Once-Daily Extended-Release Tacrolimus vs. Twice-Daily Tacrolimus: Impact on T-Cell Subpopulations and Markers of Renal Tubule-toxicity in Kidney Transplant Patients
Phase: PHASE3
Conditions: End Stage Renal Disease, Rejection of Renal Transplant
Sponsor: Lorenzo Gallon

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PROTOCOL TITLE:  
ONCE-DAILY EXTENDED-RELEASE TACROLIMUS VS. TWICE-DAILY 
TACROLIMUS: IMPACT ON T-CELL SUBPOPULATIONS AND MARKERS OF 
RENAL TUBULE-TOXICITY IN KIDNEY TRANSPLANT PATIENTS 
PRINCIPAL INVESTIGATOR:
Lorenzo Gallon, MD
Professor, Medicine and Surgery
Director, Translational Medicine FCVRI; Director, International Relations DOM
Director, Transplant Nephrology Fellowship
Phone: (312) 695-4457
Email: l-gallon@northwestern.edu
CO-INVESTIGATORS:
Joseph R Leventhal MD PhD
Fowler McCormick Professor of Surgery; Director of Kidney Transplantation 
Office: (312) 695-9019 
Email: jleventh@nm.org 
&
M. Javeed Ansari, MD
Assistant Professor of Medicine
Office: (312) 695-0602 
Email: jansari@northwestern.edu 
&
James M. Mathew, PhD 
Associate Professor of Surgery & Microbiology-Immunology; 
Director: Immune Monitoring Core
Office: (312) 908-5180 
James-mathew@northwestern.edu
ADDRESS:
Comprehensive Transplant Center
Northwestern University Feinberg School of Medicine
Arkes Family Pavilion, Suite 19
676 North St. Clair Street
Chicago, IL, 60611-2923
SPONSOR:
Lorenzo Gallon, MD
Professor, Medicine and Surgery
Director, Translational Medicine FCVRI; Director, International Relations DOM
Director, Transplant Nephrology Fellowship
Phone: (312) 695-4457
Email: l-gallon@northwestern.edu
 
VERSION NUMBER: 3.3
VERSION DATE: October 3, 2019
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OBJECTIVES AND HYPOTHESIS:
The overall aim of the present study is to prospectively investigate the impact of two 
maintenance CNI immunosuppressive regimens (both prednisone-free, with MMF) on 
subpopulations of T and B cells and alloreactive T cells as well as on renal allograft function at 
week 2, 3 months and 12 months post-transplantation.
1.
To compare renal toxicity of once-daily LCP-Tacro/Envarsus XR vs. twice/day Tacrolimus by 
measured acute kidney injury markers.
2.
To compare the effect on T and B cell subpopulations of once-daily LCP-Tacro/Envarsus XR 
vs. twice /day Tacrolimus.  Specific studies to include prospective, serial flow cytometric 
analysis of T and B cell subpopulations, as well as high throughput sequencing T cell 
receptor repertoire analysis monitoring for presence or absence of donor-reactive T cells.
Our hypothesis is that that more consistent exposure with LCP-Tacro/Envarsus® XR avoiding 
under immunosuppression will prevent the emergence of alloreactive T cells, development of 
effector memory T cells, and a reduced incidence of immunologic allograft injury. We also posit 
that avoidance of the peaks associated with conventional twice daily tacrolimus will spare the 
renal allograft from drug induced nephrotoxicity. 
BACKGROUND:
Kidney transplantation is the treatment of choice for most patients with end-stage renal disease. 
Lifelong immunosuppressive therapies are required to prevent organ rejection. However, long 
term exposure to immunosuppressive therapy after kidney transplantation can place patients at 
risk for multiple adverse events. The optimal immunosuppressive therapy is not well 
established. Tacrolimus, a calcineurin inhibitor (CNI) is highly effective in preventing acute 
rejection after organ transplantation (2). It is used as part of the immunosuppression regimen for 
the majority of kidney and liver transplant recipients (3). However, treatment with current 
formulation of Tacrolimus generates high peaks and low troughs in drug concentrations in the 
blood. It is known that high exposure to CNI is associated with renal toxicities and adverse 
events (4).  New once-daily dosage formulations are now developed with the hope of minimizing 
side effects while maintaining excellent outcomes (5-8). 
LCP-Tacro/Envarsus® XR, a new once-daily formulation of tacrolimus, was approved by the 
Figure 1 Mean whole-blood tacrolimus concentration in patients on days 7, 14 & 21 vs time 
(1)
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FDA in 2015 for conversion from twice-daily tacrolimus in kidney transplant recipients. It is a 
prolonged-release tacrolimus formulation, utilizing a MeltDose drug delivery technology 
designed to improve the bioavailability of drugs with low water solubility (1). Recent clinical data 
demonstrated that once-daily LCP-Tacro/Envarsus XR has improved pharmacokinetic 
bioavailability, rapid achievement of therapeutic trough levels, less fluctuation and swing in 
whole blood concentration, non-inferior efficacy and similar safety, with lower tacrolimus dose 
than other tacrolimus formulations (1, 9-13). (Figure1). 
INCLUSION AND EXCLUSION CRITERIA:
The target population is adult recipients of immediately functioning living and deceased donor 
renal allografts. Immediate function will be defined as the absence of the need for hemodialysis 
in the first week following renal transplantation.
Inclusion Criteria:
1. Patients who are males or females aged 18-65 years. 
2. Donors aged 18-65 years. 
3. No prior organ transplant 
4. Patients who are single-organ recipients (kidney only). 
5. Women who are of childbearing potential must have a negative serum pregnancy test 
before transplantation and agree to use a medically acceptable method of contraception 
throughout the treatment period. 
6. Subject (recipient) is able to understand the consent form and give written informed 
consent 
Exclusion Criteria 
1.
Delayed graft function (please see above).
2.
Known sensitivity or contraindication to alemtuzumab, Envarsus® XR, tacrolimus or 
MMF.
3.
Use of the following induction medications: basiliximab and rituximab.
4.
Patient with significant or active infection. 
5.
Patients with a positive flow cytometric crossmatch using donor lymphocytes and 
recipient serum. 
6.
Patients with PRA > 40%
7.
Patients with current or historic donor specific antibodies 
8.
Body Mass Index (BMI) of < 18 or > 35 
9.
Patients who are pregnant or nursing mothers. 
10. Patients whose life expectancy is severely limited by diseases other than renal 
disease. 
11. Ongoing active substance abuse, drug or alcohol. 
12. Major ongoing psychiatric illness or recent history of noncompliance. 
13. Significant cardiovascular disease (e.g.): 
a.
Significant non-correctable coronary artery disease; 
b.
Ejection fraction below 30%; 
c.
History of recent myocardial infarction. 
14. Malignancy within 3 years, excluding non-melanoma skin cancers. 
15. Serologic evidence of infection with HIV or HBVs-Ag positive. 
16. Patients with a screening/baseline total white blood cell count < 4,000/mm3; platelet 
count < 100,000/mm3; triglyceride > 400 mg/dl; total cholesterol > 300 mg/dl. 
17. Investigational drug within 30 days prior to transplant surgery.
18. Anti-T cell therapy within 30 days prior to transplant surgery.
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19. Diagnosis of atypical-Hemolytic Uremic Syndrome (aHUS).
20. Subjects transplanted with a Hepatitis C NAT-positive kidney.
NUMBER OF PARTICIPANTS:
Prospective randomized single center open label study of 2 groups of kidney transplant patients

Group 1 : standard of care (SOC) control group will receive tacrolimus twice-daily (n=25)

Group 2 : LCP-Tacro/Envarsus® XR group will receive LCPT tablets once daily (n=25)
RECRUITMENT METHODS:
The investigators will recruit patients in a manner that is sensitive to the inclusion of women and 
members of minority groups into this study. The renal failure population awaiting transplantation 
on the participating program waiting lists is representative of the renal failure population in 
general. Subjects will be recruited from the patients who present themselves to Northwestern 
University, Kovler Organ Transplantation Clinic, located in Arkes Pavilion, 676 N. St. Clair 
Street, Suite 1900, Chicago, IL 60611.
Subject Information and Consent 
Prior to any testing under this protocol, including screening tests and evaluations, written 
informed consent will be obtained from the subject in accordance with local practice and 
regulations. Whenever possible, the investigator(s) will also be involved in this procedure. 
The background of the proposed study and the benefits and risks of the procedures and 
study will be explained to the subject. A copy of the informed consent document signed and 
dated by the subject will be given to the subject. Confirmation of a subject’s informed 
consent will be documented in the subject’s medical records prior to any testing under this 
protocol. 
Avoidance of Coercion 
It will be made clear to all patients that they can receive transplantation therapy (contingent 
on their medical suitability) without participation in this trial.
STUDY TIMELINES:
We expect the total duration for this study not to exceed 2 years from the date of the last 
subject’s enrollment. Each individual subject will be part of the study for 12 months after 
transplantation.
STUDY ENDPOINTS:
Primary endpoints: 
One of the 2 primary end points Kidney transplant function assessed as kidney injury 
markers in urine samples collected at 12-hour and 24-hour trough levels and 2-hour peak 
levels at 2nd week, 3rd month,12th month post transplantation, and at any for-cause biopsy 
event. The injury markers will be assessed using Myriad-RBM KidneyMAP panel 
https://myriadrbm.com/products-services/humanmap-services/kidneymap/. 
The other primary end point is immune activation assessed through serial flow cytometric 
immunophenotyping for immune cells including T and B cell subpopulations as well as 
donor-reactive TCR repertoire analysis in biopsy, blood and urine.
Secondary endpoints:
Kidney biopsy will be performed per SOC at month 3 and 12 post-transplant. Biopsies will 
be evaluated and compared between two groups for CNI-related toxicities. Correlations 
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between kidney injury markers and biopsy data at 3 and 12 months post-transplant will be 
performed (details are below).
Antibody responses and correlation with CNI exposure by SOC donor specific antibody 
(DSA) assessment at months 3 and 12 post-transplant (performed at the Clinical 
Immunology Laboratory of the CTC).
PROCEDURES INVOLVED:
Study Design: 
Donors: 
Blood samples (50ml) from donor subjects will be obtained before 
transplantation. These donor leukocytes will be used as stimulator cells in studies of 
functional activity of recipient T-cells. These samples will be processed in the research 
laboratory of co-investigator, Dr. James Mathew.
Renal Transplant Recipients: All recipients will receive the same induction immunotherapy 
at the time of transplant. 
Mycophenolate (MMF, Myfortic®) 
The MPA/Myfortic will be initiated at 720- 1250 mg BID orally. The first dose will be 
given, per SOC. The agent will be given in an open label fashion. Once discharged from 
the hospital, the subject will obtain this medication via a prescription at a pharmacy of 
their choice. 
Alemtuzumab (Campath-1H®) 
One dose will be 30mg given IV, and administered intra-operatively during renal 
transplantation. The agent will be given in an open label fashion and will only be 
administered in the hospital. 
Corticosteroids 
Intravenous corticosteroids (prednisolone, Solumedrol®) 
Time: Dose 
Preop (Intraop) 500mg 
POD#1 250mg 
POD#2 125mg 
No further steroids will be given post-transplant unless indicated by the following medical 
conditions: acute renal allograft rejection, renal diseases necessitating the use of 
steroids, and other systemic diseases such as rheumatoid arthritis (RA), systemic lupus 
erythematosus (SLE), and asthma.
Tacrolimus (Prograf®) 
Tacrolimus will be given 1-2 mg BID, per SOC. The dose will be modified to achieve 12 
hour trough concentrations of 8-12 ng/ml. The agent will be given in an open label 
fashion. Half of the study subjects (randomized; n=25) will be on this drug treatment 
LCP-Tacrolimus/Envarsus® XR (Study related drug)
LCP-Tacro/Envarsus® XR a new once-daily formulation of tacrolimus, was approved by 
the FDA in 2015 for conversion from twice-daily tacrolimus (above) in kidney transplant 
recipients. It is a prolonged-release tacrolimus formulation, utilizing a MeltDose drug 
delivery technology designed to improve the bioavailability of drugs with low water 
solubility. The other half of the study subjects (randomized; n=25) will be on this drug 
treatment. LCP-Tacro/Envarsus XR will be given 5 mg QD to achieve a 24-hour trough 
concentration of 8-12 ng/ml.
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Study Drugs
None of the drugs given in conjunction with this study are provided by the study sponsor, 
including LCP-tacro/Envarsus XR.
Recipient Samples collection: 
Blood, urine and biopsies will be collected from the kidney transplant recipients as 
shown in Figure 2 (bottom). 
Enrollment and randomization at 1:1 in the trial
Renal Transplant
Renal Transplant
Induction: Alemtuzumab (Campath) + Rapid Steroid elimination
Standard Tacrolimus 
+ MMF (n=25)
LCP-Tacrolimus/Envarsus XR 
+ MMF (n=25)
50 ml of blood samples at baseline (pre-Tx), 3 and 12 months post-Tx*
(For FACS and donor-reactive TcR & B cell analyses. Also SOC DSA estimation.)
Up to 200 cc of urine at 2 weeks, 3 months and 12 months post-Tx*
(for renal injury markers & donor-reactive TcR analyses)
Kidney tissue samples at baseline, 3 and 12 months post-Tx*
(for SOC immuno-histochemical and experimental TcR Repertoire assessments)
At any for-cause biopsy, the blood, urine and tissue samples will be collected.
*Note: Sample collection may be adjusted by +/- 10 days, with the exception of the
2nd week urine sample, which may be adjusted by +/- 7 days.
Figure 2: Flow Chart of the Study
Kidney transplant function: 
For subjects receiving Standard Tacorolimus + MMF urine samples will be collected at 
12-hour trough levels and 2-hour peak levels at 2nd week, 3rd month, 12th month post 
transplantation, and at any for-cause biopsy event. 
For subjects receiving LCP-Tacrolimus/Envarsus XR + MMF, urine samples will be 
collected at 24-hour trough and 2-hour peak levels at 2nd week, 3rd month, 12th month 
post transplantation, and at any for-cause biopsy event.
Urine will be used for the analysis of kidney injury markers using Myriad-RBM 
KidneyMAP panel https://myriadrbm.com/products-services/humanmap-
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services/kidneymap/. [The samples will be used also for TcR repertoire analysis; (please 
see below).]
Blood Immunophenotyping. 
The approach will be to conduct a multi-parameter flow cytometric immunophenotyping 
of peripheral blood before and at 3 and 12 months post-transplant to analyze cellular 
changes. 
Methods (14-16): Our well-established 5-color flow cytometric analyses is performed 
using whole blood to delineate the cellular makeup in terms of both percentages and 
absolute numbers (Table 1). RBCs are lysed before flow acquisition. The analysis is 
for defining the T cell subsets, monocytes and natural killer cells (Tube 2), the B cells 
including naïve, transitional, memory and effector-memory subsets (Tube 3) and the 
dendritic cells including immature, monocytoid and plasmacytoid fractions (Tube 4). 
In addition to the whole blood analyses, PBMC are isolated from heparinized 40 cc 
blood samples by Ficoll-Hypaque gradients. Surface markers are identified with 
indicated monoclonal antibodies, and after fixation and permeabilization the 
intracellular protein ligands are detected with antibodies shown on the bottom row 
(Table 2). This panel delineates expression of markers for activated, effector, 
memory, anergic and exhausted cell fractions as well as myriad sub-sets of 
regulatory T and B cells. 
Donor-reactive TCR repertoire analysis The approach will be to identify and estimate 
the total T cell clonal repertoire that a patient is capable of mounting against the donor 
by performing MLR and purifying reactive cells followed by ImmunoSEQ analysis (17) 
prior to transplant. Then after transplant, monitor for the absence, presence or 
amplification of the pre-identified T cell clones in the post-transplant biopsies, blood 
and/or urine samples. This is a novel innovative technology that we have developed for 
profiling recipient’s immune status against the donor to correlate with the status of the 
graft. The methodological approach is follows:
Table 1: Whole blood immunophenotyping panel
Tube 1
Tube 2
Tube 3
Tube 4
IgG-FITC
CD4-FITC
CD38-FITC
CD3, 14, 16, 19, 20, 56 FITC
IgG-PE
CD14-PE
IgD-PE & IgM-PE
CD33-PE
IgG-ECD
CD56-ECD
CD19-ECD
HLA-DR-ECD
IgG-PC5
CD3-PC5
CD27-PC5
CD11b-PC5
IgG-PC7
CD8-PC7
CD24-PC7
CD123-PC7
Table 2: Immunophenotyping in using isolated PBMC (surface and 
intracellular)
Tube 5
Tube 6
Tube 7
Tube 8
Tube 9
Staining
CD4-ECD
IgD/M-PE
CD3-ECD
CD49b 
FITC
KLRG-1 
PE
CD25-PC5
CD19-ECD
CD28-PC5
LAG-3 PE
PD1-ECD
CD127-PC7
CD27-PC5
CD8-PC7
CD4 ECD
CD3-PC5
CTLA4-FITC
CD24-PC7
CD45RA -FITC
CD8  -PC7
Surface
FoxP3-PE
IL-10 -FITC
FoxP3-PE
FoxP3 PC5 
& IL10 PC7
Ki-67 FITC
Intra-
cellular
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Selection of donor-reactive T cells: Recipient pre-transplant peripheral blood 
mononuclear cells (PBMC) will be labeled with CFSE dye and then stimulated with 
PKH266-labled donor irradiated PBMC in MLR. After 7 days, the proliferating responder 
cells that have diluted the CFSE are flow-sorted (at NU Flow Core) as shown in Figure 3 
(top middle). Based on previous results, it is expected that the proliferating cells will be 
>90% CD3+ T cells, distributed into CD4+ or CD8+ cells, with minimal contamination with 
CD19+ B cells or CD14+ monocytes but some CD56+ NK cells. Both the CD3+CD4+ and 
CD3+CD8+ cell subsets are sorted out separately and is shipped to Adaptive 
Biotechnologies in Seattle.
ImmunoSEQ analysis of donor-reactive T cells:  At Adaptive Biotechnologies total 
genomic DNA is purified from the flow-sorted CD3+CD4+ and CD3+CD8+ cell subsets 
from each culture using Qiagen DNeasy Blood extraction Kit following the 
manufacturer’s protocol. Then, a multiplex PCR assay that is capable of specifically 
amplifying rearranged 
TCRB genes from a 
complex background 
followed by deep 
sequencing is 
performed (Figure 4). 
The data is analyzed 
measuring clonality, 
proportion and 
diversity of the T cell 
repertoire (18-20). 
These represent the 
total T cell repertoire 
the recipient is capable 
of reacting to the 
donor. We have 
denoted the 
performance of the 
pre-transplant MLR, 
isolation of donor reactive clones and ImmunoSEQ analysis, collectively as 
AlloSEQ. 
Figure 3.  Flow Sorting and 
phenotypic subset analysis 
of donor reactive clones. 
Bulk MLRs were prepared and 
the cellular makeup of 
responder cell populations 
were delineated after 7 days in 
culture using fluorochrome 
coupled monoclonal 
antibodies. The cells were 
analyzed first by gating on 
lymphocytes and then after 
gating on CFSE diluted 
proliferating responder cells. 
The CFSE diluted donor 
reactive T cells were sorted for 
ImmunoSEQ analysis.
Figure 
4.  
TCRβ 
sequencing assay.  
(A) The assay is designed 
to identify the V D and J 
segments in recombined 
CDR3 TCRβ genes  
(B) Forward primers were 
designed to amplify all 45 
TCRβ 
V 
segments.   
Reverse primers were 
designed to each of the 
13 TCRβ J segments.  
(C) 
Custom 
software 
tools 
have 
been 
developed 
to 
verify, 
collapse, 
align 
and 
catalog 
the 
CDR3 
sequences for calculation 
of repertoire diversity.
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Enumeration of donor reactive T cell clones in post-transplant kidney biopsies, 
blood and urine: 
The transplant biopsy represents a sampling of the allograft and rejection is manifested 
as cellular infiltration, mostly of donor reactive T cells and B cells, classically detected 
through immunohistopathological staining and microscopic examination. An alternate 
approach that we have developed is to monitor the graft for infiltrating donor-reactive 
clonotype identified through AlloSEQ (17). This would entail isolation of total genomic 
DNA from the biopsies, performance of immunoSEQ analysis as above and monitoring 
for the presence and copy number of donor-reactive clones identified through the pre-
transplant AlloSEQ, and then correlating with the severity of rejection monitored through 
immunohistopathological examinations. 
We had proposed that transplant rejection can more easily be diagnosed through the 
sampling of the urine pellet or peripheral blood for clonal frequency and abundance of 
donor-reactive T cells pre-identified through AlloSEQ. This is to monitor rejection in a 
non-invasive manner.
We have already established the technology (17) and other investigators have used it to 
show that donor reactive clones are reduced or deleted in tolerant kidney transplants 
(21). Therefore, these proposed studies monitoring post-transplant graft rejection or its 
absence can be greatly leveraged to innovatively assess the relative efficacies of the 
drugs being compared. 
Analysis of HLA and Donor-Specific B cells 
Measurement of B cells in the peripheral blood with potential to make HLA antibodies 
against graft donor antigens might provide a way of analyzing antibody formation and 
shed light on the balance between production, inhibition and sequestration at any given 
time in the post-transplant period. Perry and coworkers have recently reported HLA 
antibody production in vitro by B cells from the bone marrow of sensitized kidney 
transplant recipients. T and B-cell ELISPOT have also been used to measure HLA 
specific B cell frequency against a given antigen. ELISPOT assay does not measure the 
frequency of cells that actually bind the antigen and only measures biological events 
such as cytokine release or production of immunoglobulin after differentiation in vitro. 
Furthermore, the estimated frequency is restricted to the cells that are able to be 
selectively stimulated by the antigen depending on read-out and thus may lead to an 
underestimation of the actual frequency of committed cells. Another approach based on 
the structural similarity between B cell receptor and immunoglobulin binding sites, it is 
postulated that HLA-specific B cells (HSB) should bind to HLA molecules with specificity 
comparable to that of the secreted immunoglobulin. Identification of HLA specific B cells 
by staining through binding of the B cell receptor using fluorescently labeled tetramers of 
identified HLA class I specificities has been described. A solid-phase assay utilizing 
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color-coded microspheres, each coated with a unique HLA antigen has become the 
main multiplex assay used for the detection of allo-specific antibodies against donor 
antigens. Its multiplexing power enables the analysis of the antibodies directed to all 
available synthetic HLA class I and class II alleles in one assay. Utilizing these single 
antigen HLA beads (SAB) Degauque et al. described a novel assay to identify HLA-
specific B cells in transplant recipients (22). We have further developed this novel 
multiplex bead-based assay to identify HSB (schematically described in Figures 5 and 
6), and propose here to study the circulating HSB in kidney transplant recipients with 
chronic AMR. 
Figure 5: Assay Design
Proposed studies: 
1. Frequency of Donor-specific B cells (DSB) by Novel HLA Bead Multiplex Assay: We 
will analyze the serial samples of peripheral blood mononuclear cells (PBMC) using 
our novel HLA-bead multiplex assay to identify and determine the changes in 
frequency of total B, HLA-specific B (HSB) and DSB cells in KT recipients with cAMR 
in each study arm. 
Figure 6: Analysis Strategy
2. Immunophenotyping of HLA-specific B cells and Donor-Specific B cells by Flow 
Cytometry: We will analyze serial samples of PBMC, before and at 3 and 12 months 
post-transplant, using multi-parameter flow-cytometry for immunophenotyping of (i) 
total B, HSB and DSB cells with surface expression of IgD, CD24, CD27 and CD38. 
DATA AND SPECIMEN BANKING:
Samples collected will be banked at the Immune Monitoring Core (IMC) (under the leadership of 
Co-I Dr. James Mathew) of the Comprehensive Transplant Center (CTC) of Northwestern 
University. The blood immunophenotyping will be performed prospectively on fresh samples.  
The pre-transplant donor-specific MLR and flow-sorting of reactive T cells will also be performed 
with fresh samples.  Cell pellets thus obtained (for AlloSEQ analysis) as well as post-transplant 
PBMC, urine sample pellets and biopsies will be frozen and sent blinded (de-identified) to 
Adaptive Biotechnologies, Seattle for DNA purification and ImmunoSEQ analyses. Leftover 
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samples of biopsies (paraffin-block after IHC staining), PBMC and urine pellets will be used for 
RNA isolation and gene expression profiling in a future study.  
DATA AND SPECIMEN MANAGEMENT:
Statistical analysis:
The primary endpoints of the study are graft function and subpopulations of T cells, 
including regulatory T cells and B cell subpopulations as well as immune functions at 3 and 
12 months post kidney transplant. 
The secondary endpoints of the study include 1) incidence of acute rejection, graft loss, and 
death at 3 and 12 months post kidney transplant; 2) allograft immunohistopathology profiles 
at 3 and 12 months post kidney transplant.
We will run univariate and bivariate analyses for all variables prior to our main analyses. We 
will evaluate continuous variables using t-tests or F-tests and categorical variables using 2. 
We will examine data distributions and test all variables for linear relationships or non-linear 
relationships.
Descriptive statistics (means, standard deviation, frequency) and correlations (both Pearson 
and Spearman) among variables will be calculated for preliminary data assessment.
Necessary transformation and imputations will be carried out based on the raw data 
distribution.
As missing data are inevitable in a longitudinal study, we will determine whether missing 
data are MCAR (data are missing completely at random), MAR (data are missing at 
random), or NMAR (not missing at random). If missing data are MCAR or MAR, it is likely 
that the standard multivariate computations using PROC MI (multiple imputations) will not 
result in biased standard error estimates. However, if missing data are NMAR, we will use 
the "pattern mixture" approach to compute a weighted average of the parameters that are 
associated with the missing data to estimate what the data would have been.
For the primary objective, we hypothesize that subjects receiving LCP-Tacro/Envarsus® XR 
will exhibit significantly greater graft function and lower frequency / abundance of AlloSeq 
pre-identified donor-reactive T cell clones in the graft, peripheral blood and /or urine at 3 and 
12 month post-transplant as compared to the standard of care tacrolimus group. A variety of 
covariance structures (first order regressive, compound symmetry, Toeplitz, variance 
components, unstructured) will be carefully examined and compared using the best fitting 
statistics, such as Akaike’s Information (AIC) and Bayesian Information Criteria (BIC). These 
statistics are likelihood functions and can be compared across models. If, in spite of 
randomization, distributions of subjects’ baseline characteristics are found to be different 
between the two experimental groups, these variables will be entered as covariates in the 
statistical analysis.
For the secondary endpoints, we will use logistic regression, Kaplan-Meier method, and Cox 
proportional hazards models to analyze time-to-event outcomes (acute rejection, graft 
failure, and death). For allograft immunohistopathology outcomes, we will first develop a 
comprehensive immune profile of each participant by longitudinal analysis of frequency of T 
and B cell subsets and function. We will examine how these correlate with 
immunohistopathology and alloantibody production using Pearson or Spearman 
correlations. We will compare correlation coefficients between the two experimental groups 
using Z -tests to determine the distinct effects of each treatment arm. We will also compare 
the trajectory changes in cell populations and antibody production over 3 and 12 months 
using both repeated measures ANOVA and random mixed effect models. All tests will be 
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two-sided, and an error rate of α <0.05 will be considered statistically significant. All data 
analyses will be performed using SAS 9.2 statistical software (SAS Inc., Cary, NC).
Sample size and power:  
This proposal bases its sample size calculations on published data demonstrating increased 
peaks and troughs with conventional tacrolimus and absence of such peaks and troughs 
with LCP-Tacro/Envarsus® XR (1) and correlation with high doses and nephrotoxicity with 
conventional tacrolimus (4). Using a two-tailed α of 0.05, the anticipated effect sizes 
corresponding to each specific aim between the two treatment arms were calculated using a 
minimum of 80% statistical power and assumed 10% attrition at 12 months.
For the sample size of n= 25 in each group, the proposed study will have a power of 0.82 
and 0.99 to detect effect size of 1.0 and 2.0 for graft function and immune activation (in 
terms of T cell clonal repertoire change), respectively, between the two treatment arms. 
Because this is a pilot study, we do not consider the statistical power of secondary 
outcomes; It is anticipated that a much larger sample size will be needed in order to achieve 
statistical power for the secondary endpoints in this proposed study.
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 a properly constituted independent 
Institutional Review Board (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).  Those regulations 
require a signed subject authorization informing the subject of the following: 1) What 
protected health information (PHI) will be collected from subjects in this study 2) Who will 
have access to that information and why 3) Who will use or disclose that information 4) The 
rights of a research subject to revoke their authorization for use of their PHI. 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 IRB for the study.  The formal consent of a subject, using the 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. After study initiation, the electronic case report form (eCRF) will be the primary 
data collection instrument for the study.  Only the study staff 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 participants. All clinical data will be kept 
completely confidential. The PI, Dr. Lorenzo Gallon and the Co-Is Drs. Joseph Leventhal 
and Dr. Javeed Ansari or their designees will be responsible for the clinical data 
management.
PROVISIONS TO MONITOR THE DATA TO ENSURE THE SAFETY OF PARTICIPANTS:
Safety:
Safety Endpoints: The primary safety endpoints will be assessed on the AEs and SAEs that 
are observed throughout the trial. For the purpose of this clinical trial, the National Cancer 
Institute Common Terminology Criteria for Adverse Events v4.0 3(CTCAE), dated June 14, 
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2010, will be used to grade all adverse events. The collection of AE’s and SAE’s is limited in 
this study to those Grade 3 or higher that are related to study mandated procedures.
Recording of Adverse Events
At each contact with the subject, the investigator/member of investigator’s team will seek 
information on adverse events by specific questioning and, as appropriate, by examination. 
Information on all adverse events will be recorded in the source document. All clearly related 
signs, symptoms, and abnormal diagnostic procedures results should be recorded in the 
source document, though should be grouped under one diagnosis.
All adverse events occurring during the study period that meet the criteria above will be 
recorded. The clinical course of each event should be followed until resolution, stabilization, 
or until it has been determined that the study treatment or participation is not the cause. 
Serious adverse events that are still ongoing at the end of the study period must be followed 
up to determine the final outcome. Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study participation 
will be recorded and reported according to IRB requirements.
Stopping Rules
Subjects developing post-transplant infections (i.e., UTI, CMV, HSV, EBV, HCV, HBV, HIV, 
PCP), that in the opinion of the investigator are detrimental to the subject and his/her 
participation in this research trial, will be stopped from participating in this study (even 
though these adverse events expected to be lower in the experimental arm of the study).
Patient Authorization: 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 a properly constituted independent 
Institutional Review Board (IRB), (Northwestern University Institutional Review Board), in 
agreement with local legal prescriptions, for formal approval of the study conduct. The 
decision of the IRB concerning the conduct of the study will be made in writing to the 
investigator and a copy of this decision will be provided to the investigator/sponsor before 
commencement of this study. The investigator will place a list of IRB members and their 
affiliate in the regulatory binder for the clinical trial. 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.
WITHDRAWAL OF PARTICIPANTS:
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 IRB may also discontinue the study at 
any point. The subjects will be fully informed of these actions. If participants 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. 
RISKS TO PARTICIPANTS:
The risks to the subjects are limited to the risks related to venipuncture and at least two passes 
of renal biopsies. Also, the routine risk of rejection with either of the drugs is expected; in fact, 
lower risk of nephrotoxicity is anticipated with LCP-Tacro/Envarsus® XR.
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The National Cancer Institute Common Terminology Criteria for Adverse Events v4.03 
(CTCAE), dated June 14, 2010 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.  
Serious adverse events that are determined to be unexpected and at least possibly related to 
study participation will be submitted to the IRB within 5 business days of the investigator’s 
knowledge of the event. Follow-up information will be submitted to the IRB as required. The 
IRB/Office for the Protection of Research Subjects at NU is under the direction of Nathalia 
Henry, MS, CIP, CHRC, (312) 503- 2578.
POTENTIAL BENEFITS TO PARTICIPANTS:
It is expected that participants in the experimental arm of LCP-Tacro/Envarsus® XR treatment 
will have lower nephrotoxicity, immune activation and associated improved clinical outcomes. 
These may be direct long-term benefits for participants, if they happen to be randomized into 
the study arm.
ECONOMIC BURDEN TO PARTICIPANTS:
None other than their own routine SOC expenses. A parking voucher will be given to the 
participants at their study visits.  
VULNERABLE POPULATIONS:
None (Please see Inclusion and Exclusion Criteria)
SHARING OF RESULTS WITH PARTICIPANTS:
At the end of the study, at the PIs discretion, the participants may be informed of the incidental 
findings. This study will be registered at clinicaltrials.gov.
PRIOR APPROVALS:
All required approvals such as laboratory, radiation safety, or biosafety are already in place. 
SETTING:
Renal transplantations will be performed by the co-I, Dr. Joseph Leventhal and his surgical 
colleagues at Northwestern Memorial Hospital. The clinical follow-up will be conducted as 
standard of care by the PI, Dr. Lorenzo Gallon and the transplant nephrologists including Dr. 
Javeed Ansari with the help of transplant nurse coordinator (TNC). The immune monitoring 
studies will be directed by Co-I, Dr. James Mathew with the help of Drs. Jie He and Xuemei 
Huang, two senior technologists in the Immune Monitoring Core of the CTC. Blinded (de-
identified) samples will also be sent to Adaptive Biotechnologies, Seattle for ImmunoSEQ 
analyses. Leftover samples, if any, are expected to be discarded at the end of the study.  
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RESOURCES AVAILABLE:
Successful completion of the project requires the following: a) Team of dedicated investigators: 
Drs. Gallon and Ansari are transplant nephrologists and Dr. Leventhal is a transplant surgeon 
and they have dedicated their careers to translational research to refine the management of 
renal transplant recipients. They will actively recruit all patients and, assisted by an RN research 
coordinator at the CTC, perform all of the clinical assessments in this cohort. They will maintain 
the clinical database with help from the assistant coordinators. They will work closely with the 
co-I, Dr. James Mathew and his trained technologists in the Immune Monitoring Core of the 
CTC performing and interpreting the assays. Dr. Lihui Zhao will perform the statistical analysis 
of the immunophenotyping and Dr. Yashpal Kanwar will interpret the biopsies and IHC staining. 
Northwestern has formed a collaborative partnership with Adaptive Biotechnologies, Seattle for 
ImmunoSEQ analyses to identify TcR repertoire usage in donor-specific immune responses. A 
similar partnership has been established with Myriad-RBM for the analysis of  kidney injury 
markers using their KidneyMAP panel,  b) Ideal patient population: The Northwestern CTC 
transplant center provide the ideal infrastructure and performs a high number (>220) of living 
and deceased donor kidney transplant procedures/year to conduct this prospective trial and to 
analyze fresh patient samples. This readily supports recruiting 50 patients within one year 
enrollment period, even when considering the inclusion/exclusion criteria and other concurrent 
studies; c) Institutional support: Dr. Michael Abecassis, division head of the CTC will provide the 
full infrastructural support to conduct all clinical and laboratory portions of the study. In 
summary, the investigators, patient availability, and environment afford the highest chance of 
success in this proposal. 
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