Clinical trial NCT02723591
Conditions: Kidney Transplantation
Keywords: FK506 Kidney Transplantation Tacrolimus Astagraf XL
Sponsor: Astellas Pharma Global Development, Inc.
Start date: 2016-09-09; Completion date: 2019-06-14
Enrollment: 599 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study compared the incidence of a two-part composite endpoint consisting of de novo donor specific antibody (DSA) formation or a designation of immune activation (IA) on peripheral blood molecular profiling in participants maintained on twice daily, immediate-release tacrolimus versus those maintained on Astagraf XL in the first year post-transplant.

Detailed description:
This was an exploratory, two year (shortened to 1 year due to a stopping rule necessitated by the adaptive design), prospective, randomized, multi-center, open-label trial examining long-term kidney transplant outcomes through the use of an adaptive design and a two-part, composite surrogate endpoint. Specifically, it was designed to compare the effects of twice daily, immediate-release tacrolimus and once daily Astagraf XL on DSA formation and the development of a peripheral blood molecular profile indicating the presence of IA in de novo kidney transplant recipients during the first year following transplantation. For the purposes of this study, IA was defined as a positive molecular signature using a molecular assay in all participants.

Participants were screened prior to surgery and randomized 1:1 to receive immediate-release tacrolimus, administered twice daily, or Astagraf XL, as a component of a standard immunosuppression maintenance regimen also consisting of corticosteroids (if given per institutional protocol) and mycophenolate mofetil (MMF) (or Myfortic® equivalent). Investigators were encouraged to start participants on the randomized study treatment (immediate release tacrolimus or Astagraf XL) within 48 hours of transplantation (pre-transplant administration of study treatment was not allowed). However, if medically indicated per the treating physician's discretion, initiation of study treatment was delayed for up to seven days post-transplant.

Arms: arm 1: Participants received tacrolimus extended release (Astagraf XL) at a starting dose of 0.15 milligram per kilogram (mg/kg), once daily, orally within 48 hours of transplantation (per the treating physician's discretion) for up to 1 year. Dose adjustments were allowed such that participants receiving tacrolimus maintained a minimal trough concentration of 6 nanogram per milliliter (ng/mL) at all times during the study. arm 2: Participants received tacrolimus immediate release as per the institutionally-derived protocol, BID, orally within 48 hours of transplantation (per the treating physician's discretion) for up to 1 year. Dose adjustments were allowed such that participants receiving tacrolimus maintained a minimal trough concentration of 6 ng/mL at all times during the study.
Interventions: intervention 1: Tacrolimus intervention 2: Tacrolimus immediate release
Intervention descriptions: intervention 1: Oral Capsule intervention 2: Oral Capsule

Protocol documents:
Prot_000.pdf: 
Astagraf XL® to Understand the Impact of 
Immunosuppression on De Novo DSA Development and 
Chronic Immune Activation in Kidney Transplantation
ISN/Protocol IDTX-MA-3004
ClinicalTrials.gov Identifier: NCT02723591
Date of Protocol v2.1: 25 May 2018
Sponsor: Astellas Pharma Global Development, Inc. (APGD)
Medical Affairs, Americas
1 Astellas Way
Northbrook, IL 60062


Sponsor: APGD, Medical Affairs, Americas
ISN/Protocol IDTX-MA-3004
- CONFIDENTIAL -
25 May 2018 
V2.1, Incorporating Non-Substantial Amendment 2
Astellas
Page 1 of 124
Astagraf XL® to Understand the Impact of Immunosuppression 
on De Novo DSA Development and Chronic Immune Activation 
in Kidney Transplantation
™
Protocol for Phase 4 Study of Astagraf XL®
(tacrolimus extended-release capsules)
ISN/Protocol IDTX-MA-3004
Version 2.1
Incorporating Non-Substantial Amendment 2
May 25, 2018
IND 64148
Sponsor:
Astellas Pharma Global Development, Inc. (APGD)
Medical Affairs, Americas
1 Astellas Way
Northbrook, IL 60062
Investigator:  Investigator information is on file at Astellas.
This confidential document is the property of the Sponsor.  No unpublished information contained in this 
document may be disclosed without prior written approval of the Sponsor.


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Table of Contents
I.
SIGNATURES ······················································································ 8
II.
CONTACT DETAILS OF KEY SPONSOR’S PERSONNEL····························10
III.
LIST OF ABBREVIATIONS AND DEFINITION OF KEY TERMS ··················11
IV.
SYNOPSIS··························································································16
V.
FLOW CHART AND SCHEDULE OF ASSESSMENTS·································30
1
INTRODUCTION·················································································34
1.1
Background ····················································································34
1.2
Clinical Data···················································································37
1.2.1
Clinical Summary ·······································································37
1.2.2
Clinical Studies of Efficacy and Safety···············································39
1.2.3
Whole Blood Trough Concentrations Can Be Used to Target the Same 
Systemic Exposure for Both Prograf and Astagraf··································40
1.2.4
Increased Potential for Adverse Clinical Outcomes with Too High or Too 
Low Tacrolimus Exposure ·····························································40
1.2.5
Association Between the Potential for Adverse Clinical Outcomes and 
Increased Tacrolimus Exposure Variability··········································41
1.3
Summary of Key Safety Information for Study Drugs ···································43
1.4
Risk-Benefit Assessment·····································································43
2
STUDY OBJECTIVE(S), DESIGN, AND ENDPOINTS ··································44
2.1
Study Objectives ··············································································44
2.1.1
Primary Objective ·······································································44
2.1.2
Secondary Objectives ···································································44
2.1.3
Exploratory Objectives ·································································45
2.2
Study Design and Dose Rationale···························································46
2.2.1
Study Design·············································································46
2.2.2
Dose Rationale···········································································48
2.3
Endpoints·······················································································49
2.3.1
Primary Endpoints·······································································49
2.3.2
Secondary Endpoints····································································49
2.3.3
Exploratory Endpoints ··································································50
3
STUDY POPULATION··········································································51
3.1
Selection of Study Population ·······························································51


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3.2
Inclusion Criteria··············································································51
3.3
Exclusion Criteria ·············································································53
4
TREATMENT(S)··················································································55
4.1
Identification of Investigational Product(s) ················································55
4.1.1
Test Drug(s)··············································································55
4.1.2
Comparative Drug(s)····································································55
4.2
Packaging and Labeling ······································································56
4.3
Study Drug Handling ·········································································56
4.4
Blinding ························································································57
4.5
Assignment and Allocation ··································································57
5
TREATMENTS AND EVALUATION························································57
5.1
Dosing and Administration of Study Drug(s) and Other Medication(s)················57
5.1.1
Dose/Dose Regimen and Administration Period ····································57
5.1.2
Increase or Reduction in Dose of the Study Drug(s)································59
5.1.3
Previous and Concomitant Treatment (Medication and Non-Medication 
Therapy) ··················································································59
5.1.4
Treatment Compliance··································································59
5.1.5
Restrictions During the Study··························································60
5.2
Demographics and Baseline Characteristics ···············································60
5.2.1
Demographics············································································60
5.2.2
Medical History··········································································60
5.2.3
Transplant Information ·································································60
5.2.4
Height and Body Weight ·······························································61
5.2.5
Diagnosis of the Target Disease, Severity, and Duration of Disease··············61
5.3
Efficacy Assessment··········································································61
5.3.1
Efficacy···················································································61
5.3.2
Donor Specific Antibody·······························································61
5.3.3
Antibody Persistence····································································62
5.3.4
C1q-binding DSA ·······································································62
5.3.5
IgG3 Isotyping ···········································································62
5.3.6
Requirement for Antibody Removal ··················································62
5.3.7
Histopathology···········································································62
5.3.8
Molecular Endpoints ····································································63
5.3.9
Estimated Glomerular Filtration Rate·················································63


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5.3.10
Patient Survival··········································································63
5.3.11
Graft Survival············································································63
5.3.12
Acute Rejection··········································································63
5.4
Safety Assessment ············································································64
5.4.1
Vital Signs················································································64
5.4.2
Adverse Events ··········································································64
5.4.2.1
Adverse Events of Possible Hepatic Origin ····································64
5.4.3
Laboratory and Pathological Assessments ···········································64
5.4.4
Physical Examination···································································65
5.5
Adverse Events and Other Safety Aspects ·················································65
5.5.1
Definition of Adverse Events (AEs) ··················································65
5.5.2
Definition of Serious Adverse Events (SAEs) ·······································65
5.5.3
Criteria for Causal Relationship to the Study Drug ·································66
5.5.4
Criteria for Defining the Severity of an Adverse Event ····························67
5.5.5
Reporting of Serious Adverse Events (SAEs) ·······································67
5.5.6
Follow-up of Adverse Events··························································68
5.5.7
Monitoring of Common Serious Adverse Events ···································68
5.5.8
Procedure in Case of Pregnancy·······················································69
5.5.9
Emergency Procedures and Management of Overdose ·····························69
5.5.10
Supply of New Information Affecting the Conduct of the Study··················70
5.6
Test Drug Concentration ·····································································70
5.7
Other Measurements, Assessments or Methods ···········································70
5.7.1
Sample for the Analysis of Validated Gene Expression Profiling in the 
Peripheral Blood·········································································70
5.7.2
Blood Sample for DSA, C1q-binding DSA Analysis, and IgG3 DSA 
Isotyping··················································································71
5.7.3
Storage of Donor Tissue for DNA Typing ···········································71
5.7.4
Banking of Blood for Future Studies··················································72
5.8
Total Amount of Blood·······································································72
6
DISCONTINUATION············································································72
6.1
Discontinuation of Individual Subject(s)···················································72
6.2
Discontinuation of the Site···································································73
6.3
Discontinuation of the Study·································································73
7
STATISTICAL METHODOLOGY ···························································74


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7.1
Sample Size····················································································74
7.2
Analysis Set····················································································74
7.2.1
Full Analysis Set (FAS) ································································74
7.2.2
Modified Full Analysis Set (mFAS) ··················································74
7.2.3
Per Protocol Set (PPS) ··································································75
7.2.4
Safety Analysis Set (SAF)······························································75
7.2.5
Pharmacokinetic Analysis Set (PKAS) ···············································75
7.3
Demographics and Other Baseline Characteristics········································75
7.4
Analysis of Efficacy ··········································································75
7.4.1
Analysis of Primary Endpoint ·························································75
7.4.1.1
Primary Analysis···································································75
7.4.1.2
Sensitivity Analysis ·······························································76
7.4.1.3
Secondary Analysis································································76
7.4.1.4
Subgroup Analyses of the Primary Endpoint···································76
7.4.2
Analysis of Secondary Endpoints ·····················································76
7.4.3
Analysis of Exploratory Endpoints····················································78
7.5
Analysis of Safety·············································································79
7.5.1
Adverse Events ··········································································79
7.5.2
Laboratory Assessments································································79
7.5.3
Histopathology···········································································80
7.6
Analysis of Tacrolimus Dose and Trough Concentrations·······························80
7.7
Protocol Deviations and Other Analyses···················································80
7.8
Interim Analysis (and Early Discontinuation of the Clinical Study)····················81
7.8.1
Formal Stopping Rule···································································81
7.9
Handling of Missing Data, Outliers, Visit Windows, and Other Information ·········82
8
OPERATIONAL AND ADMINISTRATIVE CONSIDERATIONS ····················83
8.1
Procedure for Clinical Study Quality Control ·············································83
8.1.1
Data Collection ··········································································83
8.1.2
Specification of Source Documents···················································84
8.1.3
Clinical Study Monitoring······························································84
8.1.4
Direct Access to Source Data/Documents············································85
8.1.5
Data Management ·······································································85
8.1.6
Protocol Deviations ·····································································85
8.1.7
End of Trial in All Participating Countries···········································86


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8.2
Ethics and Protection of Subject Confidentiality··········································86
8.2.1
Institutional Review Board (IRB) / Independent Ethics Committee (IEC) / 
Competent Authorities (CA) ···························································86
8.2.2
Ethical Conduct of the Study···························································87
8.2.3
Informed Consent of Subjects ·························································87
8.2.3.1
Subject Information and Consent/Assent ·······································87
8.2.3.2
Supply of New and Important Information Influencing the Subject’s 
Consent and Revision of the Written Information ·····························87
8.2.4
Subject Confidentiality ·································································88
8.3
Administrative Matters ·······································································88
8.3.1
Arrangement for Use of Information and Publication of the Clinical Study ·····88
8.3.2
Documents and Records Related to the Clinical Study ·····························89
8.3.3
Protocol Amendment and/or Revision················································89
8.3.4
Signatory Investigator for Clinical Study Report····································89
9
QUALITY ASSURANCE········································································90
10
STUDY ORGANIZATION······································································90
10.1
Independent Data-Monitoring Committee (IDMC) | Data and Safety Monitoring 
Board (DSMB) | Monitoring Committee | Other Evaluation Committee(s) ···········90
10.2
Adjudication Board: MFI Adjudication Committee ······································90
11
REFERENCES ····················································································91
12
APPENDICES ··················································································· 100
12.1
List of Excluded Concomitant Medications·············································· 100
12.2
Liver Safety Monitoring and Assessment ················································ 101
12.3
Common Serious Adverse Events························································· 104
12.4
Interim Decision Rule Details ····························································· 105
12.5
Interim Stopping Rules Risk Analysis···················································· 107
13
ATTACHMENT 1: NON-SUBSTANTIAL AMENDMENT 2 ························· 110
14
SPONSOR’S SIGNATURE ··································································· 124


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List of In-Text Tables
Table 1
Schedule of Assessments·····································································31
List of In-Text Figures
Figure 1
Correlation between AUC and Cmin of Advagraf (tacrolimus QD) and Prograf 
(tacrolimus BID) in heart transplant recipients ············································40
Figure 2
Variability in tacrolimus trough levels associated with increased risk of graft 
loss in adolescents·············································································42
Figure 3
Application of Stopping Rule································································82


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I.
SIGNATURES
1.  SPONSOR’S SIGNATURE
Astagraf XL® to Understand the Impact of Immunosuppression on De Novo DSA 
Development and Chronic Immune Activation in Kidney Transplantation
ISN/Protocol IDTX-MA-3004 / 
Version 2.1 / Incorporating Non-Substantial Amendment 2 / dated May 25, 2018
Required signatures (e.g., Protocol authors, Sponsor’s reviewers and contributors, etc.) are 
located in Section 14, Signatures; e-signatures (when applicable) are located at the end of 
this document. 


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2.  INVESTIGATOR’S SIGNATURE
Astagraf XL® to Understand the Impact of Immunosuppression on De Novo DSA 
Development and Chronic Immune Activation in Kidney Transplantation
ISN/Protocol IDTX-MA-3004 / 
Version 2.1 / Incorporating Non-Substantial Amendment 2 / dated May 25, 2018
I have read all pages of this clinical study protocol for which Astellas is the Sponsor. I agree 
to conduct the study as outlined in the protocol and to comply with all the terms and 
conditions set out therein. I confirm that I will conduct the study in accordance with ICH 
GCP guidelines and applicable local regulations. I will also ensure that sub-investigator(s) 
and other relevant members of my staff have access to copies of this protocol and the ICH 
GCP guidelines to enable them to work in accordance with the provisions of these 
documents.
Principal Investigator:
Signature:
<Insert name and qualifications of the Investigator>
Printed Name:
Date (DD Mmm YYYY)
Address:




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III.
LIST OF ABBREVIATIONS AND DEFINITION OF KEY TERMS
List of Abbreviations
Abbreviations
Description of abbreviations
ABMR
Antibody-mediated Rejection
ABO
Blood Group System (A, B, AB, and O)
ADNR
Acute Dysfunction/No Rejection
AE
Adverse event
Ag
Antigen
ALP
Alkaline Phosphatase
ALT
Alanine Aminotransferase (GPT)
Anti-HBs
Hepatitis B surface antibody
APGD
Astellas Pharma Global Development, Inc.
AST
Aspartate Aminotransferase (GOT)
ATP
Adenosine 5'-Triphosphate
AUST
Astellas US Technology
BKV
BK Virus
BPAR
Biopsy-Proven Acute Rejection
C1q
Complement Component 1, Q Subcomponent
C4d
Complement Split Product, 4d
CAN
Chronic Allograft Nephropathy
CD52
Cambridge Pathology Antigen 1
CG
Chronic Glomerulopathy
CI
Confidence Intervals
CIT
Cold Ischemia Time
CLIA
Clinical Laboratory Improvement Amendments
CMV
Cytomegalovirus
CNI
Calcineurin Inhibitors
cPRA
Calculated Panel Reactivity Antibody
CR
Chronic Rejection
CRF
Case Report Form
CRO
Contract Research Organization
CSR
Clinical Study Report
CV
Coefficient of Variation
CYP3A
Cytochrome P450 3A System
DCD
Donation after Circulatory Death
DD
Deceased Donor
DeKAF
Deterioration of Kidney Allograft Trial
DILI
Drug-induced Liver Injury
DLDA
Diagonal Linear Discriminant Analysis
DSA
Donor-Specific Antibody
EBV
Epstein–Barr Virus
ECD
Extended Criteria Donor
ECG
Electrocardiogram


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Abbreviations
Description of abbreviations
eCRF
Electronic Case Report Form
EDC
Electronic Data Capture
eGFR
Estimated Glomerular Filtration Rate
FAS
Full Analysis Set
GCP
Good Clinical Practice
GEE
General Estimating Equations
GMP
Good Manufacturing Practices
H&E
Hematoxylin and Eosin
HBc
Hepatitis B Virus Core 
HBsAg
Hepatitis B Surface Antigen
HBV
Hepatitis B Virus
HCV
Hepatitis C Virus
HgA1c
Hemoglobin A1c
HIPAA
Health Insurance Portability and Accountability Act
HIV
Human Immunodeficiency Virus
HLA
Human Leukocyte Antigen
HLA-DP
Human Leukocyte Antigen, DP locus
HLA-DQ
Human Leukocyte Antigen, Class II, DQ locus
IA
Immune Activation
IB
Investigator’s Brochure
ICF
Informed Consent Form
ICH 
International Conference on Harmonization of Technical Requirements for 
Registration of Pharmaceuticals for Human Use
IEC
Independent Ethics Committee
IFTA
Interstitial Fibrosis and Tubular Atrophy
IgG3
Immunoglobulin G
IgM
Immunoglobulin M
IL-2
Interleukin-2
IND
Investigational New Drug
INR
International Normalized Ratio
IPV
Intrapatient Variability
IQ
Immune Quiescence
IRB
Institutional Review Board
IRT
Interactive Response Technology
ISN
International Study Number
IVIG
Intravenous Immunoglobulin
KDPI
Kidney Donor Profile Index
KM
Kaplan-Meier
LA-CRF
Liver Abnormality Case Report Form
LD
Living Donor
LFT
Liver Function Tests
m2
Meter squared


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Abbreviations
Description of abbreviations
MA-A
Medical Affairs, Americas
MDRD-4
Modification of Diet in Renal Disease (4 variable)
MFC
Multiparameter flow cytometry
MFI
Mean Fluorescence Intensity
MHC
Major Histocompatibility Complex
MMF
Mycophenolate Mofetil
mFAS
Modified Full Analysis Set
mTOR
Mammalian target of rapamycin
NASH
Non-alcoholic steatohepatitis
NCBI
National Center for Biotechnology Information
NGT
Nasogastric Tube
NIH
National Institutes of Health
NTI
Narrow Therapeutic Index
OPO
Organ Procurement Organization
OPTN
Organ Procurement and Transplantation Network
PAS
Periodic Acid Schiff
PBS
Phosphate Buffered Saline
PCA
Principle Component Analysis
PD
Pharmacodynamics
PHI
Personal Health Information
PI
Package Insert
PK
Pharmacokinetics
PKAS
Pharmacokinetic Analysis Set
PPS
Per Protocol Set
PRCA
Pure Red Cell Aplasia
PRES
Posterior Reversible Encephalopathy Syndrome
PTC
Peritubular Capillaries
RMA
Robust Multi-array Average
SAE
Serious Adverse Event
SAF
Safety Analysis Set
SAP
Statistical Analysis Plan
SDV
Source Document Verification
SGOT
Serum Glutamic Oxaloacetic Transaminase
SOC
Standard of Care
SOP
Standard Operating Procedure
SPA
Solid Phase Assay
SPGT
Serum Glutamic Pyruvic Transaminase
SRTR
Scientific Registry of Transplant Recipients
SUSAR
Suspected Unexpected Serious Adverse Reactions
TacSD
Standard Deviation of Tacrolimus
TB
Tuberculosis
TBL
Total Bilirubin


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Abbreviations
Description of abbreviations
T-cell
Thymus Lymphocytes
TCMR
T-cell Mediated Rejection
TDM
Therapeutic Drug Monitoring
TG
Transplant Glomerulopathy
TGI
Transplant Genomics Inc.
TLFs
Tables, Listings, and Figures
TMF
Trial Master File
TX
Transplant eXcellent
ULN
Upper Limit of Normal
UNOS
United Network of Organ Sharing
USP
United States Pharmacopeia
WIT
Warm Ischemia Time
XM
Crossmatching


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Definition of Key Study Terms
Terms
Definition of terms
Baseline
Observed values/findings which are regarded as the observed starting point for 
comparison. 
Enroll
To register or enter into a clinical trial.  NOTE: Once a subject has been 
enrolled, the clinical trial protocol applies to the subject. 
Intervention
The drug, therapy or process under investigation in a clinical study that is 
believed to have an effect on outcomes of interest in a study (e.g., health-
related quality of life, efficacy, safety, pharmacoeconomics). 
Investigational 
period
Period of time where major interests of protocol objectives are observed, and 
where the test drug or comparative drug (sometimes without randomization) is 
usually given to a subject, and continues until the last assessment after 
completing administration of the test drug or comparative drug.
Screening period
Period of time before entering the investigational period, usually from the time 
of starting a subject signing consent until just before the test drug or 
comparative drug (sometimes without randomization) is given to a subject.
Randomization
The process of assigning trial subjects to treatment or control groups using an 
element of chance to determine assignments in order to reduce bias.
Screening
A process of active consideration of potential subjects for enrollment in a trial.
Screen failure
Potential subject who did not meet one or more criteria required for 
participation in a trial.
Study period
Period of time from the first site initiation date to the last site completing the 
study.
Variable
Any quantity that varies; any attribute, phenomenon or event that can have 
different qualitative or quantitative values.


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IV.
SYNOPSIS
Date and Version # of Protocol Synopsis:
2.0, October 6, 2017
Sponsor:  Astellas Medical Affairs, Americas
Protocol Number: IDTX-MA-3004
Name of Study Drug: 
Tacrolimus extended-release (Astagraf XL®)
Phase of Development:  4
Title of Study:  ASTOUND
Astagraf XL® to Understand the Impact of Immunosuppression on De Novo DSA Development and 
Chronic Immune Activation in Kidney Transplantation 
Planned Study Period: 
From 1Q FY2016 to 3Q FY2019
Study Objective(s): 
Primary Objective:   

Compare the incidence of a two-part composite endpoint consisting of de novo donor specific 
antibody (DSA) formation or a designation of “immune activation (IA)” on peripheral blood 
molecular profiling in patients maintained on twice daily, immediate-release tacrolimus 
versus those maintained on Astagraf XL® in the first two years post-transplant.
Secondary Objectives:

To assess the risk factors for each of the following outcomes: DSA formation;, IA; transplant 
glomerulopathy (TG); acute and chronic forms of antibody-mediated rejection (ABMR); 
C1q-binding DSA; HLA-DQ DSA; DSA IgG3 isotype; requirement for and type of antibody 
reduction required; various threshold levels of estimated glomerular filtration rate (eGFR) 
(less than 30, 40, and 50 mL/min/1.73 m2); and the four components of the traditional 
composite endpoint, consisting of graft loss, mortality, biopsy-proven acute rejection 
(BPAR), and loss to follow-up; and additionally, the persistence of DSA and IA. 

Assess and compare, between treatment groups, the association/correlation of the appearance 
of DSA with the development of IA on molecular profiling.

Compare, between treatment cohorts, the incidence and hazards of each of the following, 
occurring over the course of one year, and, where appropriate, at 24 months post-
transplantation: DSA, HLA-DQ DSA, C1q-binding DSA, DSA IgG3 isotype, IA, TG, select 
BANFF histology grades (acute and chronic forms of ABMR, acute and chronic active 
TCMR, borderline changes, interstitial fibrosis and tubular atrophy [IFTA]), graft loss, 
mortality, and BPAR.

Compare between cohorts the incidence of various thresholds of eGFR (less than 30, 40, 
50 mL/min/1.73 m2, and a five-point decline).

Compare the distribution of ordinal categories of antibody strength (weak, moderate, strong) 
between treatment groups across the study time course as well as at each time point.

Compare raw mean fluorescence intensity (MFI) scores between treatment groups at each 
time point and across the duration of the study.

Examine and compare histopathology between the treatment cohorts in biopsies obtained for 
cause and for maintenance during the course of clinical care. 

Examine and compare outcomes as a function of tacrolimus manufacturer and the number of 
switches between immediate-release tacrolimus products.


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
Summarize adverse events (AEs) between study groups.

Compare the change in eGFR over the study duration beginning from 30 days post-transplant 
between groups, and in patients who develop DSA or IA.

Compare the persistence of the development of IA on molecular profiling between the two 
cohorts across the study duration in those patients who develop IA on molecular profiling.
Exploratory Objectives:

Compare the frequency of those patients experiencing either component of a two-part 
composite endpoint consisting of DSA formation and the presence of TG (cg > 0) on 
centrally-interpreted, institutional protocol (maintenance) biopsy and / or biopsy obtained for 
cause during the first year post-transplant and/or up to the conclusion of the study.

To assess and compare, between treatment groups, the association/correlation of incidence 
and strength of DSA, and the incidences of HLA-DQ DSA, C1q-binding DSA, and DSA IgG3
isotype with each other, and individually, with each of the following: IA, TG, ABMR (both 
chronic forms and acute), Banff histology, various thresholds of eGFR (less than 30, 40, and 
50 mL/min/1.73 m2), eGFR change over time, TCMR, graft loss, and mortality.

Examine the proportion of patients with the IA designation who have normal and abnormal 
creatinine.  

In patients with an IA designation and abnormal creatinine, examine biopsies (if available) 
for concordance.

To assess the association/correlation of the histopathology with DSA, C1q-binding DSA, 
DSA IgG3 isotype, molecular phenotyping, graft loss, and measures of renal function in each 
cohort in patients for whom one year institutionally-derived, protocol (maintenance) biopsies 
are available.

To assess and compare, between treatment groups, the association/correlation of the 
coefficient of variation (CV) and standard deviation (SD) of tacrolimus trough concentrations 
with each of the following: the composite endpoint of DSA and IA, DSA formation and IA
individually, TG, ABMR, C1q-binding DSA, HLA-DQ DSA, DSA IgG3 isotype, 
histopathology, renal dysfunction, and the components of the traditional composite endpoint.

To compare dose changes and intrapatient variability (IPV) between groups as a function of 
tacrolimus trough level after the first 6 weeks following transplantation in patients on stable 
doses of immediate-release tacrolimus and Astagraf XL.

To assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with each of the following: TG, chronic forms of ABMR, C1q-binding 
DSA, HLA-DQ DSA, DSA IgG3 isotype, IA, histopathology, graft loss, various thresholds of 
eGFR (< 30, 40, and 50 mL/min/1.73 m2), acute ABMR, TCMR, and mortality in patients 
who develop IA on molecular profiling in each cohort.

To compare the MFI shift to a lower category (and magnitude of shift) between groups 
(including C1q MFI shift), in those patients who develop DSA during the course of the study.

To examine percent MFI reduction within each subgroup over time in each treatment arm 
(including C1q MFI reduction) in those patients who develop DSA during the course of the 
study. 

To map expression results to known immunological pathways implicated in immune-
mediated, kidney transplant tissue injury.


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
To determine the longitudinal consequences of DSA formation and immune activation, 
perform de-identified, long-term follow-up through participating transplant centers in select 
study participants who provide the appropriate informed consent by future linking to the 
Scientific Registry of Transplant Recipients (SRTR) after completion of ASTOUND.

To examine the economic burden of treating patients with molecular evidence of immune 
activation and DSA formation using Medicare claims data or commercial payer cost data in 
patients required to transfer from Medicare to private insurance.

To compare outcomes in like patients between cohorts managed with maintenance protocol 
biopsy vs. biopsy for cause with respect to the composite endpoint, the components thereof, 
complications, and resource utilization.
Planned Total Number of Study Centers and Location(s):  
Approximately 25-30 centers
US
Study Population:  
Living or deceased donor kidney transplant recipients 16 to 70 years of age. 
Number of Subjects to be Enrolled / Randomized:  
550 patients to be enrolled.
Study Design Overview:  
This is an exploratory, two year, prospective, randomized, multi-center, open-label trial examining 
long-term kidney transplant outcomes through the use of an adaptive design and a two-part, 
composite surrogate endpoint.  Specifically, it is designed to compare the effects of twice daily, 
immediate-release tacrolimus and once daily Astagraf XL on DSA formation and the development of 
a peripheral blood molecular profile indicating the presence of immune activation (IA) in de novo 
kidney transplant recipients during the first two years following transplantation.  For the purposes of 
this study, IA will be defined as a positive molecular signature using the Trugraf v2.0 molecular assay 
(Transplant Genomics, Inc., Pleasanton, CA) in all patients.
Patients will be screened prior to surgery and randomized 1:1 to receive immediate-release 
tacrolimus, administered twice daily, or Astagraf XL, as a component of a standard 
immunosuppression maintenance regimen also consisting of corticosteroids (complete steroid 
avoidance not allowed – steroid taper and withdrawal permitted if part of institutional protocol) and 
mycophenolate mofetil (MMF) (or Myfortic® equivalent). Investigators are encouraged to start 
subjects on the randomized study treatment (immediate release tacrolimus or Astagraf XL) within 48 
hours of transplantation (pre-transplant administration of study treatment is not allowed). However, if 
medically indicated per the treating physician’s discretion, initiation of study treatment may be 
delayed for up to seven days post-transplant. 
IF medically indicated, subjects who are unable to tolerate oral administration of their assigned study 
drug may be administered tacrolimus via an alternative route (for Astagraf XL treatment arm: 
intravenous [IV] tacrolimus; for immediate release tacrolimus: IV tacrolimus or nasogastric tube 
[NGT]) for up to seven consecutive days. Dosing via alternative routes should be discontinued as 
soon as the subject can tolerate oral administration, and is not allowed for more than seven 
consecutive days. Please see Section 5.1.1 and 4.1 for further details regarding non-oral 
administration of tacrolimus.  


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Given the variability of incidence rates for DSA reported in the literature, a stopping rule has been 
incorporated herein to mitigate the risk of continuing the study under incorrect prior assumptions 
relating to the true incidence rate of DSA formation. To apply the rule, the incidence rate of the 
primary endpoint in the control group will be examined once 50% of control patients have completed 
one year of therapy. Early termination of the study, or extension to a second year, will be predicated 
on this assessment (see Formal Stopping Rules below).
Induction therapy is required for all participants, and will be administered (dosing, type of agent) per 
center protocol.  Intravenous corticosteroids will be administered prior to revascularization as part of 
the initial induction regimen, with dose and duration of therapy also determined by the participating 
transplant center’s standard protocol.  Thereafter, patients will be followed for up to 2 years during 
the open-label study period.  Immediate-release tacrolimus will be obtained from pharmacies per the 
local standard of care.  After discharge from the hospital, the patients randomized to immediate-
release tacrolimus will receive study-provided vouchers and will be permitted to receive any 
immediate-release tacrolimus product available to them through normal dispensing mechanisms.  
Astagraf XL will be supplied by Astellas and dispensed by the participating institution.
Initial pre-transplant cross-matching (XM) will be performed per local protocol.  Initial XM serum 
will be collected and stored centrally to facilitate the later assessment of pre-formed antibody on an as 
needed basis.  Thereafter, antibody screening and molecular phenotyping will be performed by a 
central lab at one month, 3 months, 6 months, 9 months, 12 months, and 24 months.  Antibody status, 
including HLA-DSA isotype and C1q-binding DSA status, will also be recorded when measured 
locally during the course of clinical care. 
For central laboratory analysis, initial FlowPRA® (One Lambda, Canoga Park, CA) screening will be 
used to test for the presence of anti-HLA antibodies.  Once identified, single antigen testing will be 
used to identify DSA specificity with an MFI greater than or approaching 1000 used to determine 
antibody positivity.  In borderline cases, a three- member adjudication board of independent, qualified 
HLA Lab Directors will determine positivity based on relevant clinical data.  In samples that meet 
threshold criteria for DSA positivity, additional classification will be performed to determine the 
degree of antibody strength.  By performing a single dilution (1:16) of DSA-containing samples, the 
alloimmune response can be further elucidated [Tambur et al, 2015].  Samples in which MFI 
disappears (becomes < 1000 MFI) will be regarded as “weak.”  Samples increasing to > 10,000 MFI 
upon dilution will be regarded as having significant prozone and will be regarded as “strong.”  
Samples which may increase or decrease, but nevertheless remain between 1000 and 10,000 MFI will 
be regarded as “moderate.”  In certain cases, samples with evidence of fluorescence that are 
nevertheless below MFI criteria for DSA positivity will also be subject to dilution.  Samples 
increasing to above threshold criteria following dilution will be regarded as positive and assumed to 
be under the influence of a prozone effect that impairs antibody detection.  For statistical purposes, 
the DSA with the highest MFI level obtained during the course of the study will be used for statistical 
comparisons between groups.  However, the strongest antibody observed at each HLA-locus will be 
reported separately and tracked for patients who are DSA positive [Tambur et al, 2015].  In all cases, 
positive DSA and/or molecular testing results will be communicated to the patient’s treating physician 
when available (DSA) or at the end of study (molecular testing). Testing will be performed in batches. 
De-identified typing reports (donor and recipient) will be requested from the participating centers to 
inform the central laboratory testing. De-identified results of recent local pre-transplant DSA testing 
(if performed per SOC) will also be requested to facilitate interpretation of central DSA testing 
results. 


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Follow-up visits will be performed quarterly during the first year of follow-up to collect standard 
clinical data.  For follow-up beyond the first year, urinary protein and tacrolimus trough 
concentrations obtained closest to targeted date of visit, per SOC, should be recorded. The two closest 
serum creatinine levels to the study visit, obtained at least one month apart, if performed per SOC, 
should also be recorded. Additionally, all local kidney biopsy results, AEs, and concomitant 
medications pertaining to AEs will be recorded.  At the terminal study visit at 24 months, the most 
recently obtained lab results as well as any episodes of rejection, need for antibody reduction, graft 
loss, and deaths occurring beyond the first year will be detailed in the eCRF.  Final testing for DSA 
and molecular profiling will also be performed at the 24 month visit.
Pathology results [hematoxylin and eosin (H&E), light microscopy, and immunofluorescence] will be 
recorded in the eCRF when local kidney biopsies are performed and interpreted during the course of 
clinical care.  De-identified copies of all kidney pathology reports must be submitted to Astellas. For 
purposes of data recording, substantiation by local biopsy will be required for cases of suspected 
rejection.  For all participants who undergo at least one kidney biopsy during the first year post-
transplant (either for cause or per institutional protocol), the kidney biopsy performed closest to the 
12 month visit (but no later than 14 months post-transplant) will be submitted for central pathological 
grading.  All biopsies that are submitted for central pathology review will be evaluated and scored 
using the most recent version of the 2007 Update to the Banff ’97 Classification by a central 
pathologist blinded to clinical results and patient identifiers [Solez et al, 2008].  
Inclusion/Exclusion Criteria:  
Inclusion:
Subject is eligible for the study if all of the following apply.
1.
Kidney transplant patient ≥ 16 years and ≤ 70 years old.
2.
Institutional Review Board (IRB)-/Independent Ethics Committee (IEC)-approved written
Informed Consent and privacy language as per national regulations (e.g., HIPAA 
Authorization for US sites) must be obtained from the subject or legally authorized 
representative prior to any study-related procedures (including withdrawal of prohibited 
medication, if applicable). For subjects < 18 years old, informed consent must be obtained 
from the subject’s parent(s) or legal guardian(s); subject Assent (where appropriate) must be 
given.
3.
Recipient of a de novo kidney from a living or deceased donor. Note: Recipient of an en bloc
deceased donor kidney transplant from a pediatric donor ≥5 years of age AND weighing 
greater than 20 kg is allowed.  
4.
If deceased donor, a Kidney Donor Profile Index (KDPI) ≤ 85 [donation after circulatory 
death (DCD) and what was previously known as extended criteria donor (ECD) organ 
recipients are eligible for enrollment provided KDPI ≤ 85].
5.
Removed 
6.
At least one antigen mismatch at major MHC (class I or class II).
7.
Willingness to comply with study protocol.
8.
Subject agrees not to participate in another investigational drug study while on treatment.


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9.
Female subject must be either:
a.
Of non-child-bearing potential, 
i.
Post-menopausal (defined as at least 1 year without any menses) prior to screening, 
or 
ii.
Documented surgically sterile or status post-hysterectomy
b.
Or, if of childbearing potential, 
i.
Agree not to try to become pregnant during the study and for 90 days after the final 
study drug administration
ii.
And have a negative serum or urine pregnancy test within 7 days prior to transplant 
procedure
iii.
And, if heterosexually active, agree to consistently use two forms of highly 
effective birth control (at least one of which must be a barrier method) which 
includes consistent and correct usage of established oral contraception, established 
intrauterine device or intrauterine system, or barrier methods of contraception: 
condom or occlusive cap (diaphragm or cervical/vault caps) with spermicidal 
foam/gel/film/cream/suppository or vasectomy in the male partner, starting at 
screening and throughout the study period and for 90 days after the final study drug 
administration.
10. Male subject and their female spouse/partners who are of childbearing potential must be using 
highly effective contraception consisting of two forms of birth control (one of which must be 
a barrier method) starting at screening and continuing throughout the study period and for 90 
days after the final study drug administration (acceptable forms of birth control are listed in 
Inclusion Criterion 9). 
11. Male subject must not donate sperm starting at screening throughout the study period and for 
90 days after the final study drug administration.
12. Female subject must agree not to breastfeed starting at screening and throughout the study 
period, and for 90 days after the final study drug administration.
13. Female subject must not donate ova starting at screening and throughout the study period, and 
for 90 days after the final study drug administration.
14. Will be receiving induction immunotherapy (either T-cell depleting agent, anti-CD52
monoclonal antibody, or IL-2 co-stimulation blocker), with dose and frequency of the chosen 
induction agent determined by local standard of care. Steroid-only induction therapy does not 
satisfy this criterion. 
Waivers to the inclusion criteria will NOT be allowed.
Previous kidney transplants will be permitted.
Patients who are receiving a secondary transplant and who previously received Astagraf XL or who 
are currently on Astagraf XL as a component of maintenance immunosuppression and re-listed for 
transplant will be eligible to enroll in ASTOUND and will be randomized at the time of transplant to 
either cohort.  


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Exclusion:  
Subject will be excluded from participation if any of the following apply.
1.
Patient is known to have a positive test for latent tuberculosis (TB) and has not previously 
received adequate anti-microbial therapy or would require TB prophylaxis after transplant.
2.
Uncontrolled concomitant infection or any unstable medical condition that could interfere 
with study objectives.
3.
Significant liver disease, defined as having, during the past 28 days, consistently elevated 
AST (SGOT) and/or ALT (SPGT) levels greater than 3 times the upper value of the normal 
range of the investigational site.
4.
Patient currently taking or maintained on another form of extended-release tacrolimus 
following his/her transplant procedure.
5.
Patient who will be maintained on a non-tacrolimus-based maintenance immunosuppressive 
regimen following his/her transplant procedure. 
6.
Patient currently taking, having taken within 30 days, or who will be maintained on an mTOR 
inhibitor following his/her transplant procedure.
7.
Use of an investigational study drug in the 30 days prior to the transplant procedure.
8.
Contraindication or hypersensitivity to drugs or any of their components that constitute the 
immunosuppression regimen.
9.
6 Ag match or zero mismatch at major MHC (class I or class II).
10. Receipt of an ABO-incompatible organ. Note: A2 donor to O recipient or A2 donor to B 
recipient is considered ABO-compatible and not excluded by this criterion. 
11. Removed
12. Removed
13. The presence of current or historic, pre-formed anti-HLA DSA against the current donor
(evidence of pre-formed, non-donor HLA is not exclusionary) as defined by a subject 
meeting any of the following criteria*:
a.
Positive virtual crossmatch, 
b.
Positive T- or B-cell crossmatch by NIH antiglobulin lymphocytotoxicity method,** 
c.
Positive T- or B-cell flow cytometry crossmatch defined by the MFC criteria used by the 
center’s HLA lab for their local proficiency testing,**
d.
An MFI greater than or approaching 1000 using flow cytometry/Luminex-based, specific 
anti-HLA antibody testing.
* Patients are eligible to enroll with a negative virtual crossmatch if used in lieu of a physical 
crossmatch, if, in the opinion of the patient’s attending physician, use of such is required to 
obviate the accrual of excessive ischemia time.  However, continued participation is 
predicated on the performance of the physical crossmatch within 48 hours of transplant. If the 
physical crossmatch is positive, the subject will be discontinued. 
** If b or c above are positive secondary to a suspected positive auto-crossmatch, that is not 
exclusionary as long as a and d above are not met.
14. Receipt of desensitization, antibody-removal, anti-B-cell, or anti-plasma cell therapy in the 90 
days preceding the transplant procedure. 


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15. Planned initiation (prior to transplant) of desensitization, antibody-removal, anti-B-cell, or 
anti-plasma cell therapy within 7 days of the transplant procedure.
16. Donor or recipient with known hepatitis C infection (HCV antibody positive), HIV infection 
(HIV antibody positive), acute hepatitis B infection (HBsAg positive, anti-HBc positive, IgM 
anti-HBc positive, anti-HBs negative) chronic hepatitis B infection (HBsAg positive, anti-
HBc positive, IgM anti-HBc negative, anti-HBs negative), or equivocal hepatitis B status 
(HBsAg negative, anti-HBc positive, anti-HBs negative). Patients (donor or recipient) who 
have normal liver function tests (LFT) and who are either hepatitis C positive with a negative 
viral load or have natural or vaccine-acquired immunity from hepatitis B are not excluded by 
this criterion.
17. Primary focal segmental glomerulosclerosis.
18. Subject has a current malignancy or history of malignancy (within the past 5 years), except 
non-metastatic basal or squamous cell carcinoma of the skin or carcinoma-in-situ of the 
cervix that has been successfully treated.
19. Recipient of multi-organ or dual kidney transplants (inclusive of current transplant and any 
prior non-renal transplants). Note: Patients with prior kidney transplants are eligible.
20. Recipient of an en bloc, pediatric deceased donor kidney from a donor less than 5 years of age 
OR weighing less than 20 kg.
21. Prior graft loss secondary to CMV or BK nephropathy.
22. Prior history of invasive organ disease in the presence of CMV or BKV or clinically 
significant CMV viremia.
23. History of clinically significant (per investigator’s discretion) BK viruria
24. Any condition which, in the investigator’s opinion, makes the subject unsuitable for study 
participation.
25. Planned complete steroid avoidance (Steroid initiation and subsequent taper / withdrawal will 
be allowed and will be under the purview of the treating physician.)  
26. Planned receipt of post-transplant prophylactic HCV treatment.
Waivers to the exclusion criteria will NOT be allowed.
Investigational Product(s):
Tacrolimus, extended-release, oral (Astagraf XL); 0.5 mg, 1 mg, 5 mg capsules
Dose(s):  
Tacrolimus, extended-release (Astagraf XL); administered once daily at initial weight-based dose of 
0.15 mg/kg.  Dosing and monitoring thereafter predicated on clinical judgment to effect a minimum 
whole blood tacrolimus concentration of at least 6 ng/mL. A prolonged significant reduction of 
tacrolimus or withdrawal of tacrolimus will result in study discontinuation (see discontinuation 
criteria). 
Mode of Administration:
Tacrolimus, extended-release (Astagraf XL); oral
IF medically indicated, subjects randomized to the Astagraf XL treatment arm who are unable to 
tolerate oral administration may be administered intravenous (IV) tacrolimus. In such cases, the 
patient must resume conventional oral Astagraf XL as soon as possible. Administration of IV
tacrolimus may not exceed a period of seven consecutive days. It is important to note that Astagraf 
XL cannot be given intravenously. Astagraf XL should not be given via NGT due to its extended 
release characteristics. IV tacrolimus will not be provided by the Sponsor. 


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Comparative Drug(s):
Tacrolimus, immediate-release, oral; 0.5 mg, 1 mg, 5 mg capsules 
Dose(s):
Tacrolimus, immediate-release; administered twice daily per clinical judgment of supervising  
physician (dosing and monitoring in accordance with center protocol) to effect a minimum whole 
blood tacrolimus concentration of at least 6 ng/mL. A prolonged significant reduction of tacrolimus or 
withdrawal of tacrolimus will result in study discontinuation (see discontinuation criteria). 
Mode of Administration:
Tacrolimus, immediate-release; oral
IF medically indicated, subjects randomized to the immediate-release tacrolimus treatment arm who 
are unable to tolerate oral administration may be administered tacrolimus via NGT or IV, for no more 
than seven days. In such cases, the patient must resume conventional oral immediate release 
tacrolimus as soon as possible. Administration of immediate release tacrolimus via NGT or IV may 
not exceed a period of seven days. Additionally, a subject may not receive more than seven 
consecutive days of combined NGT and IV administration in cases where a subject is switched from 
one therapy to another. IV tacrolimus will not be provided by the Sponsor.
Drug(s) for Screening: 
Not applicable.
Advanced Measures to Treat Rejections: 
The need for advanced therapy to treat rejection will be determined by the supervising physician and 
predicated on the emergence of steroid-refractory antibody-mediated rejection.  Typical rescue drugs 
employed under these conditions include, but are not limited to anti-thymocyte globulin, intravenous 
immunoglobulin (IVIG), rituximab, bortezomib, and eculizumab.
Concomitant Medication Restrictions or Requirements:   
All patients will receive induction immunotherapy (either T-cell depleting agent, anti-CD52 
monoclonal antibody, or IL-2 co-stimulation blocker – steroid-only induction therapy is not allowed).  
The dose and frequency of the chosen induction agent will be determined by the patient’s treating 
physician and administered in conjunction with the participating transplant center’s de novo kidney 
immunosuppression protocol.  Anti-CMV, fungal, bacterial, and pneumocystis prophylaxis will be 
determined per local standard of care.  
Non-tacrolimus based immunosuppressive regimens (i.e. cyclosporine, everolimus, sirolimus, 
belatacept), as well as all forms of extended-release tacrolimus other than Astagraf XL, are prohibited 
during the course of the study. As a clarifying statement, all patients must be maintained on MMF or 
its equivalent during the course of the study.  Due to the influence the following medications have on 
tacrolimus blood concentrations, antiviral medications used in HCV and HIV treatment, as well as 
isoniazid, Rifampin, ethambutol, and pyrazinamide are also prohibited.
Duration of Treatment:  
Study enrollees will receive study medication (single daily dose of Astagraf XL or standard of care 
twice daily, immediate-release tacrolimus) for up to two years following their transplant procedure. In 
cases where delayed renal function is suspected following transplantation, therapy initiation with 
either tacrolimus agent can be delayed for up to 7 days.  


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Formal Stopping Rules: 
A decision to terminate the study early or extend to a second year will be predicated on the event rate 
in the control arm once 50% of control patients have completed one year of therapy.  At that time, the 
estimated proportion will be used to calculate the Beta-binomial posterior Bayesian probability that 
the primary endpoint’s incidence rate will, indeed, be at least 20%.  
If this probability is ≥ 70%, the following rules apply:
1) The study will continue to enroll to completion. 
2) The study duration will be such that patients will complete at least one year of study visits.
a)
Patients who have not yet completed one year of study (at the time of the interim 
analysis) will complete the study at the 12m visit.
b) Patients who have already completed the 12m visit (at the time of the interim 
analysis) will complete the study at their next scheduled study visit. 
3) Data analysis will be performed once all patients complete 12 months of study.
If this probability is < 70% and ≥ 30%:
1) The study will continue to enroll to completion.
2) The study duration will be 24 months; patients will complete the study at the 24 month visit.
3) Data analysis will be performed once all patients complete 24 months of therapy.
If this probability is < 30%, the study will be terminated at that time (50% of patients completing 
12 months of therapy).
1) The study will be terminated upon completion of the interim analysis (done when 50% of 
patients have completed 12 months of therapy). 
2) Patients will be discontinued from the study at their next scheduled study visit. 
Discontinuation Criteria from Study for Individual Subjects:

Subject develops allograft loss (as defined by subject death, retransplantation, transplant 
nephrectomy, or return to dialysis of ≥ 6 consecutive weeks duration);

Subject develops unacceptable toxicity or is withdrawn at the discretion of the patient’s 
supervising physician;

Subject is lost to follow-up despite reasonable efforts by the investigator to locate the subject;

Subject withdraws consent for further treatment or expires during the course of the study;

Subject begins taking any prohibited medications;

Subject undergoes a second organ transplant;

Conversion to a non-tacrolimus-based maintenance regimen in either study arm if required to 
manage toxicities;

Removal from an MMF-containing immunosuppressive regimen (or its equivalent);

Failure to achieve a tacrolimus whole blood concentration ≥ 6 ng/mL for 4 consecutive weeks 
during the first 6 weeks following transplantation;

Permanent withdrawal of tacrolimus; 


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
Interruption or prolonged significant reduction of tacrolimus (as an example, a 50% reduction 
or dose at a level of less than 3.5ng/mL) for periods exceeding 4 weeks; 

Conversion by the investigator to the therapy of the other study treatment arm (i.e., subject 
randomized to Astagraf XL is converted to immediate release tacrolimus OR subject 
randomized to immediate release tacrolimus is converted to Astagraf XL. Cases of 
inadvertent and temporary conversion [i.e., dispensing error] do not meet this discontinuation 
criterion); 

Subject is not initiated on Astagraf XL or immediate-release tacrolimus within 7 days of 
transplant;

Gross non-compliance with protocol: The medical monitor or investigator may request 
permanent study discontinuation in the event of a major protocol deviation such as 
administration of prohibited concomitant medication, lack of cooperation, or noncompliance.
Endpoints for Evaluation:  
Primary:
The primary endpoint is the combined incidence of either DSA or IA on peripheral blood molecular 
profiling at one year or at the conclusion of the study.  For this purpose, DSA will be considered as a 
categorical (binary) variable with positivity determined at a threshold criteria approaching MFI = 
1000 at any time during the study.  For reporting purposes, IA will be considered either present or 
absent using the Trugraf™ v2.0 molecular assay.  For the purposes of the study, a negative 
designation (Trugraf TX Normal) will be referred to as Immune Quiescence (IQ).  Due to operating 
characteristics of the assay, a positive designation will be considered evidence of Immune Activation 
(IA) in all patients.
The modified Full Analysis Set (mFAS) will be used for the primary endpoint analysis.
Secondary:
The assessment and comparison of the incidence of DSA between treatment groups will rely on the 
binary variable of DSA positivity at a threshold criteria approaching an MFI of 1000.  Comparisons of 
the cohorts with respect to the various molecular designations as well as correlating the results with 
DSA will rely on the categorical (binary) variable of positivity using the TrugrafTM v2.0 molecular 
assay. 
Comparisons between treatment cohorts regarding incidence will rely upon assessments for each of 
the following results at any point in the study:

DSA

IA)

TG

Acute and chronic forms of ABMR 

C1q-binding DSA

HLA-DQ DSA

DSA IgG3 isotype

Required antibody reduction 


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
eGFR at various thresholds (less than 30, 40, and 50 mL/min/1.73 m2) and whether a five-
point decline in eGFR occurs

Graft loss (defined as subject death, retransplantation, transplant nephrectomy, or a return to 
dialysis for at least a 6 week duration)

Death

BPAR (inclusive of ABMR and TCMR)

Loss to follow-up
Time-to first occurrence will be assessed for each of the following: DSA, HLA-DQ DSA, C1q-
binding DSA, DSA IgG3 isotype, IA, TG, select BANFF histology grades (acute and chronic forms 
of ABMR, acute and chronic active TCMR, borderline changes, and IFTA), mortality, and local 
BPAR.
The frequency of the type of antibody reduction employed will be assessed.
Patient MFI and eGFR values will be assessed over time.
Assessments of histopathology in biopsies will be made using the following endpoints:

Incidence of ci, ct, and ptc scores greater than one

Biopsy scores for g, t, v, i, cg, ct, ci, cv, ah, ptc, and mm 
The safety endpoint for AEs will be the incidence of each type of AE based on system organ class and 
using the preferred term from the most recent version of the Medical Dictionary for Regulatory 
Activities (MedDRA).
Persistence of DSA and IA will be additional endpoints for comparisons in patients who develop IA 
and DSA on molecular profiling.
Exploratory: 

The incidence of a second two-part composite endpoint encompassing the incidence of 
acquiring DSA or positive evidence of TG on a centrally-interpreted biopsy during the first 
year post-transplant or by the conclusion of the study

Expression results, mapped to known immunological pathways implicated in immune-
mediated, kidney transplant tissue injury.

With appropriate informed consent, long-term graft and patient survival in study participants 
by future linking to the Scientific Registry of Transplant Recipients (SRTR) following 
completion of ASTOUND.

The economic burden of treating patients with molecular evidence of immune activation and 
DSA formation may be assessed using Medicare claims data (or commercial payer cost data 
in patients required to transfer from Medicare to private insurance).

Time to first DSA or IA positive test results

Outcomes in like patients between cohorts managed with maintenance protocol biopsy vs. 
biopsy for cause with respect to the composite endpoint, the components thereof, 
complications, and resource utilization.


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Statistical Methods:   
Sample Size Justification: 
The sample size for this study was determined based upon a comparison of the rate of the two-part 
composite endpoint in the Astagraf XL group compared to the immediate-release tacrolimus group.  
Assuming a rate of 20% would be observed for the immediate-release tacrolimus group compared to a 
rate of 10% for the Astagraf XL group, 220 subjects per group are needed to achieve an 80% power to 
detect a difference between Astagraf XL and immediate-release tacrolimus with a two-sided alpha 
level of 0.05.  The study will enroll 275 subjects/group (550 total) to allow for 20% dropout.
Efficacy:  
The analysis of efficacy will be conducted using the mFAS.  
The primary efficacy endpoint is the incidence of any one of the following in the first two years post-
transplant: 

DSA formation 

IA
The primary endpoint will be analyzed at a single time point per Section 7.4.1
The incidence of the primary endpoint will be summarized by treatment group with frequencies and 
percentages.  Differences between treatment groups will be analyzed using logistic regression and 
controlled using pre-defined covariates.
For subjects with missing or incomplete endpoint data who did not permanently switch therapy or 
were discontinued from the study prior to DSA/IA event (who are lost to follow up, or those who 
expire for any reason), negative DSA/IA values will be imputed unless there is an otherwise positive 
result.  
The hypothesis for comparison is as follows:

H0: The probability of incidence for the primary endpoint is the same between patients 
receiving Astagraf XL and patients receiving immediate-release tacrolimus.

H1: The probability of incidence for the primary endpoint is not the same between patients 
receiving Astagraf XL and immediate-release tacrolimus.
Hypothesis testing will be performed using a two-sided test with a 0.05 significance level.  The 95% 
confidence interval for the odds ratio of the primary endpoint between Astagraf XL and twice daily 
tacrolimus will be included. 
Analyses of treatment group differences for secondary endpoints will be evaluated by time and 
overall.  The scope of these analyses will include the following: DSA formation, IA, TG, acute and 
chronic forms of ABMR, C1q-binding DSA, HLA-DQ DSA, DSA IgG3 isotype, the requirement for 
antibody reduction therapy, chronic graft dysfunction, graft loss, local BPAR, mortality, loss to 
follow-up, ordinal antibody strength, MFI score, histopathology assessments, and tacrolimus trough 
levels.  


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Pharmacokinetics:  
Not applicable.
Pharmacodynamics:  
Not applicable.
Safety:
Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA).  
The number and percentage of AEs, SAEs, AEs leading to discontinuation, and AEs related to study 
drug will be summarized by system organ class, preferred term and treatment group. The number and 
percentage of AEs by severity will also be summarized. All AEs will be listed.
Interim Analyses: 
Not applicable.




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Table 1
Schedule of Assessments
Study Day
Screening
(-21 to 0)1
01
30
90
180
270
365
4552
5452
6352
730 3
Unscheduled 
Visit4
Week
-3 to 0
0
4
12
24
36
52
64
76
88
104
n/a
Window (days)
± 21
± 21
± 21
±21
± 21
± 21
± 21
± 21
± 21
n/a
Screening: Inclusion/Exclusion Criteria
X
Informed Consent/Assent
X
Demographics & Medical History
X
X5
Height, Pre-op Weight6
X
Concomitant Medications7
X
X
X
X
X
X
X
X8
X8
X8
X8
X
Physical Exam
X
Transplant Information and Donor 
Information/HLA typing9
X
Randomization10
X
Astagraf XL Arm: Study Drug Dispensing11
X
X
X
X
X
X
X
X
X
X
BID Tacrolimus Arm: Study Drug 
Dispensing12
X                                                                                                                                                                       X
Clinical Labs Recording
X13
X13
X13
X13
X13
X13
X14
X14
X14
X14
Kidney Biopsy Results 15
X                                                                                                                                                                                                  X
Outcomes Review16
X
X
X
X
X
X
X
Adverse Events Recording17
X                                                                                                                                                                                                X
Blood Samples for DSA/anti- HLA/IgG3
isotyping, C1q-binding DSA18
X19
X
X
X
X
X
X
Blood Samples for Molecular Diagnostics18
X
X
X
X
X
X
Blood Sample for Potential Future Analysis 
(optional)18, 20
X
X
X
X
X
X
BK Viremia Review21
X                                                                                                                                                                                               X
Footnotes appear on next page


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1.
Per convention, Day 0 of transplant is the date of transplant completion (revascularization).  Assessments may be done on day -1 for transplant procedures that span midnight. 
Screening and day 0 may occur on the same day.  In all cases, blood obtained for baseline antibody testing must be obtained prior to surgery.  For subjects that have a separate 
screening visit prior to day of transplant, inclusion/exclusion criteria will be reassessed prior to transplant to ensure continued eligibility.
2.
Visits in 2nd year of follow-up to include quarterly drug dispensing visits for Astagraf XL cohort at which time AEs will be recorded in the eCRF.  For patients receiving 
immediate-release tacrolimus in the control arm, AEs will be assessed by quarterly phone interview in the 2nd year and recorded in the eCRF.  
3.
Study visit at end of 2nd year to include measurement of DSA and molecular markers in both control and experimental groups along with the following clinical history:  serum 
creatinine; urinary protein; interval biopsy results; and interval history of infection, BPAR, graft loss, or mortality.
4.
Unscheduled visits may be performed at any time during the study whenever necessary to assess for or follow-up on adverse events, or if deemed necessary by the investigator.
5.
For subjects whose screening visit occurred prior to day 0, any updates to medical history should be recorded at day 0 visit.
6.
Height will only be obtained at the screening visit and dry weight will only be obtained at screening and pre-operatively on day 0.
7.
Includes, but not limited to, concomitant immunosuppressant medication, tacrolimus dose/formulation(s)/manufacturer(s), recording of all anti-rejection therapy, and P450
inducers/inhibitors.
8.
For the 2nd year of follow-up, only concomitant medications administered for a corresponding AE should be recorded in the eCRF.
9.
Transplant information: type of transplant (living related, living non-related, DD, and whether organ was obtained in the setting of DCD), number of prior transplants and reason 
for prior graft loss (if applicable), total cold and warm ischemia time in hours and minutes, donor KDPI, ABO blood typing, HLA typing of donor and recipient, degree of HLA 
mismatch between donor and recipient, most recent panel reactive antibody testing (most recent cPRA level), and pre-transplant MFI status.  Donor viral serology information 
(HBV, HCV, CMV and EBV), age, sex, height, weight, donor cause of death, ethnicity, ex vivo perfusion parameters, ABO typing, and results of any pre-implantation biopsies of 
the donor kidney.
10. Occurs at the pre-op visit, prior to transplant, if living donor (within 7 days).  Randomization to occur upon hospital admission for deceased donors or for living donor recipients 
whose Screening and Day 0 visit occur on the same day.
11. Astagraf XL will be supplied by Astellas and dispensed by the participating institution. At the final study visit, subjects will return unused Astagraf XL. Additional Astagraf XL 
will not be provided to subjects at the final study visit. 
12. After discharge from the hospital, patients randomized to receive twice daily immediate-release tacrolimus will receive study-provided vouchers and will be permitted to receive 
any immediate-release tacrolimus product available to them through normal dispensing mechanisms. At the final study visit, subjects will return unused vouchers; following the 
final study visit, vouchers previously provided to the subjects will be deactivated to prevent additional use. Additional vouchers will not be provided to subjects at the final study 
visit. 
13. Recording of hematology, serum chemistries, urinalysis (including urine protein), tacrolimus trough concentrations, HgA1c, and whole blood tacrolimus concentrations obtained 
per SOC.  See Section 5.4.3 for the components of the routine clinical labs that will be recorded. For all labs except whole blood tacrolimus concentrations and creatinine, only
those labs obtained closest to targeted date of visit need to be recorded.  For the Day 0 visit, pre-transplant labs should be recorded. For whole blood tacrolimus concentrations, at 
the Day 0 and Day 30 visits, only the concentration obtained closest to the targeted date of visit needs to be recorded. For the 90, 180, 270, and 365 day visits, all available 
outpatient tacrolimus concentration assessments done (per SOC) and available in the centralized medical records since the previous study visit should be recorded. For creatinine 
assessments, the assessment done closest to the targeted date of visit (obtained per SOC) as well as an additional assessment done at least one month prior to that assessment (if 
obtained per SOC) will be recorded on the eCRF. 
Footnotes continued on next page


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14. For visits following day 365, only serum creatinine levels, urinary protein, and tacrolimus trough concentrations obtained as part of SOC should be recorded. For all labs except 
creatinine, only labs obtained closest to targeted date of visit need to be recorded.  For creatinine assessments, the assessment done closest to the targeted date of visit (obtained per 
SOC) as well as an additional assessment done at least one month prior to that assessment (if obtained per SOC) will be recorded on the eCRF. 
15. All kidney biopsy results obtained during the study should be recorded in the eCRF.  For all participants who undergo at least one kidney biopsy during the first year post-
transplant (either for cause or per institutional protocol), the kidney biopsy performed closest to the 12 month visit (but no later than 14 months post-transplant) will be submitted 
for central pathological grading.  Additionally, de-identified copies of all kidney pathology reports must be submitted to Astellas.
16. Inclusive of patient survival status, cause of death, rejection episodes, new diagnoses, and pathology review.
17. Adverse event collection will begin once informed consent/assent has been signed and continue throughout the subject’s participation in the study.  All new SAEs occurring up to 
7 days after the end of study visit must be reported. Note: The transplant procedure that occurs on Day 0 is not considered an AE or an SAE.
18. Assessed via central labs.
19. On Day 0, sample drawn prior to transplant procedure and stored to confirm pre-op DSA (will be analyzed only as needed). Sample may be drawn on Day -1 for morning 
transplant procedures. De-identified copies of local typing reports (for donor and recipient typing) and recent local pre-transplant DSA testing results (if performed) will also be 
submitted to the central lab. 
20. Patients will have the option of having an additional sample collected at each phlebotomy session for storage and analysis in future trials of transplant outcomes and biomarker 
discovery.
21. If a subject develops clinically significant BK viremia (as assessed per SOC) during study participation, the peak viremia level obtained per SOC will be retrospectively recorded 
in the eCRF at the time of subject’s study discontinuation or completion. 


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1
INTRODUCTION
1.1
Background
Despite lower rates of acute rejection and short-term improvements in patient and graft 
survival, the rate of late allograft loss following kidney transplantation has remained 
unchanged for more than 30 years [Lamb et al, 2011; Kaneku and Terasaki, 2006].  During 
this time, chronic allograft nephropathy (CAN) became recognized as the major cause of late 
kidney allograft failure [Loupy et al, 2009; Seron & Moreso, 2007; Myers et al, 1984] and 
numerous attempts were made to correlate the progressive fibrosis and vascular injury seen 
on biopsy with the use of calcineurin inhibitors (CNIs) [Nankivell et al, 2003].  However, 
over the past three decades, the link between chronic nephrotoxicity and the use of CNIs has 
proven largely elusive.  For example, in reports examining independent risk factors for 
chronic histological change, low CNI levels were often cited [Shihab et al, 2008; Ekberg, 
Grinyo, et al, 2007; Naesens et al, 2007; Nankivell et al, 2004; Seron et al, 2002].  Similarly, 
when investigators have attempted to minimize, avoid, or withdraw the use of tacrolimus 
during the course of a randomized clinical trial, they have routinely been confronted with 
inferior outcomes in the reduced tacrolimus/tacrolimus avoidance arms [de Sandes-Frietas et 
al, 2015; Hricik et al, 2015; Flechner et al, 2011; Weir et al, 2011; Durrbach et al, 2010; 
Vincenti et al, 2010; Lebranchu et al, 2009; Dean et al, 2008; Ekberg, Tedesco-Silva, et al, 
2007; Srinvias et al, 2007; Ciancio et al, 2006; Larson et al, 2006; Medez et al, 2005; Meier-
Kreische et al, 2005; Halloran, 2000; Andoh et al, 1996].
Late allograft injury as evidenced by moderate-to-severe arteriolar hyalinosis (a traditional 
hallmark of CNI nephrotoxicity) is found to a similar extent in patients never exposed to 
CNIs at 5 years [Stegall et al, 2011].  Likewise, when 1317 cases of allograft loss were 
re-examined by El-Zoghby and colleagues, alloimmunity was found to be the leading cause 
of kidney graft failure, prompting a reclassification of all biopsies with an original diagnosis 
of idiopathic interstitial fibrosis or CNI toxicity [2009].  There have also been several recent 
studies in which biopsies were performed in the setting of new-onset graft dysfunction.  In 
the Deterioration of Kidney Allograft (DeKAF) Trial, the risk of graft failure was unrelated 
to immunosuppression and actually intensified in patients presenting with DSA.  In fact, the 
severity of clinical injury strongly correlated with the magnitude of antibody response 
[Gaston et al, 2010].  In another large study, the Edmonton group demonstrated that the 
majority of kidney failures were not attributable to CNI use, but rather, rejection, non-
adherence, and the presence of alloantibody.  In this study and others, the incidence of 
allograft dysfunction hastened by the presence of antibody increased steadily over time, 
becoming more noticeable with increasing time from transplant [Wiebe et al, 2015; Sellares 
et al, 2012].   
Indeed, the link between subclinical immune-mediated graft injury and progressive
worsening of graft function is now firmly established.  However, given that the appearance of 
chronic renal allograft injury may also take years to manifest, any randomized trial involving 
new forms of therapy and utilizing this parameter as an endpoint may still be difficult to 
conduct from a practical standpoint [Stegall et al, 2015].  For this reason, the identification of 


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an appropriate surrogate endpoint becomes an important consideration.  In the context of 
long-term investigations, a surrogate offers a theoretical advantage as a study endpoint in lieu 
of a parameter such as graft loss; the primary benefit being to decrease the interval between 
therapeutic intervention and the ability to determine efficacy [Stegall et al, 2015].  
Unfortunately, many prospective biomarkers are relatively non-specific, poorly-correlated, or 
may be causally influenced by a disease process without, itself, influencing the desired 
clinical outcome [Stegall et al, 2015; Fleming, 2005].  Therefore, in order to establish that the 
effect of a particular intervention on a clinical efficacy endpoint is reliably predicted by the 
effect of that intervention on a surrogate, Prentice identified two conditions that, if 
satisfactorily validated, would be sufficient to authenticate any proposed biomarker as an 
appropriate surrogate.  First and foremost, the biological marker must be correlated with the 
clinical endpoint – in this case, graft failure.  Secondly, it must fully capture the net effect of 
an intervention (immunosuppression) on the clinical efficacy endpoint [Prentice, 1989].
Histologic changes, such as transplant glomerulopathy (TG) or subclinical inflammation, are 
moderately good predictors of subsequent graft loss [Issa et al, 2008; Cosio et al, 2005].  
Patients with graft fibrosis, especially with inflammatory cell infiltrates, have worse 
outcomes and return to dialysis and/or undergo repeat transplantation with greater frequency.  
Likewise, subclinical histologic abnormalities found only on protocol biopsies have been 
associated with the development of chronic injury and decreased renal function over time 
[Loupy et al, 2009; Moreso et al, 2006; Nankivell et al, 2004; Shishido et al, 2003; Nankivell 
et al, 2001; Kirk et al, 1999; Rush et al, 1994].  
While some transplant centers routinely perform protocol biopsies as part of their standard of 
care (SOC), others do not, despite the fact that there is compelling evidence that subclinical 
acute rejection present in the graft leads to chronic rejection and early graft loss.  For this 
reason, a search for molecular biomarkers which can predict these phenomenon has been 
undertaken in the allograft, the urine, and the blood [El Ters et al, 2013; Li et al, 2013; Loupy 
et al, 2009; Kee et al, 2006; Moreso et al, 2006; Nankivell et al, 2004; Shishido et al, 2003; 
Nankivell et al, 2001; Kirk et al, 1999], the latter two of which having been proposed as a 
minimally invasive way to diagnose and monitor subclinical rejection, to optimize and 
personalize immunosuppression decisions, and as a means to potentially reduce the incidence 
of chronic rejection, which, in turn, could lead to significantly improved graft survival and 
function over the long-term.  In fact, the need to complement histopathological grading with 
an interpretation of accompanying molecular signals was recently identified at the 2013 
Banff Conference on Allograft Pathology [Haas et al, 2014].  However, up to now, relatively 
few individual molecular transcripts have been shown to correlate with chronic injury.  
Interestingly, an approach examining global genomic gene expression profiling in the 
peripheral blood was recently described.  Using multiple, three-way classifier tools to 
validate 200 of the highest value probesets, the groups at Northwestern and Scripps have 
identified a variety of molecular signatures in the peripheral blood.  Mapping these gene 
signatures to what is concomitantly revealed in biopsy specimens has demonstrated a 
sensitivity ranging from 82% to 100%, a specificity of 76% to 95%, a positive predictive 
value of 76% to 95%, and a negative predictive value of 79% to 100% [Kurian et al, 2014].  


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If similar methodology can be used to predict subclinical acute rejection, before the 
appearance of kidney dysfunction from the cumulative effects of tissue injury, this, too, could
satisfy the necessary conditions for use as a surrogate marker.
Given the wealth of accumulated evidence for fulfillment of the Prentice criteria, DSA also 
seems to be a relatively good surrogate marker to examine the effects of chronic antibody-
mediated injury.  For example, the disease process itself, but also the contribution of DSA, 
has been well-elucidated with regard to chronic, alloimmune injury [Einecke et al, 2009; Sis 
et al, 2007].  Further strengthening its role as a surrogate, DSA appears to act late in the 
biological injury pathway, and based on clinical insight and empirical evidence, is likely the 
predominant mechanism precipitating chronic, immune-mediated graft injury [Jordan & Vo, 
2014; Loupy et al, 2012].  In this regard, studies examining the impact of DSA on transplant 
outcomes allude to a quantitative epidemiologic relationship as well [Marfo et al, 2014; 
Alachkar et al, 2012; Zachary & Reinsmoen, 2011; Lefaucheur et al, 2010].  Additionally, 
newer reports are also beginning to link antibody formation to non-adherence [Wiebe et al, 
2015; Wiebe et al, 2012].  The “real world” evidence linking DSA to at least two primary 
clinical outcome measures [antibody-mediated rejection (ABMR) and graft survival] has 
been extensive as well as consistent.  For example, DSA dramatically accelerates the 
progression of histological lesions [Hill et al, 2011], is independently associated with TG 
[Issa et al, 2008; Gloor et al, 2007], and is a strong predictor of both ABMR and graft failure 
[Guidicelli et al, 2015; Wiebe et al, 2015; DeVos et al, 2014; Kim et al, 2014; Banasik et al, 
2013; Everly et al, 2013; Freitas et al, 2013; DeVos et al, 2012; Ginevri et al, 2012; Sellares 
et al, 2012; Wiebe et al, 2012; Willicombe et al, 2012; Cooper et al, 2011; Ntokou et al, 
2011; Gaston et al, 2010; El-Zoghby et al, 2009; Hidalgo et al, 2009; Lachmann et al, 2009; 
Mao et al, 2007; Cardarelli et al, 2005; Campos et al, 2006; Zhang et al, 2005].  It is also 
highly pragmatic that the measurement of DSA is relatively non-invasive and unlike biopsy, 
can be performed sequentially in the same patient. 
Recent publications describe that the major causes for de novo development of DSA relate 
primarily to medication non-adherence and immunosuppression minimization or elimination
protocols [Matas & Gaston, 2015; Nickerson & Rush, 2015; Jordan & Vo, 2014; Issa et al, 
2013].  Therefore, strategies which improve adherence and optimize exposure to 
immunosuppression should be evaluated to reduce the consequences of alloimmune-mediated 
injury.  Astagraf XL® is an oral, extended-release formulation of tacrolimus developed to 
enable a once daily dosing regimen with a similar safety and efficacy profile as Prograf®.  
Available in the European Union as Advagraf® since 2007, its decreased intrapatient 
variability (IPV) [Stifft et al, 2014; Kurnatowska et al, 2011; Wu et al, 2011] and flatter 
kinetics [van Hooff et al, 2012; Alloway et al, 2011] should ensure a more consistent 
immunosuppression exposure over the long-term and the once daily dosing has been 
described as improving medication adherence [Sabbatini et al, 2014; Doesch et al, 2013; 
Eberlin et al, 2013; Kuypers et al, 2013].  Given the negative impact of IPV on kidney 
transplant outcomes [Sapir-Pichhadze et al, 2014; Pollock-Barziv et al, 2010] and the 
association of non-adherence with DSA production [Wiebe et al, 2015; Wiebe et al, 2012], 


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the ability to target both with a single agent may be a highly relevant mechanism to improve 
long-term outcomes.
To this end, Astellas proposes a prospective, randomized, controlled trial comparing two 
immunosuppressant agents, specifically powered and designed to detect a difference in 
surrogates of chronic immune-mediated graft loss: DSA and real-time molecular profiling 
from individual patient, peripheral blood samples.  Both components will be combined as 
part of a two-part composite endpoint designed to test the hypothesis that the consistency of 
immunosuppression with Astagraf XL decreases the development of DSA and reduces the 
molecular signature of immune activation (IA).  Accordingly, this study will perform tests of 
correlation between DSA and peripheral blood molecular profiling as well as correlate each 
with information gained from SOC or institutional-derived protocol biopsies.  In the course 
of this study, patients in the control arm will receive immediate-release formulations of 
tacrolimus in accordance with local SOC dispensing practices, thus preserving the real-world 
conditions in which the patient could receive any manufacturer’s approved product.  In this 
manner, it is hoped that this research will improve the understanding of chronic alloimmune 
injury and provide greater insight into how tacrolimus exposure impacts DSA production.
1.2
Clinical Data
1.2.1
Clinical Summary
Pharmacokinetics of Tacrolimus (Prograf) in Humans
Following oral administration, tacrolimus is generally rapidly absorbed (tmax of 
approximately 1.5 hours).  The mean oral bioavailability is low, approximately 20% to 25%, 
with a large amount of variation between individuals.  The drug binds strongly to 
erythrocytes resulting in the distribution ratio of whole blood/plasma concentrations of 
tacrolimus of approximately 20:1. In plasma, the drug is highly bound to plasma proteins 
(> 98.8%), mainly to serum albumin and alpha-1-acid glycoprotein.  Tacrolimus is also 
extensively distributed in the body as indicated by a large steady state volume of distribution.
Systemically available tacrolimus is cleared by hepatic metabolism, specifically, from 
isoforms of the CYP3A system, a fact that contributes to a significant first pass effect.  
Pre-systemic metabolism by the gastrointestinal CYP3A and the ATP-binding cassette 
transporter protein (ABC-transporter), p-glycoprotein, has also been demonstrated and is 
considered responsible for the limited oral bioavailability of tacrolimus.
Tacrolimus is primarily eliminated in the bile; urinary excretion amounts to < 2% of the dose 
and < 1% as unchanged drug.
As tacrolimus is metabolized via CYP3A system enzymes, concomitant use of substances 
known to inhibit or induce CYP3A system enzymes may influence the metabolism of 
tacrolimus and thereby increase or decrease blood levels.  Tacrolimus, itself, is an inhibitor of 
CYP3A4; thus, concomitant use of tacrolimus with other drugs known to be metabolized by 
CYP3A4-dependent pathways may, in turn, affect the metabolism of such drugs (e.g., 
cortisone, testosterone).  Tacrolimus is also extensively bound to plasma proteins; therefore, 


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interactions with other drugs known to have high affinity for plasma proteins should be 
considered (e.g., NSAIDs, oral anticoagulants or oral antidiabetics).
Pharmacokinetics of Tacrolimus (Astagraf XL/Advagraf) in Humans
Biopharmaceutics in Healthy Volunteers
Absorption is variable and the mean oral bioavailability of tacrolimus (investigated with the 
Prograf formulation) is in the range of 20% to 25% (individual range in adult patients of 6% 
to 43%).  The oral bioavailability of tacrolimus for Advagraf or Prograf was approximately 
35% lower in the evening relative to morning administration.  Bioavailability was also 
reduced when Advagraf or Prograf capsules were administered with, or up to, 1.5 hours after 
consumption of food.  It is therefore recommended that Advagraf or Astagraf XL capsules be 
taken consistently in the morning either with or without food.
Pharmacokinetics in De Novo Transplant Patients
Two phase 2 pharmacokinetic studies in de novo liver and de novo kidney transplant patients 
have been performed in order to compare the pharmacokinetics of tacrolimus for the 
Advagraf and Prograf formulations.  The pharmacokinetic data from the study in de novo 
kidney transplant recipients revealed that the systemic exposure to tacrolimus on day 1 was 
lower for Advagraf than for Prograf despite similar doses.  By day 4, systemic exposure as 
measured by trough levels was similar for both formulations.
To obtain more data on the pharmacokinetics of tacrolimus for Advagraf during the early 
post-transplant period, a phase 3 study protocol, in which the first dose of tacrolimus was 
given preoperatively, included a pharmacokinetic substudy which was conducted in selected 
centers.  This pharmacokinetic substudy aimed to obtain information on the pharmacokinetics 
of tacrolimus for Prograf and Advagraf during the first 2 weeks after transplantation in de 
novo kidney transplant patients.  The data for all patients who completed the study as planned 
and who provided 4 complete blood concentration-time profiles (day 1, day 3, day 7, and day 
14) without major protocol deviations or factors which could influence the pharmacokinetic 
evaluations showed that the systemic exposure to tacrolimus (ln[AUC24]) on day 1 was 
approximately 16% lower for Advagraf than for Prograf, although the mean total daily dose 
(mg/kg) was approximately the same.  On days 3, 7 and 14, the ln(AUC24) for Advagraf was 
5%, 22% and 22% higher than that for Prograf, with the 90% Confidence Intervals (CIs) for 
ln(AUC24) being outside the equivalence range of 80% to 125%.
However, the mean total daily doses (mg/kg) of Advagraf were approximately the same as 
the corresponding mean Prograf doses at the times of the day 1, day 3, and day 14 profiles. 
These data suggest that systemic exposure is similar by day 3.
Across all studies where stable kidney, liver, and heart transplant recipients were converted 
from twice daily Prograf to once daily Advagraf on a 1:1 (mg:mg) total daily dose basis, the 
geometric mean ratios of systemic exposure of tacrolimus (Advagraf:Prograf) ranged from
88.8% to 100.9%.  The 90% CIs across these studies were all contained within the 
equivalence limits of 80% to 125%.


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There was a high correlation between AUC24 and trough levels in de novo kidney transplant 
recipients (correlation coefficients of 0.83 and 0.87 in Studies FG-506E-12-01 and FG-506E-
12-03, respectively) and in stable kidney, liver, and heart transplant recipients converted from 
Prograf to Advagraf (correlation coefficients of 0.86 to 0.934 in kidney transplant recipients, 
0.88 to 0.90 in liver transplant recipients and 0.94 in heart transplant recipients).  The slope 
of the line of best fit was also similar for both Advagraf and Prograf, indicating that for 
therapeutic drug monitoring (TDM), the same target trough level range is applicable for both 
formulations.
1.2.2
Clinical Studies of Efficacy and Safety
Tacrolimus as Prograf has been proven to be effective in adult and pediatric liver, kidney, 
and heart transplant recipients.  Several controlled clinical trials have confirmed that the 
efficacy of Prograf in this population is therapeutically equivalent, in terms of preventing 
acute rejection, to a cyclosporine-based regimen.
The efficacy of Advagraf in adult de novo kidney transplant recipients was evaluated in 
3 phase 3/3b clinical studies that provided comparative efficacy for 1 year (Studies 02-0-158 
and FG-506E-12-03) and 6 months (Study PMR-EC-1210).  The 6-week data from phase 2 
pharmacokinetic Study FG-506E-12-01 provides additional support of efficacy.  Efficacy in 
adult de novo liver transplant recipients was evaluated in phase 3 Study FG-506E-11-03 for 
patients treated for at least 1 year.  
Data from the studies in adult de novo kidney transplant recipients indicate that Advagraf is 
non-inferior to Prograf in both the short and longer-term prevention of kidney allograft 
rejection and in extending allograft survival.  Efficacy was maintained in terms of patient and 
graft survival and prevention of biopsy-proven acute rejection (BPAR).  This was seen at 
3 years post-transplant in patients for the phase 3 de novo studies (Studies FG-506E-12-03 
and FG-506E-11-03) from the Advagraf treatment group who were included in the long-term 
follow-up Study FG-506-14-02.
Overall Safety Profile
The safety profile of Prograf has been established worldwide in over 100,000 transplant 
patients treated since its introduction in 1993.  Clinically important events include 
impairment of renal function, abnormal glucose metabolism, and neurological disorders. 
Many of the adverse drug reactions are reversible and/or responded to dose reduction. 
Transplant patients on immunosuppressive therapy are also known to be at increased risk of 
developing post-transplant lymphoproliferative disorders and other malignancies.  This risk is 
considered to be related to the overall immunosuppressive burden.
The safety of Advagraf was evaluated in 4201 transplant patients in 16 phase 2/3 studies, of 
who 2801 received Advagraf.
The adverse events observed following administration of Advagraf in the three large phase 
3/3b studies in de novo kidney transplant recipients (Study 02-0-158, Study FG-506E-12-03 
and Study PMR-EC-1210 [OSAKA]), and in the two phase 2 pharmacokinetic studies in de 
novo kidney (Study FG-506E-12-01) transplant recipients were consistent with the 


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established safety profile of Prograf.  No new adverse events that would suggest a clinical 
concern were identified, with Advagraf having a similar safety profile to that of Prograf, 
despite small differences in individual adverse events.
The safety profile of Advagraf and Prograf were also similar in stable kidney, liver, and heart 
transplant recipients converted from twice daily Prograf to once daily Advagraf.
1.2.3
Whole Blood Trough Concentrations Can Be Used to Target the Same Systemic 
Exposure for Both Prograf and Astagraf
Tacrolimus is an active substance with a narrow therapeutic index (NTI).  Like other NTI 
drugs, tacrolimus requires regular monitoring to establish and maintain appropriate drug 
exposure.  Immunosuppressive therapy with once daily tacrolimus is usually initiated at a 
dose between 0.1 mg/kg per day and 0.3 mg/kg per day in orally divided doses.  Subsequent 
doses are adjusted on the basis of clinical signs aided by tacrolimus whole blood trough 
concentration monitoring.  To ensure that appropriate systemic exposure is achieved, the 
prevailing practice is to conduct TDM based on the trough whole blood concentrations 
(Cmin).  Although tacrolimus systemic exposure (AUC) is the critical parameter for both 
safety and efficacy in transplant recipients [Kuypers et al, 2004; Undre et al, 1999], AUC 
monitoring is not feasible in clinical practice.  For this reason, Cmin (i.e., trough 
concentration) is used as a surrogate for overall exposure.  Moreover, as shown in Figure 1, 
the relationship between Cmin and AUC is the same for both Prograf and Astagraf XL.  
Therefore, for both formulations, the established trough concentration goals can be used to 
target the same systemic exposure level.  It is not known whether this is the case for other 
formulations. 
Figure 1
Correlation between AUC and Cmin of Advagraf (tacrolimus QD) and 
Prograf (tacrolimus BID) in heart transplant recipients 
Reproduced from Alloway et al, 2011.
1.2.4
Increased Potential for Adverse Clinical Outcomes with Too High or Too Low 
Tacrolimus Exposure
Several studies have shown that with increasing tacrolimus exposure, the decreased risk of 
acute rejection is offset by a potential for increased risk of toxicity and infection [(Cockfield 
et al, 2014; Kuypers et al, 2004) reviewed in (Staatz & Tett, 2004)].  Additional studies have 


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shown that modest, short-term changes in tacrolimus exposure may result in adverse clinical 
outcomes.  These studies, explained in greater detail below, demonstrated: 
●
A significantly (26.9%) lower AUC in patients who experienced acute rejection at 
day 2/3 post-kidney transplant, compared with those who remained rejection-free 
(157 ng∙h/mL vs. 215 ng∙h/mL, P = 0.007) [Undre et al, 1999];
●
Increased risk of BPAR in patients with AUC < 150 ng∙h/mL at day 7 post-kidney 
transplant in comparison to those above this threshold [Kuypers et al, 2004];
●
A 7.2% increased risk of acute rejection with every 1 ng/mL decrease in tacrolimus 
trough concentration within the first 6 months following kidney transplant; an additional 
23% increased risk of acute rejection with every 1 ng/mL decrease in trough between 
3 and 6 months [Israni et al, 2013];
●
Significantly increased incidence of BPAR in de novo kidney transplant recipients 
receiving low-dose tacrolimus ER compared with those receiving standard-dose 
tacrolimus ER (actual tacrolimus exposure differed by 30% to 40%) [Cockfield et al, 
2014];
●
Significantly higher mean AUC in de novo kidney transplant recipients who had 
infection at day 7 and week 6, compared with those who remained infection-free (197.4 
± 70.5 ng∙h/mL vs. 160.5 ± 47.9 ng∙h/mL, P = 0.01 at day 7; 177.5 ± 28.3 ng∙h/mL vs. 
149.2 ± 31.3 ng∙h/mL, P = 0.02 at week 6) [Kuypers et al, 2004].
Based on the AUCs associated with these adverse events, it appears that risk of acute 
rejection and infection increased outside of the range of 150–177 ng∙h/mL. 
Similar to the studies described above, the association between early acute rejection (within 
the first 6 months of transplantation) and decreased tacrolimus trough concentration was 
shown in a post hoc analysis of the prospective DeKAF study.  DeKAF was a landmark study 
that has shaped our understanding of post-transplantation immunosuppression and long-term 
kidney transplant function.  The authors of this analysis reported that for every 1 ng/mL 
decrease in tacrolimus trough concentration, a 7.2% increased risk of acute rejection within 
the first 6 months could be expected post-transplant (P = 0.03).  Between months 3 and 6, 
when centers typically reduce tacrolimus exposure to limit long-term side effects, there was 
an additional 23% increased risk of acute rejection with each 1 ng/mL decrease in tacrolimus 
trough level (P = 0.008) [Israni et al, 2013].  The latter result is especially concerning, as 
stable patients are typically monitored no more than once in 3 months.
Together, these studies show that small deviations in tacrolimus exposure places patients at 
increased risk of acute rejection and opportunistic infection, highlighting the fine balance 
between tacrolimus under- and overexposure.
1.2.5
Association Between the Potential for Adverse Clinical Outcomes and 
Increased Tacrolimus Exposure Variability
High IPV in tacrolimus concentration over time may put the patient at risk of over- or 
under-immunosuppression. Tacrolimus IPV has been assessed in several studies enrolling 
stable transplant patients, in which the coefficient of variation (CV) of tacrolimus trough 


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concentration is consistently calculated to be approximately 15%.  The following section 
describes study results suggesting that increased IPV is associated with adverse long-term 
(≥6 month post-transplant) clinical outcomes.
Study Details
Borra et al. reported that high tacrolimus IPV was associated with significantly higher risk of 
graft failure at ≥ 1 year post-transplant, compared with those with low IPV (P = 0.003).  IPV 
was calculated from a retrospective analysis of 6–12 month post-transplant trough 
measurements in 297 kidney transplant recipients.  The median tacrolimus IPV in this study 
(14.9%) was used as the cut-off for high vs. low variability.  Notably, mean ± SD tacrolimus 
concentration was similar between patients who experienced or did not experience graft 
failure (6.9 ± 2.5 ng/mL vs. 7.4 ± 2.9 ng/mL, P = NS).  However, multivariate analysis 
showed that IPV was indeed an independent predictor of graft failure [2010]. 
Another retrospective analysis in kidney transplant recipients demonstrated a similar 
association between high variability and poor graft function.  In a retrospective analysis of 
200 kidney transplant patients, Makowski et al. reported a significant association between 
high tacrolimus IPV, defined here as CV > 40%, and increased development of delayed graft 
function and diminished renal function [2013].
Pollock-Barziv et al. reported that increased standard deviation of tacrolimus (TacSD) was an 
independent predictor of acute rejection at ≥ 6 months post-transplant.  In this retrospective 
analysis of 144 adolescent heart, kidney, liver, or lung transplant patients, TacSD was 
calculated from trough level measurements since transplant.  In recipients with TacSD < 2 at 
least 6 months post-transplant, graft survival was significantly better than in those with 
TacSD > 2 [Figure 2]. Moreover, each 1-point increase in TacSD > 2 of tacrolimus trough 
was associated with a 58% increase in risk of graft loss (P = 0.003) [2010].
Figure 2
Variability in tacrolimus trough levels associated with increased risk of 
graft loss in adolescents
Reproduced from Pollock-Barziv et al, 2010


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1.3
Summary of Key Safety Information for Study Drugs
As with other potent immunosuppressive agents, patients receiving tacrolimus are at an 
increased risk for infections (viral, bacterial, fungal, and protozoal).  Likewise, the course of 
pre-existing infections may be aggravated.  Overall, infections have been reported frequently 
in patients being treated with tacrolimus.  Both generalized and localized infections can 
occur.  Patients receiving immunosuppressive agents are also at an increased risk of 
developing malignancies, particularly of the skin.  Benign as well as malignant neoplasms, 
including Epstein-Barr virus (EBV)-associated lymphoproliferative disorders, have been 
reported in association with tacrolimus treatment.
Tacrolimus can result in renal function impairment in post-transplant patients due to its 
vasoconstrictive effect on renal vasculature.  Acute renal impairment, that is usually 
reversible, can result in high serum creatinine, hyperkalemia, decreased secretion of urea, and 
hyperuricemia.
Additional adverse reactions reported in patients receiving tacrolimus include allergic and 
anaphylactoid reactions, ventricular hypertrophy or hypertrophy of the septum (reported as 
cardiomyopathies; most cases have been reversible), encephalopathies (such as posterior 
reversible encephalopathy syndrome [PRES], and pure red cell aplasia [PRCA]).  In the case 
of PRCA, all patients reported risk factors such as parvovirus B19 infection, underlying 
disease, or concomitant medications associated with PRCA.  Gastrointestinal perforation has 
also been reported in patients treated with tacrolimus, although all cases were considered a 
complication of transplant surgery or accompanied by infection, diverticulum, or malignant 
neoplasm.  Tacrolimus may prolong the QT interval and may cause Torsade de Pointes. 
For additional information regarding possible adverse drug reactions related to tacrolimus or 
Astagraf XL, please refer to the Package Inserts (PIs) for Astagraf XL or the various 
immediate-release tacrolimus products.
1.4
Risk-Benefit Assessment
The research proposed herein compares two FDA-approved medications indicated for the 
prophylaxis of acute rejection in kidney transplantation: 1) tacrolimus, extended-release 
(Astagraf XL) and 2) tacrolimus, immediate-release.  Tacrolimus (in the twice daily 
formulation of Prograf) has been extensively studied and used in clinical practice for many 
years.  The safety profile of tacrolimus has been well-established.  The use of tacrolimus in 
the current study is consistent with the recommendations described in the approved package 
inserts, and as such, is not expected to shift the known risk-benefit profile in the transplant 
population under study.  Clinical studies have confirmed that Astagraf XL is as efficacious as 
Prograf, with a similar safety profile [Trunecka et al, 2008; Fischer et al, 2007; Alloway, 
2005; Undre, 2005]. 


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2
STUDY OBJECTIVE(S), DESIGN, AND ENDPOINTS
2.1
Study Objectives
2.1.1
Primary Objective
The primary objective is to compare the incidence of a two-part composite endpoint 
consisting of de novo DSA formation or a designation of immune activation (IA) on 
peripheral blood molecular profiling in patients maintained on twice daily, immediate-release 
tacrolimus versus those maintained on Astagraf XL in the first two years post-transplant.
2.1.2
Secondary Objectives
The secondary objectives of the study are to:
●
To assess the risk factors for each of the following outcomes: DSA formation; IA;
transplant glomerulopathy (TG); acute and chronic forms of antibody-mediated rejection 
(ABMR); C1q-binding DSA; HLA-DQ DSA; DSA IgG3 isotype; requirement for and type 
of antibody reduction required; various threshold levels of estimated glomerular filtration 
rate (eGFR) (less than 30, 40, and 50 mL/min/1.73 m2); and the four components of the 
traditional composite endpoint, consisting of graft loss, mortality, biopsy-proven acute 
rejection (BPAR), and loss to follow-up; and additionally, the persistence of DSA and IA.
●
Assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with the development of IA on molecular profiling.
●
Compare, between treatment cohorts, the incidence and hazards of each of the following, 
occurring over the course of one year and, where appropriate, at 24 months post-
transplantation: DSA, HLA-DQ DSA, C1q-binding DSA, DSA IgG3 isotype, IA, TG, 
select BANFF histology grades (acute and chronic forms of ABMR, acute and chronic 
active TCMR, borderline changes, and IFTA), graft loss, mortality, and BPAR.
●
Compare between cohorts the incidence of various thresholds of eGFR (less than 30, 40, 
50 mL/min/1.73 m2, and a five-point decline).
●
Compare the distribution of ordinal categories of antibody strength (weak, moderate, 
strong) between treatment groups across the study time course as well as at each time 
point.
●
Compare raw MFI scores between treatment groups at each time point and across the 
duration of the study.
●
Examine and compare histopathology between the treatment cohorts in biopsies obtained 
for cause and for maintenance during the course of clinical care. 
●
Examine and compare outcomes as a function of tacrolimus manufacturer and the 
number of switches between immediate-release tacrolimus products.
●
Summarize AEs between study groups.
●
Compare the change in eGFR over the study duration beginning from 30 days post-
transplant between groups, and in patients who develop DSA or IA.


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●
Compare the persistence of the development of IA on molecular profiling between the 
two cohorts across the study duration in those patients who develop IA on molecular 
profiling.
2.1.3
Exploratory Objectives
Exploratory objectives are:
●
Compare the frequency of those patients experiencing either component of a two-part 
composite endpoint consisting of DSA formation and the presence of TG (cg > 0) on 
centrally-interpreted institutional protocol (maintenance) biopsy and / or biopsy obtained 
for cause during the first year post-transplant  and/or up to the conclusion of the study.
●
To assess and compare, between treatment groups, the association/correlation of 
incidence and strength of DSA, and the incidences of HLA-DQ DSA, C1q-binding DSA, 
and DSA IgG3 isotype with each other, and individually, with each of the following: IA,
TG, ABMR (both chronic forms and acute), Banff histology, various thresholds of eGFR 
(less than 30, 40, and 50 mL/min/1.73 m2), eGFR change over time, TCMR, graft loss, 
and mortality.  
●
Examine the proportion of patients with the IA designation who have normal and 
abnormal creatinine.   
●
In patients with an IA designation and abnormal creatinine, examine biopsies (if 
available) for concordance.
●
To assess the association/correlation of the histopathology with DSA, C1q-binding DSA, 
DSA IgG3 isotype, molecular phenotyping, graft loss, and measures of renal function in 
each cohort in patients for whom one year institutionally-derived, protocol (maintenance) 
biopsies are available.
●
To assess and compare, between treatment groups, the association/correlation of the CV 
and SD of tacrolimus trough concentrations with each of the following: the composite 
endpoint of DSA and IA,  DSA formation and IA individually, TG, ABMR, C1q-binding 
DSA, HLA-DQ DSA, DSA IgG3 isotype, histopathology, renal dysfunction, and the 
components of the traditional composite endpoint.
●
To compare dose changes and IPV between groups as a function of tacrolimus trough 
level after the first 6 weeks following transplantation in patients on stable doses of 
immediate-release tacrolimus and Astagraf XL.
●
To assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with each of the following: TG, chronic forms of ABMR, C1q-
binding DSA, HLA-DQ DSA, DSA IgG3 isotype, IA, histopathology, graft loss, various 
thresholds of eGFR (< 30, 40, and 50 mL/min/1.73 m2), acute ABMR, TCMR, and 
mortality in patients who develop IA on molecular profiling in each cohort.
●
To compare the MFI shift to a lower category (and magnitude of shift) between groups 
(including C1q MFI shift), in those patients who develop DSA during the course of the 
study.


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●
To examine percent MFI reduction within each subgroup over time in each treatment 
arm (including C1q MFI reduction) in those patients who develop DSA during the 
course of the study. 
●
To map expression results to known immunological pathways implicated in immune-
mediated, kidney transplant tissue injury.
●
To determine the longitudinal consequences of DSA formation and immune activation, 
perform de-identified, long-term follow-up through participating transplant centers in 
select study participants who provide the appropriate informed consent by future linking 
to the Scientific Registry of Transplant Recipients (SRTR) after completion of 
ASTOUND.
●
To examine the economic burden of treating patients with molecular evidence of 
immune activation and DSA formation using Medicare claims data or commercial payer 
cost data in patients required to transfer from Medicare to private insurance.
●
To compare outcomes in like patients between cohorts managed with maintenance 
protocol biopsy vs. biopsy for cause with respect to the composite endpoint, the 
components thereof, complications, and resource utilization.
2.2
Study Design and Dose Rationale
2.2.1
Study Design
This is an exploratory, two year, prospective, randomized, multi-center, open-label trial 
examining long-term kidney transplant outcomes through the use of an adaptive design and a 
two-part, composite surrogate endpoint.  Specifically, it is designed to compare the effects of 
twice daily, immediate-release tacrolimus and once daily Astagraf XL on DSA formation and 
the development of a peripheral blood molecular profile indicating the presence of immune 
activation (IA) in de novo kidney transplant recipients during the first two years following 
transplantation. For the purposes of this study, IA will be defined as a positive molecular 
signature using the Trugraf™v.2.0 molecular assay (Transplant Genomics, Inc., Pleasanton, 
CA) in all patients.
Patients will be screened prior to surgery and randomized 1:1 to receive  immediate-release 
tacrolimus, administered twice daily, or Astagraf XL, as a component of a standard 
immunosuppression maintenance regimen also consisting of corticosteroids (complete steroid 
avoidance not allowed – steroid taper and withdrawal permitted if part of  institutional 
protocol) and mycophenolate mofetil (MMF) (or Myfortic® equivalent).  Investigators are 
encouraged to start subjects on the randomized study treatment (immediate release tacrolimus 
or Astagraf XL) within 48 hours of transplantation (pre-transplant administration of study 
treatment is not allowed). However, if medically indicated, per the treating physician’s 
discretion, initiation of study treatment may be delayed for up to seven days post-transplant. 
IF medically indicated, subjects who are unable to tolerate oral administration of their 
assigned study drug may be administered tacrolimus via an alternative route (for Astagraf XL 
treatment arm: intravenous [IV] tacrolimus; for immediate release tacrolimus: IV tacrolimus 


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or nasogastric tube [NGT]) for up to seven consecutive days. Dosing via alternative routes 
should be discontinued as soon as the subject can tolerate standard oral administration, and is 
not allowed for more than seven consecutive days. Please see Section 5.1.1 and 4.1 for 
further details regarding non-oral administration of tacrolimus.  
Given the variability of incidence rates for DSA reported in the literature, a stopping rule has 
been incorporated herein to mitigate the risk of continuing the study under incorrect prior 
assumptions relating to the true incidence rate of DSA formation (Section 7.1).  To apply the
rule, the incidence rate of the primary endpoint in the control group will be examined once 
50% of control patients have completed one year of therapy.  Early termination of the study, 
or extension to a second year, will be predicated on this assessment (see Section 7.8.1).
Induction therapy is required for all participants, and will be administered (dosing, type of 
agent) per center protocol.  Intravenous corticosteroids will be administered prior to 
revascularization as part of the initial induction regimen, with dose and duration of therapy 
also determined by the participating transplant center’s standard protocol.  Thereafter, 
patients will be followed for up to 2 years during the open-label study period.  Immediate-
release tacrolimus will be obtained from pharmacies per the local standard of care.  After 
discharge from the hospital, the patients randomized to immediate-release tacrolimus will 
receive study-provided vouchers and will be permitted to receive any immediate-release 
tacrolimus product available to them through normal dispensing mechanisms.  Astagraf XL 
will be supplied by Astellas and dispensed by the participating institution.
Initial pre-transplant cross-matching (XM) will be performed per local protocol.  Initial XM 
serum will be collected and stored centrally to facilitate the later assessment of pre-formed 
antibody on an as needed basis.  Thereafter, antibody screening and molecular phenotyping 
will be performed by a central lab at one month, 3 months, 6 months, 9 months, 12 months, 
and 24 months.  Antibody status, including HLA-DSA isotype and C1q-binding DSA status, 
will also be recorded when measured locally during the course of clinical care. 
For central laboratory analysis, initial FlowPRA® (One Lambda, Canoga Park, CA) screening 
will be used to test for the presence of anti-HLA antibodies.  Once identified, single antigen 
testing will be used to identify DSA specificity with an MFI greater than or approaching 
1000  used to determine antibody positivity.  In borderline cases, a three-member 
adjudication board of independent, qualified HLA Lab Directors will determine positivity 
based on relevant clinical data.  In samples that meet threshold criteria for DSA positivity, 
additional classification will be performed to determine the degree of antibody strength.  By 
performing a single dilution (1:16) of DSA-containing samples, the alloimmune response can 
be further elucidated [Tambur et al, 2015].  Samples in which MFI disappears (becomes < 
1000 MFI) will be regarded as “weak.”  Samples increasing to > 10,000 MFI upon dilution 
will be regarded as having significant prozone and will be regarded as “strong.”  Samples 
which may increase or decrease, but nevertheless remain between 1000 and 10,000 MFI will 
be regarded as “moderate.”  In certain cases, samples with evidence of fluorescence that are 
nevertheless below MFI criteria for DSA positivity will also be subject to dilution.  Samples 
increasing to above threshold criteria following dilution will be regarded as positive and 


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assumed to be under the influence of a prozone effect that impairs antibody detection.  For 
statistical purposes, the DSA with the highest MFI level obtained during the course of the 
study will be used for statistical comparisons between groups.  However, the strongest 
antibody observed at each HLA-locus will be reported separately and tracked for patients 
who are DSA positive [Tambur et al, 2015].  In all cases, positive DSA and/or molecular 
testing results will be communicated to the patient’s treating physician when available (DSA) 
or at the end of study (molecular testing). Testing will be performed in batches. De-identified 
typing reports (donor and recipient) will be requested from the participating centers to inform 
the central laboratory testing. De-identified results of recent local pre-transplant DSA testing 
(if performed per SOC) will also be requested to facilitate interpretation of central DSA 
testing results. 
Follow-up visits will be performed quarterly during the first year of follow-up to collect 
standard clinical data.  For follow-up beyond the first year, urinary protein, and tacrolimus 
trough concentrations obtained closest to targeted date of visit, per SOC, should be recorded. 
The two closest serum creatinine levels to the study visit, obtained at least one month apart, if 
performed per SOC, should also be recorded. Additionally, all local kidney biopsy results, 
AEs, and concomitant medications pertaining to AEs will be recorded.  At the terminal study 
visit at 24 months, the most recently obtained lab results as well as any episodes of rejection, 
need for antibody reduction, graft loss, and deaths occurring beyond the first year will be 
detailed in the eCRF.  Final testing for DSA and molecular profiling will also be performed 
at the 24 month visit.
Pathology results [hematoxylin and eosin (H&E), light microscopy, and immunofluorescence] 
will be recorded in the eCRF when local kidney biopsies are performed and interpreted during 
the course of clinical care.  De-identified copies of all kidney pathology reports must be 
submitted to Astellas. For purposes of data recording, substantiation by local biopsy will be 
required for cases of suspected rejection.  For all participants who undergo at least one kidney 
biopsy during the first year post-transplant (either for cause or per institutional protocol), the 
kidney biopsy performed closest to the 12 month visit (but no later than 14 months post-
transplant) will be submitted for central pathological grading.  All biopsies that are submitted 
for central pathology review will be evaluated and scored using the most recent version of the 
2007 Update to the Banff ’97 Classification by a central pathologist blinded to clinical results 
and patient identifiers [Solez et al, 2008]. 
2.2.2
Dose Rationale
In line with recommendations from the US PI, the initial dose of Astagraf XL should be 
weight-based in order to avoid the possibility of under-immunosuppression and ranged 
between 0.1 mg/kg/day and 0.2 mg/kg/day in the two pivotal trials considered by the FDA 
for Astagraf XL approval [Silva et al, 2007; Kramer et al, 2010].  For the purposes of the 
ASTOUND study, patients randomized to the Astagraf XL treatment arm will be 
administered an initial, weight-based dose of 0.15 mg/kg/day.  Dry weight, or estimated 
dry weight, if dry weight is not available, should be used to calculate the initial dose. 
Subsequent dosing should be based on clinical assessments of rejection and tolerability in 


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each individual patient and should be aided by whole blood trough concentration monitoring.  
To assist in dose titration of Astagraf XL, it is important to note that the relationship between 
Cmin and AUC has been validated for both Prograf and Astagraf XL, allowing practitioners to 
rely on their clinical experience with Prograf to appropriately titrate Astagraf XL.  For the 
purpose of this study, immediate-release tacrolimus will be dosed in accordance with local 
center protocol.
2.3
Endpoints 
2.3.1
Primary Endpoints
The primary endpoint is the combined incidence of either DSA or IA on peripheral blood 
molecular profiling at one year or at the conclusion of the study.  For this purpose, DSA will 
be considered as a categorical (binary) variable with positivity determined at a threshold 
criteria approaching MFI = 1000 at any time during the study.  For reporting purposes, IA 
will be considered either present or absent using the Trugraf™ v2.0 molecular assay.  For the 
purposes of the study, a negative designation (Trugraf TX Normal) will be referred to as 
Immune Quiescence (IQ).  Due to operating characteristics of the assay, a positive 
designation will be considered evidence of Immune Activation (IA) in all patients.
The mFAS will be used for the primary endpoint analysis.
2.3.2
Secondary Endpoints 
The assessment and comparison of the incidence of DSA between treatment groups will rely 
on the binary variable of DSA positivity at a threshold criteria approaching an MFI of 1000.  
Comparisons of the cohorts with respect to the various molecular designations as well as 
correlating the results with DSA will rely on the categorical (binary) variable of positivity 
using the Trugraf™ v2.0 molecular assay.  Comparisons between treatment cohorts regarding 
incidence will rely upon assessments for each of the following results at any point in the study:
●
DSA 
●
IA 
●
TG
●
Acute and chronic forms of ABMR
●
C1q-binding DSA
●
HLA-DQ DSA
●
DSA IgG3 isotype
●
Required antibody reduction
●
eGFR at various thresholds (less than 30, 40, and 50 mL/min/1.73 m2, and whether a 
five-point decline in eGFR occurs
●
Graft loss (defined as subject death, retransplantation, transplant nephrectomy, or a 
return to dialysis for at least a 6 week duration)
●
Death
●
BPAR (inclusive of ABMR and TCMR)
●
Loss to follow-up


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Time-to first occurrence will be assessed for each of the following: DSA, HLA-DQ DSA, 
C1q-binding DSA, DSA IgG3 isotype, IA, TG, select BANFF histology grades (acute and 
chronic forms of ABMR, acute and chronic active TCMR, borderline changes, and IFTA),
mortality, and local BPAR.
The frequency of the type of antibody reduction employed will be assessed.
Patient MFI and eGFR values will be assessed over time.
Assessments of histopathology in biopsies will be made using the following endpoints:
●
Incidence of ci, ct, and ptc scores greater than one
●
Biopsy scores for g, t, v, i, cg, ct, ci, cv, ah, ptc, and mm 
The safety endpoint for AEs will be the incidence of each type of AE based on system organ 
class and using the preferred term from the most recent version of the Medical Dictionary for 
Regulatory Activities (MedDRA).
Persistence of DSA and IA will be the additional endpoints for comparisons in patients who 
develop IA and DSA on molecular profiling.
2.3.3
Exploratory Endpoints
Additional exploratory endpoints include the following:
●
A second two-part composite endpoint encompassing the incidence of acquiring DSA or 
positive evidence of TG on biopsy during the first year post-transplant or by the 
conclusion of the study.
●
Expression results, mapped to known immunological pathways implicated in immune-
mediated, kidney transplant tissue injury.
●
With appropriate informed consent, long-term graft and patient survival in de-identified 
study participants by future linking to the Scientific Registry of Transplant Recipients 
(SRTR) following completion of ASTOUND.
●
The economic burden of treating patients with molecular evidence of immune activation 
and DSA formation using Medicare claims data (or commercial payer cost data in 
patients required to transfer from Medicare to private insurance).
●
Outcomes in like patients between cohorts managed with maintenance protocol biopsy 
vs. biopsy for cause with respect to the composite endpoint, the components thereof, 
complications, and resource utilization.


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3
STUDY POPULATION
3.1
Selection of Study Population 
This study will enroll 550 living or deceased donor kidney transplant recipients, 16 to 70 years 
of age.  Patients will be screened and randomized 1:1 prior to surgery to receive, twice daily, 
immediate-release tacrolimus or Astagraf XL as a component of a standard immunosuppression 
maintenance regimen also consisting of corticosteroids (complete steroid avoidance not allowed 
– steroid taper and withdrawal permitted if given per institutional protocol) and MMF (or 
Myfortic equivalent) (see Section 4.5 for randomization details).  After discharge from the 
hospital, patients randomized to receive twice-daily immediate-release tacrolimus will receive 
study-provided vouchers and will be permitted to receive any immediate-release tacrolimus 
product available to them through normal dispensing mechanisms.  Patients randomized to 
receive Astagraf XL will receive this treatment for the duration of the study and will not be 
permitted to use other extended-release tacrolimus formulations. 
Upon request from the investigator, the Sponsor’s medical monitor will consider requests for 
patient re-screenings on a case-by-case basis.  
3.2
Inclusion Criteria
Subject is eligible for the study if all of the following apply.
1.
Kidney transplant patient ≥16 years and ≤ 70 years old.
2.
Institutional Review Board (IRB)-/Independent Ethics Committee (IEC)-approved 
written Informed Consent and privacy language as per national regulations (e.g., HIPAA 
Authorization for US sites) must be obtained from the subject or legally authorized 
representative prior to any study-related procedures (including withdrawal of prohibited 
medication, if applicable).  For subjects < 18 years old, informed consent must be 
obtained from the subject’s parent(s) or legal guardian(s); subject Assent (where 
appropriate) must be given.
3.
Recipient of a de novo kidney from a living or deceased donor. Note: Recipient of an en 
bloc deceased donor kidney transplant from a pediatric donor ≥5 years of age AND 
weighing greater than 20 kg is allowed. 
4.
If deceased donor, a Kidney Donor Profile Index (KDPI) ≤ 85 [donation after circulatory 
death (DCD) and what was previously known as extended criteria donor (ECD) organ 
recipients are eligible for enrollment provided KDPI ≤ 85] 
5.
Removed
6.
At least one antigen mismatch at major MHC (class I or class II).
7.
Willingness to comply with study protocol.
8.
Subject agrees not to participate in another investigational drug study while on treatment.


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9.
Female subject must be either:
a.
Of non-child-bearing potential
i. Post-menopausal (defined as at least 1 year without any menses) prior to 
screening, or 
ii. Documented surgically sterile or status post-hysterectomy
b. Or, if of childbearing potential, 
i. Agree not to try to become pregnant during the study and for 90 days after 
the final study drug administration
ii. And have a negative serum or urine pregnancy test within 7 days prior to 
transplant procedure
iii. And, if heterosexually active, agree to consistently use two forms of highly 
effective birth control (at least one of which must be a barrier method) 
which includes consistent and correct usage of established oral 
contraception, established intrauterine device or intrauterine system, or 
barrier methods of contraception: condom or occlusive cap (diaphragm or 
cervical/vault caps) with spermicidal foam/gel/film/cream/suppository or 
vasectomy in the male partner, starting at screening and throughout the 
study period and for 90 days after the final study drug administration.
10. Male subject and their female spouse/partners who are of childbearing potential must be 
using highly effective contraception consisting of two forms of birth control (one of 
which must be a barrier method) starting at screening and continuing throughout the 
study period and for 90 days after the final study drug administration (acceptable forms 
of birth control are listed in Inclusion Criterion 9). 
11. Male subject must not donate sperm starting at screening throughout the study period 
and for 90 days after the final study drug administration.
12. Female subject must agree not to breastfeed starting at screening and throughout the 
study period, and for 90 days after the final study drug administration.
13. Female subject must not donate ova starting at screening and throughout the study period, 
and for 90 days after the final study drug administration.
14. Will be receiving induction immunotherapy (either T-cell depleting agent, anti-CD52 
monoclonal antibody, or IL-2 co-stimulation blocker), with dose and frequency of the 
chosen induction agent determined by local standard of care. Steroid-only induction does 
not satisfy this criterion. 
Waivers to the inclusion criteria will NOT be allowed.
Previous kidney transplants will be permitted.
Patients who are receiving a secondary transplant and who previously received Astagraf XL 
or who are currently on Astagraf XL as a component of maintenance immunosuppression and 
re-listed for transplant will be eligible to enroll in ASTOUND and will be randomized at the 
time of transplant to either cohort.


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3.3
Exclusion Criteria
Subject will be excluded from participation if any of the following apply.
1.
Patient is known to have a positive test for latent tuberculosis (TB) and has not 
previously received adequate anti-microbial therapy or would require TB prophylaxis 
after transplant.
2.
Uncontrolled concomitant infection or any unstable medical condition that could 
interfere with study objectives.
3.
Significant liver disease, defined as having, during the past 28 days, consistently 
elevated AST (SGOT) and/or ALT (SPGT) levels greater than 3 times the upper value of 
the normal range of the investigational site.
4.
Patient currently taking or maintained on another form of extended-release tacrolimus 
following his/her transplant procedure.
5.
Patient who will be maintained on a non-tacrolimus-based maintenance 
immunosuppressive regimen following his/her transplant procedure. 
6.
Patient currently taking, having taken within 30 days, or who will be maintained on an 
mTOR inhibitor following his/her transplant procedure.
7.
Use of an investigational study drug in the 30 days prior to the transplant procedure.
8.
Contraindication or hypersensitivity to drugs or any of their components that constitute 
the immunosuppression regimen.
9.
6 Ag match or zero mismatch at major MHC (class I or class II).
10. Receipt of an ABO-incompatible organ. Note: A2 donor to O recipient or A2 donor to B 
recipient is considered ABO-compatible and not excluded by this criterion.
11. Removed
12. Removed
13. The presence of current or historic, pre-formed anti-HLA DSA against the current donor
(evidence of pre-formed, non-donor HLA is not exclusionary) as defined by a subject meeting 
any of the following criteria*:
a. Positive virtual crossmatch, 
b. Positive T- or B-cell crossmatch by NIH antiglobulin lymphocytotoxicity
method,**
c. Positive T- or B-cell flow cytometry crossmatch defined by the MFC criteria used 
by the center’s HLA lab for their local proficiency testing,**
d. An MFI greater than or approaching 1000 using flow cytometry/Luminex-based, 
specific anti-HLA antibody testing.
*Patients are eligible to enroll with a negative virtual crossmatch if used in lieu of a 
physical crossmatch, if, in the opinion of the patient’s attending physician, use of such 
is required to obviate the accrual of excessive ischemia time.  However, continued 


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participation is predicated on the performance of the physical crossmatch within 
48 hours of transplant. If the physical crossmatch is positive, the subject will be 
discontinued.
** If b or c above are positive secondary to a suspected positive auto-crossmatch, that 
is not exclusionary as long as a and b above are not met. 
14. Receipt of desensitization, antibody-removal, anti-B-cell, or anti-plasma cell therapy in 
the 90 days preceding the transplant procedure. 
15. Planned initiation (prior to transplant) of desensitization, antibody-removal, anti-B-cell, 
or anti-plasma cell therapy within 7 days of the transplant procedure.
16. Donor or recipient with known hepatitis C infection (HCV antibody positive), HIV 
infection (HIV antibody positive), acute hepatitis B infection (HBsAg positive, anti-HBc 
positive, IgM anti-HBc positive, anti-HBs negative) chronic hepatitis B infection 
(HBsAg positive, anti-HBC positive, IgM anti-HBC negative, anti-HBs negative), or 
equivocal hepatitis B status (HBsAg negative, anti-HBc positive, anti-HBs negative). 
Patients (donor or recipient) who have normal liver function tests (LFT) and who are 
either hepatitis C positive with a negative viral load or have natural or vaccine-acquired 
immunity from hepatitis B are not excluded by this criterion.
17. Primary focal segmental glomerulosclerosis.
18. Subject has a current malignancy or history of malignancy (within the past 5 years), 
except non-metastatic basal or squamous cell carcinoma of the skin or carcinoma-in-situ 
of the cervix that has been successfully treated.
19. Recipient of multi-organ or dual kidney transplants (inclusive of current transplant and 
any prior non-renal transplants).  Note: Patients with prior kidney transplants are 
eligible.
20. Recipient of an en bloc, pediatric deceased donor kidney from a donor less than 5 years 
of age OR weighing less than 20 kg.
21. Prior graft loss secondary to CMV or BK nephropathy.  
22. Prior history of invasive organ disease in the presence of CMV or BKV or clinically 
significant CMV viremia.
23. History of clinically significant (per investigator’s discretion) BK viruria.
24. Any condition which, in the investigator’s opinion, makes the subject unsuitable for 
study participation.
25. Planned complete steroid avoidance. (Steroid initiation and subsequent taper / 
withdrawal will allowed and will be under the purview of the treating physician.)
26. Planned receipt of post-transplant, prophylactic HCV treatment. 
Waivers to the exclusion criteria will NOT be allowed.


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4
TREATMENT(S)
4.1
Identification of Investigational Product(s)
4.1.1
Test Drug(s)
Astellas will provide the following for patients randomized to the Astagraf XL treatment arm 
for the study duration of up to 24 months:
Astagraf XL 0.5 mg
Oblong capsule with a light yellow cap and orange body.  Capsule is branded with red “647” 
on capsule body and “0.5 mg” on capsule cap.  The capsule is supplied in 30-count short, 
square bottles with brown caps.
Astagraf XL 1 mg
Oblong capsule with a white cap and orange body.  Capsule is branded with red “677” on 
capsule body and “1 mg” on capsule cap.  The capsule is supplied in 30-count short, square 
bottles with blue caps.
Astagraf XL 5 mg
Oblong capsule with a grayish-red cap and orange body.  Capsule is branded with red “687” 
on capsule body and “5 mg” on capsule cap. The capsule is supplied in 30-count short, square 
bottles with orange caps.
IF medically indicated, subjects randomized to the Astagraf XL treatment arm who are 
unable to tolerate oral administration of Astagraf XL, may be administered tacrolimus via IV 
solution (Prograf solution for intravenous use), administered as a 24-hour continuous 
infusion. Administration of IV tacrolimus should be discontinued as soon as the subject can 
tolerate oral administration, and may not exceed a period of seven consecutive days. 
Astagraf XL cannot be given intravenously. Astagraf XL should not be given via NGT 
due to its extended release characteristics. IV tacrolimus will not be provided by the 
Sponsor. 
4.1.2
Comparative Drug(s) 
Tacrolimus, immediate-release, oral; 0.5 mg, 1 mg, 5 mg capsules to be obtained by the 
transplanting center during a participant’s inpatient stay, and by the patients themselves, 
thereafter, at retail pharmacies via a study-provided voucher for the study duration of up to 
24 months.
IF medically indicated, subjects randomized to the immediate release tacrolimus treatment 
arm who are unable to tolerate oral administration may be administered tacrolimus via either 
IV solution, (Prograf solution for intravenous use) administered as a 24-hour continuous 
infusion, or via NGT for no more than seven consecutive days. Dosing via these routes 
should be discontinued as soon as the subject can tolerate oral administration, and may not 
exceed a period of seven consecutive days. Additionally, a subject may not receive more than 


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seven days of combined NGT and IV administration in cases where a subject is switched 
from one therapy to another. Intravenous tacrolimus will not be provided by the Sponsor.
4.2
Packaging and Labeling
Astagraf XL will be prepared, packaged, and labeled under the responsibility of qualified 
staff at AUST in accordance with APGD-AUST Standard Operating Procedures (SOPs), 
Good Manufacturing Practice (GMP) guidelines, ICH GCP guidelines, and applicable local 
laws/regulations.
Each bottle will bear a label conforming to regulatory guidelines, Good Manufacturing 
Practice and local laws and regulations which identifies the contents as investigational drug.
Tacrolimus, immediate-release will be obtained by the transplanting center during a 
participant’s inpatient stay and by the patients, themselves, thereafter, at retail pharmacies via 
a study-provided voucher.
Astagraf XL should be stored at 25°C (77°F); excursions permitted to 15°C-30°C (59°F to 
86°F) [see United States Pharmacopeia (USP) Controlled Room Temperature].
Tacrolimus, immediate-release should be stored according to the product label.
4.3
Study Drug Handling 
Current ICH GCP Guidelines require the investigator to ensure that study drug deliveries 
from the Sponsor are received by the investigator/or designee and
●
that such deliveries are recorded,
●
that study drug is handled and stored according to labeled storage conditions,
●
that study drug with appropriate expiry/retest and is only dispensed to study subjects in 
accordance with the protocol, and
●
that any unused study drug is returned to the Sponsor.
Drug inventory and accountability records for the study drugs will be kept by the 
investigator/or designee. Study drug accountability throughout the study must be documented 
and reconciled. The following guidelines are therefore pertinent:
●
The investigator agrees not to supply study drugs/vouchers to any persons except the 
eligible subjects in this study in accordance with the protocol.
●
The investigator or designee will keep the study drugs in a pharmacy or other locked and
secure storage facility under controlled storage conditions, accessible only to those 
authorized by the investigator to dispense these test drugs.
●
A study drug inventory will be maintained by the investigator or designee. The inventory 
will include details of material received and a clear record of when they were dispensed 
and to which subject.
●
At the conclusion or termination of this study, the investigator or designee agrees to 
conduct a final drug supply inventory and to record the results of this inventory on the 
Drug Accountability Record. It must be possible to reconcile delivery records with those 
of used and/or returned medication. Any discrepancies must be accounted for and 


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documented. Appropriate forms of deliveries and returns must be signed by the site staff 
delegated this responsibility.
●
The site must return study drug to the Sponsor or designee at the end of the study (or, 
with prior Sponsor approval, after interim reconciliation by the monitor) or upon 
expiration.
It is prohibited to remotely dispense study Astagraf XL (i.e., by mail or courier) to subjects 
unless there are exceptional circumstances and prior approval from the Sponsor is received in 
writing. 
4.4
Blinding
This section is not applicable as this is an open-label study.
4.5
Assignment and Allocation
Randomization will be performed via Interactive Response Technology (IRT).  Prior to the 
initiation of the study treatment, the site staff will contact the IRT in order to determine the 
randomly assigned treatment.  Specific procedures for randomization through the IRT are 
contained in the study procedures manual.
●
The randomization allocation will consist of a 1:1 ratio for twice daily, immediate-
release tacrolimus or Astagraf XL.  The randomization will be stratified according to the 
following three factors:  Use of alemtuzumab (Campath) (yes/no)
●
KDPI [3 levels: N/A (living donors) vs. ≤ 50 vs. > 50]
●
HLA Class II mismatch (yes/no)
Additionally, enrollment of Campath users will be capped to not exceed 20% of the study 
population.
5
TREATMENTS AND EVALUATION
5.1
Dosing and Administration of Study Drug(s) and Other 
Medication(s)
5.1.1
Dose/Dose Regimen and Administration Period
Study participants will receive either Astagraf XL (single daily dose) or SOC, twice daily, 
immediate-release tacrolimus for up to 730 consecutive days (2 years) following their 
transplant procedure. Investigators are encouraged to start subjects on the randomized study 
treatment (immediate release tacrolimus or Astagraf XL) within 48 hours of transplantation. 
If medically indicated, therapy initiation with either tacrolimus agent can be delayed for up to 
7 days. Administration of the randomized study treatment prior to the transplant is not 
allowed for the purposes of the current study.  
When possible, patients should receive their daily dose of Astagraf XL using the fewest 
number of pills possible (i.e., a patient on 15 mg of Astagraf XL daily should ideally receive 
three 5 mg tablets). Because of its extended-release pharmacokinetics, it is critical that 
Astagraf XL be initiated at a dose of 0.15/mg/kg/day as a single daily dose.  Failure to 


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dose patients in this manner will result in a protocol violation.  Dry weight (or estimated 
dry weight, if dry weight is not available) should be used for the initial dosing. There is no 
dosing adjustment for subjects with gastric sleeves. After the initial dose, Astagraf XL dosing
should be titrated according to the clinical judgment of the attending physician and 
appropriate TDM of tacrolimus trough concentrations, typically occurring 48 hours after a 
dose change.  This dosing should account for the fact that when identical trough levels are 
targeted, systemic exposure to tacrolimus is the same for both Prograf and Astagraf XL.
Patients receiving immediate-release tacrolimus should be initiated on therapy per center 
protocol.
For the purposes of this study, patients receiving Astagraf XL or immediate-release 
tacrolimus should maintain a minimal tacrolimus trough concentration of 6 ng/mL at all 
times during the study.  The failure to do so for four out of 6 weeks (continuous) during the 
first 6 weeks after transplantation will result in a subject level discontinuation.  Patients 
requiring a reduction in tacrolimus levels in either cohort for treatment of infection,
malignant disease, or other complication for four out of the first 6 weeks after transplantation 
will also result in a subject level discontinuation.  Patients whose tacrolimus is withdrawn or 
who have a prolonged significant reduction of tacrolimus levels will be discontinued from the 
study (see Section 6.1). 
Patients meeting inclusion criteria at the time of randomization and not initiated on Astagraf 
XL or immediate-release tacrolimus within 7 days of transplant will be discontinued.
IF medically indicated, subjects in either treatment arm who are unable to tolerate oral 
administration of their study drug may be administered tacrolimus via an alternative route. 
Subjects randomized to the Astagraf XL treatment arm may be administered IV tacrolimus.
Note, however, that Astagraf XL cannot be given intravenously. Likewise, Astagraf XL 
should not be given via NGT due to its extended release characteristics. Subjects randomized 
to the immediate release tacrolimus arm may be administered tacrolimus via IV or NGT. 
Dosing via alternative routes should be discontinued as soon as the subject can tolerate oral 
administration, and may not be done for more than seven consecutive days. Additionally, for 
subjects in the immediate release tacrolimus arm, a subject may not receive more than seven 
consecutive days of combined NGT and IV administration in cases where a subject is 
switched from one therapy to another.  
Intravenous tacrolimus will not be provided by the Sponsor. 
Instructions for NGT Administration of Immediate Release Tacrolimus
NGT administration of immediate release tacrolimus is only allowed for subjects randomized 
to the immediate release tacrolimus treatment arm. If a decision is made to administer 
immediate release tacrolimus via NGT, the capsules may be opened and the contents mixed 
with 50 ml of water to form a suspension. The suspension should be drawn into a syringe and 
administered via the NGT. The container should then be refilled with another 50 ml of water, 
which should be drawn into the same syringe to flush the NGT tube and the container.   After 
administration, the NGT may taken off suction in line with hospital practice. Clinical studies 


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have recommended removing the NGT from suction for 45 to 60 minutes after medication 
administration [Marubashi et al, 2012; PMR-EC-1106].
Appropriate safety precautions should be taken when preparing a suspension of immediate 
release tacrolimus. 
Healthcare professionals should bear in mind that tacrolimus in any form is not compatible 
with PVC (polyvinylchloride). Tubing, syringes and other equipment used to prepare a 
suspension of immediate release tacrolimus capsule contents should not contain PVC.
5.1.2
Increase or Reduction in Dose of the Study Drug(s)
After initial dosing, subsequent dosing in patients receiving either Astagraf XL or 
immediate-release tacrolimus should be predicated on clinical experience, patient tolerability, 
and monitoring of whole blood tacrolimus drug concentrations, the measurement of which, 
along with target tacrolimus levels, should be guided by individual center protocol and 
currently approved US Package Insert.  For the ASTOUND study, the lower limit of 
acceptable tacrolimus trough concentrations in both the Astagraf XL and immediate-release
tacrolimus treatment arms is 6 ng/mL for the duration of the study.  If a prolonged period of 
reduced immunosuppression will be required to treat infection, sepsis, delayed wound 
healing, or other circumstances, patients should be removed from the study as per the 
discontinuation criteria (Section 6.1).
5.1.3
Previous and Concomitant Treatment (Medication and Non-Medication 
Therapy)
Previous medication (all medications taken up to 30 days prior to transplant) and concomitant 
medication will be entered on the eCRF.  All patients will receive induction immunotherapy 
(either T-cell depleting agent, IL-2 co-stimulation blocker, or anti-CD52 monoclonal 
antibody – steroid only induction therapy is not allowed).  The dose and frequency of the 
chosen induction agent will be determined by the patient’s treating physician and 
administered in conjunction with the participating transplant center’s de novo kidney 
immunosuppression protocol.  Anti-CMV, fungal, bacterial, and pneumocystis prophylaxis 
will be determined per local standard of care.  As a participant in ASTOUND, in addition to 
tacrolimus, study subjects will need to concomitantly receive MMF (or Myfortic equivalent) 
and, if given per institutional protocol, corticosteroids (dose and duration of therapy of both 
medications to be determined according to clinical judgment of the patient’s attending 
physician) as their maintenance immunosuppressive regimen.
5.1.4
Treatment Compliance
Study subjects should be counseled on the need to exhibit 100% compliance with their 
medication regimen in a manner that reflects a center’s own standard of care.  As this study is 
designed to assess the real-world impact of either once-daily Astagraf XL or a twice daily 
tacrolimus regimen, investigators or their designee should not change how they counsel 
patients on compliance based on their patient’s randomization status or participation in 
ASTOUND.


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Astagraf XL will be monitored by the accounting of unused medication returned by the 
subject at quarterly visits.  Patients receiving immediate-release tacrolimus will be monitored 
by a third party vendor (via voucher system).  Manufacturer, dose, number of pills dispensed, 
and the date of prescription refills will be captured using a voucher system with a built-in 
reporting mechanism.
5.1.5
Restrictions During the Study 
The following are prohibited medications during ASTOUND (see Appendix 12.1):
1) Extended-release formulations of tacrolimus, other than Astagraf XL 
2) Non-tacrolimus based immunosuppressive regimens (i.e., cyclosporine, everolimus, 
sirolimus, belatacept)
3) Antiviral medications used in HCV treatment
4) Antiviral medications used in HIV treatment
5) Isoniazid
6) Rifampin
7) Ethambutol
8) Pyrazinamide
5.2
Demographics and Baseline Characteristics
5.2.1
Demographics
The following demographic information will be collected during screening: date of birth, sex, 
race, and ethnicity.
5.2.2
Medical History
Site personnel will collect and record information regarding the subject’s medical history (if 
available) at screening and any updated information will be recorded on study day 0.  
Medical history will include: subject etiology of renal failure, viral serology (e.g., HBV, 
HCV, CMV, EBV), duration and severity of renal disease, and diabetes history (as 
applicable) at enrollment.
5.2.3
Transplant Information
The following transplant information will be obtained at the Day 0 visit: type of transplant 
(living related, living non-related, DD, and whether organ was obtained in the setting of 
DCD), number of prior transplants and reason for prior graft loss (if applicable), total cold 
and warm ischemia time in hours and minutes, KDPI, ABO blood typing, HLA typing of 
donor and recipient (obtained via de-identified copies of the donor and recipient HLA typing 
reports, submitted to the Central HLA laboratory), degree of HLA mismatch between donor 
and recipient, and most recent panel reactive antibody testing (most recent cPRA level).  
Donor viral serology information (HBV, HCV, CMV and EBV), if available, will be 
collected and recorded.  Additional donor information will include age, sex, height, weight, 


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donor cause of death, ethnicity of the donor, ex vivo perfusion parameters, ABO typing, and 
results of any pre-implantation biopsies of the donor kidney. De-identified copies of recent 
local pre-transplant DSA testing results (if performed) will b also be obtained. 
5.2.4
Height and Body Weight
Height and dry weight will be recorded at screening. Dry weight will also be collected 
pre-operatively on study day 0.
5.2.5
Diagnosis of the Target Disease, Severity, and Duration of Disease
Medical history, including etiology of renal failure, will be collected during screening.
5.3
Efficacy Assessment
5.3.1
Efficacy
5.3.2
Donor Specific Antibody
Class I and Class II DSA will be assessed by a central HLA lab at 1, 3, 6, 9, 12, and 
24 months in all patients, and as needed at baseline to confirm local testing results.  
Otherwise, initial DSA screening prior to transplant will be performed locally as per standard 
of care with results reported on the eCRF.  For the purpose of assessing the impact of the 
proposed intervention (Astagraf XL vs. twice daily, immediate-release tacrolimus), detection 
of DSA at any time during the course of the study will be treated as a positive result.  For the 
purposes of the study, DSA positivity will be defined at an MFI threshold approaching 1000.  
An independent adjudication board will take into account relevant clinical information to 
qualify samples in borderline cases.  In cases where DSA is quantifiable, but nevertheless at 
an MFI < 1000, patient serum will be diluted and re-measured to mitigate the possible impact 
of the prozone phenomenon [Tambur et al, 2015].  Samples with new MFI levels above the 
target threshold will subsequently be regarded as positive.  
In each case of quantifiable DSA or C1q-binding DSA at or above the MFI threshold, patient 
sera may be diluted 1:16 and re-assessed [Tambur et al, 2015].  MFI readings remaining 
above 10,000 following dilution or those increasing to over 10,000 following dilution (thus 
indicating prozone activity) will be regarded as “strong.”  Samples with MFI values from 
1000 to 10,000 will be designated “moderate.”  Serum samples that demonstrate
disappearance of MFI or reduction to < 1000 following dilution will be regarded as “weak.”
All borderline MFI threshold results will be reviewed by the Adjudication Board (see 
Section 10.2) in conjunction with relevant clinical information to confirm that the results 
have been categorized correctly. The Adjudication Board will also 1) review and adjudicate 
all cases antibody formation (per central HLA lab assessment) post-transplantation and 2) 
determine the need for ancillary testing of pre-transplant sera in equivocal cases in which 
such testing could indicate the presence of pre-transplant DSA.


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5.3.3
Antibody Persistence 
DSA and C1q-binding DSA will be regarded as persistent under the following conditions: 
1) DSA is detected and remains above the threshold for positivity (MFI = 1000) for two 
consecutive or non-consecutive measurements, or 2) the new appearance of a DSA at the 
threshold for positivity when preceded by a DSA of a different specificity that has 
subsequently become non-detectable.
5.3.4
C1q-binding DSA
C1q-binding DSA will be assessed in all patients who are DSA positive.  Results will be 
reported as positive or negative.  MFI values and DSA strength will also be recorded.
5.3.5
IgG3 Isotyping
IgG3 isotyping will be performed in patients who are DSA positive.  Results will be reported 
as positive or negative.
5.3.6
Requirement for Antibody Removal
The number of plasmapheresis sessions will be tracked in patients who develop DSA.  The 
need for IV corticosteroids, IVIG (grams), thymoglobulin (mg), eculizumab, and bortezomib 
will be recorded per patient.
5.3.7
Histopathology
For all participants who undergo at least one kidney biopsy during the first year post-
transplant (either for cause or per institutional protocol), the kidney biopsy performed closest 
to the 12 month visit (but no later than 14 months post-transplant) will be submitted 
forcentral histopathology grading. During the central histopathology grading, published 
criteria will be used to diagnose the following: 1) antibody mediated changes, 2) acute 
cellular and ABMR, 3) borderline changes (suspicious acute TCMR), and 4) IFTA.  The 
diagnosis of acute and chronic forms of ABMR will be based on C4d positivity with prior or 
current evidence of DSA during the study or evidence of chronic tissue injury, such as 
glomerular double contours, and/or peritubular capillary basement membrane multilayering 
and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries, also 
in-line with published criteria [Solez et al, 2008].  Biopsies will be regarded as suspicious for 
ABMR if C4d is not demonstrated in the biopsy, but morphologic evidence of tissue injury is 
nonetheless present, coincident with, or following DSA development [Solez et al, 2008].  
Specimens will be similarly scored for g, t, v, i, cg, ct, ci, cv, ah, ptc, and mm in the manner 
previously described [Racusen et al, 1999].  Specimens will be regarded as positive for a 
particular feature if they receive a score > 0 for that particular characteristic. TG will be 
defined, for the purposes of this study, as cg > 0.
For specimens that are read locally, the above information will be extracted from the official 
biopsy report for inclusion in the eCRF.


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5.3.8
Molecular Endpoints 
Validated molecular signatures will be used to classify patients dichotomously at each blood 
sampling (i.e., 1, 3, 6, 9, 12, and 24 months following transplantation) as “IA” or Immune 
Quiescence (IQ). It is assumed that other causes for the IA signature other than tacrolimus 
exposure will be equally and randomly distributed between the 2 groups. The frequency of 
IA and IQ signatures will be compared between cohorts receiving Astagraf XL and 
immediate-release tacrolimus. The use of clinical and biopsy-proven samples from over 
500 patients at 7 clinical centers in the United States and one from Brazil has enabled 
creation of locked Transplant Genomics Inc. (TGI) classifiers based on core data sets for 
each possible molecular phenotype. Thus, each new patient in the ASTOUND study will be 
treated as an unknown and run against each of the core data sets for classification using a 
TGI automated workflow based on multiple R Bioconductor tools and enabling scripts.
For the purposes of the study, assessment and comparison of the incidence of the molecular 
endpoint will rely on a binary molecular designation in accordance with the TruGraf v2.0 
molecular assay.  A negative designation (Trugraf TX Normal) will be referred to as Immune 
Quiescence (IQ).  Due to operating characteristics of the assay, a positive designation will be 
considered evidence of Immune Activation (IA) in all patients.
The above-mentioned classifier has already been “locked” during the discovery process to 
avoid over-training a classifier on new data. As a dichotomous variable, ‘IQ’ will represent 
any patient sample classified as not positive. In turn, ‘IA’ will represent any patient sample 
not classified as ’IQ’. Biopsies, when available, will be used to inform the molecular 
diagnosis when alternative pathology is recognized (i.e. recurrent disease).
5.3.9
Estimated Glomerular Filtration Rate
Estimated glomerular filtration rate (eGFR) will be calculated at day 0 and months 1, 3, 6, 9, 
12, and 24 (where available) using the Modification of Diet in Renal Disease (4 variable –
MDRD) criteria.
5.3.10
Patient Survival
Patient survival is any subject that is known to be alive at the study conclusion.
5.3.11
Graft Survival
Graft survival is defined as any subject that does not fit the following definition of graft loss: 
subject death, retransplantation, transplant nephrectomy, or return to dialysis for a period of 
≥6 weeks by study end.
5.3.12
Acute Rejection
For study purposes, diagnoses of rejection require biopsy confirmation.  Both acute TCMR 
and ABMR (acute and chronic) will be assessed and graded according to published criteria 
[Solez et al, 2008].


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5.4
Safety Assessment
5.4.1
Vital Signs
Vital signs will not be collected for this study. 
5.4.2
Adverse Events
Adverse event collection will begin once informed consent/assent has been signed and 
continue throughout the subject’s participation in the study. The kidney transplant surgery at 
Day 0 is not considered an AE or SAE. See Section 5.5 Adverse Events and Other Safety 
Aspects for information regarding adverse event collection and data handling. 
5.4.2.1 Adverse Events of Possible Hepatic Origin
See Appendix 12.2 Liver Safety Monitoring and Assessment for detailed information on 
liver abnormalities, monitoring and assessment, if the AE for a subject enrolled in a study 
and receiving study drug is accompanied by increases in liver function testing (LFT, e.g.: 
AST, ALT, bilirubin, etc.) or is suspected to be due to hepatic dysfunction.
Subjects with AEs of hepatic origin accompanied by LFT abnormalities should be carefully 
monitored. 
5.4.3
Laboratory and Pathological Assessments
A subset of the routine laboratory assessments obtained per SOC, including creatinine,
hemoglobin, hematocrit, platelet count, white blood cell count, serum sodium, potassium, 
blood urea nitrogen, glucose, urinalysis (including urinary protein), tacrolimus trough 
concentrations, and HgA1c will be recorded on the eCRF.  Of these, excepting creatinine and 
tacrolimus trough concentrations, only the assessments done closest to the targeted dates of 
visits will be recorded on the eCRF. 
For creatinine assessments, the measurement done closest to the targeted visit date (obtained 
per SOC) as well as an additional assessment done at least one month prior to that assessment 
(if obtained per SOC) will be recorded on the eCRF. 
For tacrolimus trough concentrations, only the assessments (as per SOC) done closest to the 
targeted visit dates will be recorded for the visits occurring at baseline, Day 30, Day 455, 
Day 545, Day 635, and day 730. For the visits occurring at Day 90, Day 180, Day 270, and 
Day 365, all available outpatient tacrolimus concentration assessments done (per SOC) and 
available in the centralized medical records since the previous study visit should be recorded. 
All kidney transplant biopsy results obtained during the study will also be recorded on the 
eCRF. 
For subjects who develop clinically significant BK viremia (as assessed per standard of care) 
during study participation, the peak viremia level obtained per standard of care will be 
retrospectively recorded in the eCRF at the time of the subject’s study discontinuation or 
completion. 


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Additionally, blood will be collected at the baseline visit, and the study visits at months 1, 3, 
6, 9, 12, and 24 for the following central laboratory assessments:
●
DSA and determination of MFI and antibody strength, IgG3 isotyping, and C1q-binding
DSA capability
●
Molecular diagnostics (not collected at baseline)
●
Long term storage for potential future analyses (optional; not collected at baseline)
Please refer to Sections 5.7 and 5.8 for more information regarding sample collection.
5.4.4
Physical Examination
A complete physical exam consisting of an examination of general appearance, eyes, nose, 
throat, neck (including thyroid), lymph nodes, chest lungs, cardiovascular, abdominal, skin, 
extremities, musculoskeletal, and neurological system including mental status will be 
conducted at screening.  Any abnormal findings must be assessed and documented as not 
clinically significant if a subject is to be enrolled in the study.  The investigator or qualified 
designee will conduct the exam, determine findings, and assess any abnormalities as to 
clinical significance.
5.5
Adverse Events and Other Safety Aspects
5.5.1
Definition of Adverse Events (AEs)
An AE is defined as any untoward medical occurrence in a subject administered a study drug 
or who has undergone a study procedure which does not necessarily have a causal 
relationship with this treatment.  An AE can therefore be any unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the medicinal (investigational) 
product. 
An abnormality identified during a medical test (e.g., laboratory parameter, vital sign, ECG 
data, physical exam) should be defined as an AE only if the abnormality meets one of the 
following criteria:
●
Induces clinical signs or symptoms
●
Requires active intervention
●
Requires interruption or discontinuation of study medication
●
The abnormality or investigational value is clinically significant in the opinion of the 
investigator.
5.5.2
Definition of Serious Adverse Events (SAEs)
An adverse event is considered “serious” if, in the view of either the investigator or Sponsor, 
it results in any of the following outcomes:
●
Results in death
●
Is life threatening (an adverse event is considered “life-threatening” if, in the view of 
either the investigator or Sponsor, its occurrence places the subject at immediate risk of 


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death. It does not include an adverse event that, had it occurred in a more severe form, 
might have caused death)
●
Results in persistent or significant disability/incapacity or substantial disruption of the 
ability to conduct normal life functions 
●
Results in congenital anomaly, or birth defect
●
Requires inpatient hospitalization or leads to prolongation of hospitalization 
(hospitalization for treatment/observation/examination caused by AE is to be considered 
as serious) 
●
Other medically important events
Medical and scientific judgment should be exercised in deciding whether expedited reporting 
is appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization, but may jeopardize the 
subject or may require intervention to prevent one of the other outcomes listed in the 
definition above. These events, including those that may result in disability/incapacity, 
should also usually be considered serious. Examples of such events are intensive treatment in 
an emergency room or at home for allergic bronchospasm; blood dyscrasias or convulsions 
that do not result in hospitalization; or development of drug dependency or drug abuse.
Special situations on the medicinal products administered to the subject as part of the study 
(e.g., study drug, comparator, background therapy) that may require expedited reporting 
and/or safety evaluation include, but are not limited to:
●
Overdose of the medicinal product(s)
●
Suspected abuse/misuse of the medicinal product(s)
●
Drug exposure during pregnancy or via breast milk
●
Medication error involving the medicinal product(s) (with or without subject/patient 
exposure to the Sponsor medicinal product, e.g., name confusion)
All of the special situations noted above should be recorded on the eCRF. Any special 
situations that also meets the criteria for an SAE should be recorded on the AE page of the 
eCRF and marked ‘serious’ and the SAE worksheet. 
The Sponsor has a list of events that they classify as “always serious” events.  If an adverse 
event is reported that is considered to be an event per this classification as “always serious”, 
additional information on the event may be requested.
5.5.3
Criteria for Causal Relationship to the Study Drug
Adverse events that fall under either "Possible" or "Probable" should be defined as "adverse 
events whose relationship to the study drugs could not be ruled out".
Causal relationship to 
the study drug
Criteria for causal relationship
Not Related
A clinical event, including laboratory test abnormality, with a temporal 
relationship to drug administration which makes a causal relationship 
improbable, and/or in which other drugs, chemicals, or underlying disease 
provide plausible explanations.


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Causal relationship to 
the study drug
Criteria for causal relationship
Possible
A clinical event, including laboratory test abnormality, with a reasonable 
time sequence to administration of the drug, but which could also be 
explained by concurrent disease or other drugs or chemicals. Information 
on drug withdrawal may be lacking or unclear.
Probable
A clinical event, including laboratory test abnormality, with a reasonable 
time sequence to administration of the drug, unlikely to be attributed to 
concurrent disease or other drugs or chemicals, and which follows a 
clinically reasonable response on re- administration (rechallenge) or 
withdrawal (dechallenge).
5.5.4
Criteria for Defining the Severity of an Adverse Event
The investigator will use the following definitions to rate the severity of each adverse event
●
Mild:
No disruption of normal daily activities (Asymptomatic, or mild symptoms, 
clinical or diagnostic observations noted; intervention not indicated.)
●
Moderate:Affect normal daily activities (Local or noninvasive intervention indicated.)
●
Severe:
Inability to perform daily activities (Medically significant but not immediately 
life threatening, hospitalization or prolonged hospitalization.)
5.5.5
Reporting of Serious Adverse Events (SAEs)
In the case of a serious adverse event (SAE), the investigator must contact the Sponsor by 
telephone or fax immediately (within 24 hours of awareness).
The investigator should complete and submit an SAE Worksheet containing all information 
that is required by the Regulatory Authorities to the Sponsor by fax immediately (within 
24 hours of awareness). If the faxing of an SAE Worksheet is not possible or is not possible 
within 24 hours, the local drug safety contact should be informed by phone.
For contact details, see Section II Contact Details of Key Sponsor's Personnel. Please fax the 
SAE Worksheet to:
Astellas Global 
Pharmacovigilance 
Email: Safety-US@astellas.com
North America telefax numbers: 888-396-3750 
(alternate 847-317-1241)
If there are any questions, or if clarification is needed regarding the SAE, please contact the 
Sponsor's Medical Monitor/Expert or his/her designee (see Section II Contact Details of Key 
Sponsor’s Personnel). 
Follow-up information for the event should be sent promptly (within 7 days of the initial 
notification.
Full details of the SAE should be recorded on the medical records and on the eCRF.


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The following minimum information is required:
●
ISN/Study number, 
●
Subject number, sex, and age,
●
The date of report,
●
A description of the SAE (event, seriousness of the event), and
●
Causal relationship to the study drug.
The Sponsor or Sponsor's designee will submit expedited safety reports (i.e., IND Safety 
Reports) to the regulatory agencies (i.e., FDA) as necessary, and will inform the investigators 
of such regulatory reports. Investigators must submit safety reports as required by their 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC) within timelines set 
by regional regulations (i.e., EU, (e)CTD, FDA).  Documentation of the submission to and 
receipt by the IRB/IEC of expedited safety reports should be retained by the site.
The Sponsor will notify all investigators responsible for ongoing clinical studies with the 
study drug of all SAEs which require submission per local requirements IRB/IEC/ head of 
the study site.
The investigators should provide written documentation of IRB/IEC notification for each 
report to the Sponsor.
You may contact the Sponsor's Medical Monitor/Expert for any other problem related to the 
safety, welfare, or rights of the subject.
All AEs occurring after informed consent/assent is signed should be collected on the eCRF. 
The transplant operation on Day 0 is not considered an AE or SAE. 
All new SAEs occurring up to 7 days after the end of study visit must be reported. SAEs 
starting after the subject’s end of study visit but within 7 days shall be recorded via the SAE 
form only. After the 7 day period, Adverse Drug Reactions (non-serious and serious AEs 
related to systemic tacrolimus) will be captured through spontaneous reporting (please see 
Packet Insert for reporting information). 
5.5.6
Follow-up of Adverse Events
All AEs occurring during the study are to be followed up until resolved or judged to be no 
longer clinically significant, or until they become chronic to the extent that they can be fully 
characterized. If during AE follow-up, the adverse event progresses to an "SAE", or if a 
subject experiences a new SAE, the investigator must immediately report the information to 
the Sponsor. Please refer to Appendix 12.2 Liver Safety Monitoring and Assessment for 
detailed instructions on Drug Induced Liver Injury (DILI).
5.5.7
Monitoring of Common Serious Adverse Events
Common serious adverse events are SAEs commonly anticipated to occur in the study 
population independent of drug exposure. SAEs classified as “common” are provided in 
Appendix 12.3 Common Serious Adverse Events for your reference. The list does NOT 
change your reporting obligations or prevent the need to report an AE meeting the definition 


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of an SAE as detailed above. The purpose of this list is to alert you that some events reported 
as SAEs may not require expedited reporting to the FDA based on the classification of 
“common serious adverse events” as specified in Appendix 12.3 Common Serious Adverse 
Events. The Sponsor will monitor these events throughout the course of the study for any 
change in frequency. Any changes to this list will be communicated to the participating 
investigational sites and regulatory authorities as appropriate. Investigators must report 
individual occurrences of these events as stated in Section 5.5.5 Reporting of Serious 
Adverse Events. 
5.5.8
Procedure in Case of Pregnancy
If a female subject or partner of a male subject becomes pregnant during the study dosing 
period or within 30 days from the discontinuation of dosing, the investigator should report 
the information to the Sponsor as if it is an SAE. The expected date of delivery or expected 
date of the end of the pregnancy, last menstruation, estimated conception date, pregnancy 
result and neonatal data etc., should be included in this information.
The investigator will follow the medical status of the mother, as well as the fetus, as if the 
pregnancy is an SAE and will report the outcome to the Sponsor.
When the outcome of the pregnancy falls under the criteria for SAEs [spontaneous abortion, 
induced abortion, stillbirth, death of newborn, congenital anomaly (including anomaly in a 
miscarried fetus)], the investigator should respond in accordance with the report procedure 
for SAEs.  Additional information regarding the outcome of a pregnancy (which is 
categorized as an SAE) is mentioned below.
●
"Spontaneous abortion" includes miscarriage, abortion, and missed abortion
●
Death of an infant within 1 month after birth should be reported as an SAE regardless of 
its relationship with the study drug
●
If an infant dies more than 1 month after the birth, it should be reported if a relationship 
between the death and intrauterine exposure to the study drug is judged as "possible" by 
the investigator
●
In the case of a delivery of a living newborn, the "normality" of the infant is evaluated at 
the birth
●
Unless a congenital anomaly are identified prior to spontaneous abortion or miscarriage, 
the embryo or fetus should be assessed for congenital defects by visual examination
If during the conduct of a clinical trial, a male subject makes his partner pregnant, the subject 
should report the pregnancy to the investigator. The investigator will report the pregnancy to 
the Sponsor as an SAE.
5.5.9
Emergency Procedures and Management of Overdose
Experience with overdosage of tacrolimus is limited. Several cases of accidental overdosage 
have been reported; symptoms have included tremor, headache, nausea and vomiting, 
infections, urticaria, lethargy, increased blood urea nitrogen and elevated serum creatinine 
concentrations, and an increase in alanine aminotransferase levels. No specific antidote to 
systemic tacrolimus therapy is available. If overdosage occurs, general supportive measures 


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and symptomatic treatment should be conducted. Based on its high molecular weight, poor 
aqueous solubility, and extensive erythrocyte and plasma protein binding, it is anticipated 
that systemic tacrolimus will not be dialyzable. In isolated patients with very high plasma 
levels, hemofiltration or diafiltration have been effective in reducing toxic tacrolimus 
concentrations. In cases of oral intoxication, gastric lavage and/or the use of adsorbents (such 
as activated charcoal) may be helpful, if used shortly after intake.
5.5.10
Supply of New Information Affecting the Conduct of the Study
When new information becomes available necessary for conducting the clinical study 
properly, the Sponsor will inform all investigators involved in the clinical study as well as the 
regulatory authorities. Investigators should inform the IRB/IEC of such information when 
needed. 
5.6
Test Drug Concentration
Not applicable.
5.7
Other Measurements, Assessments or Methods
5.7.1
Sample for the Analysis of Validated Gene Expression Profiling in the 
Peripheral Blood
Peripheral blood for the analysis of validated gene expression profiling will be collected at 
the study visits occurring 1, 3, 6, 9, 12, and 24 months following transplantation. Validated 
gene expression profiling analyses will be completed by Transplant Genomics (TGI).   Whole 
blood will be collected at each of the above mentioned visits and shipped for central analysis, 
in accordance with the central laboratory manual. 
Affymetrix Hu133PM Peg Arrays will be used in conjunction with a fully automated 
GeneAtlas instrument, which interrogates > 28,000 well-annotated genes along with 
Expressed Sequence Tags and other sequences representing over 47,000 analytes, such as 
long, non-coding RNAs from RefSeq and Ensembl.  TGI automated bioinformatics 
workflows will be used for analysis of array-based RNA expression profiling in addition to 
multiple other tools including quantile frozen robust multi-array average (RMA) 
normalization, batch detection and correction, outlier detection by Principle Component 
Analysis (PCA), signal filtering by log2 histograms, differential expression by ANOVA 
methods with multiple correction testing, and multi-class prediction using DLDA validated 
by an independent classification tool, Vector Machines.  Mapping to functional biological 
pathways will also be done using multiple tools including Gene Ontology, Ingenuity’s 
Pathway Analysis™ (Ingenuity Systems, Redwood, CA), TGI’s proprietary tool called 
ImmuneMap, WikiPathways, GSEA Broad, Panther, and National Center for Biotechnology 
Information (NCBI) databases such as DAVID.  Visualization of networks and hub genes 
will be done with Cytoscape.  Reports on functional pathway mapping will be prepared and 
provided to Astellas by TGI.


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5.7.2
Blood Sample for DSA, C1q-binding DSA Analysis, and IgG3 DSA Isotyping
Blood samples will be collected and shipped, in accordance with the central laboratory 
manual, for analysis at a central lab at baseline (prior to transplant; this baseline sample will 
be stored centrally for analysis as-needed), and at the study visits occurring 1, 3, 6, 9, 12 and 
24 months post-transplant.  These samples will be screened for anti-HLA class-I and class-II 
antibodies using Flow PRA.  For samples testing positive, the Luminex solid phase assay 
(SPA) will be used to determine antibody specificity, using LabScreen® Single Antigen 
Detection Tests and the C1q Screen® (both One Lambda, Inc.) following the manufacturer’s 
recommendations to obtain neat MFI values for DSA.  C1q MFI will also be determined for 
those samples testing positive for DSA.  Results from the centrally-performed Single Antigen 
Detection Tests on recipient serum will be compared to the donor HLA typing report 
furnished by the participating center to ascertain the presence of donor-specific anti-HLA 
antibody.  De-identified local typing reports will be used to confirm the degree of HLA 
mismatch between donor and recipient. De-identified local recent pre-transplant DSA testing 
results (if performed) will be used to inform interpretation of the central DSA results. 
Samples positive for DSA per study protocol will be tested a second time after diluting the 
serum 1:16 with phosphate buffered saline (PBS) to provide a semi-quantitative measure of 
antibody strength.  Samples in which MFI disappears (becomes < 1000 MFI) will be 
regarded as “weak.”  Samples increasing to > 10,000 MFI upon dilution will be regarded as 
having significant prozone and will be regarded as “strong.” Samples which may increase or 
decrease, but nevertheless remain between 1000 and 10,000 MFI will be regarded as 
“moderate.”  Samples with evidence of fluorescence that are nevertheless below MFI criteria 
for DSA positivity will also be subject to dilution.  Samples increasing to above threshold 
criteria following dilution will regarded as positive and assumed to be under the influence of 
a prozone effect that impairs antibody detection.  For statistical purposes, the antibody with 
the highest MFI level obtained during the course of the study will be used for statistical 
comparisons between groups.  However, the strongest antibody observed at each HLA-locus 
will be reported separately and tracked for patients who are DSA positive.
C1q-binding DSA, MFI, and DSA IgG3 isotyping will be performed in accordance with 
established protocol [Lefaucheur et al, 2015; Honger et al, 2011].
5.7.3
Storage of Donor Tissue for DNA Typing
In order to determine the presence of HLA-DSA with a high degree of accuracy, it is 
expected that in some rare situations, additional typing will be required using donor DNA 
due to the fact that frequently, only partial donor typing is available through the United 
Network of Organ Sharing (UNOS).  This will be especially important in situations where 
antibodies might arise to HLA-DP, a locus that is infrequently typed in donors.  Other 
examples may be situations where a donor is not typed at the level of resolution necessary to 
discern when a recipient has antibodies to some alleles of an antigen group and not others.  
Thus, to accommodate such contingencies, organ procurement organizations (OPOs) and 
HLA labs are required to maintain donor/recipient material by their respective regulatory 
bodies.  In most cases, the relationships maintained by transplant centers with their local 


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OPO or HLA lab affords them access to this material if it is needed for subsequent, additional 
HLA typing.  If this situation should arise in the course of ASTOUND, the participating 
transplant center will be asked to request such material from their OPO or HLA lab for 
additional donor HLA typing, the results of which will also be communicated to a patient’s 
participating transplant physician.  If additional typing is sought for recipients of living donor 
organs, donors will be approached and separately consented, as needed.
5.7.4
Banking of Blood for Future Studies
Patients participating in ASTOUND will be provided, through the informed consent/assent 
process, the option of having an additional sample collected at each phlebotomy session for 
storage and analysis in future trials of transplant outcomes and biomarker discovery.  In the 
event additional investigations are contemplated, patients will be re-consented for subsequent 
use of their biological material.  Samples will be stored for a maximum of 5 years and then 
destroyed thereafter.  Samples will be stored at -80°C at a facility designated by the Sponsor.
5.8
Total Amount of Blood
Prior to transplant, participants will have one 10 mL tube of blood collected.  
At each post-transplant study-designated phlebotomy session, participants will have one 
10 mL tube and two or three 2.5 mL tubes of blood. The third 2.5 mL tube of blood will only 
be collected from patients consenting to additional blood storage.  Seven study-specific 
phlebotomy sessions have been incorporated into ASTOUND and timed to what is likely to 
coincide with a participant’s regularly scheduled therapeutic drug monitoring (TDM).  The 
total amount of additional blood for research purposes to be collected from an individual 
study participant over the course of two years is expected to be between 100 and 115 mL.
6
DISCONTINUATION
6.1
Discontinuation of Individual Subject(s) 
A discontinuation is a subject who enrolled in the study and for whom study treatment is 
permanently discontinued prematurely for any reason.  
The subject is  free to withdraw from the study treatment and/or study for any reason and at 
any time without giving reason for doing so and without penalty or prejudice. The 
investigator is also free to terminate a subject's involvement in the study at any time if the 
subject's clinical condition warrants it.
If a subject is discontinued from the study with an ongoing AE or an unresolved laboratory 
result that is significantly outside of the reference range, the investigator will attempt to 
provide follow-up until the condition stabilizes or no longer is clinically significant.  
However, other than pertinent information regarding the AE (i.e., follow-up laboratory 
values, AE information updates, etc.), no additional clinical information will be collected 
following the discontinuation. 


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Discontinuation Criteria from Study for Individual Subjects:
●
Subject develops allograft loss (as defined by subject death, retransplantation, transplant 
nephrectomy, or return to dialysis of ≥ 6 consecutive weeks duration);
●
Subject develops unacceptable toxicity or is withdrawn at the discretion of the patient’s 
supervising physician;
●
Subject is lost to follow-up despite reasonable efforts by the investigator to locate the 
subject;
●
Subject withdraws consent for further treatment or expires during the course of the study;
●
Subject begins taking any prohibited medications (see Section 5.1.5);
●
Subject undergoes a second organ transplant;
●
Conversion to a non-tacrolimus-based maintenance regimen in either study arm if 
required to manage toxicities;
●
Removal from an MMF-containing immusosuppressive regimen (or its equivalent);
●
Failure to achieve a tacrolimus whole blood concentration ≥ 6 ng/mL for 4 consecutive 
weeks during the first 6 weeks following transplantation;
●
Permanent withdrawal of tacrolimus;
●
Interruption or prolonged significant reduction of tacrolimus (as an example, a 50% 
reduction or dose at a level of less than 3.5ng/mL) for periods exceeding 4 weeks;
●
Conversion by the investigator to the therapy of the other study treatment arm (i.e., 
subject randomized to Astagraf XL is converted to immediate release tacrolimus OR 
subject randomized to immediate release tacrolimus is converted to Astagraf XL. Cases 
of inadvertent and temporary conversion [i.e., dispensing error] do not meet this 
discontinuation criterion ); 
●
Subject is not initiated on Astagraf XL or immediate-release tacrolimus within 7 days of 
transplant;
●
Gross non-compliance with protocol: The medical monitor or investigator may request 
permanent study discontinuation in the event of a major protocol deviation such as 
administration of prohibited concomitant medication, lack of cooperation, or 
noncompliance.
6.2
Discontinuation of the Site
If an investigator intends to discontinue participation in the study, the investigator must 
immediately inform the Sponsor.
6.3
Discontinuation of the Study
The Sponsor may terminate this study prematurely, either in its entirety or at any study site, 
for reasonable cause provided that written notice is submitted in advance of the intended 
termination. Advance notice is not required if the study is stopped due to safety concerns. If 
the Sponsor terminates the study for safety reasons, the Sponsor will immediately notify the 
investigator and subsequently provide written instructions for study termination.


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7
STATISTICAL METHODOLOGY
The statistical analysis will be coordinated by the responsible Astellas Medical Affairs, 
Americas (MA-A) biostatistician. A Statistical Analysis Plan (SAP) will be written to 
provide details of the analysis, along with specifications for tables, listings and figures to be 
produced.  The SAP will be finalized before the database soft lock at the latest.  Any changes 
from the analyses planned in the SAP will be justified in the Clinical Study Report (CSR).
Prior to Database Lock, a Final Review of Data and TLFs Meeting will be held to allow a 
review of the clinical trial data and to verify the data that will be used for analysis set 
classification.  If required, consequences for the statistical analysis will be discussed and 
documented.  A meeting to determine analysis set classifications may also be held prior to 
database lock.
In general, all data will be summarized by treatment group with descriptive statistics (number 
of subjects, mean, standard deviation, minimum, median, maximum, and interquartile range) 
for continuous endpoints, and frequency and percentage for categorical endpoints.
7.1
Sample Size
The sample size for this study was determined based upon a comparison of the rate of the 
two-part composite endpoint in the Astagraf XL group compared to the immediate-release 
tacrolimus group.  Assuming a rate of 20% would be observed for the immediate-release 
tacrolimus group compared to a rate of 10% for the Astagraf XL group, 220 subjects per 
group are needed to achieve an 80% power to detect a difference between Astagraf XL and 
immediate-release tacrolimus with a two-sided alpha level of 0.05.  The study will enroll 
275 subjects/group (550 total) to allow for 20% dropout.
7.2
Analysis Set
Detailed criteria for analysis sets will be laid out in Classification Specifications and the 
allocation of subjects to analysis sets will be determined prior to database hard-lock.
7.2.1
Full Analysis Set (FAS)
The full analysis set will consist of all subjects who are randomized and receive at least one 
dose of study drug (Astagraf XL or immediate-release tacrolimus). Subjects with  pre-
transplant cross-match samples that later demonstrate pre-formed antibody (undetected at the 
time of transplant), as assessed by the adjudication board, will be included in the FAS but not 
in the modified FAS (see next section).
7.2.2
Modified Full Analysis Set (mFAS)
The modified full analysis set will consist of all subjects who are randomized and receive at 
least one dose of study drug (Astagraf XL or immediate-release tacrolimus), and whose pre-
transplant cross-match antibody samples do not demonstrate pre-formed DSA for the 
duration of the study.  This will be the primary analysis set for efficacy assessments.


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7.2.3
Per Protocol Set (PPS)
The per protocol set will consist of the subset of the FAS who do not experience any major 
protocol deviations.  Final adjudication and assessment of the classification for any protocol 
deviations will be conducted prior to database hard-lock.  Further criteria may be defined in 
the SAP.  
7.2.4
Safety Analysis Set (SAF)
For the statistical summary and analysis of safety data, the safety analysis set (SAF) will be 
used.  The SAF consists of all subjects who enrolled into the study and took at least one dose 
of study medication.
7.2.5
Pharmacokinetic Analysis Set (PKAS)
Not applicable. 
7.3
Demographics and Other Baseline Characteristics 
Demographics and other baseline characteristics will be summarized by treatment group for 
the SAF.  Descriptive statistics will be included for continuous endpoints, and frequency and 
percentage for categorical endpoints.
7.4
Analysis of Efficacy 
The analysis of Efficacy will be conducted using the mFAS.  
7.4.1
Analysis of Primary Endpoint 
7.4.1.1 Primary Analysis
The primary efficacy endpoint is the combined incidence of either DSA or IA on molecular 
blood profiling at one year or at the conclusion of the study.
The primary endpoint will be analyzed at a single time point, corresponding to end of study 
and with respect to the formal stopping rule (see Section 7.8.1). The primary analysis of this 
endpoint will be based on the mFAS. The incidence of the primary endpoint will be 
summarized by treatment group with frequencies and percentages.  Differences between 
treatment groups will be analyzed using logistic regression with the following covariates: 
treatment group (Astagraf XL group versus the immediate-release group), the pre-defined 
randomization stratification factors, recipient age, race, and gender; type of induction 
therapy, delayed graft function, and pre-transplant cPRA.
For subjects with missing or incomplete endpoint data who did not permanently switch 
therapy or were discontinued from the study prior to a DSA/IA event, who are lost to follow 
up, or those who expire for any reason), negative DSA/ IA values will be imputed unless 
there is an otherwise positive result.  
The hypothesis for comparison is as follows:
H0: The probability of incidence for the primary endpoint is the same between patients 
receiving Astagraf XL and patients receiving immediate-release tacrolimus.


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H1: The probability of incidence for the primary endpoint is not the same between patients 
receiving Astagraf XL and those receiving immediate-release tacrolimus.
Hypothesis testing will be performed using a two-sided test with a 0.05 significance level. 
The 95% confidence interval for the odds ratio of the primary endpoint between Astagraf XL 
and twice daily tacrolimus will be included.  
7.4.1.2 Sensitivity Analysis 
A sensitivity analysis using the FAS will be conducted on the primary endpoint.  The same 
logistic regression analysis described in Section 7.4.1.1 will be used.  
Additionally, a sensitivity analysis for mFAS patients will be performed exclusively using
assessments with a basis in normal creatinine function. 
7.4.1.3 Secondary Analysis 
A secondary analysis using the PPS will be conducted on the primary endpoint data.  The 
same logistic regression analysis described in Section 7.4.1.1 will be used.  
7.4.1.4 Subgroup Analyses of the Primary Endpoint
Subgroup analyses of the primary endpoint, using the mFAS, will be conducted by patient 
age, sex, and race as well as donor race.
7.4.2
Analysis of Secondary Endpoints 
Risk factor analyses will be conducted for all 15 binary assessments.  In addition to the 
treatment effect, the risk factors assessed will include: manufacturer and type of immediate-
release tacrolimus; medication compliance, prior transplantation recipient, IPV of tacrolimus 
whole blood concentrations; absolute tacrolimus whole blood concentration dose and 
duration of MMF (or Myfortic equivalent); amount and duration of post-transplant 
corticosteroid use, type and amount of induction therapy used; age, sex, race, cold ischemia 
time (CIT), warm ischemia time (WIT), KDPI, DCD status, LD vs. DD status, recipient pre-
transplant dialysis status > 3 months (yes/no), donor race, pre-transplant donor kidney ex vivo
perfusion parameters, degree of HLA mismatch, and pre-transplant cPRA; and the presence 
of post-transplant bacterial and viral infection (including BK and CMV).  Additional risk 
factors may be included per the SAP.
Binary endpoints will be summarized with frequencies and percentages by treatment group 
and time point.  The incidence of events will be analyzed for treatment group differences at 
each time point, in addition to overall.  Analysis of treatment group differences will be 
implemented with generalized estimating equations (GEEs) using random effect(s), as 
necessary, to account for the temporal structure of data collection.  This analysis applies to 
each of the following incidence-based endpoints:
1.
DSA formation 
2.
IA 
3.
TG


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4.
Acute and chronic forms of ABMR
5.
C1q-binding DSA
6.
HLA-DQ DSA
7.
DSA IgG3 isotype
8.
Requirement for antibody reduction therapy
9.
eGFR at various thresholds (less than 30, 40, 50 mL/min/1.73 m2 at one year or later; 
and a five-point decline)
10. Graft loss (defined as subject death, retransplantation, transplant nephrectomy, or a 
return to dialysis for at least a 6 week duration)
11. Mortality
12. BPAR (inclusive of ABMR and TCMR)
13. The four-part traditional composite endpoint: graft loss, local BPAR, mortality, and lost 
to follow-up (note – lost to follow-up will be summarized descriptively only)
GEE models used for analysis of treatment group differences will use the same covariates 
detailed in Section 7.4.1.1 with the exception of biopsy-based endpoints (i.e., TG and 
ABMR).  In addition to the set of covariates from Section 7.4.1.1, biopsy-based endpoints 
will also include the covariates of CIT, WIT, KDPI, DCD status, and the three ex vivo 
perfusion parameters recorded pre-transplant.  Similar to the primary endpoint, additional 
changes/details for the set of covariates will be included in the SAP.
For study purposes, lost to follow-up is defined as a subject failing to complete their final 
study visit no more than 30 days prior to the scheduled visit, and without a prior incidence of 
local BPAR, graft loss, or death.  The incidence of lost to follow-up will be summarized with 
frequencies and percentage by treatment group.
The assessments of time to first occurrence for DSA, HLA-DQ DSA, C1q-binding DSA, 
DSA IgG3 isotype, IA, TG, select BANFF histology grades (acute and chronic forms of 
ABMR, acute and chronic active TCMR, borderline changes, and IFTA), graft loss, 
mortality, local BPAR, and the four-part traditional composite endpoint will be summarized 
from the date of transplant. For patients who did not experience the endpoint event and/or 
were lost to follow-up or died prior to reporting the event, they will be censored at the date of 
their last known assessment/follow-up/death.  Summaries will include estimable quartiles of 
time-to-first event with 95% confidence interval, Kaplan-Meier (KM) figures, and hazard 
ratios with 95% confidence interval.  In the event that the median times are not estimable, the 
proportion of subjects with no DSA formation at the one and two year time points (as 
applicable) will be summarized by treatment group.  Treatment group differences will be 
assessed with Cox regression models that account for the same covariates as used in the 
analogous GEE models.
The type of antibody reduction therapy will be summarized at each time point using 
frequencies and percentages.  Analysis of treatment group differences will be conducted 


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using multinomial logistic regression and GEEs with the same covariates used in the primary 
endpoint’s model and random effects(s), as necessary.
Ordinal assessments of antibody strength (weak, moderate, and strong) will be summarized at 
each time point using frequencies and percentages.  Analysis of treatment group differences 
will be done by time point and overall using a proportional odds model and GEEs with the 
same covariates used in the primary endpoint’s model and random effect(s), as necessary, to 
account for the temporal structure of data collection.
The continuous endpoint of MFI score will be summarized with descriptive statistics by time 
point.  Due to the expected non-normality of errors, an analysis of treatment group 
differences will be implemented with a semi-parametric, mixed repeated measures model on 
rank scores using a random effect of subject and the same covariates included in the analysis 
of Section 7.4.1.1   Least square mean (LS Mean) estimates will be provided at each time 
point and treatment group differences will be assessed at each time point and overall.
Histopathology assessments will be summarized with both descriptive statistics and 
frequencies and percentages.  Raw biopsy scores, including g, t, v, i, cg, ct, ci, cv, ah, ptc, and 
mm will be summarized as ordinal outcomes.  Analysis of treatment group differences will 
be performed using the same proportional odds model and GEEs implemented for the 
analysis of antibody strength.  The incidence of binary outcomes including ci, ct, and ptc 
scores greater than 1 will be summarized with frequencies and percentages.  Analysis of 
treatment group differences will be done consistent with the models using GEEs for the other 
binary endpoints.  For biopsy-based analyses, additional covariates will be added to the 
existing set in order to account for CIT, WIT, KDPI, DCD status, and the three ex vivo 
perfusion parameters recorded pre-transplant.  
Assessments of association between DSA and IA will be analyzed overall and by treatment 
group with logistic regression models using DSA formation as the response and IA as 
predictor. 
For patients who develop IA or DSA, change in eGFR from the time of diagnosis will be 
summarized with descriptive statistics by treatment group and by time point.  An analysis of 
treatment group differences will also be made using the same mixed, repeated measures 
model that is fit for the raw MFI scores.
The binary endpoints (bulleted early in this section) and the persistence of DSA will be 
analyzed under the subgroups of patients who developed DSA and patients who developed 
IA.  Treatment group differences by time point and overall will be assessed with the same 
GEE models and covariates detailed in this section.
7.4.3
Analysis of Exploratory Endpoints 
Analysis will be performed on the secondary composite endpoint consisting of DSA 
formation and the presence of TG (cg > 0).  The same analysis from Section 7.4.1.1 will be 
conducted for treatment group differences.  The analysis will be performed using the mFAS 
population. 


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The associations between each of HLA-DQ DSA incidence, ordinal DSA strength, DSA 
formation, C1q-binding DSA, DSA IgG3 isotype will be analyzed with respect to each other 
and with IA, TG, acute and chronic ABMR, histopathology, eGFR at various thresholds (less 
than 30, 40, and 50 mL/min/1.73 m2), ABMR (acute and chronic), TCMR, graft loss, and 
mortality.  Binary GEE models and proportional odds models will be fit for incidence and 
ordinal strength of HLA-DQ DSA, respectively.  Associations will be analyzed between 
treatment groups and overall as well as across time points.  Analogous methods will be used to 
assess the association between DSA and each component of the traditional composite endpoint.
The incidence of patients with normal and abnormal creatinine assessments and an IA 
designation will be summarized with descriptive statistics.  Concordance between the IA and 
biopsy results will be summarized with descriptive statistics for each of the normal and 
abnormal creatinine assessments.  Analysis of concordance between the IA and biopsy results 
will be conducted using Cohen’s Kappa for each of the abnormal and normal creatinine 
assessments.
Percent MFI reduction will be summarized with descriptive statistics by time point.  Analysis 
of treatment group differences will be done using the same semi-parametric, mixed, repeated 
measures model implemented for raw MFI scores.  MFI shift will be summarized with 
frequencies and percentages and analyzed for treatment group differences using the same 
proportional odds model used to analyze DSA antibody strength.
Mapping of expression results to known immunological pathways implicated in immune-
mediated, kidney transplant injury will be performed using multiple tools including Gene 
Ontology, Ingenuity’s Pathway Analysis™ (Ingenuity Systems, Redwood, CA), TGI’s 
proprietary tool called ImmuneMap, WikiPathways, GSEA Broad, Panther, and NCBI 
databases such as DAVID.  Visualization of networks and hub genes will be done with 
Cytoscape. Reports on functional pathway mapping will be prepared and provided to Astellas 
by TGI.
Additional sensitivity and secondary analyses for secondary and exploratory endpoints may 
be conducted using the PPS or FAS. 
7.5
Analysis of Safety
7.5.1
Adverse Events
Adverse events will be coded using the MedDRA.  The number and percentage of AEs, 
SAEs, AEs leading to discontinuation, and AEs related to study drug will be summarized by 
system organ class, preferred term and treatment group. The number and percentage of AEs 
by severity will also be summarized. All AEs will be listed.
7.5.2
Laboratory Assessments 
For quantitative laboratory measurements obtained during standard of care, descriptive 
statistics will be used to summarize results and change from baseline by treatment group and 
time point. Shifts relative to normal ranges from baseline to each time point during the 


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treatment period with regard to lab tests will also be tabulated.  Laboratory data will be 
displayed in listings.
7.5.3
Histopathology
Data collected based on light microscopy, electron microscopy, and immunofluorescent 
images will be summarized by treatment group, pathologic designation, and time point 
stratified by local and central readings.
7.6
Analysis of Tacrolimus Dose and Trough Concentrations
Descriptive statistics (e.g., n, mean, standard deviation, minimum, median, maximum) as 
well as interquartile range, coefficient of variation, and geometric mean will be provided for 
tacrolimus dose and whole blood concentrations of tacrolimus for both study cohorts. 
Tacrolimus concentrations will be summarized with descriptive statistics.  Assumptions and 
methods for estimation and time point windowing will be detailed in the SAP as appropriate.  
Tacrolimus trough by-patient CV and SD estimates will be assessed for association across time 
and overall with each of the following: the primary endpoint, the secondary composite endpoint 
of DSA and IA, DSA formation and IA individually, TG, chronic ABMR, C1q-binding DSA, 
HLA-DQ DSA, DSA IgG3 isotype, histopathology, renal dysfunction, and the components of 
the traditional composite endpoint.  Analysis details will be included in the SAP.
IPV of dose-adjusted tacrolimus trough levels after the 6 weeks following transplantation will 
be assessed for treatment group differences.  The SAP will detail the analysis methods and 
time points.  
The association between whole blood concentrations and the following endpoints will be 
assessed: DSA presence, DSA strength, C1q-binding DSA, IgG3, IA,eGFR, histopathology, 
and conventional measures of transplant outcomes.
Additionally, the effect of switching product manufacturers during the study will be assessed 
with tacrolimus concentration.  Changes will be assessed between treatment groups and for 
patients taking bid tacrolimus with the bid tacrolimus group analyzed for differences between 
those who did and did not switch manufacturers during the study.  
7.7
Protocol Deviations and Other Analyses 
Protocol deviations as defined in Section 8.1.6 will be summarized for all randomized 
subjects by treatment group and total as well as by site. A data listing will be provided by site 
and subject. The protocol deviation criteria will be uniquely identified in the summary table 
and listing. The unique identifiers will be as follows:
PD1 - Entered into the study even though they did not satisfy entry criteria, 
PD2 - Developed withdrawal criteria during the study and was not withdrawn,
PD3 - Received wrong treatment or incorrect dose,
PD4 - Received excluded concomitant treatment.


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7.8
Interim Analysis (and Early Discontinuation of the Clinical Study)
No formal interim analysis is planned.
7.8.1
Formal Stopping Rule
Given the variability of incidence rates for DSA reported in the literature, a stopping rule has 
been incorporated into ASTOUND to mitigate the risk of continuing the study under 
incorrect prior assumptions (Section 7.1).  This stopping rule will be based on the incidence 
of the primary endpoint (combined incidence of either DSA or IA). 
Patients will remain on treatment for 12 months or potentially longer (up to a maximum of 
24 months).  Nevertheless, in order to minimize the risk of conducting a study that could be, in 
the end, underpowered, a decision tree [Figure 3] will be actuated once 50% of control patients 
have completed one year of therapy.  If, based on the enclosed algorithm, the primary 
endpoint’s event rate in the control arm at that time is high enough for efficacy to be assessed at 
one year for the totality of subjects entering the study, the trial will continue enrolling and those 
subjects who, up until that point have completed <12 months of therapy, will be permitted to 
remain on such until study completion (1 year).  However, it is acknowledged that in deploying 
a potential stopping rule once 50% of patients have completed one year of therapy, that a certain 
percentage of patients may have progressed beyond one year of therapy by the time the decision 
algorithm is applied.  If this situation should occur, only the one year data for these patients will 
be included in the efficacy assessment, the exception being if the decision algorithm dictates 
that the study should continue for an additional year, in which case, the efficacy assessment will 
be made at 24 months for all patients.  
For the decision algorithm, the estimated proportion will be used to calculate the Beta-binomial 
posterior Bayesian probability that the primary endpoint’s incidence rate will, indeed, be at least 
20% (see Appendix 12.4 for assumption details).  
As mentioned above, a decision to terminate the study early or extend to a second year will be 
predicated on the event rate in the control arm once 50% of control patients have completed one 
year of therapy.  At that time, the estimated proportion will be used to calculate the Beta-
binomial posterior Bayesian probability that the primary endpoint’s incidence rate will, indeed, 
be at least 20%.
If this probability is ≥ 70%, the following rules apply:
1) The study will continue to enroll to completion. 
2) The study duration will be such that patients will complete at least one year of study 
visits.
a) Patients who have not yet completed one year of study (at the time of the interim 
analysis) will complete the study at the 12m visit.
b) Patients who have already completed the 12m visit (at the time of the interim 
analysis) will complete the study at their next scheduled study visit. 
3) Data analysis will be performed once all patients complete 12 months of study.


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If this probability is < 70% and ≥ 30%:
1) The study will continue to enroll to completion.
2) The study duration will be 24 months; patients will complete the study at the 24 month 
visit.
3) Data analysis will be performed once all patients complete 24 months of therapy.
If this probability is < 30%, the study will be terminated at that time (50% of patients 
completing 12 months of therapy).
1) The study will be terminated upon completion of the interim analysis (done when 50% of 
patients have completed 12 months of therapy). 
2) Patients will be discontinued from the study at their next scheduled study visit. 
Figure 3
Application of Stopping Rule
7.9
Handling of Missing Data, Outliers, Visit Windows, and Other 
Information
In the primary efficacy analysis, other than imputation specifically stated in Section 7.4.1.1, 
there is no planned imputation of missing values.  
See the SAP for details of the definitions for windows to be used for analyses by visit.


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8
OPERATIONAL AND ADMINISTRATIVE CONSIDERATIONS
8.1
Procedure for Clinical Study Quality Control
8.1.1
Data Collection
The investigator or site designee will enter data collected using an Electronic Data Capture 
(EDC) system. In the interest of collecting data in the most efficient manner, the investigator 
or site designee should record data (including local laboratory values, as applicable) in the 
electronic case report form (eCRF) within 5 days after the subject visit.
The investigator or site designee is responsible to ensure that all data in the eCRFs and 
queries are accurate and complete and that all entries are verifiable with source documents. 
These documents should be appropriately maintained by the site.
The monitor should verify the data in the eCRFs with source documents according to the 
study monitoring plan. 
Laboratory testing performed during the course of routine clinical care, including HLA 
typing and pre-transplant cross-match results, will be performed according to local standards. 
De-identified copies of HLA typing reports (donor and recipient) as well as recent pre-
transplant local DSA testing results (if performed per SOC) will be sent to the central lab.
For the antibody and molecular assessments conducted by the central laboratory (performed at 
baseline, months 1, 3, 6, 9, 12, and 24), results will be compiled and the data electronically 
transferred to Astellas or its designee for inclusion in the clinical study database. The results of 
these central assessments for each participant will be shared with the corresponding 
Investigator. The results of the antibody assessments will be provided, as available (testing 
performed in batches), on an on-going basis, throughout the study.  The results of the molecular 
assessments will not be shared with the corresponding Investigator until the end of the study.
For all participants who undergo at least one kidney biopsy during the first year post-
transplant (either for cause or per institutional protocol), the kidney biopsy performed closest 
to the 12 month visit (but no later than 14 months post-transplant) will be submitted for 
central pathology review for pathological grading. It is preferred that centers submit whole 
slide digital imaging using a standard protocol on at least Hematoxlyin and Eosin (H&E), 
Periodic Acid Schiff (PAS), Trichrome-stained slides, silver slides, immunoperoxidase C4d, 
and control immunoperoxidase slides for C4d (as available). If an institution does not do 
whole slide imaging, then the paraffin section slides must also be submitted for central 
review along with the available digital images. Absent the availability of digital pathology 
transmission capabilities, centers will submit representative H&E, PAS, Trichrome-stained, 
silver, immunoperoxidase C4d, and control immunoperoxidase C4d slides (as available) for 
central pathology review. The results for each participant with a central pathology review 
will be shared with the corresponding Investigator at the end of study, upon Investigator/site 
request.
For screen failures, the demographic data, reason for failing, informed consent/assent, 
inclusion and exclusion criteria, and AEs will be collected in the eCRF. 


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The investigator or designee must record all protocol-required data in the provided electronic 
Case Report Forms (eCRF).  In the interest of collecting data in the most efficient manner, 
the investigator or site designee should record data (including laboratory values, if 
applicable) onto the eCRF as soon as possible after the subject visit.  ECRFs and any 
supporting documents should be available for retrieval by the Sponsor/delegated CRO at any 
given time.  The monitor should verify the data in the eCRFs with source documents to 
confirm that there are no inconsistencies between them.  
If any inconsistency is detected on the collected eCRFs, the monitor or data manager should 
query the investigator/sub-investigator.  The investigator/sub-investigator should provide an 
answer to the query and provide the resolved query to the Sponsor.
The monitor should verify the revised data of the eCRFs with source documents and confirm 
that there are no inconsistencies between them, and also check that appropriate records on the 
correction/addition of data are maintained.
8.1.2
Specification of Source Documents
Source data must be available at the site to document the existence of the study subjects and 
to substantiate the integrity of study data collected. Source data must include the original 
documents relating to the study, as well as the medical treatment and medical history of the 
subject.  
The following information should be included in the source medical records:
●
Demographic data (age, sex, race, ethnicity, height and body weight)
●
cPRA
●
Cross-match results
●
Separate donor source data including donor demographics, KDPI of donor kidney and 
donor HLA typing.
●
Inclusion and exclusion criteria details
●
Participation in study and original signed and dated informed consent/assent forms 
●
Visit dates
●
Medical history and physical examination details
●
Key efficacy and safety data, if applicable (as specified in the protocol)
●
Adverse events (including causality) and concomitant medication 
●
Results of relevant examinations (e.g., ECG charts, X-ray films etc.)
●
Laboratory printouts (if applicable)
●
Dispensing and return of study drug details
●
Reason for premature discontinuation (if applicable)
●
Randomization number (if applicable)
8.1.3
Clinical Study Monitoring
Astellas or Astellas’ delegated CRO is responsible for monitoring the clinical study to ensure 
that subjects’ human rights, safety, and well-being are protected, that the study is properly 
conducted in adherence to the current protocol and GCP, and that study data reported by the 


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investigator/sub-investigator are accurate and complete and that they are verifiable with 
study-related records such as source documents.  Astellas or Astellas’ delegated CRO is 
responsible for assigning a study monitor(s) to this study to ensure proper study oversight.  
Monitoring will occur in accordance with planned monitoring procedures.
8.1.4
Direct Access to Source Data/Documents
The investigator and the study site must accept monitoring and auditing by Astellas or its 
delegated CRO as well as inspections from the IRB/IEC and relevant regulatory authorities. 
In these instances, they must provide all study-related records, such as source documents 
(refer to Section 8.1.2 Specification of Source Documents) when they are requested by 
Astellas monitors and auditors, the IRB/IEC, or regulatory authorities. The confidentiality of 
the subject's identities shall be well protected consistent with local and national regulations 
when the source documents are subject to direct access.  
8.1.5
Data Management
Data Management will be in accordance with the standard operating procedures (SOPs) of 
Astellas or Astellas’ designated CRO. Data analysis will be performed in adherence to 
Astellas data standards. All study specific processes and definitions will be documented by
Data Management. ECRF completion will be described in the eCRF instructions. Coding of 
medical terms and medications will be performed using MedDRA and World Health 
Organization (WHO) Drug Dictionary respectively.
8.1.6
Protocol Deviations 
A protocol deviation is generally an unplanned excursion from the protocol that is not 
implemented or intended as a systematic change. The investigator is responsible for ensuring 
the study is conducted in accordance with the procedures and evaluations described in this 
protocol and for protecting the rights, safety, and welfare of subjects. The investigator should 
not implement any deviation from, or changes to, the protocol, unless it is necessary to 
eliminate an immediate hazard to trial subjects.  
A protocol waiver is a documented prospective approval of a request from an investigator to 
deviate from the protocol.  Protocol waivers are strictly prohibited.
For the purposes of this protocol, deviations requiring notification to Sponsor are defined as 
any subject who: 
●
Entered into the study even though they did not satisfy entry criteria. 
●
Developed withdrawal criteria during the study and not withdrawn
●
Received wrong treatment or incorrect dose. 
●
Received excluded concomitant treatment. 
When a deviation from the protocol is identified for an individual subject, the investigator or 
designee must ensure Astellas is notified.  Astellas will follow-up with the investigator, as 
applicable, to assess the deviation and the possible impact to the safety and/or efficacy of the 
subject and to determine subject continuation in the study. 


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If a deviation impacts the safety of a subject, the investigator must contact the Sponsor 
immediately.
The investigator will also assure that deviations meeting IRB/IEC and applicable regulatory 
authorities’ criteria are documented and communicated appropriately. All documentation and 
communications to the IRB/IEC and applicable regulatory authorities will be provided to the 
Sponsor and maintained within the Trial Master File (TMF).  
NOTE: Other deviations outside of the categories defined above that are required to be 
reported by the IRB/IEC in accordance with local requirements will be reported, as 
applicable.
8.1.7
End of Trial in All Participating Countries
The end of trial in all participating countries is defined as the Last Subject’s Last Visit.
8.2
Ethics and Protection of Subject Confidentiality 
8.2.1
Institutional Review Board (IRB) / Independent Ethics Committee (IEC) / 
Competent Authorities (CA)
Good Clinical Practice (GCP) requires that the clinical protocol, any protocol amendments, 
the Investigator’s Brochure, the informed consent, all other forms of subject information 
related to the study (e.g., advertisements used to recruit subjects), and any other necessary 
documents be reviewed by an IEC/IRB.  The IEC/IRB will review the ethical, scientific and 
medical appropriateness of the study before it is conducted.  IEC/IRB approval of the 
protocol, informed consent/assent, and subject information and/or advertising, as relevant, 
will be obtained prior to the authorization of drug shipment to a study site.
Any substantial amendments to the protocol will require IEC/IRB approval prior to 
implementation of the changes made to the study design at the site.  The investigator will be 
required to submit, maintain, and archive study essential documents according to ICH GCP.
Any serious adverse events that meet reporting criteria, as dictated by local regulations, will 
be reported to both responsible Ethics Committees and Regulatory Agencies, as required. 
During the conduct of the study, the investigator should promptly provide written reports 
(e.g., ICH Expedited Reports, and any additional reports required by local regulations) to the 
IEC/IRB of any changes that affect the conduct of the study and/or increase the risk to 
subjects. Written documentation of the submission to the IEC/IRB should also be provided to 
Sponsor.
If required by local regulations, the investigator shall make accurate and adequate written
progress reports to the IEC/IRB at appropriate intervals, not exceeding one year. The 
investigator shall make an accurate and adequate final report to the IRB/IEC within 90 days
after the close-out visit for APGD-sponsored studies, or for APEB/APEL-sponsored studies 
within one year after last subject out (LSO) or termination of the study.


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8.2.2
Ethical Conduct of the Study
The study will be conducted in accordance with the protocol, ICH guidelines, applicable
regulations and guidelines governing clinical study conduct and the ethical principles that 
have their origin in the Declaration of Helsinki. 
8.2.3
Informed Consent of Subjects
8.2.3.1 Subject Information and Consent/Assent
The investigator or his/her representative will explain the nature of the study to the subject or 
his/her parents/guardian (if applicable) or legal representative, and answer all questions 
regarding this study.  Prior to any study-related screening procedures being performed on the 
subject, the informed consent (and assent, if applicable) statement will be reviewed and 
signed and dated by the subject or his/her parents/guardian or legal representative, the person 
who administered the informed consent, and any other signatories according to local 
requirements. A copy of the signed informed consent form (and assent, if applicable) will be 
given to the subject and the original will be placed in the subject’s medical record. An entry 
must also be made in the subject’s dated source documents to confirm that informed consent 
(and assent, if applicable) was obtained prior to any study-related procedures and that the 
subject received a signed copy.
The signed consent forms (and assent, if applicable) will be retained by the investigator and 
made available (for review only) to the study monitor, regulatory authorities and other 
applicable individuals upon request.
8.2.3.2 Supply of New and Important Information Influencing the Subject’s Consent 
and Revision of the Written Information
1.
The investigator or his/her representative will immediately inform the subject orally 
whenever new information becomes available that may be relevant to the subject’s 
consent or may influence the subject’s willingness to continue to participate in the study 
(e.g., report of serious drug adverse drug reaction). The communication must be 
documented in the subject’s medical records and must document whether the subject is 
willing to remain in the study or not.
2.
The investigator must update their ICF and submit it for approval to the IRB/IEC. The 
investigator or his/her representative must obtain written informed consent (and assent, if 
applicable) from the subject on all updated ICFs throughout their participation in the 
study. The investigator or his/her designee must re-consent subjects with the updated 
ICF even if relevant information was provided orally. The investigator or his/her 
representative who obtained the written informed consent and the subject should sign 
and date the informed consent (and assent, if applicable) form. A copy of the signed 
informed consent (and assent, if applicable) form will be given to the subject and the 
original will be placed in the subject’s medical record. An entry must be made in the 
subject’s records documenting the re-consent process.


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8.2.4
Subject Confidentiality
Individual subject medical information obtained as a result of this study is considered 
confidential and disclosure to third parties is prohibited. Such medical information may be 
given only after approval of the subject to the subject's physician or to other appropriate 
medical personnel responsible for the subject's well-being.
For the purpose of fulfilling exploratory objectives, ASTOUND has made allowances for the 
conduct of future studies by which the long-term outcomes of patients testing positive for 
DSA or IA, as well as the health care costs associated with those outcomes, are examined.  
For those patients agreeing to contribute to the possible future study of health care costs, 
Astellas would receive de-identified Medicare claims or commercial payer costs data from 
the participating transplant centers. If this follow up study is conducted, it will be done in 
accordance with a separate, IRB-approved study protocol.  
For those patients agreeing to contribute to the possible future study of long-term outcomes, 
ASTOUND will make use of each patient’s unique transplant identifier, maintained by the 
OPTN for recording of data within the SRTR and UNOS databases.  This identifier is 
maintained by each individual transplant center and permits access to the aforementioned 
databases for required reporting purposes.  ASTOUND will not ask for or maintain a record 
of this identifier.  In the event future long-term follow-up studies are warranted, individual 
transplant centers and investigators who participated in ASTOUND will be asked, subject to 
separate IRB approval, to query the respective databases in the context of those studies, with 
reporting of de-identified results (graft survival, patient survival, and immune-mediated 
phenomenon) to a central study location.  Astellas shall not disclose any confidential 
information on subjects obtained during the performance of their duties in the clinical study 
without justifiable reasons.
Astellas affirms the subject's right to protection against invasion of privacy. Only a subject 
identification number (different and separate than that maintained by the OPTN) and/or 
initials will identify subject data retrieved by Astellas.  However, Astellas requires the 
investigator to permit Astellas, its representative(s), the IRB/IEC and when necessary, and 
representatives of the regulatory health authorities to review and/or to copy any medical 
records relevant to the study.
Astellas will ensure that the use and disclosure of protected health information (PHI) 
obtained during a research study complies with the federal and/or regional legislation related 
to the privacy and protection of personal information (i.e., HIPAA).
8.3
Administrative Matters
8.3.1
Arrangement for Use of Information and Publication of the Clinical Study
Information concerning the study drug, patent applications, processes, unpublished scientific 
data, the Investigator's Brochure, and other pertinent information is confidential and remains 
the property of the Sponsor. Details should be disclosed only to the persons involved in the 
approval or conduct of the study. The investigator may use this information for the purpose 


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of the study only. It is understood by the investigator that the Sponsor will use the 
information obtained during the clinical study in connection with the development of the 
drug and therefore may disclose it as required to other clinical investigators or to regulatory 
agencies. In order to allow for the use of the information derived from this clinical study, the 
investigator understands that he/she has an obligation to provide the Sponsor with all data 
obtained during the study.
Publication of the study results is discussed in the Clinical Study Agreement.
8.3.2
Documents and Records Related to the Clinical Study
The investigator will archive all study data (e.g., Subject Identification Code List, source
data, eCRFs, and Investigator's File) and relevant correspondence. These documents are to be 
kept on file for the appropriate term determined by local regulation (for US sites, two years 
after approval of the NDA or discontinuation of the IND). The Sponsor will notify the 
site/investigator if the NDA/MAA/J-NDA is approved or if the IND/IMPD/CHIKEN 
TODOKE is discontinued.  The investigator agrees to obtain the Astellas’ agreement prior to 
disposal, moving, or transferring of any study-related records. Astellas will archive and retain 
all documents pertaining to the study according to local regulations. 
Data generated by the methods described in the protocol will be recorded in the subjects' 
medical records and/or study progress notes. All data will be entered on the eCRFs supplied 
for each subject.
8.3.3
Protocol Amendment and/or Revision
Any changes to the study that arise after approval of the protocol must be documented as 
protocol amendments/substantial amendments and/or /non-substantial amendments.
Depending on the nature of the amendment, either IRB/IEC/CA approval or notification may 
be required. The changes will become effective only after the approval of Astellas, the 
investigator, the regulatory authority, and the IRB/IEC (if applicable).
Amendments to this protocol must be signed by Astellas and the investigator. Written 
verification of IRB/IEC approval will be obtained before any amendment is implemented 
which affects subject safety or the evaluation of safety, and/or efficacy. Modifications to the 
protocol that are administrative in nature do not require IRB/IEC approval, but will be 
submitted to the IRB/IEC for their information, if required by local regulations.
If there are changes to the Informed Consent/Assent, written verification of IRB/IEC 
approval must be forwarded to the Sponsor.  An approved copy of the new Informed 
Consent/Assent must also be forwarded to Astellas.
8.3.4
Signatory Investigator for Clinical Study Report
ICH E3 guidelines recommend and EU Directive 2001/83/EC requires that a final study 
report which forms part of a marketing authorization application be signed by the
representative for the Coordinating Investigator(s) or the Principal Investigator(s). The 
representative for the Coordinating Investigator (s) or the Principal Investigator(s) will have 
the responsibility to review the final study results to confirm to the best of his/her knowledge 


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it accurately describes the conduct and results of the study.  The representative for
Coordinating Investigator(s) or the Principal Investigator(s) will be selected from the 
participating investigators by Astellas prior to database lock.
9
QUALITY ASSURANCE
Astellas is implementing and maintaining quality assurance and quality control systems with 
written SOPs to ensure that trials are conducted and data are generated, documented, 
recorded, and reported in compliance with the protocol, GCP, and applicable regulatory 
requirement(s).
Astellas or Astellas’ designee may arrange to audit the clinical study at any or all 
investigational sites and facilities. The audit may include on-site review of regulatory
documents, case report forms, and source documents. Direct access to these documents will 
be required by the auditors.
10
STUDY ORGANIZATION
10.1
Independent Data-Monitoring Committee (IDMC) | Data and Safety 
Monitoring Board (DSMB) | Monitoring Committee | Other 
Evaluation Committee(s)
Not applicable
10.2
Adjudication Board: MFI Adjudication Committee
An Adjudication Board will be formed to review all borderline MFI threshold results that 
occur during the study to confirm that the results have been categorized correctly.  The Board 
will consist of three independent members of appropriate expertise who are not directly 
involved in the clinical study and who are blinded to the treatment allocation. Information 
related to the borderline threshold results will be sent to the committee for blinded 
assessment. A separate charter will describe which results will require adjudication and the 
specification of the result-related documents, listings, data flow, method(s) for data collection 
and data transfer.
The Adjudication Board will  1) review and adjudicate all cases of antibody formation (per 
central HLA lab assessment) post-transplantation and 2) determine the need for ancillary 
testing of pre-transplant sera in equivocal cases in which such testing could indicate the 
presence of pre-transplant DSA.  


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11
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immunogenicity of HLA class II graft molecules. Transpl Int. 2011 Nov;24(11):1084-93.  
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tacrolimus blood levels increases the risk of late rejection and graft loss after solid organ 
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1106.


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Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, et al. The Banff 97 working 
classification of renal allograft pathology. Kidney Int. 1999 Feb;55(2):713-23.
Rush DN, Henry SF, Jeffery JR, Schroeder TJ, Gough J. Histological findings in early routine 
biopsies of stable renal allograft recipients. Transplantation. 1994 Jan;57(2):208-11.
Sabbatini M, Garofalo G, Borrelli S, Vitale S, Torino M, Capone D, et al. Efficacy of reduced pill 
burden on therapeutic adherence to calcineurin inhibitors in renal transplant recipients: an 
observational study. Patient Prefer Adherence. 2014;8:73-81. 
Sapir-Pichhadze R, Wang Y, Famure O, Li Y, Kim SJ. Time-dependent variability in tacrolimus 
trough blood levels is a risk factor for late kidney transplant failure. Kidney Int. 2014 
Jun;85(6):1404-11.
Sellares J, de Frietas DG, Megel M, Reeve J, Einecke G, Sis B, et al. Understanding the causes of 
kidney transplant failure: the dominant role of antibody-mediated rejection and nonadherence. 
Am J Transplant. 2012 Feb;12(2):388-99.
Seron D, Moreso F. Protocol biopsies in renal transplantation: prognostic value of structural 
monitoring. Kidney Int. 2007 Sep;72(6):690-7.
Seron D, Moreso F, Fulladosa X, Hueso M, Carrera M, Grinyo JM. Reliability of chronic allograft 
nephropathy diagnosis in sequential protocol biopsies. Kidney Int. 2002;61:727-33.
Shihab F, Waid TH, Conti DJ, Yang H, Holman MJ, Mulloy LC, et al. Conversion from cyclosporine 
to tacrolimus in patients at risk for chronic renal allograft failure: 60-month results of the 
CRAF Study. Transplantation. 2008 May;85(9):1261-9.
Shishido S, Asanuma H, Nakai H, Mori Y, Satoh H, Kamimaki I, et al. The impact of repeated 
subclinical acute rejection on the progression of chronic allograft nephropathy. J Am Soc 
Nephrol. 2003 Apr;14(4):1046-52.
Silva HT Jr, Yang HC, Aboulijoud M, Kuo PC, Wisemandle K, Bhattacharya P, et al. One-year 
results with extended-release tacrolimus/MMF, tacrolimus/MMF and cyclosporine/MMF in de 
novo kidney transplant recipients. Am J Transplant. 2007 Mar;7(3):595-608.
Sis B, Campbell PM, Mueller T, Hunter C, Cockfield SM, Cruz J, et al. Transplant glomerulopathy, 
late antibody-mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. 
Am J Transplant. 2007 Jul;7(7):1743-52.
Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, PF et al. Banff 07 classification of renal 
allograft pathology: updates and future directions. Am J Transplant. 2008 Apr;8(4):1-8.
Srinvias TR, Schold JD, Guerra G, Eagan A, Bucci CM, Meier-Kriesche HU. Mycophenolate 
mofetil/sirolimus compared to other common immunosuppressive regimens in kidney 
transplantation. Am J Transplant. 2007 Mar;7(3):586-94.


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Staatz CE, Tett SE. Clinical pharmacokinetics and pharmacodynamics of tacrolimus in solid organ 
transplantation. Clin Pharmacokinet. 2004;43(10):623-53.
Stegall MD, Gaston RS, Cosio FG, Matas A. Through a glass darkly:  seeking clarity in preventing 
late kidney transplant failure. J Am Soc Nephrol. 2015 Jan;26(1):20-9.
Stegall MD, Park WD, Larson TS, Gloor JM, Cornell LD, Sethi S, et al. The histology of solitary 
renal allograft at 1 and 5 years after transplantation. Am J Transplant. 2011 Apr;11(4):698-707.
Stifft F, Stolk LM, Undre N, van Hooff JP, Christiaans MHL. Lower variability in 24-hour exposure 
during once-daily compared to twice-daily tacrolimus formulation in kidney transplantation. 
Transplantation. 2014 Apr;97(7):775-80. 
Tambur AR, Herrera ND, Haarberg KM, Cusick MF, Gordon RA, Leventhal JR, et al.  Assessing 
antibody strength: comparison of MFI, C1q, and titer information. Am J Transplant. Epub 2015 
Apr 30. doi 10.1111/ajt.13295.
Trunecka P, Boillot O, Langrehr J, Pinna AD, Fischer L, Ericzon BG. Tacrolimus once-daily 
prolonged release versus tacrolimus twice-daily in combination with steroids - A phase III 
multicentre liver study: 864. Transplantation. 2008;86:301-302.
Undre NA. For the Tacrolimus Modified Release Kidney Study Group. Use of a once daily modified 
release tacrolimus regimen in de novo kidney transplant recipients [abstract]. Am J Transplant. 
2005;5:190. Abs 132.
Undre NA. Tacrolimus Modified Release Liver Study Group. Use of a once daily modified release 
tacrolimus regimen in de novo liver transplant recipients [abstract]. Am J Transplant. 
2005;5:374. Abs 859.
Undre NA, van Hooff J, Christiaans M, Vanrenterghem Y, Donck J, Heeman U, et al. Low systemic 
exposure to tacrolimus correlates with acute rejection. Transplant Proc. 1999 Feb-Mar;31(1-
2):296-8.
van Hooff J, Van der Walt I, Kallmeyer J, Miller D, Dawood S, Moosa MR, et al. Pharmacokinetics 
in stable kidney transplant recipients after conversion from twice-daily to once-daily tacrolimus 
formulations. Ther Drug Monit. 2012 Feb;34(1):46-52.
Vincenti F, Charpentier B, Vanrenterghem Y, Rostaing L, Bresnahan B, Darji P, et al. A phase III 
study of belatacept-based immunosuppression regimens versus cyclosporine in renal transplant 
recipients (BENEFIT study). Am J Transplant. 2010 Mar;10(3):535-46.
Weir MR, Mulgaonkar S, Chan L, Shidban H, Waid TH, Preston D, et al. Mycophenolate mofetil-
based immunosuppression with sirolimus in renal transplantation: a randomized, controlled 
Spare-the-Nephron trial. Kidney Int. 2011 Apr;79(8):897-907.
Wiebe C, Gibson IW, Blydt-Hansen TD, Karpinski M, Ho J, Storsley LJ, et al.  Evolution and clinical 
pathologic correlations of de novo donor-specific HLA antibody post kidney transplant.  Am J 
Transplant. 2012 May;12(5):1157-67.


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Wiebe C, Gibson IW, Blydt-Hansen TD, Pochinco D, Birk PE, Ho J, et al. Rates and determinants of 
progression to graft failure in kidney allograft recipients with de novo donor-specific antibody. 
Am J Transplant. Epub 2015 Jun 10. doi 10.1111/ajt.13347.
Willicombe M, Brookes P, Sergeant R, Santos-Nunez E, Steggar C, Galliford J, et al. De novo DQ 
donor-specific antibodies are associated with a significant risk of antibody-mediated rejection 
and transplant glomerulopathy. Transplantation. 2012 Jul;94(2):172-7.
Wu MJ, Cheng CY, Chen CH, Wu WP, Cheng CH, Yu DM, et al. Lower variability of tacrolimus 
trough concentration after conversion from prograf to advagraf in stable kidney transplant 
recipients. Transplantation. 2011 Sep;92(6):648-52. 
Zachary A and Reinsmoen N. Quantifying HLA antibodies in patients undergoing desensitization. 
Curr Opin Organ Transplant. 2011 Aug;16(4):410-5.
Zhang Q, Liang LW, Gjertson DW, Lassman C, Wilkinson AH, Kendrick E, et al. Development of 
posttransplant antidonor HLA antibodies is associated with acute humoral rejection and early 
graft dysfunction. Transplantation. 2005 Mar;79(5):591-8.
Company Reports:
Astagraf XL US Package Insert. Northbrook, IL: Astellas Pharma US, Inc.; June 2015.


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12
APPENDICES
12.1
List of Excluded Concomitant Medications
The following are prohibited medications during ASTOUND:
1) Extended-release formulations of tacrolimus, other than Astagraf XL 
2) Non-tacrolimus based immunosuppressive regimens (i.e. cyclosporine, everolimus, 
sirolimus, belatacept)
3) Antiviral medications used in HCV treatment
4) Antiviral medications used in HIV treatment
5) Isoniazid
6) Rifampin
7) Ethambutol
8) Pyrazinamide


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12.2
Liver Safety Monitoring and Assessment 
Any subject enrolled in a clinical study with active drug therapy and reveals an increase of 
serum aminotransferases (AT) to > 3 × ULN, or bilirubin > 2 × ULN, should undergo 
detailed testing for liver enzymes (including at least ALT, AST, ALP, and TBL). Testing 
should be repeated within 48-72 hours of notification of the test results. For studies for which 
a central laboratory is used, alerts will be generated by the central lab regarding moderate and 
severe liver abnormality to inform the investigator, study monitor and study team. Subjects 
should be asked if they have any symptoms suggestive of hepatobiliary dysfunction.
Definition of Liver Abnormalities
Confirmed abnormalities will be characterized as moderate and severe where ULN:
ALT or AST
Total Bilirubin
Moderate
> 3× ULN 
or
> 2× ULN
Severe*
> 3× ULN
and
> 2× ULN
In addition, the subject should be considered to have severe hepatic abnormalities for any of 
the following:
●
ALT or AST > 8× ULN
●
ALT or AST > 5× ULN for more than 2 weeks  
●
ALT or AST > 3× ULN and INR > 1.5 (If INR testing is applicable/evaluated).
●
ALT or AST > 3× ULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash and/or eosinophilia (> 5%). 
The investigator may determine that abnormal liver function results, other than as described 
above, may qualify as moderate or severe abnormalities and require additional monitoring 
and follow-up.
Follow-up Procedures
Confirmed moderate and severe abnormalities in hepatic functions should be thoroughly 
characterized by obtaining appropriate expert consultations, detailed pertinent history, 
physical examination and laboratory tests. The site should complete the Liver Abnormality 
Case Report Form (LA-CRF) that has been developed globally and can be activated for any 
study> or an appropriate document. Subjects with confirmed abnormal liver function testing 
should be followed as described below.
Confirmed moderately abnormal LFTs should be repeated 2-3 times weekly then weekly or 
less if abnormalities stabilize or the study drug has been discontinued and the subject is 
asymptomatic.  
Severe hepatic liver function abnormalities as defined above, in the absence of another 
etiology, may be considered an important medical event and may be reported as a Serious 
Adverse Event (SAE).  The Sponsor should be contacted and informed of all subjects for 
whom severe hepatic liver function abnormalities possibly attributable to study drug are 
observed.


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To further assess abnormal hepatic laboratory findings, the investigator is expected to:
●
Obtain a more detailed history of symptoms and prior or concurrent diseases.  Symptoms 
and new onset-diseases should be recorded as ‘adverse events’ on the AE page of the
eCRF. Illnesses and conditions such as hypotensive events, and decompensated cardiac 
disease that may lead to secondary liver abnormalities should be noted. Non-alcoholic 
steatohepatitis (NASH) is seen in obese hyperlipoproteinemic, and/or diabetic patients 
and may be associated with fluctuating aminotransferase levels.   The investigator should 
ensure that the medical history form captures any illness that pre-dates study enrollment 
that may be relevant in assessing hepatic function. 
●
Obtain a history of concomitant drug use (including non-prescription medication, 
complementary and alternative medications), alcohol use, recreational drug use, and 
special diets.  Medications, including dose, should be entered on the concomitant 
medication page of the eCRF.  Information on alcohol, other substance use, and diet 
should be entered on the LA-CRF or an appropriate document.
●
Obtain a history of exposure to environmental chemical agents.
●
Based on the subject’s history, other testing may be appropriate including: 
o
acute viral hepatitis (A,B, C, D, E or other infectious agents) 
o
ultrasound or other imaging to assess biliary tract disease
o
other laboratory tests including INR, direct bilirubin
●
Consider gastroenterology or hepatology consultations.
●
Submit results for any additional testing and possible etiology on the LA-CRF or an 
appropriate document.
Study Discontinuation
In the absence of an explanation for increased LFT’s, such as viral hepatitis, pre-existing or 
acute liver disease or exposure to other agents associated with liver injury, the subject may be 
discontinued from the study. The investigator may determine that it is not in the subject’s 
best interest to continue study enrollment. Discontinuation of treatment should be considered 
if:
●
ALT or AST > 8 × ULN
●
ALT or AST > 5 × ULN for more than 2 weeks 
●
ALT or AST > 3 × ULN and TBL > 2 × ULN or INR > 1.5) (If INR testing is 
applicable/evaluated)
●
ALT or AST > 3 × ULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash and/or eosinophilia (> 5%). 
In addition, if close monitoring for a subject with moderate or severe hepatic laboratory tests 
is not possible, drug should be discontinued.
*Hy’s Law Definition-Drug-induced jaundice caused by hepatocellular injury, without a 
significant obstructive component, has a high rate of bad outcomes, from 10–50% mortality 
(or transplant).” The two “requirements” for Hy’s Law are: 1) Evidence that a drug can cause 
hepatocellular-type injury, generally shown by an increase in transaminase elevations higher 


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3 times the upper limit of normal (“2 x ULN elevations are too common in treated and 
untreated patients to be discriminating”). 2) Cases of increased bilirubin (at least 2 x ULN) 
with concurrent transaminase elevations at least 3x ULN and no evidence of intra- or extra-
hepatic bilirubin obstruction (elevated alkaline phosphatase) or Gilbert’s syndrome. [Temple 
R. Hy's law: predicting serious hepatotoxicity. Pharmacoepidemiol Drug Saf 2006 
Apr;15(4):241-3.]
Reference
Guidance for Industry titled “Drug-Induced Liver Injury: Premarketing Clinical Evaluation” 
issued by FDA on July 2009.  


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12.3
Common Serious Adverse Events
The following list of adverse events are considered common for the renal transplant study 
population and single occurrences of the AEs if serious, unexpected and related will not be 
reported in an expedited manner to the FDA and investigators.  Frequency of these events 
will be analyzed according to the protocol Section 7.5 and/or the Statistical Analysis Plan. If 
aggregate analysis of these events indicate that they occur more frequently with study drug an 
expedited report will be submitted to FDA as well as investigators.
1. Kidney transplant rejection
2. Kidney allograft loss
3. Increase in blood creatinine
4. Diabetes mellitus
5. Hyperglycemia
6. Increase in blood glucose


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12.4
Interim Decision Rule Details
The Interim report stopping rules for this study are based on a Bayesian methodology of the 
Beta-Binomial posterior distribution.  
The prior distribution used as the basis of the posterior is the Beta(α=2, β=8).  This prior was 
chosen due to its expectation (mean) of 0.20, and therefore remains consistent with the 
assumption that the twice daily tacrolimus DSA/IA composite endpoint incidence rate is 0.20 
(Section 7.1).  The variance of the distribution was also analyzed for robustness to the scaling 
of α and β, holding the expectation constant (mean of 0.20).  Note that in this case, the 
variance of the Beta distribution is inversely related to the scaling of parameters.  The 
decision rule cutoff values found in Section 7.8.1 remain robust to scaling of the parameters 
for anything from 1/1000 to unity (results not shown).  Decreasing the variance beyond what 
is associated with a Beta(α=2, β=8) distribution was not considered in order to maintain the 
conservative nature of weighting prior information.  
Let x be the number of occurrences of the DSA/IA composite primary endpoint, n be the 
number of evaluable subjects at the time of analysis, and p be the actual probability of the 
primary endpoint’s occurrence in the population of twice daily tacrolimus use.  Therefore, the 
posterior probability is based on the assumption that x given n and p is distributed as 
	
(, ).  Therefore given the prior distribution of |, ~	( = 2,  = 8) and a 
binomial distribution for the frequency of the DSA/IA primary endpoint 
(|, ~	
[, ]), then by Bayes’ rule the conditional distribution of p follows as a 
Beta-Binomial posterior of |, , , ~	(2 + , 8 +  −).
The following table [Table 1] illustrates the possible posterior probabilities of p ≥ 0.2 for the 
twice daily tacrolimus group given 50% of the planned subjects completed the 12 month 
follow-up visit (n = 110).  In particular, the distribution in this case is |, , , ~	(2 +
, 118 −).


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Table 1
50% of Subjects Complete 12 Month Visit (n=110)
Incidence of DSA/IA
Observed Proportion
Posterior Probabilities
x
p=x/110
P(p≥0.20|x,n,alpha,beta)
10
0.091
0.001
11
0.1
0.003
12
0.109
0.006
13
0.118
0.013
14
0.127
0.024
15
0.136
0.042
16
0.146
0.07
17
0.155
0.11
18
0.164
0.162
19
0.173
0.228
20
0.182
0.305
21
0.191
0.391
22
0.2
0.482
23
0.209
0.572
24
0.218
0.659
25
0.227
0.737
26
0.236
0.804
27
0.246
0.859
28
0.255
0.902
29
0.264
0.934
30
0.273
0.958
31
0.282
0.974
32
0.291
0.984
33
0.3
0.991
34
0.309
0.995
35
0.318
0.997
36
0.327
0.999


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12.5
Interim Stopping Rules Risk Analysis
The purpose of this appendix is to provide an assessment of favorable scientific outcomes 
that are possible under the conditions of early termination of the IDTX-MA-3004 study.  This 
analysis explores how it is possible to have a favorable scientific outcome if the study would 
be terminated early due to the immediate-release tacrolimus arm having a DSA/IA incidence 
rate less than 0.20.
All analyses in this report are based on SAS version 9.3, a 2-sided test, significance level of 
0.05, and power of at least 0.80.  Additionally, the conservative distributional assumptions of 
Fisher’s Exact Test are used.
The following figure and table illustrate the difference in statistical significance tests between
having a sample of 220 subjects per treatment group (study data from either the one year or 
two year study design) versus 110 subjects (early termination study data).  In this case, the 
difference between the boundaries of statistical significance for the two possible sample sizes 
illustrates the risk of the study having insufficient power to statistically detect a difference 
between the treatment arms due to  early termination of the study.  
For example, the first line of Table 1 represents a potential scenario where the immediate-
release tacrolimus cohort has a 0.19 incidence of DSA/ IA. In this scenario, if the sample size 
per treatment group is N=220, statistical significance will be met with at least 80% power if 
the Astagraf XL cohort’s incidence is 0.093 or less.  However, if the size of each treatment 
group is N=110, statistical significance will be met with at least 80% power if Astagraf XL 
cohort’s incidence of DSA/IA is less than or equal to 0.059.  Therefore, in this example 
scenario, the range between 0.059 and 0.093 for DSA/IA incidence in the Astagraf XL cohort 
shows the risk of study failure due to early study termination – i.e. had the study not 
terminated early, the Astagraf XL cohort’s DSA/IA incidence would be statistically 
distinguishable from the immediate-release tacrolimus cohort’s incidence; however, with the 
early termination, there is not sufficient statistical power to detect a difference in DSA/IA
incidence between the two cohorts.  
The calculations in the following table and figure show the incidence boundaries for at least 
80% power under incidence rates of DSA/IA in the immediate-release tacrolimus arm of 
0.04 to 0.19.  Values of Astagraf XL less than immediate-release tacrolimus values were 
investigated exclusively, as values of Astagraf XL being larger than immediate-release 
tacrolimus are not of scientific interest for this study.




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Figure 1
Astagraf XL and immediate-release tacrolimus DSA/IA proportions that 
are statistically significant with 220 and 110 subjects per group and at 
least 80% power.
Figure 1 illustrates statistical significance with 80% power on the continuum for all points 
falling above each of the respective lines.  For example, any points above the red line 
represent outcomes of statistical significance with at least 80% power for the full study 
sample of 220 subjects.  The same applies to the black line representing N=110 as the early 
termination sample size.  Therefore, the risk of early termination for the study is all points 
between each of the two lines in the figure.
This area of risk has been identified and mitigated with the current stopping rules in 
Section 7.8.1 of the study protocol.
Control DSA/IA Proportion
 0 04
 0 05
 0 06
 0 07
 0 08
 0 09
 0 10
 0 11
 0 12
 0 13
 0 14
 0 15
 0 16
 0 17
 0 18
 0 19
Astagraf XL DSA/IA Proportion
0 000
0 010
0 020
0 030
0 040
0 050
0 060
0 070
0 080
0 090
0 100
Statistical Significance with Control Rate < 0.20
Power = 80%, Significance = 0.05, Two-Sided Testing
Sample_Size
110
220


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13
ATTACHMENT 1: NON-SUBSTANTIAL AMENDMENT 2
ISN/Protocol IDTX-MA-3004
Non-Substantial Amendment 2
May 25, 2018
I.  The purpose of this amendment is:
Non-Substantial Changes
1. Revision of molecular profiling categories 
DESCRIPTION OF CHANGE
Sub-categories for the molecular profiling endpoints of cAR (clinical acute rejection) and 
subAR (subacute rejection) were removed throughout the protocol.  
RATIONALE:
Since the approval of the previous protocol amendment, Transplant Genomics, Inc. (the 
vendor performing the molecular categorization analyses) have revised their molecular 
categorizations; the changes in the amendment, therefore, reflect the categorizations available 
in the most contemporary version of Transplant Genomics’ TruGraf v2.0 test.  
2. Addition of two exploratory objectives to compare the molecular profiling results 
with creatinine and (where available) biopsy results 
DESCRIPTION OF CHANGE
Two exploratory objectives were added to assess creatinine results in subjects with the 
immune activation (IA) designation and to compare biopsy results for concordance, where 
available, in patients with an IA designation and abnormal creatinine. 
RATIONALE:
Given that the TruGraf 2.0 test has only been formally validated in patients with normal renal 
function, additional exploratory objectives were included to improve the confidence of results 
in all study patients (which also includes patients with abnormal creatinines).
3. Clarification of the requirement that all subjects must be taking Mycophenolate 
Mofetil (MMF) or its equivalent while on study
DESCRIPTION OF CHANGE
Language was inserted into the concomitant medications section and discontinuation criteria 
to clarify that patients must be maintained on MMF or its equivalent throughout the study. 
RATIONALE
Correction of previous oversight; MMF or its equivalent is given with tacrolimus per 
standard practice / product labeling requirements. 
4. Addition of discontinuation criteria related to tacrolimus discontinuation or 


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interruption
DESCRIPTION OF CHANGE
Discontinuation criteria were added to include discontinuation for subjects who permanently 
withdraw from tacrolimus or who have a significant reduction or interruption of tacrolimus 
for a period exceeding four weeks. 
RATIONALE:
In the previous amendment, the requirement for a minimum tacrolimus trough concentration 
was removed (due to variation in standard of care therapeutic drug monitoring levels at 
participating centers). This raised questions about whether subjects whose tacrolimus was 
intentionally reduced (e.g., temporarily, due to infection or other medical reasons) should be 
discontinued. These revised discontinuation criteria address those questions.  
5. Clarification that Astagraf XL may not be remotely dispensed without prior written 
approval from the Sponsor 
DESCRIPTION OF CHANGE
Language inserted to confirm that, due to study drug handling requirements, remotely 
dispensing Astagraf XL (for example, by mail or by courier) is prohibited without prior 
written approval from the Sponsor. 
RATIONALE
Several participating centers have provided feedback that language related to study drug 
handling is not sufficiently clear in the protocol for them to discern whether Astagraf XL 
may be sent directly to subjects via courier. Language was added to prevent centers from 
couriering study Astagraf XL to study subjects without adequate quality assurance. 
6. Minor edits to improve clarity of language
DESCRIPTION OF CHANGE
Grammatical, consistency, and other minor edits throughout the document. 
RATIONALE:
The changes are intended to facilitate better understanding and implementation of the study 
protocol.


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II Amendment Summary of Changes: Non-Substantial
1. Revision of molecular profiling categories 
III LIST OF ABBREVIATIONS AND DEFINITION OF KEY TERMS
Page 11, List of Abbreviations and Definitions of Key Terms
DELETED:
cAR
Clinical Acute Rejection
1. Revision of molecular profiling categories 
Page 13, List of Abbreviations and Definitions of Key Terms
DELETED:
subAR
Subacute Rejection
1. Revision of molecular profiling categories 
IV Synopsis, Study Objective(s) and 2.1 Study Objectives 
Page 15; IV. Synopsis – Study Objective(s) 
Page 42; Section 2.1.2 Secondary Objectives
WAS:

To assess the risk factors for each of the following outcomes: DSA formation; IA (and 
separately for cAR [clinical acute rejection] and subAR [subacute rejection]); transplant 
glomerulopathy (TG); acute and chronic forms of antibody-mediated rejection (ABMR); 
C1q-binding DSA; HLA-DQ DSA; DSA IgG3 isotype; requirement for and type of 
antibody reduction required; various threshold levels of estimated glomerular filtration 
rate (eGFR) (less than 30, 40, and 50 mL/min/1.73 m2); and the four components of the 
traditional composite endpoint, consisting of graft loss, mortality, biopsy-proven acute 
rejection (BPAR), and loss to follow-up; and additionally, the persistence of DSA and 
IA.
IS AMENDED TO:

To assess the risk factors for each of the following outcomes: DSA formation;, IA (and 
separately for cAR [clinical acute rejection] and subAR [subacute rejection]); transplant 
glomerulopathy (TG); acute and chronic forms of antibody-mediated rejection (ABMR); 
C1q-binding DSA; HLA-DQ DSA; DSA IgG3 isotype; requirement for and type of 
antibody reduction required; various threshold levels of estimated glomerular filtration 
rate (eGFR) (less than 30, 40, and 50 mL/min/1.73 m2); and the four components of the 
traditional composite endpoint, consisting of graft loss, mortality, biopsy-proven acute 
rejection (BPAR), and loss to follow-up; and additionally, the persistence of DSA and 
IA.


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1. Revision of molecular profiling categories 
IV Synopsis, Study Objective(s) and 2.1 Study Objectives
Page 16; IV. Synopsis – Study Objective(s) 
Page 43; Section 2.1.2 Secondary Objectives
WAS:

Compare the persistence of the development of cAR and subAR on molecular profiling 
between the two cohorts across the study duration in those patients who develop IA on 
molecular profiling.
IS AMENDED TO:

Compare the persistence of the development of cAR and subAR IA on molecular 
profiling between the two cohorts across the study duration in those patients who 
develop IA on molecular profiling.
1. Revision of molecular profiling categories 
IV Synopsis, Study Objective(s) and 2.1 Study Objectives
Page 16; IV. Synopsis – Study Objective(s) 
Page 43; Section 2.1.3 Exploratory Objectives
WAS:

To assess and compare, between treatment groups, the association/correlation of 
incidence and strength of DSA, and the incidences of HLA-DQ DSA, C1q-binding DSA, 
and DSA IgG3 isotype with each other, and individually, with each of the following: IA
(and components thereof), TG, ABMR (both chronic forms and acute), Banff histology, 
various thresholds of eGFR (less than 30, 40, and 50 mL/min/1.73 m2), eGFR change 
over time, TCMR, graft loss, and mortality.
IS AMENDED TO:

To assess and compare, between treatment groups, the association/correlation of 
incidence and strength of DSA, and the incidences of HLA-DQ DSA, C1q-binding DSA, 
and DSA IgG3 isotype with each other, and individually, with each of the following: IA
(and components thereof), TG, ABMR (both chronic forms and acute), Banff histology, 
various thresholds of eGFR (less than 30, 40, and 50 mL/min/1.73 m2), eGFR change 
over time, TCMR, graft loss, and mortality.
1. Revision of molecular profiling categories 
IV Synopsis, Study Objective(s) and 2.1 Study Objectives
Page 16; IV. Synopsis – Study Objective(s) 
Page 43; Section 2.1.3  Exploratory Objectives
ADDED:

Examine the proportion of patients with the IA designation who have normal and 
abnormal creatinine.   

In patients with an IA designation and abnormal creatinine, examine biopsies (if 
available) for concordance.


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1. Revision of molecular profiling categories 
IV Synopsis, Study Objective(s) and 2.1 Study Objectives
Page 16; IV. Synopsis – Study Objective(s) 
Page 43; Section 2.1.3 Exploratory Objectives
WAS:

To assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with each of the following: TG, chronic forms of ABMR, C1q-
binding DSA, HLA-DQ DSA, DSA IgG3 isotype, IA (and components thereof), 
histopathology, graft loss, various thresholds of eGFR (< 30, 40, and 
50 mL/min/1.73 m2), acute ABMR, TCMR, and mortality in patients who develop IA on 
molecular profiling in each cohort.
IS AMENDED TO:

To assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with each of the following: TG, chronic forms of ABMR, C1q-
binding DSA, HLA-DQ DSA, DSA IgG3 isotype, IA, (and components thereof), 
histopathology, graft loss, various thresholds of eGFR (< 30, 40, and 
50 mL/min/1.73 m2), acute ABMR, TCMR, and mortality in patients who develop IA on 
molecular profiling in each cohort.
1. Revision of molecular profiling categories 
IV Synopsis, Study Design Overview and 2.2 Study Design and Dose Rationale
Page 17; IV. Synopsis – Study Design Overview
Page 44; Section 2.2.1 Study Design 
WAS:
For the purposes of this study, IA will be defined as a molecular signature indicating either 
clinical acute rejection (cAR) or subacute rejection (subAR). 
IS AMENDED TO:
For the purposes of this study, IA will be defined as a positive molecular signature indicating 
either clinical acute rejection (cAR) or subacute rejection (subAR) using the Trugraf v2.0 
molecular assay (Transplant Genomics, Inc., Pleasanton, CA) in all patients.
1. Revision of molecular profiling categories 
IV Synopsis, Endpoints for Evaluation and 2.3 Endpoints
Page 24; IV. Synopsis – Endpoints for Evaluation (Primary)
Page 47; Section 2.3.1 Primary Endpoints
WAS:
For reporting purposes, IA will be considered either present or absent.
IS AMENDED TO:
For reporting purposes, IA will be considered either present or absent using the Trugraf™ 
v2.0 molecular assay.  For the purposes of the study, a negative designation (Trugraf 


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TX Normal) will be referred to as Immune Quiescence (IQ).  Due to operating 
characteristics of the assay, a positive designation will be considered evidence of 
Immune Activation (IA) in all patients.
1. Revision of molecular profiling categories 
IV Synopsis, Endpoints for Evaluation and 2.3 Endpoints
Page 24; IV. Synopsis – Endpoints for Evaluation (Secondary)
Page 47; Section 2.3.2 Secondary Endpoints
WAS:
Comparisons of the cohorts with respect to the various molecular designations as well as 
correlating the results with DSA will rely on the categorical (binary) variable of positivity for 
the following molecular designations:  Transplant Normal (TX), otherwise referred to as 
Immune Quiescence (IQ); and Clinical Acute Rejection [cAR, inclusive of Sub-acute Acute 
Rejection (subAR)], also referred to as Immune Activation (IA). A designation of subAR will 
be given to patients who exhibit cAR on peripheral blood molecular profiling in the setting of 
normal renal function as defined by a stable serum creatinine ranging from between 0.7 and 
2.4 ng/mL on two separate measurements at least one month apart (stability further defined 
by no more than a 20% difference between the two individual measurements).  Biopsies, 
when available, will be used to inform the molecular diagnosis when alternative pathology is 
recognized (i.e. recurrent disease). 
Comparisons between treatment cohorts regarding incidence will rely upon assessments for 
each of the following results at any point in the study:

DSA

IA (inclusive of cAR and subAR)

TG
IS AMENDED TO:
Comparisons of the cohorts with respect to the various molecular designations as well as 
correlating the results with DSA will rely on the categorical (binary) variable of positivity
using the TrugrafTM v2.0 molecular assay. for the following molecular designations:  
Transplant Normal (TX), otherwise referred to as Immune Quiescence (IQ); and Clinical 
Acute Rejection [cAR, inclusive of Sub acute Acute Rejection (subAR)], also referred to as 
Immune Activation (IA). A designation of subAR will be given to patients who exhibit cAR 
on peripheral blood molecular profiling in the setting of normal renal function as defined by a 
stable serum creatinine ranging from between 0.7 and 2.4 ng/mL on two separate 
measurements at least one month apart (stability further defined by no more than a 20% 
difference between the two individual measurements).  Biopsies, when available, will be used 
to inform the molecular diagnosis when alternative pathology is recognized (i.e. recurrent 
disease). 
Comparisons between treatment cohorts regarding incidence will rely upon assessments for 
each of the following results at any point in the study:

DSA

IA (inclusive of cAR and subAR)


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
TG
1. Revision of molecular profiling categories 
IV Synopsis, Endpoints for Evaluation and 2.3 Endpoints
Page 25; IV. Synopsis – Endpoints for Evaluation (Secondary)
Page 48; Section 2.3.2 Secondary Endpoints
WAS:
Persistence of DSA and IA will be additional endpoints for comparisons in patients who 
develop IA and DSA on molecular profiling, with specific focus on subAR.
IS AMENDED TO:
Persistence of DSA and IA will be additional endpoints for comparisons in patients who 
develop IA and DSA on molecular profiling, with specific focus on subAR.
1. Revision of molecular profiling categories 
IV Synopsis, Statistical Methods
Page 26; IV. Synopsis – Statistical Methods (Efficacy)
WAS:
The primary efficacy endpoint is the incidence of any one of the following in the first two 
years post-transplant: 

DSA formation 

IA (cAR and subAR)
IS AMENDED TO:
The primary efficacy endpoint is the incidence of any one of the following in the first two 
years post-transplant: 

DSA formation 

IA (cAR and subAR)
1. Revision of molecular profiling categories 
IV Synopsis, Statistical Methods
Page 27; IV. Synopsis – Statistical Methods (Efficacy)
WAS:
The scope of these analyses will include the following: DSA formation, IA (and components 
thereof), TG, acute and chronic forms of ABMR, C1q-binding DSA, HLA-DQ DSA, DSA 
IgG3 isotype, the requirement for antibody reduction therapy, chronic graft dysfunction, graft 
loss, local BPAR, mortality, loss to follow-up, ordinal antibody strength, MFI score, 
histopathology assessments, and tacrolimus trough levels.  
IS AMENDED TO:
The scope of these analyses will include the following: DSA formation, IA (and components 
thereof), TG, acute and chronic forms of ABMR, C1q-binding DSA, HLA-DQ DSA, DSA 


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IgG3 isotype, the requirement for antibody reduction therapy, chronic graft dysfunction, graft 
loss, local BPAR, mortality, loss to follow-up, ordinal antibody strength, MFI score, 
histopathology assessments, and tacrolimus trough levels.  
1. Revision of molecular profiling categories 
1.1 Background
Page 33; Section 1.1 Background
WAS:
Using multiple, three-way classifier tools to validate 200 of the highest value probesets, the 
groups at Northwestern and Scripps have identified molecular signatures in the peripheral 
blood for the following five clinical phenotypes: 1) clinical acute rejection (cAR), 2) subAR, 
3) immune quiescence (Transplant eXcellent [TX]), 4) acute dysfunction/no rejection 
(ADNR), and 5) chronic rejection (CR)… 
IS AMENDED TO:
Using multiple, three-way classifier tools to validate 200 of the highest value probesets, the 
groups at Northwestern and Scripps have identified a variety of molecular signatures in the 
peripheral blood. for the following five clinical phenotypes: 1) clinical acute rejection (cAR), 
2) subAR, 3) immune quiescence (Transplant eXcellent [TX]), 4) acute dysfunction/no 
rejection (ADNR), and 5) chronic rejection (CR).
1. Revision of molecular profiling categories 
5.3 Efficacy Assessment
Page 60; Section 5.3.8 Molecular Endpoints
WAS:
5.3.8 Molecular Endpoints (cAR, subAR, and TX)
IS AMENDED TO:
5.3.8 Molecular Endpoints (cAR, subAR, and TX)
1. Revision of molecular profiling categories 
5.3 Efficacy Assessment
Page 61; Section 5.3.8, Molecular Endpoints
WAS:
Each patient will be classified into one of the three currently validated phenotypes (TX/cAR/  
subAR, see above). Patients with the cAR designation in the setting of a normal creatinine 
(as defined by a value between 0.7 and 2.4 mg/dL and stable based on at least two reads over 
one month apart where stable is defined as no more than 20% difference between the 
creatinine levels) will be further given the sub-designation of subAR.  Classification by 
phenotypes will be done with every scheduled blood test completed. A final phenotype will 
be created when patients complete the protocol at 1 year and / or at the conclusion of the 
study. The posterior probabilities for each new classification (essentially equivalent to CIs) 
will also be recorded. The above-mentioned classifiers have already been “locked” during 


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the discovery process to avoid over-training a classifier on new data. ‘IA’ signatures will be 
recorded. As a dichotomous variable, ‘IA’ will represent any patient sample classified as 
cAR or subAR by the Diagonal Linear Discriminant Analysis (DLDA) and Vector Machines 
classifiers. In turn, ‘IQ’ will represent any patient sample classified as TX.
IS AMENDED TO:
Each patient will be classified into one of the three currently validated phenotypes (TX/cAR/  
subAR, see above). Patients with the cAR designation in the setting of a normal creatinine 
(as defined by a value between 0.7 and 2.4 mg/dL and stable based on at least two reads over 
one month apart where stable is defined as no more than 20% difference between the 
creatinine levels) will be further given the sub designation of subAR.  Classification by 
phenotypes will be done with every scheduled blood test completed. A final phenotype will 
be created when patients complete the protocol at 1 year and / or at the conclusion of the 
study. The posterior probabilities for each new classification (essentially equivalent to CIs) 
will also be recorded. For the purposes of the study, assessment and comparison of the 
incidence of the molecular endpoint will rely on a binary molecular designation in 
accordance with the TruGraf v2.0 molecular assay.  A negative designation (Trugraf 
TX Normal) will be referred to as Immune Quiescence (IQ).  Due to operating 
characteristics of the assay, a positive designation will be considered evidence of 
Immune Activation (IA) in all patients.
The above-mentioned classifiers have has already been “locked” during the discovery 
process to avoid over-training a classifier on new data. ‘IA’ signatures will be recorded. As a 
dichotomous variable, ‘IA’ ‘IQ’ will represent any patient sample classified as cAR or 
subAR not positive. by the Diagonal Linear Discriminant Analysis (DLDA) and Vector 
Machines classifiers. In turn, ‘IQ’ ‘IA’ will represent any patient sample not classified as 
TX ‘IQ’.
1. Revision of molecular profiling categories 
5.4 Safety Assessment
Page 63; Section 5.4.3 Laboratory and Pathological Assessments
WAS:
●
Molecular diagnostics (cAR, subAR, and TX; not collected at baseline)
IS AMENDED TO:
●
Molecular diagnostics (cAR, subAR, and TX; not collected at baseline)
1. Revision of molecular profiling categories 
7.4 Analysis of Efficacy
Page 75; Section 7.4.2 Analysis of Secondary Endpoints
WAS:
1. DSA formation 


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2. IA (and components thereof)
3. TG
IS AMENDED TO:
1. DSA formation 
2. IA (and components thereof)
3. TG
1. Revision of molecular profiling categories 
7.4 Analysis of Efficacy
Page 76; Section 7.4.2 Analysis of Secondary Endpoints
WAS:
Assessments of association between DSA and IA (and its subcomponents) will be analyzed 
overall and by treatment group with logistic regression models using DSA formation as the 
response and IA as predictor.
IS AMENDED TO:
Assessments of association between DSA and IA (and its subcomponents) will be analyzed 
overall and by treatment group with logistic regression models using DSA formation as the 
response and IA as predictor.
1. Revision of molecular profiling categories 
7.4 Analysis of Efficacy
Page 77; Section 7.4.3 Analysis of Exploratory Endpoints
WAS:
The associations between each of HLA-DQ DSA incidence, ordinal DSA strength, DSA 
formation, C1q-binding DSA, DSA IgG3 isotype will be analyzed with respect to each other 
and with IA, TG, acute and chronic ABMR, histopathology, cAR (and subAR), eGFR at 
various thresholds (less than 30, 40, and 50 mL/min/1.73 m2), ABMR (acute and chronic), 
TCMR, graft loss, and mortality. 
IS AMENDED TO:
The associations between each of HLA-DQ DSA incidence, ordinal DSA strength, DSA 
formation, C1q-binding DSA, DSA IgG3 isotype will be analyzed with respect to each other 
and with IA, TG, acute and chronic ABMR, histopathology, cAR (and subAR), eGFR at 
various thresholds (less than 30, 40, and 50 mL/min/1.73 m2), ABMR (acute and chronic), 
TCMR, graft loss, and mortality.  
1. Revision of molecular profiling categories 
7.6 Analysis of Tacrolimus Dose and Trough Concentrations


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Page 78; Section 7.6 Analysis of Tacrolimus Dose and Trough Concentrations
WAS:
The association between whole blood concentrations and the following endpoints will be 
assessed: DSA presence, DSA strength, C1q-binding DSA, IgG3, IA (cAR / subAR), eGFR, 
histopathology, and conventional measures of transplant outcomes.
IS AMENDED TO:
The association between whole blood concentrations and the following endpoints will be 
assessed: DSA presence, DSA strength, C1q-binding DSA, IgG3, IA (cAR / subAR), eGFR, 
histopathology, and conventional measures of transplant outcomes.
2. Added two exploratory objectives to compare the molecular profiling results with 
creatinine and (where available) biopsy results.
IV Synopsis, Study Design Overview and 2.2 Study Design and Dose Rationale
Page 18; IV. Synopsis – Study Design Overview
Page 46; Section 2.2.1 Study Design
WAS:
Up to two serum creatinine levels obtained at least one month apart, if performed per SOC, 
should also be recorded.
IS AMENDED TO:
Up to The two closest serum creatinine levels to the study visit, obtained at least one month 
apart, if performed per SOC, should also be recorded.
2. Added two exploratory objectives to compare the molecular profiling results with 
creatinine and (where available) biopsy results
7.4 Analysis of Efficacy
Page 74; Section 7.4.1.2 Sensitivity Analysis
ADDED:
Additionally, a sensitivity analysis for mFAS patients will be performed exclusively 
using assessments with a basis in normal creatinine function.
2. Added two exploratory objectives to compare the molecular profiling results with 
creatinine and (where available) biopsy results
7.4 Analysis of Efficacy
Page 78; Section 7.4.2 Analysis of Secondary Endpoints
ADDED:


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The incidence of patients with normal and abnormal creatinine assessments and an IA 
designation will be summarized with descriptive statistics.  Concordance between the IA 
and biopsy results will be summarized with descriptive statistics for each of the normal 
and abnormal creatinine assessments.  Analysis of concordance between the IA and 
biopsy results will be conducted using Cohen’s Kappa for each of the abnormal and 
normal creatinine assessments.
3. Clarification of the requirements for subjects to be taking MMF or its equivalent.
IV Synopsis, Concomitant Medication Restrictions or Requirements
Page 23; IV. Synopsis – Concomitant Medication Restrictions or Requirements
WAS:
Non-tacrolimus based immunosuppressive regimens (i.e. cyclosporine, everolimus, sirolimus, 
belatacept), as well as all forms of extended-release tacrolimus other than Astagraf XL, are 
prohibited during the course of the study. Due to the influence these medications have on 
tacrolimus blood concentrations, antiviral medications used in HCV and HIV treatment, as 
well as isoniazid, Rifampin, ethambutol, and pyrazinamide are also prohibited.
IS AMENDED TO:
Non-tacrolimus based immunosuppressive regimens (i.e. cyclosporine, everolimus, sirolimus, 
belatacept), as well as all forms of extended-release tacrolimus other than Astagraf XL, are 
prohibited during the course of the study. As a clarifying statement, all patients must be 
maintained on MMF or its equivalent during the course of the study.  Due to the 
influence these the following medications have on tacrolimus blood concentrations, antiviral 
medications used in HCV and HIV treatment, as well as isoniazid, Rifampin, ethambutol, and 
pyrazinamide are also prohibited.
3. Clarification of the requirements for subjects to be taking MMF or its equivalent.
IV Synopsis, Formal Stopping Rules and 6.1 Discontinuation of Individual Subject(s)
Page 24; IV. Synopsis – Discontinuation Criteria from Study for Individual Subjects
Page 71; Section 6.1 Discontinuation of Individual Subject(s)
ADDED:

Removal from an MMF-containing immunosuppressive regimen (or its 
equivalent);
4. Addition of discontinuation criteria for subjects who permanently withdraw from 
tacrolimus or who have a prolonged significant reduction or interruption of tacrolimus 
for a period of greater than four weeks.
IV Synopsis, Investigational Product(s) & Comparative Drug(s) 
Page 22; IV. Synopsis – Investigational Product(s)  
Page 22; IV. Synopsis – Comparative Drug(s) 
ADDED:
A prolonged significant reduction of tacrolimus or withdrawal of tacrolimus will result 


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Page 122 of 124
in study discontinuation (see discontinuation criteria). 
4. Addition of discontinuation criteria for subjects who permanently withdraw from 
tacrolimus or who have a prolonged significant reduction or interruption of tacrolimus 
for a period of greater than four weeks.
IV Synopsis, Formal Stopping Rules and 6.1 Discontinuation of Individual Subject(s)
Page 24; IV. Synopsis – Discontinuation Criteria from Study for Individual Subjects
Page 71; Section 6.1, Discontinuation of Individual Subject(s)
WAS:

With regard to subjects receiving Astagraf XL,  failure to achieve a tacrolimus whole 
blood concentration ≥ 6 ng/mL for 4 consecutive weeks during the first 6 weeks 
following transplantation;

With regard to subjects receiving immediate-release tacrolimus, failure to achieve a 
tacrolimus whole blood concentration ≥6 ng/mL for 4 consecutive weeks during the first 
6 weeks following transplantation;
IS AMENDED TO:

Removal from an MMF-containing immunosuppressive regimen (or its equivalent);

With regard to subjects receiving Astagraf XL, failure to achieve a tacrolimus whole 
blood concentration ≥ 6 ng/mL for 4 consecutive weeks during the first 6 weeks 
following transplantation;

With regard to subjects receiving immediate release tacrolimus, fFailure to achieve a 
tacrolimus whole blood concentration ≥ 6 ng/mL for 4 consecutive weeks during the first 
6 weeks following transplantation;

Permanent withdrawal of tacrolimus; 

Interruption or prolonged significant reduction of tacrolimus (as an example, a 
50% reduction or dose at a level of less than 3.5ng/mL) for periods exceeding 4 
weeks; 
4. Addition of discontinuation criteria for subjects who permanently withdraw from 
tacrolimus or who have a prolonged significant reduction or interruption of tacrolimus 
for a period of greater than four weeks.
5.1 Dosing and Administration of Study Drug(s) and Other Medication(s)
Page 56; Section 5.1.1 Dose/Dose Regimen and Administration Period
ADDED:
Patients whose tacrolimus is withdrawn or who have a prolonged significant reduction of 
tacrolimus levels will be discontinued from the study (see Section 6.1).
4. Addition of discontinuation criteria for subjects who permanently withdraw from 
tacrolimus or who have a prolonged significant reduction or interruption of tacrolimus 
for a period of greater than four weeks.
5.1 Dosing and Administration of Study Drug(s) and Other Medication(s)
Page 57; Section 5.1.2 Increase or Reduction in Dose of Study Drug(s)


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ADDED:
If a prolonged period of reduced immunosuppression will be required to treat infection, 
sepsis, delayed wound healing, or other circumstances, patients should be removed from 
the study as per the discontinuation criteria (Section 6.1).
5. Confirmation that study Astagraf XL may not be remotely dispensed without prior 
written approval from the Sponsor.
4.3 Study Drug Handling
Page 55; Section 4.3 Study Drug Handling
ADDED:
It is prohibited to remotely dispense study Astagraf XL (i.e. by mail or courier) to 
subjects unless there are exceptional circumstances and prior approval from the 
Sponsor is received in writing. 
III  Non-Substantial Amendment Rationale:
Rationale for Non-Substantial Designation
The changes incorporated into this amendment do not increase any potential harm or affect
the safety of the subject. The revisions are meant to provide clarity for protocol procedures
and allow for enrollment of appropriate subjects.



SAP_001.pdf: 
Astagraf XL® to Understand the Impact of 
Immunosuppression on De Novo DSA Development and 
Chronic Immune Activation in Kidney Transplantation
ISN/Protocol IDTX-MA-3004
ClinicalTrials.gov Identifier: NCT02723591
Date of SAP v2.0: 9 Dec 2019
Sponsor: Astellas Pharma Global Development (APGD)
Medical Affairs, Americas
1 Astellas Way
Northbrook, IL 60062


Sponsor: Astellas Pharma Global Development, Inc. 
SAP Version:  2.00 
ISN/Protocol: IDTX-MA-3004 
09 Dec 2019 
Astellas 
 
Page 1 of 38 
 
 
 
STATISTICAL ANALYSIS PLAN 
 
Version 2.00, 09 December 2019 
 
Astagraf XL® to Understand the Impact of Immunosuppression on De Novo DSA 
Development and Chronic Immune Activation in Kidney Transplantation 
 
 
 
ISN/Protocol: IDTX-MA-3004 
 
IND number: 64148 
 
 
 
Sponsor: 
Astellas Pharma Global Development, Inc. (APGD) 
Medical Affairs, Americas 
1 Astellas Way 
Northbrook, IL 60062 
 
 
 
 
 
 
 
 
 
This confidential document is the property of the sponsor.  No unpublished information 
contained in this document may be disclosed without prior written approval of the sponsor. 


Sponsor: Astellas Pharma Global Development, Inc. 
SAP Version:  2.00 
ISN/Protocol: IDTX-MA-3004 
09 Dec 2019 
Astellas 
 
Page 2 of 38 
Table of Contents 
I. 
LIST OF ABBREVIATIONS AND KEY TERMS ··········································· 4 
1 
INTRODUCTION ·················································································· 6 
2 
FLOW CHART AND VISIT SCHEDULE ···················································· 7 
3 
STUDY OBJECTIVE(S) AND DESIGN ······················································ 11 
3.1 
Study Objective(s) ············································································ 11 
3.1.1 
Primary Objective ······································································· 11 
3.1.2 
Secondary Objectives ··································································· 11 
3.1.3 
Exploratory Objectives ································································· 12 
3.2 
Study Design ··················································································· 12 
3.3 
Randomization················································································· 14 
4 
SAMPLE SIZE ···················································································· 15 
5 
ANALYSIS SETS ················································································· 15 
5.1 
Full Analysis Set (FAS) ······································································ 15 
5.2 
Modified Full Analysis Set (mFAS) ························································ 15 
5.3 
Per Protocol Set (PPS) ········································································ 15 
5.4 
Safety Analysis Set (SAF) ··································································· 16 
5.5 
Biopsy Analysis Set (BAS) ·································································· 16 
5.6 
Pharmacokinetics Analysis Set (PKAS) ···················································· 16 
5.7 
Pharmacodynamic Analysis Set (PDAS) ··················································· 16 
6 
ANALYSIS VARIABLES ······································································· 16 
6.1 
Efficacy Endpoints ············································································ 16 
6.1.1 
Primary Efficacy Endpoint(s) ·························································· 16 
6.1.2 
Secondary Efficacy Endpoints ························································· 16 
6.1.3 
Exploratory Efficacy Endpoints ······················································· 19 
6.1.4 
Other Efficacy Variables ······························································· 19 
6.2 
Safety Variables ··············································································· 19 
6.3 
Pharmacokinetic Variables ··································································· 20 
6.4 
Pharmacodynamic Variables ································································ 20 
6.5 
Other Variables ················································································ 20 
7 
STATISTICAL METHODOLOGY ··························································· 22 
7.1 
General Considerations ······································································· 22 


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7.2 
Study Population ·············································································· 23 
7.2.1 
Disposition of Subjects ································································· 23 
7.2.2 
Protocol Deviations ····································································· 23 
7.2.3 
Demographic and Other Baseline Characteristics ··································· 24 
7.2.4 
Previous and Concomitant Medications ·············································· 25 
7.3 
Study Drugs ···················································································· 26 
7.3.1 
Exposure ·················································································· 26 
7.3.2 
Treatment Compliance ·································································· 26 
7.3.3 
Tacrolimus Trough Concentrations ··················································· 27 
7.4 
Analysis of Efficacy ·········································································· 27 
7.4.1 
Analysis of Primary Endpoint(s) ······················································ 27 
7.4.2 
Analysis of Secondary Endpoints ····················································· 29 
7.4.3 
Analysis of Exploratory Endpoints ···················································· 31 
7.4.4 
Analysis of Other Variables ···························································· 31 
7.5 
Analysis of Safety ············································································· 31 
7.5.1 
Adverse Events ·········································································· 31 
7.5.2 
Clinical Laboratory Evaluation ························································ 32 
7.5.3 
Vital Signs ················································································ 33 
7.5.4 
Electrocardiograms (ECGs) ···························································· 33 
7.5.5 
Other Safety-Related Observations ··················································· 33 
7.6 
Analysis of PK················································································· 33 
7.7 
Analysis of PD················································································· 33 
7.8 
Other Analyses ················································································ 33 
7.9 
Interim Analysis ··············································································· 34 
7.10 
Handling of Missing Data, Outliers, Visit Windows, and Other Information ········· 34 
7.10.1 
Missing Data ············································································· 34 
7.10.2 
Outliers ··················································································· 34 
7.10.3 
Visit Windows ··········································································· 34 
8 
DOCUMENT REVISION HISTORY ························································· 36 
9 
REFERENCES ···················································································· 37 
10 
AUTHOR AND APPROVER SIGNATORIES ·············································· 38 
 
 
 


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I. 
LIST OF ABBREVIATIONS AND KEY TERMS 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
ABMR 
Antibody –Mediated Rejection 
AE 
Adverse Event 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Transaminase 
API 
Astellas Pharma Inc 
ASCM 
Analysis Set Classification Meeting  
AST 
Aspartate Transaminase 
ATC 
Anatomical Therapeutic Chemical 
BAS 
Biopsy Analysis Set 
BID 
Twice Daily 
BMI 
Body Mass Index 
BPAR 
Biopsy-Proven Acute Rejection 
CI 
Confidence Intervals 
CIT 
Cold Ischemia Time 
C1q 
Complement Component 1, Q Subcomponent 
CRF 
Case Report Form 
cPRA 
Calculated Panel Reactivity Antibody 
CSR 
Clinical Study Report 
DCD 
Donation after Circulatory Death 
DSA 
Donor Specific Antibody 
ECD 
Extended Criteria Donor 
ECG 
Electrocardiogram 
eGFR 
Estimated Glomerular Filtration Rate 
ESRD 
End of Stage Renal Disease 
FAS 
Full Analysis Set 
GD 
Global Development 
HLA 
Human Leukocyte Antigen 
IA 
Immune Activation 
ICH 
International Conference on Harmonization 
IFTA 
Interstitial Fibrosis and Tubular Atrophy 
IgG3 
Immunoglobulin G 
IPV 
Intrapatient Variability 
IRT 
Interactive Response Technology 
IVIG 
Intravenous Immunoglobulin 
KDPI 
Kidney Donor Profile Index 
MDRD-4 
Modification of Diet in Renal Disease 
MedDRA 
Medical Dictionary for Regulatory Activities 
mFAS 
Modified Full Analysis Set 
MFI 
Mean Fluorescence Intensity 
MMF 
Mycophenolate Mofetil 
PD 
Pharmacodynamic 
PD1-x 
Protocol Deviation 1-x 
PDAS 
Pharmacodynamic Analysis Set 
PK 
Pharmacokinetic 
PKAS 
Pharmacokinetics Analysis Set 
PPS 
Per-Protocol Analysis Set 


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Abbreviations 
Description of abbreviations 
PT 
Preferred Term 
SAF 
Safety Analysis Set 
SAP 
Statistical Analysis Plan 
SAS  
Statistical Analysis Software 
SoC 
Standard of Care 
SOC 
System Organ Class 
TBL 
Total Bilirubin 
TCMR 
T-cell Mediated Rejection 
TEAE 
Treatment Emergent Adverse Event 
TG 
Transplant Glomerulopathy 
TGI 
Transplant Genomics Inc. 
TLF 
Tables, Listings and Figures 
ULN 
Upper Limit of Normal 
WHO-DD 
World Health Organization Drug Dictionary 
WIT 
Warm Ischemia Time 
XM 
Cross-Matching 
 
List of Key Terms 
Terms 
Definition of terms 
Endpoint 
A variable that pertains to the trial objectives 
Variable 
Any quantity that varies; any attribute, phenomenon or event that can have 
different qualitative or quantitative values. 
 


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1 
INTRODUCTION 
This Statistical Analysis Plan (SAP) contains a more technical and detailed elaboration of the 
principal features of the analysis described in the protocol, and includes detailed procedures 
for executing the statistical analysis of the primary and secondary endpoints and other data.  
This statistical analysis is coordinated by the responsible biostatistician of Global 
Development (GD), Astellas Pharma Inc. (API).  Any changes from the analyses planned in 
the SAP will be justified in the Clinical Study Report (CSR). 
Prior to database hard lock, a final review of data and TLFs meeting will be held to allow a 
review of the clinical trial data and to verify the data that will be used for analysis set 
classification.  If required, consequences for the statistical analysis will be discussed and 
documented.  A meeting to determine analysis set classifications may also be held prior to 
database hard lock.


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2 
FLOW CHART AND VISIT SCHEDULE 
 
 
 


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3 
STUDY OBJECTIVE(S) AND DESIGN 
3.1 
Study Objective(s) 
3.1.1 
Primary Objective 
The primary objective is to compare the incidence of a two-part composite endpoint 
consisting of de novo donor specific antibody (DSA) formation or a designation of immune 
activation (IA) on peripheral blood molecular profiling in subjects maintained on twice daily, 
immediate-release tacrolimus versus those maintained on Astagraf XL in the first year post-
transplant. 
3.1.2 
Secondary Objectives 
The secondary objectives of the study, as stated in the protocol, have been further clarified 
and adjusted to more closely address the research questions and in consideration of data 
availability.  They are to: 
• Assess the risk factors for each of the following outcomes: Interstitial Fibrosis and 
Tubular Atrophy (IFTA), IFTA and Inflammation, biopsy-proven acute rejection  
(BPAR), the four-part traditional composite endpoint, early mycophenolate mofetil  
(MMF) interruption/discontinuation, and estimated glomerular filtration rate (eGFR) 
status based on three thresholds 30, 40, and 50 at one year. 
• 
Assess and compare, between treatment groups, the association/correlation of the 
appearance of DSA with the development of IA on molecular profiling at one year. 
• 
Assess and compare, between treatment groups, the incidence of each of the following, 
occurring over the course of one year : De novo DSA, C1q-binding DSA, DSA IgG3 
isotype, IA, transplant glomerulopathy (TG), select BANFF histology grades (acute and 
chronic forms of antibody-mediated rejection (ABMR), acute and chronic active T-cell 
mediated rejection (TCMR), borderline changes, and IFTA), graft loss, mortality, and 
BPAR. 
• 
Compare, between treatment groups, the incidence of various thresholds of eGFR (less 
than 30, 40, 50 mL/min/1.73 m2) at Day 365 visit and a five-point decline from Day 30 
to Day 365). 
• 
Summarize the distribution of ordinal categories of antibody strength (weak, moderate, 
strong) between treatment groups across the 1-year study time course. 
• 
Summarize maximum Mean Fluorescence Intensity (MFI) scores by treatment group 
across the duration of the study. 
• 
Examine histopathology in cases where biopsies were taken for maintenance (up to 
fourteen months) between treatment groups. In cases where biopsies were performed for 
cause, they will be examined separately.  
• 
Summarize tacrolimus manufacturer and the number of switches between immediate-
release tacrolimus products by the occurrence of study composite endpoint. 
• 
Summarize tacrolimus manufacturer and the number of switches by the status of renal 
dysfunction.  
• 
Summarize adverse events (AEs) between treatment groups. 


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• 
Summarize the absolute and percent changes in eGFR over the study duration beginning 
from 30 days post-transplant between groups, and in subjects who develop DSA or IA.  
• 
Compare the persistence of the development of IA on molecular profiling between two 
treatment groups across the study duration in those subjects who develop IA on 
molecular profiling. 
 
3.1.3 
Exploratory Objectives 
Exploratory objectives are: 
• 
Compare the frequency of those subjects experiencing either component of a two-part 
composite endpoint consisting of DSA formation and the presence of TG (cg > 0) on 
centrally-interpreted institutionally-derived protocol (maintenance) biopsy and /or biopsy 
obtained for cause during the first year post-transplant. 
• 
Examine the proportion with IA designation in subject groups at various thresholds of 
eGFR (<30, 40, and 50 mL/min/1.73 m2), and those with threshold of serum creatinine 
(> 1.2 mg/dL) at one year.    
• 
Assess and compare, between treatment groups, the association/correlation of the 
coefficient of variation (CV) and standard deviation (SD) of tacrolimus trough 
concentrations with each of the following: the composite endpoint of DSA and IA, DSA 
formation and IA individually, TG, ABMR, IFTA, renal dysfunction, various thresholds 
of eGFR (< 30, 40, and 50 mL/min/1.73 m2), and the four components of the traditional 
composite endpoint. 
• 
Summarize intrapatient variability (IPV) by treatment group in subjects with at least 
three non-missing tacrolimus trough level assessments after the first six weeks following 
transplantation and on or before Day 365. 
3.2 
Study Design 
This is an exploratory, two year, prospective, randomized, multi-center, open-label trial 
examining long-term kidney transplant outcomes through the use of an adaptive design and a 
two-part, composite surrogate endpoint.  Specifically, it is designed to compare the effects of 
twice daily, immediate-release tacrolimus and once daily Astagraf XL on DSA formation and 
the development of a peripheral blood molecular profile indicating the presence of IA in de 
novo kidney transplant recipients during the first two years following transplantation.  For 
the purposes of this study, IA will be defined as a positive molecular signature using the 
Transplant Genomics (TGI) Trugraf™ v.2.0 molecular assay (Transplant Genomics, Inc., 
Pleasanton, CA).   
This study will enroll 550 living or deceased donor kidney transplant recipients, 16 to 70 
years of age.  Subjects will be screened prior to surgery and randomized 1:1 to receive 
immediate-release tacrolimus, administered twice daily, or Astagraf XL, as a component of a 
standard immunosuppression maintenance regimen also consisting of corticosteroids 
(complete steroid avoidance not allowed – steroid taper and withdrawal permitted if part of 
institutional protocol) and MMF (or Myfortic® equivalent). Investigators are encouraged to 


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start subjects on the randomized study treatment (immediate release tacrolimus or Astagraf 
XL) within 48 hours of transplantation (pre-transplant administration of study treatment is 
not allowed).  However, if medically indicated per the treating physician’s discretion, 
initiation of study treatment may be delayed for up to seven days post-transplant.   
Given the variability of incidence rates for DSA reported in the literature, a stopping rule has 
been incorporated herein to mitigate the risk of continuing the study under incorrect prior 
assumptions relating to the true incidence rate of DSA formation (Section 4).  To apply the 
rule, the incidence rate of the primary endpoint in the control group will be examined once 
50% of control subjects have completed one year of therapy.  Early termination of the study, 
or extension to a second year, will be predicated on this assessment (see Section 7.9). 
Induction therapy is required for all participants, and will be administered (dosing, type of 
agent) per center protocol.  Intravenous corticosteroids will be administered prior to 
revascularization as part of the initial induction regimen, with dose and duration of therapy 
also determined by the participating transplant center’s standard protocol.  Thereafter, 
subjects will be followed for up to two years during the open-label study period.  
Initial pre-transplant cross-matching (XM) will be performed per local protocol.  Initial XM 
serum will be collected and stored centrally to facilitate the later assessment of pre-formed 
antibody on an as needed basis.  Thereafter, antibody screening and molecular phenotyping 
will be performed by a central lab at one month, three months, six months, nine months, 12 
months, and 24 months.  Antibody status, including HLA-DSA isotype and C1q-binding 
DSA status, will also be recorded when measured locally during the course of clinical care. 
For central laboratory analysis, initial FlowPRA® (One Lambda, Canoga Park, CA) screening 
will be used to test for the presence of anti-HLA antibodies.  Once identified, single antigen 
testing will be used to identify DSA specificity with an MFI greater than or approaching 
1000 used to determine antibody positivity.  For borderline cases, a three member 
adjudication panel of independent, qualified, HLA Lab Directors will determine positivity 
based on relevant clinical data.  In samples that meet threshold criteria for DSA positivity, 
additional classification will be performed to determine the degree of antibody strength.  By 
performing a single dilution (1:16) of DSA-containing samples, the alloimmune response can 
be further elucidated.  Samples in which MFI disappears (becomes < 1000 MFI) will be 
regarded as “weak.”  Samples increasing to > 10,000 MFI upon dilution will be regarded as 
having significant prozone and will be regarded as “strong.”  Samples which may increase or 
decrease, but nevertheless remain between 1000 and 10,000 MFI will be regarded as 
“moderate.”  In certain cases, samples with evidence of fluorescence that are nevertheless 
below MFI criteria for DSA positivity will also be subject to dilution.  Samples increasing to 
above threshold criteria following dilution will be regarded as positive and assumed to be 
under the influence of a prozone effect that impairs antibody detection.  For statistical 
purposes, the DSA with the highest MFI level obtained during the course of the study will be 
used for statistical comparisons of DSA positivity between groups.  However, the strongest 
antibody observed at each HLA-locus will be reported separately and tracked for subjects 
who are DSA positive.  In all cases, positive DSA and/or molecular testing results will be 


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communicated to the subject’s treating physician when available (DSA) or at the end of study 
(molecular testing).  Testing will be performed in batches.  De-identified typing reports 
(donor and recipient) will be requested from the participating centers to inform the central 
laboratory testing.  De-identified results of recent local pre-transplant DSA testing (if 
performed per standard of care [SoC]) will also be requested to facilitate interpretation of 
central DSA testing results.   
Follow-up visits will be performed quarterly during the first year of follow-up to collect 
standard clinical data.  For follow-up beyond the first year, urinary protein, and tacrolimus 
trough concentrations obtained closest to targeted date of visit, per SoC, should be recorded.  
Up to two serum creatinine levels obtained at least one month apart, if performed per SoC, 
should also be recorded.  Additionally, all local kidney biopsy results, AEs, and concomitant 
medications pertaining to AEs will be recorded.  If the study progresses to two years, at the 
terminal study visit at 24 months, the most recently obtained lab results as well as any 
episodes of rejection, need for antibody reduction, graft loss, and deaths occurring beyond 
the first year will be detailed in the electronic Case Report Form (eCRF).  Final testing for 
DSA and molecular profiling will also be performed at the 24 month visit. 
Pathology results [hematoxylin and eosin (H&E), light microscopy, and 
immunofluorescence] will be appended to the eCRF when local kidney biopsies are 
performed and interpreted during the course of clinical care.  De-identified copies of all 
kidney pathology reports must be submitted to Astellas.  For all participants who undergo at 
least one kidney biopsy during the first year post-transplant (either for cause or per 
institutional protocol), the kidney biopsy performed closest to the 12 month visit (but no later 
than 14 months post-transplant) will be submitted for central pathological grading.  All 
biopsies that are submitted for central pathology review will be evaluated and scored using 
the most recent version of the 2007 Update to the Banff ’97 Classification by a central 
pathologist blinded to clinical results and subject identifiers. 
3.3 
Randomization 
Randomization will be carried out using permuted blocks.  The randomization allocation will 
consist of a 1:1 ratio for twice daily, immediate-release tacrolimus or Astagraf XL.  The 
randomization will be stratified according to the following three factors: 
• 
Planned use of alemtuzumab (Campath) (yes/no) 
• 
Kidney donor profile index (KDPI) [three levels: N/A (living donors) vs. ≤ 50% vs. > 
50%] 
• 
HLA Class II mismatch (yes/no) 
Additionally, enrollment of Campath users will be capped to not exceed 20% of the study 
population. 
Subjects enrolled will be randomized using one central list via Interactive Response 
Technology (IRT).  Prior to the initiation of the study treatment, the site staff will contact the 
IRT in order to determine the randomly assigned treatment.  Specific procedures for 
randomization through the IRT are contained in the study procedures manual.  Each subject 


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will be assigned a randomization number that is five digits long.  The first two digits are 
chosen to be fixed for each strata; and the last three digits correspond to the sequential 
subject number (001 to 550) for the study subjects in each strata. 
4 
SAMPLE SIZE 
The sample size for this study was determined based upon a comparison of the rate of the 
two-part composite endpoint in the Astagraf XL group compared to the immediate-release 
tacrolimus group.  Assuming a rate of 20% would be observed for the immediate-release 
tacrolimus group compared to a rate of 10% for the Astagraf XL group, 220 subjects per 
group are needed to achieve an 80% power to detect a difference between Astagraf XL and 
immediate-release tacrolimus with a two-sided alpha level of 0.05.  The study will enroll 275 
subjects/group (550 total) to allow for 20% dropout. 
5 
ANALYSIS SETS 
In accordance with International Conference on Harmonization (ICH) recommendations in 
guidelines E3 and E9, the following analysis sets will be used for the analyses. 
Detailed criteria for analysis sets will be laid out in Analysis Dataset Specifications and the 
allocation of subjects to analysis sets will be finalized prior to database hard lock.  
5.1 
Full Analysis Set (FAS) 
The full analysis set (FAS) will consist of all subjects who are randomized and receive at 
least one dose of study drug (Astagraf XL or immediate-release tacrolimus).  Subjects will be 
included in the FAS based on randomized study drug assignment.  This population will be 
used for selected demographic and baseline summaries and for sensitivity analysis of primary 
efficacy endpoint. 
5.2 
Modified Full Analysis Set (mFAS) 
The modified full analysis set (mFAS) consists of all subjects who are randomized and 
received at least one dose of study drug (Astagraf XL or immediate-release tacrolimus), and 
whose pre-transplant cross-match antibody samples do not demonstrate pre-formed DSA for 
the duration of the study.  This will be the primary analysis set for efficacy assessments, and 
subjects will be included based on randomized study drug assignment. 
The selection of subjects for the mFAS will be confirmed in the Analysis Set Classification 
Meeting (ASCM) / Data Review Meeting.  
The mFAS will be used for summaries and primary analyses of efficacy data, as well as 
selected demographic and baseline characteristics. 
5.3 
Per Protocol Set (PPS) 
The Per-Protocol Set (PPS) includes all subjects of the mFAS who do not experience any 
protocol deviation criteria defined in Section 7.2.2. 


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Final judgments on exclusion of subjects from the PPS will be made at the ASCM and data 
review meeting, held prior to database lock.  
The PPS will be used for secondary analyses of efficacy data.  
5.4 
Safety Analysis Set (SAF) 
The Safety Analysis Set (SAF) consists of all subjects who enrolled into the study and took at 
least one dose of study medication.  
The SAF will be used for summaries of all safety, tolerability and medication compliance 
related variables, and subjects will be included based on actual study medication received. 
5.5 
Biopsy Analysis Set (BAS) 
The Biopsy Analysis Dataset (BAS) consists of all mFAS subjects who had at least one post-
transplant central pathology assessment. 
The Biopsy Analysis Set will be used for summaries of all secondary and exploratory 
efficacy analyses that need to use central pathology findings. 
5.6 
Pharmacokinetics Analysis Set (PKAS) 
Not applicable 
5.7 
Pharmacodynamic Analysis Set (PDAS) 
Not applicable 
6 
ANALYSIS VARIABLES 
6.1 
Efficacy Endpoints 
Following the decision from the interim analysis (see Section 7.9), subjects will complete at 
least one year of study visits, and data analysis will be performed once all subjects complete 
12 months of study.  Accordingly, efficacy analyses will base on data reported within the one 
year period. 
6.1.1 
Primary Efficacy Endpoint(s) 
The primary endpoint is a composite endpoint of either the presence of DSA or IA (as 
assessed by a central lab) on peripheral blood molecular profiling by one year post-
transplant.  
6.1.2 
Secondary Efficacy Endpoints 
Comparisons between treatment groups regarding incidence will rely upon assessments for 
the following results: 
• 
De novo DSA formation over the course of 1 year 
o De novo DSA (positive / negative / indeterminate) up to Day 365 
o Class I and Class II DSA will be assessed by a central HLA lab at 1, 3, 6, 9, and 
12 months.  Overall, Class I and Class II DSA occurrence (positive / negative) of 


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the period from transplant to Month 12 will be assessed based on assessments at 
each visit. 
o Endpoints for the subset of DSA positive subjects: 
▪ initial MFI values and antibody strength (mild, moderate, strong) based on 
MFI 
▪ post-transplant peak MFI and antibody strength  
▪ DSA persistent (yes/no) will be assessed by sponsor based on DSA lab data 
▪ C1q-binding DSA (positive/negative) 
▪ DSA IgG3 isotype (positive/negative) 
For de novo DSA positive subjects, their initial MFI values and antibody strength, peak 
MFI and antibody strength, and DSA persistence (yes/no) will be assessed by sponsor 
based on DSA lab data.   
• 
IA 
o Immune Activation (IA) from Day 1 to Day 365 visit 
o Immune Activation (IA) during the period from Day 30 to Day 365 visit 
IA will be regarded as persistent if IA is detected for positivity for any two 
measurements on or before Day 365 visit. 
• 
TG during the first year 
The presence of TG will be reported where TG is defined as cg > 0 on centrally-
interpreted institutional protocol biopsy or biopsy obtained for cause during the first year 
post-transplant with +2 months visit window. 
• 
Microcirculatory Inflammantion during the first year 
The presence of microcirculatory inflammation will be reported where microcirculatory 
inflammantion is defined as g + ptc >=2 on centrally-interpreted institutional protocol 
biopsy or biopsy obtained for cause during the first year post-transplant, with +2 months 
visit window.  
• 
IFTA and Inflammantion during the first year 
The presence of IFTA and inflammation will be reported where IFTA and inflammation 
is defined as IFTA positive and inflammation positive (i >0) on centrally-interpreted 
institutional protocol biopsy or biopsy obtained for cause during the first year post-
transplant, with +2 months visit window. 
• 
Thresholds for eGFR 
o Values less than 30, 40, and 50 mL/min/1.73 m2 


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These threshold endpoints will be calculated for months 1, 3, 6, 9, and 12using the 
Modification of Diet in Renal Disease (four variable-MDRD) criteria.  Assessments 
are positive or negative. 
o Five-point decline in eGFR 
The decline in eGFR will be calculated with respect to the eGFR value at Day 30 
visit. 
• 
Traditional transplantation composite endpoint 
Incidence will be assessed at month 12 for the following: 
o Graft loss (defined as retransplantation, transplant nephrectomy, or a return to dialysis 
for at least a six week duration, or subject death) 
o Death 
o BPAR – positivity will be determined by local biopsy, central pathology, or reported 
adverse events.  
o Composite occurrence of either graft loss, death, BPAR, and loss-to-follow-up 
• 
Histopathology grading within 1 year 
Central histopathology grading over 1 year will be diagnosed according to the BANFF 
diagnostic categories and subcategories that include the following:   
o Normal 
o Antibody mediated changes 
▪ C4d deposition without morphologic evidence of active rejection 
▪ Acute ABMR (grades: I, II, and III) 
▪ Chronic active ABMR 
▪ ABMR: either Acute ABMR or Chronic Active ABMR 
o Borderline changes 
o TCMR 
▪ Acute TCMR (grades: IA, IB, IIA, IIB, and III) 
▪ Chronic active TCMR 
o IFTA (grades: I, II, and III) 
o Other: all categorical findings will be summarized. 
o In addition to the categories above, specimens will be given individual scores for the 
following: glomerulitis (g), tubulitis (t), intimal arteritis (v), mononuclear cell 
interstitial inflammation (i), early allograft Glomerulopathy (cg), tubular atrophy (ct), 
interstitial fibrosis (ci), vascular fibrous intimal thickening (cv), arteriolar hyaline 
thickening (ah), peritubular capillaries (ptc) and mesangial matrix (mm). 


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6.1.3 
Exploratory Efficacy Endpoints 
Additional exploratory endpoints include the following: 
• The validation of peripheral blood molecular profiling will employ the use of a second 
two-part composite endpoint encompassing the incidence of acquiring DSA or positive 
evidence of TG on biopsy during the first year post-transplant or by the conclusion of the 
study. 
6.1.4 
Other Efficacy Variables 
Not applicable 
6.2 
Safety Variables 
Safety will be assessed by evaluation of the following variables: 
• 
Treatment-emergent adverse events (TEAEs; frequency, severity, seriousness, and 
relationship to study drug).  
TEAE is defined as an AE observed on or after the day of starting the administration of 
the test drug/comparative drug.  If a subject experiences an event both during the pre-
investigational period and during the investigational period, the event will be considered 
as TEAE only if it has worsened in severity (i.e., it is reported with a new start date).  
All AEs collected that begin within seven days after taking the last dose of study drug 
will also be counted as TEAE.  Based on conservative rule, all AEs that could not be 
determined as started before transplant or after seven days of the last study dose will be 
assumed to be TEAEs. 
A drug-related TEAE is defined as any TEAE with at least a possible relationship to 
study treatment, as assessed by the investigator.  
For TEAE (and serious TEAE), the exposure-adjusted incidence rate (EAIR) will be 
calculated using following formula: 
EAIR (%) = 100 * n / sum of exposure 
In the formula, n is the number of subjects with an event (e.g. one SOC/PT category). 
Sum of exposure is the total exposure time of all subjects in the group. For subject with 
one event, exposure time = time from transplant to the event; for subject with multiple 
events, exposure time = time from transplant to the earliest event; for subject with no 
event, exposure time = time from transplant to end of study. Because of the large sample 
size of the study, time unit is set to Year. 
• 
The safety endpoint for AEs will be the incidence of each type of AE based on system 
organ class and using the preferred term from the Medical Dictionary for Regulatory 
Activities (MedDRA) version 20.1. 
• 
Clinical laboratory variables (hematology, biochemistry including liver enzymes and 
total bilirubin, urinalysis, coagulation, and tacrolimus trough levels). 


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6.3 
Pharmacokinetic Variables 
Not applicable 
6.4 
Pharmacodynamic Variables 
Not applicable 
6.5 
Other Variables 
Baseline  
Baseline renal function (serum creatinine, eGFR) will be defined as the value measured at the 
Day 30 visit.  For all other data, the baseline value will be defined as the last observation 
collected prior to or at the time of reperfusion of the transplanted kidney. 
Extended Criteria Donor (ECD) 
The donor characteristics that define an ECD kidney include age > or = 60 years, or age 50-
59 years plus two of the following: cerebrovascular accident as the cause of death, 
preexisting hypertension, or terminal serum creatinine greater than 1.5 mg/dl. 
Study Days  
For the purpose of analyzing time to event endpoints, the time-in-study for each subject will 
be measured relative to Day 0, the day of transplantation; defined as the date in which 
reperfusion to the transplanted organ is established.  From this starting point, the day of 
reperfusion will be considered the first day in study for each subject for analysis and the 
study days will be calculated as: 
Date of event - Date of reperfusion  
Study Treatment Dose Increased, Decreased, Interrupted, or Treatment Arm Switched  
During the whole study treatment period, a subject is considered as: 
• Dose Increased – at least one treatment record has higher dose than that of at least 
one previous record 
• Dose Decreased – at least one treatment record has lower dose than that of at least 
one previous record 
• Dose Interrupted – at least one gap (i.e. non-treated days between two records) is >= 
7 days 
• Arm Switched – at least one treatment record with non-assigned treatment (i.e. not 
the randomized treatment group) 
Time on Treatment (days) 
For each subject, the length of time from first dose of study medication to date of 
discontinuation from study treatment will be calculated in days, using the following formula: 
(‘Date last dose of study drug’ - ‘Date first dose’) + 1 
Actual Treatment Exposure (days) 


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For each subject, Actual Treatment Exposure (days) will be the sum of treatment days of all 
treatment records = Sum of all treatment records’ (‘Stop Date’ – ‘Start Date’ + 1), where the 
treatment record must have Dose > 0. 
Average Daily Dose (mg/day) 
Average Daily Dose = Total dose (mg) of study drug / Actual Treatment Exposure (day). 
Intrapatient Variability (IPV) of Tacrolimus Trough Level 
If the subject had at least three non-missing tacrolimus trough concentration assessments 
between Week 6 (Day 42) and Day 365, the IPV will be estimated from CV%, geometric 
CV%, and SD: 
CV% = (σ/μ) x 100, where σ is standard deviation and μ is mean of the tacrolimus trough 
levels 
Geometric CV% = sqrt(exp(σlog2)-1) x 100, where σlog is the standard deviation of log-
transformed tacrolimus concentration value. 
SD = standard deviation 
Above three IPVs will also be calculated from dose-adjusted tacrolimus concentration:  
Dose-adjusted Tacrolimus Concentration = Tacrolimus Concentration / Tacrolimus Dose of 
that Day. 
Switching of Tacrolimus Manufacturer 
The study medication of the BID Immediate-release tacrolimus can be from different 
manufacturers. A manufacturer switch is counted if the manufacturer(s) of record (based on 
NDC codes) at one dispensing date are not completely same as those of the previous 
dispensing. 
Compliance 
The two common measures of medication adherence using refill data are the medication 
possession ratio (MPR) and proportion of days covered (PDC).  Although the MPR has been 
used to define medication adherence in kidney transplantation, the PDC seems to be the 
estimator of choice for CMS and pharmacies.  This is due to the fact that the PDC estimator 
is considered a more conservative measure of refill record-based adherence.   
 
PDC is the percentage of days covered with medication, calculated based on dispensing days 
from the pharmacy, as shown in the following formula:     
 
PDC = (Number of days in period "covered"
Number of days in period
) × 100% 
 
PDC threshold for compliance is typically set at 80% of days covered.  This means that 
someone missing 5 or fewer days’ worth of medication in an average month is considered 
compliant. 
 


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Steps to calculate individual PDC based on dispensing data are provided below:   
• Step 1:  Determine subject’s measurement period, defined as the index 
prescription date until graft loss, death or end of 12-month follow-up period.  
Index prescription date can be the first fill date after discharge from the hospital 
for kidney transplantation.   
• Step 2:  Within the measurement period, count the days the subject was covered 
(2-daily, immediate release) based on the prescription fill date and days of 
supply.  If prescriptions for the same drug overlap, then adjust the prescription 
start date to be the day after the previous fill has ended.  
• Step 3:  Divide the number of covered days found in Step 2 by the number of 
days found in Step 1.  Multiply this number by 100 to obtain the PDC (as a 
percentage) for each subject.  
• Step 4:  Count the number of subjects who had a PDC greater than 80% and 
then divide by the total number of eligible subject. 
MPR is a ratio between number of days of medication supply and number of days in the refill 
interval. MPR is calculated as: 
 
MPR = (Total number of days of medication supplied 
Number of days in period
) × 100% 
 
The number of days in period (e.g., 1 year) is the sum of the number of days from first 
dispensing (ie., first fill date after discharge from the hospital for kidney transplantation) up 
to the date of last dispensation plus the number of days’ supply obtained at the last 
dispensation (e.g., 30 days).  Any prescription days’ supply that spans the 1-year observation 
period will be truncated at 365 days.  MPR threshold for compliance is typically set at 80%. 
For this study, the study drug dispense data is only available for the BID Immediate-Release 
Tacrolimus group. PDC, MPR, and their compliance rates can only be calculated for that 
treatment group. 
Previous and concomitant medication 
Previous medication is defined as medications which started and stopped prior to transplant.  
Concomitant medication is defined as medications with at least one dose taken between the 
date of transplant (inclusive) and the date of last dose (inclusive) of study drug. 
7 
STATISTICAL METHODOLOGY 
7.1 
General Considerations 
All numeric and categorical efficacy and safety endpoints will be descriptively summarized. 
For continuous variables, descriptive statistics will include the number of subjects (n), mean, 


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standard deviation, median, minimum and maximum.  Frequencies and percentages will be 
displayed for categorical data.     
Summaries based on the FAS, mFAS, PPS, and BAS (e.g. disposition, baseline and efficacy 
data) will be presented by planned treatment group, unless specifically stated otherwise.  
Unless otherwise stated, all analyses will be performed using data pooled across sites.  
Subject listings of all data from the CRFs as well as any derived variables will be presented. 
All statistical comparisons will be made using two sided tests at the α = 0.05 significance 
level unless specifically stated otherwise.  All null hypotheses will be of no treatment 
difference, all alternative hypotheses will be two-sided, unless specifically stated otherwise. 
P-values will not be adjusted for multiple comparisons. In the event that any analysis set 
sample size falls below 15 in any treatment group, descriptive statistics will exclusively be 
reported with no p-values or statistical inferences.  In addition, logistic regression analysis 
and other regression model analysis as planned in the following sections will not be 
performed for outcome variables that have an event rate of less than 10% (i.e. reported by 
fewer than 57 subjects). 
Unless otherwise specified, all data processing, summarization, and analyses will be 
performed using SAS® Version 9.4 or higher.   
A hard database lock will occur prior to the final analysis.  
7.2 
Study Population 
7.2.1 
Disposition of Subjects 
The following subject data will be summarized overall and by treatment group.  Unless 
otherwise noted, the data will be summarized using the randomized treatment group: 
• 
Number of subjects who provided informed consent, who discontinued before 
randomization, who were randomized, who received a transplant, and who had at least 
one post-baseline assessment; 
• 
Number and percentage of subjects in each analysis set; 
• 
Number and percentage of subjects who discontinued (actual) tacrolimus treatment for 
any reason, with primary reason for treatment discontinuation presented; and 
• 
Number and percentage of subjects excluded from the PPS by reason for exclusion. 
A subject listing for early discontinuations and reasons for discontinuation will be provided.   
7.2.2 
Protocol Deviations 
Protocol deviations as defined in the study protocol (Section 8.1.6 Protocol Deviations) will 
be assessed for all randomized subjects.  The number and percentage of subjects with major 
protocol deviations will be summarized by criterion, (randomized) treatment group, and 
overall.  Protocol deviations will also be summarized by study site.  Subjects with more than 
one major deviation from the same criterion will be summarized once for the corresponding 
criterion.  Any subjects who have more than one major deviation will be counted once in the 
overall summary.  A data listing will be provided for all deviations by site and subject.    


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All deviations will be assessed as either major or minor according to a final review meeting 
prior to unblinding. 
The protocol deviation criteria will be uniquely identified in the summary table and listing.  
The unique identifiers will be as follows: 
• 
PD1 - Entered into the study even though they did not satisfy entry criteria,  
• 
PD2 - Developed withdrawal criteria during the study and was not withdrawn, 
• 
PD3 - Received wrong treatment or incorrect dose, 
• 
PD4 - Received excluded concomitant treatment.   
7.2.3 
Demographic and Other Baseline Characteristics 
Demographic and other baseline characteristics will be summarized using descriptive 
statistics, presented overall and by randomized treatment group. 
Number and percentage of subjects randomized in each site will be presented by treatment 
group for the SAF.  
7.2.3.1 Recipient Demographics  
Descriptive statistics for age (years), age categories (<=17, 19-34, 35-49, >=50), weight (kg), 
body mass index (BMI, kg/m2) and height (cm) will be presented.  Frequency tabulations for 
sex, ethnicity, age group (defined in section 7.8) and race will be presented.  This will be 
done for the non-randomized subjects, as well as for the SAF, FAS, mFAS, PPS, and BAS by 
treatment group.  A listing of demographic characteristics will also be provided.  
7.2.3.2 Donor Demographics and Baseline Characteristics 
Descriptive statistics for donor demographic and baseline characteristics of age (years), 
weight (kg), height (cm), BMI (kg/m2), KDPI will be presented.  Frequency tabulations for 
age groups (<=17, 18-34, 35-49, >=50), extended criteria donor (ECD), race, ethnicity, 
Living/Deceased donor, deceased donor type, cause of death of deceased donor, Donation 
after Circulatory Death (DCD), will be presented.  This will be done for the FAS, mFAS, 
SAF, PPS, and BAS by treatment group.  A listing of demographic characteristics will also 
be provided.  
7.2.3.3 Recipient Baseline Disease Characteristics  
Baseline characteristics, including primary etiology of end of stage renal disease (ESRD), 
duration of ESRD prior to transplantation (months), on dialysis prior to current transplant, 
duration of dialysis prior to transplant, type of dialysis, weight before transplant, and reason 
for dialysis will be summarized using the FAS by treatment group.  
7.2.3.4 Baseline Viral Serology  
The number and percent of subjects for each recipient/donor pair with the following results at 
baseline: (Positive – Positive, Positive – Negative, Negative – Positive, Negative – Negative) 


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for viral serology test Hepatitis B Core Antibody IgG and Hepatitis C Antibody will be 
summarized using the FAS by treatment group.  
All baseline viral serology data will be listed. 
7.2.3.5 Transplant Related Information  
The following transplant-related information will be summarized using the FAS by treatment 
group: 
• 
Number and percent of previous kidney transplants 
• 
Reason for most recent graft loss 
• 
Type of transplant 
• 
ABO compatibility for each recipient/donor pair  
• 
Calculated Panel reactive antibody (cPRA) 
• 
Organ storage method 
• 
Any complications in organ preservation or transplant procedure? 
• 
Cold ischemic time 
• Warm ischemic time  
• 
Test used for recipient histocompatibility 
• 
Crossmatch results 
• 
Pre-transplant donor kidney biopsy performed (yes/no) 
7.2.3.6 DSA Testing 
The following transplant-related information will be summarized using the FAS by treatment 
group:  
• 
Method used 
• 
Any evidence of DSA positivity 
• 
Single Antigen Bead Testing performed 
7.2.3.7 Medical History 
Targeted diabetes medical history and non-diabetic medical history will be collected.  The 
number and percent of subjects with each targeted diabetes medical history condition will be 
summarized using the SAF by treatment group.  All non-diabetic medical history conditions 
are coded in MedDRA version 20.1, and will be summarized by System Organ Class (SOC) 
and Preferred Term (PT) as well as by PT alone, by treatment group for the SAF.  All 
medical history data will be listed.    
7.2.4 
Previous and Concomitant Medications 
Previous medications will be coded using the version September 2017 of the World Health 
Organization Drug Dictionary (WHO-DD), and will be included in data listings.  
Concomitant medications will be summarized for each treatment group by therapeutic 
subgroup (ATC 2nd level) and chemical subgroup (ATC 4th level) for the SAF.  Subjects 
taking the same medication multiple times will be counted once per medication.   


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Immunosuppression medications including mycophenolic acid (MMF, EC-MPS, or generic 
total daily dose [mg]), corticosteroids usage (total daily dose [mg]), and antithymocyte 
globulin (total daily dose [mg]) will be summarized. 
Separate listings for concomitant and immunosuppression medications will be provided.   
7.3 
Study Drugs 
7.3.1 
Exposure 
The following information on drug exposure according to the details in Section 6.5 will be 
presented for each treatment group for the SAF: 
• 
Descriptive statistics for cumulative amount of the drug that a subject was exposed to 
and average daily dose; and 
• 
Number and percent of subjects with dose increases, decreases, or interruption by 
treatment group. 
Duration of exposure will be summarized in two ways. 
• 
Descriptive statistics will be presented by treatment group. 
• 
Exposure time will be categorized according to the following categories by treatment 
group: 
o 
less than 7 days 
o 
at least 7 days, less than 14 days  
o 
at least 14 days, less than 28 days  
o 
at least 28 days, less than 42 days 
o 
at least 42 days, less than 60 days 
o 
at least 60 days, less than 91 days 
o 
at least 91 days, less than 182 days 
o 
at least 182 days, less than 364 days 
o 
364 days or more 
o 
Unknown. 
Counts and percentages of subjects in each of these categories will be summarized for 
each treatment group for the SAF. 
Additionally, descriptive statistics (counts and percentages) will be used to assess for the BID 
Immediate-release Tacrolimus treatment group for the SAF the distribution of use for the 
different tacrolimus formulations in the study (i.e., generic formulations of immediate release 
tacrolimus) and the frequency of manufacturer switching for the study. 
7.3.2 
Treatment Compliance 
Treatment Compliance is only available for the BID Immediate-release tacrolimus treatment 
group. See section 6.5.  All analyses planned in the Protocol to compare compliance between 
treatment groups will not be conducted.  
PDC, MPR rates and their compliance values will be descriptively summarized for the BID 
Immediate-release Tacrolimus treatment group. 


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7.3.3 
Tacrolimus Trough Concentrations 
Since tacrolimus trough concentration is not assessed at scheduled visits and each subject 
may have different numbers of assessments. Following analyses will be conducted: 
1) Based on all tacrolimus trough concentration assessments from D30 to Day 365, the 
number of assessments, the mean and median trough level of each subject will be 
calculated and summarized by treatment group. 
2) Number of excursions of tacrolimus trough concentrations above 120% or below 80% 
of the average trough level of each subject will be tabulated as a proportion of the all 
tacrolimus trough concentrations reported during the study:  proportion of tacrolimus 
trough concentrations above 120% or below 80% of the average trough level = 100 x 
(number of tacrolimus trough concentrations above 120% or below 80% of the 
average trough level/total number of tacrolimus trough concentrations).  
3) IPV estimated from CV, geometric CV, and SD, and Dose-adjusted IPVs (see section 
6.5) will be summarized by treatment group. The summaries will be provided for SAF 
and subgroups of subjects with TG, microcirculatory inflammantion, or BPAR. 
4) IPVs will also be visually presented in Box plots. 
Mean tacrolimus level, number of excursions of tacrolimus trough concentrations above 
120% or below 80% of the average trough level, and IPV estimated by CV% will also be 
used as covariates in the risk factor analysis. See section 7.4.2.  
IPVs will also be estimated from the mixed model and functional regression model (Kim et 
al., 2019). The methods and results will be presented in a separate report, and are not covered 
by this SAP.  
7.4 
Analysis of Efficacy  
The analysis of Efficacy will be conducted using the mFAS, with sensitivity analyses using 
the FAS and PPS. All efficacy analyses related to central pathology findings will be 
conducted using BAS. Risk factor analysis will be conducted for both mFAS and PPS. 
7.4.1 
Analysis of Primary Endpoint(s) 
7.4.1.1 Primary Analysis of Primary Endpoint 
The primary analysis will be performed on the primary composite endpoint of combined 
incidence of either De Novo DSA or IA on molecular blood profiling at one year.  Both De 
novo DSA (Positive/Negative) and IA (Present/Absent) will be considered binary. DSA 
positivity is assessed by the Transplant Immunology Lab of Northwestern University. 
In the case where MFI signal is greater than 1000, and DSA is suspected, but the formation 
of De Novo DSA cannot be confirmed due to inadequate donor typing, the formation of De 
Novo DSA will be labeled indeterminate. If subject discontinues the study between D0 and 
D30, and no testing results are available, then De Novo DSA will be assumed to be negative. 
If subject does not have a specimen collection, then De Novo DSA will be assumed missing 
and will be excluded from primary efficacy analysis, but will be assumed to be negative in 




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H0: The proportion of subjects with the primary endpoint is the same between subjects 
receiving Astagraf XL and subjects receiving immediate-release tacrolimus. 
H1: The proportion of subjects with the primary endpoint is not the same between subjects 
receiving Astagraf XL and those receiving immediate-release tacrolimus. 
7.4.1.2 Sensitivity Analyses of Primary Endpoint 
A sensitivity analysis, based on missing data imputation, will be included for the mFAS.  
Subjects that have missing or incomplete data who did not permanently switch therapy or 
were discontinued from the study prior to a DSA/IA event, who are lost to follow up, or those 
who expire for any reason, negative DSA/IA values will be imputed. 
A sensitivity analysis using the FAS will be conducted on the primary endpoint.  The logistic 
regression analysis described in Section 7.4.1.1 will be used.  
A secondary analysis using the PPS will be conducted on the primary endpoint data.  The 
logistic regression analysis described in Section 7.4.1.1 will be used.  
Additional sensitivity analysis under different assumptions may be conducted in the future to 
address specific research questions. 
7.4.2 
Analysis of Secondary Endpoints  
Risk factor analysis will be conducted in two parts.  First, is to identify risk factors for 
outcomes based on factors measured at time of transplantation (baseline) including the pre-
defined stratification factors.  Last, is to identify risk factors for outcomes based on factors 
that are measured post-randomization and therefore, are subject to change e.g., average daily 
dose of MPA treatment or cumulative dose of corticosteroids.  All models will include 
randomized treatment, irrespective of statistical significance.  Models to assess risk factors 
for binary or categorical variables will be done using logistic regression.  Odds ratio and 95% 
CI will be used as measures of strength of association and precision, respectively.   
Risk factor analyses will be conducted for eight binary variables: IFTA, IFTA and 
Inflammation, BPAR, the four-part traditional composite endpoint, early MMF 
interruption/discontinuation, and eGFR status based on three thresholds 30, 40, and 50. The 
biopsy data are based on the central pathology results over the course of one year plus 2 
months window. eGFR status variables are based on the assessment at Day 365 visit. 
Each risk factor analysis model will include treatment group and a main factor: either De 
novo DSA, IA, or the composite of De novo DSA and IA. Following additional covariates 
will be considered for the two types of models, and a multicollinearity assessment will be 
proceeded for the final variable selection: 
Risk factors measured at time of transplantation include: 
• 
Cold ischemic time  
• 
Warm ischemic time  


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• 
KDPI% <= 50% (yes/no) 
• 
KDPI% > 50% (yes/no) 
• 
HLA Class II mismatch (yes/no) 
• 
Donor status (living/deceased) 
• 
Donation after circulatory death (yes/no) 
• 
Donor Age 
• 
Donor-Recipient sex mismatch (match/mismatch) 
• 
Donor-Recipient race mismatch (match/mismatch) 
• 
Preservation by cold storage (yes/no) 
Post randomization risk factors include: 
• 
Cumulative Dose of Corticosteroids 
• 
Average daily dose of MPA 
• 
Mean dose level of tacrolimus 
• 
Mean tacrolimus trough level 
• 
Number of excursions below 80% or above 120% of the average tacrolimus trough level 
• 
IPV estimated by CV% 
• 
Post-transplant BK virus infection 
Following secondary endpoints will be summarized with frequencies and percentages by 
treatment group.  The incidence of events will be analyzed for treatment group differences. 
Analysis of treatment group differences will be implemented from logistic regression model 
with the same covariates of primary efficacy analysis. 
1. 
De novo DSA formation 
2. 
IA  
3. 
C1q-binding DSA 
4. 
DSA IgG3 isotype 
5. 
Persistence of DSA 
6. 
Persistence of IA 
7. 
TG (cg>0) 
8. 
Microcirculatory Inflammation (g+ptc>2) 
9. 
IFTA 
10. IFTA and Inflammation (IFTA and ci>0) 
11. Acute and chronic forms of ABMR 
12. eGFR at various thresholds (less than 30, 40, 50 mL/min/1.73 m2 at one year; and a 
five-point decline from Day 30 to Day 365) 
13. Graft loss  
14. Mortality 


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15. BPAR 
16. The four-part traditional composite endpoint 
Separate risk factor analyses will be conducted for BID Immediate-release Tacrolimus 
treatment group only. Manufacturer will be included as additional covariate.  
Ordinal assessments of antibody peak strength (weak, moderate, and strong) will be 
summarized based on the results over the course of one year using frequencies and 
percentages. Analysis of treatment group difference will be done using Fisher’s Exact Test. 
The continuous endpoint of Peak MFI score will be summarized with descriptive statistics 
based on the results over the course of one year by antibody.    
Histopathology assessments will be summarized with both descriptive statistics and 
frequencies and percentages.  Raw biopsy scores, including g, t, v, i, cg, ct, ci, cv, ah, ptc, 
and mm will be summarized as ordinal outcomes.  Analysis of treatment group differences 
will be performed using Fisher’s Exact Test. The incidence of binary outcomes including ci, 
ct, and ptc scores greater than 1 will be summarized with frequencies and percentages.  
Analysis of treatment group differences will be performed using Fisher’s Exact test. 
Assessments of association between De novo DSA and IA will be analyzed with logistic 
regression models using DSA formation as the response and IA as predictor using the same 
covariates detailed in Section 7.4.1.1. 
Mean of eGFR by treatment group will be plotted across visits.  
7.4.3 
Analysis of Exploratory Endpoints  
Analysis will be performed on the secondary composite endpoint consisting of DSA 
formation and the presence of TG (cg > 0).  Logistic model with the same covariates of the 
primary analysis will be conducted for treatment group differences with the incidence of 
DSA or TG as the response and treatment group as the predictor.  The analysis will be 
performed using the biopsy analysis set.  
7.4.4 
Analysis of Other Variables  
Not applicable 
7.5 
Analysis of Safety 
All analysis of safety will be presented by treatment group for SAF, unless specified 
otherwise.   
7.5.1 
Adverse Events 
The coding dictionary for this study will be MedDRA.  All AEs will be summarized by SOC 
and PT. 
The following lists the TEAE summaries that will be provided: 
• 
Number of TEAEs, 
• 
Number and percentage of subjects with TEAEs, 


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• 
Number and percentage of TEAEs by relationship, 
• 
Number and percentage of subjects with TEAEs by relationship, 
• 
Number and percentage of TEAEs by intensity, 
• 
Number and percentage of subjects with TEAEs by intensity, 
• 
Number and percentage of TEAEs occurring in at least 3.5% of any treatment group,   
• 
Number and percentage of subjects with TEAEs occurring in at least 3.5% of any 
treatment group,  
• 
Exposure adjusted incidence rate (EAIR) of TEAEs, 
• 
Number of TEAEs leading to permanent discontinuation of study drug, 
• 
Number and percentage of subjects with TEAEs leading to permanent discontinuation of 
study drug,  
• 
Number of serious TEAEs, 
• 
Number and percentage of subjects with serious TEAEs, 
• 
Number and percentage of serious TEAEs by relationship, 
• 
Number and percentage of subjects with serious TEAEs by relationship, 
• 
Exposure adjusted incidence rate (EAIR) of serious TEAEs,  
• 
Number of deaths. 
The number and percentage of subjects with TEAEs, as classified by PT only, will be 
summarized for each treatment group. 
If a TEAE has missing grade of intensity or relationship, the highest level of assessment, i.e. 
Severe for intensity and Probable for relationship, will be used for the table summary. The 
data listings will still present original missing assessments.     
7.5.2 
Clinical Laboratory Evaluation 
The baseline renal function (serum creatinine, eGFR) will be defined as the value measured 
at the Day 30 visit.  For all other data, the baseline value will be defined as the last 
observation collected prior to reperfusion of the transplanted kidney.   
Quantitative clinical laboratory variables, i.e. hematology and biochemistry, will be 
summarized using mean, standard deviation, minimum, maximum and median for each 
treatment group at each visit.  Additionally, a within-subject change will be calculated as the 
post-baseline measurement minus the baseline measurement and summarized in the same 
way.  
Urinalysis and coagulation results will be presented in data listing. 
7.5.2.1 Liver Enzymes and Total Bilirubin  
The following potentially clinically significant (PCS) criteria for liver tests – defined as 
Alkaline Phosphatase (ALP), Alanine Transaminase (ALT), total bilirubin (TBL), Aspartate 
Transaminase (AST) and their combination are defined.  The subject’s highest value during 
the investigational period will be used.  
Parameter  
Criteria 
ALT  
 
> 3xULN 


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> 5xULN 
 
 
 
> 10xULN 
 
 
 
> 20xULN 
AST 
 
> 3xULN 
 
 
 
> 5xULN 
 
 
 
> 10xULN 
 
 
 
> 20xULN 
ALT or AST 
> 3xULN 
Total Bilirubin 
> 2xULN 
ALP 
 
> 1.5xULN 
ALT and/or AST AND Total Bilirubin (*) 
(ALT and/or AST > 3xULN) and  
total bilirubin > 2xULN 
(*) Combination of values measured within same sample 
The PCS of liver enzymes and total Bilirubin data will be summarized and listed. 
7.5.3 
Vital Signs 
Vital signs will not be collected for this study.    
7.5.4 
Electrocardiograms (ECGs) 
ECG data will not be collected as stand-alone data.  Any abnormal ECG results will be 
reported as AEs.  No separate ECG results will be presented. 
7.5.5 
Other Safety-Related Observations 
For the plasma exchange therapy with reason of Post-Transplant AMR Treatment, the 
number and percentage of subjects who received therapy, and the total number of sessions 
will be reported by treatment group.  
The number and percent of subjects who undergo any antibody removal therapy or get any 
transfusions during the study will be reported by treatment group 
7.6 
Analysis of PK 
Not applicable 
7.7 
Analysis of PD 
Not applicable 
7.8 
Other Analyses 
Not applicable 


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7.9 
Interim Analysis  
An interim analysis was conducted after 50% of control subjects had completed one year of 
therapy, following the procedures as described in the protocol and SAP version 1. The results 
of the interim analysis lead to the following decision: 
1. 
The study would continue to enroll to completion. 
2. 
The study duration would be such that subjects will complete at least one year of study 
visits. 
a. 
Subjects who had not yet completed one year of study (at the time of the interim 
analysis) would complete the study at the 12m visit. 
b. 
Subjects who had already completed the 12m visit (at the time of the interim 
analysis) would complete the study at their next scheduled study visit. 
3. 
Data analysis would be performed once all subjects complete 12 months of study. 
 
7.10 
Handling of Missing Data, Outliers, Visit Windows, and Other 
Information 
7.10.1 Missing Data 
Generally, all missing efficacy endpoints (e.g. DSA, IA, pathological findings, eGFR) will 
not be imputed.  Only for the sensitivity analyses, e.g. the sensitivity analysis of primary 
endpoint (section 7.4.1.2), the indetermined endpoint will be imputed as Negative. 
In AE summary tables, the missing AE severity and relationship to study drug will be 
replaced with the highest level of severity and relationship: “Severe” and “Probable”, 
respectively. In AE data listing, original missing data will be displayed. 
As a general principle, no imputation of missing data for other variables will be done. 
7.10.2 Outliers 
Unless otherwise specified, all values will be included in the analyses. 
7.10.3 Visit Windows 
Visit windows are defined in Section 2 (Flow Chart and Visit Schedule).  For all data 
collected by visit, the assessments will be presented per the scheduled visits. For data 
collected by date (e.g. start/stop dates) or collected at unscheduled visits, following rules are 
applied: 
• Study treatment and Tacrolimus trough level – All data from Day 30 to Day 365 will be 
used in calculation. In the calculation of IPVs, only data after Day 42 (i.e. 6 weeks) will be 
used. 
• Central pathology – All data from Day 0 to Day 426 (Month 14 = 365 + 61) will be included 
in the calculation of endpoints related to central pathology, e.g. IFTA, Acute Rejection, 
etc. 


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• Serum creatinine and eGFR – If the scheduled Day 30 assessment is missing, it will be 
imputed using the unscheduled assessment within Day 30 +/- 14 window that is closest to 
Day 30; If the scheduled Day 365 assessment is missing, it will be imputed using the 
unscheduled assessment within Day 365 +/- 61 window that is closest to Day 365. 
• All other primary and secondary endpoints will use the data up to Day 386. 
• Death, graft loss, immunosuppressant total dose, and other safety endpoints - Day 365 will 
be used as cut point for the one-year analysis. 


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8 
DOCUMENT REVISION HISTORY  
Version 
Date 
Changes 
Comment/rationale for change 
1.00 
12-JUL-2018 
NA 
NA 
2.00 
25-OCT-2019 
1. Define a biopsy set 
To facilitate analyses of biopsy based 
endpoints by selecting subjects who 
undergo at least one kidney biopsy. 
 
 
2. Define protocol 
deviation based on ICH 
guidance.  Clarify PPS 
definition. 
To follow ICH guidance on defining 
protocol deviations and a PPS.  PPS is 
further clarified as subjects of the mFAS 
who do not experience any protocol 
deviation criteria defined in Section 7.2.2.  
This will allow for a more meaningful 
comparison of the results between mFAS 
and PPS since mFAS is the analysis set 
for primary efficacy analyses. 
 
 
3. Revise the plan for 
risk factor analysis 
To prevent from overfitting of logistic 
regression models when the expected 
number of incidences is very small.  A 
large number of pre-specified risk factors 
and predictors can potentially affect 
accuracy of the estimated parameters in 
the case of low event rates. 
 
 
4.Define prior 
medication 
To be more clinically relevant, previous 
medication is defined as medications 
which started and stopped prior to 
transplant. 
 
 
5. Time-to-first 
occurrence for DSA, 
HLA-DQ DSA, C11-
binding DSA, DSA IgG3 
type, IA, TG, etc. 
When it is not possible to define the exact 
date of occurrence due to the way the data 
is collected, the analysis of time-to-first 
occurrence cannot provide exact 
information. 
 
 
6. Revise the analysis of 
tacrolimus dose and 
trough concentration 
To provide clarification and details. 
 
 
7. Revise the analysis of 
plasma exchange 
therapy 
Given that the low number of incidence is 
expected, assessing the treatment 
differences are not practically applicable. 
Descriptive statistics will be summarized. 
 
 
8. Add the estimation 
exposure-adjusted 
incidence rate (EAIR) 
for TEAEs and SAEs in 
the safety analysis plan 
The crude percent estimation of AEs can 
result in the increased percent rates for 
TEAEs and SAE particularly in the later 
phase of the study. 
 
 
9. Exploratory endpoints 
and analyses 
The exploratory endpoints and analyses 
have been adjusted based on available 
data, to more closely address the clinical 
and research questions. 
 


Sponsor: Astellas Pharma Global Development, Inc. 
SAP Version:  2.00 
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Page 37 of 38 
9 
REFERENCES 
1. 
Tambur AR, Herrera ND, Haarberg KM, Cusick MF, Gordon RA, Leventhal JR, et al. 
Assessing antibody strength: comparison of MFI, C1q, and titer information. Am J 
Transplant. Epub 2015 Apr 30. doi 10.1111/ajt.13295. 
2. 
Solez K, Colvin RB, Racusen LC, Haas M, Sis B, Mengel M, PF et al. Banff 07 
classification of renal allograft pathology: updates and future directions. Am J 
Transplant. 2008 Apr;8(4):1-8. 
3. 
Kim J, Wilson S, Undre N, Fei S, Kristy R, Schwartz J. A Novel, Dose-Adjusted 
Tacrolimus Trough Concentration (TTC) Model for Prediction and Variance 
Estimation. Am J Transplant. 2017;17 (suppl 3). 
4. 
Kim, J., A. Maity, and A. M. Staicu (2016). General functional concurrent model. 
Unpublished manuscript (under review). 
5. 
Scheipl, F., A.-M. Staicu, and S. Greven (2015). Functional additive mixed models. 
Journal of Computational and Graphical Statistics 24, 477-501. 
6. 
Shuker et al.  (2015) Intra-patient variability in tacrolimus exposure: Causes, 
consequences for clinical management. Transplantation Reviews. 29: 78-84. 
 
 






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