Conditions: Kidney Transplantation
Keywords: renal transplantation conditioning regimen BMT high dose post-transplant cyclophosphamide (PT/Cy) renal allograft tolerance immunosuppression withdrawal
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 4
Healthy volunteers: no

Brief summary:
The primary objective of this study is to assess the ability of bone marrow transplantation (BMT) and high-dose post-transplantation cyclophosphamide (PT/Cy) to induce renal allograft tolerance and thus enable discontinuation of immunosuppressive therapy in haploidentical living related donor renal transplant recipients.

Detailed description:
Transplantation is a good treatment for people with end-stage kidney disease. However, there is still much to learn about how to best care for the transplanted kidney and keep it working for a long time. Unless a person receiving a kidney from someone else takes drugs that reduce immune function, the kidney will be rejected. Those drugs must be continued life-long and cause many issues. Therefore, tolerance of the transplanted kidney, without chronic rejection and without the need for permanent immunosuppressive drug treatment, is a highly desirable goal. If this can be achieved, it would make "one kidney for life" possible.

The study treatment includes several days of study medications followed by a kidney and bone marrow transplant. After the transplant, the study treatment will continue with a few more doses of study medications and then anti-rejection medication is started. After a while, the anti-rejection medication is slowly stopped. Researchers will examine blood and tissue samples and try to identify genetic markers for certain conditions like chimerism, response to therapy, and tolerance.

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

Arms:
arm 1: Reduced-intensity conditioning regimen (RICG), bone marrow transplantation (BMT), high dose post-transplant cyclophosphamide (PT/Cy) and Standard of Care (SOC).

Participants will receive: ATG (pre-transplant), pre-medicated with acetaminophen, diphenhydramine; steroid taper of methylprednisolone; fludarabine (2-6 days before transplant), and low-dose cyclophosphamide (pre- transplant); total body irradiation the day before transplant. Participants will receive a living renal transplant followed by BMT. High-dose cyclophosphamide will be given on days 3 and 4 post-transplant with MESNA. Filgrastim will be given on day 5 post-transplant and continue until absolute neutrophil recovery. Standard immunosuppression of tacrolimus, MMF, and prednisone will begin on day 5 post-transplant and be given ≥26 weeks post-transplant. Eligible participants will be gradually withdrawn from medication over a period of 24-40 weeks.

Interventions: intervention 1: anti-thymocyte globulin intervention 2: Fludarabine intervention 3: Cyclophosphamide intervention 4: Total Body Irradiation intervention 5: acetaminophen intervention 6: diphenhydramine intervention 7: methylprednisolone intervention 8: bone marrow transplant intervention 9: MESNA intervention 10: mycophenolate mofetil intervention 11: prednisone intervention 12: filgrastim
Intervention descriptions: intervention 1: An initial dose of 0.5 mg/kg IV will be administered over 6 hours on Day -9. Thereafter the daily dose will be increased to 2 mg/kg IV given over 4 hours on Days -8 and -7. No more than 150 mg of ATG may be administered per day. intervention 2: Fludarabine at dose 30 mg/m\^2 will be administered daily by intravenous infusion over 30 minutes on Day -6 to Day -2. intervention 3: 1. Low dose pre-transplant cyclophosphamide will be administered intravenously (IV) over 1- 2 hours, (depending on volume) on Days -6 and -5. The dose of pre-transplant cyclophosphamide is 14.5 mg/kg/day.
2. High dose cyclophosphamide \[50mg/kg (Ideal Body Weight)\] will be administered on Day 3 post-transplant (within 60 to 72 hours of marrow infusion) and on Day 4 post-transplant. Cyclophosphamide will be given IV over 1-2 hours depending on volume. intervention 4: Total body irradiation, consisting of 200 centigray (cGy) Anterior-Posterior/Posterior-Anterior (AP/PA) with 4 Megavolts (MV) or 6 MV photons at 8-12 cGy/min at the point of prescription will be administered in a single day on Day -1. intervention 5: 650 mg orally prior to antithymocyte globulin infusion. intervention 6: 25mg diphenhydramine orally prior to antithymocyte globulin infusion. intervention 7: On Days -9 to -7 methylprednisolone 1mg/kg IV 1 hour prior ATG. This dose may be repeated once 3 hours after the first dose of steroids. On Day -6 and -5, methylprednisolone 0.75 mg/kg/ IV as a single dose; on Days -4 and -3, methylprednisolone 0.5 mg/kg/ IV as a single dose; on Day -2 methylprednisolone 0.25 mg/kg/ IV as a single dose. intervention 8: Unprocessed, unmanipulated bone marrow will be harvested from the donor and infused into the recipient on Day 0. intervention 9: A series of MESNA doses will be administered for each dose of high dose, post-transplant cyclophosphamide. The total daily dose of MESNA is equal to 80% of the total daily dose of cyclophosphamide. intervention 10: MMF will be administered at a dose of 15 mg/kg orally three times per day based upon actual body weight, with the maximum of 3 grams a day from Day 5 to 35. The dose will then be reduced to the standard 1 g twice daily thereafter. intervention 11: Prednisone will be administered at a dose of 10 mg orally daily from Day 5 for 12 weeks. Thereafter the dose will be reduced to 5 mg orally daily. intervention 12: All recipients will receive 5 microgram/kg per day of filgrastim as a single, subcutaneous injection from Day 5 post-transplant until the absolute neutrophil count is greater than 1000/µl on three consecutive measurements over at least 2 days.

Protocol document:
Prot_000.pdf: 
Immune 
Tolerance
Network
ACCEPTOR
Bone Marrow Transplantation and High Dose Post-Transplant 
Cyclophosphamide for Chimerism Induction and Renal Allograft Tolerance
Protocol ITN054ST
Version 5.0 (March 28, 2016)
IND # 118,975
 
 
 
 
 
This clinical study is supported and conducted by the Immune Tolerance Network, which is sponsored by 
the National Institute of Allergy and Infectious Diseases.
This document is confidential. It is provided for review only to Investigators, potential Investigators, 
consultants, study staff, and applicable independent ethics committees or institutional review boards.It is 
understood that the contents of this document will not be disclosed to others without written authorization 
from ITN and NIAID unless it is necessary to obtain informed consent from potential study participants.


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Protocol Approval
Trial ID:  ITN054ST
Protocol Version: 5.0
Dated: March 28, 2016
IND # 118,975
Protocol Chair:  
Title: Bone Marrow Transplantation and High Dose Post-Transplant Cyclophosphamide
for Chimerism Induction and Renal Allograft Tolerance
I confirm that I have read the above protocol in the latest version. I understand it, and I will work 
according to the principles of good clinical practice (GCP) as described in the US Code of Federal 
Regulations (CFR) - 45 CFR part 46 and 21 CFR parts 50, 56, and 312, and in the International
Conference on Harmonization (ICH) document Guidance for Industry: E6 Good Clinical Practice: 
Consolidated Guidance dated April 1996. Further, I will conduct the study in keeping with local 
legal and regulatory requirements.
As the Principal Investigator, I agree to carry out the study by the criteria written in the protocol 
and understand that no changes can be made to this protocol without written permission of the 
NIAID.
__________________________________________
Protocol Chair                                  (Print)
__________________________________________         _________________
Protocol Chair                                    (Sign)                      Date


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TABLE OF CONTENTS
TABLE OF CONTENTS......................................................................................................................3
LIST OF TABLES...............................................................................................................................8
LIST OF FIGURES .............................................................................................................................8
SYNOPSIS .........................................................................................................................................9
LIST OF ABBREVIATIONS.............................................................................................................. 17
1.
BACKGROUND AND RATIONALE........................................................................................... 20
1.1
BACKGROUND .....................................................................................................................20
1.2
PRECLINICAL AND CLINICAL EXPERIENCE ......................................................................... 22
1.2.1
Preclinical Studies ..........................................................................................................................................................22
1.2.2
Clinical Experience ........................................................................................................................................................22
1.3
SUMMARY OF KNOWN AND POTENTIAL RISKS FOR HUMAN PARTICIPANTS ...................... 29
1.3.1
Risks Associated with Study Procedures for the Recipient.....................................................................................29
1.3.2
Risks Associated with Study Procedures for the Donor..........................................................................................31
1.3.3
Risks Associated with Study Medication for the Recipient ....................................................................................31
1.3.4
Risks Associated with Study Regimen for the Recipient ........................................................................................35
2.
OBJECTIVES ...........................................................................................................................36
2.1
PRIMARY OBJECTIVE ........................................................................................................... 36
2.2
SECONDARY OBJECTIVES.................................................................................................... 37
3.
STUDY DESIGN .......................................................................................................................37
3.1
DESCRIPTION.......................................................................................................................37
3.1.1
Amendment to Allow Sensitized Participants...........................................................................................................37
3.1.2
Accrual..............................................................................................................................................................................38
3.1.3
Informed Consent for Participation .............................................................................................................................38
3.1.4
Interim Safety Review ...................................................................................................................................................38
3.2
STUDY REGIMEN .................................................................................................................40
3.2.1
Recipient Induction and Maintenance Therapy.........................................................................................................40
3.2.2
Donor Therapy and Procedures....................................................................................................................................41
3.3
IMMUNOSUPPRESSION WITHDRAWAL ................................................................................ 41
3.3.1
Withdrawal of MMF and Prednisone..........................................................................................................................41
3.3.2
Withdrawal of Tacrolimus ............................................................................................................................................43
3.4
STUDY DURATION ............................................................................................................... 44
3.5
STUDY ENDPOINTS .............................................................................................................. 45
3.5.1
Primary Endpoint............................................................................................................................................................45


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3.5.2
Secondary Endpoints......................................................................................................................................................45
3.6
STOPPING GUIDELINES ........................................................................................................ 46
3.6.1
General Stopping Guidelines........................................................................................................................................46
3.6.2
Stopping Guidelines for Rejection...............................................................................................................................47
3.6.3
Ongoing Review .............................................................................................................................................................47
4.
ELIGIBILITY...........................................................................................................................48
4.1
INCLUSION CRITERIA .......................................................................................................... 48
4.1.1
Recipient...........................................................................................................................................................................48
4.1.2
Donor................................................................................................................................................................................49
4.2
EXCLUSION CRITERIA.......................................................................................................... 49
4.2.1
Recipient...........................................................................................................................................................................49
4.2.2
Donor................................................................................................................................................................................50
4.3
PREMATURE DISCONTINUATION OF STUDY THERAPY........................................................ 51
4.3.1
Follow–up for Participants Prematurely Discontinued from Study Therapy.......................................................51
4.4
PREMATURE TERMINATION OF A PARTICIPANT FROM THE STUDY.................................... 52
4.4.1
Follow-up for Participants Prematurely Terminated from the Study ....................................................................52
4.5
REPLACEMENT OF STUDY PARTICIPANTS........................................................................... 52
5.
STUDY MEDICATIONS AND PROCEDURES ............................................................................ 52
5.1
OVERVIEW OF CONDITIONING AND POST-TRANSPLANT REGIMEN..................................... 52
5.2
CONDITIONING REGIMEN FOR RECIPIENTS ......................................................................... 53
5.2.1
Indwelling central venous catheter..............................................................................................................................53
5.2.2
Preparative regimen........................................................................................................................................................53
5.2.3
High Dose Post-Transplant Cyclophosphamide (Days 3, 4)...................................................................................54
5.2.4
MESNA (Days 3, 4).......................................................................................................................................................55
5.3
MAINTENANCE THERAPY (DAY 5 THROUGH ELIGIBILITY ASSESSMENT FOR
IMMUNOSUPPRESSION WITHDRAWAL) .............................................................................. 55
5.3.1
MMF.................................................................................................................................................................................55
5.3.2
Prednisone........................................................................................................................................................................55
5.3.3
Tacrolimus .......................................................................................................................................................................55
5.3.4
Filgrastim (Day 5 to ANC Reconstitution)................................................................................................................56
5.4
STUDY REGIMEN FOR DONORS............................................................................................ 56
5.4.1
Bone Marrow Harvest (Day 0).....................................................................................................................................56
5.5
MODIFICATION OF RECIPIENT STUDY MEDICATIONS ......................................................... 56
5.5.1
Bacterial Prophylaxis .....................................................................................................................................................56
5.5.2
Tacrolimus .......................................................................................................................................................................56
5.5.3
Mycophenolic Compounds...........................................................................................................................................56


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5.5.4
Corticosteroids ................................................................................................................................................................57
5.6
PROPHYLACTIC MEDICATIONS AND MONITORING ............................................................. 57
5.6.1
Pneumocystis Jiroveci (PCP) Prophylaxis .................................................................................................................57
5.6.2
Fungal and Yeast Prophylaxis ......................................................................................................................................58
5.6.3
CMV Prophylaxis and Monitoring ..............................................................................................................................58
5.6.4
CMV Treatment ..............................................................................................................................................................58
5.6.5
Treatment of Herpes Infection......................................................................................................................................59
5.7
MONITORING FOR BK VIREMIA ........................................................................................... 59
5.8
MONITORING FOR EBV REACTIVATION............................................................................... 59
5.9
PROHIBITED MEDICATIONS AND VACCINES ....................................................................... 60
5.10
CONCOMITANT MEDICATIONS ............................................................................................ 60
5.11
BLOOD REPLACEMENT THERAPY........................................................................................ 60
6.
STUDY PROCEDURES .............................................................................................................60
6.1
VISIT WINDOWS...................................................................................................................60
6.1.1
Scheduled Visits..............................................................................................................................................................60
6.2
GENERAL ASSESSMENTS ..................................................................................................... 65
6.3
CLINICAL LABORATORY ASSESSMENTS ............................................................................. 65
6.4
MECHANISTIC ASSESSMENTS.............................................................................................. 66
6.5
ASSESSMENT AND MANAGEMENT OF ALLOGRAFT DYSFUNCTION AND REJECTION.......... 67
6.5.1
Definition of Allograft Dysfunction............................................................................................................................67
6.5.2
Indications for Increased Monitoring and For-cause Biopsy..................................................................................67
6.5.3
Modification of Creatinine Threshold for Performance of a For-cause Biopsy ..................................................68
6.5.4
Diagnosis of Rejection...................................................................................................................................................68
6.5.5
Treatment and Management of Participants with Rejection ...................................................................................69
6.5.6
Types of Kidney Biopsies.............................................................................................................................................69
6.5.7
Use and Interpretation of Kidney Biopsies ................................................................................................................70
6.5.8
Biopsy Technique and Handling..................................................................................................................................71
6.5.9
Recording of Biopsy Interpretations............................................................................................................................71
6.6
MONITORING AND MANAGEMENT OF DONOR SPECIFIC ANTIBODIES ................................ 71
6.7
GRAFT-VERSUS-HOST DISEASE ........................................................................................... 72
6.7.1
Acute GVHD...................................................................................................................................................................72
6.7.2
Chronic GVHD ...............................................................................................................................................................73
6.8
ENGRAFTMENT SYNDROME ................................................................................................ 74
7.
MECHANISTIC ASSAYS .......................................................................................................... 75
7.1
RATIONALE FOR IMMUNE STUDIES..................................................................................... 75
7.2
PLANNED MECHANISTIC ASSAYS........................................................................................ 75


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7.2.1
Histological Assessments of Tolerance Mechanisms...............................................................................................75
7.2.2
Gene Expression in peripheral blood and biopsies...................................................................................................76
7.2.3
Multi-Parameter Flow Cytometry (MFC) ..................................................................................................................78
7.2.4
Alloreactive T Cell and Plasma Cytokine Assays.....................................................................................................80
7.2.5
Alloreactive B Cell Assays and B cell Repertoire Analysis....................................................................................80
7.2.6
Blood Chimerism............................................................................................................................................................81
7.2.7
Anti-donor HLA Alloantibody and Flow Cross Match............................................................................................82
7.2.8
HLA Typing.....................................................................................................................................................................83
7.3
OVERVIEW OF DATA ANALYSIS .......................................................................................... 83
7.3.1
Individual based longitudinal profiling.......................................................................................................................83
7.3.2
Exploratory analysis.......................................................................................................................................................84
7.4
FUTURE/UNPLANNED STUDIES............................................................................................ 84
7.5
SPECIMEN LOGISTICS .......................................................................................................... 84
7.6
SPECIMEN TRACKING PROCEDURES.................................................................................... 84
7.7
SPECIMEN STORAGE ............................................................................................................ 85
8.
ADVERSE EVENTS ..................................................................................................................85
8.1
OVERVIEW ...........................................................................................................................85
8.2
DEFINITIONS........................................................................................................................86
8.2.1
Adverse Event .................................................................................................................................................................86
8.2.2
Adverse Reaction and Suspected Adverse Reaction ................................................................................................86
8.2.3
Serious Adverse Event or Serious Suspected Adverse Reaction............................................................................86
8.2.4
Unexpected Adverse Events.........................................................................................................................................87
8.3
COLLECTING AND RECORDING ADVERSE EVENTS.............................................................. 87
8.3.1
Methods of Collection....................................................................................................................................................87
8.3.2
Severity of Adverse Events to be Collected...............................................................................................................87
8.3.3
Recording Method..........................................................................................................................................................88
8.4
GRADING AND ATTRIBUTION OF ADVERSE EVENTS ........................................................... 89
8.4.1
Grading Criteria ..............................................................................................................................................................89
8.4.2
Attribution Definitions...................................................................................................................................................89
8.5
REPORTING SERIOUS ADVERSE EVENTS ............................................................................. 90
8.5.1
Reporting SAEs to the IND Sponsor...........................................................................................................................90
8.5.2
Reporting SAEs to Health Authorities........................................................................................................................91
8.5.3
Reporting SAEs to IRBs and Ethics Committees .....................................................................................................91
8.5.4
Reporting SAEs to the DSMB......................................................................................................................................91
8.5.5
Reporting Pregnancy......................................................................................................................................................91
9.
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN ................................................. 91


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9.1
ANALYSIS SAMPLES ............................................................................................................ 91
9.2
ANALYSIS PLAN...................................................................................................................92
9.2.1
Primary Endpoint............................................................................................................................................................92
9.2.2
Secondary Endpoints......................................................................................................................................................92
9.2.3
Safety Analysis................................................................................................................................................................92
9.2.4
Baseline Characteristics and Demographics ..............................................................................................................93
9.2.5
Medical History...............................................................................................................................................................93
9.2.6
Use of Medications.........................................................................................................................................................93
9.2.7
Study Completion ...........................................................................................................................................................93
9.3
SAMPLE SIZE........................................................................................................................93
9.4
INTERIM ANALYSIS .............................................................................................................94
9.5
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL PLAN................................... 94
10.
ACCESS TO SOURCE DATA/DOCUMENTS.............................................................................. 94
11.
QUALITY CONTROL AND QUALITY ASSURANCE ................................................................. 94
12.
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL PRACTICE ........ 95
12.1
STATEMENT OF COMPLIANCE ............................................................................................. 95
12.2
INFORMED CONSENT ........................................................................................................... 95
12.3
PRIVACY AND CONFIDENTIALITY....................................................................................... 95
13.
PUBLICATION POLICY........................................................................................................... 96
14.
REFERENCES..........................................................................................................................97
APPENDIX 1A. PRE-TRANSPLANT THROUGH TRANSPLANT: RECIPIENT ................................. 105
APPENDIX 1B. PRE-TRANSPLANT THROUGH POST-TRANSPLANT: DONOR.............................. 109
APPENDIX 1C. REINITIATION OF CONDITIONING: PRE-TRANSPLANT THROUGH TRANSPLANT: 
RECIPIENT ....................................................................................................................... 111
APPENDIX 1D. REINITIATION OF CONDITIONING: PRE-TRANSPLANT THROUGH POST-
TRANSPLANT: DONOR..................................................................................................... 114
APPENDIX 2. POST-TRANSPLANT THROUGH EVALUATION FOR WITHDRAWAL 
ELIGIBILITY .................................................................................................................... 116
APPENDIX 3. IMMUNOSUPPRESSION WITHDRAWAL (INITIATED BETWEEN 26 AND 3 WEEKS 
POST-TRANSPLANT) ........................................................................................................ 119
APPENDIX 4. POST IMMUNOSUPPRESSION WITHDRAWAL FOLLOW-UP (INITIATED BETWEEN
52 AND 62 WEEKS POST-TRANSPLANT)........................................................................... 123
APPENDIX 5. SAFETY FOLLOW-UP: RECIPIENTS ....................................................................... 125
APPENDIX 6. ACUTE GVHD ASSESSMENT WORKSHEET ............................................................ 128


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APPENDIX 7. CHRONIC GVHD ASSESSMENT WORKSHEET ....................................................... 131
APPENDIX 8. STUDY REGIMEN WITH DE-SENSITIZATION SCHEDULE FOR SENSITIZED 
RECIPIENTS ..................................................................................................................... 134
APPENDIX 9. DE-SENSITIZATION SCHEDULE OF EVENTS PER JHH STANDARD OF CARE ....... 135
APPENDIX 10. REINITIATION OF CONDITIONING REGIMEN ..................................................... 136
LIST OF TABLES
Table 1. Outcomes of reduced intensity allogeneic BMT for hemoglobinopathy ...................................24
Table 2. Rapid Oral TMP-SMX desensitization protocol.....................................................................58
Table 3. Designated study windows...................................................................................................61
Table 4. Consensus conference on clinical grading of acute GVHD.....................................................73
Table 5. Clinical Grading of Chronic GVHD......................................................................................74
Table 6. Proposed Multiplex IHC Staining Panel................................................................................76
Table 7. Thirteen (13) of 25 genes used to derive renal tolerance signatures in 3 clinical studies............77
Table 8. Frozen/banked specimen flow cytometry panel design and list of suggested cellular antigens ...79
Table 9. Attribution of adverse events................................................................................................90
LIST OF FIGURES
Figure 1 Schema for reduced intensity allogeneic BMT for hemoglobinopathy .....................................23
Figure 2: Donor chimerism early after transplantation among patients who experienced bone marrow graft 
loss......................................................................................................................................26
Figure 3: Daily WBC counts, maximum temperatures, and serum creatinines in patients undergoing 
reduced intensity conditioning allogeneic stem cell transplant for hemoglobinopathy................27
Figure 4: Schematic of trial design with decision points for safety review.............................................39
Figure 5: Study regimen for unsensitized recipients ............................................................................41
Figure 6: Study regimen for sensitized recipients .............................................................................. 134


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SYNOPSIS
Title
Bone Marrow Transplantation and High Dose Post Transplant 
Cyclophosphamide for Chimerism Induction and Renal Allograft Tolerance
IND Sponsor
NIAID
Conducted by
Immune Tolerance Network
Protocol Chair
Co-PIs
Accrual 
Objective
The accrual goal for this study is six renal transplant recipient-donor pairs
who meet the per-protocol (PP) analysis sample definition.
Study 
Treatment
Recipient Induction and Maintenance Therapy
Study Treatment for Unsensitized Recipients
For unsensitized recipients, antithymocyte globulin (ATG) will be given from 
Day -9 to Day -7; fludarabine from Day -4 to Day -2 and low-dose 
cyclophosphamide on Day -4 and Day -3. Participants will undergo 
hemodialysis 6 to 8 hours after each fludarabine dose. Participants will 
undergo total body irradiation (TBI) on Day -1. Renal transplantation 
followed by bone marrow infusion will occur on Day 0. High dose post-
transplant cyclophosphamide (PT/Cy) will be administered on Days 3 and 4 
with hydration, sodium-2-mercaptoethane sulfonate (MESNA) and anti-
emetics. Filgrastim will be administered daily starting on Day 5 until absolute 
neutrophil count (ANC) recovery. Standard maintenance immunosuppression 
consisting of tacrolimus, with target trough level of 8-10 ng/ml, and 
mycophenolate mofetil (MMF) 15 mg/kg three times daily, and prednisone 
10mg daily, will be started on Day 5.
On Days -9 through -7 ATG will be administered daily at a specialized 
outpatient Bone Marrow Transplant (BMT) unit. On Day -4 participants will be 
admitted to the inpatient BMT unit for the three-day course of fludarabine to 
ensure precise timing of dosing and dialysis as well as monitoring of side 
effects. Because fludarabine is cleared by the kidneys, these end stage renal 
disease (ESRD) participants will require daily dialysis to avoid the potentially 
fatal side effect of neurotoxicity. Participants will remain hospitalized from the 
first day of fludarabine administration through the kidney and bone marrow 
transplant. Recipient hospitalization will then continue through post-operative 
Day 7 under the care of the surgeon and the nephrologist in keeping with the 
site’s standard renal transplantation procedures. Recipients will then be 
discharged to a specialized outpatient BMT unit on Day 8 and followed per 


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standard of post-non-myeloablative bone marrow transplant care for 60 days 
post-transplant.
Study Treatment for Sensitized Recipients
Sensitized participants with detectable DSA at screening who meet inclusion 
criterion 5b will undergo de-sensitization per Johns Hopkins Hospital (JHH)
standard of care (Appendices 8 and 9) prior to receiving the first dose of study 
therapy. They will receive the same induction and maintenance therapy as 
unsensitized recipients but with continued de-sensitization therapy and 
additional monitoring for recurrence of DSA. Please see Appendix 8 for the 
Study Regimen with De-sensitization Schedule for Sensitized Recipients and 
Appendix 9 for the De-sensitization Schedule of Events for Sensitized 
Recipients.
Donor Therapy and Procedures
Donor nephrectomy immediately followed by bone marrow harvest will take 
place under the same anesthetic on Day 0, with renal transplantation followed 
by bone marrow infusion into the recipient also on Day 0.
Immuno-
suppression 
Withdrawal
Participants will receive standard maintenance immunosuppression for at least 
26 weeks post-transplant. Those participants demonstrating no evidence of 
rejection will first undergo withdrawal of MMF and prednisone over a 4- to 8-
week period. Withdrawal from MMF and prednisone may be initiated no 
earlier than Week 26 and no later than Week 34 post-transplant. After 8 
weeks on stable tacrolimus monotherapy, participants demonstrating no 
evidence of rejection, as described in section 3.3.2, will undergo withdrawal 
of tacrolimus. The tacrolimus dose will be reduced in a stepwise fashion over 
a period of no fewer than 12 weeks and no greater than 16 weeks with the 
goal of complete discontinuation no later than 66 weeks post-transplant.
Participants who successfully complete immunosuppression withdrawal will 
then undergo 4 years of follow-up. Participants who fail to initiate or to 
complete immunosuppression withdrawal will undergo 2 years of safety 
follow-up.
Study Design
This trial is a phase II, single arm, open-label, single center pilot study to 
assess a reduced-intensity conditioning regimen, bone marrow transplantation 
and high dose PT/Cy in six recipients of renal allografts from Human 
Leukocyte Antigen-haploidentical (HLA-haploidentical) living related 
donors.
Study Duration
Total study duration will be 361 weeks (6.9 years).
Primary 
Objective
The primary objective of this trial is to assess the ability of bone marrow 
transplantation and high dose PT/Cy to induce renal allograft tolerance and 
thus enable discontinuation of immunosuppressive therapy in haploidentical 
living related donor renal transplant recipients. 


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Secondary 
Objectives
1.
To assess the safety of the study regimen.
2.
To assess whether maintenance of donor chimerism is required for 
sustained allograft tolerance.
3.
To explore mechanistic assays which might predict successful 
immunosuppression withdrawal.
Primary
Endpoint
The primary endpoint is the proportion of participants who achieve 
operational tolerance, defined as remaining off all immunosuppression 52
weeks after completion of immunosuppression withdrawal with:
a) no evidence of biopsy-proven allograft rejection and
b) acceptable renal function, defined as a serum creatinine that has increased 
no more than 25% above baseline (see section 6.5.1 for baseline thresholds) at 
the primary endpoint visit.
All participants who successfully complete immunosuppression withdrawal 
will undergo a protocol biopsy at this time point to assess the primary 
endpoint.
Secondary 
Endpoints
Safety
1.
The incidence, severity and duration of graft versus host disease 
(GVHD) in transplanted participants.
2.
The incidence and duration of engraftment syndrome in transplanted 
participants.
3.
The proportion of transplanted participants who die.
4.
The proportion of transplanted participants with acute renal allograft 
rejection demonstrated by a biopsy or clinically if a biopsy cannot be 
performed. If participant has allograft dysfunction as defined in 
section 6.5 and cannot undergo biopsy he or she will be presumed to 
have rejection without biopsy confirmation.
5.
The histological severity of biopsies demonstrating acute rejection as 
measured by Banff Grade per Banff 2007 Classification Renal 
Allograft Pathology1.
6.
The proportion of transplanted participants with chronic T cell-
mediated or antibody-mediated rejection. This assessment should also 
include progressive interstitial fibrosis/tubular atrophy (IF/TA), 
transplant glomerulopathy or chronic obliterative arteriopathy without 
an alternative, non-rejection-related cause. See Banff 2007 
Classification Renal Allograft Pathology for definition of terms1.
7.
Time from transplant to the first episode of acute rejection requiring 
treatment.


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8.
The incidence, severity and duration of adverse events including 
infection, wound complications, post-transplant diabetes, 
hemorrhagic cystitis and malignancy. 
9.
The proportion of transplanted participants who develop donor 
specific antibody:
a.
after initiation of immunosuppression withdrawal
b.
at any time during trial participation
10. The time to absolute neutrophil recovery. This is defined as the 
interval from the neutrophil nadir to the first day of three consecutive 
GDLO\ QHXWURSKLO FRXQWV per μL. The neutrophil nadir is defined 
as the first day post-transplant on which the absolute neutrophil count 
(ANC) is below 500 per μL.
11. The time to platelet count recovery. This is defined as the interval 
from transplant to the first day of a platelet count of 20,000 per μL
without a prior platelet transfusion in the preceding seven days.
Efficacy
1.
The proportion of transplanted participants who remain off 
immunosuppression for at least 52 weeks including those in whom 
the 52 week biopsy was not performed.
2.
The proportion of participants who remain free from return to 
immunosuppression for the duration of the study.
Mechanistic
1.
The correlation of operational tolerance with the extent and durability 
of donor hematopoietic and T cell chimerism as measured by serial 
short tandem repeat analysis of recipients’ peripheral blood 
mononuclear cells (PBMCs) and T cells.
2.
The correlation of operational tolerance with other biomarkers such as 
cell subsets or gene expression.
The following secondary endpoints pertaining to safety and efficacy will 
be assessed only in participants who complete tacrolimus withdrawal:
1.
Immunosuppression-free duration, defined as time from completion 
of tacrolimus to end of trial participation or to time of restarting 
immunosuppression.
2.
Time from completion of tacrolimus withdrawal to first episode of 
acute rejection or presumed acute rejection, defined per Banff 2007 
Classification Renal Allograft Pathology1.
3.
Time from completion of tacrolimus withdrawal to first diagnosis of 
chronic T cell mediated or antibody-mediated rejection. This 
assessment should also include IF/TA, transplant glomerulopathy or 
chronic obliterative arteriopathy without an alternative, non-rejection 


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related cause. See Banff 2007 Classification Renal Allograft 
Pathology1.
Inclusion 
Criteria
Recipient
Recipient participants must meet all of the following criteria to be eligible for 
this study:
1.
Recipient of a first renal allograft from an HLA-haploidentical, living 
related donor. The donor and recipient must be HLA identical for at least 
one allele (using high resolution DNA based typing) at the following 
genetic loci: HLA-A, HLA-B, HLA-C and, HLA-DRB1. Fulfillment of 
this criterion shall be considered sufficient evidence that the donor and 
recipient share one HLA haplotype.
2.
Age 18 to 65 years.
3.
Single solid organ recipients (kidney only).
4.
ABO compatibility with donor.
5.
DSA will be assessed by the local laboratory 30 days or less prior to 
transplant using solid phase micro particle technology (by Luminex®
phenotype panel or Luminex single antigen bead test.) The following 
criteria apply:
a.
Participants without detectable DSA will be deemed eligible if 
they meet other entry criteria.
b.
Participants with detectable DSA and a positive flow cytometric 
crossmatch may undergo de-sensitization per standard of care if 
they are cytotoxic crossmatch negative. Such participants must 
demonstrate a negative flow cytometric crossmatch by day -9 in 
order to receive the first dose of study therapy (ATG). 
Participants who do not demonstrate an acceptable response to 
de-sensitization by day -9 will be considered screen failures and 
will be terminated from the study.
c.
Participants with a positive cytotoxicity crossmatch will be 
excluded. 
6.
Removed in protocol version 5.0
7.
Removed in protocol version 5.0
8.
Normal estimated left ventricular ejection fraction and no history of 
ischemic heart disease requiring revascularization, unless cleared by a 
cardiologist.
9.
Forced expiratory volume (FEV1) and forced vital capacity (FVC) > 40% 
of predicted at the screening visit.
10. Serological evidence of prior Epstein-Barr virus (EBV) infection as 
documented by positive IgG and negative IgM antibodies against EBV.


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11. For women of childbearing potential, a negative serum or urine pregnancy 
test with sensitivity less than 50 mIU/m within 72 hours before the start of 
study medication.
12. Use of two forms of contraception with less than a 5% failure rate or 
abstinence by all transplanted participants for 18 months after the first 
dose of study therapy. For the first 60 days post-transplant, recipients 
should be encouraged to use non-hormonal contraceptives due to the 
potential adverse effect of hormones on bone marrow engraftment. If
using a barrier method, a double barrier method should be used.
13. Ability to receive oral medication.
14. Ability to understand and provide informed consent.
15. All participants must demonstrate a negative QuantiFERON® (QFT) 
assay result within 52 weeks of transplant regardless of PPD status. 
Participants with a positive QFT assay will not be eligible for the study 
unless they have completed treatment for latent TB and have a negative 
chest x-ray. QFT testing done within 52 weeks before transplant is 
acceptable as long as there is documentation of the results. Prior 
recipients of a BCG vaccination are not exempt.
Donor
Donor participants must meet all of the following criteria to be eligible for this 
study:
1.
HLA-haploidentical, first-degree relatives or half-siblings of the recipient 
participant at the allele or allele group. The donor and recipient must be 
HLA identical for at least one allele (using high resolution DNA based 
typing) at the following genetic loci: HLA-A, HLA-B, HLA-C, and HLA-
DRB1. Fulfillment of this criterion shall be considered sufficient evidence 
that the donor and recipient share one HLA haplotype.
2.
Age 18 to 65 years.
3.
Creatinine clearance >80 ml/minute as measured from a 24 hour urine 
collection within 26 weeks of the screening visit. If a serum creatinine 
drawn at the screening visit is > 20% higher than the serum creatinine 
drawn at the time of the 24 hour urine collection, the creatinine clearance 
must be re-evaluated by a repeat 24 hour urine test. If the new value is 
PJG/ WKHGRQRUZLOO EHH[FOXGHG.
4.
Meets institutional selection criteria for organ and bone marrow donation.
5.
Ability to understand and provide informed consent for all study
procedures including kidney transplant and bone marrow harvest.
6.
Serologic evidence of prior EBV infection as documented by positive IgG 
and negative IgM antibodies against EBV.


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Exclusion 
Criteria
Recipient
Recipient participants who meet any of the following criteria will not be 
eligible for this study:
1.
Underlying renal disease with a high risk of disease recurrence in the 
transplanted kidney, including:
a.
focal segmental glomerulosclerosis (FSGS).
b.
type I or II membranoproliferative glomerulonephritis.
c.
hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura.
2.
Clinically important genital/urinary tract dysfunction.
3.
Body mass index (BMI) > 40.
4.
Women who are breastfeeding.
5.
History of cancer within the last 5 years, except for nonmelanoma skin 
cancer, stage 1 renal cell carcinoma, stage 1 prostate cancers cured by 
local resection and any curatively treated carcinomas in situ.
6.
History of positive HIV-1 or HIV-2 serologies or nucleic acid test.
7.
Evidence of hepatitis B infection.
Participants demonstrating any one of the following will be excluded:
a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM.
c.
positive anti-HBc IgG
d.
positive HBV PCR
8.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV 
RNA PCR. All positive HCV antibody results must be assessed by an EIA 
assay and confirmed by a quantitative serum HCV RNA assay. 
Participants with positive HCV antibodies but undetectable serum HCV 
RNA may be considered for eligibility. Participants with negative anti-
HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV 
serologies.
9.
History of active tuberculosis (TB). 
10. Any active, severe local or systemic infection at the screening visit.
11. Autoimmune disease requiring immunosuppressive drugs for 
maintenance.
12. Use of investigational drug, other than the study medications specified by 
the protocol, within 30 days of transplantation.
13. Receipt of a live vaccine within 30 days of receipt of study therapy.
14. The presence of any medical condition that the Investigator deems 
incompatible with participation in the trial.
15. Positive cytotoxic crossmatch.
16. Calculated PRA greater than 90%.
Donor
Donor participants who meet any of the following criteria will not be eligible 
for this study:
1.
History of type I or type II diabetes mellitus.


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2.
History of severe cardiovascular disease, defined as New York Heart 
Association Class III or IV2.
3.
History of blood product donation to recipient.
4.
History of positive HIV-1 or HIV-2 serology or nucleic acid test.
5.
Evidence of hepatitis B infection.
Participants demonstrating any one of the following will be excluded:
a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM.
c.
positive anti-HBc IgG
d.
positive HBV PCR
6.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV 
RNA PCR. All positive HCV antibody results must be assessed by an 
EIA assay and confirmed by a quantitative serum HCV RNA assay. 
Participants with positive HCV antibodies but undetectable serum HCV 
RNA may be considered for eligibility. Participants with negative anti-
HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV 
serologies.
7.
Autoimmune disease requiring immunosuppressive drugs for 
maintenance.
8.
The presence of any medical condition that the Investigator deems 
incompatible with participation in the trial. 


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LIST OF ABBREVIATIONS
Abs
Antibodies
AKI
acute kidney injury
AE
adverse event
ALT
alanine aminotransferase
ANC
absolute neutrophil count
AST
aspartate aminotransferase
AUC
area under the curve
BMT
bone marrow transplant
CBC
complete blood count
CFR
Code of Federal Regulations
CHF
congestive heart failure
CKBMT
combined kidney and bone marrow transplant 
CLS
capillary leak syndrome
CRF
case report form
CMV
Cytomegalovirus
CMVIG
CMV intravenous immune globulin
CRO
contract research organization
CRS
cytokine release syndrome
Cy
Cyclophosphamide
DSA
donor specific antibody
DSMB
Data and Safety Monitoring Board
ESRD
end stage renal disease
FCXM
flow cytometry crossmatch
FDA
US Food and Drug Administration 
FCB
for cause biopsy


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FSGS
focal segmental glomerulosclerosis
GCP
good clinical practice
GI
Gastrointestinal
HSCT
hematopoietic stem cell transplantation
IARs
infusion associated reactions 
IBW
ideal body weight
ICH
International Conference on Harmonization
IRB
institutional review board
IS
Immunosuppression
ISW
immunosuppression withdrawal
ITN
Immune Tolerance Network
JHU
Johns Hopkins University
LDCy
Low dose cyclophosphamide
MedDRA
Medical Dictionary for Regulatory Activities
MESNA
sodium-2-mercaptoethane sulfonate
MHC
major histocompatibility complex
MMF
mycophenolate mofetil
NCI-
CTCAE v. 
4.0
National Cancer Institute Common Terminology Criteria for Adverse Events
version 4.0
PP
per protocol
PML
progressive multifocal leukoencephelopathy
PT/Cy, 
HDCy
post-transplant cyclophosphamide, high dose cyclophosphamide
PTLD
post-transplant lymphoproliferative disease
RB
repeat biopsy
SAE
serious adverse event
SAR
suspected adverse reaction


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SIADH
syndrome of inappropriate anti-diuretic hormone
SOT
solid organ transplantation
TBI
total body irradiation
TPE
therapeutic plasma exchange
WHO
World Health Organization


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1.
BACKGROUND AND RATIONALE
1.1
BACKGROUND
Despite substantial progress in living donor transplantation leading to one year graft survival rates 
exceeding 95%, the problem of chronic rejection and the complications of long-term pharmacologic 
immunosuppression remain3,4. For these reasons, the achievement of durable transplantation tolerance, as 
reflected by the maintenance of stable graft function off all immunosuppressive medications, is a 
paramount objective. Based upon early observations that natural5 or induced 6,7 hematopoietic chimeras 
are tolerant of solid organ allografts from the same donor8, there has been substantial interest in 
combining solid organ and bone marrow or hematopoietic stem cell transplantation (HSCT) from the 
same donor to achieve donor hematopoietic chimerism and durable tolerance of the solid organ. Indeed, 
case reports have shown that patients who develop kidney or liver failure after undergoing allogeneic 
stem cell transplantation for a hematologic disorder can be successfully transplanted from the same donor 
and weaned off immunosuppression without experiencing graft rejection9. However, the morbidity and 
mortality of lethal conditioning and allogeneic HSCT have precluded its use specifically for the induction 
of chimerism and solid organ allograft tolerance.
Study investigators at Johns Hopkins have recently developed non-myeloablative conditioning regimens 
that permit the sustained engraftment of partially HLA-mismatched (HLA-haploidentical) bone marrow 
from first-degree relatives with minimal toxicities. To mitigate the risks of graft rejection and graft-
versus-host disease (GVHD), the regimen includes the IV administration of high dose post-transplantation 
cyclophosphamide (PT/Cy) on days 3 and 4 after transplantation to selectively deplete or inactivate 
proliferating alloreactive cells while permitting immune reconstitution from cyclophosphamide-resistant, 
non-alloreactive lymphocytes present in the donor graft. They have used this regimen extensively in 
patients with hematological malignancies and have piloted a similar regimen in sickle cell and 
thalassemia patients as described in section 1.2.2.
Cyclophosphamide-induced transplantation tolerance: Mechanisms of action
High-dose cyclophosphamide, when administered in a narrow window after transplantation, depletes 
alloreactive T cells from the donor and host and can inhibit both GVHD and graft rejection10-13. As a form 
of drug-induced immunologic tolerance14, the strategy of giving high-dose cyclophosphamide after 
transplantation takes advantage of the heightened cytotoxic sensitivity of proliferating, alloreactive T cells 
over non-alloreactive, resting T cells to being killed by a DNA-damaging agent12,15. Importantly, 
hematopoietic stem cells are relatively quiescent16 and express high levels of aldehyde dehydrogenase, 
which likely confer cellular resistance to cyclophosphamide17. These findings explain how high dose 
PT/Cy can be used to achieve selective depletion of alloreactive T cells without substantially delaying 
hematologic recovery in the short or long term after transplantation.
Prior experience with combined bone marrow and solid organ transplantation
There has been previous clinical experience with both sequential and combined bone marrow transplant
and solid organ transplant (SOT). Several investigators have reported results with combined bone marrow 
or hematopoietic stem cell and solid organ transplantation from the same donor. A 1998 review of the 
published literature18 identified four patients who were treated with allogeneic bone marrow 
transplantation for a hematologic disorder, developed end-stage renal disease (ESRD), and then received 
a kidney allograft from the original stem cell donor19-21. At the time of reporting, there were no episodes 
of kidney failure or rejection and all patients were off all immunosuppressive drugs with no evidence of 
recurrent hematologic disorder or renal dysfunction. These isolated case reports provided the “proof of 


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principle” that durable donor hematopoietic chimerism permits the safe discontinuation of pharmacologic 
immunosuppression following transplantation of a kidney from the same donor.
However the considerable toxicities of bone marrow transplant have militated against its use solely as an 
adjunct to SOT to achieve immunosuppression-free tolerance. Spitzer and colleagues performed 
combined kidney and bone marrow transplants for seven patients with multiple myeloma and myeloma-
related ESRD22-24. Interestingly, three patients developed operational tolerance of the transplanted organ 
and were successfully weaned off all immunosuppression even though donor hematopoietic chimerism 
was transient. In a subsequent trial sponsored by the ITN, the group at Massachusetts General Hospital 
treated five ESRD patients with non-myeloablative conditioning and combined kidney and bone marrow 
transplantation from HLA-haploidentical related donors (ClinicalTrials.gov Identifier: NCT00801632)25.
Although donor chimerism was lost in all five patients within the first 21 days after combined 
transplantation, four were successfully weaned off immunosuppression.
These investigators have recently updated the outcomes of these five patients plus an additional five 
patients receiving treatment in a similar transplantation protocol26. Nine of the ten patients developed 
acute kidney injury (AKI), with or without fever and fluid retention, as part of an idiopathic capillary leak 
syndrome (CLS) that is similar clinically to engraftment syndrome seen after autologous or allogeneic 
HSCT27. Altogether, seven patients were successfully weaned off immunosuppression with stable renal 
function. However one successfully weaned patient subsequently was returned to immunosuppression for 
recurrence of his underlying disease, IgA nephropathy. A second successfully weaned participant was 
restarted on MMF after 6 years off immunosuppression for chronic humoral rejection, with persistent 
DSA and c4d+ positive staining on biopsy. Early c4d negative transplant glomerulopathy and low level 
DSA have been detected in a third patient who has remained off immunosuppression for 6.8 years. Thus 
as of early 2013, 5 of 10 patients remain off immunosuppression. Two patients have lost their grafts (one 
due to idiopathic CLS and AKI),and a third had to be placed back on immunosuppression due to an 
episode of acute rejection following immunosuppression withdrawal26.
High Dose Post Transplant Cyclophosphamide in Combined Kidney and Hematopoietic Stem cell 
Transplantation
Investigators at Northwestern have reported successful induction of durable hematopoietic chimerism in 
highly HLA-mismatched, living donor kidney transplant patients using combined donor hematopoietic 
stem cell and kidney transplant. The conditioning regimen described: 3 doses of fludarabine (30mg/kg), 
200cGy total body irradiation, pre-transplant cyclophosphamide and HSCs, followed by high dose 
cyclophosphamide 50mg/kg on day 3 and maintenance therapy with MMF and tacrolimus, differs from 
this trial’s planned regimen mainly in the co-administration of proprietary, tolerogenic ‘facilitator cells’ 
with the hematopoietic stem cell graft. These investigators initially reported durable, multi-lineage 
hematopoietic chimerism in 5 of 8 study participants. These five chimeric patients demonstrated 
immunocompetence and donor-specific hypo-responsiveness and were successfully withdrawn from all 
immunosuppression 1 year after transplantation without allograft rejection28. One participant achieved 
100% donor chimerism at month 2 but developed viral sepsis, renal artery thrombosis and subsequent 
renal allograft loss. Two participants developed transient chimerism and were maintained on 
immunosuppression. One participant experienced sub-clinical Banff IA rejection and was returned to 
immunosuppression. Staining for c4d was negative. None of the participants produced DSA, experienced 
GVHD or engraftment syndrome. The study regimen was well-tolerated, with outpatient management 
after post-operative day 228.
These investigators recently reported long-term follow-up in a total of 15 HLA mismatched kidney 
recipients who underwent combined hematopoietic stem cell and renal transplant under the regimen
described above. Ten of these 15 patients have achieved durable full or mixed hematopoietic chimerism 


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without GVHD or engraftment syndrome. Eight of these 10 achieved durable, high level (>90%) 
hematopoietic chimerism. Six of the 8 have successfully completed immunosuppression withdrawn 
without allograft rejection or graft loss (range of between 10 and 22 months off IS). The two remaining 
patients with high level chimerism are currently undergoing immunosuppression withdrawal. Two 
subjects achieved sustained, mixed chimerism. Three participants achieved transient chimerism. Of 
interest, 2 of these transiently chimeric recipients subsequently experienced subclinical rejection (Banff 
IA) during immunosuppression withdrawal despite evidence of donor-specific hyporesponsiveness by
MLR and CML. None of the participants have produced DSA. The incidence of serious infections has 
been low. No CMV infections or opportunistic fungal infections have been reported29. The ITN 054ST 
trial does not include use of ‘facilitator cells’. Nevertheless, given the similarities in study regimens, these 
results provide an encouraging backdrop for the current protocol with regard to efficacy and safety.
In summary operational tolerance can be achieved across an HLA barrier following combined, kidney and
bone marrow transplantation. Preliminary results suggest that durable, high level donor chimerism may be 
associated with tolerance of the kidney allograft, though longer term follow-up is required to confirm this 
finding. This goal must be balanced against the toxicities of bone marrow transplantation, including the 
risk of GVHD. Operational tolerance is also achievable in the setting of transient, mixed chimerism.
However, a subset of patients experienced acute kidney injury, acute rejection and ultimately, graft loss.
Thus it remains unclear whether hematopoietic chimerism is required to achieve tolerance and whether 
the extent or durability of chimerism impacts the likelihood of reaching this objective.
1.2
PRECLINICAL AND CLINICAL EXPERIENCE
1.2.1
Preclinical Studies
For over a decade, the Johns Hopkins investigators have been developing minimally toxic regimens for 
crossing the HLA barrier in HSCT by incorporating the method of cyclophosphamide-induced
immunologic tolerance.
Initial pre-clinical efforts were devoted to using PT/Cy to overcome the HLA barrier in HSCT. A non-
myeloablative regimen comprised of pre-transplantation fludarabine, an immunosuppressive purine 
analog, and 200 cGy total body irradiation and PT/Cy was developed in a mouse model of MHC-
mismatched BMT. This regimen was sufficient to induce stable engraftment of donor cells in 100% of 
transplanted mice. The conditioning regimen did not ablate recipient hematopoiesis, since autologous 
hematopoietic recovery occurred in mice that received the conditioning and high dose PT/Cy but did not 
receive an allogeneic bone marrow infusion. Furthermore, high dose PT/Cy given on day 2 post-
transplantation mitigated both the incidence and severity of acute GVHD in MHC mismatched donor-
recipient pairs11. These results provided the pre-clinical rationale to translate this regimen to the clinic for 
patients with advanced hematologic malignancies30,31.
1.2.2
Clinical Experience
The study investigators’ extensive experience in performing HLA-haploidentical bone marrow 
transplantation incorporating high dose PT/Cy is summarized below.
The Hopkins group has now treated over 400 patients with non-myeloablative, HLA-haploidentical bone
marrow transplantation and high dose PT/Cy and has recently completed a 16-center phase 2 trial of this 
approach through the NHLBI-sponsored Blood and Marrow Transplant Clinical Trials Network. Patients 
(n=50) had either high-risk acute leukemia in remission or relapsed lymphoma and lacked a suitably 
HLA-matched related or unrelated donor. Of note, 49 out of 50 patients (98%) had stable engraftment of 
donor cells and there was only one case of primary graft failure. The cumulative incidence of grades II-IV 


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acute GVHD was 32%, but there were no cases of severe (grades III-IV) GVHD, a 13% cumulative 
incidence of chronic GVHD, and no GVHD-related mortality. Non-relapse mortality (NRM) at one year 
after transplantation was 7%, which is comparable to or lower than the incidence of NRM after reduced 
intensity BMT from HLA-matched donors32. These data suggest that high dose PT/Cy achieves selective 
depletion of alloreactive T cells in vivo, resulting in acceptably low morbidity and mortality in older 
adults with advanced hematologic malignancies. Further, Johns Hopkins’ single center results 
demonstrating the low toxicity of this platform, including the tolerability of high dose PT/Cy, were easily 
replicated by multiple centers without a need for a “learning curve.”
In light of the low toxicity of HLA-haploidentical transplantation for patients with hematologic 
malignancies, the Hopkins investigators have opened a protocol of reduced intensity transplantation
from HLA-matched or haploidentical first-degree relatives for patients with sickle cell anemia or 
beta thalassemia and have reported preliminary results33. The treatment regimen, illustrated below 
in Figure 1, is nearly identical to the planned regimen for this study, ITN054ST, with 3 exceptions: 
a) participants 12 through 26 received sirolimus instead of tacrolimus due to concerns about 
tacrolimus-associated posterior reversible encephalopathy (PRES) and b) participants 15 through 26
received bone marrow from filgrastim (G-CSF)-treated donors and c) the number of fludarabine 
doses is being reduced from five to three. Three days of fludarabine dosing is selected because there 
are no clear data favoring one duration over the other for non-myeloablative conditioning in 
general; concern exists regarding the potential for neurotoxicity in ESRD patients on dialysis; and 
recent data from the Northwestern trial show good engraftment results using only three days of 
fludarabine.
-8
-4
-9
-7 -6 -5
-3
3
4
5
0
-1
-2
10 20
BMT 
Day
30 
50
T cell-replete 
Bone Marrow 
Infusion
TBI
200cGy
ATG
MMF 15mg/kg po tid
Tacrolimus or Sirolimus
40
60
Cy
14.5 mg/kg/day
180
1
2
0.5
2 
mg/kg
2 
Fludarabine
30mg/m2/day 
Cy 
50 mg/kg/day
360
Patient # 
ATG
Donor Rx
Post - BMT
1 -2 
No
None
Tacrolimus
3 - 11
Yes
None
Tacrolimus
12 - 14
Yes
None
Tacrolimus
15 - 20
Yes
Filgrastim (G-CSF) Tacrolimus
Figure 1 Schema for reduced intensity allogeneic BMT for hemoglobinopathy
Since initial results were reported in October 2012 seven additional patients have been enrolled in the 
sickle cell trial for a total of 26 participants. Detailed clinical outcomes for 26 hemoglobinopathy patients 
transplanted as of May 2014 are provided in the table below:


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Table 1. Outcomes of reduced intensity allogeneic BMT for hemoglobinopathy
Pt 
# 
Age 
at 
BMT 
Hb 
type 
HLA 
mismatch 
GVH/HVG 
Days to 
PMNs> 
500 μl 
Donor 
G-CSF 
stim 
GVHD 
Complications 
Off 
IS 
Chimerism 
Whole 
Blood/Tcell 
(BMT day) 
Last % 
HbA 
(BMT 
day) 
Last 
Hct 
1 
33 
SC 
5/5 
14 
No 
No 
 
Yes 
100/ND (739) 
52 (245) 
38 
2 
20 
SS 
5/5 
14 
No 
No 
PRES 
Yes 
0/ND (80) 
44 
(1579) 
18 
3 
31 
SS 
4/0 
14 
No 
No 
PRES 
Yes 
100/100 
(1083) 
56 (795) 
42 
4 
33 
SS 
Matched 
12 
No 
No 
 
No 
85/70 (1601) 
53.9 
(1601) 
42 
5 
21 
SS 
0/5 
31 
No 
No 
 
No 
0/0 (1019) 
44 (755) 
33 
6 
27 
SS 
2/5 
15 
No 
No 
Dental abscess (d-
3) 
Yes 
92/100 (732) 
93 (948) 
40 
7 
31 
SC 
5/4 
27 
No 
No 
 
Yes 
100/ND (1483) 
55 
(1483) 
36 
8 
16 
SS 
3/3 
25 
No 
No 
 
Yes 
0/0 (49) 
84 (480) 
23 
9 
18 
SS 
4/3 
23 
No 
No 
CMV reactivation 
No 
8/13 (1362) 
24 
(1133) 
25 
10 
25 
SS 
5/0 
17 
No 
No 
PRES 
Yes 
100/100 
(1082) 
55 
(1082) 
33 
11 
46 
SS a-
thal 
Matched 
29 
No 
Transient 
Acute Gr I 
skin 
 
No 
89/49 (397) 
64 (397) 
37 
12 
42 
SS 
4/3 
41 
No 
No 
 
Yes 
0/0 (367) 
92 
(1232) 
29 
13 
31 
SS 
Matched 
30 
No 
No 
 
No 
48/40 (1201) 
51 
(1237) 
37 
14 
28 
SS 
3/4 
29 
No 
No 
CMV reactivation, 
RSV, TB 
Yes 
0/0 (68) 
95 (61) 
30 
15 
20 
SS 
5/3 
28 
Yes 
No 
 
No 
0/0 (157) 
55 (784) 
24 
16 
21 
SS 
5/4 
21 
Yes 
No 
 
Yes 
100/100 (728) 
100 
(364) 
32 
17 
15 
SS 
3/3 
35 
Yes 
No 
EBV pneumoniits 
Yes 
100/100 (758) 
55 (366) 
35 
18 
26 
SS 
ND 
25 
Yes 
No 
 
No 
43/13 (608) 
88 (608) 
34 
19 
26 
SS 
ND 
19 
Yes 
Mild 
chronic 
skin 
EBV reactivation 
Yes 
>95/100 (459) 
57 (368) 
45 
20 
38 
SS 
ND 
30 
Yes 
No 
Shingles 
No 
33/57 (531) 
50 (531) 
28 
21 
8 
SS 
ND 
24 
Yes 
No 
 
No 
49/36 (263) 
56 (172) 
33 
22 
14 
SS 
ND 
23 
Yes 
No 
 
No 
31/19 (315) 
28 (315) 
26 
23 
28 
SS 
ND 
23 
Yes 
Ext 
chronic 
(skin and 
lung) 
DEATH 
No 
100/100 (181) 
75 (71) 
25 
24 
35 
SS 
ND 
28 
Yes 
No 
 
No 
0/0 (194) 
91 (56) 
26 
25 
20 
SS 
ND 
21 
Yes 
Acute Gr 
III GI 
 
No 
100/93 (238) 
92 (238) 
35 
26 
12 
SS 
ND 
19 
Yes 
No 
 
No 
0/0 (61) 
37 (103) 
25 
Abbreviations
BMT
Bone marrow transplant
EBV
Epstein-barr virus
Hb
hemoglobin
GVH
graft-versus-host direction
HVG
host-versus-graft direction
PMN
polymorphonuclear leukocyte
GVHD
graft-versus-host disease
IS
immunosuppressive
Hct
hematocrit
PRES
posterior reversible encephalopathy syndrome
CMV
cytomegalovirus
RSV
respiratory syncytial virus
pulm TB
pulmonary tuberculosis
ND
Not done
** patients 4, 11 and 13 are HLA-matched sibling transplants. All other donor-recipient pairs are HLA haploidentical. 


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Ten recipients of HLA-haploidentical bone marrow (Table 1, patients 1, 3, 6, 7, 10, 16, 17, 19, 
23 and 25, in yellow) achieved full or near full donor chimerism. Of these ten, 8 are off 
immunosuppression. The remaining 2 patients developed GVHD (details below). Nine 
recipients of HLA-haploidentical bone marrow (patients 2, 5, 8, 9, (blue) 12, 14, 15, 24 and 
26, in red) have experienced graft failure, one of whom (patient 12) was re-transplanted 
unsuccessfully from the same donor. These 9 patients have all experienced return of 
autologous hematopoiesis. Seven recipients (patients 4, 11, 13, 18, 20, 21 and 22, in green) 
demonstrate stable mixed hematopoietic chimerism. These 7 patients remain free of sickle cell 
disease but remain on continued immunosuppression per the choice of the treating physician.
Patient 9 (in blue) has stable low level donor chimerism (~5%) over 2 years post-
transplantation. She remains on immunosuppression and continues to experience sickle cell 
crises.
With regard to PRES, three cases occurred in the initial cohort (patients 2,3 and 10) but no 
further incidents of PRES have occurred after sirolimus was substituted for tacrolimus.
With regard to infection, 3 patients (9, 19 and 19) experienced CMV reactivation but not 
CMV disease. Patient 17 developed EBV pneumonitis. Patient 14 developed RSV and was 
later found to have Mycobacterium tuberculosis on bronchoscopy. Patient 20 developed 
shingles. Patient 19 experienced EBV viremia that responded to a single dose of rituximab.
Among 26 patients transplanted, there have been up to four incidents of GVHD:
x
Patient 11 developed a transient skin rash localized to the forehead which resolved 
spontaneously and was considered to be questionable Grade 1 cutaneous acute 
GVHD.
x
Patient 19 developed a mild, chronic GVHD skin rash, responsive to treatment.
x
Patient 23 developed extensive, severe chronic skin and pulmonary GVHD and 
ultimately died.
x
Patient 25 developed acute Grade III GVHD of the gastrointestinal tract, ongoing.
The first 14 patients received bone marrow from untreated donors whereas the last 12 patients 
received bone marrow from donors treated with filgrastim for 5 days preceding bone marrow 
collection. Of the 14 initial patients, five achieved full donor hematopoietic chimerism and 
one case of GVHD was reported. Of the 12 patients who received marrow from filgrastim-
stimulated donors, five patients achieved full donor chimerism but three cases of GVHD were 
reported, including one case of severe GVHD with multi-organ involvement and death. The
single reported case of GVHD in the recipients of untreated marrow occurred in a patient who 
developed a limited facial rash that was not biopsied and resolved without treatment. This 
patient receieved a bone marrow graft from an HLA-identical sibling. Though the numbers are 
small it is possible that filgrastim treatment of donors may be associated with an increased risk 
of GVHD.
All of the patients treated on this trial had a history of receiving multiple transfusions of red 
blood cells and were therefore highly alloimmunized. It is therefore notable that durable 
hematopoietic chimerism has been achieved in roughly 40 percent of evaluable patients. PRES
(Posterior Reversible Encephalopathy Syndrome) was seen much more frequently than has 
been the case in hematologic malignancies patients, likely because of prior neurologic injury 
associated with sickle cell disease33.


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Figure 2 shows percent donor chimerism early after transplantation among patients who 
eventually lost their bone marrow grafts.
Figure 2: Donor chimerism early after transplantation among patients who experienced 
bone marrow graft loss
Four of five of these patients still had detectable chimerism by day 30 after transplantation. It
will be of interest to determine whether transient hematopoietic chimerism will result in durable 
renal allograft tolerance or whether sustained chimerism is necessary. The rate of durable 
hematopoietic chimerism in renal recipients treated with this regimen should be similar or 
greater to what we observed in the hemoglobinopathy patients, in view of their prior extensive 
blood product sensitization.
PT/Cy abrogates engraftment syndrome
Figure 3 shows daily white blood cell (WBC) counts, maximum temperatures, and serum 
creatinines in the first 30 days after transplantation. Interestingly, all but six patients developed 
fevers>38.0°C between days 2-4 after transplantation (middle panel, shaded bar), and all fevers 
resolved within 24 hours following the last dose of PT/Cy. Interestingly, fever did not recur 
(middle panel) and kidney functioned remained stable (lower panel) during WBC recovery 
(upper panel), and serum creatinine never exceeded 2.0 mg/dl. These data suggest that PT/Cy 
may limit or abrogate engraftment syndrome by depleting alloreactive T cells early after 
transplantation34.


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Red lines indicate patients who experienced graft failure.
Figure 3: Daily WBC counts, maximum temperatures, and serum creatinines in patients undergoing
reduced intensity conditioning allogeneic stem cell transplant for hemoglobinopathy
Toxicity of high-dose cyclophosphamide
The feasibility of high-dose cyclophosphamide is based on extensive experience with this
approach in a variety of clinical settings, including combined bone marrow and renal 


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transplant as described above28. High-dose cyclophosphamide produces a hypocellular 
marrow with killing of the differentiated progeny of hematopoietic stem cells (HSCs), but the 
HSCs themselves survive by virtue of high levels of the enzyme aldehyde dehydrogenase,
which detoxifies the active metabolite of cyclophosphamide13,17,35,36. High-dose 
cyclophosphamide can therefore be expected to eliminate mature elements of the immune 
system, while allowing full bone marrow recovery from the surviving stem cell pool.
Both hypotheses have been substantiated in several human clinical trials conducted at Johns 
Hopkins University (JHU). Durable remission has been achieved in a high proportion of 
patients with auto-immune diseases, including aplastic anemia, systemic lupus erythematous,
and myasthenia gravis37-40. These results were achieved with acceptable toxicity, largely 
limited to a period of myelosuppression, even in patients with significant pre-existing 
infectious problems or other co-morbidities. Further favorable toxicity data and additional 
evidence for the recovery of blood cells after high-dose cyclophosphamide are evident from 
the results of a recent study of 81 patients in which high-dose cyclophosphamide, without 
stem cell rescue, was used for the treatment of low-grade and mantle cell non-Hodgkin's 
lymphoma.41 (This approach administers significant dose intensification while obviating the 
problem of disease re-infusion that may result from autologous stem cell rescue). All patients 
experienced full hematopoietic recovery, with a median time to neutrophil count > 500/μL of 
15 days. There were no deaths. Treatment was given in an outpatient setting, with fewer than 
half of the patients ever hospitalized, and when so, primarily for neutropenic fever, which 
developed in one third. All patients had received several cycles of chemotherapy immediately 
prior to high-dose cyclophosphamide, and there was no age limit to enrollment. All patients 
were supported with granulocyte colony stimulating factor (G-CSF), blood product support 
when needed, antibiotic prophylaxis, effective preventive anti-emetics, and urothelial 
protection with MESNA.
The Northwestern regimen containing high dose PT/Cy was generally well tolerated in a 
similar donor-recipient population, with median ANC recovery of 10 days (range 2 to 14) and 
outpatient management on post-operative day 2 for most patients. One participant developed 
viral sepsis and hypotension requiring hospitalization and intubation and one participant 
experienced VZV reactivation. Additional significant infections include histoplasmosis in one 
subject, BK viremia without nephropathy and 3 episodes of single dermatome herpes zoster29.
The use of high dose PT/Cy may present specific problems in patients with ESRD in the 
immediate post-transplant period. Although early renal function is likely after living-related 
donor transplantation, delayed graft function or renal dysfunction associated with engraftment 
syndrome is not an obstacle to the use of high-dose cyclophosphamide. The drug is inactive 
until it has been metabolized by hepatic microsomal enzymes, forming the major 
intermediates aldophosphamide and 4-hydroxycyclophosphamide. Approximately 15% of the 
drug is excreted unchanged in the urine, and a variety of inactive metabolites are also 
excreted through the urine. A large proportion of a cyclophosphamide dose is eliminated by 
hepatic metabolism. The clinical toxicity of cyclophosphamide, even at high doses, appears to 
be independent of renal function42,43. The available literature is limited44,45. The half-life in 
hours of cyclophosphamide is slightly prolonged in patients with renal failure compared to 
those with normal renal function (10 versus 7.5 hours). Cyclophosphamide has a protein 
binding percentage of 14% and a volume of distribution of 0.5-1.0 L/Kg, making it suitable 
for removal by dialysis if necessary. Recommendations exist for dose reduction of 
cyclophosphamide by 25% for patients on hemodialysis46,47. However, a detailed study of the 
pharmacokinetics of high-dose cyclophosphamide was performed at Johns Hopkins in one 


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patient with ESRD and compared to a patient with normal renal function42,48-51. The 
metabolism and AUC achieved in both patients were similar. In the ESRD patient, 
cyclophosphamide was completely metabolized by the liver and no dose adjustment was 
required. Therefore additional toxicity or significant problems with the dosing of 
cyclophosphamide is not anticipated, even in patients with minimal or no renal function and 
who continue to require hemodialysis in the immediate post-transplant period. It is possible 
that wound healing could be adversely affected by high-dose cyclophosphamide. This has not 
been the case among post-transplant FSGS patients at Hopkins treated chronically with 
cyclophosphamide. Also, because the graft is placed in the retroperitoneum through a small 
incision, wound problems tend to have less dire consequences, rarely result in an open 
abdomen, and are quite amenable to wound VAC coverage or local tissue transfer, if needed.
1.3
SUMMARY OF KNOWN AND POTENTIAL RISKS FOR HUMAN
PARTICIPANTS
1.3.1
Risks Associated with Study Procedures for the Recipient
1.3.1.1
Bone M arrow Transfusion
The donor bone marrow will be transfused into the recipient through an intravenous catheter 
in the chest or neck. The risks of transfusion include chills, fever, headache, nausea, vomiting 
and dyspnea. These side effects can be ameliorated by administration of antihistamine and 
analgesic pre-medications.
1.3.1.2 Total Body Irradiation
Recipients will receive a single dose of 200 cGY total body irradiation on Day -1. This is 
considered a low dose of radiation and thus is not usually associated with side effects common 
at higher doses such as nausea and vomiting, alopecia and mouth sores. Low dose total body 
irradiation is associated with minimal to no short term side effects with the exception of 
possible edema in the radiation field and transient cytopenias. Long term side effects of total 
body irradiation include infertility. In addition there is an increased risk of lung fibrosis, 
pericarditis, thyroid disease, and cataracts. The additional risk of secondary malignancy due to 
TBI is detailed below.
1.3.1.2.1
Secondary malignancy as a long-term risk of TBI-containing 
conditioning.
Data regarding subsequent malignancies after TBI-containing conditioning regimens are 
almost all in the setting of malignant disease and reflect the overall risk attributable to the 
cumulative chemotherapy and radiation therapy used to treat the malignancy, as well as any 
contribution from the conditioning regimen.
A retrospective study of 1626 pediatric patients treated with autologous bone marrow 
transplantation showed a secondary malignancy cumulative incidence of 2.6% at 10 years, 
with no significant difference between TBI-containing and non-TBI containing conditioning 
regimens52. Leukemia is very uncommon after allogeneic transplantation, and most of the 
malignancies have been solid tumors or EBV-related lymphoproliferative disorders. A study 
of 2062 patients who had undergone allogeneic hematopoietic stem cell transplantation in 
Japan showed a cumulative incidence of solid tumors of 2.4% at 10 years. There was no 


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difference between TBI-containing and non-TBI containing regimens53. A study of 1036 long-
term bone marrow transplant survivors conducted by the European Cooperative Group for 
Blood and Marrow Transplantation, Late Effects Project Group, found a cumulative second 
malignancy incidence of 3.5% at 10 years, with no difference between TBI-containing and 
non-TBI containing regimens. Increasing donor or recipient age, a female donor, and the use 
of immunosuppressive drugs to manage graft versus host disease were all identified as factors 
related to increased risk54. A study of 926 patients who had undergone allogeneic 
hematopoietic stem cell transplantation in British Columbia found a 10 year cumulative 
incidence of secondary malignancy of 2.3%, all solid tumors. Basal cell skin cancer and 
carcinoma-in-situ were excluded. The relative risk compared to the age-matched general 
population was 1.85. Approximately half the patients had received TBI, dosed at < 1200 cGy.
No difference in secondary malignancy incidence was evident between the group that had and 
the group that had not received TBI55. Earlier studies have related the risk of secondary 
malignancies to TBI dose, particularly to doses >1000 cGy, more than 5 times the low-dose 
TBI (200 cGy) used in this protocol54,56,57.
In summary the short term risk of TBI are less commonly seen with the relatively low dose of 
radiation used in this regimen. The risk of secondary malignancy is approximately 2.5% at 10 
years in participants who undergo hematopoietic stem cell transplantation, but does not appear 
increased by TBI. Other long term risks of TBI such as infertility, pulmonary fibrosis etc. have 
been described; these risks are above and beyond the risks associated with standard renal 
transplant.
1.3.1.3
Renal Biopsies
Renal biopsies will be obtained in the event of suspected allograft dysfunction. In addition, 
surveillance protocol renal biopsies will be performed at pre-specified time points to monitor 
the status of the graft, including before initiation of immunosuppression withdrawal, to 
exclude the presence of subclinical rejection. These biopsies will also be used for performing 
mechanistic evaluations.
Obtaining biopsy samples using ultrasound guidance and automated needles had an incidence 
of complications requiring intervention of 0.2% (3/1302) while minor complications (i.e., self-
limited hematuria, small hematoma, non-significant arteriovenous fistula) occurred in 6.8% 
(89/1302) of patients in two series comprised of adults and children. There were no allograft 
losses or patient deaths.
Significant bleeding following renal biopsy may require blood component therapy for 
participants with significant anemia, coagulopathy, or thrombocytopenia.
1.3.1.4
Risk Associated with Immunosuppression Withdrawal
Inherent in the withdrawal of immunosuppression is the increased risk of precipitating graft 
rejection. This risk will be mitigated by the study design, which includes careful patient 
selection and close follow-up during and after drug withdrawal. Rejection may occur with a 
higher frequency or with greater severity than it does with conventional immunosuppression.
The protocol provides for frequent clinical evaluation of participants during the period of 
active administration of this novel immunosuppressive combination and for continued follow-
up monitoring through approximately 5 years after enrollment.


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1.3.2
Risks Associated with Study Procedures for the Donor
1.3.2.1
Bone M arrow Harvest
Bone marrow harvest will occur under local or general anesthesia. Bone marrow stem cells 
will be obtained by using a large needle to draw cells from the bone marrow in the iliac crests, 
the hip bone. The risks associated with bone marrow harvest include pain and bleeding at the 
harvest site, adverse reactions to anesthesia and infection. Rarely, mechanical injury to the 
underlying bone, nerves, or soft tissue can occur. Factors associated with more complications 
are use of regional anesthesia, longer duration of harvesting, female gender and older age.
1.3.3
Risks Associated with Study Medication for the Recipient
1.3.3.1
Fludarabine
Clinical toxicities of fludarabine monophosphate include: myelosuppression, primarily 
lymphopenia and granulocytopenia, alopecia, rash, dermatitis, nausea, vomiting, anorexia, 
mucositis, stomatitis, diarrhea, somnolence, fatigue, peripheral neuropathy, mental status 
changes, cortical blindness, hepatocellular toxicity with elevation in serum transaminases, and 
interstitial pneumonitis. These effects may be reversible when the drug is discontinued. The 
use of fludarabine has been associated with an increased risk of secondary malignancy as 
described in section 1.3.3.
For additional information on the risk associated with fludarabine, refer to the fludarabine 
drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=6f1094e3-4c28-40ae-a82f-
37cdfa865f49
1.3.3.2
Cyclophosphamide
The clinical toxicities associated with cyclophosphamide are more severe at higher doses such 
as those used in this study regimen. The most common side effects of high dose 
cyclophosphamide include nausea and vomiting, transient alopecia and neutropenic fever.
Treatment is associated with leukopenia, including neutropenia, which can be prolonged at 
high doses, resulting in increased rates of infection. Anemia and thrombocytopenia have been 
reported but are less common. Cyclophosphamide is associated with headaches and dizziness, 
elevated liver function tests, hemorrhagic cystitis, cardiotoxicity and syndrome of 
inappropriate anti-diuretic hormone (SIADH). Long term risks include infertility, malignancy
and interstitial pulmonary fibrosis. Cyclophosphamide may interfere with normal wound 
healing. Cyclophosphamide can cause fetal harm when administered to pregnant women and 
has been associated with birth abnormalities.
For additional information on the risk associated with cyclophosphamide, refer to the 
cyclophosphamide drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=591d9955-3d9c-4cdc-a308-
2f1288376b9f
1.3.3.3
Antithymocyte Globulin
Risks associated with rabbit antithymocyte globulin (ATG) (Thymoglobulin®, Sanofi) include 
immune-mediated reactions, infections, reactivation of infection, malaise, dizziness, sepsis, 


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thrombocytopenia, leukopenia, and increased incidence of malignancies. Occasional reactions 
are observed at the infusion site including pain, swelling and erythema.
Immune-mediated reactions
Serious immune-mediated reactions have been reported with the use of thymoglobulin and 
consist of anaphylaxis or severe cytokine release syndrome (CRS). Fatal anaphylaxis has been 
reported. Severe, acute infusion-associated reactions (IARs) are consistent with CRS which is 
attributed to the release of cytokines by activated monocytes and lymphocytes. Severe acute 
CRS can cause serious cardiorespiratory events and/or death.
Infection
ATG is routinely used in combination with other immunosuppressive agents. Infections 
(bacterial, fungal, viral and protozoan), reactivation of infection (particularly cytomegalovirus 
(CMV)), and sepsis have been reported after ATG administration in combination with 
multiple immunosuppressive agents. Severe acute infections can be fatal.
Malignancy
Use of immunosuppressive agents, including ATG, may increase the incidence of 
malignancies, including lymphoma or post-transplant lymphoproliferative disease (PTLD).
Carcinogenesis, Mutagenesis, Impairment of Fertility
The carcinogenic and mutagenic potential of thymoglobulin and its potential to impair fertility 
have not been studied.
In 82 kidney-transplant recipients receiving ATG 1.5 mg/kg/day for 7–14 days, the principal 
adverse events were fever (52%) and chills (47%) associated with the infusions, leucopenia 
(47%) and thrombocytopenia (30%). CMV infection occurred in 13% of the recipients and 
PTLD in 2% of the recipients. Neutropenia has been described; anaphylaxis has been reported 
rarely.
ATG is contraindicated in patients with a history of allergy or anaphylaxis to rabbit proteins or 
to any product excipients, or who have active acute or chronic infections that contraindicate 
any additional immunosuppression.
For additional information on the risk associated with rabbit antithymocyte globulin, refer to 
the rabbit antithymocyte globulin drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=bbd8ab99-552e-4b81-aca4-
6b0c7af8b9ae
1.3.3.4
Filgrastim (G-CSF)
The most common side effects of filgrastim injection are bone pain, muscle pain and 
headaches. These symptoms can usually be relieved by non-aspirin analgesics such as 
acetaminophen. Nausea, fatigue and low grade fevers have been reported. Some patients 
experience swelling, erythema or itching at the injection site. Rare but serious adverse events 
associated with filgrastim include splenic rupture and acute respiratory syndrome, both 
associated with the rapid proliferation of neutrophils. Filgrastim has not been studied in 
pregnant women and its effect on the developing fetus is unknown.


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For further information on the risks associated with filgrastim, please refer to filgrastim drug 
label at the following website: 
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=3bc802bd-76b4-4f45-8571-
a436ec26228e
1.3.3.5
M ycophenolate M ofetil/M ycophenolic Acid
The following risks are associated with the use of mycophenolate mofetil (MMF). The risks 
associated with mycophenolic acid are identical; therefore only those associated with MMF 
are listed below:
x
Increased risk of developing lymphomas, lymphoproliferative diseases and other 
malignancies especially of the skin in patients receiving MMF as part of an 
immunosuppressive regimen. The risk appears to be related to the intensity and 
duration of immunosuppression rather than to the use of any specific agent.
x
Increased susceptibility to infection, including opportunistic infections, fatal 
infections and sepsis.
x
Increased risk of first trimester pregnancy loss and congenital malformations, 
especially of the external ear, face, limbs, heart esophagus and kidneys.
x
Increased risk of neutropenia and leukopenia.
x
Increased risk of pure red cell aplasia
x
Increased risk of gastrointestinal bleeding.
x
Immunosuppressed patients are at increased risk for opportunistic infections, 
including activation of latent viral infections. These include cases of progressive 
multifocal leukoencephalopathy (PML) and BK virus-associated nephropathy which 
have been observed in patients receiving immunosuppressants, including 
mycophenolate mofetil.
Common side effects associated with use of MMF include diarrhea, abdominal pain, nausea 
and vomiting, headache and drug induced fever.
For further information about the risks of MMF, please refer to the MMF drug label at the 
following website: http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=209efe01-7908-
46e4-a964-0665078c0acd#druglabelcontent.
1.3.3.6
Prednisone
Side effects of prednisone include convulsions, headache, vertigo, mood swings, psychosis, 
congestive heart failure, hypertension, salt and water retention, increased potassium excretion, 
Cushing syndrome, menstrual irregularities, hyperglycemia, GI irritation, peptic ulcer, weight 
gain. Dermatologic effects may include thin skin, petechiae, ecchymosis, facial erythema, 
poor wound healing, hirsutism and urticaria. Muscle weakness, loss of muscle mass and 
osteoporosis may also occur. Ophthalmologic complications may include increased intraocular 
pressure, glaucoma, exophthalmos and cataracts. Other complications may include 
immunosuppression and increased susceptibility to infection.


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For further information about the risks associated with prednisone, please refer to prednisone 
drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=3115aef0-fd50-4ec8-a064-
3effb695f3f2#druglabelcontent .
1.3.3.7
Tacrolimus
The following risks are associated with the administration of tacrolimus:
x
Hypertension.
x
Hyperkalemia.
x
Nephrotoxicity.
x
Neurotoxicity.
x
Post-transplant insulin-independent diabetes mellitus.
x
Myocardial hypertrophy (in most cases reversible upon dose reduction).
x
Increased risk of renal insufficiency in patients with hepatic impairment.
x
Increased susceptibility to infection.
Posterior reversible encephalopathy syndrome (PRES)58 when used in the setting of bone 
marrow or solid organ transplantation.For further information on the risks of tacrolimus, 
please refer to the tacrolimus drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=bd447ffa-9196-4c3c-accf-
5adf29b84665#druglabelcontent
1.3.3.8
Sirolimus
The following risks are associated with use of sirolimus:
x
Delayed or poor wound healing
x
cytopenias
x
Lymphocoele
x
Peripheral edema
x
Angioedema
x
Hyperlipidemia,
x
Proteinuria
x
Mouth ulcers
x
Severe gastrointestinal adverse effects
x
Interstitial pneumonitis
x
Increased susceptibility to infection


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x
Increased risk of lymphoma
For additional information about the risks of sirolimus, refer to the Rapamune® package insert 
at the following website: http://labeling.pfizer.com/showlabeling.aspx?id=139.
1.3.4
Risks Associated with Study Regimen for the Recipient
This section describes risks to the recipients associated with the combined use of study drugs 
and study procedures specified in this trial. These risks extend beyond the risks associated 
with standard kidney transplantation.
1.3.4.1
Neutropenic Fever
The median time to neutrophil recovery (ANC> 500 cells/uL) was 24 days (range 0 to 35 
days) in 17 patients with sickle cell disease who received this study regimen33. Recipients may 
experience fevers during this period of neutrophil nadir and recovery. These episodes will be 
managed with supportive care and may require hospitalization of the recipient.
1.3.4.2 Infection
Recipients will be at increased risk of infection due to immunosuppression associated with the 
study regimen and TBI. Patients will receive appropriate viral, fungal and PCP prophylaxis as 
described in section 5.6. Asymptomatic viral reactivation (CMV, EBV) without active disease
has been reported in sickle cell and malignancy patients treated with this regimen30,32. One 
case of M. tuberculosis reactivation was observed in the sickle cell cohort. Invasive 
Aspergillus infections occurred in 5 of 68 (7%) malignancy patients treated with this regimen,
including 2 infections resulting in death31.
1.3.4.3
Graft versus Host Disease
Study recipients may be at increased risk of both acute and chronic GVHD as a result of 
undergoing the conditioning regimen and bone marrow transplantation. However the exact 
risk of GVHD in this study population is unclear. A lower rate of GVHD (4 cases in 26
patients, 15%) was observed in the sickle cell patients who received this regimen compared to 
the rate of GVHD in patients with hematological malignancies (27%). Among 26 sickle cell 
patients:
x
One patient developed a transient skin rash localized to the forehead which resolved 
without treatment and was considered to be questionable Grade 1 cutaneous acute 
GVHD.
x
One patient developed a mild, chronic GVHD skin rash, responsive to treatment.
x
One patient developed extensive, severe chronic GVHD involving the skin and lungs, 
and ultimately died.
x
One patient developed Grade III acute GVHD involving the gastrointestinal tract.
In contrast, the incidence of acute GVHD in 210 patients with hematological malignancies 
treated with this regimen was 27%, with a 5 % incidence of severe (Grade III-IV) GVHD. The 
incidence of chronic GVHD in these patients was 13%59. The first renal transplant recipient in 
the ITN054ST trial experienced a grade 2 GVHD limited to the skin and responsive to 
treatment. We hypothesize that the incidence of GVHD in renal transplant recipients who are 


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chemotherapy-naïve will be closer to the low incidence in sickle cell patients than the 
malignancy patients, but this is unknown.
1.3.4.4
Carcinogenicity
The conditioning regimen, consisting of fludarabine, cyclophosphamide, and total body 
irradiation (TBI) has the potential for causing malignancies at a later date, including 
hematologic malignancies such as myelodysplastic syndrome and leukemia, and also solid 
tumors. Standard renal transplantation and the immunosuppressive drugs typically used, 
including antithymocyte globulin (ATG) , tacrolimus, and mycophenolate (MMF) also carries 
the risk of subsequent malignancies, including lymphomas, Kaposi’s sarcoma, and squamous 
carcinomas.
The largest study of malignancies following allogeneic stem cell transplantation to date, 
involving 28,874 patients60 found that patients developed new solid cancers at about twice the 
frequency expected in the general population. The risk was three-fold at 15 years or more 
following transplantation. Analysis of risk following TBI showed a strong relationship with 
age, with patients > 30 years showing little elevation in risk (RR 1.08), but patients <30 years 
showing a 9 to 10 fold increase in risk. A separate study of secondary malignancies occurring 
after fludarabine-based non-myeloablative conditioning61 showed a 10 year cumulative 
incidence of 5.6%, twice the rate expected in a normal population, with a related 10 year 
mortality rate of 2.4%.
Standard renal transplantation and immunosuppression to prevent rejection has been 
associated with an age-related increased risk in malignancy compared to the general 
population of 15-20 times greater risk in the pediatric population, 1.8-5 times greater risk in 
patients 35 to 65 years old, and 2 times the risk in patients over the age of 6562.
1.3.4.5
Reproductive Risk
Fertility
The effect of the non-myeloablative conditioning regimen and post-transplant high-dose 
cyclophosphamide on subsequent male and female fertility is not well defined. It is clear that 
fertility can be preserved after such therapy63, but the rate of infertility is not clearly 
determined. Female infertility after high dose cyclophosphamide alone appears to be related to 
age, with younger patients being more likely to preserve fertility. Male patients considering 
this protocol will be advised to bank sperm; female patients will be offered consultation in the 
fertility clinic to explore possible interventions that may reduce the risk of infertility.
Teratogenicity
Cyclophosphamide, TBI and MMF all have the potential for fetal toxicity. Effective 
contraception is required for both male and female participants in the trial.
2.
OBJECTIVES
2.1
PRIMARY OBJECTIVE
The primary objective of this trial is to assess the ability of bone marrow transplantation and 
high dose PT/Cy to induce renal allograft tolerance and thus enable discontinuation of 
immunosuppressive therapy in haploidentical living related donor renal transplant recipients.


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2.2
SECONDARY OBJECTIVES
1. To assess the safety of the study regimen.
2. To assess whether maintenance of donor chimerism is required for sustained allograft 
tolerance.
3. To explore mechanistic assays which might predict successful immunosuppression
withdrawal. 
3.
STUDY DESIGN
3.1
DESCRIPTION
This trial is a phase II, single arm, open-label, single center pilot study to assess a reduced-
intensity conditioning regimen, bone marrow transplantation and high dose PT/Cy in six
recipients of renal allografts from HLA-haploidentical living related donors. The proposed 
trial design is depicted in Figure 4.
The proposed treatment regimen is described in detail in section 1.1 and is depicted in Figure 
5: Study regimen for unsensitized recipients. Briefly, it consists of a non-myeloablative pre-
transplant conditioning regimen followed by bone marrow and kidney transplantation as 
described in section 3.2.1 and Figure 5: Study regimen for unsensitized recipients. Recipients 
will receive high dose PT/Cy and maintenance immunosuppression. This will be followed by 
immunosuppression withdrawal at six months in eligible participants with completion of 
withdrawal at 12 months post-transplant. The primary endpoint is assessed at 52 weeks after 
completion of immunosuppression withdrawal.
3.1.1
Amendment to Allow Sensitized Participants
3.1.1.1
Rationale
Version 5.0 of the protocol allows the inclusion of sensitized recipients of a first renal 
transplant (see section 4). The rationale for this change was as follows: 1) Broadening the 
entry criteria may improve recruitment. 2) Allowing sensitized patients will improve the 
equipoise of the study. Such sensitized patients have less favorable outcomes with standard 
transplantation and therefore would benefit more from undergoing the study regimen than an 
unsensitized patient. 3) Inclusion of such patients does not affect the safety of the study 
regimen.64 4) The site (JHH) has extensive experience with de-sensitization protocols and has 
demonstrated that it can be performed safely in selected candidates.65
3.1.1.2
Study Flow for Sensitized Participants
Sensitized participants will undergo screening per protocol. Once it is confirmed that they 
have met all entry criteria (section 4) these participants will undergo de-sensitization per 
standard of care at JHH (see Appendix 8. Study Regimen with De-sensitization Schedule for 
Sensitized Recipients and Appendix 9. De-sensitization Schedule of Events per JHH Standard 
of Care). Adequacy of the response to de-sensitization will be assessed prior to the first dose 
of study therapy (ATG). Participants who fail to achieve an adequate response to de-
sensitization, defined as a negative flow cytometric crossmatch by day -9, will be considered 
screen failures and will be terminated from the study. There is no upper limit on screen 


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failures. The Johns Hopkins Hospital (JHH) standard-of-care de-sensitization algorithms are 
described in Appendices 8-9.
3.1.2
Accrual
The accrual goal for this study is six renal transplant recipient-donor pairs who fulfill the per 
protocol (PP) analysis sample definition (see section 9.1). Enrollment is defined as the signing 
of informed consent. Enrolled participants who do not fulfill eligibility criteria will be 
considered screen failures and will be terminated from the study. There is no upper limit on 
screen failures. A sufficient number of recipient-donor pairs will be enrolled in order to 
achieve the accrual goal.
Enrolled participants who receive any form of study therapy but who do not fulfill the PP 
analysis sample definition will remain in the study and will undergo safety follow-up per 
Appendix 5. Safety follow-up: Recipients. These participants will be included in the safety
analysis but will not count towards the accrual goal. A minimum of 60 days must elapse
between transplantation of each subsequent participant.
3.1.3
Informed Consent for Participation
Informed consent will be obtained from each study participant at two points during the study:
x
Study enrollment: A consent addressing all aspects of the study will be obtained before 
any protocol-directed procedures are performed.
x
Before initiation of tacarolimus withdrawal: Study participants will reaffirm consent 
prior to withdrawal of tacrolimus.
Informed consent will also be obtained from the donor and will be completed prior to 
performance of donor screening procedures.
3.1.4
Interim Safety Review
Accrual of six participants will occur in two stages with a pre-specified interim safety 
analysis. Treatment will consist of the regimen described in section 3.2 for at least the first 
three participants. An interim safety review will be performed after the third participant 
reaches three months post-transplant and the first and second participants are at least six 
months post-transplant. Immunosuppression withdrawal may proceed in eligible participants 
before completion of the safety review. Further enrollment will halt until the safety review is 
completed. The decision to proceed with further accrual will be evaluated by the NIAID
Transplant Data and Safety Monitoring Board (DSMB). The acceptability of the regimen will 
be evaluated based on the stopping guidelines listed in section 3.6 as well as the DSMB’s 
discretion.
If one or more stopping guideline is met or the DSMB deems that the toxicity of the regimen 
is unacceptable, enrollment of the second cohort of three participants will proceed but the pre-
transplant conditioning regimen consisting of fludarabine, low-dose cyclophosphamide and 
total body irradiation will be omitted. This decision would also be strongly bolstered by the 
observation of allograft tolerance after transient chimerism in the initial patient group.
Otherwise the accrual of the next three participants will resume.


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ATG
Conditioning Regimen
Renal Transplant + Bone Marrow 
Transplant3
High Dose Cyclophosphamide
Post – Transplant Treatment
Safety Review2
STOP
Renal Transplant + Bone Marrow 
Transplant
ATG
Post-Transplant Treatment
High Dose Cyclophosphamide
IS Withdrawal
ATG
Renal Tranplant + Bone Marrow 
Transplant
Conditioning Regimen 
Post-Transplant Treatment
High Dose Cyclophosphamide
IS Withdrawal
Acceptable toxicity
Unacceptable toxicity
IS Withdrawal1
Standard 
Follow-up; 
2 years
Follow-up per 
Protocol; 
4 years
Fail
Success
Participants 
1-3 
Participants 
4-6 
Follow-up per 
Protocol;
 4 years
Success
Standard 
Follow-up; 
2 years
Fail
Follow-up per 
Protocol;
 4 years
Standard 
Follow-up;
2 years
Fail
Success
1. IS withdrawal may proceed while the Safety Review is ongoing.
2. The Safety review will occur after 3rd participant completes 3 months and 1st and 2nd participants are at least 6 months post-transplant.
3. A minimum of 60 days must elapse between transplantation of each subsequent participant. 
Figure 4: Schematic of trial design with decision points for safety review


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3.2
STUDY REGIMEN
3.2.1
Recipient Induction and Maintenance Therapy
3.2.1.1
Unsensitized Recipients
The treatment regimen for unsensitized participants is outlined in Figure 5. Antithymocyte 
globulin (ATG) will be given from Day -9 to Day -7; fludarabine from Days -4 to Day -2 and 
low-dose cyclophosphamide on Day -4 and Day -3. Participants will undergo hemodialysis 6 
to 8 hours after each fludarabine dose. Participants will undergo TBI on Day -1. Renal 
transplantation followed by bone marrow infusion will occur on Day 0. High dose PT/Cy will 
be administered on Days 3 and 4 with hydration, MESNA and anti-emetics. Filgrastim will be 
administered daily starting on Day 5 until absolute neutrophil count (ANC) recovery. Standard 
maintenance immunosuppression consisting of tacrolimus, with target trough level of 8-10
ng/ml, and MMF 15 mg/kg three times daily and prednisone 10mg daily will be started on 
Day 5.
On Days -9 through -7 ATG will be administered daily at a specialized outpatient BMT unit. 
On Day -4 participants will be admitted to the inpatient BMT unit for the three-day course of 
fludarabine to ensure precise timing of dosing and dialysis as well as monitoring of side 
effects. Because fludarabine is cleared by the kidneys, these ESRD participants will require 
daily dialysis to avoid the potentially fatal side effect of neurotoxicity. Participants will remain 
hospitalized from the first day of fludarabine administration through the kidney and bone 
marrow transplant. Recipient hospitalization will then continue through post-operative Day 7
under the care of the surgeon and the nephrologist in keeping with the site’s standard renal 
transplantation procedures. Recipients will then be discharged to a specialized outpatient BMT 
unit on Day 8 and followed per standard of post-non-myeloablative bone marrow transplant 
care for 60 days post-transplant.
3.2.1.2
Sensitized Recipients
Sensitized participants with detectable DSA at screening who meet inclusion criterion 5b will 
undergo de-sensitization per JHH standard of care (Appendices 8 and 9) prior to receiving the 
first dose of study therapy. They will receive the same induction and maintenance therapy as 
unsensitized recipients but with continued de-sensitization therapy and additional monitoring 
for recurrence of DSA. Please see Appendix 8 for the Study Regimen with De-sensitization 
Schedule for Sensitized Recipients and Appendix 9 for the De-sensitization Schedule of 
Events for Sensitized Recipients.
3.2.1.3
Reinitiating conditioning regimen
If the recipient receives any part of the conditioning regimen but is unable to undergo 
combined kidney and bone marrow transplant (CKBMT) due to unanticipated issues with the 
tissue and organ harvest from the donor, he/she will be allowed the option of undergoing a 
second course of conditioning in order to proceed with the combined bone marrow and kidney 
transplant (CKBMT) per protocol. The recipient may undergo CKBMT using the same donor 
or a different donor. Please see Appendices 1C, 1D and 10 for further details.


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Recipients may reinitiate the conditioning regimen no more than once. Recipients who are 
unable to undergo CKBMT after reinitiating conditioning will move to Safety Follow up 
(Appendix 5).
3.2.2
Donor Therapy and Procedures
Donor nephrectomy immediately followed by bone marrow harvest will take place under the 
same anesthetic on Day 0, with renal transplantation followed by bone marrow infusion into 
the recipient also on Day 0.
Figure 5: Study regimen for unsensitized recipients
3.3
IMMUNOSUPPRESSION WITHDRAWAL
Participants will receive standard maintenance immunosuppression for at least 26 weeks post-
transplant. Those participants demonstrating no evidence of rejection will first undergo 
withdrawal of MMF and prednisone over a 4- to 8-week period. Withdrawal from MMF and 
prednisone may be initiated no earlier than Week 26 and no later than Week 34 post-
transplant. After 8 weeks on stable tacrolimus monotherapy, participants demonstrating no 
evidence of rejection, as described in section 3.3.2, will undergo withdrawal of tacrolimus. 
The tacrolimus dose will be reduced in a stepwise fashion over a period of no fewer than 12 
weeks and no greater than 16 weeks with the goal of complete discontinuation no later than 66
weeks post-transplant.
Participants who successfully complete immunosuppression withdrawal will then undergo 4 
years of follow-up. Participants who fail to initiate or to complete immunosuppression 
withdrawal will undergo 2 years of safety follow-up.
3.3.1
Withdrawal of MMF and Prednisone
3.3.1.1
Eligibility
After at least 22 weeks on tacrolimus, MMF and prednisone triple therapy, participants may
be evaluated for eligibility for withdrawal of MMF and prednisone. MMF and prednisone 
withdrawal may be initiated as early as week 26 in eligible participants. To be eligible for 
MMF and prednisone withdrawal participants must meet the following criteria:


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1.
A renal biopsy that demonstrates the absence of rejection of any type including acute 
humoral rejection, acute cellular rejection and chronic rejection as defined in section 
6.5.4.1 within the 4 weeks prior to initiation of withdrawal.
2.
No history of biopsy-proven rejection or clinically presumed rejection since 
transplantation.
3.
No history of biopsy-confirmed indeterminate rejection (less than Banff Grade IA) on
the withdrawal eligibility biopsy that has not resolved, as assessed by repeat biopsy
performed within 1 month of diagnosis.
4.
A history of stable renal function. Stable renal function is defined as no unexplained 
increases in serum creatinine of greater than 25% relative to baseline for a 72 hour time 
interval. Baseline is defined as the mean of the three lowest creatinines during Weeks 2 
to 4 post-transplant, excluding days on dialysis.
5.
An estimated GFR > 40 mL/min/1.73m2 as calculated by CKD-EPI equation.
6.
Must demonstrate absence of donor-specific antibody (DSA) as determined by solid 
phase micro particle technology (by Luminex® phenotype panel or Luminex single 
antigen bead test) performed within 4 weeks prior to initiating withdrawal; as assessed 
by local laboratories.
7.
No evidence of active GVHD at the time of initiation of withdrawal.
3.3.1.2
Initiation and Completion of Withdrawal of M M F and Prednisone
MMF and prednisone withdrawal must be initiated between Week 26 and Week 34 post-
transplant (see Appendix 3. Immunosuppression Withdrawal (Initiated Between 26 and 3 
Weeks Post-Transplant)).
MMF and prednisone withdrawal will occur simultaneously according to a standard algorithm. 
Withdrawal must occur over a minimum of 4 weeks and a maximum of 8 weeks, with 
scheduled dose reductions every 2 weeks (QOW) as follows:
x
The daily dose of prednisone will be reduced by 2.5 mg every 2 weeks until off.
x
The daily dose of MMF will be reduced by 500 mg every 2 weeks until off. This dose 
reduction will be achieved by a 250 mg reduction per BID dose. Similarly, the daily 
dose of mycophenolic acid will be reduced by 320 mg every 2 weeks until off. This 
dose reduction will be achieved by a 180 mg reduction per BID dose.
x
No more than one drug should be completely discontinued on the same day.
3.3.1.3
Failure to Start or to Complete Withdrawal of M M F and Prednisone
Participants who do not start MMF and prednisone withdrawal by the end of week 34 post-
transplant will continue on study medication. At Week 34, these participants will move to
Appendix 5 and complete all safety follow-up visits per the schedule of events.


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Participants who experience rejection as described in section 6.5 will be ineligible for 
continuation of MMF and prednisone withdrawal. These participants will convert to site-
specific immunosuppression and will undergo safety follow-up per Appendix 5.
Participants who attempt, but fail to complete MMF and prednisone withdrawal for any 
reason, including rejection, will convert to site-specific immunosuppression and will complete 
all safety follow-up visits per the schedule of events in Appendix 5.
Participants who successfully complete immunosuppression withdrawal but subsequently 
return to maintenance immunosuppression for any reason (i.e., rejection, recurrent disease) 
will move to Appendix 5 and complete all safety follow-up visits per the schedule of events.
Withdrawal from MMF and prednisone may not be reattempted.
3.3.2
Withdrawal of Tacrolimus
3.3.2.1
Eligibility
After at least 34 weeks on tacrolimus therapy including at least 4 weeks on tacrolimus 
monotherapy, participants will be evaluated for eligibility for tacrolimus withdrawal.
Withdrawal from tacrolimus may be initiated no earlier than week 38 and no later than week 
50 post-transplant. To be eligible for withdrawal of tacrolimus, participants must meet the 
following criteria:
1.
A minimum of 34 weeks on tacrolimus therapy at the time of the eligibility evaluation.
2.
A renal biopsy that demonstrates the absence of rejection of any type, including acute 
humoral rejection, acute cellular rejection, and chronic rejection as defined in section 
6.5.4.1, within the 4 weeks prior to initiation of withdrawal.
3.
No history of biopsy-proven rejection or clinically presumed acute rejection without 
biopsy confirmation since transplantation.
4.
No history of biopsy-confirmed indeterminate rejection (less than Banff Grade IA) on
the withdrawal eligibility biopsy that has not resolved, as assessed by RB performed 
within 4 weeks of diagnosis.
5.
History of stable renal function. Stable renal function is defined as no unexplained 
increases in serum creatinine of greater than 25% relative to baseline sustained over a
72-hour time interval. Baseline is defined as the mean of the three lowest creatinines 
during Weeks 2 to 4 post-transplant, excluding days on dialysis.
6.
An estimated GFR > 40 mL/min/1.73m2 as calculated by CK-EPI equation.
7.
Eligible participants must demonstrate absence of donor-specific antibody as 
determined by solid phase micro particle technology (by Luminex® phenotype panel or 
Luminex single antigen bead test) performed within 4 weeks prior to initiating 
withdrawal, as assessed by local laboratories.
8.
No evidence of active GVHD at the time of initiation of withdrawal.
9.
Participant informed consent for withdrawal of tacrolimus.


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3.3.2.2
Initiation and Completion of Tacrolimus Withdrawal
Tacrolimus will be withdrawn in a stepwise fashion at an approximate rate of 25% of the pre-
withdrawal dose every 3 weeks. The dose of tacrolimus may not be reduced by more than 50% 
of the starting dose per dose reduction. Participants will be allowed a window of an additional 
1 week per 3-week interval before proceeding to the next dose reduction, especially if 
increases in creatinine are observed.
x
Initiation of tacrolimus withdrawal is defined as the day of first dose reduction made 
with intent to withdraw.
x
Completion of tacrolimus withdrawal is defined as the first day on which tacrolimus
is intentionally no longer administered after initiation of withdrawal.
Tacrolimus withdrawal:
x
May not be initiated earlier than Week 38 or later than Week 50 post-transplant.
x
Must be completed in no fewer than 12 weeks and no more than 16 weeks after 
initiation.
x
Will be discontinued if the participant experiences acute rejection as defined in section 
6.5.4.
May not be reattempted if discontinuation of withdrawal was due to renal allograft rejection.
All participants who meet the eligibility criteria for tacrolimus withdrawal will undergo study 
visits as specified in Appendix 2.
3.3.2.3
Failure to Start or to Complete Tacrolimus Withdrawal
Participants who do not attempt to start tacrolimus withdrawal by Week 50 post-transplant 
will continue on study medication and will complete all study visits for safety follow-up per 
the schedule of events in Appendix 5.
Participants who experience rejection will be ineligible for continuation of tacrolimus
withdrawal. They will convert to site-specific immunosuppression and will undergo safety
follow-up per Appendix 5.
Participants who attempt but fail to complete tacrolimus withdrawal for any reason including 
rejection will convert to site-specific immunosuppression and will complete all study visits for 
safety follow-up per the schedule of events in Appendix 5.
3.4
STUDY DURATION
Total study duration will be approximately 361 weeks (6.9 years).
x
The enrollment phase will be 79 weeks (1.5 years) and includes enrollment activity of 
71 weeks plus an allowance for the safety review between the enrollment of the third 
and fourth participants of as much as eight weeks.
x
The duration of the study for an individual participant may range from 271 to 383 weeks
depending on the length of time required for successful completion of 
immunosuppression withdrawal and includes 216 weeks of post-withdrawal follow-up.


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x
The study primary endpoint, measured 52 weeks after the last participant’s completion 
of immunosuppression withdrawal, could be achieved as early as 181 weeks (3.5 years),
or as late as 197 weeks (3.8 years) after enrollment of the first participant.
3.5
STUDY ENDPOINTS
3.5.1
Primary Endpoint
The primary endpoint is the proportion of participants who achieve operational tolerance, 
defined as remaining off all immunosuppression 52 weeks after completion of 
immunosuppression withdrawal with:
a)
no evidence of biopsy-proven allograft rejection and
b)
acceptable renal function, defined as a serum creatinine that has increased no more than 
25% above baseline (see section 6.5.1 for baseline thresholds) at the primary endpoint 
visit.
All participants who successfully complete immunosuppression withdrawal will undergo a 
protocol biopsy at this time point to assess the primary endpoint.
3.5.2
Secondary Endpoints
Safety
1.
The incidence, severity and duration of GVHD in transplanted participants.
2.
The incidence and duration of engraftment syndrome in transplanted participants.
3.
The proportion of transplanted participants who die.
4.
The proportion of transplanted participants with acute renal allograft rejection
demonstrated by a biopsy or clinically if a biopsy cannot be performed. If participant 
has allograft dysfunction as defined in section 6.5 and cannot undergo biopsy he or she
will be presumed to have rejection without biopsy confirmation.
5.
The histological severity of biopsies demonstrating acute rejection as measured by 
Banff Grade per Banff 2007 Classification Renal Allograft Pathology1.
6.
The proportion of transplanted participants with chronic T cell-mediated or antibody-
mediated rejection. This assessment should also include progressive interstitial 
fibrosis/tubular atrophy (IF/TA), transplant glomerulopathy or chronic obliterative 
arteriopathy without an alternative, non-rejection-related cause. See Banff 2007 
Classification Renal Allograft Pathology for definition of terms1.
7.
Time from transplant to the first episode of acute rejection requiring treatment.
8.
The incidence, severity and duration of adverse events including infection, wound 
complications, post-transplant diabetes, hemorrhagic cystitis and malignancy. 
9.
The proportion of transplanted participants who develop donor specific antibody:
a.
after initiation of immunosuppression withdrawal
b.
at any time during trial participation


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10. The time to absolute neutrophil recovery. This is defined as the interval from the 
neutrophil nadir to the first day of three consecutive daily neutrRSKLO FRXQWV per 
μL. The neutrophil nadir is defined as the first day post-transplant on which the absolute 
neutrophil count (ANC) is below 500 per μL.
11. The time to platelet count recovery. This is defined as the interval from transplant to the 
first day of a platelet count of 20,000 per μLwithout a prior platelet transfusion in the 
preceding seven days.
Efficacy
1.
The proportion of transplanted participants who remain off immunosuppression for at 
least 52 weeks including those in whom the 52 week biopsy was not performed.
2.
The proportion of participants who remain free from return to immunosuppression for 
the duration of the study.
Mechanistic
1.
The correlation of operational tolerance with the extent and durability of donor 
hematopoietic and T cell chimerism as measured by serial short tandem repeat analysis 
of recipient peripheral blood mononuclear cells (PBMCs) and T cells.
2.
The correlation of operational tolerance with other biomarkers such as cell subsets or 
gene expression.
The following secondary endpoints pertaining to safety and efficacy will be assessed only 
in participants who complete tacrolimus withdrawal:
1.
Immunosuppression-free duration, defined as time from completion of tacrolimus to end 
of trial participation or to time of restarting immunosuppression.
2.
Time from completion of tacrolimus withdrawal to first episode of acute rejection or 
presumed acute rejection, defined per Banff 2007 Classification Renal Allograft 
Pathology1.
3.
Time from completion of tacrolimus withdrawal to first diagnosis of chronic T cell 
mediated or antibody-mediated rejection. This assessment should also include 
progressive interstitial fibrosis/tubular atrophy (IF/TA), transplant glomerulopathy or 
chronic obliterative arteriopathy without an alternative, non-rejection related cause. See 
Banff 2007 Classification Renal Allograft Pathology1.
3.6
STOPPING GUIDELINES
If any one of the criteria listed below is met, study enrollment will be suspended and 
participants will be maintained on their current immunosuppressive treatment regimens (i.e., 
further reduction of immunosuppressive agents will be suspended) pending expedited review 
of all pertinent data by the NIAID Transplant DSMB, NIAID DAIT and the ITN.
3.6.1
General Stopping Guidelines
General stopping criteria include any of the following observed in any study participant:


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x
Death.
x
Grade 4 infection. 
x
Grade 3 or 4 acute GVHD or steroid refractory grade 2 acute GVHD (see Table 4 for 
grading criteria for acute GVHD). Steroid-refractory acute GVHD is defined as acute
GVHD that progresses despite 48 hours of treatment with methylprednisolone (2
mg/kg/day) or that does not improve despite 4 days of methylprednisolone (2
mg/kg/day).
x
Any occurrence of severe chronic GVHD (see Table 5 for grading criteria for chronic 
GVHD).
x
Renal graft loss defined as any of the following:
o
Institution of chronic dialysis (at least 6 consecutive weeks, excluding 
participants with delayed graft function).
o
Transplant nephrectomy.
o
Re-transplantation.
x
PTLD.
x
Progressive multifocal encephalopathy.
x
Failure to achieve neutrophil count recovery; recovery is defined DVDQ$1& per 
μL, by day 30 following the post-transplant neutrophil nadir. The neutrophil nadir is 
defined as the first day post-transplant on which the absolute neutrophil count (ANC) is 
below 500 per μL.
x
Irreversible grade 4 organ toxicity.
3.6.2
Stopping Guidelines for Rejection
Stopping criteria for rejection include:
x
The occurrence of rejection of the renal allograft in more than one participant prior to 
initiation of immunosuppression withdrawal.
x
The occurrence of rejection of the renal allograft in more than two participants at any 
time in the trial.
For the purposes of assessing the stopping rule, renal allograft rejection will be defined to 
include: acute humoral rejection, acute cellular rejection, and chronic rejection as defined in 
section 6.5.4.1 as well as presumed rejection without biopsy confirmation as defined in section 
6.5.4.2). See section 6.5 for guidelines for use of kidney biopsies to determine rates of acute 
rejection.
3.6.3
Ongoing Review
The Protocol Chair, the ITN Clinical Trial Physician, the NIAID Medical Monitor and the 
NIAID Transplant Data and Safety Monitoring Board (DSMB) will periodically review safety 
data. Enrollment of participants in the trial and further immunosuppression withdrawal for 
current trial participants will be suspended at any time if any of these reviews concludes that 
there are significant safety concerns.


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4.
ELIGIBILITY
4.1
INCLUSION CRITERIA
4.1.1
Recipient
Recipient participants must meet all of the following criteria to be eligible for this study:
1.
Recipient of a first renal allograft from an HLA-haploidentical, living related donor.
The donor and recipient must be HLA identical for at least one allele (using high 
resolution DNA based typing) at the following genetic loci: HLA-A, HLA-B, HLA-C, 
and HLA-DRB1. Fulfillment of this criterion shall be considered sufficient evidence 
that the donor and recipient share one HLA haplotype.
2.
Age 18 to 65 years.
3.
Single solid organ recipients (kidney only).
4.
ABO compatibility with donor.
5.
DSA will be assessed by the local laboratory 30 days or less prior to transplant using 
solid phase micro particle technology (by Luminex® phenotype panel or Luminex
single antigen bead test.) The following criteria apply:
a.
Participants without detectable DSA will be deemed eligible if they meet other 
entry criteria.
b.
Participants with detectable DSA and a positive flow cytometric crossmatch may 
undergo de-sensitization per standard of care if they are cytotoxic crossmatch 
negative.Such participants must demonstrate a negative flow cytometric 
crossmatch by day -9 in order to receive the first dose of study therapy (ATG). 
Participants who do not demonstrate an acceptable response to de-sensitization by
day -9 will be considered screen failures and will be terminated from the study.
c.
Participants with a positive cytotoxicity crossmatch will be excluded. 
6.
Removed in protocol version 5.0.
7.
Removed in protocol verision 5.0.
8.
Normal estimated left ventricular ejection fraction and no history of ischemic heart 
disease requiring revascularization, unless cleared by a cardiologist.
9.
FEV1 and FVC > 40% of predicted at the screening visit.
10. Serological evidence of prior EBV infection as documented by positive IgG and 
negative IgM antibodies against EBV.
11. For women of childbearing potential, a negative serum or urine pregnancy test with 
sensitivity less than 50 mIU/m within 72 hours before the start of study medication.
12. Use of two forms of contraception with less than a 5% failure rate or abstinence by all 
transplanted participants for 18 months after the first dose of study therapy. For the 
first 60 days post-transplant, recipients should be encouraged to use non-hormonal 


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contraceptives due to the potential adverse effect of hormones on bone marrow 
engraftment. If using a barrier method, a double barrier method should be used.
13. Ability to receive oral medication.
14. Ability to understand and provide informed consent.
15. All participants must demonstrate a negative QuantiFERON® (QFT) assay result 
within 52 weeks of transplant regardless of PPD status. Participants with a positive 
QFT assay must complete treatment for latent TB and have a negative chest x-ray. 
QFT testing done within 52 weeks before transplant is acceptable as long as there is 
documentation of the results. Prior recipients of a BCG vaccination are not exempt.
4.1.2
Donor
Donor participants must meet all of the following criteria to be eligible for this study:
1.
HLA-haploidentical, first-degree relatives or half-siblings of the recipient participant
at the allele or allele group. The donor and recipient must be HLA identical for at least 
one allele (using high resolution DNA based typing) at the following genetic loci: 
HLA-A, HLA-B, HLA-C and HLA-DRB1. Fulfillment of this criterion shall be 
considered sufficient evidence that the donor and recipient share one HLA haplotype.
2.
Age 18 to 65 years.
3.
Creatinine clearance >80 ml/minute as measured from a 24 hour urine collection 
within 26 weeks of the screening visit. If a serum creatinine drawn at the screening 
visit is > 20% higher than the serum creatinine drawn at the time of the 24 hour urine 
collection, the creatinine clearance must be re-evaluated by a repeat 24 hour urine test. 
,IWKHQHZYDOXHLVPJG/ WKHGRQRUZLOO EHH[FOXGHG
4.
Meets institutional selection criteria for organ and bone marrow donation.
5.
Ability to understand and provide informed consent for all study procedures including 
kidney transplant and bone marrow harvest.
6.
Serologic evidence of prior EBV infection as documented by positive IgG and 
negative IgM antibodies against EBV.
4.2
EXCLUSION CRITERIA
4.2.1
Recipient
Recipient participants who meet any of the following criteria will not be eligible for this study:
1.
Underlying renal disease with a high risk of disease recurrence in the transplanted 
kidney, including:
a.focal segmental glomerulosclerosis (FSGS).
b.type I or II membranoproliferative glomerulonephritis.
c. hemolytic-uremic syndrome/thrombotic thrombocytopenic purpura.


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2.
Clinically important genital/urinary tract dysfunction.
3.
Body mass index (BMI) > 40.
4.
Women who are breastfeeding.
5.
History of cancer within the last 5 years, except for nonmelanoma skin cancer, stage 1 
renal cell carcinoma, stage 1 prostate cancers cured by local resection and any 
curatively treated carcinomas in situ.
6.
History of positive HIV-1 or HIV-2 serologies or nucleic acid test.
7.
Evidence of hepatitis B infection. Participants demonstrating any one of the following 
will be excluded:
a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM.
c.
positive anti-HBc IgG
d.
positive HBV PCR
8.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV RNA PCR. All 
positive HCV antibody results must be assessed by an EIA assay and confirmed by a 
quantitative serum HCV RNA assay. Participants with positive HCV antibodies but 
undetectable serum HCV RNA may be considered for eligibility. Participants with 
negative anti-HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV serologies.
9.
History of active tuberculosis (TB).
10. Any active, severe local or systemic infection at the screening visit.
11. Autoimmune disease requiring immunosuppressive drugs for maintenance.
12. Use of investigational drug, other than the study medications specified by the 
protocol, within 30 days of transplantation.
13. Receipt of a live vaccine within 30 days of receipt of study therapy.
14. The presence of any medical condition that the Investigator deems incompatible with 
participation in the trial.
15. Positive cytotoxic crossmatch.
16. Calculated PRA greater than 90%.
4.2.2
Donor
Donor participants who meet any of the following criteria will not be eligible for this study:
1.
History of type I or type II diabetes mellitus.
2.
History of severe cardiovascular disease, defined as New York Heart Association
Class III or IV.
3.
History of blood product donation to recipient.
4.
History of positive HIV-1 or HIV-2 serology or nucleic acid test.
5.
Evidence of hepatitis B infection. Participants demonstrating any one of the following 
will be excluded:


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a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM
c.
positive anti-HBc IgG
d.
positive HBV PCR
6.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV RNA PCR. All 
positive HCV antibody results must be assessed by an EIA assay and confirmed by a 
quantitative serum HCV RNA assay. Participants with positive HCV antibodies but 
undetectable serum HCV RNA may be considered for eligibility. Participants with 
negative anti-HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV serologies.
7.
Autoimmune disease requiring immunosuppressive drugs for maintenance.
8.
The presence of any medical condition that the Investigator deems incompatible with 
participation in the trial.
4.3
PREMATURE DISCONTINUATION OF STUDY THERAPY
Study therapy is defined as initiation of any study-mandated therapies and study procedures 
described in section 5.
Study therapy will be discontinued for the following reasons:
x
Hypersensitivity. If a participant develops a persistent, life threatening hypersensitivity 
reaction to ATG, fludarabine or cyclophosphamide despite the measures described in 
section 5.2 before receiving the full dose of study drug.
x
Adverse experience. If a participant suffers from an adverse experience that, in the 
judgment of the Principal Investigator or the Medical Monitor, may present an 
unacceptable consequence or risk to the participant.
x
Intercurrent illness or infection. If during the course of the study a participant develops 
an illness or infection that is not associated with the condition under study and that 
requires treatment not consistent with protocol requirements; or, if a participant 
develops an intercurrent illness that in the judgment of the Principal Investigator
justifies discontinuation.
x
Protocol violation. If a participant cannot comply with the study protocol and the 
protocol deviations are sufficient to jeopardize his or her well-being or the integrity of 
the study.
x
Pregnancy.
x
Investigator discretion. If the Investigator determines that the study therapy is no longer 
in the best interests of the participant.
x
If the participant is unwilling or unable to continue on study therapy.
4.3.1
Follow–up for Participants Prematurely Discontinued from 
Study Therapy
All participants who discontinue study therapy will be asked to complete Safety Follow-up
procedures as specified in Appendix 5.


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4.4
PREMATURE TERMINATION OF A PARTICIPANT FROM THE 
STUDY
Participants will be prematurely terminated from the study for the following reasons:
Withdrawal of consent. If for any reason a participant withdraws consent during the 
study they will be terminated from the study.
Failure to return. Participants who do not return for visits and who do not respond to 
repeated attempts by the site staff to have them return will be considered lost to follow-
up.
4.4.1
Follow-up for Participants Prematurely Terminated from the 
Study
Participants who wish to withdraw consent will be asked to complete the Safety Follow-up
schedule in Appendix 5. If they decline they will be asked to undergo a study termination visit 
containing the assessments listed in visit 401 of Appendix 5. If they decline they will be 
terminated from the study with no further study data.
4.5
REPLACEMENT OF STUDY PARTICIPANTS
Enrolled participants who receive any form of study therapy, including ATG, fludarabine, 
cyclophosphamide and TBI, but who do not fulfill the PP analysis sample definition will be 
replaced. Participants who meet the PP analysis sample definition (see section 9.1) but who 
for any reason are no longer being treated according to the study protocol will undergo safety 
follow-up per Appendix 5 and will not be replaced. Participants who meet the PP analysis 
sample definition but who prematurely terminate from the study (section 4.4) will not be 
replaced.
5.
STUDY MEDICATIONS AND PROCEDURES
5.1
OVERVIEW OF CONDITIONING AND POST-TRANSPLANT 
REGIMEN
The treatment regimen is outlined in Figure 5.
The recipient will receive Antithymocyte globulin (ATG) from Day -9 to Day -7; fludarabine 
and low-dose cyclophosphamide on Days -4 and -3; fludarabine on Days -4, -3 and -2. 
Recipients will undergo hemodialysis 6 to 8 hours after each fludarabine dose. Recipients will 
undergo TBI on Day -1.
Donor nephrectomy and bone marrow harvest will take place under the same anesthetic on 
Day 0, with renal transplantation and bone marrow infusion into the recipient also on Day 0.
The recipient will receive high dose PT/Cy on Days 3 and 4 with hydration, MESNA and anti-
emetics. Standard maintenance immunosuppression consisting of tacrolimus, with target 
trough level of 8 to 12 ng/ml, MMF, 15 mg/kg three times daily (maximum daily dose of 3
grams per day), and prednisone, 5 mg twice daily, will be started on Day 5.


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5.2
CONDITIONING REGIMEN FOR RECIPIENTS
The following sections list study medications and procedures in chronological order of 
administration.
5.2.1
Indwelling central venous catheter
Placement of a double lumen central venous catheter will be required for administration of IV 
medications and transfusion of blood products including donor bone marrow.
5.2.2
Preparative regimen
5.2.2.1 Antithymocyte Globulin (Days -9, -8, -7)
Antithymocyte globulin (ATG) will be administered daily at a specialized outpatient BMT 
unit as an intravenous infusion on Days -9, -8 and -7. ATG will be infused through a 0.22 
micro filter with premedications: acetaminophen 650 mg orally and diphenhydramine 25mg 
orally. An anaphylaxis kit must be kept at bedside during ATG administration. An initial dose 
of 0.5 mg/kg IV will be administered over 6 hours on Day -9. Thereafter the daily dose will be 
increased to 2 mg/kg IV given over 4 hours on Days -8 and -7. No more than 150 mg of ATG 
may be administered per day.
All participants must receive at least 2 full doses of ATG to continue on study therapy. A full 
dose is defined as at least 80% of the dose volume. Participants who fail to receive at least 2
full ATG doses within the first week post-transplant (Day -9 through Day -7) will undergo 
safety follow-up (see Appendix 5. Safety Follow-up: Recipients).
A steroid taper will be given to prevent reactions to ATG as follows: On Days -9 to -7
methylprednisolone 1mg/kg IV 1 hour prior ATG. This dose may be repeated once 3 hours 
after the first dose of steroids. On Day -6 and -5, methylprednisolone 0.75 mg/kg/ IV as a 
single dose; on Days -4 and -3, methylprednisolone 0.5 mg/kg/ IV as a single dose; on Day -2,
methylprednisolone 0.25 mg/kg/ IV as a single dose.
For additional information on the preparation, storage and handling of ATG, refer to the 
package insert for Thymoglobulin® (Genzyme) at the following website: 
http://www.thymoglobulin.com/home/thymo_pdf_pi.pdf.
5.2.2.2 Fludarabine (Days -4 through -2)
Participants will be admitted to the inpatient bone marrow unit for administration of 
fludarabine. Fludarabine will be administered daily by intravenous infusion over 30 minutes
on Day -4 to Day -2. The daily dose will be 30 mg/m2. Hemodialysis will be performed no
earlier than 6 hours after and no later than 12 hours after each dose.
For additional information on the preparation, storage and handling of fludarabine, refer to the 
package insert for fludarabine at the following website: 
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=6f1094e3-4c28-40ae-a82f-
37cdfa865f49
5.2.2.3 Cyclophosphamide (Days -4, -3)
Cyclophosphamide will be administered as an intravenous infusion over 1-2 hours, (depending 
on volume) on Days -4 and -3. The dose of pre-transplantation cyclophosphamide is 14.5 


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mg/kg/day. Dose is calculated based on ideal body weight (IBW). Actual body weight should 
be used to dose cyclophosphamide in the case that actual body weight is less than ideal body 
weight. Body weight and height will be measured directly. Ideal body weight will be 
calculated using the following:
Males IBW = 50kg + 2.3kg/inch over 5 feet
Females IBW = 45.5kg + 2.3kg/inch over 5 feet.
Note: Hydration and MESNA will not be required for the Day -4 and -3 pre-BMT 
cyclophosphamide doses.
For additional information on the preparation, storage and handling of cyclophosphamide,
refer to the cyclophosphamide drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=591d9955-3d9c-4cdc-a308-
2f1288376b9f
5.2.2.4 Total Body Irradiation (Day -1)
Total body irradiation, consisting of 200 cGy AP/PA with 4MV or 6MV photons at 8-12
cGy/min at the point of prescription (average separation of measurements at mediastinum, 
abdomen, and hips), will be administered in a single day on Day -1.
5.2.2.5
Bone M arrow Infusion (Day 0)
Unprocessed, unmanipulated bone marrow will be harvested from the donor and infused into 
the recipient on Day 0. The nucleated cell target range will be between 8 to 16 x 108/kg of 
recipient ideal body weight with the volume not to exceed 20 mL/kg of donor's weight once 
the minimal target of 8 x 108/kg has been reached. All efforts will be made to assure a cell 
count of at least 2-4 x 108/kg of recipient ideal body weight. However there is no minimum 
donor bone marrow cell count threshold; the bone marrow infusion will proceed regardless of 
donor cell counts recovered.
5.2.3
High Dose Post-Transplant Cyclophosphamide (Days 3, 4)
High dose post-transplant cyclophosphamide (PT/Cy) [50mg/kg (Ideal Body Weight)] will be 
administered on Day 3 post-transplant (within 60 to 72 hours of marrow infusion) and on Day 
4 post-transplant. Cyclophosphamide will be given as an IV infusion over 1-2 hours
depending on volume. Participants requiring hemodialysis for renal insufficiency on days 3 
and 4 post-transplant will undergo hemodialysis no earlier than 12 hours after each 
cyclophosphamide dose.
For additional information on the preparation, storage and handling of cyclophosphamide,
refer to the cyclophosphamide drug label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=591d9955-3d9c-4cdc-a308-
2f1288376b9f
It is crucial that no immunosuppressive agents are given from the time of the transplant until 
24 hours after the completion of the post-transplant cyclophosphamide. This includes steroids 
as anti-emetics.


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5.2.4
MESNA (Days 3, 4)
A series of MESNA doses will be administered for each dose of high dose, post-transplant 
cyclophosphamide. MESNA will be given intravenously in divided doses 30 minutes before
and at 3, 6, and 8 hours after cyclophosphamide administration, or administered per 
institutional standards. The MESNA dose will be based on the cyclophosphamide dose being 
given. The total daily dose of MESNA is equal to 80% of the total daily dose of 
cyclophosphamide. For participants with renal insufficiency requiring hemodialysis, 
continuous bladder irrigation with normal saline at 200 ml/hour via a 3-way Foley catheter 
will be used instead of MESNA for hemorrhagic cystitis prevention. Continuous bladder 
irrigation will be continued for at least 12 hours after the last dose of high dose , post-
transplant cyclophosphamide66.
5.3
MAINTENANCE THERAPY (DAY 5 THROUGH ELIGIBILITY 
ASSESSMENT FOR IMMUNOSUPPRESSION WITHDRAWAL)
5.3.1
MMF
MMF will be administered at a dose of 15 mg/kg orally three times per day based upon actual 
body weight, with the maximum of 3 grams a day from Day 5 to 35. The dose will then be 
reduced to the standard 1 gram twice daily thereafter. Participants may not tolerate three 
grams a day due to GI side effects. A reduced dose of 500 or 750 mg twice daily will be 
allowed for those who are intolerant. Mycophenolic acid (Myfortic®) may be substituted for 
severe MMF intolerance.
For additional information about the risks of MMF, refer to the drug label at the following 
website: http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=209efe01-7908-46e4-a964-
0665078c0acd#druglabelcontent.
5.3.2
Prednisone
Prednisone will be administered at a dose of 10 mg orally daily from Day 5 for 12 weeks. 
Thereafter the dose will be reduced to 5 mg orally daily.
For further information about the risks associated with prednisone, please refer to the drug 
label at the following website:
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=3115aef0-fd50-4ec8-a064-
3effb695f3f2#druglabelcontent .
5.3.3
Tacrolimus
The first dose of tacrolimus will be administered orally on Day 5. The participant will receive 
tacrolimus daily to sustain target trough levels of between 8 to 12 ng/ml. Intravenous 
administration of tacrolimus will not be allowed due to its association with vasoconstriction 
and renal allograft loss.
For additional information on preparation, storage and handling of tacrolimus, refer to the 
drug label at the following website: 
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=bd447ffa-9196-4c3c-accf-
5adf29b84665#druglabelcontent


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5.3.4
Filgrastim (Day 5 to ANC Reconstitution)
All recipients will receive 5 ȝJNJSHUGD\RIILOJUDVWLP DVDVLQJOH, subcutaneous injection 
from Day -5 until the ANC is greater than 1000/μl on three consecutive measurements over at 
least 2 days31.
For additional information on preparation, storage and handling of filgrastim, refer to the drug 
label at the following website: 
http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=3bc802bd-76b4-4f45-8571-
a436ec26228e
5.4
STUDY REGIMEN FOR DONORS
5.4.1
Bone Marrow Harvest (Day 0)
Unprocessed, unmanipulated bone marrow will be harvested from the donor and infused into 
the recipient on Day 0. The nucleated cell target range will be between 8 to 16 x 108/kg of 
recipient ideal body weight with the volume not to exceed 20 mL/kg of donor's weight once 
the minimal target of 8 x 108/kg has been reached. All efforts will be made to assure a cell 
count of at least 2-4 x 108/kg of recipient ideal body weight. However there is no minimum 
donor bone marrow cell count threshold; the bone marrow infusion will proceed regardless of 
donor cell counts recovered.
5.5
MODIFICATION OF RECIPIENT STUDY MEDICATIONS
5.5.1
Bacterial Prophylaxis
Allograft recipients will receive ciprofloxacin 500 mg orally twice daily, starting on Day 1, 
until the $1&PP3 for three consecutive days.
5.5.2
Tacrolimus
In the event of toxicity related to tacrolimus (nephrotoxicity, neurotoxicity hyperglycemia, 
etc.), the dose of tacrolimus may be adjusted at the discretion of the Investigator to maintain 
trough levels lower than those specified in section 5.3.3.
In the event of development of posterior reversible encephalopathy syndrome (PRES)58
sirolimus will be substituted for tacrolimus at the discretion of the investigator. Sirolimus will
be dosed to achieve a goal trough level of 8 to 10 ng/ml for the first 3 months of therapy.
Thereafter the dose may be lowered to achieve a goal trough level of 6 to 8 ng/ml. All effort 
should be made to avoid sirolimus use in the first 10 days post-transplant to avoid its adverse 
effect on wound healing.
5.5.3
Mycophenolic Compounds
The dose of MMF or mycophenolic acid may be adjusted for intolerance or leukopenia. A
reduced dose of MMF 500 or 750 mg twice daily will be allowed for those who are intolerant.
Mycophenolic acid (Myfortic®) may be substituted for severe MMF intolerance. The 
maximum allowable dose of MMF is 3g daily; for mycophenolic acid, it is 1440 mg daily.


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5.5.4
Corticosteroids
On Days -9 to -7 a second dose of methylprednisolone 1mg/kg IV may be given at the 
Investigator’s discretion once 3 hours after the first dose of intravenous methylprednisolone,
especially in cases of ATG infusion reaction (section 5.2.2). Otherwise the dose of study-
mandated corticosteroids, including the steroid taper after ATG infusion and the course of 
maintenance prednisone, may not be modified.
5.6
PROPHYLACTIC MEDICATIONS AND MONITORING
Prophylactic medications associated with adverse events such as leukopenia or rash may be 
held or dose-adjusted per institutional standard until the episode has resolved.
5.6.1
Pneumocystis Jiroveci (PCP) Prophylaxis
Allograft recipients will receive prophylaxis for Pneumocystis jiroveci (PCP). Participants will 
receive trimethoprim sulfa (TMP-SMX) , one single- or double-strength tablet at least 3-times 
weekly, beginning on Day 21 or when the ANC 1000/mm3 for three consecutive days.
Pneumocystis jiroveci prophylaxis will continue until Day 365. For sulfa intolerant 
participants the following options are available:
1.
Sulfa desensitization followed TMP-SMX single-strength, one tablet orally every 
day until Day 365.
2.
Dapsone 100 mg orally every day until Day 365.
3.
Pentamidine 300 mg inhaled every month until Day 365.
4.
Atovaquone 1500 mg/day
For participants who are known to be allergic to or intolerant of sulfa compounds, de-
sensitization will be allowed. Desensitization to TMP-SMX should be considered when 
there are no reasonable or available alternatives and the patient has not experienced severe 
reactions (e.g., Stevens-Johnson syndrome) to sulfa drugs. The following algorithm will be 
followed:
Use commercially available pediatric suspension (containing TMP 8 mg and SMX 40 mg 
per mL), followed by double-strength tablets, as follows:


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Table 2. Rapid Oral TMP-SMX desensitization protocol
TMP (mg)
SMX (mg) PO
Dose plus formulation
Day 1*
9 AM
0.8
4
1 mL 1:10 dilution
11 AM
1.6
8
2 mL 1:10 dilution
1 PM
4
20
5 mL 1:10 dilution
5 PM
8
40
10 mL 1:10 dilution
Day 2
9 AM
16
80
2 mL undiluted suspension
3 PM
32
160
4 mL undiluted suspension
9 PM
40
200
5 mL undiluted suspension
Day 3
9 AM
80
400
1 TMP-SMX SS tab
*The solution used on Day 1 may be prepared from the oral suspension (which contains 40 mg 
of trimethoprim and 200 mg of sulfamethoxazole per 5 mL) by combining 2 mL of suspension 
and 18 mL of water67.
Pneumocystis jiro prophylaxis will be reinstituted for 12 weeks after receiving treatment for 
an episode of rejection occurring after the first 52 weeks of the transplant.
5.6.2
Fungal and Yeast Prophylaxis
Allograft recipients will receive fluconazole 400 mg orally daily, starting on Day 1, for fungal 
and yeast prophylaxis until the ANC 1000/mm3 for three consecutive days.
5.6.3
CMV Prophylaxis and Monitoring
Allograft recipients, regardless of CMV serological status, will receive valacyclovir 500 mg 
orally three times per day, starting on Day -9 until Day 365. If participants cannot tolerate oral 
medications, acyclovir 250mg/m2 IV every 12 hours may be substituted for valacyclovir. The 
dose will be adjusted for renal dysfunction.
CMV viremia will be monitored by PCR and documented weekly beginning once the total 
white blood cell count is greater than 1000/mm3. Monitoring of CMV viremia will continue 
weekly until Day 100; then every other week until Day 365.
5.6.4
CMV Treatment
Patients who are viremic will be treated pre-emptively with ganciclovir (5 mg/kg IV every 12 
hours) for at least, 14 days, at the discretion of the physician, and then with maintenance 
ganciclovir (5 mg/kg IV daily) until CMV testing is negative for at least 2 weeks.
Consideration should be given to administration of CMV hyperimmune globulin (Cytogam®), 
concomitant with ganciclovir at a dose of 150 mg/kg IV every other day times 4 doses and 
then weekly thereafter if the patient continues to be treated with ganciclovir for persistent 


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viremia. If the patient is diagnosed with CMV disease, more frequent CMV hyperimmune 
globulin administration may be considered. The dose will be adjusted for renal dysfunction.
Unless the patient is being treated for CMV infection or CMV disease, ganciclovir should be 
discontinued for development of neutropenia (ANC<500). If the participant is neutropenic, G-
CSF may be used if clinically indicated. Ganciclovir should then be restarted ZKHQWKH$1&
500, if appropriate. Ganciclovir will be dose-adjusted appropriately for renal failure.
Initial treatment of CMV disease will be for a minimum of 2 weeks with intravenous 
ganciclovir at a dose of 5 mg/kg twice daily (with dosage adjustment for renal dysfunction), 
oral valganciclovir 900 mg orally twice daily, or other approved antiviral medication per 
institutional standards. CMV hyperimmune globulin (150 mg/kg every 4 weeks) will be added 
for seronegative participants at the discretion of the Investigator. The endpoint of intravenous 
therapy is the documented clearance of virus from the blood as demonstrated by CMV 
quantitative PCR assay.
The risk of subsequent relapse is 15-20%, so treatment may be continued for up to three
months, at the discretion of the physician. With three months of oral therapy following 
clearance of viremia, the rate of relapse is decreased substantially.
In participants with relapsing infection, initial treatment will be repeated at the discretion of 
the physician. CMV hyperimmune globulin (at 150 mg/kg IV once and 100 mg/kg IV monthly
for 3-6 months) is usually administered in conjunction with the intravenous ganciclovir, oral 
valganciclovir, or other approved therapy for seronegative recipients at the discretion of the 
Investigator.
5.6.5
Treatment of Herpes Infection
All participants will receive anti-viral prophylaxis for herpes infection per institutional 
standard for 26 weeks post-transplant.
5.7
MONITORING FOR BK VIREMIA
All participants who receive at least 1 full dose of ATG (see section 5.2.2) will be monitored 
for BK viremia by quantitative BK viral PCR. Participants who complete immunosuppression 
withdrawal will be monitored at least 1 year post-transplant. Participants who remain on 
immunosuppression and are moved to Safety Follow-up will be monitored at least 2 years 
post-transplant. For-cause biopsy visits will include a test for BK viremia. BK viremia will be 
monitored at the following timepoints: monthly for months 1 through 6 post-transplant; at 
months 9 and 12 post-transplant; at 18 and 24 months post-transplant for participants who 
remain on immunosuppression. Treatment of BK viremia will be left to the discretion of the 
site PI.
5.8
MONITORING FOR EBV REACTIVATION
All participants who receive at least 1 full dose of ATG (see section 5.2.2) will be monitored 
for EBV viremia by quantitative EBV PCR at the time points specified in Appendix 1 through 
Appendix 5. Safety Follow-up: Recipients. Treatment of EBV viremia will be left to the 
discretion of the site PI.


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5.9
PROHIBITED MEDICATIONS AND VACCINES
x
All immunosuppressive medications including oral corticosteroids are prohibited from 
the day of transplant to 24 hours after the last day on which high dose 
cyclophosphamide is administered.
x
6KRUWGD\VFRXUVHVRIRUDOFRUWLFRVWHURLGVDUHSHUPLWWHGRQO\ IRUWKHWUHDWPHQWRI
unrelated illnesses such as asthma, allergy, etc. These should be avoided unless 
absolutely necessary.
x
Otherwise, all immunosuppressive medications other than those used as specified in the 
protocol are prohibited during immunosuppression withdrawal unless rejection is 
suspected or diagnosed.
x
Topical or inhaled corticosteroids or steroid mouthwashes will not be considered 
immunosuppressive medications.
x
Live vaccines are prohibited from 30 days prior to the first dose of study therapy 
through the end of study participation.
x
Any other investigational products are prohibited for the duration of study participation.
5.10
CONCOMITANT MEDICATIONS
All recipient concomitant medications will be documented at the screening Visit 1 (see 
Appendix 1A. Pre-Transplant Through Transplant: Recipient). At all subsequent visits, only 
immunosuppressive medications, prophylactic antibiotics, anti-viral and anti-fungal 
medications will be recorded.
5.11
BLOOD REPLACEMENT THERAPY
If blood component replacement therapy is required for any reason, leukocyte-filtered blood 
components will be administered. If clinically indicated, blood components will be irradiated.
6.
STUDY PROCEDURES
6.1
VISIT WINDOWS
6.1.1
Scheduled Visits
The schedule of events for this trial can be found in Appendix 1A. Pre-Transplant Through 
Transplant: Recipient through Appendix 5. Safety follow-up: Recipients. For both the 
recipient and donor, the initial screening visit, Visit 1, must occur within 30 days of the first 
administration of study drug. All other scheduled study visits must occur within the time 
limits specified below:


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Table 3. Designated study windows
Appendix
Trial Phase
Visit Window
1.A
Pre-transplant through 
transplant - Recipient
Visits should take place on the day indicated in the 
schedule of events
Assessments listed in the screening visit should take 
place within 30 days of the first administration of 
study drug unless otherwise indicated
Screening laboratory assessments
x
hematology, chemistry and pregnancy 
assessments must be completed within 30 
days of Day -9
x
viral serology assessments: results of CMV, 
EBV, HIV-1 and 2, Hep B and HCV testing 
must be reported by day -14
x
previously performed high resolution 
molecular HLA typing, ABO typing and 
positive viral serologies may be used, 
provided the respective assays were 
performed using the same technology 
currently in use by the site laboratory 
x
cytocytoxic crossmatch
x
calculated PRA
If visit 2 falls on a weekend, baseline specimens may 
be collected up to 6 days prior to the visit 
1.B
Pre-transplant through 
post-transplant - Donor
Visits pre-transplant through transplant should take 
place on the day indicated in the schedule of events
If visit 2 falls on a weekend, baseline specimens may 
be collected up to 6 days prior to the visit
Assessments listed in the screening visit should take
place within 30 days of transplant (Day 0) unless 
otherwise indicated
Screening laboratory assessments
x
hematology, chemistry and pregnancy 
assessments must be completed within 30 
days of Day 0


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Appendix
Trial Phase
Visit Window
x
viral serology assessments:CMV, EBV, HIV-
1 and 2, Hep B and HCV must be completed 
on day -14, +/- 2 days
x
previously performed high resolution 
molecular HLA typing, ABO typing and 
positive viral serologies may be used,
provided the respective assays were 
performed using the same technology 
currently in use by the site laboratory
The Day 7 post-transplant visit has a +3-day window
The Day 56 post-transplant visit has a +14-day 
window
1.C
Reinitiation of 
Conditioning: Pre-
Transplant Through 
Transplant: Recipient
Visits 501 through 504 has a +7-day window
Visits 505 through 514 should take place on the day 
indicated in the schedule of events
Assessments listed in Visit 505 should take place 
within 30 days of the first administration of study drug
during the reinitiation of conditioning, unless 
otherwise indicated
For eligibility laboratory assessments that must be 
repeated, the following guidelines apply
x
hematology, chemistry and pregnancy 
assessments must be completed within 30 
days of Day -9
x
viral serology assessments: results of CMV, 
EBV, HIV-1 and 2, Hep B and HCV testing 
must be reported by day -14.
x
previously performed high resolution 
molecular HLA typing, ABO typing and 
positive viral serologies may be used, 
provided the respective assays were 


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Appendix
Trial Phase
Visit Window
performed using the same technology 
currently in use by the site laboratory 
x
cytocytoxic crossmatch
x
calculated PRA
If Visit 506 falls on a weekend, baseline specimens 
may be collected up to 6 days prior to the visit
1.D
Reinitiation of 
Conditioning: Pre-
Transplant Through Post-
Transplant: Donor
Visits pre-transplant through transplant should take 
place on the day indicated in the schedule of events
If visit 506 falls on a weekend, baseline specimens 
may be collected up to 6 days prior to the visit
Assessments listed in the screening visit should take 
place within 30 days of transplant (Day 0) unless 
otherwise indicated
Screening laboratory assessments
x
hematology, chemistry and pregnancy 
assessments must be completed within 30 
days of Day 0
x
viral serology assessments: CMV, EBV, HIV-
1 and 2, Hep B and HCV must be completed 
on day -14, +/- 2 days
x
previously performed high resolution 
molecular HLA typing, ABO typing and 
positive viral serologies may be used, 
provided the respective assays were 
performed using the same technology 
currently in use by the site laboratory
The Day 7 post-transplant visit has a +3-day window
The Day 56 post-transplant visit has a +14-day 
window
2
Post-transplant through 
evaluation for withdrawal 
eligibility
Visits 101-104
Day indicated
Visits 105-106
+/- 2 days
Visit 107
+/- 7 days
The Initiation of IS withdrawal eligibility biopsymay 
take place as early as Week 22 or as late as Week 30


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Appendix
Trial Phase
Visit Window
post-transplant at Investigator discretion, based on 
participant medical status
3
Immunosuppression 
withdrawal
Visits 201-214
+/- 7 days
Visits 215-218 (if 
needed)
+/- 7 days
The Initiation of tacrolimus withdrawal eligibility 
biopsy may take place as early as Week 34 or as late 
as Week 46 post-transplant based on progress of 
immunosuppression withdrawal and at Investigator 
discretion, based on participant medical status
4
Post immunosuppression 
withdrawal follow-up
Visits 301-305
+/- 7 days
Visits 306-322
+/- 2 weeks
The Week 52 post IS withdrawal completion biopsy
should take place 52 weeks after completion of 
immunosuppression withdrawal +/- 2 weeks
The Week 104 post IS withdrawal completion biopsy
should take place 104 weeks after completion of 
immunosuppression withdrawal +/- 2 weeks
The Week 208 post IS withdrawal completion biopsy
should take place 208 weeks after completion of 
immunosuppression withdrawal +/- 2 weeks
5
Safety follow-up for 
participants who are 
ineligible or have failed 
immunosuppression 
withdrawal. All 
participants who receive 
the preparative regimen 
will be followed for at 
least 2 years as indicated 
in Appendix 5. Safety 
Follow-up: Recipients.
Visit 401
Participants who experience a 
rejection event should begin 
safety follow-up within 4-6 weeks 
of the date of diagnosis of 
rejection. Participants who move 
to safety follow-up following
non-rejection events should begin 
this schedule within 2 weeks of 
the determination of their 
ineligibility for further progress in 
the protocol.
Visits 402-410
+/- 2 weeks


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6.2
GENERAL ASSESSMENTS
Recipient Assessments:
x
Informed consent
x
Medical, demographic, and blood products from donor history
x
Complete physical examination including height
x
Limited physical examination (to include: respiratory, cardiovascular, gastrointestinal, 
skin, neurologic and renal/urinary systems)
x
Vital signs – Weight, temperature, blood pressure, respiration, and pulse
x
Cardiac function – echocardiogram
x
Pulmonary function – chest x-ray and spirometry
x
Acute GVHD assessment – staged and graded as in section 6.7
x
Chronic GVHD assessment – staged and graded as in section 6.6
x
Screening for status change since prior visit including assessment of change in 
administration of blood products; adverse events and concomitant medications.
Donor Assessments:
x
Informed consent
x
Medical and demographic history including screening for any autoimmune disease 
requiring immunosuppressive drugs for maintenance
x
Physical examination including height and weight
x
Vital signs – temperature, blood pressure, respiration, and pulse
x
Screening for status change since prior visit including assessment of change in 
administration of blood products; adverse events and concomitant medications.
6.3
CLINICAL LABORATORY ASSESSMENTS
Recipient Assessments:
x
Hematology – CBC with differential and platelets
x
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, BUN, creatinine, glucose, albumin,
total bilirubin, AST, ALT, alkaline phosphatase)
x
Urine or serum pregnancy test
x
CMV and EBV serologies
x
BK virus (blood)


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x
HIV-1 and 2 serologies and nucleic acid test
x
Anti-hepatitis C (HCV) antibodies, hepatitis B surface antigen (HBsAg), total hepatitis 
B core antibody (anti-HBc IgM and IgG), hepatitis B surface antibody (anti-HBsAb)
x
ABO typing
x
High resolution molecular HLA typing and DNA (A, B, C, DRB1, DRB3-5, DQA1, 
DQB1, DPB1)
x
HLA alloantibodies includes DSA, flow PRA
x
CMV and EBV viral load by PCR
x
T and B cell lymphocyte crossmatch
x
Cytotoxic crossmatch
x
Calculated PRA
x
Tacrolimus levels
x
Kidney biopsy
Donor Assessments:
x
High resolution molecular HLA typing and DNA (A, B, C, DRB1, DRB3-5, DQA1, 
DQB1, DPB1)
x
CMV and EBV serology
x
HIV-1 and 2 serologies and nucleic acid test
x
Anti-hepatitis C (HCV) antibodies, hepatitis B surface antigen (HBsAg), total hepatitis 
B core antibody (anti-HBc IgM and IgG), hepatitis B surface antibody (anti-HBsAb)
x
ABO typing
x
Hematology – CBC with differential and platelets
x
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, BUN, creatinine, glucose, albumin,
total bilirubin, AST, ALT, alkaline phosphatase)
x
PBMC's for T and B cell lymphocyte crossmatch
6.4
MECHANISTIC ASSESSMENTS
Recipient Assessments:
x
Blood chimerism (whole blood and CD3+ T cell)
x
T cell assays (MLR, CML)
x
Plasma cytokines
x
B cell assays (HLA tetramer selection and culture)


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x
Whole blood – gene expression profiling
x
Kidney biopsy – histology
x
Kidney biopsy – gene expression profiling
x
B cell repertoire sequencing
x
Flow cytometry panel staining (frozen PBMC)
x
PBMC collection
Donor Assessments:
x
Blood chimerism (whole blood and CD3+ T cell)
x
T cell assays (MLR, CML)
x
B cell assays (HLA tetramer selection and culture)
x
PBMC collection
See section 7 for detailed discussion of additional mechanistic assays.
6.5
ASSESSMENT AND MANAGEMENT OF ALLOGRAFT 
DYSFUNCTION AND REJECTION
6.5.1
Definition of Allograft Dysfunction
Allograft dysfunction is defined as a sustained increase in creatinine levels of 25% or greater 
over 72 hours relative to baseline. Baseline thresholds are defined as follows:
x
Prior to and During Immunosuppression Withdrawal: Baseline is defined as the mean of 
the three lowest creatinines during weeks 2 to 4 post-transplant, excluding days on 
dialysis.
x
After Completion of Immunosuppression Withdrawal: Baseline is defined as the mean 
of the three lowest creatinines in the 8 weeks prior to the final dose of 
immunosuppression.
6.5.2
Indications for Increased Monitoring and For-cause Biopsy
If an episode of allograft dysfunction occurs, a serum creatinine must be drawn and recorded 
every 2 weeks for at least 1 month after the episode begins. If allograft dysfunction is 
unexplained and persists for more than 72 hours, a renal biopsy must be performed to rule out 
acute rejection. Unexplained is defined as demonstration of patent vessels and no significant 
hydronephrosis by sonogram or other imaging method and the absence of other factors known 
to affect renal function (i.e., dehydration, urosepsis, drug toxicity, polyoma viral infection).
If a biopsy is performed for allograft dysfunction, all mechanistic assessments listed for Visit 
308, Appendix 3. Immunosuppression Withdrawal (Initiated Between 26 and 3 Weeks Post-
Transplant) should be performed. If the biopsy does not demonstrate rejection, further 
evaluation for other causes of renal dysfunction will be performed.


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If a transient increase in creatinine occurs as defined by a level that meets the threshold for 
allograft dysfunction but is not sustained for 72 hours, a follow-up creatinine level must be 
drawn one week after the last elevated value to confirm return to baseline. If the repeat 
creatinine remains above the threshold, and is unexplained, a biopsy must be performed.
6.5.3
Modification of Creatinine Threshold for Performance of a For-
cause Biopsy
The baseline creatinine values used to determine thresholds for performance of a for-cause 
biopsy may be modified if a participant’s creatinine remains persistently elevated (i.e., above 
threshold levels greater than 72 hours) despite a biopsy demonstrating the absence of 
rejection. Such modification requires approval by the NIAID Medical Monitor, the ITN 
Clinical Trial Physician, the ITN Therapeutic Area Advisor and the Protocol Chair after 
review of the clinical history and biopsy findings. This biopsy will be reviewed by both local 
and central pathologists and the worst read will be used. The new baseline will be defined as 
the mean of the three consecutive values immediately prior to the date of the for cause biopsy
(FCB).
NOTE: These adjusted baseline values will be used to determine thresholds for repeat biopsy
(RB) only. For the purposes of calculating rates of allograft dysfunction or sustained renal 
impairment previously defined baseline values will be used.
6.5.4
Diagnosis of Rejection
6.5.4.1
Biopsy-Proven Rejection
Unless medically contraindicated, all episodes of acute rejection must be confirmed by renal 
allograft biopsy. For all biopsies performed to evaluate rejection, C4d staining should be 
performed using the most sensitive method. Biopsies will be read by both local and central 
pathology laboratories. For cases where rejection is diagnosed a Banff score should be 
assigned by both pathologists according to the 2007 Banff Classification of Renal Allograft 
Pathology1.
See section 6.5.7 for guidelines on use and interpretation of kidney biopsies.
The types of rejections are defined as follows:
x
Acute humoral (antibody-mediated) rejection is defined according to the 2007 Banff 
Classification of Renal Allograft Pathology1.
x
Acute cellular rejection is defined by a Banff score of Grade IA or above per the 2007 
Banff Classification of Renal Allograft Pathology1.
x
Chronic rejection includes both chronic T cell -mediated and chronic antibody-mediated 
rejection as defined according to the 2007 Banff Classification of Renal Allograft 
Pathology1. This assessment should also include progressive interstitial fibrosis/tubular 
atrophy (IF/TA), transplant glomerulopathy or chronic obliterative arteriopathy without 
an alternative, non-rejection-related cause. See Banff 2007 Classification Renal 
Allograft Pathology for definition of terms1.


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Biopsy-proven indeterminate (borderline) acute cellular rejection, defined by a Banff score 
between normal and less than Banff Grade IA, will not be classified as rejection.
6.5.4.2
Rejection without Biopsy Confirmation
If a participant is unable to undergo a biopsy to evaluate unexplained allograft dysfunction or 
to confirm suspected rejection, this event will be classified as rejection without biopsy 
confirmation.
If there is unresolvable disagreement with regard to this classification, an adjudication 
committee will be convened. This committee, consisting of the NIAID Medical Monitor, the 
ITN Clinical Trial Physician and the Protocol Chairs, will determine whether the event should 
be classified as rejection.
A participant will be considered to have experienced presumed rejection regardless of biopsy 
findings if he or she receives more than a single dose of steroids for the purpose of treating 
rejection.
6.5.5
Treatment and Management of Participants with Rejection
Immunosuppression according to institutional standard will be reinstituted for all participants 
who experience rejection as confirmed by local pathology read. These participants will 
undergo safety follow-up per Appendix 5. Safety Follow-up: Recipients.
All participants who experience presumed rejection without biopsy confirmation (see section 
6.5.4.2) will also resume immunosuppression according to institutional standard and will 
undergo safety follow-up per Appendix 5. Safety Follow-up: Recipients.
All participants with biopsy-confirmed rejection assessed by either central or local pathology 
reading will undergo a repeat biopsy (RB) 4 weeks after diagnosis to document that the 
rejection has resolved (see section 6.5.7.3) .
6.5.6
Types of Kidney Biopsies
The following types of renal biopsies are defined for this trial:
6.5.6.1 Protocol biopsies
Participants will undergo renal biopsies at times specified in the schedule of events. These 
biopsies will be used to monitor renal function, to screen for subclinical rejection, to 
determine eligibility for immunosuppression withdrawal and for mechanistic analyses.
6.5.6.2
For-cause biopsies
For-cause biopsies (FCB) will be obtained to confirm suspected rejection following 
unexplained allograft dysfunction and for performing mechanistic analyses.
6.5.6.3
Repeat biopsies
Participants will undergo a repeat biopsy (RB) at a specified interval after an initial protocol 
biopsy or for-cause biopsy (FCB) to confirm the resolution of histological abnormalities or to 
ensure that none have evolved.


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6.5.7
Use and Interpretation of Kidney Biopsies
All renal biopsies will be interpreted and analyzed by both local and central pathology 
laboratories. The guidelines for the use of these pathology reports are outlined below.
6.5.7.1 Protocol Biopsies
For purposes of determining study outcomes such as eligibility for immunosuppression 
withdrawal or endpoint classification, the local and central pathologist will each report a Banff 
score according to the 2007 Banff classification1. If significant discrepancies between the two 
readings are identified, the two pathologists will be asked to conduct a collaborative 
consultation. This consultation may include the exchange of images or biopsy material. A 
‘significant discrepancy’ is defined as greater than or equal to one letter grade difference in 
Banff score (i.e., IA versus IB) or reports that result in discrepancies in eligibility or endpoint 
classification. If discrepancies cannot be resolved through this collaborative consultation, an
independent third party will be used for adjudication of biopsy classification. In cases where 
there is no local read the central read will be used.
For the purposes of clinical management the local pathology read will be used.
6.5.7.1.1
Biopsy for Determination of the Primary Endpoint
A protocol biopsy is required for determination of the primary endpoint. Participants who 
decline to undergo the post immunosuppression withdrawal protocol biopsy, as designated in 
Appendix 3. Immunosuppression Withdrawal (Initiated Between 26 and 3 Weeks Post-
Transplant), will be considered to have failed the primary endpoint and will be restarted on 
immunosuppression at the discretion of the site Investigator. Participants will be counseled on 
the risks of remaining off immunosuppression without appropriate surveillance biopsies.
6.5.7.2
For-cause Biopsies
For all FCBs the local pathology read will be used for clinical management and determination 
of study outcomes such as continued immunosuppression withdrawal. For the purposes of 
analysis the central and local findings will be considered in conjunction with any clinical 
treatment to determine rates of rejection. Significant discrepancies between central and local 
readings will be adjudicated by a third party pathologist.
6.5.7.3
Repeat Biopsies
Participants will undergo a RB after an initial biopsy under the circumstances listed below.
6.5.7.3.1
Biopsy-confirmed Rejection
All participants with biopsy-confirmed rejection assessed by either central or local pathology 
reading will undergo a RB 4 weeks after diagnosis to document that the rejection has resolved. 
This RB will be interpreted and analyzed using the guidelines FCB (see section 6.5.7.2).
6.5.7.3.2
Indeterminate Rejection on Withdrawal Eligibility Biopsy
Participants with indeterminate acute cellular rejection (<IA) as the worst read on eligibility 
biopsy for immunosuppression withdrawal as read by either local or central pathology lab will 
undergo a RB within 4 weeks of their initial biopsy unless medically contraindicated. The 
results of this biopsy will be used to determine eligibility for immunosuppression withdrawal. 


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These biopsies will be considered protocol biopsies and will be interpreted and analyzed using 
the guidelines above for all other protocol biopsies.
6.5.7.3.3
Development of DSA off Immunosuppression
A participant who undergoes a FCB for the development of DSA off immunosuppression after 
successful completion of immunosuppression withdrawal will undergo a RB 3 months after 
the DSA biopsy is obtained (see section 6.6). This RB will be interpreted and analyzed using 
the guidelines for FCB (see section 6.5.7.2).
6.5.7.4
Determination of Acute Rejection Rate for Stopping Guidelines
For the purposes of determining rates of acute rejection for stopping guidelines both local and 
central pathology reads will be used as described above; the local read will be used for for-
cause biopsies and the worst read will be used for protocol biopsies. In addition all episodes of 
presumed rejection without biopsy confirmation will count towards this determination.
6.5.8
Biopsy Technique and Handling
The Day 0 protocol biopsy will be performed as a core biopsy on the back table prior to 
transplantation of the kidney into the recipient. Otherwise, the protocol and for-cause biopsies 
will be performed percutaneously under ultrasound guidance with a 15- to 18-gauge needle. 
Use of a 16-gauge needle or larger is preferred. A minimum of two core biopsies should be 
obtained with a minimum of 1 cm of tissue per core biopsy. Where possible, trained personnel 
should confirm, by visual inspection, that biopsy tissue contains glomeruli (cortex). If 
insufficient tissue is obtained, cortical tissue should be allocated first to the ITN Pathology 
core and then to processing for gene expression studies. A consent specific for this procedure 
will be obtained according to the guidelines at each study site.
6.5.9
Recording of Biopsy Interpretations
The local pathology read shall be recorded on the eCRF by the site. The central pathology read 
will be recorded by the ITN central pathology core laboratory and the data will be transferred 
at regular intervals to the clinical database.
6.6
MONITORING AND MANAGEMENT OF DONOR SPECIFIC 
ANTIBODIES
Participants will be monitored at regular, frequent intervals for the occurrence of HLA 
alloantibodies including donor-specific antibodies (DSA) per SOE (see Appendix 1A. Pre-
Transplant Through Transplant: Recipient through Appendix 5. Safety Follow-up: 
Recipients).
Participants who develop DSA prior to initiation of immunosuppression withdrawal will be 
deemed ineligible for withdrawal.
Participants who develop DSA during immunosuppression withdrawal or while on tacrolimus 
monotherapy will restart immunosuppression and will move to safety follow up per Appendix 
5. Safety Follow-up: Recipients
Participants who develop DSA while off immunosuppression after successful completion of 
immunosuppression withdrawal will undergo a FCB. Immunosuppression will be restarted


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and the participant will move to safety follow-up (Appendix 5. Safety Follow-up: Recipients)
if any of the below are observed by the local pathologist:
x
Diffuse c4d+ (defined as positive staining of >50% of the PTCs stained) AND either:
a.
Glomerulitis %DQIIg1 or
b.
7UDQVSODQWJORPHUXORSDWK\ %DQIIFJ, or
x
Rejection of any type (see section 6.5.4.1).
Other concerning biopsy findings, including but not limited to capillaritis or inflammation 
with focal or absent c4d staining, will prompt a review by a committee consisting of the 
protocol chairs, the NIAID Medical Monitor, the ITN Clinical Trial Physician and the central 
pathologist. Immunosuppression will be restarted per committee recommendation.
All participants who develop DSA off immunosuppression will undergo a RB 3 months after 
the initial FCB and the same criteria for restarting immunosuppression will apply.
6.7
GRAFT-VERSUS-HOST DISEASE
All participants will be monitored closely for GVHD. All episodes of GVHD will be graded. 
Treatment of GVHD will be initiated per standard of care at the discretion of the investigator.
6.7.1
Acute GVHD
Acute GVHD will be graded by standard criteria (see 
Table 4)68. The occurrence of acute GVHD will be assessed at all study visits between 
transplant and Day 200 post-transplant using an acute GVHD assessment worksheet
(Appendix 6. Acute GVHD Assessment Worksheet ). Time to onset, organ involvement,
maximum grade and dates of treatment will be recorded. All suspected cases of acute GVHD 
greater than grade 1with skin involvement must be evaluated histologically by a skin biopsy.
Biopsies from other sites or organs will be obtained as clinically indicated. 


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Table 4. Consensus conference on clinical grading of acute GVHD
Grading of acute GVHD
Stage Skin
Liver
Intestinal Tract
1
Maculopapular rash <25% of 
body surface
Bilirubin 2-3 mg/dL
> 500 ml diarrhea/day
2
Maculopapular rash 25 - 50% 
of body surface
Bilirubin 3-6 mg/dL l
> 1000 ml diarrhea/day
3
Generalized erythroderma
Bilirubin 6-15 mg/dL
> 1500 ml diarrhea/day
4
Generalized erythroderma with 
bullous formation and 
desquamation
Bilirubin > 15 mg/dL
Severe abdominal pain, 
with or without ileus
6.7.2
Chronic GVHD
Chronic GVHD will be graded by NIH consensus criteria (see Table 5)69. The occurrence of 
chronic GVHD will be assessed in all participants, regardless of donor chimerism, at every 
study visit between 60 days and 2 years post-transplant using the Chronic GVHD Assessment 
Worksheet (see Appendix 7. Chronic GVHD Assessment Worksheet) . If a participant’s 
chronic GVHD persists beyond 2 years post-transplant or if he or she is diagnosed with 
chronic GVHD >2 years post transplant, the chronic GVHD Assessment Worksheet will be 
graded at every subsequent study visit until resolution of the episode or until the end of study 
participation.
Time to onset and date of biopsy proof (if any) will be recorded, as well as dates and types of 
treatment.
Grade Overall Clinical Grading of Severity of Acute GVHD
I
Stage 1 - 2 skin rash; no gut involvement; no liver involvement; no decrease in clinical 
performance
II
Stage 1 - 3 skin rash; stage 1 gut involvement or stage 1 liver involvement (or both); 
mild decrease in clinical performance
III
Stage 2 - 3 skin rash; stage 2 - 3 gut involvement or 2 - 4 liver involvement (or both); 
marked decrease in clinical performance
IV
Similar to Grade III with stage 2 - 4 organ involvement and extreme decrease in clinical 
performance


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Table 5. Clinical Grading of Chronic GVHD
Grade 
Overall Clinical Grading of Severity of Chronic GVHD based on Global Scoring of 
Individual Organs 
Mild
Involves only 1 or 2 organs or sites (except the lung) with no clinically significant 
functional impairment (maximum score of 1 in all affected organs or sites).
Moderate
x At least 1 organ or site with clinically significant but no major disability (maximum 
score of 2 in any affected organ or site) OR
x Three or more organs or sites with no clinically significant functional impairment 
(maximum score of 1 in all affected organs or sites).
Severe
x Major disability caused by chronic GVHD (score of 3 in any organ or site).
x A lung score of 2 or greater will also be considered severe chronic GVHD
6.8
ENGRAFTMENT SYNDROME
Engraftment syndrome will be assessed by the clinical study team based on the following 
criteria:
1.
Symptoms consistent with engraftment syndrome ( see below)
2.
A renal biopsy consistent with engraftment syndrome70
Symptoms:
The presence of all symptoms or symptoms 1, 2 and 4 will trigger a for-cause biopsy to 
confirm engraftment syndrome:
1. Temperature >39 deg C
2. Erythroderma/rash > 50% body surface
3. Non-cardiogenic pulmonary edema/pulmonary infiltrates
4. Creatinine >4
occurring in the time interval between day 0 and 1 week after recovery of ANC,without 
apparent other cause.
Biopsy:
Engraftment syndrome will be confirmed by the presence of histological features on renal 
biopsy consistent with this diagnosis70. These may include but are not limited to:
1. Prominent interstitial haemorrhage, congestion and edema
2. Acute tubular injury
3. Minimal or no interstitial inflammation or tubulitis
4. Occasional intracapillary mononuclear or polymorphonuclear cells


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7.
MECHANISTIC ASSAYS
7.1
RATIONALE FOR IMMUNE STUDIES
A battery of mechanistic assays will measure the immune response in pre- and post-transplant 
samples from patients undergoing combined kidney and HSCT. Results from these assays will 
correlate the degree of hematopoietic chimerism to the rate and success in achieving clinical 
tolerance. Additionally, these assays will identify potential biomarkers that are predictive of
patient success for immunosuppression withdrawal (ISW). Biomarkers may be most easily 
identified after immunosuppression withdrawal since the process of withdrawal or its outcome 
may well create or magnify differences between the two populations (immunosuppression 
withdrawal-success and immunosuppression withdrawal-failure). For the derived biomarker(s) 
to be clinically useful as an indicator, the differences must be appreciable in specimens 
collected at baseline, prior to attempted withdrawal.
The central hypothesis of the study is that stable or rising T cell chimerism greater than 50% at 
six months post transplantation is sufficient, but not necessary,for evidence of hypo-
responsiveness to host and donor antigens in vitro and for kidney allograft tolerance without 
maintenance immunosuppression. The exact mechanism(s) responsible for cyclophosphamide 
induced tolerance is not clear, but may include deletion of alloreactive clones and/or up-
regulation of regulatory T cells. It is additionally hypothesized that hematopoietic chimerism 
plus cyclophosphamide-induced tolerance affects B-cell responses producing a durable 
elimination of HLA antibody, thereby increasing the probability for successful kidney 
transplantation. It is unclear whether the B cell response is a direct response to the study 
regimen or is an indirect result of targeting T cells and the subsequent modulation of the 
adaptive immune response. The assays described below have been designed to test these 
hypotheses and monitor the patient’s overall immune response including changes in HLA 
antibody, peripheral blood cell subsets, immune response in biopsy samples, cellular 
activation, T and B cell alloreactivity and changes in gene expression etc.
7.2
PLANNED MECHANISTIC ASSAYS
7.2.1
Histological Assessments of Tolerance Mechanisms
Biopsies are used to identify the cause or process responsible for allograft dysfunction. In the 
case of patients with normally functioning allografts, biopsies are used to monitor structural 
integrity of long surviving allograft as well as response to the treatment. Renal biopsies are 
used to determine the absence of acute, intermediate and chronic rejection at intervals before 
and after immunosuppression withdrawal. Biopsies are thus necessary to rule out 
misclassification errors that would compromise subsequent attempts to identify “biomarkers 
of tolerance”.
Multiplex antigen labeling (immunohistochemistry IHC) by the ITN core pathology laboratory 
will be used to determine state of the allograft tissue during immunosuppression withdrawal at 
baseline and longitudinally in banked specimens. Suggested staining panels are described in
Table 6 with their key attributes and will be finalized once longitudinal banked specimens are 
available. We will test the hypothesis that the renal allograft itself plays a critical role in 
development and maintenance of tolerance. It may achieve this via gradual erosion of donor 
reactivity through inefficient stimulation by donor antigen presenting cells and a 
microenvironment hostile to effector and memory cell development and maintenance. In a 


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recently completed ITN liver transplant trial (ITN029ST), patients immunosuppression 
withdrawal status correlated with differences in C4d expression, JGT cell ratio, and MHC class 
II positive cell frequency71 (and unpublished observations). Additional ITN studies have 
demonstrated increased CD20, FoxP3, and CD3 gene expression in urinary cell sediment of 
tolerant kidney transplant recipients compared to SI or healthy controls (ITN507ST)72.
Detailed biopsy analysis using these markers and additional IHC panels proposed in Table 6
will evaluate factors associated with donor reactive cells, absence of inflammation (effector T
cells), and increased ratios of regulatory cells and effector to memory cell ratio in the 
transplant organ niche. The analyses will reflect systemic immune reactivity toward the 
allograft and the allograft’s ability to suppress the immune response. These findings will be 
interpreted in conjunction with other cellular, molecular, and immunologic assays performed 
within this trial. 
Table 6. Proposed Multiplex IHC Staining Panel
1.
Ki-67 (proliferation marker)
2.
CD68 (pan monocyte, macrophage, and dendritic cell marker)/ DC-SIGN (myeloid 
dendritic cell marker); check for infiltration of CD68+ macrophages (DC-SIGN 
negative) with ISW
3.
CD8 (T subset)
4.
CD20 (B cell infiltrates)
5.
NKG2D (NK, CD8+ activated, NK1.1+ T, some myeloid cells)
6.
CD34 (hematopoietic precursors, capillary endothelial cells)
7.
Myeloperoxidase (MPO, heme protein secreted by activated phagocytes)
8.
C4d (deposition suggests antibody mediated rejection; Is C4D deposition associated 
with failed ISW?) 
9.
CD3/CD45RO/CD45RA (ratio of naïve to memory T cells, do increased memory cells 
associate with failed IS withdrawal?)
10. CD3/Ȗį-Ȗį-2 (Is increased ratio of Ȗį-Ȗį-2 associated with ISW or tolerance)
11. HLA-DR/CD31 (is expression of HLA-DR associated with failed ISW?)
12. CD4/Tbet/GATA-3/IL-17/FoxP3 (regulatory T cells)
13. IL10/TGFȕHLA-DR (is increased IL-10 expression associated with transplant 
tolerance?) 
14. CD56/PD-1/CD3 (ratio of CD3+, CD56+, and PD-1+ lymphocytes and whether changes 
in NKT cells are associated with operational tolerance?)
15. CD5/CD19/CD27/IgG (ratios of naïve to memory B and B1:B2; do increased numbers 
of memory or regulatory B contribute to allograft acceptance or rejection?)
16. CD3/CD45RO/CD45RA (ratio of naïve to memory T cells, test if increased numbers of 
memory cells are associated with failed ISW?)
7.2.2
Gene Expression in peripheral blood and biopsies
Total RNA and miRNA may be extracted and batch processed from banked specimens for 
gene expression experiments. As patient numbers are insufficient for de novo gene discovery, 
only a-priori hypothesis based assays are feasible. Ongoing studies in peripheral blood of 
renal transplant recipients by the ITN and a European consortium identified 25 immune-
function related genes that differentiated operationally tolerant patients from patients receiving 


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continued immunosuppression72-74. Table 7 lists the 3 genes published by the ITN72 and an
additional 10 genes published from RISET. Expression of these genes, and additional genes 
reported with a strong association to tolerance, may be assessed using samples from this trial. 
Follow up work by the ITN demonstrates that the published ITN renal signature is broadly 
indicative of tolerance irrespective of the therapeutic intervention (unpublished data). It will 
therefore be informative to test if these reported tolerance signatures differ based on the 
presence or absence of hematopoietic chimerism and/or the induction regime used in this 
protocol.
Biopsy tissue, if available, for gene expression studies, may be compared pre- and post-
withdrawal and between immunosuppression withdrawal success and failure patients. A gene 
panel for biopsy and micro-RNA will be finalized before the start of the experiment based on 
current literature.
Drug effects may confound mechanistic observations; therefore, care needs to be taken in the 
interpretation of data comparing patients on immunosuppression versus those that have 
withdrawn from immunosuppression. This increases complexity given that greater and more 
frequent gene expression differences are typically seen between pre- and post-
immunosuppression withdrawal samples. Comparisons at baseline will be important since 
patients will be on the same or similar treatment regimens and a detectable signature at the 
baseline would allow selection of the patients most likely to be successful in 
immunosuppression withdrawal. Gene expression levels will be quantified in whole blood 
(and biopsy tissue if available) by RT-PCR in conjunction with samples from the ITN 
biorepository to assess comparative levels of the known “tolerance” signatures.
Table 7. Thirteen (13) of 25 genes used to derive renal tolerance signatures in 3 clinical 
studies
Genes*1
Description
ITN/ 
RISET
1.
IGKV1D
-13
Part of the immunoglobulin kappa variable cluster (IGKV@), 1D-13
involved in antigen binding*2.
ITN
2.
IGKV4-1
Part of the immunoglobulin kappa variable cluster (IGKV@), involved in 
antigen binding 4-1*2.
ITN
3.
IGLL1
Immunoglobulin lambda like polypeptide-1 is found on the surface of 
proB and preB cells. Gene encodes one of the surrogate light chain 
subunits of the preB cell receptor. 
ITN
4.
TLR5
Fundamental role in pathogen recognition (bacterial flagellins) and 
activation of innate immunity by recognition of pathogen-associated 
molecular patterns that are expressed on infectious agents. Acts via 
MYD88 and TRAF6, leading to NF-ț%DFWLYDWLRQ F\WRNLQHVHFUHWLRQ
and the inflammatory response. It is highly expressed in peripheral blood 
leukocytes, particularly monocytes.
RISET
5.
PNOC
Secreted protein that binds the opioid receptor–like receptor (OPRL1). 
Altered plasma levels have been reported in patients with various pain 
states, depression, and liver disease.
RISET 


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Genes*1
Description
ITN/ 
RISET
6.
SH2D1B
Single SH2-domain adapter that binds to specific tyrosine residues in 
cytoplasmic tail of SLAM and related receptors. Signals downstream of 
CD84 (upregulated in memory B). Stimulated B cells undergo early 
apoptotic events in the presence of SH2D1B. 
RISET 
7.
SLC8A1
Transmembrane protein that plays a fundamental role in Ca2+ refilling in 
the endoplasmic reticulum. In macrophages and monocytes, it restores 
Ca2+ signals that induce TNF-ĮSURGXFWLRQ
RISET 
8.
H3ST1
Member of the heparan sulfate biosynthetic enzyme family, is a rate-
limiting enzyme for synthesis of anticoagulant heparin
RISET
9.
FCRL1
Membrane protein, considered as a B cell co-receptor. Specifically 
expressed by mature B lineage cells, with higher expression in naive 
versus memory B cells.
RISET 
10.
FCRL2
Membrane protein, expressed preferentially by memory B cells.
RISET 
11.
MS4A1
B lymphocyte–specific, cell-surface molecule involved in B cell 
activation and differentiation.
RISET 
12.
TCL1A
Located downstream of the BCR signaling pathway. Overexpression 
prolongs naive B cell survival.
RISET 
13.
CD79B
Part of the B cell antigen receptor (BCR) complex
RISET 
*1: Twelve (12) additional genes from recent ITN507ST and RISET clinical study results are unpublished at 
this time and therefore not included in this table, but will likely be included in these studies.
*2: During lymphoid differentiation into Ab. producing cells, an IG kappa variable region is transcriptionally 
activated by rearrangement linking it to a kappa constant (C kappa) region gene that is transcribed prior to 
somatic mutation.
7.2.3
Multi-Parameter Flow Cytometry (MFC)
In an ITN study (FACTOR, ITN507ST) renal allograft tolerance was strongly associated with 
a baseline B cell signature using flow cytometry as well as gene expression assays72. The 
baseline flow cytometry B-cell signature was associated with elevated numbers of peripheral 
blood total B cells, and sub-populations of naive and transitional B cells in tolerant 
participants when compared to participants on standard immunosuppression.
In a recently completed, unpublished, frozen flow cytometry analysis of ITN507ST 
longitudinal samples, the tolerance associated B cell phenotype (higher frequencies of total B 
cell, naïve B cells and T1/ 2 transitional B cells when compared to the standard 
immunosuppression group), was reproducible and persisted longitudinally. The group on 
standard immunosuppression displayed higher frequencies of activated B memory cells. These 
results highlight a critical role for B cells in regulating alloimmunity.
Peripheral blood will be evaluated using flow cytometry on frozen/banked specimens to 
identify specific cells or cellular subsets that correlate with tolerance or other clinical 


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phenotype. Frozen flow, performed on banked specimens, has the advantage that it can be 
batch processed, allowing for the same reagents to be used for each sample. This typically 
generating tighter data sets for longitudinal samples. A second significant advantage of 
working with stored samples for clinical trials of significant duration is that the most relevant, 
up-to-date antibody staining panels can be used.
Longitudinal changes in T cells, B cells, NK cells, and monocytes will be evaluated using 
panels of antibodies directed at cell surface markers, nuclear factors, and intracellular 
cytokines. Cell populations of interest include lymphoid naïve, memory and transitional 
subsets, dendritic cell subsets (plasmacytoid, pDC and myeloid, mDC) and NK cells as well as 
activated lymphoid subsets. We hypothesize that cyclophosphamide will induce tolerance 
through the up-regulation of regulatory T cells. Antibody panels will be finalized and 
validated by the ITN flow core prior to starting assays. Examples of panels that may be used 
for analysis are listed in Table 8.
Table 8. Frozen/banked specimen flow cytometry panel design and list of suggested 
cellular antigens
Data analyses will identify differences in baseline immune phenotype between participants 
who succeed and those who failed immunosuppression withdrawal. Longitudinal changes in 
cell phenotype will be assessed during and after immunosuppression withdrawal and will be 
correlated with successful versus failed withdrawal. Frozen flow markers used previously to
analyze the B cell subset of ITN507ST participant longitudinal visits (see B cell panel above)
can be included in this study to allow direct comparison between these two trials. We expect 
that the flow panels and analyses planned in this study will expand our understanding of 
mechanisms of immune tolerance as well as compare results of combined kidney and bone 
marrow transplant (CKBMT, this trial) with kidney transplant alone (ITN507ST).
Function
1
2
3
4
5
6
7
8
9
10
11
12
T sub-set, 
T regs
CCR
7
CD12
7
CD25
CD3
CD45
RA
CD45
RO
CD4
HLA-
Dr
CD8
FoxP
3
Helio
s
Live/ 
Dead
B Cell sub-
set*1
CD3
CD19
IgD
CD27
CD24
CD38
MTG
CD21
CD23
CD95
9G4
Live/ 
Dead
NK cells
NKG
2D
CD12
2
NKp4
4
CD3
CD56
CD4
HLA-
Dr
CD8
CD16
Live/ 
Dead
B cell 
subsets
IgD
CD24
CD10
CD19
CD38
IgM
HLA-
Dr
CD27
CD20
Live/
Dead
B cell 
cytokines
IgD
CD24
CD20
IFNg
CD38
IgM
IL-10
CD27
TGFb
Live/
Dead
TLR5, DC 
and 
monocytes
CD11
c
CD12
3
CD20
CD3
CD56
CD16
HLA-
Dr
CD14
TLR5
Live/
Dead
*1: This panel was used to analyze B-cell subsets in banked baseline and longitudinal samples of ITN507ST and will be 
included in current trial to compare CKBMT patients with ITN507ST kidney transplant recipients.


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7.2.4
Alloreactive T Cell and Plasma Cytokine Assays
T cells are central mediators of anti-donor immunity that can cause direct (cell mediated) or 
indirect (through B cells and innate immune mechanisms) allograft damage through pre-
existing recipient alloreactive antibodies. Frequencies of proliferating alloreactive T cell can 
be measured in vitro when performed in limiting dilutions of mixed lymphocyte responses 
(MLR, CD4) or cell mediated lympholysis (CML, CD8) assays, done at pre-transplant and 
longitudinal intervals. In addition, T cell alloreactivity can also be measured by monitoring 
specific cytokine levels directly from the patient’s peripheral blood, ex vivo. These multiplex 
cytokine panels can be designed to measure the alloreactivity mediated by direct or indirect 
pathways and can define the phenotype of corresponding T cell (such as Th1 or Th2). Taken 
together, these in vitro and ex vivo assays provide a comprehensive readout of the alloreactive 
T cells and their state of tolerance throughout the immune system.
Whole PBMCs and isolated CD3+ T cells from real-time specimens will be compared in MLR 
and CML assays. These methods have been successfully developed and tested at the Human 
Immunology Core Lab of the JHU (Dr. Alan Hess, Director, Human Immunology Core 
Laboratory). Depletion of CD25+ (marker of lymphocyte activation) cells was shown to 
enhance third party CML and MLR responses in ITN036ST, a renal transplant study, and may 
be used in addition to above75. Donor specific suppressive activity of regulatory cells may be 
present pre-transplant or induced post-transplant and these can be measured in vitro. Donor 
antigens and/or third party stimulators will be cultured with recipient PBMC from pre-
transplant and post-transplant collections for these “stimulated suppression assays”. In 
addition, supernatants will be collected after five days of the mixed lymphocyte culture and 
analyzed for the levels of multiple cytokines using multiplex assays, e.g. the Bio-Rad Pro 
Human Cytokine 17-plex assay:(IL-1˟, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, 
IL-13, IL-17, G-CSF, GM-CSF, IFN-ˠ, MCP-1, MIP1˟, and TNF-˞).
These experiments, in conjunction with other mechanistic assays,will test whether tolerance 
in CKBMT recipients is due to deletion or anergy of donor reactive T cells. Results of 
cytokine assays from mixed lymphocyte cultures will be related with cytokine levels in 
patient’s serum/plasma taken at the onset of fever between days 2-5 after transplantation, 
effectively representing an in vivo MLR. The same cytokine panels, as those used for the MLR 
described above, will be tested in the patient’s serum/plasma samples. We will compare levels 
of individual cytokines between patients with, versus without, sustained donor hematopoietic 
chimerism to determine if there is a cytokine pattern that predicts donor hematopoietic 
engraftment and kidney allograft tolerance.
7.2.5
Alloreactive B Cell Assays and B cell Repertoire Analysis
Activation of alloreactive B cells or pathogenic alloantibodies can contribute to graft rejection 
either directly or indirectly, in addition to alloreactive T cell response. Antibodies to donor 
HLA antigens are a major barrier to transplant acceptance and characterizing these specific 
anti-HLA-antibody secreting B cells provides a method to identify humorally sensitized 
patients. These HLA-specific B cells can be identified/ phenotyped, enumerated and isolated 
by staining with HLA tetramers76,77.
B cells specific for mismatched HLA antigens (including: HLA-A1, A2, A11, A24, B7, or 
B15) will be quantified and isolated (by fluorescent PE labeled tetramers) from both the 
recipient and donor (real-time specimens) at pre-specified time points per the schedules of 
events (see appendices 1 through 5). Whenever possible, HLA tetramers for mismatched, 


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autologous, and third party antigens will be used. These methods have been successfully 
developed and tested at the Immunogenetics Laboratory of the JHU and will be carried out 
there75-77. Isolated HLA-specific B lymphocytes (and B lymphocytes isolated from PBMC) 
will be tested for their ability to respond to alloantigenic challenge by flow cytometric 
measurement of activation markers78 and may also be tested for possible deletion or expansion 
of particular clones using high throughput DNA sequencing of the B cell receptor 79.
Experimentally, multiplex PCR may be run to amplify rearranged BCR loci from the genomic 
DNA or cDNA. The amplified products that uniquely identify the V and J segments of the 
corresponding CDR3 region may then be sequenced on the Illumina platform and compared to 
germline sequences catalogued on public websites (e.g. International ImmunoGenetics 
database). The aligned sequences or groups of sequences reflect the B cell composition in the 
original sample and can be analyzed statistically by their V-J recombination frequency, BCR 
diversity, distribution of BCR clonotype frequencies that represent clonal expansions or 
deletions etc.
The ITN and Dr. Maria H. Fuentes (Kings College London and ITN collaborator) have 
consistently and independently72,80 seen expansion of B cell populations in renal transplant 
tolerant participants versus participants on SI. During these analyses, a consistent “B cell gene 
signature” was also observed from several immunoglobulin variable genes regions (IGKV1D-
13, IGKV4-1 and 4 other unpublished genes). The expanded B cells may preferentially use 
these variable region immunoglobulin gene segments. We may therefore, test for hyper 
expansion of the Ig variable regions (heavy and light chains) as well as somatic mutations 
within such clonal expansions that may represent clonal cell lineage. These analyses may be 
done using longitudinal samples, and the results correlated with other mechanistic data as well 
as clinical outcomes. These BCR sequencing experiments would need to be combined with 
other ITN renal transplant studies such as ITN507ST or ITN036ST to generate sufficient 
sample size.
7.2.6
Blood Chimerism
There is increasing evidence to support that patients with hematopoietic chimerism are 
tolerant of solid organ allografts from the same donor and can be weaned off 
immunosuppression even when chimerism is transient. In a recent renal transplant study at 
ITN, 4 out of 5 patients with end stage renal disease (ESRD) that received CKBMT after non-
myeloablative regimen had transient hematopoietic chimerism (21 days) and were 
successfully weaned off all immunosuppression25. In longitudinal analysis of these 5 patients 
along with 5 additional patients treated similarly (ITN036ST), 7 out of the total 10 patients 
with transient hematopoietic chimerism were successfully weaned off drugs although 
idiopathic capillary leak syndrome was observed in 9 of the 10 patients. It is unclear whether 
sustained hematopoietic chimerism will result in durable renal allograft tolerance.
A similar treatment regimen as that described in this protocol was shown to achieve full, or 
near full, donor chimerism in 5 (also completely off immunosuppression) of 19 patients with 
sickle cell anemia or thalassemia, receiving transplantation from HLA matched or HLA-
haploidentical first degree relatives81. For 5 of 19 patients that experienced graft failure in that 
study, donor chimerism was lost early after transplantation. Furthermore, durable, but not full, 
hematopoietic chimerism was achieved in >50% of the patients. We expect to induce durable 
chimerism in most of the patients transplanted during this trial, and at least temporary 
chimerism.


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Experimentally, a buccal swab will be collected from the donor and recipient pre-transplant to 
measure degree of donor chimerism at the Pathology Molecular Diagnostics Lab of the JHU 
labs. Following transplantation, peripheral blood from transplant recipients and CD3+ cells 
separated from peripheral blood using the RoboSep® automated instrument (StemCell 
Technologies) will be used to measure the degree of chimerism. Fifteen microsatellite markers 
and the amelogenin locus will be amplified by PCR using AmpFlSTR® Identifiler PCR 
Amplification Kit (Applied Biosystems). The resulting PCR products will be analyzed by 
capillary electrophoresis and peak heights of the informative alleles compared to calculate a 
percentage engraftment82,83. The true limit of detection for an individual reaction is both locus 
and PCR dependent. The formal limit of detection is 5%. A micro chimerism assay with 
improved sensitivity is also being developed by this lab and may be adopted if validated prior 
to the start of this study. The degree and durability of chimerism will be correlated with organ 
allograft tolerance. Chimerism levels may be correlated with T and B cell phenotype, 
alloreactivity, and possibly gene expression to identify predictors and mechanisms of 
tolerance.
7.2.7
Anti-donor HLA Alloantibody and Flow Cross Match
HLA Abs have been strongly implicated as a risk factor in kidney transplant recipients, their 
presence at baseline may preclude successful immunosuppression withdrawal. Moreover, after 
initiation of immunosuppression withdrawal, monitoring the increasing breadth and/or 
strength of alloantibodies particularly if they are directed against donor antigens, may be 
important as this may signal the presence of an immune response that will be deleterious to 
long-term allograft histology.
Serum samples collected from participants will therefore be evaluated for alloantibodies,
including donor-specific alloantibodies (DSA), in real time at the Immunogenetics Laboratory 
of the JHU. Assessments will be performed prior to immunosuppression withdrawal and at 
multiple time points during and after immunosuppression withdrawal. An initial screen will 
determine if the participant has detectable alloantibodies. If the screening assay is positive, 
antibody specificities will then be determined using beads coated with single HLA-antigens.
HLA typing of donor and recipient will be necessary to assign donor specificity. Whole blood 
will be collected from all donors and recipients for HLA typing. Details of HLA typing 
assessment are provided below.
The flow cytometry crossmatch (FCXM) is a highly sensitive method for confirming the 
presence of DSA. FCXM testing will be performed selectively, that is, only after observing 
DSA in participant serum. Experimentally, three color FCXM is performed by incubating 
donor cells or surrogates with recipient serum followed by addition of a fluoresceinated 
(FITC) goat, anti-human polyclonal immunoglobulin. A phycoerythrin (PE) labeled 
monoclonal antibody that detects B cells along with chlorophyll protein (PerCP)-conjugated 
monoclonal antibody that detects T cells will be analyzed by flow cytometry and expressed as 
positive or negative based on a shift in median channel fluorescence intensity (MFI) of the test 
serum with respect to negative control. This method can also be adapted to assess anti-
monocyte Abs if needed. If necessary, pronase treatments to reduce non-specific background 
staining or dithiothreitol (DTT) treatments to eliminate interference from IgM class Abs can 
be included.


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Results from Alloantibody and FCXM may be used to evaluate: 1) whether de novo anti-HLA 
alloantibody correlates with successful IS withdrawal or failure, 2) whether donor-specific 
alloantibody (DSA) correlates with IS withdrawal success or failure, 3) compare temporal 
patterns of recipient anti-HLA antibodies between IS withdrawal success and failure 
participants; and longitudinally with chimerism durability, 4) whether baseline anti-HLA 
antibody and/or donor-specific anti-HLA antibody profiles classify participants with donor 
chimerism strength and durability as well as with other clinical outcomes 5) determine 
relationship between serum HLA alloantibody strength and tissue immune histopathological 
measurements, such as complement pathway products or activated lymphocytes.
7.2.8
HLA Typing
HLA typing information will be collected as standard of clinical care for transplantation. 
Information will be used to assess presence of DSA and will be done on early/ baseline 
specimens collected in this trial from both allograft donors and recipients. Whole blood 
collected from participants (recipients and donors) will be used to perform sequence-based 
HLA-typing (HLA-A, B, C, DRB1, DRB3-5, DQA1, DQB1, DPB1) in real time at the 
Immunogenetics Laboratory of the JHU.
7.3
OVERVIEW OF DATA ANALYSIS
This study is designed to assess ability of a novel conditioning regimen, bone marrow 
transplantation and high dose PT/Cy to enable discontinuation of immunosuppressive therapy,
as well as to assess safety in haploidentical living related donor renal transplant recipients. The 
study size (total of 6 participants) is limited by considerations of safety of the novel testing 
regimen that may include risk of graft-versus-host disease (GVHD), graft rejection, graft loss, 
infection and malignancy etc. The accrual of six participants is planned in two stages (3:3) 
with a pre-specified interim safety analysis. If safety of conditioning regimen is deemed 
unacceptable, the experimental pre-transplant conditioning regimen may be omitted. The 
change, if it occurs, will effectively create two participant subgroups (with and without 
conditioning regimen), and may impact the scope of data analysis. Based on the nature of the 
study, the primary mechanistic data analysis strategy is individual participant-based 
longitudinal profiling which includes graphic plotting and descriptive statistics etc. 
Exploratory analysis is also planned to discover potential molecular and cellular patterns that 
might be linked to withdrawal outcome, explained below.
7.3.1
Individual based longitudinal profiling
Mechanistic samples from patients enrolled in this study will be assayed as described in the 
individual sections above with the aim to identify specific cell phenotypes, donor chimerism, 
intracellular responses and /or gene sets (tissue and/or peripheral blood) that correlate with 
successful immunosuppression withdrawal or predict failure thereof. These assay data may 
also be assessed in support of study safety evaluation of the experimental pre-transplant 
regimen (such as by flow cytometry or alloreactive assays). Graphic plots will facilitate 
longitudinal monitoring of relevant cellular populations, immune cell repertoire, donor 
chimerism, DSA and gene expression etc., in conjunction with the treatment process, adverse 
events and clinical outcomes. Descriptive statistics will be provided as appropriate.


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7.3.2
Exploratory analysis
The study participants may be grouped based on primary clinical outcome (withdrawal 
success vs. failure) and/or degree of chimerism. However, the classification variables will be 
obtained at the end of the study (withdrawal outcome) or after marrow transplant (donor 
chimerism) rather than pre-defined before participant recruitment. Hence, the limited and 
uncertain sample size of these groups limits the use of formal statistical hypothesis testing 
approaches. The primary objective of exploratory analysis is to discover molecular, cellular, 
and immunogenetic patterns among the six participants at clinical milestones during the study 
that may provide insights into potential association with donor chimerism and/ or withdrawal 
outcome. Pattern recognition methods including hierarchical clustering and principal 
component analysis may be used for the analysis. The exploratory analysis will be conducted 
on individual assay datasets (flow cytometry, gene expression, histological assessment assays, 
etc.) as well as integrated multi-assay data.
7.4
FUTURE/UNPLANNED STUDIES
Specimens stored during the trial may be used in future assays to reevaluate biological 
responses as research tests are developed over time. Additionally, samples may be used for 
assays/ experiments outside the scope of this proposal, such as investigation of differences in 
the T cell receptor (TCR) repertoire as evaluated by sequencing, proteomics or other assays
that may emerge and be compelling. Note that with only 6 participants in this study, it is likely 
that any future work would be in conjunction with other studies. Re-evaluations or new assays 
will only be performed on samples of participants who have consented for future research. The 
ITN sample sharing policy will apply for the provision of samples to study or outside 
investigators (www.immunetolerance.org). Any research conducted using stored samples for 
future use may also need appropriate regulatory approval, such as Institutional Review Board 
per the study consent.
7.5
SPECIMEN LOGISTICS
The clinical site will be trained in collection, processing, shipment, and tracking of 
mechanistic research specimens. The ITN will monitor specimen quality, shipping compliance 
etc. and retrain the clinical site if not producing optimum quality mechanistic samples. Johns 
Hopkins University clinical site will process all mechanistic samples according to the ITN 
standard procedures and use the ITN Specimen Tracking System (STS) software to identify 
and track all mechanistic specimens. The site is required to have its own calibrated laboratory 
equipment for use in standard ITN procedures, such as a centrifuge for spinning primary blood 
tubes, a micropipettor to aliquot specimens, and -70 or 80° C freezer to store frozen specimens 
until they can be shipped. JHU will use an appropriate courier service for shipping specimens 
to ITN repositories/ core labs, per ITN standard procedures. All shipping will conform to 
Department of Transportation regulations (49 CFR 173.199) for Diagnostic Specimens.
7.6
SPECIMEN TRACKING PROCEDURES
The ITN will track all mechanistic specimens until the final disposition of all material is 
known. Samples will remain in either JHU or ITN repositories until used for assays or 
destroyed.


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7.7
SPECIMEN STORAGE
Samples will be stored under specific conditions to maintain long-term sample integrity, as 
well as specimen tracking from receipt to shipment to alternate locations. The ITN specimen 
tracking system will be used to track date of shipment, location shipped to, carrier, items 
shipped, amount shipped, barcode numbers, protocol number and associated comments about 
each individual specimen. Storage temperature, location, processing, aliquoting, and 
freeze/thaw events may also be recorded.
If the study subject allows storage for future studies, the subject’s specimens will be stored 
indefinitely. The subject can change their mind at any time and have their stored specimens 
destroyed by notifying the study physician in writing. In such cases, the site coordinator would 
send all requests for sample destruction to the ITN. The site will receive confirmation that the 
specimen was destroyed as requested. If the subject’s samples have already been analyzed, 
then the data will be used as part of the overall analysis. The subject can only request to have 
samples destroyed if they still exist, i.e. have not already been used in an experiment.
Specimens can only be transferred from JHU or the ITN repository to another destination with 
appropriate authorization per ITN standard procedures. Some items checked prior to 
authorization include: purpose for accessing/transferring the specimen (within study assay as 
defined by the protocol or future studies), evaluation of subject consent for the purpose 
provided, verification of specimen identifiers, and quality and quantity of the specimen.
8.
ADVERSE EVENTS
8.1
OVERVIEW
The Principal Investigator is responsible for the detection and documentation of events 
meeting the criteria and definition of an AE (adverse event) or SAE (serious adverse event) as 
described in sections 8.2.1 and 8.2.3 in this protocol. All AEs and SAEs will be recorded in 
the source documents and on the appropriate electronic CRF(s). All data will be reviewed 
periodically by the DSMB, which may provide recommendations to NIAID about 
withdrawing any participant and/or terminating the study because of safety concerns.
Adverse events that are classified as serious according to the definition of health authorities 
must be reported promptly and appropriately to the NIAID, ITN, Principal Investigators in the 
trial, IRBs and health authorities. This section defines the types of AEs and outlines the 
procedures for appropriately collecting, grading, recording and reporting them. Information in 
this section complies with 21CFR 312; ICH Guideline E2A: Clinical Safety Data 
Management: Definitions and Standards for Expedited Reporting; and ICH Guideline E-6: 
Guidelines for Good Clinical Practice; and applies the standards set forth in the National 
Cancer Institute (NCI), Common Terminology Criteria for Adverse Events, Version 4.0 (May 
28, 2009).


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8.2
DEFINITIONS
8.2.1
Adverse Event
An AE is any occurrence or worsening of an undesirable or unintended sign, symptom, 
laboratory finding, or disease that occurs during participation in the trial. An AE for the
recipient will be followed until it resolves or until 30 days after the recipient terminates from 
the study, whichever comes first. An AE for the donor will be followed until it resolves or 
until 48 hours after the final donor study visit, whichever comes first. All AEs will be reported 
as specified in section 8.3.2.1 whether they are or are not related to disease progression or 
study participation.
8.2.2
Adverse Reaction and Suspected Adverse Reaction
An adverse reaction means any adverse event caused by a drug. Adverse reactions are a subset 
of all suspected adverse reactions for which there is reason to conclude that the drug caused 
the event.
Suspected adverse reaction (SAR) means any adverse event for which there is a reasonable 
possibility that the drug caused the adverse event. For the purposes of safety reporting, 
‘reasonable possibility’ means there is evidence to suggest a causal relationship between the 
drug and the adverse event. A suspected adverse reaction implies a lesser degree of certainty 
about causality than adverse reaction, which means any adverse event caused by a drug (21 
CFR 312.32(a)).
8.2.3
Serious Adverse Event or Serious Suspected Adverse Reaction
An AE or SAR is considered “serious” if, in the view of either the Investigator or NIAID
Medical Monitor, it results in any of the following outcomes (21 CFR 312.32(a)):
x
Death: A death that occurs during the study or that comes to the attention of the 
Principal Investigator during the protocol-defined follow-up period as defined in
section 8.3.2.2 must be reported whether it is considered treatment-related or not.
x
A life-threatening event: An AE or SAR is considered “life-threatening” if, in the 
view of either the Investigator or NIAID Medical Monitor, its occurrence places the 
subject at immediate risk of death. It does not include an AE or SAR that, had it 
occurred in a more severe form, might have caused death.
x
Inpatient hospitalization or prolongation of existing hospitalization.
x
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions.
x
An event that requires intervention to prevent permanent impairment or damage. An 
important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical 


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judgment, it may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the outcomes listed above.
x
Congenital anomaly or birth defect.
8.2.4
Unexpected Adverse Events
A SAR is considered “unexpected” if it is not identified in the package insert and/or drug 
label, investigator brochure, or protocol, or is not listed at the specificity, or severity that has 
been observed (21 CFR 312.32(a)).
8.3
COLLECTING AND RECORDING ADVERSE EVENTS
8.3.1
Methods of Collection
Adverse events for recipients will be collected from the time the participant receives the first 
dose of study medication as specified in section 8.3.2. Adverse events will be followed until 
the time an event is resolved or until 30 days after the recipient completes or terminates from 
the study, whichever comes first. The methods for collecting AEs will include:
x
Observing the participant.
x
Questioning the participant in an objective manner.
x
Receiving an unsolicited complaint from the participant.
An abnormal value or result from a clinical or laboratory evaluation (e.g., a radiograph, an 
ultrasound, or an electrocardiogram) can also indicate an AE if it is determined by the 
Investigator to be clinically significant. If this is the case, it must be recorded in the source 
document and as an AE on the appropriate AE form(s). The evaluation that produced the value 
or result should be repeated until that value or result returns to normal or can be explained and 
the participant’s safety is not at risk.
8.3.2
Severity of Adverse Events to be Collected
8.3.2.1
Adverse Events
All AE grades will be defined per NCI-CTCAE version 4.0 criteria unless otherwise specified.
Recipient
With the following exceptions, all AEs of grade 3 or higher will be collected from the time at 
which the participant receives the first dose of ATGuntil the time the participant completes or 
prematurely withdraws from the study:
x
During the first 30 days post-bone marrow transplant, expected hematological 
abnormalities associated with the non-myeloablative conditioning regimen, such as 
cytopenias, will not be collected as AEs. Rather, participants will undergo frequent 
complete blood counts as specified in the SOE (see Appendix 2. Post-Transplant Through


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Evaluation for Withdrawal Eligibility). Delayed hematological recovery will be captured 
as an AE as follows:
o
Delayed ANC recovery is defined as failure to achieve neutrophil recovery by day 30 
following the post-transplant neutrophil nadir. The neutrophil nadir is defined as the 
first day post-transplant on which the ANC is below 500 per μL.
o
Neutrophil recovery is defined as greater than or equal to 500/uL, the first of three 
consecutive measurements over at least two days. 
o
Delayed platelet recovery is defined as failure of recovery by post-transplant day 30.
o
Platelet recovery is defined as greater than or equal to 20,000/uL with no history of 
platelet transfusions over the prior seven days.
x
Hematological abnormalities will be collected as AEs per NCI-CTCAE v. 4.0 criteria once 
participants have achieved platelet and neutrophil recovery.
x
All episodes of renal allograft rejection as defined in section 3.6.2 and GVHD as defined 
in section 6.7, regardless of grade, will be collected as AEs.
Donor
Only AEs related to bone marrow harvest and study-mandated blood draws will be collected 
for the donor.
8.3.2.2
Serious Adverse Events
Serious AEs, regardless of grade, will be collected from the time at which the recipient 
receives the first dose of ATG until 30 days after the recipient completes, or prematurely 
terminates, from the study. Serious AEs, regardless of grade, will be collected from the time 
the donor undergoes the first study-mandated blood draw until 48 hours after the final donor 
study visit.
8.3.3
Recording Method
8.3.3.1
Adverse Events
Throughout the study, the Investigator will record AEs on the appropriate eCRF regardless of 
their relation to study participation. The Investigator will treat participants experiencing AEs
appropriately and observe them at suitable intervals until their symptoms resolve or their 
status stabilizes.
8.3.3.2
Serious Adverse Events
Serious AEs will be recorded on the AE eCRF and on the SAE eCRF, and health authorities 
will be notified as outlined in section 8.5.


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8.4
GRADING AND ATTRIBUTION OF ADVERSE EVENTS
8.4.1
Grading Criteria
The study site will grade the severity of AEs experienced by study participants according to 
the criteria set forth in the National Cancer Institute’s Common Terminology Criteria for 
Adverse Events Version 4.0 (published May 28, 2009). This document (referred to herein as 
the “NCI-CTCAE v. 4.0 manual”) provides a common language to describe levels of severity, 
to analyze and interpret data, and to articulate the clinical significance of all AEs.
Severity of adverse events will be graded on a scale from 1 to 5 according to the following 
standards in the NCI-CTCAE v. 4.0 manual:
x
Grade 1 = mild adverse event.
x
Grade 2 = moderate adverse event.
x
Grade 3 = severe and undesirable adverse event.
x
Grade 4 = life-threatening or disabling adverse event.
x
Grade 5 = death.
For additional information and a printable version of the NCI-CTCAE v. 4.0 manual, go to 
http://ctep.cancer.gov/reporting/ctc.html.
8.4.2
Attribution Definitions
Adverse events will be categorized for their relation to one or more of the following:
x
bone marrow transplantation
x
study medications
x
immunosuppression withdrawal including withdrawal from mycophenolate 
compounds, prednisone and tacrolimus
x
other protocol-directed study procedures
The Principal Investigator will do the initial determination of the relation, or attribution, of an 
AE to study participation and will record the initial determination on the appropriate eCRF 
and/or SAE reporting form. The relation of an AE to study participation will be determined 
using definitions in Table 9. Final determination of attribution for safety reporting will be 
decided by NIAID Medical Monitor.


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Table 9. Attribution of adverse events
Code
Descriptor
Relationship (to primary investigational product 
and/or other concurrent mandated study therapy)
Unrelated Categories 
1
Unrelated
The adverse event is clearly not related.
2
Unlikely
The adverse event is unlikely related.
Related Categories
3
Possible
The adverse event has a reasonable possibility 
to be related; there is evidence to suggest a 
causal relationship.
4
Probable
The adverse event is likely related.
5
Definite
The adverse event is clearly related.
8.5
REPORTING SERIOUS ADVERSE EVENTS
8.5.1
Reporting SAEs to the IND Sponsor
The following process for reporting a SAE ensures compliance with 21CFR 312 and ICH 
guidelines. After learning that a participant has experienced an SAE, the Investigator or 
designee will report the SAE via the electronic SAE report form (SAE eCRF) within 24 hours 
of becoming aware of the event to the sponsor. Initial SAE eCRF should include as much 
information as possible, but at a minimum must include the following:
x
AE term.
x
Relationship to study-mandated therapy and procedures.
x
Reason why the event is serious.
x
Supplementary CRF pages that are current at the time of SAE reporting: medical 
history, concomitant medications, demographics, study drug administration, death.
As additional details become available, the SAE eCRF should be updated and submitted to the 
sponsor. Every time the SAE eCRF is submitted, it should be electronically signed by the 
Investigator or sub-Investigator.
For additional information regarding SAE reporting, contact Rho Product Safety:


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8.5.2
Reporting SAEs to Health Authorities
After the SAE has been reported by the site Investigator and assessed by the IND sponsor, the 
IND sponsor must report an event to the appropriate health authorities based on the following 
criteria:
x
Standard reporting. This option applies if the AE is classified as one of the 
following and will be reported in the IND annual report:
o
Serious, expected, suspected adverse reactions (see sections 8.2.3 and 8.2.4).
o
Serious and not a suspected adverse reaction (see section 8.2.3).
x
Expedited reporting. This option applies if the AE is considered serious, related to 
study medications described in section 5, and unexpected per section 8.2.4.
These events must be reported by the sponsor to the appropriate health authorities 
within 15 calendar days; fatal or life-threatening events must be reported within 7 
calendar days.
8.5.3
Reporting SAEs to IRBs and Ethics Committees
All Investigators must report IND Safety Reports and related safety information to their 
respective IRBs or Ethics Committees as mandated by them.
8.5.4
Reporting SAEs to the DSMB
The NIAID and ITN will provide the DSMB with data of all SAEs on an ongoing basis.
8.5.5
Reporting Pregnancy
The Investigator should be informed immediately of any pregnancy in a study participant or a 
partner of a study participant and all available pregnancy information should be entered into 
the electronic data capture (EDC) system within 24 hours of becoming aware of the event. The 
Investigator should counsel the participant and discuss the risks of continuing with the 
pregnancy and the possible effects on the fetus. Monitoring of the participant should continue 
until the conclusion of the pregnancy. Follow-up information detailing the outcome of the 
pregnancy should be entered into the EDC system as it becomes available. Any premature 
termination of the pregnancy will be reported.
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or 
elective termination of a pregnancy for medical reasons will be recorded as an AE or SAE as 
described in sections 8.2.1 and 8.2.3.
9.
STATISTICAL CONSIDERATIONS AND ANALYTICAL PLAN
9.1
ANALYSIS SAMPLES
Safety sample: All participants who provide informed consent for study participation and 
receive any form of study therapy, including ATG, cyclophosphamide, fludarabine, 
tacrolimus, prednisone, or mycophenolate compounds.


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Per protocol (PP) sample: All participants who receive a combined bone marrow and renal 
allograft transplant and at least one dose of high dose PT/Cy.
9.2
ANALYSIS PLAN
The principal features of the plan for statistical data analysis are outlined in this protocol and 
will be described in greater detail in the statistical analysis plan (SAP).
Analysis of study data will be conducted to address all objectives of the trial and other 
interrelationships among all data elements of interest to the Investigators and of relevance to 
the objectives of the study. Analyses by gender, age, and race/ethnicity, as appropriate, are 
also planned.
Primary analysis of treatment effect will be conducted on the PP sample.
9.2.1
Primary Endpoint
The primary endpoint is defined in section 3.5.1.
The primary endpoint will be analyzed using the PP sample and descriptively summarized 
with a point estimate and two-sided, 95% exact binomial confidence interval. All 
participants who fail to complete immunosuppression withdrawal, regardless of reason, will be 
considered to have failed the primary endpoint. Participants who restart immunosuppression 
or experience acute rejection after 52 weeks off immunosuppression will be considered 
successfully withdrawn for the purpose of the primary endpoint.
A sensitivity analysis of the primary endpoint will be performed comparing sensitized versus 
unsensitized participants.
9.2.2
Secondary Endpoints
x
The secondary endpoints are defined in section 3.5.2. These will be analyzed using the 
PP sample.
x
Proportion endpoints will be descriptively summarized using frequency tables with 
frequencies and percentages and 95% exact binomial confidence intervals.
x
Time-to-event endpoints will be assessed using Kaplan-Meier survival estimates and 
associated two-sided 95% confidence intervals. Subjects lost due to competing risks 
or being lost to follow-up will be censored at the time of occurrence of these events.
x
Continuous endpoints will be summarized using descriptive statistics (n, mean, 
standard deviation, median, minimum, maximum).
x
Categorical variables will be summarized using counts (n) and percentages (%).
9.2.3
Safety Analysis
The safety analyses will be performed using the safety sample. All adverse events will be 
classified by body system and preferred term according to the MedDRA dictionary. Frequency 


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tables by category of event (e.g. serious, related to study therapy) and by NCI-CTCAE v. 4.0
grade will be presented. Selected laboratory values will be summarized and displayed 
graphically.
9.2.3.1
Donor Safety Analysis: Selected Adverse Events
The incidence of the following selected donor AEs related to study procedures will be 
summarized:
x
Pain, bleeding and infection associated with the bone marrow harvest.
x
The requirement for blood product transfusion.
9.2.4
Baseline Characteristics and Demographics
Baseline and demographic characteristics will be collected for all transplanted participants.
Demographic data will include age, race, sex, ethnicity, body weight and height; these data 
will be presented in the following manner:
x
Continuous data (i.e., age, body weight and height) will be summarized descriptively 
by mean, standard deviation, median, and range.
x
Categorical data (i.e., sex and race) will be presented as frequencies and percentages.
9.2.5
Medical History
Medical history within the past 12 months, including the existence of current signs and 
symptoms, will be collected for each body system and summarized in tabular form.
9.2.6
Use of Medications
All prophylactic antibiotics, immunosuppressive medications, and steroids taken by or 
administered to study participants throughout the study will be collected.
9.2.7
Study Completion
The percentage of participants who complete the study, losses to follow-up, times to lost to 
follow-up and reasons for termination (e.g., adverse events) will be presented.
9.3
SAMPLE SIZE
Given a sample size of 6 for this pilot study, there is insufficient power for hypothesis testing. 
Thus the analyses will be primarily descriptive. For example, if 5 participants successfully 
withdraw from immunosuppression, the point estimate and 95% exact binomial confidence 
interval for the proportion of participants successfully withdrawn will be 0.83 (0.36, 0.996). If
3 subjects are successfully withdrawn, the resulting estimate and 95% confidence interval will 
be 0.50 (0.12, 0.88).


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9.4
INTERIM ANALYSIS
No formal interim efficacy analysis is planned for this study. The DSMB will receive periodic 
safety reports on participants who have received a transplant along with all study therapy 
discontinuation cases. The DSMB may request modifications to the protocol based on its 
review of the findings.
Additionally, a safety review will be performed after the third transplanted participant has 
been followed for 3 months post-transplant, as described in section 3.1.4.
9.5
REPORTING DEVIATIONS FROM THE ORIGINAL STATISTICAL 
PLAN
The principal features of both the study design and the plan for statistical data analysis are 
outlined in this protocol and in the statistical analysis plan (SAP). Any change in these 
features requires either a protocol or an SAP amendment, which is subject to review by study 
sponsor(s), and the health authorities. These changes will be described in the final study report 
as appropriate.
10.
ACCESS TO SOURCE DATA/DOCUMENTS
The investigational sites participating in this study will maintain the highest degree of 
confidentiality permitted for the clinical and research information obtained from participants 
in this clinical trial. Medical and research records should be maintained at each site in the 
strictest confidence. However, as a part of the quality assurance and legal responsibilities of an 
investigation, the investigational sites must permit authorized representatives of the ITN, 
sponsor, and health authorities to examine (and to copy when required by applicable law) 
clinical records for the purposes of quality assurance reviews, audits, and evaluation of the 
study safety and progress. Unless required by the laws permitting copying of records, only the 
coded identity associated with documents or other participant data may be copied (and any 
personally identifying information must be obscured). Authorized representatives as noted 
above are bound to maintain the strict confidentiality of medical and research information that 
may be linked to identified individuals. The investigational sites will normally be notified in 
advance of auditing visits.
11.
QUALITY CONTROL AND QUALITY ASSURANCE
The Investigator is required to keep accurate records to ensure that the conduct of the study is 
fully documented. The Investigator is required to ensure that all CRFs are completed for every 
participant entered in the trial.
The sponsor is responsible for regular inspection of the conduct of the trial, for verifying 
adherence to the protocol, and for confirming the completeness, consistency, and accuracy of 
all documented data.
The CRFs will be completed online via a web-based electronic data capture (EDC) system that 
has been validated and is compliant with Part 11 Title 21 of the Code of Federal Regulations.


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Study staff at the site will enter information into the electronic CRFs, and the data will be 
stored remotely at a central database. Data quality will be ensured through the EDC system’s 
continuous monitoring of data and real-time detection and correction of errors. All elements of 
data entry (i.e., time, date, verbatim text, and the name of the person performing the data 
entry) will be recorded in an electronic audit trail to allow all changes in the database to be 
monitored and maintained in accordance with federal regulations.
12.
ETHICAL CONSIDERATIONS AND COMPLIANCE WITH GOOD CLINICAL 
PRACTICE
12.1
STATEMENT OF COMPLIANCE
This trial will be conducted in compliance with the protocol, current Good Clinical Practice 
(GCP) guidelines—adopting the principles of the Declaration of Helsinki—and all applicable 
regulatory requirements.
Prior to study initiation, the protocol and the informed consent documents will be reviewed 
and approved by the sponsor and an appropriate ethics review committee or institutional 
review board (IRB). Any amendments to the protocol or consent materials must also be 
approved by the Sponsor, the IRB and submitted to FDA before they are implemented.
12.2
INFORMED CONSENT
The informed consent form is a means of providing information about the trial to a prospective 
participant and allows for an informed decision about participation in the study. All 
participants (or their legally acceptable representative) must read, sign, and date a consent 
form before participating in the study, taking the study drug, and/or undergoing any study-
specific procedures. If a participant does not speak and read English, the consent materials 
must be translated into the appropriate language.
The informed consent form must be updated or revised whenever important new safety 
information is available, whenever the protocol is amended, and/or whenever any new
information becomes available that may affect participation in the trial.
A copy of the informed consent will be given to a prospective participant for review. The 
attending physician, in the presence of a witness, will review the consent and answer 
questions. The participant will be informed that participation is voluntary and that he/she may 
withdraw from the study at any time, for any reason.
12.3
PRIVACY AND CONFIDENTIALITY
A participant’s privacy and confidentiality will be respected throughout the study. Each 
participant will be assigned a sequential identification number. This number, rather than the 
participant’s name, will be used to collect, store, and report participant information.


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13.
PUBLICATION POLICY
The ITN policy on publication of study results will apply to this study. Authorized participants 
may find details regarding the policy statement on the ITN internet website at 
http://www.immunetolerance.org.


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14.
REFERENCES
1.
Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal 
allograft pathology: updates and future directions. American journal of 
transplantation : official journal of the American Society of 
Transplantation and the American Society of Transplant Surgeons. 
2008;8(4):753-760.
2.
association CCftnyh. Nomenclature and Criteria for the Diagnosis of 
Diseases of the Heart and Great Vessels Ninth Edition. 1994.
3.
Brennan. Long-term trends in allograft survival. Adv Chronic Kidney 
Dis. 2006 Jan;13(1):7-11.
4.
Marcen R. Immunosuppressive Drugs in Kidney Transplantation: 
Impact on Patient Survival, and Incidence of Cardiovascular Disease, 
Malignancy and Infection.[Review]. Drugs. November 12, 
2009;69(16):2227-2243.
5.
Owen RD. Immunogenetic Consequences of Vascular Anastomoses 
between Bovine Twins. Science. 1945;102(2651):400-401.
6.
Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of 
foreign cells. Nature. 1953;172(4379):603-606.
7.
Sharabi Y, Sachs DH. Mixed chimerism and permanent specific 
transplantation tolerance induced by a nonlethal preparative regimen. 
The Journal of experimental medicine. 1989;169(2):493-502.
8.
Anderson D, Billingham RE, Lampkin GH, Medawar PB. The use of 
skin grafting to distinguish between monozygotic and dizygotic twins in 
cattle. Heredity. 1951;5(3):379-397.
9.
Koenecke C, Hertenstein B, Schetelig J, et al. Solid organ 
transplantation after allogeneic hematopoietic stem cell transplantation: 
a retrospective, multicenter study of the EBMT. American journal of 
transplantation : official journal of the American Society of 
Transplantation and the American Society of Transplant Surgeons. 
2010;10(8):1897-1906.
10.
Eto M, Mayumi H, Tomita Y, et al. Specific destruction of host-reactive 
mature T cells of donor origin prevents graft-versus-host disease in 
cyclophosphamide-induced tolerant mice. J Immunol. 
1991;146(5):1402-1409.
11.
Luznik L, Jalla S, Engstrom LW, Iannone R, Fuchs EJ. Durable 
engraftment of major histocompatibility complex-incompatible cells after 
nonmyeloablative conditioning with fludarabine, low-dose total body 
irradiation, and posttransplantation cyclophosphamide. Blood. 
2001;98(12):3456-3464.


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12.
Mayumi H, Umesue M, Nomoto K. Cyclophosphamide-induced 
immunological tolerance: an overview. Immunobiology.
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13.
Emadi A, Jones RJ, Brodsky RA. Cyclophosphamide and cancer: 
golden anniversary. Nature reviews. Clinical oncology. 2009;6(11):638-
647.
14.
Schwartz R, Dameshek W. Drug-induced immunological tolerance. 
Nature. 1959;183(4676):1682-1683.
15.
Ross DB, Komanduri KV, Levy RB. Post-Transplant Cyclophosphamide 
(PTC) Gvhd Prophylaxis: Kinetics of Proliferation of Donor T Cells 
Affects Susceptibility to PTC Administration. Blood. 2011;118(21):1721-
1722.
16.
Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation 
and cell cycle kinetics of long-term self-renewing hematopoietic stem 
cells. Proceedings of the National Academy of Sciences of the United 
States of America. 1999;96(6):3120-3125.
17.
Jones RJ, Barber JP, Vala MS, et al. Assessment of aldehyde 
dehydrogenase in viable cells. Blood. 1995;85(10):2742-2746.
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Dey B, Sykes M, Spitzer TR. Outcomes of recipients of both bone 
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1998;77(5):355-369.
19.
Helg C, Chapuis B, Bolle JF, et al. Renal transplantation without 
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20.
Sayegh MH, Fine NA, Smith JL, Rennke HG, Milford EL, Tilney NL. 
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21.
Sorof JM, Koerper MA, Portale AA, Potter D, DeSantes K, Cowan M. 
Renal transplantation without chronic immunosuppression after T cell-
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22.
Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and 
tolerance following combined kidney and nonmyeloablative marrow 
transplantation: in vivo and in vitro analyses. American journal of 
transplantation : official journal of the American Society of 
Transplantation and the American Society of Transplant Surgeons. 
2006;6(9):2121-2133.


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23.
Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al. Combined 
histocompatibility leukocyte antigen-matched donor bone marrow and 
renal transplantation for multiple myeloma with end stage renal disease: 
the induction of allograft tolerance through mixed lymphohematopoietic 
chimerism. Transplantation. 1999;68(4):480-484.
24.
Spitzer TR, Sykes M, Tolkoff-Rubin N, et al. Long-term follow-up of 
recipients of combined human leukocyte antigen-matched bone marrow 
and kidney transplantation for multiple myeloma with end-stage renal 
disease. Transplantation. 2011;91(6):672-676.
25.
Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal 
transplantation without maintenance immunosuppression. The New 
England journal of medicine. 2008;358(4):353-361.
26.
Kawai T, Sachs DH, Sykes M, Cosimi AB. HLA-mismatched renal 
transplantation without maintenance immunosuppression. The New 
England journal of medicine. 2013;368(19):1850-1852.
27.
Spitzer TR. Engraftment syndrome following hematopoietic stem cell 
transplantation. Bone marrow transplantation. 2001;27(9):893-898.
28.
Leventhal J, Abecassis M, Miller J, et al. Chimerism and tolerance 
without GVHD or engraftment syndrome in HLA-mismatched combined 
kidney and hematopoietic stem cell transplantation. Science 
translational medicine. 2012;4(124):124ra128.
29.
Leventhal J AM, Miller J, Gallon L, Tollerud D, Elliott MJ, Bozulic LD, 
Houston C, Sustento-Reodica N, Ildstad ST. Tolerance induction in 
HLA disparate living donor kidney transplantation by donor stem cell 
infusion: durable chimerism predicts outcome. Transplantation. 
2013;95(1):169-176.
30.
Kasamon YL, Luznik L, Leffell MS, et al. Nonmyeloablative HLA-
haploidentical bone marrow transplantation with high-dose 
posttransplantation cyclophosphamide: effect of HLA disparity on 
outcome. Biology of blood and marrow transplantation : journal of the 
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2010;16(4):482-489.
31.
Luznik L, O'Donnell PV, Symons HJ, et al. HLA-haploidentical bone 
marrow transplantation for hematologic malignancies using 
nonmyeloablative conditioning and high-dose, posttransplantation 
cyclophosphamide. Biology of blood and marrow transplantation : 
journal of the American Society for Blood and Marrow Transplantation. 
2008;14(6):641-650.


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32.
Brunstein CG, Fuchs EJ, Carter SL, et al. Alternative donor 
transplantation after reduced intensity conditioning: results of parallel 
phase 2 trials using partially HLA-mismatched related bone marrow or 
unrelated double umbilical cord blood grafts. Blood. 2011;118(2):282-
288.
33.
Bolanos-Meade J, Fuchs EJ, Luznik L, et al. HLA-haploidentical bone 
marrow transplantation with posttransplant cyclophosphamide expands 
the donor pool for patients with sickle cell disease. Blood. 
2012;120(22):4285-4291.
34.
Grosso D, Carabasi M, Filicko-O'Hara J, et al. A 2-step approach to 
myeloablative haploidentical stem cell transplantation: a phase 1/2 trial 
performed with optimized T-cell dosing. Blood. 2011;118(17):4732-
4739.
35.
Gordon MY, Goldman JM, Gordon-Smith EC. 4-
Hydroperoxycyclophosphamide inhibits proliferation by human 
granulocyte-macrophage colony-forming cells (GM-CFC) but spares 
more primitive progenitor cells. Leukemia research. 1985;9(8):1017-
1021.
36.
Kastan MB, Schlaffer E, Russo JE, Colvin OM, Civin CI, Hilton J. Direct 
demonstration of elevated aldehyde dehydrogenase in human 
hematopoietic progenitor cells. Blood. 1990;75(10):1947-1950.
37.
Brodsky RA, Fuller AK, Ratner LE, Leffell MS, Jones RJ. Elimination of 
alloantibodies by immunoablative high-dose cyclophosphamide. 
Transplantation. 2001;71(3):482-484.
38.
Brodsky RA, Sensenbrenner LL, Smith BD, et al. Durable treatment-
free remission after high-dose cyclophosphamide therapy for previously 
untreated severe aplastic anemia. Annals of internal medicine. 
2001;135(7):477-483.
39.
Drachman DB, Jones RJ, Brodsky RA. Treatment of refractory 
myasthenia: "rebooting" with high-dose cyclophosphamide. Annals of 
neurology. 2003;53(1):29-34.
40.
Petri M, Jones RJ, Brodsky RA. High-dose cyclophosphamide without 
stem cell transplantation in systemic lupus erythematosus. Arthritis and 
rheumatism. 2003;48(1):166-173.
41.
Gladstone DE, Bolanos-Meade J, Huff CA, et al. High-dose 
cyclophosphamide and rituximab without stem cell transplant: a 
feasibility study for low grade B-cell, transformed and mantle cell 
lymphomas. Leukemia & lymphoma. 2011;52(11):2076-2081.


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42.
Grochow LB, Colvin M. Clinical pharmacokinetics of cyclophosphamide. 
Clinical pharmacokinetics. 1979;4(5):380-394.
43.
Humphrey RL, Kvols LK. Influence of Renal-Insufficiency on 
Cyclophosphamide Induced Hematopoietic Depression and Recovery. 
P Am Assoc Canc Res. 1974;15(Mar):84-84.
44.
Ballester OF, Tummala R, Janssen WE, et al. High-dose chemotherapy 
and autologous peripheral blood stem cell transplantation in patients 
with multiple myeloma and renal insufficiency. Bone marrow 
transplantation. 1997;20(8):653-656.
45.
Bischoff ME, Blau W, Wagner T, et al. Total body irradiation and 
cyclophosphamide is a conditioning regimen for unrelated bone marrow 
transplantation in a patient with chronic myelogenous leukemia and 
renal failure on hemodialysis. Bone marrow transplantation. 
1998;22(6):591-593.
46.
Aronoff GR. Drug prescribing in renal failure : dosing guidelines for 
adults. 4th ed. Philadelphia, Pa.: American College of Physicians; 1999.
47.
Henrich WL. Principles and practice of dialysis. 2nd ed. Baltimore: 
Williams & Wilkins; 1999.
48.
Colvin M. The comparative pharmacology of cyclophosphamide and 
ifosfamide. Seminars in oncology. 1982;9(4 Suppl 1):2-7.
49.
Colvin M, Hilton J. Pharmacology of cyclophosphamide and 
metabolites. Cancer treatment reports. 1981;65 Suppl 3:89-95.
50.
Colvin M, Padgett CA, Fenselau C. A biologically active metabolite of 
cyclophosphamide. Cancer research. 1973;33(4):915-918.
51.
Jardine I, Fenselau C, Appler M, Kan MN, Brundrett RB, Colvin M. 
Quantitation by gas chromatography-chemical ionization mass 
spectrometry of cyclophosphamide, phosphoramide mustard, and 
nornitrogen mustard in the plasma and urine of patients receiving 
cyclophosphamide therapy. Cancer research. 1978;38(2):408-415.
52.
Danner-Koptik KE, Majhail NS, Brazauskas R, et al. Second 
malignancies after autologous hematopoietic cell transplantation in 
children. Bone marrow transplantation. 2013;48(3):363-368.
53.
Yokota A, Ozawa S, Masanori T, et al. Secondary solid tumors after 
allogeneic hematopoietic SCT in Japan. Bone marrow transplantation. 
2012;47(1):95-100.
54.
Kolb HJ, Socie G, Duell T, et al. Malignant neoplasms in long-term 
survivors of bone marrow transplantation. Late Effects Working Party of 
the European Cooperative Group for Blood and Marrow Transplantation 


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and the European Late Effect Project Group. Annals of internal 
medicine. 1999;131(10):738-744.
55.
Gallagher G, Forrest DL. Second solid cancers after allogeneic 
hematopoietic stem cell transplantation. Cancer. 2007;109(1):84-92.
56.
Socie G, Henry-Amar M, Bacigalupo A, et al. Malignant tumors 
occurring after treatment of aplastic anemia. European Bone Marrow 
Transplantation-Severe Aplastic Anaemia Working Party. The New 
England journal of medicine. 1993;329(16):1152-1157.
57.
Witherspoon RP, Fisher LD, Schoch G, et al. Secondary cancers after 
bone marrow transplantation for leukemia or aplastic anemia. The New 
England journal of medicine. 1989;321(12):784-789.
58.
Bartynski WS. Posterior reversible encephalopathy syndrome, part 1: 
fundamental imaging and clinical features. AJNR. American journal of 
neuroradiology. 2008;29(6):1036-1042.
59.
Munchel A, Kesserwan C, Symons HJ, et al. Nonmyeloablative, HLA-
haploidentical bone marrow transplantation with high dose, post-
transplantation cyclophosphamide. Pediatric reports. 2011;3 Suppl 
2:e15.
60.
Rizzo JD, Curtis RE, Socie G, et al. Solid cancers after allogeneic 
hematopoietic cell transplantation. Blood. 2009;113(5):1175-1183.
61.
Shimoni A, Shem-Tov N, Chetrit A, et al. Secondary malignancies after 
allogeneic stem-cell transplantation in the era of reduced-intensity 
conditioning; the incidence is not reduced. Leukemia : official journal of 
the Leukemia Society of America, Leukemia Research Fund, U.K. 
2012.
62.
KDIGO clinical practice guideline for the care of kidney transplant 
recipients. American journal of transplantation : official journal of the 
American Society of Transplantation and the American Society of 
Transplant Surgeons. 2009;9 Suppl 3:S1-155.
63.
Sanders JE, Hawley J, Levy W, et al. Pregnancies following high-dose 
cyclophosphamide with or without high-dose busulfan or total-body 
irradiation and bone marrow transplantation. Blood. 1996;87(7):3045-
3052.
64.
Gladstone DE, Zachary AA, Fuchs EJ, et al. Partially mismatched 
transplantation and human leukocyte antigen donor-specific antibodies. 
Biol Blood Marrow Transplant. 2013;19(4):647-652.
65.
Montgomery RA. Renal transplantation across HLA and ABO antibody 
barriers: integrating paired donation into desensitization protocols. Am J 
Transplant. 2010;10(3):449-457.


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66.
Vose JM, Reed EC, Pippert GC, et al. Mesna compared with 
continuous bladder irrigation as uroprotection during high-dose 
chemotherapy and transplantation: a randomized trial. Journal of 
clinical oncology : official journal of the American Society of Clinical 
Oncology. 1993;11(7):1306-1310.
67.
Caumes E, Guermonprez G, Lecomte C, Katlama C, Bricaire F. 
Efficacy and safety of desensitization with sulfamethoxazole and 
trimethoprim in 48 previously hypersensitive patients infected with 
human immunodeficiency virus. Archives of dermatology. 
1997;133(4):465-469.
68.
Rowlings PA, Przepiorka D, Klein JP, et al. IBMTR Severity Index for 
grading acute graft-versus-host disease: retrospective comparison with 
Glucksberg grade. British journal of haematology. 1997;97(4):855-864.
69.
Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health 
consensus development project on criteria for clinical trials in chronic 
graft-versus-host disease: I. Diagnosis and staging working group 
report. Biology of blood and marrow transplantation : journal of the 
American Society for Blood and Marrow Transplantation. 
2005;11(12):945-956.
70.
Farris AB, Taheri D, Kawai T, et al. Acute renal endothelial injury during 
marrow recovery in a cohort of combined kidney and bone marrow 
allografts. American journal of transplantation : official journal of the 
American Society of Transplantation and the American Society of 
Transplant Surgeons. 2011;11(7):1464-1477.
71.
Feng S, Ekong UD, Lobritto SJ, et al. Complete immunosuppression 
withdrawal and subsequent allograft function among pediatric recipients 
of parental living donor liver transplants. JAMA : the journal of the 
American Medical Association. 2012;307(3):283-293.
72.
Newell KA, Asare A, Kirk AD, et al. Identification of a B cell signature 
associated with renal transplant tolerance in humans. The Journal of 
clinical investigation. 2010;120(6):1836-1847.
73.
Sagoo P, Perucha E, Sawitzki B, et al. Development of a cross-platform 
biomarker signature to detect renal transplant tolerance in humans. The 
Journal of clinical investigation. 2010;120(6):1848-1861.
74.
Canaud G, Bruneval P, Noel LH, et al. Glomerular collapse associated 
with subtotal renal infarction in kidney transplant recipients with multiple 
renal arteries. American journal of kidney diseases : the official journal 
of the National Kidney Foundation. 2010;55(3):558-565.


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75.
Andreola G, Chittenden M, Shaffer J, et al. Mechanisms of donor-
specific tolerance in recipients of haploidentical combined bone 
marrow/kidney transplantation. American journal of transplantation : 
official journal of the American Society of Transplantation and the 
American Society of Transplant Surgeons. 2011;11(6):1236-1247.
76.
Zachary AA, Kopchaliiska D, Montgomery RA, Leffell MS. HLA-specific 
B cells: I. A method for their detection, quantification, and isolation 
using HLA tetramers. Transplantation. 2007;83(7):982-988.
77.
Zachary AA, Kopchaliiska D, Montgomery RA, Melancon JK, Leffell MS. 
HLA-specific B cells: II. Application to transplantation. Transplantation. 
2007;83(7):989-994.
78.
Gauld SB, Benschop RJ, Merrell KT, Cambier JC. Maintenance of B 
cell anergy requires constant antigen receptor occupancy and signaling. 
Nature immunology. 2005;6(11):1160-1167.
79.
Robins HS, Campregher PV, Srivastava SK, et al. Comprehensive 
assessment of T-cell receptor beta-chain diversity in alphabeta T cells. 
Blood. 2009;114(19):4099-4107.
80.
Loupy A, Suberbielle-Boissel C, Hill GS, et al. Outcome of subclinical 
antibody-mediated rejection in kidney transplant recipients with 
preformed donor-specific antibodies. American journal of 
transplantation : official journal of the American Society of 
Transplantation and the American Society of Transplant Surgeons. 
2009;9(11):2561-2570.
81.
Pallet N, Fougeray S, Beaune P, Legendre C, Thervet E, Anglicheau D. 
Endoplasmic reticulum stress: an unrecognized actor in solid organ 
transplantation. Transplantation. 2009;88(5):605-613.
82.
Thiede C, Bornhauser M, Oelschlagel U, et al. Sequential monitoring of 
chimerism and detection of minimal residual disease after allogeneic 
blood stem cell transplantation (BSCT) using multiplex PCR 
amplification of short tandem repeat-markers. Leukemia : official journal 
of the Leukemia Society of America, Leukemia Research Fund, U.K. 
2001;15(2):293-302.
83.
Thiede C, Bornhauser M, Ehninger G. Strategies and clinical 
implications of chimerism diagnostics after allogeneic hematopoietic 
stem cell transplantation. Acta haematologica. 2004;112(1-2):16-23.


Protocol ITN054ST
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APPENDIX 1A. PRE-TRANSPLANT THROUGH TRANSPLANT: RECIPIENT
Study Day
Screen1, 2
-14
to -9
-8
-7
Days of 
rest
-4
-3
-2
-1
0
Visit (Windows: Visits should take 
place on the day indicated in this 
schedule.)
1
2
3
4
5
6
7
8
9
10
11
1A - GENERAL ASSESSMENTS
Informed consent
X
Medical and demographic history
including history of blood 
products donation and 
concomitant medications
X
Complete physical examination
including height
X
Limited physical exam3
X
X
X
X
X
X
X
X
Vital signs including w eight
X
X
X
X
X
X
X
X
X
Cardiac function –
echocardiogram
X
Pulmonary function – chest 
radiography and spirometry
X
1 Recipient screening must occur within 30 days of first administration of study medication. Study 
medication dosing is initiated on day -9. The screening assessments in Visit 1 or Visit 505 may be 
performed over multiple days within the 30 day window.
2 All standard-of-care laboratory assessments (hematology, chemistry, pregnancy and viral serologies) 
must be completed within 30 days of day -9. High resolution molecular HLA typing and ABO typing, 
and any positive viral serology obtained per site standard of care, may be completed greater than 30 
days prior to Visit -9,with no upper window, provided the respective assays were performed using the 
same technology currently in use by the site laboratory.
3 To include assessment of at least the following body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary.


Protocol ITN054ST
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ACCEPTOR
Study Day
Screen1, 2
-14
to -9
-8
-7
Days of 
rest
-4
-3
-2
-1
0
Visit (Windows: Visits should take 
place on the day indicated in this 
schedule.)
1
2
3
4
5
6
7
8
9
10
11
Status change since prior visit4
X
X
X
X
X
X
X
X
1A - LOCAL CLINICAL LABORATORY ASSESSMENTS5
Hematology – CBC w ith 
differential and platelets
X
X
X
X
X
X
X
X
X
Comprehensive Metabolic Panel 
(Na, K, Cl, HCO3, BUN, 
creatinine, glucose, albumin, total 
bilirubin, AST, ALT, alkaline 
phosphatase)
X
X
X
X
X
X
X
X
X
Urine or serum pregnancy test
X
X6
CMV and EBV serologies
X7
HIV-1 and 2 serologies and 
nucleic acid test
X7
Anti-hepatitis C (HCV) 
antibodies 8
X7
4 Includes assessment of change in administration of blood products; adverse events and concomitant 
medications. (See section 5.10 for specific concomitant medications to be assessed.)Changes should be 
recorded in eCRFs as appropriate.
5 All blood collections should be performed prior to any study drug administration, if blood collection 
and drug administration occur on the same day.
6 Pregnancy test must be repeated within 72 hours of first study drug administration.
7 Collect screening blood samples for serologies such that results are available by day -14. Previously 
performed viral serologies will be used for eligibility criteria.
8 All positive HCV antibody results must be assessed by an EIA assay and confirmed by a quantitative 
serum HCV RNA assay. Participants with positive HCV antibodies but undetectable serum HCV RNA 
may be considered for eligibility. Participants with negative anti-HCV antibodies but unexplained liver 
enzyme abnormalities must undergo a quantitative serum RNA assay to rule out false negative HCV 
serologies.


Protocol ITN054ST
Version 5.0
March 28, 2016
ACCEPTOR
Study Day
Screen1, 2
-14
to -9
-8
-7
Days of 
rest
-4
-3
-2
-1
0
Visit (Windows: Visits should take 
place on the day indicated in this 
schedule.)
1
2
3
4
5
6
7
8
9
10
11
Hepatitis B surface antigen 
(HBsAg), total hepatitis B core 
antibody (anti-HBc IgM and IgG), 
hepatitis B surface antibody (anti-
HBsAb) 9
X7
Quantiferon test
X
ABO typing10
X
High resolution molecular HLA 
typing and DNA (A, B, C, DRB1,
DRB3-5, DQA1, DQB1, DPB1) 11
X
HLA alloantibodies includes DSA, 
flow  PRA
X
T and B cell lymphocyte 
crossmatch
X12
X12
Cytotoxic crossmatch (CDC)
X
Calculated PRA
X
Kidney biopsy
X
1A - LOCAL MECHANISTIC ASSESSMENTS5,13
Chimerism (buccal swab)
X
T cell assays (MLR, CML)
X
B cell assays (HLA tetramer 
selection and culture)
X
B cell sequencing
X
9 Participants will undergo a quantitative HBV DNA PCR test per institutional standard and will be 
excluded if HBV DNA is detectable.
10 ABO typing to be taken from medical records.
11 High resolution HLA typing to be performed on DNA sample banked from routine initial evalutation.
12 Screening T and B cell lymphocyte crossmatch to be performed as close to day -30 as possible. Final T 
and B cell lymphocyte crossmatch to be performed on day -14, before recipient receives study drug.
13 To be collected no later than study day -9 once candidate has been successfully screened for eligibility 
criteria.


Protocol ITN054ST
Version 5.0
March 28, 2016
ACCEPTOR
Study Day
Screen1, 2
-14
to -9
-8
-7
Days of 
rest
-4
-3
-2
-1
0
Visit (Windows: Visits should take 
place on the day indicated in this 
schedule.)
1
2
3
4
5
6
7
8
9
10
11
1A - CENTRAL MECHANISTIC ASSESSMENTS5
Plasma cytokines
X
Flow  cytometry panel staining 
(frozen PBMC)
X
PBMC collection
X
Whole blood – gene expression 
profiling
X
Kidney biopsy – histology
X
Kidney biopsy – gene expression 
profiling
X
1A - STUDY TREATMENT
Hemodialysis (6-12 hours after 
fludarabine dosing)
X
X
X
Total body irradiation
X
Renal transplantation
X
Bone marrow  infusion
X
1A - STUDY MEDICATION
ATG14
X
X
X
Methylprednisolone (ATG 
premedication)
X
X
X
X
X
X
Fludarabine
X
X
X
Low  dose cyclophosphamide
X
X
14 ATG to be administered along with premedication as specified in section 5.2.2.


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APPENDIX 1B. PRE-TRANSPLANT THROUGH POST-TRANSPLANT: DONOR
Study Day
Screen15,2
-14 to -9
-1
0
7
56
Visit
(Visits pre-transplant through transplant should take 
place on the day indicated in the schedule of events;
Day 7 post-transplant visit has a +3-day w indow; Day 
56 post-transplant visit has a +14-day w indow.)
1
2
10
11
104
106
1B - GENERAL ASSESSMENTS
Informed consent
X
Medical and demographic history
X
Screening for any autoimmune disease requiring 
immunosuppressive drugs for maintenance
X
Complete physical examination including height 
X
Vital signs including w eight
X
Status change since prior visit16,17
X
X
X
X
X
1B - LOCAL CLINICAL LABORATORY ASSESSMENTS5
Hematology – CBC w ith differential and platelets
X
X
X
X
X
X
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, 
BUN, creatinine, glucose, albumin, total bilirubin, 
AST, ALT, alkaline phosphatase)
X
X
X
X
X
X
CMV and EBV serology
X7
HIV-1 and 2 serologies and nucleic acid test
X7
Anti-hepatitis C (HCV) antibodies 8
X7
Hepatitis B surface antigen (HBsAg), total hepatitis B 
core antibody (anti-HBc IgM and IgG), hepatitis B 
surface antibody (anti-HBsAb)9
X7
ABO typing10
X
High resolution molecular HLA typing and DNA (A, 
B, C, DRB1, DRB3-5, DQA1, DQB1, DPB1) 11
X
15 Donor screening must occur within 30 days of Day 0. The screening assessments in Visit 1 or Visit 
505 may be performed over multiple days within the visit study window.
16 Includes assessment of change in adverse events, concomitant medications and administration of blood 
products. (See section 5.10 for specified concomitant medications.) Changes should be recorded in 
eCRFs as appropriate.
17 Only AEs related to bone marrow harvest and study-mandated blood draw will be collected for the 
donor.


Immune Tolerance Network                                CONFIDENTIAL
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Study Day
Screen15,2
-14 to -9
-1
0
7
56
Visit
(Visits pre-transplant through transplant should take 
place on the day indicated in the schedule of events;
Day 7 post-transplant visit has a +3-day w indow; Day 
56 post-transplant visit has a +14-day w indow.)
1
2
10
11
104
106
T and B cell lymphocyte crossmatch
X12
X12
PBMC’s for future T and B cell lymphocyte 
crossmatches
X18
X
1B - LOCAL MECHANISTIC ASSESSMENTS5
Chimerism (buccal swab)
X18
T cell assays (MLR, CML)
X18
B cell assays (HLA tetramer selection and culture)
X
1B - CENTRAL MECHANISTIC ASSESSMENTS
PBMC collection
X
1B - STUDY TREATMENT
Bone marrow  harvest and nephrectomy
X
18 Collect donor baseline blood samples on day -14 (+/-2 days).


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APPENDIX 1C. REINITIATION OF CONDITIONING: PRE-TRANSPLANT 
THROUGH TRANSPLANT: RECIPIENT
Post 1st Attempt Conditioning
2nd Attempt Conditioning19
Transplant
Study Day
+2-6
w eeks
+12 
weeks
+18 
weeks
+24-
26
weeks
Eligibility
1,2,20
-14
to -9
-8
-7
Days of 
rest (-6
and -5)
-4
-3
-2
-1
0
Visit
501
50220
50320
50420
505
506
507
508
509
510
511
512
513
514
11
1C - GENERAL ASSESSM ENTS
Informed consent
X
Medical and 
demographic history
including history of 
blood products donation 
and concomitant 
medications
X
Complete physical 
examination including 
height
X
Limited physical exam3
X
X
X
X
X
X
X
X
X
X
X
X
Vital signs including 
w eight
X
X
X
X
X
X
X
X
X
X
X
X
X
Cardiac function –
echocardiogram21
X
Pulmonary function –
chest radiography and
spirometry21
X
Status change since 
prior visit4
X
X
X
X
X
X
X
X
X
X
X
X
X
1C - LOCAL CLINICAL LABORATORY ASSESSMENTS5
Hematology – CBC w ith 
differential and platelets
X
X
X
X
X
X
X
X
X
X
X
X
X
19 Visits 506-513 are to be completed per investigators discretion. The determination of when to 
administer reconditioning and how much reconditioning, if any, is necessary to proceed with the 
transplantation will be left to the discretion of the investigator and may be adjusted based on the amount 
of conditioning initially received and the time elapsed since the last dose. 
20 Visits 502, 503, 504 are to be completed as needed between conditioning attempts. Eligibility 
assessment can occur any time after complete ANC recovery.
21 Pulmonary function and cardiac tests may be repeated if deemed necessary by the investigator.


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ACCEPTOR
Post 1st Attempt Conditioning
2nd Attempt Conditioning19
Transplant
Study Day
+2-6
w eeks
+12 
weeks
+18 
weeks
+24-
26
weeks
Eligibility
1,2,20
-14
to -9
-8
-7
Days of 
rest (-6
and -5)
-4
-3
-2
-1
0
Visit
501
50220
50320
50420
505
506
507
508
509
510
511
512
513
514
11
Comprehensive 
Metabolic Panel (Na, K, 
Cl, HCO3, BUN, 
creatinine, glucose, 
albumin, total bilirubin, 
AST, ALT, alkaline 
phosphatase)
X
X
X
X
X
X
X
X
X
X
X
X
X
Urine or serum 
pregnancy test
X
X6
CMV and EBV 
serologies
X7
HIV-1 and 2 serologies 
and nucleic acid test
X7
Anti-hepatitis C (HCV) 
antibodies8
X7
Hepatitis B surface 
antigen (HBsAg), total 
hepatitis B core 
antibody (anti-HBc IgM 
and IgG), hepatitis B 
surface antibody (anti-
HBsAb)9
X7
Chest x-ray 22
X
ABO typing10
High resolution 
molecular HLA typing 
and DNA (A, B, C, 
DRB1, DRB3-5, DQA1, 
DQB1, DPB1) 11
HLA alloantibodies
includes DSA, flow  PRA
X
T and B cell lymphocyte 
crossmatch
X12
X12
Cytotoxic crossmatch 
(CDC)
X
Calculated PRA
X
22 Chest x-ray will only occur for those recipients who were previously Quantiferon positive.


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ACCEPTOR
Post 1st Attempt Conditioning
2nd Attempt Conditioning19
Transplant
Study Day
+2-6
w eeks
+12 
weeks
+18 
weeks
+24-
26
weeks
Eligibility
1,2,20
-14
to -9
-8
-7
Days of 
rest (-6
and -5)
-4
-3
-2
-1
0
Visit
501
50220
50320
50420
505
506
507
508
509
510
511
512
513
514
11
Kidney biopsy
X
1C - LOCAL MECHANISTIC ASSESSMENTS5,13
Chimerism (buccal 
sw ab)
X23
T cell assays (MLR, 
CML)
X23
B cell assays (HLA 
tetramer selection and 
culture)
X23
B cell sequencing
X23
1C - CENTRAL MECHANISTIC ASSESSMENTS5
Plasma cytokines
X23
Flow  cytometry panel 
staining (frozen PBMC)
X23
PBMC collection
X23
Whole blood – gene
expression profiling
X23
Kidney biopsy –
histology
X
Kidney biopsy – gene
expression profiling
X
1C - STUDY TREATMENT
Hemodialysis (6-12
hours after fludarabine 
dosing)
X
X
X
Total body irradiation
X
Renal transplantation
X
Bone marrow  infusion
X
1C - STUDY MEDICATION
ATG14
X
X
X
Methylprednisolone 
(ATG premedication)
X
X
X
X
X
X
Fludarabine
X
X
X
Low  dose 
cyclophosphamide
X
X
23 To be collected only if first attempted collection is inadequate.


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APPENDIX 1D. REINITIATION OF CONDITIONING: PRE-TRANSPLANT 
THROUGH POST-TRANSPLANT: DONOR
(Recipient’s) 2nd Attempt 
Conditioning24
Transplant
Post-Transplant
Study Day
Screen15,2
-14 to -9
-1
0
7
56
Visit
505
506
514
11
104
106
1D - GENERAL ASSESSMENTS
Informed consent
X
Medical and demographic history
X
Screening for any autoimmune disease requiring 
immunosuppressive drugs for maintenance
X
Complete physical examination including height 
X
Vital signs including w eight
X
Status change since prior visit16,17
X
X
X
X
X
1D - LOCAL CLINICAL ASSESSMENTS5
Hematology – CBC w ith differential and platelets
X
X
X
X
X
X
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, 
BUN, creatinine, glucose, albumin, total bilirubin, 
AST, ALT, alkaline phosphatase)
X
X
X
X
X
X
CMV and EBV serology
X7
HIV-1 and 2 serologies and nucleic acid test
X7
Anti-hepatitis C (HCV) antibodies8
X7
Hepatitis B surface antigen (HBsAg), total hepatitis B 
core antibody (anti-HBc IgM and IgG), hepatitis B 
surface antibody (anti-HBsAb)9
X7
ABO typing10
X
High resolution molecular HLA typing and DNA (A, 
B, C, DRB1, DRB3-5, DQA1, DQB1, DPB1)11
X
T and B cell lymphocyte crossmatch
X12
X12
PBMC’s for future T and B cell lymphocyte 
crossmatches
X18
X
1D - LOCAL MECHANISTIC ASSESSMENTS5
Chimerism (buccal swab)
X18
T cell assays (MLR, CML)
X18
B cell assays (HLA tetramer selection and culture)
X
1D - CENTRAL MECHANISTIC ASSESSMENTS
24 Visit 506  to be completed per investigators discretion. 


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ACCEPTOR
(Recipient’s) 2nd Attempt 
Conditioning24
Transplant
Post-Transplant
Study Day
Screen15,2
-14 to -9
-1
0
7
56
Visit
505
506
514
11
104
106
PBMC collection
X
1D - STUDY TREATMENT
Bone marrow  harvest and nephrectomy
X


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APPENDIX 2. POST-TRANSPLANT THROUGH EVALUATION FOR WITHDRAWAL 
ELIGIBILITY
Post-transplant Week
0
0
0
0
4
8
22
30
Post-transplant Day
3
4
5
7
28
56
154
210
Post-transplant Visit (Windows: Visits 101-104 to 
be conducted on day indicated; Visits 105-106 +/-
2 days; Visit 107 +/- 7 days)
101
102
103
104
105
106
1071
108A,B
2 - GENERAL ASSESSMENTS
Vital signs including w eight
X
X
X
X
X
X
X
X
Limited physical exam2
X
X
X
X
X
X
X
X
Status change since prior visit3
X
X
X
X
X
X
X
X
Acute GVHD assessment4
X
X
X
X
X
X
X
X
Chronic GVHD assessment5
X
X
X
Absolute neutrophil count recovery 6
X
1 Participants may be evaluated for prednisone and MMF IS withdrawal as early as Week 22 (Day 154) 
post-transplant. Immunosuppression withdrawal may begin as early as Week 26 (Day 182) as per 
Appendix 3. Participants who are not ready to undergo eligibility biopsy at Week 22, should undergo 
routine study visit at this time point and may return for eligibility evaluation no later than Week 30 
(Day 210, Visit 108) post-transplant.If the eligibility biopsy must be repeated, perform all assessments 
indicated for Visit 108.
2 To include assessment of at least the following body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary.
3 Includes assessment of change in administration of blood products,adverse events and concomitant 
medications. (See section 5.10 for specific concomitant medications to be assessed.) Changes should be 
recorded in eCRFs as appropriate.
4 Acute GVHD will be staged and graded as in section 6.7, post-transplant Day 0 through approximately 
Day 200, at the visits indicated.
5 Chronic GVHD will be staged and graded as in section 6.7.2, approximately post-transplant Day 60 
through approximately 2 years post-transplant, at the visits indicated, using the Chronic GVHD 
Assessment Worksheet (Appendix 7. Chronic GVHD Assessment Worksheet).If a participant’s 
chronic GVHD persists beyond 2 years post-transplant or if he/she is diagnosed with chronic GVHD 
>2 years post-transplant, the chronic GVHD Assessment Worksheet will be graded at every subsequent 
study visit until resolution of the episode, or until the end of study participation.
6 Time of absolute neutrophil count (ANC) recovery to be captured on the Absolute Neutrophil Count 
recovery eCRF.


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Post-transplant Week
0
0
0
0
4
8
22
30
Post-transplant Day
3
4
5
7
28
56
154
210
Post-transplant Visit (Windows: Visits 101-104 to 
be conducted on day indicated; Visits 105-106 +/-
2 days; Visit 107 +/- 7 days)
101
102
103
104
105
106
1071
108A,B
Platelet count recovery7
X
2 - LOCAL CLINICAL LABORATORY ASSESSMENTS8
Hematology – CBC w ith differential and platelets 9
X
X
X
X
Comprehensive Metabolic Panel (Na, K, Cl, HCO3, 
BUN, creatinine, glucose, albumin, total bilirubin, 
AST, ALT, alkaline phosphatase) 10
X
X
X
X
BK virus (blood)
X
X
X
X
Tacrolimus levels
X
X
X
X
HLA alloantibodies includes DSA, flow  PRA
X
X
X
X
X
CMV and EBV viral load11
X
X
X
Kidney biopsy 12
X13
2 - LOCAL MECHANISTIC ASSESSMENTS8,14
7 Time to platelet count recovery to be captured on the Platelet Count Recovery eCRF.
8 All blood collections should be performed prior to any study drug administration, if blood collection 
and drug administration occur on the same day.
9 Values from more frequent, standard of care testing on non-study visit days may also be recorded in the 
participant’s eCRF study record.
10 Creatinine values used to establish baseline value during weeks 2-4 post-transplant will be recorded on 
the Baseline Creatinine eCRF.
11 CMV viremia (by PCR) will be monitored and documented weekly post-transplant beginning once the 
total white blood cell count is greater than 1000/mm3. Monitoring of CMV viremia will continue 
weekly post-transplant until Day 100; then every other week until Day 365.
12 In the event of a for cause biopsy or repeat biopsy, follow assessments listed in Appendix 4 visit 308.
13 The immunosuppression withdrawal eligibility biopsy may take place as early as week 22 or as late as 
week 30 post-transplant.
14 Mechanistic draws must be completed prior to biopsy procedures on visits when both biopsies and 
mechanistic draws are being performed.


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Post-transplant Week
0
0
0
0
4
8
22
30
Post-transplant Day
3
4
5
7
28
56
154
210
Post-transplant Visit (Windows: Visits 101-104 to 
be conducted on day indicated; Visits 105-106 +/-
2 days; Visit 107 +/- 7 days)
101
102
103
104
105
106
1071
108A,B
Blood chimerism (w hole blood and CD3+ T cell)
X
X
X15
T cell assays (MLR, CML)
X15
B cell assays (HLA tetramer selection and culture)
X15
B cell repertoire sequencing
X15
2 - CENTRAL MECHANISTIC ASSESSMENTS8,14
Plasma cytokines
X
X
X15
Flow  cytometry panel staining (frozen PBMC)
X
X
X15
PBMC collection
X
X
X15
Whole blood – gene expression profiling
X
X
X15
Kidney biopsy – histology
X15
Kidney biopsy – gene expression profiling
X15
2 - STUDY MEDICATION
Tacrolimus, prednisone and mycophenolate 
mofetil16
X
X
X
X
X
X
High dose cyclophosphamide17
X
X
Filgrastim18
X
X
X
15 To be collected at the visit in which the immunosuppression withdrawal eligibility biopsy is 
performed.
16 Maintenance immunosuppressive therapy to be initiated on Day 5 post-transplant per section 5.3. It is 
crucial that no immunosuppressive agents are given from the time of the transplant until 24 hours after 
the completion of the post-transplant cyclophosphamide. This includes steroids as anti-emetics.
Intravenous administration of tacrolimus will not be allowed.
17 To be administered with MESNA and anti-emetics as outlined in section 5.2.3 on post-transplant Days 
3 and 4 (within 48-72 hours of marrow infusion).
18 Filgrastim 5 Pg/kg/day sc will be administered from day 5 until the absolute neutrophil count is greater 
than 1000/Pl on three consecutive measurements over at least 2 days.


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APPENDIX 3. IMMUNOSUPPRESSION WITHDRAWAL (INITIATED BETWEEN 26
AND 34 WEEKS POST-TRANSPLANT)
Immunosuppression
Withdrawal Week
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Immunosuppression
Withdrawal Visit
(Window s: +/- 7 days)
201 202 203 204 205 206
207 208
209 210
211 212
213 214 215 216 217
218
3 - GENERAL ASSESSMENTS
Informed consent
X43
Vital signs including 
w eight
X
X
X
X
X
X
X
X
X
Limited physical exam44
X
X
X
X
X
X
X
X
X
Status change since 
prior visit45
X46
X
X46
X
X46
X
X46
X
X46
X
X46
X
X46
X
X46
X
X46
X
Acute GVHD 
assessment47
X48
X
X48
X
Chronic GVHD 
assessment49
X48
X
X48
X
X48
X
X48
X
X48
X
X48
X
X48
X
X48
X
X48
X
43 To be obtained prior to assessment for eligibility for tacrolimus withdrawal as early as Week 8 or as 
late as Week 16 of the Immunosuppression Withdrawal schedule. 
44 To include assessment of at least the following body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary.
45 Includes assessment of change in administration of blood products, adverse events and concomitant 
medications. (See section 5.10 for specific concomitant medications to be assessed.) Changes should be 
recorded in eCRFs as appropriate.
46 Visit may be conducted by telephone, unless eligibility for withdrawal is being assessed. Participants 
eligible for immunosuppression withdrawal should be instructed on dose change for prednisone and 
MMF.
47 Acute GVHD will be staged and graded as in section 6.7 from Day 0 to approximately 200 post-
transplant,at the visits indicated.
48 Not performed if visit is conducted by telephone.
49 Chronic GVHD will be staged and graded as in section 6.7.2, approximately post-transplant Day 60 
through approximately 2 years post-transplant, at the visits indicated, using the Chronic GVHD 
Assessment Worksheet (Appendix 7. Chronic GVHD Assessment Worksheet). If a participant’s 
chronic GVHD persists beyond 2 years post-transplant or if he/she is diagnosed with chronic GVHD 
>2 years post-transplant, the chronic GVHD Assessment Worksheet will be graded at every subsequent 
study visit until resolution of the episode, or until the end of study participation.


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Immunosuppression
Withdrawal Week
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Immunosuppression
Withdrawal Visit
(Window s: +/- 7 days)
201 202 203 204 205 206
207 208
209 210
211 212
213 214 215 216 217
218
Absolute neutrophil 
count recovery 50
X
Platelet count 
recovery 51
X
3 - LOCAL CLINICAL LABORATORY ASSESSMENTS52
Hematology – CBC w ith 
differential and 
platelets 53
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Comprehensive 
Metabolic Panel (Na, K, 
Cl, HCO3, BUN, 
creatinine, glucose, 
albumin, total bilirubin, 
AST, ALT, alkaline 
phosphatase)53
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
BK virus (blood) 54
X
Tacrolimus levels53,55
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
HLA alloantibodies
includes DSA, flow  
PRA 56
X
X
X
X
X
X
X
X
X
T and B cell lymphocyte 
crossmatch
X61
50 Time of absolute neutrophil count recovery to be captured on the Absolute Neutrophil Count eCRF 
(applicable if ANC recovery is still being monitored when participant moves to Immunosuppression 
Withdrawal visits).
51 Time to platelet count recovery to be captured on the Platelet Count Recovery eCRF (applicable if 
platelet count recovery is still being monitored when participant moves to Immunosuppression 
Withdrawal visits).
52 All blood collections should be performed prior to any study drug administration.
53 For visits conducted by phone, results from the participant’s home laboratory may be used.
54 Monitoring for BK viremia should continue for up to 1 year post-transplant in participants who
complete immunosuppression withdrawal; see section 5.7 for specific time points.
55 Monitoring of tacrolimus levels may be discontinued once levels become undetectable.
56 Performed only at study site laboratory.


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Immunosuppression
Withdrawal Week
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Immunosuppression
Withdrawal Visit
(Window s: +/- 7 days)
201 202 203 204 205 206
207 208
209 210
211 212
213 214 215 216 217
218
CMV and EBV viral 
load57
X
X
X
X
X
X
X
X
X
X
X
X
Kidney biopsy 58
X59, 60
X61
3 - LOCAL MECHANISTIC ASSESSMENTS52,59
Blood chimerism (w hole 
blood and CD3+ T-Cell) 
X60
X61
T cell assays (MLR, 
CML)
X60
X61
B cell assays (HLA 
tetramer selection and 
culture)
X60
X61
B cell repertoire 
sequencing
X60
3 - CENTRAL MECHANISTIC ASSESSMENTS52,59
Plasma cytokines
X60
X61
Flow  cytometry panel 
staining (frozen PBMC)
X60
X61
PBMC collection
X60
X61
Whole blood – gene
expression profiling
X60
X61
Kidney biopsy –
histology
X60
X61
Kidney biopsy – gene
expression profiling
X60
X61
57 CMV viremia will be monitored by PCR and documented weekly post-transplant beginning once the 
total white blood cell count is greater than 1000/mm3. Monitoring of CMV will continue weekly post-
transplant until Day 100; then every other week until Day 365.
58 In the event of a for cause biopsy or repeat biopsy, follow assements listed in Appendix 4 visit 308.
59 Mechanistic draws must be completed prior to biopsy procedures on visits when both biopsies and 
mechanistic draws are being performed.
60 Tacrolimus withdrawal eligibility biopsy and associated mechanistic assessments may take place as 
early as Week 8 or as late as Week 16 of the Immunosuppression Withdrawal Schedule.
61 To be performed on the first day following the completion of immunosuppression withdrawal; the first 
day the participant takes no immunosuppressive medication. This assessment may take place as early 
as Week 24 or as late as Week 34 of the Immunosuppression Withdrawal Schedule.


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Immunosuppression
Withdrawal Week
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Immunosuppression
Withdrawal Visit
(Window s: +/- 7 days)
201 202 203 204 205 206
207 208
209 210
211 212
213 214 215 216 217
218
3 - STUDY MEDICATION
Tacrolimus 62
X
X
X
X
X
X
X
X
X
X
X
X
X21
X21
X21 X21 X21
X21
Prednisone63
X
X
X
X64 X21
Mycophenolate 
mofetil64
X
X
X
X21 X21
62 Please see section 3.3.2.1 & 3.3.2.2 for tacrolimus withdrawal eligibility criteria and 
immunosuppression withdrawal algorithm.
63 Please see section 3.3.1.2 for prednisone and MMF withdrawal algorithm.
64 Needed only if withdrawal of this medication is not complete at this time point.
A In the event of a for cause biopsy or repeat biopsy, follow assessments listed in Appendix 4 visit 308.


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APPENDIX 4. POST IMMUNOSUPPRESSION WITHDRAWAL FOLLOW-UP 
(INITIATED BETWEEN 52 AND 62 WEEKS POST-TRANSPLANT)
Post 
Immunosuppression 
Withdrawal Week
3
6
9
12
26
36
48
52
60
72
84
96
108 120 132 144 156 168 180 192 204 216
Post 
Immunosuppression 
Withdrawal Visit
(Window s: Visits 301-
305 +/- 7 days; Visits 
306-322 +/- 2 w eeks)
301 302 303 304 305 306 307 3081 309 310 311 312 313 314 315 316 317 318 319 320 321 322
4 - GENERAL ASSESSMENTS
Vital signs including 
w eight
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Limited physical exam2
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Status change since prior 
visit3
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X4
Chronic GVHD5
assessment
X
X
X
X
X
X
X
X
X
X
4 - LOCAL CLINICAL LABORATORY ASSESSMENTS
Hematology – CBC w ith 
differential and platelets
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Comprehensive 
Metabolic Panel (Na, K, 
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
1 For For Cause Biopsy (FCB) and Repeat Biopsy (RB) visits, follow the clinical and mechanistic 
assessments specified inVisit 308, with the exception of B cell repertoire sequencing which does not 
need to be performed for FCB or RB visits. If a FCB or RB is performed within 60 days of transplant 
the following local mechanistic assays will not be performed; T cell assays and B cell assays. This visit 
should also include testing for BK virus regardless of length of time since transplant.
2 To include assessment of at least the following body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary.
3 Includes assessment of change in administration of blood products, adverse events and concomitant 
medications. (See section 5.10 for specific concomitant medications to be assessed.) Changes should be 
recorded in eCRFs as appropriate.
4 Any unresolved AE will be followed until it resolves or until 30 days after a participant terminates from 
the study, whichever comes first, per section 8.2.1.
5 Chronic GVHD will be staged and graded as in section 6.7.2, approximately post-transplant Day 60 
through approximately 2 years post-transplant, at the visits indicated using the Chronic GVHD 
Assessment Worksheet (Appendix 7. Chronic GVHD Assessment Worksheet). If a participant’s 
chronic GVHD persists beyond 2 years post-transplant or if he/she is diagnosed with chronic GVHD 
>2 years post-transplant, the chronic GVHD Assessment Worksheet will be graded at every subsequent 
study visit until resolution of the episode, or until the end of study participation.


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Post 
Immunosuppression 
Withdrawal Week
3
6
9
12
26
36
48
52
60
72
84
96
108 120 132 144 156 168 180 192 204 216
Post 
Immunosuppression 
Withdrawal Visit
(Window s: Visits 301-
305 +/- 7 days; Visits 
306-322 +/- 2 w eeks)
301 302 303 304 305 306 307 3081 309 310 311 312 313 314 315 316 317 318 319 320 321 322
Cl, HCO3, BUN, 
creatinine, glucose, 
albumin, total bilirubin, 
AST, ALT, alkaline 
phosphatase)
HLA alloantibodies –
includes DSA, flow  PRA
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Kidney biopsy
X
X
X
4 - LOCAL MECHANISTIC ASSESSMENTS6
Blood chimerism (w hole
blood and 
CD3+ T cell) 
X
X
X
X
T cell assays (MLR, 
CML)
X
X
X
X
B Cell Assays (HLA 
tetramer selection and 
culture)
X
X
X
X
B cell repertoire 
sequencing
X
X
4 - CENTRAL MECHANISTIC ASSESSMENTS6
Plasma cytokines
X
X
X
X
Flow  cytometry panel 
staining (frozen PBMC)
X
X
X
X
PBMC collection
X
X
X
X
Whole blood – gene
expression profiling
X
X
X
X
Kidney biopsy – histology
X
X
X
Kidney biopsy – gene
expression profiling
X
X
X
6 Mechanistic draws must be completed prior to biopsy procedures on visits when both biopsies and 
mechanistic draws are being performed.


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1 The assessments outlined in Appendix 5 should be initiated for all unsuccessful participants who have 
completed at least one dose of ATG. Participants who experience a rejection event should begin safety 
follow-up within 4-6 weeks of the date of diagnosis of rejection. Participants who move to safety 
follow-up following non-rejection events should begin this schedule within 2 weeks of the 
determination of their ineligibility for further progress in the protocol.
2 Follow the clinical assessments specified here, Visit 401, for any participant requesting early 
termination from study.
3 Visits 411-413 to be used to ensure that all participants who receive the preparative regimen are 
followed for at least 5 years. The 3 additional visits should be completed for study participants who are 
moved to safety follow-up pre-transplant or prior to evaluation for withdrawal eligibility; who fail IS 
withdrawal; or who are moved to safety follow-up post-IS withdrawal.
4 To include assessment of at least the following body systems: respiratory, cardiovascular, 
gastrointestinal, skin, neurologic and renal/urinary.
5 Includes assessment of change in administration of blood products, adverse events and concomitant 
medications. (See section 5.10 for specific concomitant medications to be assessed.) Changes should be 
recorded in eCRFs as appropriate.
6 Any unresolved AE will be followed until it resolves or until 30 days after a participant terminates from 
the study, whichever comes first, per section 8.2.1.
7 Acute GVHD will be staged and graded as in section 6.7, on each visit in the safety follow-up schedule 
through post-transplant Day 200 or the first visit thereafter.
8 Chronic GVHD will be staged and graded as in section 6.7.2, at the first safety follow-up visit on or 
after post-transplant Day 60 and at each subsequent visit of the safety follow-up visit schedule up to the 
point 2 years post-transplant, at the visits indicate,d using the Chronic GVHD Assessment Worksheet
(Appendix 7. Chronic GVHD Assessment Worksheet). If a participant’s chronic GVHD persists 
beyond 2 years post-transplant or if he/she is diagnosed with chronic GVHD >2 years post-transplant, 


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ACCEPTOR
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the chronic GVHD Assessment Worksheet will be graded at every subsequent study visit until 
resolution of the episode, or until the end of study participation.
9 Time of absolute neutrophil count (ANC) recovery to be captured on the Absolute Neutrophil Count 
recovery eCRF (applicable if ANC recovery is still being monitored when participant moves to safety 
follow-up).
10 Time to platelet count recovery to be captured on the Platelet Count Recovery eCRF (applicable if 
platelet count recovery is still being monitored when participant moves to safety follow-up).
11 All blood collections should be performed prior to any study drug administration.
12 CMV viremia (by PCR) will be monitored and documented weekly post-transplant beginning once the 
total white blood cell count is greater than 1000/mm3. Monitoring of CMV viremia will continue 
weekly post-transplant until Day 100; then every other week until Day 365.
13 Monitoring for BK viremia should continue for up to 2 years post-transplant in participants who 
remain on immunosuppression and move to safety follow-up; see section 5.7 for specific time points.
14 Performance of per protocol kidney biopsies and mechanistic assessments is not required for 
participants who have not undergone per protocol renal transplantation.
15 Mechanistic draws must be completed prior to biopsy procedures on visits when both biopsies and 
mechanistic draws are being performed.


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ACCEPTOR
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APPENDIX 6.ACUTEGVHD ASSESSMENT WORKSHEET
Participant ID 
  
  
   
   
   
   
 
Visit #
Date
1. Record 
the highest level of organ abnormal
i
ties  during  
the assessment period:
Use the “rule  of 9s” to
 compute percentage of body
  surface area for the skin
 
assessment.
a. Skin
None
  
   
   
  Maculopapular  rash
 < 25% of body
  surface
  
   
   
  Maculopapular  
rash 25-50% of body surface
  
   
   
  General
ized erythroderma
  
   
   
  General
ized erythroderma wi
th bul
lous  
formations and desquamation
b. Intestinal Tract
  
 None
  
   
   
  > 500 ml
/day
> 1000 ml
/day
> 1500 ml
/day
> Severe abdominal  pain, wi
th or wi
thout i
leus
c. Liver
 
  
 None
  
   
   
  Bi
l
irubin 2-3 mg/dL
  
   
   
  Bi
l
irubin 3-6 mg/dL
  
   
   
  Bi
l
irubin 6-15 mg/dL
  
   
   
  Bi
l
irubin  > 15 mg/dL 
2. Overal
l Cl
inical  Grading of Severi
ty of Acute GVHD:
  
 No Acute GVHD


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 I-Stage 1-2 skin rash; no gut involvement;  no l
iver  
involvement;  no 
decrease
 in
 cl
inical
  performance
II-Stage 1-3
  
rash; stage 1
 gut involvement,
  or stage 1
 l
iver  involvement (or 
both); mi
ld decrease 
in cl
inical performance
III-Stage 2-3 skin rash; 2-3 gut 
involvement  or stage 2-4 l
iver involvement
(or both); marked decrease in cl
inical
  performance
IV-Simi
lar  to Grade III wi
th stage 2-4 organ involvement  and extreme 
decrease
 in
 cl
inical
  performance
3. Record
 biopsy
  resul
ts pertaining
  to
 GVHD for this  assessment period:
  
  
4. Treatment
 for
 GVHD
  
   
   
  No treatment necessary
  
   
   
  Tre 
a
 
tme nt 
ini
tia
 
te d  sinc 
e
 
 la 
st visi
t
  
   
   
  Treatment continued
  from
 last visi
t
  
   
   
  Treatment
 modified
  since
 last
 visi
t
  
   
   
   If participant is receiving treatment,
 specify:
a. Treatment regimen
  and
 any
 modifications
  
   
  
 Negative Positive Equivocal Not
 Done 
a.  Skin  
 
 
 
b.
 Gut
 
 
 
 
c.  Liver
 
 
 


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b.
Describe participant’s response to treatment
Comments:


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APPENDIX 7. CHRONIC GVHD ASSESSMENT WORKSHEET


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APPENDIX 8. STUDY REGIMEN WITH DE-SENSITIZATION SCHEDULE FOR 
SENSITIZED RECIPIENTS
-10
-8
-4
-9
-7 -6 -5
-3
3 4 5
26
0
-1
-2
1 
5
Screening
30 
38
5 yrs
Kidney &
Bone marrow 
transplant
Total Body 
Irradiation
ATG
HDCy,  
MESNA, 
anti-emetics
MMF
Taper
Tacrolimus
Taper
Off 
Immunosuppression
Prednisone
34
42
Taper
Fludarabine 
followed by 
hemodialysis
LDCy
Week
Day
50
1 2
*TPE/CMVIG
-11
-13
*
*
*
*
*
*
*
MMF
Tacrolimus
Figure 6: Study regimen for sensitized recipients
Sensitized participants with detectable DSA will undergo de-sensitization procedures per JHH 
standard of care as described below. Participants must demonstrate a negative flow cytometric 
crossmatch by day –9 to receive the first dose of study medication (ATG).
Sensitized participants with detectable DSA will receive therapeutic plasma exchange (TPE) 
treatments on alternating days on day -13 through day -1 prior to kidney transplantation. One 
and one-half plasma volume exchanges will be performed with each TPE treatment. Plasma 
volume will be reconstituted with human albumin, in most cases, although fresh frozen plasma 
may be used under certain circumstances. Pooled hyperimmune anti-CMV intravenous immune 
globulin (CMVIG) (Cytogam, MedImmune, Gaithersburg, MD) will be administered at 
100mg/kg after each TPE.
On day -13, participants will begin treatment with tacrolimus and mycophenolate mofetil 
(MMF). Tacrolimus will be dosed orally to a target trough level of 8-10 ng/dL. MMF will be 
dosed at 1-2 grams per 24 hours. Participants will stop tacrolimus and MMF therapy on day-7.
DSA will be monitored frequently in the immediate post-operative period. Participants who 
demonstrate an increase in DSA will receive additional TPE and CMVIG treatments per 
standard of care.


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APPENDIX 9. DE-SENSITIZATION SCHEDULE OF EVENTS PER JHH
STANDARD OF CARE16
 Appendix 1A 
Study Day 
-14 to -9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
Post-transplant 
Sensitized Recipient 
Treatment Day  
-13 
-12 
-11 
-10 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
-2 
-1 
0 
PT2 
Twice in first 
week, then 
weekly up to 2 
months, months 
3, 6, 9 & 12 
HLA alloantibodies 
includes DSA, flow 
PRA 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Therapeutic Plasma 
Exchange (TPE) 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
IVIG 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
Tacrolimus 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
MMF 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
Flow cytometric 
cross-match3,4 
X 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
16 Appendix 9 will not be captured in electronic data capture forms
2 DSA will be monitored frequently in the immediate post operative period. Participants who 
demonstrate an increase in DSA will receive additional TPE and CMVIG treatments per standard of 
care at JHH.
3 If the sensitized participant has not demonstrated a negative flow cytometric crossmatch by day -9 they 
will be considered a screen failure and terminated from the study. A negative flow cytometric 
crossmatch on either day -10 or day -9 is acceptable.
4 Flow cytometric crossmatch may be virtual or actual as needed.


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APPENDIX 10. REINITIATION OF CONDITIONING REGIMEN
Recipients who receive any part of the conditioning regimen but are unable to undergo 
combined bone marrow and kidney transplant due to unanticipated issues with the donor 
harvest may undergo a second course of conditioning in order to proceed with the combined 
bone marrow and kidney transplant (CKBMT) per protocol.  
The recipient may undergo CKBMT using the same donor or a different donor. 
The determination of when to administer reconditioning and how much reconditioning, if any, 
is necessary to proceed with the transplantation, will be left to the discretion of the investigator 
and may be adjusted based on the amount of conditioning initially received and the time 
elapsed since the last dose received by the recipient. The general intent is for reconditioning 
with the full conditioning regimen. 
Recipients may reinitiate the conditioning regimen no more than once. Recipients who are 
unable to undergo CKBMT after receiving a second course of conditioning will move to Safety 
Follow up (Appendix 5).Eligibility Criteria for Reinitiation of Conditioning.
INCLUSION CRITERIA
Recipient
If reinitiation of conditioning occurs more than 26 weeks after the first attempt, the recipient 
must fulfill all inclusion criteria in section 4.1.1, with the exception of donor and recipient 
HLA typing. In the event a new donor is used, the donor should undergo HLA typing.
If reinitiation of conditioning occurs within 26 weeks of the first conditioning attempt, recipient 
participants must meet all of the following eligibility criteria to reinitiate conditioning.
1.
&RPSOHWH$1&UHFRYHU\GHILQHGDV$1&PP3 for three consecutive 
measurements over three to five days.
2.
Recipient of a first renal allograft from an HLA-haploidentical, living related donor. 
The donor and recipient must be HLA identical for at least one allele (using high 
resolution DNA based typing) at the following genetic loci: HLA-A, HLA-B, HLA-C, 
and HLA-DRB1. Fulfillment of this criterion shall be considered sufficient evidence 
that the donor and recipient share one HLA haplotype. HLA typing reconfirmation is 
required only if a new donor is used.
3.
ABO compatibility with donor.
4.
DSA will be assessed by the local laboratory 30 days or less prior to transplant using 
solid phase micro particle technology (by Luminex® phenotype panel or Luminex 
single antigen bead test.) The following criteria apply:
a.
Participants without detectable DSA will be deemed eligible if they meet other 
entry criteria.
b.
Participants with detectable DSA and a positive flow cytometric crossmatch may 
undergo de-sensitization per standard of care if they are cytotoxic crossmatch 
negative. Such participants must demonstrate a negative flow cytometric 


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crossmatch by day -9 in order to receive the first dose of study therapy (ATG). 
Participants who do not demonstrate an acceptable response to de-sensitization by 
day -9 will be considered screen failures and will be terminated from the study.
c.
Participants with a positive cytotoxicity crossmatch will be excluded. 
5.
If recipient had a positive QuantiFERON® (QFT) assay upon study entry, recipient must 
have a negative chest x-ray prior to proceeding with reinitiation of the conditioning 
regimen. 
6.
For women of childbearing potential, a negative serum or urine pregnancy test with 
sensitivity less than 50 mIU/m within 72 hours before the start of study medication.
Pulmonary function and cardiac tests may be repeated if deemed necessary by the investigator.
Donor
Donor participants must meet all of the following criteria to be eligible, regardless of whether 
they are the initial donor or a new donor:
1.
HLA-haploidentical, first-degree relatives or half-siblings of the recipient participant at the 
allele or allele group. The donor and recipient must be HLA identical for at least one allele 
(using high resolution DNA based typing) at the following genetic loci: HLA-A, HLA-B, 
HLA-C and HLA-DRB1. Fulfillment of this criterion shall be considered sufficient 
evidence that the donor and recipient share one HLA haplotype. Reconfirmation is required 
only if a new donor is used.
2.
Age 18 to 65 years.
3.
Creatinine clearance >80 ml/minute as measured from a 24 hour urine collection within 26 
weeks of the screening visit. If a serum creatinine drawn at the screening visit is > 20% 
higher than the serum creatinine drawn at the time of the 24 hour urine collection, the 
creatinine clearance must be re-evaluated by a repeat 24 hour urine test. If the new value is 
PJG/ WKHGRQRUZLOO EHH[FOXGed. A repeat 24 hour urine collection is not required if 
the same donor is used.
4.
Meets institutional selection criteria for organ and bone marrow donation.
5.
Ability to understand and provide informed consent for all study procedures including 
kidney transplant and bone marrow harvest.
6.
Serologic evidence of prior EBV infection as documented by positive IgG and negative 
IgM antibodies against EBV. EBV status does not need to be reconfirmed if the initial 
donor is used.
EXCLUSION CRITERIA
Recipient
Recipient participants who meet any of the following criteria will not be eligible to re-initiate 
conditioning:
1.
Positive HIV-1 or HIV-2 nucleic acid test.


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2.
Evidence of hepatitis B infection. Participants demonstrating any one of the following will 
be excluded:
a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM
c.
positive anti-HBc IgG
d.
positive HBV PCR
3.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV RNA PCR. All 
positive HCV antibody results must be assessed by an EIA assay and confirmed by a 
quantitative serum HCV RNA assay. Participants with positive HCV antibodies but 
undetectable serum HCV RNA may be considered for eligibility. Participants with 
negative anti-HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV serologies.
4.
Positive cytotoxic crossmatch.
5.
Calculated PRA greater than 90%.
6.
Any active, severe local or systemic infection at the screening visit.
7.
Autoimmune disease requiring immunosuppressive drugs for maintenance.
8.
Use of investigational drug, other than the study medications specified by the protocol, 
within 30 days of transplantation.
9.
Receipt of a live vaccine within 30 days of receipt of study therapy.
10. Women who are breastfeeding.
11. The presence of any medical condition that the Investigator deems incompatible with 
participation in the trial.
Donor
Donor participants who meet any of the following criteria will not be eligible for this 
study, regardless of whether they are the initial donor or a new donor:
1.
History of type I or type II diabetes mellitus.
2.
History of severe cardiovascular disease, defined as New York Heart Association Class 
III or IV2.
3.
History of blood product donation to recipient.
4.
History of positive HIV-1 or HIV-2 serology or nucleic acid test. 
5.
Evidence of hepatitis B infection. Participants demonstrating any one of the following 
will be excluded:
a.
positive hepatitis B surface antigen (HBsAg) or
b.
positive anti-HBc IgM
c.
positive anti-HBc IgG
d.
positive HBV PCR
6.
Positive anti-hepatitis C (HCV) antibodies and a positive serum HCV RNA PCR. All 
positive HCV antibody results must be assessed by an EIA assay and confirmed by a 
quantitative serum HCV RNA assay. Participants with positive HCV antibodies but 
undetectable serum HCV RNA may be considered for eligibility. Participants with 


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ACCEPTOR
negative anti-HCV antibodies but unexplained liver enzyme abnormalities must 
undergo a quantitative serum RNA assay to rule out false negative HCV serologies.
7.
Autoimmune disease requiring immunosuppressive drugs for maintenance.
8.
The presence of any medical condition that the Investigator deems incompatible with 
participation in the trial.
Monitoring of Recipient Between Conditioning Regimens
The recipient’s blood counts will be monitored closely until complete ANC recovery, defined 
DV$1&PP3 for three consecutive measurements over three to five days. The recipient 
will receive antimicrobial prophylaxis per JHU standard of care.

SAP_001.pdf: 
 
 
 
STATISTICAL ANALYSIS PLAN 
31 August 2017 
 
 
 
 
Bone Marrow Transplantation and High Dose Post-Transplant 
Cyclophosphamide for Chimerism Induction and Renal Allograft 
Tolerance 
 
 
 
PROTOCOL NUMBER ITN054ST 
IND # 118,975 
 
 
SPONSOR 
 
This clinical study is supported and conducted by the Immune Tolerance Network, which is 
sponsored by the National Institute of Allergy and Infectious Diseases. 
 
Immune Tolerance Network 
185 Berry Street, Suite 3515 
San Francisco, CA 94107 
 
National Institute of Allergy and Infectious Diseases 
6610 Rockledge Drive, Room 3053 
Bethesda, MD, 20892-6601 
 
 
 
PREPARED BY 
 
Rho, Inc. 
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Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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DOCUMENT VERSION CONTROL 
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28 August 2017 
First Version 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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TABLE OF CONTENTS 
1. 
ANALYSIS PLAN SYNOPSIS .................................................................................. 5 
2. 
REFERENCES ......................................................................................................... 7 
 
 


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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1. ANALYSIS PLAN SYNOPSIS 
Accrual Objective 
The accrual goal for this study was six renal transplant recipient-donor pairs 
who meet the per- protocol (PP) analysis sample definition. The protocol was 
stopped after accruing one transplanted subject due to a re-assessment of the 
risks related to the study. 
Study Design 
This trial was designed to be a phase II, single arm, open-label, single center 
pilot study to assess a reduced-intensity conditioning regimen, bone marrow 
transplantation and high dose PT/Cy in six recipients of renal allografts from 
Human Leukocyte Antigen-haploidentical (HLA-haploidentical) living related 
donors  
Primary Endpoint 
The primary endpoint was designed to be the proportion of participants who 
achieved operational tolerance, defined as remaining off all immunosuppression 
52 weeks after completion of immunosuppression withdrawal with:  
a) no evidence of biopsy-proven allograft rejection and  
b) acceptable renal function, defined as a serum creatinine that has 
increased no more than 25% above baseline (see protocol section 6.5.1 
for baseline thresholds) at the primary endpoint visit.  
All participants who successfully complete immunosuppression withdrawal 
were to undergo a protocol biopsy at this time point to assess the primary 
endpoint.  
Secondary Endpoints 
Safety  
1. The incidence, severity and duration of graft versus host disease (GVHD) in 
transplanted participants.  
2. The incidence, severity and duration of engraftment syndrome in 
transplanted participants.  
3. The proportion of transplanted participants who died.  
4. The proportion of transplanted participants with acute renal allograft 
rejection demonstrated by a biopsy or clinically if a biopsy could not be 
performed. If participant had allograft dysfunction as defined in protocol 
section 6.5 and could not undergo biopsy he or she would be presumed to 
have rejection without biopsy confirmation.  
5. The histological severity of biopsies demonstrating acute rejection as 
measured by Banff Grade per Banff 2007 Classification Renal Allograft 
Pathology1.  
6. The proportion of transplanted participants with chronic T cell-mediated or 
antibody-mediated rejection. This assessment also included progressive 
interstitial fibrosis/tubular atrophy (IF/TA), transplant glomerulopathy or 
chronic obliterative arteriopathy without an alternative, non-rejection-
related cause. See Banff 2007 Classification Renal Allograft Pathology for 
definition of terms1.  
7. Time from transplant to the first episode of acute rejection requiring 
treatment.  
8. The incidence, severity and duration of adverse events including infection, 
wound complications, post-transplant diabetes, hemorrhagic cystitis and 
malignancy.  
9. The proportion of transplanted participants who developed donor specific 
antibody:  
a. 
after initiation of immunosuppression withdrawal  
b. at any time during trial participation  
10. The time to absolute neutrophil recovery. This was defined as the interval 
from the neutrophil nadir to the first day of three consecutive daily 
neutrophil counts ≥ 500 per μL. The neutrophil nadir was defined as the 
first day post-transplant on which the absolute neutrophil count (ANC) was  


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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below 500 per μL. 
11. The time to platelet count recovery. This is defined as the interval from 
transplant to the first day of a platelet count of 20,000 per μL without a 
prior platelet transfusion in the preceding seven days. 
Efficacy  
1. The proportion of transplanted participants who remained off 
immunosuppression for at least 52 weeks including those in whom the 52 
week biopsy was not performed.  
2. The proportion of participants who remained free from return to 
immunosuppression for the duration of the study.  
Mechanistic  
1. The correlation of operational tolerance with the extent and durability of 
donor hematopoietic and T cell chimerism as measured by serial short 
tandem repeat analysis of recipients’ peripheral blood mononuclear cells 
(PBMCs) and T cells.  
2. The correlation of operational tolerance with other biomarkers such as cell 
subsets or gene expression.  
The following secondary endpoints pertaining to safety and efficacy were to 
be assessed only in participants who completed tacrolimus withdrawal:  
1. Immunosuppression-free duration, defined as time from completion of 
tacrolimus to end of trial participation or to time of restarting 
immunosuppression.  
2. Time from completion of tacrolimus withdrawal to first episode of acute 
rejection or presumed acute rejection, defined per Banff 2007 Classification 
Renal Allograft Pathology1.  
3. Time from completion of tacrolimus withdrawal to first diagnosis of 
chronic T cell mediated or antibody-mediated rejection. This assessment 
also included IF/TA, transplant glomerulopathy or chronic obliterative 
arteriopathy without an alternative, non-rejection related cause. See Banff 
2007 Classification Renal Allograft Pathology1.  
Analysis Methods for 
Primary and 
Secondary Endpoints 
Due to the early study closure after one transplanted subject none of the 
endpoints will be analyzed. 
 
Safety Analysis 
All adverse events will be listed and classified by body system and preferred 
term according to MedDRA dictionary. All medication use will be listed. 
 
 
 
 


Immune Tolerance Network 
CONFIDENTIAL 
Statistical Analysis Plan 
Protocol No. ITN054ST 
 
31 August 2017 
Version:  1.0 
 
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2. REFERENCES 
1. Solez K, Colvin RB, Racusen LC, et al. Banff 07 classification of renal allograft 
pathology: updates and future directions. American journal of transplantation : official 
journal of the American Society of Transplantation and the American Society of 
Transplant Surgeons. 2008;8(4):753-760.