is not known whether everolimus is excreted in human milk.  Everolimus and/or its 
metabolites readily transferred into milk of lactating rats at a concentration 3.5 times higher 
than in maternal serum.  Because many drugs are excreted in human milk and because of the 
potential for serious adverse reactions in nursing infants from everolimus, women should avoid 
breast-feeding during treatment with Zortress.
7.4.6
Appropriateness of safety measurements
All safety measurements are appropriate as standard of care for patient management and this 
protocol.
7.4.7
Other biomarkers
Please see Exploratory Objectives above
8
Safety monitoring
It is the responsibility of the Principal Investigator and sub-investigators to oversee the safety 
of the study at their site.  This safety monitoring will include careful assessment and appropriate 
reporting of adverse events as noted above.  Medical monitoring will include a regular 
assessment of the number and type of serious adverse events.
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8.1
Adverse events
Definition of an AE: Any untoward medical occurrence in a subject administered a 
pharmaceutical product that does not necessarily have a causal relationship with the treatment. 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not considered related to the investigational medicinal 
product. Investigational Medicinal Product (IMP) includes the drug under evaluation and the 
comparator drug(s) if specified as part of the research objective, given at any time during the 
study. Medical conditions/diseases present before starting the drug of interest are only 
considered adverse events if they worsen after starting the drug of interest.
The occurrence of adverse events will be sought by non-directive questioning of the patient at 
each visit during the study. Adverse events also may be detected when they are volunteered by 
the patient during or between visits or through physical examination, laboratory test, or other 
assessments. All adverse events will be recorded in the study database including the following 
information:
1.
the severity grade (mild, moderate, severe) or (grade 1-4) 
2.
its relationship to the drug(s) of interest (suspected/not suspected)
3.
its duration (start and end dates or if continuing at final exam)
4.
whether it constitutes a serious adverse event (SAE)
8.2
Serious adverse event reporting
An SAE: is any untoward medical occurrence that at any dose:
-
results in death,
-
is life-threatening,
-
requires inpatient hospitalization or prolongation of existing hospitalization,
-
results in persistent or significant disability/incapacity,
-
is a congenital anomaly/birth defect, 
-
is otherwise a significant medical event.
This includes any SAEs likely to arise from the trial indication or progression of 
underlying/concomitant illness (es) (e.g. progression of cancer in oncology trials), unless 
specified in the protocol as study specific exemptions.
Any SAE, irrespective of causality, occurring after the subject has provided informed consent 
and until four weeks after the subject has stopped study participation must be reported unless 
otherwise stated in the protocol. SAEs occurring after four weeks from ending study 
participation should only be reported if considered by the Investigator attributable to the 
exposure to the investigational drug(s) during the trial period. This includes the period in which 
the study protocol interferes with the standard medical treatment given to a subject, even if 
study treatment has not yet started (e.g. withdrawal of previous treatment during washout 
period, change in treatment to a fixed dose of concomitant medication).
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Timelines: All serious adverse events (SAEs) from interventional clinical trials must be 
reported by the sites to Sponsor within 24 hours of occurrence of the SAE. The timelines for 
investigator initiated trials reporting to Novartis will be done as per Third Party 
Study/Investigator Initiated Trial Agreement.  
Follow-up reports: 
SAEs will be followed until resolution or until it is judged to be permanent, and an assessment 
will be made at each visit (or more frequently, if necessary) of any changes in severity, the 
suspected relationship to the drug of interest, the interventions required to treat it, and the 
outcome. 
The Sponsor shall support Novartis in the following-up of all SAEs so that complete 
information is available to maintain patient safety and also as part of any commitments by 
Novartis to any Health authority OR specific Health authority follow-up requests for the product 
under investigation.
8.3
Pregnancies
To ensure patient safety, each pregnancy in a patient (or a patient’s partner) on study drug must 
be reported to Novartis within 24 hours of learning of its occurrence. The pregnancy will be 
followed up to determine outcome, including spontaneous or voluntary termination, details of 
the birth, and the presence or absence of any birth defects, congenital abnormalities, or maternal 
and/or newborn complications.
9
Data review and database management
9.1
Data collection (data handling and record keeping)
This study is to be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312 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 Northwestern University Institutional 
Review Board (NU IRB), for formal approval of the study conduct.  
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).  
Consent:
 All subjects for this study will be provided with a printed consent form describing this study 
and providing sufficient information for subjects to make an informed decision about their 
participation in this study. 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, will be obtained before the subject is submitted to any study procedures. 
This consent form will be signed by the subject and the investigator-designated research 
professional obtaining the consent.  The subject will be given a signed copy of the informed 
consent for their records. 
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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.
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 (which is incorporated into a single 
document which includes the Northwestern University Informed Consent Form).  This 
combination document must inform 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 
should 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
Database management and quality control
Case Report Forms:
After study initiation, the electronic case report form (eCRF) will be the primary data collection 
instrument for the study.  Only the Investigators will have access to this password protected 
file. No subject identifiers will be included in the eCRF and electronic databases that are used 
to record clinical data on patients. All clinical data will be kept completely confidential.
Auditing and Inspecting:
The investigator will permit study-related monitoring, audits, and inspections by the NU IRB, 
the sponsor, 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 facilities (e.g. pharmacy, diagnostic laboratory, etc.).
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Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable University compliance and quality assurance 
offices.
Record Retention:
It is the investigator’s responsibility to retain study essential documents on site for at least 2 
years after the study has been formally terminated at the NU IRB.  After this time, it is up to 
the investigator to determine whether the documents should be stored off-site at O’Hare 
Records, located in Rosemont, IL for up to 13 more years.  Once the investigator determines 
how long to keep the records, when that time frame is up it is the responsibility of the 
investigator to ensure that all study records are properly disposed of by shredding.  
10
Data analysis 
10.1
Populations for analysis
Since there is no randomization in the study, the groups that are analyzed are those that have 
rejection before TAC-WD, those who develop rejection during/after TAC-WD, and those who 
never have rejection before or after TAC-WD. 
10.2
Analysis of the primary objective(s)
Primary objective: To identify cellular and molecular metrics of immune regulation that may 
predict successful TAC withdrawal in favor of EVL early after LT.
Overall Strategy and Rationale: The objective is to test the working hypothesis that, in a 
prospective study, cellular and genomic markers of immunoregulation become more 
pronounced after early rejection-free TAC-WD/EVL compared to failed (rejection) TAC-WD. 
Conversely, if these markers do not signify immunoregulation, we hypothesize that TAC-
WD/EVL will not be successful (i.e. rejection will ensue requiring reinstatement of TAC). The 
rationale is that the immunoregulatory mechanisms of IS therapy (EVL vs. CNI) can be more 
adequately clarified and that a higher % of patients can undergo successful TAC-WD/EVL by 
utilizing these serial, safe blood biomarkers to predict rejection, before it occurs, i.e. by 
comparing these signals right before and after TAC-WD. Our hypothesis is based on our 
previous observations that late mTOR-I conversion increased % of Treg/DCreg in blood/tissue 
sites and generated immunoregulatory proteogenomic signatures and that AR can be diagnosed 
in the peripheral blood by genomic microarrays. Therefore, the plan is to perform sequential 
flow cytometry immunophenotyping and genomic signature analysis in the blood before, during 
and after attempted TAC-WD/EVL. 
Studies Planned and Methods:
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i) Blood Immunophenotyping. The approach will be to conduct flow cytometry 
immunophenotyping of peripheral blood before TAC-WD initiation (1 to 2 months and 3 
months (± 1 month)), right at total TAC-WD (4 months (± 1 month)), 2 weeks after full TAC-
WD (4.5 months (± 1 month)) and study end (6 months (± 1 month)) post-LT to analyze cellular 
changes. Methods: The Immune Monitoring Laboratory at NU (Co-Director James Mathew, 
PhD) will perform the immunophenotyping. PBMC are isolated from heparinized 30 cc samples 
on Ficoll-Hypaque gradients. Surface markers are detected with monoclonal antibodies for the 
T cell subsets CD3, CD4, CD8, CD25, CD45RA, CTLA4 and CD127 followed by 
fixation/permeabilization/incubation with human FOXP3 antibodies and final flow cytometric 
analysis. RBC-lysed peripheral blood is also labeled with monoclonal antibodies for T cell 
subsets, naïve/memory/regulatory B cells (CD19, CD27, IgD, IgM, IL-6), monocytes (CD14) 
and NK cells (CD56) to quantify the absolute cell numbers per µL. In addition, dendritic cell 
surface markers are analyzed as follows: monocytoid vs. plasmacytoid (CD11c; CD123) ratio; 
antigen processing markers (CD83; CD205); markers inducing Tregs (ILT3; ILT4). For 
statistical analysis, we perform within subject comparisons on PBMC for both successful and 
failed TAC-WD/EVL groups using appropriate paired analysis (paired t-test or Wilcoxon 
Signed Rank test). For the main hypothesis testing, repeated measures of analysis of variance 
(RM-ANOVA) are used to test the trajectory variations between the groups on PBMC over 
time. Two-sided F-test statistics are applied; α=0.05 (stats by NU Transplant Statistician).
ii) Gene Expression Microarrays:  The approach will be to conduct parallel serial 
genomic bioassays of peripheral blood before and after attempted TAC-WD/EVL (same time 
points as immunophenotyping). Methods: The Genomics Core at Scripps Clinic (Director, 
Daniel Salomon, MD) will perform the microarray analysis. Blood will be collected in PaxGene 
tubes and sent to Scripps for the gene microarrays. Affymetrix® Hu133 Plus Peg microarrays 
will be used to test for differentially expressed genes between those developing AR vs. without 
AR during TAC WD.  We will also perform biological function and canonical pathway mapping 
of several immune/inflammatory gene pathways. For statistical analysis, we report for each 
mRNA and microRNA comparison the T-test p-value, its non-parametric analog (Kruskal 
Wallis test), the fold change, and for multi-analyte signatures, the area under the receiver 
operating curve (concordance index/C-index) and q-values by member genes/proteins. We then 
calculate the predictive accuracy, sensitivity and specificity of each signature (statistics by Dr. 
Daniel Salomon’s informatics group at Scripps Clinic)
10.2.1
Variable
The main variables are the peripheral blood Treg% (or other immunoregulatory cells) and 
mRNA AR signatures seen prior to and during either successful (no rejection) vs. failed 
(rejection) TAC-WD/EVL.
10.2.2
Statistical hypothesis, model, and method of analysis
The statistical analysis of the primary variables (immune assays) is described in detail above 
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10.2.3
Handling of missing values/censoring/discontinuations
In this study, discontinuation will usually be due to rejection occurring during the study protocol 
(TAC-WD/EVL). While unlikely as this trial is a small pilot study, patients lost to follow-up 
will be removed from the trial and analysis. 
10.2.4
Supportive analyses
None – see Exploratory Objectives above
10.3
Analysis of secondary objectives
Clinical endpoints as correlates for the success and failure of TAC-WD/EVL will serve as 
secondary endpoints. This will include but not be limited to the following at 6 months (± 1 
month) post-LT: liver allograft and renal function, EVL & TAC side effects, 
infectious/malignant complications, and patient/graft survival. At study end, all secondary 
endpoints will be statistically compared between those who succeed or fail TAC-WD/EVL.
10.3.1
Efficacy (secondary)
All potential clinical endpoints will be recorded by case report form. Since this is a small pilot 
study, the clinical outcomes will be mainly descriptive in nature, e.g. rejection rate, medication 
side effect rate, liver/renal function outcomes, infectious complications, malignancy, and 
patient/graft survival. However, we will statistically compare these secondary endpoints 
between those who succeed or fail TAC-WD/EVL by standard continuous and categorical 
statistical tests.
10.3.2
Safety/Stopping Rules
If any patient experiences a graft loss due to the protocol (TAC-WD) or ≥30% rejection is seen, 
the study will be terminated due to safety concerns.
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10.3.3
Biomarkers/Studies Planned
i) Blood Immunophenotyping. The approach will be to conduct flow cytometry 
immunophenotyping of peripheral blood before TAC-WD initiation (3 months (± 1 month)), 
right at total TAC-WD (4 months (± 1 month)), 2 weeks after full TAC-WD (4.5 months (± 1 
month)) and study end (6 months (± 1 month)) post-LT to analyze cellular changes. Methods 
19,20: The Immune Monitoring Laboratory at NU (Co-Director James Mathew, PhD) will 
perform the immunophenotyping. PBMC are isolated from heparinized 30 cc samples on Ficoll-
Hypaque gradients. Surface markers are detected with monoclonal antibodies for the T cell 
subsets CD3, CD4, CD8, CD25, CD45RA, CTLA4 and CD127 followed by 
fixation/permeabilization/incubation with human FOXP3 antibodies and final flow cytometric 
analysis. RBC-lysed peripheral blood is also labeled with monoclonal antibodies for T cell 
subsets, naïve/memory/regulatory B cells (CD19, CD27, IgD, IgM, IL-6), monocytes (CD14) 
and NK cells (CD56) to quantify the absolute cell numbers per µL. In addition, dendritic cell 
surface markers are analyzed as follows: monocytoid vs. plasmacytoid (CD11c; CD123) ratio; 
antigen processing markers (CD83; CD205); markers inducing Tregs (ILT3; ILT4). For 
statistical analysis, we perform within subject comparisons on PBMC for both successful and 
failed TAC-WD/EVL groups using appropriate paired analysis (paired t-test or Wilcoxon 
Signed Rank test). For the main hypothesis testing, repeated measures of analysis of variance 
(RM-ANOVA) are used to test the trajectory variations between the groups on PBMC over 
time. Two-sided F-test statistics are applied; α=0.05 (stats by NU Transplant Statistician).
ii) Gene Expression Microarrays:  The approach will be to conduct parallel serial 
genomic bioassays of peripheral blood before and after attempted TAC-WD/EVL (same time 
points as immunophenotyping). Methods: The Genomics Core at Scripps Clinic (Director, 
Daniel Salomon, MD) will perform the microarray analysis. Blood will be collected in PaxGene 
tubes and sent to Scripps for the gene microarrays 20,46,47,49. Affymetrix® Hu133 Plus Peg 
microarrays will be used to test for differentially expressed genes between those developing AR 
vs. without AR during TAC WD.  We will also perform biological function and canonical 
pathway mapping of several immune/inflammatory gene pathways. For statistical analysis, we 
report for each mRNA and microRNA comparison the T-test p-value, its non-parametric analog 
(Kruskal Wallis test), the fold change, and for multi-analyte signatures, the area under the 
receiver operating curve (concordance index/C-index) and q-values by member genes/proteins. 
We then calculate the predictive accuracy, sensitivity and specificity of each signature (statistics 
by Dr. Daniel Salomon’s informatics group at Scripps Clinic).
10.4
Sample size calculation
As this is an exploratory pilot study, we will enroll an initial 25 patients, 20 of who are expect 
to undergo TAC-WD/EVL. Of these, we expect 14-15 will not have AR during TAC-WD and 
4-5 are expected to have AR, based on the initial EVL clinical trial17. This will allow us to have 
preliminary information to proceed ahead to one of a few different directions, as outlined in 
Exploratory Objectives. Therefore, a power analysis was not needed for this pilot study.
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10.5
Power for analysis of critical secondary variables
Secondary clinical variables will be documented for descriptive reporting and to compare 
between those with and without AR. Given this is a small exploratory study, a power analysis 
for both primary and secondary variables were not considered necessary.
10.6
Interim analysis
Given the small size of this pilot study, an interim analysis will not be performed as clinical 
events or biomarker results will be limited at any interim time point. However, we will be 
continuously monitoring our acute rejection rates and stopping rules (e.g. any graft loss or 
rejection≥30% believed to be due to TAC-WD/EVL).
11
Ethical considerations
11.1
Regulatory and ethical compliance
This clinical study was designed and shall be implemented and reported in accordance with the 
ICH Harmonized Tripartite Guidelines for Good Clinical Practice, with applicable local 
regulations (including European Directive 2001/20/EC, US Code of Federal Regulations Title 
21, and Japanese Ministry of Health, Labor, and Welfare), and with the ethical principles laid 
down in the Declaration of Helsinki.
11.2
Informed consent procedures
Eligible patients may only be included in the study after providing written (witnessed, where 
required by law or regulation), NU IRB-approved informed consent, or, if incapable of doing 
so, after such consent has been provided by a legally acceptable representative of the patient. 
In cases where the patient’s representative gives consent, the patient should be informed about 
the study to the extent possible given his/her understanding. If the patient is capable of doing 
so, he/she should indicate assent by personally signing and dating the written informed consent 
document or a separate assent form. Informed consent must be obtained before conducting any 
study-specific procedures (i.e. all of the procedures described in the protocol). The process of 
obtaining informed consent should be documented in the patient source documents. 
Women of child bearing potential should be informed that taking the study medication may 
involve unknown risks to the fetus if pregnancy were to occur during the study and agree that 
in order to participate in the study they must adhere to the contraception requirement for the 
duration of the study. If there is any question that the patient will not reliably comply, they 
should not be entered in the study.
11.3
Responsibilities of the investigator and NU IRB
This study is to be conducted according to US and international standards of Good Clinical 
Practice (FDA Title 21 part 312 and International Conference on Harmonization guidelines), 
applicable government regulations and Northwestern University Institutional research policies 
and procedures.
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This protocol and any amendments will be submitted to the Northwestern University 
Institutional Review Board (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 before commencement of this study.  
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.  See Appendices for a copy of the Subject Informed Consent Form.  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/ investigator-designated research 
professional obtaining the consent. 
A signed and dated statement that the protocol and informed consent have been approved by 
the NU IRB must be given to Novartis before study initiation.
11.4
Publication of study protocol and results
The final study report is expected to be sent to Novartis no later than 9 months after the last 
patient visit.
The data abstracted and analyzed from this study will be submitted to scientific meetings. 
It is expected that this will be in the form of an abstract and presented as a poster or orally.  
In addition, the data will be sent to a professional peer reviewed journal for publication.
Subjects will not be identified in any way at the scientific meeting if this abstract is chosen for 
an oral or poster presentation.  Subjects will not be identified in any written format of this data 
analysis.
Neither the complete nor any part of the results of the study carried out under this protocol, nor 
any of the information provided by the sponsor (Dr. Josh Levitsky) for the purposes of 
performing the study, will be published or passed on to any third party without the consent of 
the study sponsor.  
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12
Protocol adherence
12.1
Protocol Amendments
Any change or addition to the protocol can only be made in a written protocol amendment that 
must be approved by Novartis, Health Authorities where required, and the NU IRB.  Only 
amendments that are required for patient safety may be implemented prior to NU IRB approval. 
Notwithstanding the need for approval of formal protocol amendments, the investigator is 
expected to take any immediate action required for the safety of any patient included in this 
study, even if this action represents a deviation from the protocol.
13
Figures and References 
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Appendices:
Figure 1: New CD4+CD25HighFOXP3+Treg "Recruitment" by Allo-specific Tregs 
Generated in the Presence of Media, TAC or SRL in MLR. Bulk cultures of volunteer 
responder cells were stimulated with 2 DR-matched x-irradiated cells in the presence of either 
no drugs, 5ng/ml TAC or SRL for 7 days and putative CD4+CD25HighCD127- cells were enriched 
using the Regulatory T cell Isolation Kit and AutoMACS (Miltenyi Biotech), after negatively 
selecting out CD8, CD19 and CD127. Then, in 3 separate experiments, 5x104 enriched Tregs 
or fresh autologous control PBMC were added as modulators to 5x105 autologous fresh CFSE-
labeled responding PBMC and x-irradiated (unlabeled) specific and non-specific stimulator 
PBMC. The modulator cells and x-irradiated stimulator cells were all labeled with PKH26 to 
differentiate these from the readout of CFSE labeled responding cells. After another 7 days, 5-
color flow cytometric assays were performed to estimate CD4+CD25HighFOXP3+ cells in the 
CFSE-labeled responder PBMC. Recruitment data are calculated as percentage increase in 
CD4+CD25HighFOXP3+ cell generation with the Tregs over that observed with the control fresh 
PBMC modulators (dotted line). Note that a more marked and allospecific effect of SRL 
cultured modulators occurred (green diamond bars, SRL vs. TAC and media, p=0.01). A value 
below the dotted line or a change of ≤10% from control is considered to indicate the absence 
of recruitment. 
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Figure 2: Increase in Phenotypic Regulatory T Cells and Regulatory Dendritic Cells with 
Tacrolimus to Sirolimus Conversion. Immunophenotyping flow cytometry was performed on 
peripheral blood and bone marrow aspirate specimens before (0, horizontal axis) and after 
(means of 12 and 14 weeks and of 24 and 26 weeks, horizontal axis) conversion from 
tacrolimus to sirolimus. Note the significant increase in the percentage of phenotypic Tregs 
(CD4+CD25+FOXP3+, CD4+CD25highFOXP3+) in the peripheral blood and marrow (A), as well 
as the number of peripheral blood CD123+ILT3+ILT4+ and CD11c+ILT3+ DCregs (B), following 
sirolimus conversion. * p<0.05 and ** p<0.01 compared to time point 0 (pre-conversion on 
TAC).
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Figure 3: Increased FOXP3+ cells in Liver Biopsies after Tacrolimus to Sirolimus 
Conversion: Immunohistochemical staining was performed on liver biopsy specimens before 
and after conversion from tacrolimus to sirolimus (representative example shown). Note the 
increase in the number of FOXP3+ Cells (arrows) following sirolimus conversion.
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Figure 4: Heat Map of Immune/Inflammatory Gene Transcripts (n=93) Before and After 
Tacrolimus to Sirolimus Conversion. The bottom half of the heat map (yellow bar on the 
left) represents the peripheral blood gene transcripts present in patients pre-conversion (TAC), 
while the top half (red bar on the left) represents the transcripts post-conversion (SRL). Below 
the heat map is the color-coding for the various genes represented - red represents up-
regulated genes, blue signifies down-regulated genes, and gray denotes unchanged 
transcripts. The gradation of color represents the intensity of the signal in log value.
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Figure 5: Blood and Biopsy mRNA differences in LT recipients with acute 
rejection, HCV recurrence and mixed AR/HCV-recurrence.
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