Allogeneic and Matched Unrelated Donor Stem Cell Transplantation for Congenital Immunodeficiencies or Patients With Autoinflammatory/Immunodysregulatory Conditions: Busulfan-Based Conditioning With Campath- 1H or h-ATG, Radiation, and Sirolimus
Phase: EARLY_PHASE1
Conditions: Inherited Immune Deficiencies
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

Allogeneic and Matched Unrelated Donor Stem Cell Transplantation for Congenital 
Immunodeficiencies: Busulfan-based Conditioning with Campath- 1H, Radiation, and 
Sirolimus 
 
 
National Institute of Allergy and Infectious Diseases (NIAID) / National Institutes of Health (NIH) 
 
Laboratory of Host Defenses (LHD) 
 
 
 
 
Principal Investigator:  
 
Elizabeth Kang, MD  
 
 
NIAID/LHD  
 
301-402-7567 
 
 
Associate Investigators: 
 
Harry L. Malech, MD  
 
 
NIAID/LHD  
 
301-480-6916  
Nana Kwatemaa, RN, BSN  
 
NIAID/LHD  
 
301-451-7820 
Effie Nomicos, RN, BSN 
 
 
NIAID/LHD  
 
301-402-5964 
Dianne Hilligoss, RN, CRNP  
 
NIAID/LHD  
 
301-594-5945 
Mary Garofalo, RN, BSN 
 
 
NIAID/LHD  
 
301-827-9758 
Suk See De Ravin, MD 
 
 
NIAID/LHD  
 
301-496-6772 
Corin Kelly, RN, BSN 
 
 
NIAID/LHD  
 
301-451-7906 
Sandra Anaya-O'brien, RN, MSN 
 
NIAID/LHD  
 
301-451-3088 
Deborah Citrin, MD 
 
 
NCI/ROB 
 
 
301-496-5457 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confidentiality Statement 
 
This document is confidential and is to be distributed for review only to investigators, potential investigators, consultants, study 
staff, and applicable independent ethics committees or institutional review boards.  The contents of this document shall not be 
disclosed to others without written authorization from NIAID (or others, as applicable), unless it is necessary to obtain informed 
consent from potential study participants. 
 

AlloPBSC and MUD Transplantation   
Dr. E. Kang 
Version  1.4, dated 12/20/06 
Page 2 of 72 
 
Protocol Face Sheet: 
 
Principal Investigator:   
Elizabeth Kang, MD 
Institute/Branch: 
NIAID/LHD 
Address: 
10/ CRC 6W-3752 
Phone: 
301-402-7567 
Fax: 
301-480-3502 
Protocol Number: 
07-I-0075 
Version Description: 
1.4 
Version Date  
11/27/2006  
Protocol Title: 
Allogeneic and Matched Unrelated Donor Stem Cell 
Transplantation for Congenital Immunodeficiencies: 
Busulfan-based 
Conditioning 
with 
Campath-1H, 
Radiation, and Sirolimus 
Abbreviated Title: 
AlloPBSC and MUD Transplant 
IRB Name: 
 
NIAID 
Institute Name: 
 
NIAID 
Proposed Dates: 
 
Start-12/1/2006 
End-12/1/2011 
Total Subjects to be accrued: 
30 
Ionizing Radiation Use:  
Yes 
Is tissue being collected  
for Research Purposes? 
Yes 
Location of the Study: 
Clinical Research Center 
IND No.: 
 
 
   100353 EXEMPT 
Name of Agent/Device: 
Busulfan, Campath-1H, Sirolimus, TBI 
Research Contact: 
Nana Kwatemaa, RN, BSN 
Address: 
 
Bldg 10 CRC, Room 6W-3750 
Phone: 
 
301-451-7820 
Fax: 
 
301-451-5482 
Email: 
 
nkwatemaa@niaid.nih.gov 
Medically Responsible Investigator: 
Elizabeth Kang, MD 
Institute/Branch: 
 
NIAID/LHD 
Phone: 
 
301-402-7567 
Fax: 
 
301-480-3502 
 
 
 

AlloPBSC and MUD Transplantation   
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Version  1.4, dated 12/20/06 
Page 3 of 72 
Précis 
Congenital immunodeficiencies – including chronic granulomatous disease, leukocyte adhesion 
deficiency and others – comprise a group of disorders in which the immune system fails to 
develop normally due to a genetic defect.  As a result, affected patients suffer from recurrent 
infections and have a significantly shortened life expectancy.  The current management of these 
patients is limited to close surveillance for infections, administration of prophylactic 
antimicrobials, and rapid and aggressive treatment of suspected and documented infections with 
broad-spectrum antibiotics.  Although often effective, these treatments can require long 
hospitalizations, impacting on the overall quality of life significantly, and lead to significant 
morbidity, such as renal failure and deafness.  
 
Currently, the only available cure for these disorders is bone marrow transplantation, which most 
commonly uses an HLA-matched related sibling as the donor (Allogeneic Stem Cell 
Transplantation). However, as only 30% of patients in the general population have an HLA- 
matched related sibling, allogeneic related transplantation is often not an option, resulting in the 
need for matched unrelated donor transplantation. The National Marrow Donor Program serves 
as both a national registry of volunteers who are willing to donate progenitor cells to eligible 
recipients as well as a repository of cord blood products. Despite continued improvement in the 
use of transplantation schemas – including the development of nonmyeloablative regimens – 
there remain significant morbidity and mortality associated with transplantation, in particular, 
graft versus host disease (GvHD).  
 
GvHD is a result of the graft recognizing host antigens as foreign, typically in the presence of 
inflammation, and results in a type of iatrogenic autoimmune disease. For patients with non-
malignant diseases, the aim of the transplant is solely to replace the defective or deficient cell 
population. Furthermore, as a graft versus tumor effect is not required, regimens designed to 
establish tolerance induction and/or stable mixed chimerism may be preferable for cure in this 
patient population; therefore, alternate transplant strategies can and should be used to further 
suppress the development of any GvHD effects.  To reduce the morbidity and mortality 
associated with transplantation, we propose to use a combination of uniquely designed 
conditioning regimens to achieve adequate engraftment in congenitally immunodeficient 
patients, using either alloPBSC transplantation for patients with an HLA-matched related sibling 
donor, or MUD transplantation for those without an appropriate HLA-matched related sibling 
donor. For the alloPBSC transplantation (Group 1), we propose using a novel busulfan-based, 
nonmyeloablative conditioning regimen combined with Campath-1H, an immunosuppressive 
monoclonal antibody, and sirolimus, a tolerance inducing immunosuppressant used for GvHD 
prophylaxis.  For the MUD transplantation (Group 2), we will also use a similar conditioning 
regimen, with a few modifications (due to the increased risk of graft rejection with HLA-
matched but unrelated cells) to perform matched unrelated and cord blood transplantation in 
patients with immunodeficiencies. Given its novelty, this combination will be tested in a pilot 
trial and will be compared to historical controls.  
 

AlloPBSC and MUD Transplantation   
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Version  1.4, dated 12/20/06 
Page 4 of 72 
 
TABLE OF CONTENTS 
 
PRÉCIS 
........................................................................................................................... 3 
1.0 
INTRODUCTION .......................................................................................... 10 
1.1 
Design ............................................................................................................. 10 
1.2 
Study Objectives ............................................................................................. 10 
1.2.1 
Primary Objectives.......................................................................................... 10 
1.2.2 
Secondary Objectives...................................................................................... 10 
1.3 
Background ..................................................................................................... 11 
1.3.1 
Chronic Granulomatous Disease..................................................................... 11 
1.3.2 
Leukocyte Adhesion Deficiency ..................................................................... 12 
1.3.3 
CD40 Ligand Deficiency ................................................................................ 13 
1.3.4 
National Marrow Donor Program ................................................................... 13 
1.4 
Scientific and Clinical Justification ................................................................ 14 
1.5 
Study Agent .................................................................................................... 15 
1.5.1 
Total Body Iradiation ...................................................................................... 16 
1.5.2 
Busulfan .......................................................................................................... 16 
1.5.3 
Alemtuzumab (Campath-1H).......................................................................... 17 
1.5.4 
Sirolimus (Rapamune®/rapamycin) ................................................................ 18 
1.6 
Qualifications of Investigators ........................................................................ 19 
1.7 
Conflict of Interest .......................................................................................... 19 
1.8 
Conduct of the Study ...................................................................................... 20 
2.0 
STUDY DESIGN............................................................................................ 20 
2.1 
Endpoints ........................................................................................................ 20 
2.1.1 
Primary Endpoint ............................................................................................ 20 
2.1.2 
Secondary Endpoints ...................................................................................... 20 
2.2 
Sample Size Justification ................................................................................ 20 
2.2.1 
Rationale for Subject Selection ....................................................................... 20 
2.2.2 
Donors of Allogeneic Stem Cells ................................................................... 20 
3.0 
SUBJECT ENROLLMENT............................................................................ 21 
3.1 
Patient Recruitment ......................................................................................... 21 
3.2 
Inclusion/Eligibility Criteria ........................................................................... 21 
3.2.1 
Patients (Recipient) ......................................................................................... 21 
3.2.2 
Allogeneic Donor (Sibling donor only where the collection is done at 
NIH) ................................................................................................................ 22 
3.3 
Exclusion Criteria ........................................................................................... 23 
3.3.1 
Patient (Recipient) .......................................................................................... 23 

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3.3.2 
Allogeneic Donor (Sibling donor only where the collection is done at 
NIH) ................................................................................................................ 23 
3.4 
Study Enrollment Procedures ......................................................................... 23 
3.4.1 
Consent Obtainment:  Allogeneic Recipients and Donors ............................. 23 
3.4.2 
Consent for Unrelated Donors ........................................................................ 24 
3.4.3 
Participation of Children ................................................................................. 24 
4.0 
STUDY IMPLEMENTATION ...................................................................... 25 
4.1 
Criteria for Withdrawal ................................................................................... 25 
4.1.1 
Patient’s (Recipient’s) Voluntary Withdrawal ................................................ 25 
4.1.2 
Allogeneic Donor’s Voluntary Withdrawal .................................................... 25 
4.1.3 
Involuntary Withdrawal of a Patient (Recipient) ............................................ 25 
4.1.4 
Involuntary Withdrawal of an Allogeneic Donor ........................................... 26 
4.2 
Study Method/Parameters ............................................................................... 26 
4.2.1 
Patient (Recipient) – Initial Screening ............................................................ 26 
4.2.2 
Patient (Recipient) – Baseline Testing and Evaluation................................... 26 
4.2.3 
Allogeneic Donor – Initial Screening ............................................................. 27 
4.2.4 
Allogeneic Donor Baseline – Testing and Evaluation .................................... 27 
4.2.5 
NMDP Protocol .............................................................................................. 28 
4.2.6 
Cord Blood Tests ............................................................................................ 28 
4.3 
Allogeneic Donor Intervention ....................................................................... 28 
4.3.1 
Mobilization and Collection of Allogeneic Peripheral Blood Progenitor 
Cells  (see Appendix C) .................................................................................. 28 
4.3.2 
Allogeneic Peripheral Blood Progenitor Cell Transplant ............................... 28 
4.3.3 
Unrelated Donor Product Selection ................................................................ 29 
4.3.4 
MUD Recipient Apheresis .............................................................................. 29 
4.4 
Patient (Recipient) Intervention ...................................................................... 29 
4.4.1 
Central Venous Line Placement...................................................................... 29 
4.4.2 
Treatment Plan ................................................................................................ 29 
4.4.3 
Group 1 -Preparative Regimen Schema (Daily Management) for 
Allogeneic Recipients ..................................................................................... 30 
4.4.4 
Group 2- Preparative Regimen Schema (Daily Management) for Matched 
Unrelated and Cord Blood Recipients ............................................................ 31 
4.4.5 
Post Transplant Monitoring  (Day +1 to Discharge) ...................................... 34 
4.4.6 
Hospital Discharge Criteria ............................................................................. 34 
4.4.7 
Discharge to Day 100 (Out-Patient) ............................................................... 35 
4.4.8 
Beyond Day 100 ............................................................................................. 35 
4.4.9 
Post Transplant Medical Management............................................................ 35 
4.4.10 
Management of Patient Complications ........................................................... 36 
5.0 
STUDY MODIFICATIONS ........................................................................... 37 

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6.0 
ANALYSIS OF THE RESEARCH STUDY .................................................. 37 
6.1 
Chimerism Studies .......................................................................................... 37 
6.2 
Bone Marrow Samples .................................................................................... 37 
6.3 
Transthoracic Echocardiography .................................................................... 37 
7.0 
DATA AND SAFETY MONITORING ......................................................... 37 
8.0 
ADVERSE EVENTS ...................................................................................... 38 
8.1 
Adverse Events Reporting Plan ...................................................................... 38 
8.2 
Toxicity Criteria .............................................................................................. 40 
8.3 
Recipients’ Adverse Events ............................................................................ 41 
8.3.1 
AE for the Screening Period ........................................................................... 41 
8.3.2 
Adverse Events for Enrolled Subjects ............................................................ 42 
8.4 
Donors ............................................................................................................. 43 
8.5 
Serious Adverse Events .................................................................................. 43 
8.6 
Reporting of Serious Adverse Events to the Principal Investigator ............... 44 
8.7 
Adverse Event Reporting Requirements to the NIAID IRB ........................... 44 
8.8 
Reporting Serious Adverse Events to the IND Sponsor ................................. 45 
8.9 
Reporting Serious Adverse Events to DSMB  ................................................ 45 
9.0 
HUMAN SUBJECT PROTECTIONS ........................................................... 45 
9.1 
Recruitment Plans and Procedures .................................................................. 45 
9.2 
Institutional Review Board ............................................................................. 45 
9.3 
Protocol Amendments and Study Termination ............................................... 46 
9.4 
Informed Consent............................................................................................ 46 
9.5 
Privacy and Confidentiality Provisions .......................................................... 46 
9.6 
Policy Regarding Research-Related Injuries .................................................. 46 
9.7 
Remuneration .................................................................................................. 46 
9.8 
Hazards and Discomforts – Recipient ............................................................. 47 
9.8.1 
Related to the Transplant ................................................................................ 47 
9.8.2 
Related to Radiation ........................................................................................ 47 
9.8.3 
Related to Alemtuzumab (Campath-1H, Campath®) ...................................... 48 
9.8.4 
Related to Sirolimus ........................................................................................ 49 
9.8.5 
Related to Busulfan ......................................................................................... 51 
9.9 
Hazards and Discomforts – Donor .................................................................. 52 
9.9.1 
Related to G-CSF ............................................................................................ 52 
9.9.2 
Related to Central Line Placement ................................................................. 52 
9.9.3 
Related to Apheresis ....................................................................................... 53 
9.10 
Risks in Relation to Benefit ............................................................................ 53 
9.10.1 
For Adult Transplant Subjects ........................................................................ 53 
9.10.2 
For Pediatric Donors ....................................................................................... 53 

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9.10.3 
For Pediatric Donors Involved in Laboratory Research Studies .................... 53 
9.11 
Informed consent ............................................................................................ 54 
10.0 
BIOSTATISTICAL CONSIDERATIONS ..................................................... 54 
10.1 
Stopping Rules ................................................................................................ 55 
11.0 
PHARMACEUTICALS ................................................................................. 55 
11.1 
Alemtuzumab (Campath-1H, Campath®) ....................................................... 55 
11.2 
Daclizumab (Zenapax® ) ................................................................................. 56 
11.3 
Human Recombinant Granulocyte Colony Stimulating Factor ...................... 56 
11.4 
Infliximab (Remicade® ) ................................................................................. 57 
11.5 
Sirolimus (Rapamune®) .................................................................................. 58 
11.6 
Busulfan .......................................................................................................... 58 
12.0 
BIOHAZARD CONTAINMENT ................................................................... 59 
13.0 
DATA COLLECTION, MANAGEMENT AND STORAGE ....................... 59 
13.1 
Data and Safety Monitoring Plan .................................................................... 59 
13.2 
Study Monitoring ............................................................................................ 59 
14.0 
PLAN FOR USE AND STORAGE OF BIOLOGICAL SAMPLES ............. 60 
15.0 
REFERENCES: .............................................................................................. 61 
Appendix A 
GvHD Prophlaxis: Sirolimus .......................................................................... 67 
Appendix B  Transfusion of Red Cells to Recipients of ABO Incompatible Marrow……..68 
Appendix C: Immunodeficiency AlloBMT DONOR………………………………………..69 
 
Please see the excel spreadsheet for the following appendices: 
Appendix D: Patient/Recipient AlloBMT Initial Screening 
Appendix E: Group 1: AlloBMT Recipient INPATIENT schedule of event 
Appendix F: Group 2: MUD Recipient INPATIENT schedule of events 
Appendix G: AlloBMT and MUD Recipient DISCHARGE TO DAY +100 
Appendix H: AlloBMT and MUD Recipient BEYOND DAY 100 
Appendix I: Study Drug Information Sheet, pdf format.

AlloPBSC and MUD Transplantation   
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Version  1.4, dated 12/20/06 
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ABBREVIATION LIST  
 
Abbreviation  
Text 
Ab 
Antibody  
ADA-SCID 
Adenosine deaminase deficient severe combined 
immunodeficiency  
AI 
Associate Investigator 
AlloPBSC 
Allogeneic peripheral blood stem cell (transplantation) 
ANC 
Absolute neutrophil count  
ARDS 
Acute respiratory distress syndrome 
ATG 
Anti-thymocyte globulin  
AUC 
Area under the curve  
BMT 
Bone marrow transplantation  
BUN 
Blood urea nitrogen  
CBC 
Complete blood count 
CC 
Clinical Center  
CGD 
Chronic Granulomatous Disease  
cGY 
Centigray 
CLAD  
Canine leukocyte adhesion deficiency 
CMV 
Cytomegalovirus  
CRF 
Case report form  
DHR 
Dihydrorhodamine 123 (assay)  
DLCO 
Diffusing capacity for the lungs measured using carbon monoxide  
DSMB 
Data and Safety Monitoring Board  
DTM 
Department of Transfusion Medicine  
EBV 
Epstein Barr virus 
ECOG 
Eastern Cooperative Oncology Group  
ECP 
Extracorporeal photopheresis  
ESR 
Erythrocyte sedimentation rate  
FEV-1 
Forced expiratory volume in 1 second 
FVC 
Forced vital capacity  
GCP 
Good Clinical Practice 
G-CSF 
Granulocyte colony stimulating factor  
GvHD 
Graft versus host disease  
HB 
Hematology Branch  
Hb 
Hemoglobin  
HBV 
Hepatitis B virus 
HCV 
Hepatitis C virus 
HIV 
Human immunodeficiency virus 
HLA 
Human leukocyte antigen  
HSC 
Hematopoietic stem cell 
HTLV-I/II 
Human T cell lymphotrophic virus type I, type II 
HVOD 
Hepatic veno-occlusive disease  

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Abbreviation  
Text 
ICH 
International Conference on Harmonisation  
IgM, IgG, IgA 
Immunoglobulin M, G, A 
IL-2 
Interleukin 2  
IRB 
Institutional Review Board  
IV 
Intravenous 
IVIG 
Intravenous immunoglobulin  
LAD 
Leukocyte Adhesion Deficiency  
LHD 
Laboratory of Host Defenses  
MUGA 
Multi-acquisition gated  
MUD 
Matched unrelated donor 
NCI-CTC 
National Cancer Institute-Common Toxicity Criteria  
NMDP 
National Marrow Donor Program 
NHLBI 
National Heart, Lung, and Blood Institute  
NIAID 
National Institute of Allergy and Infectious Diseases 
NIDDK 
National Institute of Diabetes and Digestive and Kidney Diseases  
NIH 
National Institutes of Health 
PBPC 
Peripheral blood progenitor cells  
PBSC 
Peripheral blood stem cells 
PCP 
Pneumocystis carinii pneumonia  
PCR 
Polymerase chain reaction 
PI 
Principal Investigator 
PO 
Latin per os (by mouth) 
PPD 
Purified protein derivative  
PUVA 
Combination of psoralen and long-wave ultraviolet radiation 
QD 
Latin quaque die (every day) 
RBC 
Red blood cells  
RCHSPB 
Regulatory Compliance and Human Subjects Protection Branch  
RPR 
Rapid plasma reagin  
SAE 
Serious adverse event  
SCID 
Severe combined immunodeficiency 
SGOT 
Serum glutamic oxaloacetic transaminase  
SGPT 
Serum glutamic pyruvic transaminase  
SOP 
Standard operating procedure  
ß-hCG 
Beta-human chorionic gonadotropin  
TBI 
Total body irradiation 
TNF-α 
Tumor necrosis factor-alpha  
UCB 
Umbilical cord blood 
 

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Version  1.4, dated 12/20/06 
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1.0 INTRODUCTION 
1.1 Design 
This is an open-label pilot study designed to evaluate whether the use of uniquely 
designed conditioning regimens reduces the occurrence of graft versus host disease 
(GvHD) after a bone marrow transplantation (BMT) for congenitally immunodeficient 
patients using a matched sibling donor (allogeneic) or matched unrelated donor 
(peripheral blood stem cells [PBSC] or cord blood), while still achieving adequate 
engraftment for normal immune function post transplant.  For this study there will be two 
groups: Group 1 will be congenitally immunodeficient patients with a matched sibling 
donor and Group 2 will be congenitally immunodeficient patients who do not have a 
matched sibling donor. For patients in Group 1, we will use a novel busulfan-based 
nonmyeloablative-conditioning regimen combined with Campath-1H (a humanized 
immunosuppressive monoclonal antibody) and graft versus host disease (GvHD) 
prophylaxis with sirolimus (a tolerance inducing immunosuppressant). For patients in 
Group 2, we will use also use a busulfan-based nonmyeloablative-conditioning regimen 
with Campath-1H, but combine it with total body irradiation (TBI) and GvHD 
prophylaxis with sirolimus. These drugs are FDA approved and all are well-known drugs 
used in various transplant regimens; however, they have neither been used in this 
combination nor at these doses (busulfan) as specified in Section 4.4.  As part of this 
study, we will also assess the level and kinetics of immune reconstitution in patients 
receiving these drugs.  
 
 
1.2 Study Objectives 
1.2.1  Primary Objectives 
The primary objective for this study is to evaluate the use of Campath-1H  and sirolimus 
in conjunction with a novel busulfan-based conditioning regimen with or without the 
addition of radiation to decrease or eliminate the occurrence of graft versus host disease 
after either allogeneic or matched unrelated (including cord blood) transplantation in 
patients with congenital immunodeficiencies.  The goal is to attain an engraftment rate of 
100% with no occurrence of Grade 3 or higher GvHD. 
 
1.2.2  Secondary Objectives 
 To further elucidate the required dose levels of busulfan as compared to historical 
controls in conditioning regimens for transplantation in general 
 To measure the engraftment rate and the engraftment kinetics using such a 
regimen 
 To assess the level and kinetics of immune reconstitution when using these 
conditioning regimens. 
 To further elucidate the factors involved in the development of graft versus host 
disease. 

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1.3 Background  
1.3.1  Chronic Granulomatous Disease 
Chronic Granulomatous Disease (CGD) is one of a group of inherited disorders affecting 
neutrophil function.  Due to a mutation in one of the genes encoding the phagocyte 
NADPH oxidase, patients with CGD are susceptible to recurrent bacterial and fungal 
infections.  Besides life threatening infections, these patients develop granulomas that can 
result in genitourinary or gastrointestinal tract obstruction.  Iatrogenic or infection related 
end organ damage has also been seen in long-term survivors of CGD.  Despite 
improvements in infection prevention and control, 2% of patients with CGD die each 
year, and few patients survive to age 50 [1]. 
 
Treatment of CGD - Prophylactic Antibiotics and Interferon gamma 
All CGD patients receive prophylactic antibiotic coverage with agents such as 
trimethoprim/sulfamethoxazole.  Interferon gamma therapy has been shown to decrease 
the incidence of infection, and is therefore also part of the prophylactic regimen.  It is, 
however, not well tolerated, and long-term compliance is rare.  When infections do arise, 
emphasis is placed on identifying the pathogen and treating with the appropriate 
antibiotic for a prolonged period of time.  When granuloma formation complicates an 
infection, a course of steroids is often effective [2]. 
 
Gene Therapy for CGD 
The genes responsible for the four forms of CGD have been cloned.  A number of small 
animal studies including xenogeneic models have shown proof of principal using vectors 
corrective for both the p47 mutation and the gp91 mutation. [3-5]  Results of a Phase I 
clinical trial of gene therapy for patients with the p47-deficient form of CGD have also 
been reported as well as a more recent study treating patients with the gp91 mutations [6].  
The authors demonstrated that autologous hematopoietic progenitors can be transduced 
with a retroviral vector containing the missing gene.  These progenitor cells were then 
safely infused back into the patient, and small numbers of gene-corrected neutrophils 
were detectable in the peripheral blood for up to 6 months.  However, without a selective 
advantage conferred upon the transduced cells, there was insufficient engraftment and/or 
proliferation to sustain a long-term cure.  More recently, a German-Swiss collaborative 
study has resulted in two patients achieving clinical benefit after receiving genetically 
transduced cells with the use of busulfan preconditioning.  Both patients maintained a 
level of 20% of corrected cells in the peripheral blood [7]. Most recently, the first patient 
succumbed to a CGD related infection felt to be secondary to poor expression of the gp91 
protein on a per cell level. We are now in the process of establishing a new clinical gene 
therapy trial for patients with X-linked CGD; however, only those patients with an 
ongoing infection, without an HLA-matched sibling donor, and without evidence of bone 
marrow dysfunction will be eligible as it is still experimental in nature and, unlike 
allogeneic transplantation, does not have a proven track record of benefit.  For patients 
without an underlying infection, and without a matched sibling donor, transplantation 
with cells obtained from an unrelated donor remains the only other treatment option. 
 
 

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Allogeneic Transplantation:  
Allogeneic transplantation has been used successfully to cure patients with CGD.  The 
vast majority of successful transplants have used myeloablative conditioning regimens.  
The toxicity and associated morbidity and mortality of these regimens, however, have 
limited the widespread use of allo transplants as a therapeutic option for many patients 
and their referring physicians.  More recent efforts using nonmyeloablative regimens 
have reduced mortality and morbidity, but these regimens have simultaneously incurred 
an increased rate of graft rejection.  In addition, despite an overall reduction in toxicity, 
patients still experienced significant GvHD  as in the study performed here, where 2 of 15 
patients died within one year of transplantation. [20]  A third patient died suddenly from 
sepsis 2 years post transplant, although there was no obvious evidence of GvHD and the 
patient had had a successful engraftment.  However of the remaining patients, including 
those who did not engraft, all are alive.   A number of these survivors have successfully 
undergone second transplants with good results. Thus, alternative regimens are needed to 
improve initial engraftment and further reduce the GvHD incidence. 
 
Matched Unrelated Donor Transplantation: 
To date, 5 patients have undergone matched unrelated donor transplantation using either 
bone marrow or cord blood;  2 patients rejected the graft, and the other 3 have had 
successful engraftment with follow up more than 1 year post transplant.  Of those 
engrafted, only 1 had GvHD.  [8, 9] 
 
1.3.2  Leukocyte Adhesion Deficiency  
Leukocyte Adhesion Deficiency (LAD) is an autosomal recessive illness resulting from a 
genetic mutation of the CD18 gene that encodes the ß2 subunit of the leukocyte integrin 
family.  As a result, the neutrophils of these patients are unable to migrate and adhere to 
sites of infection.  Children are then prone to recurrent bacterial infections and, in fact, 
are typically diagnosed at birth with an umbilical cord stump infection.  Patients are 
managed with antibiotics, and occasionally granulocyte infusions.  The severity of the 
defect, (i.e., the level of CD18 production) correlates with the severity of disease, so even 
a modest increase in production is helpful. 
 
Gene Therapy 
As with those affected by CGD, patients with LAD have been treated with gene therapy.  
Unfortunately, to date, there have been no clinical successes, again in part due to the fact 
that the corrective gene does not confer a survival advantage to the neutrophils.  
Experiments in a dog model of LAD known as CLAD (canine leukocyte adhesion 
deficiency), however, have shown proof of principal, and gene therapy remains a target 
for the cure of this disease. 
 
Allogeneic Transplantation 
Transplantation has also been used to cure a number of patients with LAD.  However, in 
the largest study of this rare disease, of the 5 patients transplanted using an HLA-matched 
sibling donor, 2 patients died from transplant complications.  Overall, of the 14 patients 
transplanted using a parental haplotype or a sibling HLA-matched donor, 5 required a 
second transplant due to graft failure, and the overall mortality was 28%.  Of note, 3 

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patients did develop mixed chimerism and remained phenotypically cured.  Additionally, 
it has been shown in the dog model that even very low levels of CD18 positive 
neutrophils are sufficient to cure the animals.  Hence nonmyeloablative-conditioning 
regimens may prove less toxic, yet still effective for this patient population. 
 
Matched Unrelated Donor Transplantation  
 
To date, there are 4 patients reported as having undergone matched unrelated donor 
transplantation for LAD.  Of these, 1 patient died from infection after a second transplant 
for graft failure.  Among the 3 survivors, 1 patient had Grade II acute GvHD limited to 
the skin, another had chronic GvHD of the skin (both of these exhibited 100% 
engraftment), while the third patient exhibited no GvHD, but had only 60% donor 
chimerism at 3 years after transplantation   [10, 11]. 
 
1.3.3  CD40 Ligand Deficiency 
CD40 ligand deficiency is an X-linked disorder of B and T cell function due to lack of 
CD40 ligand expression on T cells, which is necessary for CD40 signaling on B cells.  
This lack of cross binding between B cells and T cells leads to, among other things, a 
failure of B cell immunoglobulin class switching, resulting in elevated IgM levels and 
low to absent IgA and IgG levels.  Hence this disease is also known as Hyper IgM 
syndrome and patients are prone to unusual viral and parasitic infections due to this lack 
of humoral immunity.  In addition, the defective T cells cannot interact properly with 
antigen presenting cells, leading to an increased susceptibility to opportunistic infections, 
autoimmune disorders, and malignancies.  Treatment is limited, but includes intravenous 
immunoglobulin (IVIG) and, of course, prophylactic antibiotics, – in particular, 
sulfonamide/trimethoprim combinations for Pneumocystis carinii pneumonia (PCP) 
prophylaxis.  As a result, the median life expectancy as noted from a French study of 38 
patients, is about 25 years. [12] 
 
Allogeneic Transplantation  
Although allogeneic transplantation is the only cure available to date, using traditional 
methods the overall success rate is only 68%.  
 
Matched Unrelated Donor Transplantation 
There is 1 report detailing the results from 4 patients undergoing unrelated donor 
transplantation.  All patients are doing well except for 1 who had initial neutrophil 
engraftment, but continued to require platelet transfusion support.   This patient 
ultimately developed secondary graft failure and died after developing acute respiratory 
distress syndrome (ARDS) from progressive pulmonary aspergillosis.   Three of the 
patients developed acute GvHD and 1 also had chronic GvHD. [13] 
 
1.3.4  National Marrow Donor Program 
The National Marrow Donor Program (NMDP) was established in 1986 as the result of a 
Federal contract that was awarded to create and maintain a registry of volunteer 
hematopoietic stem cell (HSC) donors. Physicians search the NMDP Registry on behalf 
of patients in need of an HSC transplant who have no suitable matching related donor. In 

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1999 the NMDP added a Cord Blood Registry to provide more donor source options for 
patients in need of an unrelated HSC transplant. At the end of July 2003, the NMDP 
Registry listed more than 5 million volunteer donors, 28,000 cord blood units (CBU), and 
had facilitated over 16,000 unrelated HSC transplants. 
 
1.4 Scientific and Clinical Justification 
In disorders such as hematologic malignancies, the curative effect of bone marrow 
transplantation has been ascribed to the use of myeloablative chemo-radiotherapy and the 
antileukemic effect of the transplant (i.e., the graft versus leukemia effect). [14]  The assumption 
that the intensive myeloablative preparative regimen is essential for the cure of the malignancy 
went unchallenged until the demonstration by Kolb et al. (subsequently confirmed by numerous 
investigators) that donor lymphocytes alone exert a powerful antileukemic effect in the context 
of patients relapsing with myeloid leukemias after BMT. [15-18]  This observation has important 
implications.  First, it may be possible to cure some hematologic malignancies with preparative 
regimens of lower intensity, designed to immunosuppress the recipient to allow lymphocyte and 
stem cell engraftment without major cytoreduction of the malignancy by myeloablation.  Second, 
such low-intensity preparative regimens appear to have lower toxicity and may make 
transplantation appropriate in patients where procedural mortality is usually prohibitive, 
including patients with more indolent hematologic diseases, such as those with severe congenital 
anemias, as well as patients with co-morbid diseases and older patients.  Unlike patients who 
undergo allogeneic peripheral blood stem cell (AlloPBSC) transplantation for malignant 
indications, patients with non-malignant disorders, such as those with immunodeficiencies, do 
not require full and/or rapid donor engraftment for cure of the disease.  While it is generally 
accepted that GvHD is less severe in patients conditioned with low intensity preparative 
regimens, graft rejection is preferable to the development of lethal GvHD in the setting of 
immunodeficiencies.  
 
Several groups have begun to investigate this approach to improve the applicability and outcome 
following allogeneic BMT, and preliminary results have been encouraging. The first so called 
“mini-transplants” were performed by simply lowering the doses of standard agents; however, 
these doses were still sufficiently toxic to incur prolonged cytopenias, and patients continued to 
experience significant regimen-related toxicities.  Our own experience at the NIH using a 
nonmyeloablative combination of fludarabine and cyclophosphamide has included over 100 
patients, with engraftment seen in the majority, and extension to patients previously excluded 
from allogeneic transplant trials such as those infected with the human immunodeficiency virus 
has been proven feasible. [19]  However, GvHD remains a significant problem, since it occurs at 
a rate not demonstrably different from that observed with conventional AlloPBSC 
transplantation, and continues to be the principal cause of death.  This has also been seen in other 
centers developing other regimens.  The Laboratory of Host Defenses (LHD) has also used 
nonmyeloablative techniques in patients with CGD and has shown success, although there was 
still significant GvHD in some patients. [20] The regimen used here, as with the majority of 
other popular nonmyeloablative regimens, was based on the same types of drug combinations 
used previously, with only an alteration in dosing.  Newer agents and different combinations may 
prove to be more successful at truly reducing the development of graft versus host disease 
associated with transplantation. 
 

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For immunodeficient patients who lack an HLA-matched sibling donor, matched unrelated donor 
(MUD) transplantation has also been used. In a retrospective study that reviewed patients with 
various types of severe combined immunodeficiency (SCID), the survival of patients was 80% at 
168 months post transplantation.  Most deaths were related to either GvHD or infection, and 3 of 
41 patients experienced graft failures.   There was a 73% incidence of GvHD, but only 22% of 
patients had Grade 3 or higher.  As this study was retrospective, the conditioning regimens and 
GvHD prophylaxis differed amongst the centers and patients, so the effects of conditioning 
cannot be determined from this study. [21] 
 
Umbilical cord blood has been an attractive option as there is data to suggest that there is less 
risk of GvHD induction, given the immunologic immaturity of these cells.  It is also possible to 
transplant across more HLA mismatches with less risk of GvHD or rejection.  However, the 
disadvantages to using cord blood have related to the smaller number of grafted cells and the 
resultant delayed immune recovery post transplant.  In a 1997 analysis, better survival was 
associated with: age less than 6 years, weight less than 20 kg, an infusion of greater than 37 
million nucleated cells per kg, and a CMV (cytomegalovirus) negative donor. [22] Hence, cord 
blood transplant has been traditionally limited to recipients of young age and low body mass.  In 
an effort to increase the cell dose, the University of Minnesota in particular has been using more 
than one cord blood product per patient, with very promising results.  In these patients with high-
risk hematologic malignancies, the disease free survival was 57% at 1 year, and 72% overall 
survival for patients transplanted while in remission. [23] 
 
In addition, transplantation centers have been developing reduced intensity (or nonmyeloablative 
conditioning regimens) for use in MUD and umbilical cord blood transplantation, and have 
applied these methods to both adults and children.  With a median nucleated cell dose of 3.7 x 
107/kg recipient body weight, the overall engraftment was between 76% and 95% depending on 
the conditioning regimen used in a single center trial.  The incidence of GvHD was 9% and 
survival was 39% in this high-risk group of patients. [24] 
 
Matched unrelated donor and umbilical cord transplantations have also been used in patients 
with immunodeficiencies, although the number of patients is limited (See Background).  Cord 
blood from an HLA matched sibling was used in 1 patient with CGD after a busulfan based 
conditioning regimen.  Oneyear post transplant, the patient appeared to have a 
Dihydrorhodamine 123 (assay) DHR of 92% and normal T and B cell numbers.  As the risk of 
graft rejection is higher when using unrelated donor cells, the conditioning regimen for patients 
without a related donor will be modified to include low dose irradiation.  This will provide 
additional immunosuppression along with some myelosuppression.  In order to continue to 
minimize any toxicity, the busulfan dose will be reduced from 10mg/kg to 5mg/kg.   
 
 
1.5 Study Agent 
The study agents are Total Body Irradiation, Busulfan, Campath-1H, and sirolimus. 
These drugs are well-known, FDA-approved drugs used in various transplant regimens; 
however, they have not been used in this combination at these doses. 
 
Commented [JPK1]: How many patients? 
Commented [ck2]: Does this represent dihydrorhodamine 123 
(assay) as per the abbreviation list or delayed hypersensitivity 
reaction? 

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1.5.1  Total Body Irradiation: 
Preclinical and Clinical Data: In attempts to decrease transplant related toxicities, and to 
develop immunosuppressive as opposed to myeloablative regimens, many centers have now been 
exploring different combinations of regimens including the use of low dose radiation. Low dose 
radiation alone has been shown to have anti-leukemic properties as demonstrated by Sculman et 
al. where 200 centigray (cGy) was adequate for inducing remission, albeit short lived, in 
refractory patients ineligible for other standard treatments. [25] Doses of 100 to 500 cGy have 
been shown to have very little toxicity in both murine and rhesus transplant models. [26-29]  
Even patients with Fanconi anemia, who would be considered high risk for radiation induced 
toxicities, tolerate moderate dose (500 cGy) radiation based regimens. [30] While regarded as a 
method designed for the creation of “space” within the marrow in bone marrow transplantation 
(BMT) regimens, radiation has long been the fundamental treatment modality used to establish 
immunosuppression in both BMT and solid organ transplantation in animal models. [31, 32] 
There are also data obtained from canine experiments demonstrating stable mixed chimerism 
with the use of 200cGy total body irradiation alone, followed by GvHD prophylaxis with 
cyclosporin A (CysA) and mycophenolate mofetil (MMF). [33] Initial human trials using this 
regimen of 200 cGy with MMF and CysA were encouraging with approximately 20% graft 
failure.  More recent data using fludarabine, an immunosuppressive but nonmyeloablative agent, 
together with 200cGy have shown similar results in 44 patients transplanted to date  ([34] and 
personal communication, B. Sandmaier). A modest increase in the dosage of TBI from 200 cGy 
used in prior studies to 300 cGy may increase both the degree of myelosuppression and 
immunosuppression needed for an unrelated transplant without significantly altering the adverse 
effect profile.  
1.5.2  Busulfan 
Busulfan is an alkylating chemotherapeutic agent originally designed for the treatment of 
chronic myelogenous leukemia.  It was subsequently determined to have broad 
myelosuppressive effects, and was combined with cyclophosphamide as part of a 
conditioning regimen for allogeneic transplantation.  However, it also has erratic 
absorption and pharmacokinetics when used in its oral formulation and, therefore, 
requires careful monitoring and frequent dosing.  More recently, an intravenous 
formulation has been developed with more predictable pharmacokinetics.  It is known 
that busulfan does not require cell cycling (mitosis) and, therefore, can specifically target 
the quiescent hematopoietic progenitor cells, making it an even more effective 
myelosuppressive agent that is particularly well suited for transplantation conditioning.  
The toxicities are for the most part dose related and well managed with anti-emetics and 
careful monitoring. 
 
Preclinical Data 
Murine experiments:  A series of experiments have been performed using busulfan as a 
conditioning regimen in mice.  Dose-escalation studies show that between 10 and 20 
mg/kg given as a single intraperitoneal injection consistently resulted in significant levels 
of engraftment with very little to no toxicity.  In fact, all mice survived, even when 
treated with 20 mg/kg without subsequent transplantation of progenitor cells (Kang, data 
not published). 

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Rhesus experiments:  A series of transplants have also been performed using autologous 
genetically modified cells in non-human primates. [35] While reduced dosages of 4 
mg/kg and 6 mg/kg resulted in reduced toxicity, they also resulted in lower levels of 
successful engraftment.  Preliminary data using divided dosing of 4 mg/kg per day on 2 
days suggest that there may be improved engraftment but little to no increased toxicity at 
these low levels.  This would also correlate with the clinical data below. 
 
Clinical Data 
Autologous Transplantation:  Busulfan has been used in significantly reduced doses for 
autologous transplantation of genetically modified cells.  Aiuti et al., using a 4mg/kg total 
dose in the setting of adenosine deaminase deficient severe combined immunodeficiency 
(ADA-SCID), have achieved phenotypic correction in 4 of 5 patients treated to date.  The 
patient who failed treatment received a very low cell dose number. [36] 
Busulfan as a single agent was used to perform a second transplant in a patient with CGD 
who achieved only T cell engraftment despite conditioning with melphalan, Campath-1H, 
and fludarabine and multiple donor lymphocyte infusions.  As the patient continued to 
have sequelae from his underlying CGD, we decided to condition him with 10mg/kg of 
busulfan followed by a CD34+ selected graft from the original donor (NIAID Institutional 
Review Board [IRB] Protocol 04-I-0289).  Now, more than 1 year after transplantation, 
he has normal neutrophil function with 100% chimerism as measured in the peripheral 
blood and by bone marrow CD34+ cell analysis.  Thus, in this case, clinical safety and 
efficacy of this conditioning agent was successfully demonstrated.  A different protocol 
(NIAID IRB Protocol 06-I-0289) proposes to use busulfan as a single agent for 
autologous transplantation of genetically modified cells in patients without an HLA-
matched sibling.  However, in the allogeneic setting, busulfan alone would be inadequate, 
given the risk of graft rejection and also the need for some graft versus host disease 
prophylaxis.  Results from allogeneic transplant studies using lower doses of the 
intravenous formulation of busulfan have also been encouraging (Dr. S. Solomon, 
Hematology Branch (HB)/National Heart, Lung, and Blood Institute (NHLBI), personal 
communication). 
 
1.5.3  Alemtuzumab (Campath-1H): 
Alemtuzumab is a humanized monoclonal antibody directed against CD52 (which is 
abundantly expressed on all human lymphocytes), and causes T cell activation in vitro as 
well as complement-mediated lysis and antibody-dependent cellular toxicity.  As a result, 
it depletes both T and B cells efficiently in vivo.  It is currently being used in clinical 
trials as monotherapy for certain autoimmune disorders including rheumatoid arthritis 
and multiple sclerosis [37-40], T and B cell malignancies [41], treatment of solid organ 
rejection [42-44], and has recently been approved for use in chronic lymphocytic 
leukemia, a B cell malignancy, as a result of its profound immunosuppressive properties. 
[45-47] 
 
Preclinical and Clinical Data:  Alemtuzumab 
More recently, alemtuzumab has been used prospectively to prevent graft rejection in 
human renal allotransplantation.  A total of 31 patients have been transplanted using 20 
mg of alemtuzumab on Day 0 and Day 1 of transplantation in combination with half-dose 
Commented [JPK3]: 2 successive days? 

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cyclosporine, which has been shown to be ineffective when used alone.  At the 3-year 
follow up, there were no grafts lost to rejection [44].  For allogeneic bone marrow 
transplantation, the results have been equally encouraging with data suggesting that the 
use of alemtuzumab, as compared to fludarabine, reduces the risk of GvHD even in the 
unrelated donor setting [48-50].  In one study of 44 patients, including 8 patients 
receiving unmanipulated marrow from matched unrelated donors and who would 
therefore be at very high risk for developing GvHD, only 2 patients had acute GvHD, 
both of which were Grade II.  Only 1 patient developed chronic GvHD.  Follow up to this 
study has included an additional 39 patients undergoing unrelated bone marrow 
transplantation (including patients having failed a prior transplant and/or having a 
mismatch in either HLA class I or II alleles) for a total of 47 patients, with only 3 patients 
developing Grade III GvHD and none developing Grade IV. [51]  From another study of 
30 high risk patients undergoing unrelated matched bone marrow transplantation, only 5 
patients developed GvHD, none of which was greater than Grade II.  This reduced risk of 
GvHD, as well as its immunosuppressive properties, appears to be due to an in vivo T cell 
depleting effect on the incoming graft.  Unlike anti-thymocyte globulin (ATG), which is 
a nonspecific antibody directed against lymphocytes and is also used in conditioning 
regimen, alemtuzumab is better tolerated and has no risk of causing serum sickness.  
Additionally, a protocol has been initiated within the National Institute of Diabetes and 
Digestive and Kidney Diseases (NIDDK) using Campath-1H in combination with 
sirolimus and low-dose radiation.  Although patient accrual has been slow, results have 
been promising with 2 patients engrafting without any evidence of GvHD, with 1 patient 
already 1 year post transplantation.  A third patient has lost the graft but this may be 
partly due to difficulties in maintaining his sirolimus, as well as due to receiving an 
inadequate dose of radiation due to an error in protocol implementation. 
 
1.5.4  Sirolimus (Rapamune®/rapamycin): 
Sirolimus is an immunophilin drug similar to cyclosporine; however, unlike cyclosporine, 
which inhibits the phosphatase calcineurin and thereby prevents the production of 
interleukin 2 (IL-2), sirolimus prevents translation of mRNAs encoding cell-cycle 
regulators.  As a result, sirolimus only inhibits the ability of lymphocytes to proliferate in 
response to IL-2.  Powell et al. demonstrated that cells cultured and stimulated in the 
presence of sirolimus became anergic, while cells cultured in the presence of 
cyclosporine did not. [52] 
 
Animal Studies 
To confirm the in vitro data in an in vivo model, we sought to compare sirolimus to the 
standard post-transplant immunosuppressant, cyclosporine, in a nonmyeloablative setting.  
Splenocytes from F1 hybrid mice were transplanted into a parental strain – a model 
designed to promote graft rejection – using only low dose radiation and sirolimus. 
C57Bl6 recipient mice were injected with either sirolimus at 5 mg/kg or cyclosporine, 
beginning 1 day before cell infusion.  On Day 0, they were given 300 cGy total body 
irradiation (TBI) followed by 10x106 splenocytes obtained from granulocyte colony-
stimulating factor (G-CSF) mobilized BalbC/C57Bl6 hybrid donors.   
 
Commented [JPK4]: Unprocessed? 
Commented [JPK5]: Level, dosage; compliance issue? 
Commented [JPK6]: 107? 

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The immunosuppressants were continued for 4 weeks beyond cell infusion, and 
chimerism was monitored using flow cytometric analysis.  The mice who were given 
sirolimus showed moderate donor engraftment by week 2, which subsequently increased 
over time even after the discontinuation of the sirolimus, reaching levels of greater than 
75%.  In contrast, those treated with cyclosporine lost their engraftment even while 
receiving the cyclosporine.  Application of this regimen to transgenic mice expressing 
human sickle hemoglobin (a disease that is also a non-malignant hematologic disorder 
curable by transplantation) exclusively produced correction of anemia and red cell 
sickling even at very low levels of donor chimerism. 
 
In a series of experiments performed by Hale et al., sirolimus also proved superior to 
cyclosporine at prolonging skin graft survival in Class I and Class II disparate, fully 
mismatched, and xenogeneic recipients, and the use of sirolimus was superior to 
cyclosporine when added to anti-lymphocyte globulin and bone marrow in a murine 
model.  Further, mice receiving sirolimus accepted a second same-donor skin graft but 
rejected third-party grafts, demonstrating the development of tolerance.  
 
Clinical Data: 
Sirolimus has less renal toxicity than cyclosporine. A randomized trial comparing the 
addition of sirolimus at either 2 mg or 5 mg vs. azathioprine to a cyclosporine and 
prednisone regimen for prophylaxis of renal allograft rejection showed a significantly 
lower rate of acute rejection episodes at both doses of the sirolimus as compared to 
azathioprine (16.9% and 12.0% vs. 29.8%). [53]  In a similar study comparing sirolimus 
vs. cyclosporine as adjuncts to azathioprine and prednisone there were similar rates of 
graft survival and incidence of biopsy-confirmed graft rejection, (98% vs. 90% and 41% 
vs. 38% respectively), but significantly lower serum creatinine levels in the sirolimus 
group. [54]  Moreover, in renal transplant studies, sirolimus has been shown to be equally 
effective in preventing graft rejection and has been approved as an alternative to 
cyclosporine. 
 
1.6 Qualifications of Investigators 
This clinical study will be conducted by a multidisciplinary team encompassing expertise in 
immunology, infectious diseases, and hematology required for the care of patients undergoing 
bone marrow transplantation.  The care teams also are knowledgeable in the conduct of Good 
Clinical Practice (GCP) principles of clinical research and the regulatory requirements for the 
protection of human subjects.  All investigators collaborating in this study have met the training 
requirements of the Office of Human Subjects Research.  Copies of the curricula vitae to 
demonstrate the experience and qualification of all of the investigators (Principal Investigator 
[PI] and Associate Investigators [AI]) will be kept updated and on file. 
 
 
1.7 Conflict of Interest 
No reportable conflicts of interest have been identified at the present time for any of the 
investigators conducting this study.  If such conflicts of interest should develop in the future, the 
PI will take immediate corrective action and the Institutional Review Board (IRB) will be 
notified.  
Commented [JPK7]: Do you mean mg/kg? 

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1.8 Conduct of the Study 
This study will be conducted in accordance with all applicable laws and regulations, policies of 
the National Institute of Allergy and Infectious Diseases (NIAID) IRB as well as the policies of 
NIAID and NIH.  The Principal Investigator will assure that no deviation from or changes to the 
protocol will take place without prior documented approval from the IRB, except where 
necessary to eliminate an immediate hazard(s) to the study participants.  The PI will promptly 
report to the IRB any changes in research activity and all unanticipated problems involving risk 
to human subjects or others. 
 
 
2.0   STUDY DESIGN 
2.1 ENDPOINTS: 
2.1.1 Primary Endpoint 
Engraftment of allogeneic or matched unrelated (including cord blood) hematopoietic 
progenitor cells using moderate-dose busulfan and Campath-1H with or without whole 
body irradiation so as to attain phenotypic correction of congenital immunodeficiencies. 
 
2.1.2 Secondary Endpoints 
Achieve engraftment without development of graft versus host disease. 
Establish stable mixed chimerism. 
Improve rapidity of immune reconstitution. 
Determine days to neutrophil recovery. 
Measure incidence of Cytomegalovirus (CMV) reactivation. 
Measure transfusion requirements. 
 
2.2 Sample Size Justification 
2.2.1 Rationale for Subject Selection 
Over the next 5 years, a total of 30 subjects (10 sibling donors and 20 patients with 
congenital immunodeficiencies), each with a 6 out of 6 HLA-matched sibling donor or a 
suitable matched unrelated donor or cord blood product, will be enrolled in this study.  
Gender, ethnic background, and/or race will not be criteria for including or excluding 
patients during the study. 
 
2.2.2 Donors of Allogeneic Stem Cells 
 Transplantation of immunologically competent progenitor cells derived from either bone 
marrow or peripheral blood is currently the only available cure for patients with 
congenital immunodeficiencies and is considered an accepted standard clinical 
intervention for these diseases.  Such progenitor cells will be provided from any of 3 

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sources: sibling donors having a 6 out of 6 HLA-match to the recipient, or a suitably 
matched unrelated donor, or umbilical cord blood.   
Children who are < 2 years of age or who weigh ≤18 kg will be excluded as donors.  The 
risks of the apheresis procedure are related to the weight of the child, more precisely 
his/her extracorporeal volume, which is weight-dependent.  The risks have to do with (1) 
need for a central line, (2) need for an allogeneic red cell prime, and (3) need for systemic 
heparinization because the subject is too small to get citrate. Weight considerations will 
be:     
 
> 25 kg:  the procedure and associated risk is the same as that in an adult; 
however, a central line is almost always needed.    
 
19 – 25 kg:  A central line is required.  Donors may or may not need red cell 
priming (at the discretion of the Apheresis department).  Although there may be a 
need for heparinization, generally donors are anticoagulated with citrate.   
 
≤ 18 kg:  All donors in this weight range are excluded from participation, as they 
would require a central line, red cell priming, and systemic heparinization. 
 
3.0  SUBJECT ENROLLMENT 
3.1 Patient Recruitment 
Patients and their family members who may already be participating/have participated in existing 
NIH studies and are being referred by their NIH physician into the screening for inclusion in this 
study will be recruited.   
 
Participants will also be recruited from outside the existing NIH patient population.  The Clinical 
Center (CC) Patient Recruitment and Public Liaison Office serves to provide clinical study 
advertisement and contact information for both self-referring patients and physician referrals 
from outside NIH.  We expect to enroll about 3-4 patients per year into this study.  The 
participants and their relatives selected for this study will reflect gender and ethnic diversity that 
is representative of the population. 
 
 
3.2 Inclusion/Eligibility Criteria  
3.2.1 Patients (Recipient): 
 
Must have a confirmed congenital immunodeficiency  
 
Must have sufficient complications from underlying disease to warrant undergoing 
transplantation.   
 
Ages 3 years – 65 years 
 
HLA-matched family donor available or an HLA matched unrelated PBSC graft 
available, or a minimum of 4/6 HLA matched cord blood product.  (If the size of 
the cord blood graft is less than 3.0 x 107 cells, a second appropriate 4/6 or greater 
match cord blood product must be available.) 
Commented [ck8]: Recommend that this be defined a priori. 

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 
Ability to comprehend and willingness to sign the informed consent or have a 
parent/guardian consent if the donor is a minor; assent being obtained from minors 
as appropriate 
 
Negative serum beta-human chorionic gonadotropin (ß-hCG) for women of child-
bearing potential 
 
Must be HIV negative 
 
Must not be pregnant or breastfeeding 
 
Must be able to stay within one hour’s travel of the NIH for the first 3 months after 
transplantation and have a family member or other designated companion to stay 
with during the post transplant period. 
 
Must provide a durable power of attorney for health care decisions to an appropriate 
adult relative or guardian in accordance to NIH -200 “NIH Durable Power of 
Attorney for Health Care Decision Making”. 
 
If of child-bearing potential, must agree to consistently use contraception 
throughout study participation and for 3 months post-study.  Acceptable forms of 
contraception are: 
 Condoms, male or female, with or without a spermicide 
 Diaphragm or cervical cap with spermicide 
 Intrauterine device 
 Contraceptive pills or patch, Norplant®, Depo-Provera®, or other FDA-
approved contraceptive method 
 Male partner has previously undergone a vasectomy. 
 
3.2.2 Allogeneic Donor (Sibling donor only where the collection is done at NIH) 
 
HLA-matched (i.e., 6 of 6 alleles identical) family donor 
 
Ages ≥ 2 years and weight ≥ 18 kg (in so far that the weight difference between 
recipient and donor does not exceed a reasonable likelihood of being able to obtain 
an adequate cell dose from the donor with no more than 2 aphereses)   
 
Fit to receive G-CSF and give peripheral blood stem cells (normal blood counts, 
normotensive, and no history of stroke)  
 
Ability to comprehend and willing to sign informed consent or have 
parent/guardian consent if donor is a minor; assent obtained from minors as 
appropriate 
 
Must be HIV, hepatitis and syphilis negative 
 
Must not be pregnant or breastfeeding 
* Inclusion criteria for donors for matched unrelated products from the National Marrow Donor 
Program is done per NMDP protocol. 
 

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3.3 Exclusion Criteria 
3.3.1 Patient (Recipient) 
 
Age <3 years or > 65 years   
 
Eastern Cooperative Oncology Group (ECOG) performance status of 3 or more 
(See Supportive Care guidelines, available at 
http://intranettst2.cc.nih.gov/bmt/clinicalcare) 
 
Diffusion capacity of carbon monoxide (DLCO) < 60% predicted 
 
Left ventricular ejection fraction < 40%  
 
Transaminases > 5x upper limit of normal based on the patient’s clinical situation 
and at the discretion of the investigator 
 
Psychiatric disorder or mental deficiency severe enough as to make compliance 
with the BMT treatment unlikely, and/or making informed consent impossible 
 
Major anticipated illness or organ failure incompatible with survival from 
AlloPBSC transplant 
 
Pregnant or lactating 
 
HIV positive 
 
Uncontrolled seizure disorder 
 
3.3.2 Allogeneic Donor (Sibling donor only where the collection is done at NIH) 
 
Pregnant or lactating 
 
Donor unfit to receive G-CSF and undergo apheresis. (e.g., uncontrolled 
hypertension, 
history 
of 
congestive 
heart 
failure 
or 
unstable 
angina, 
thrombocytopenia, signs and symptoms of acute mononucleosis) 
 
HIV positive   
 
≤18 kg:  All donors in this weight range are excluded from participation as they 
would require a central line, red cell priming, and systemic heparinization. 
 
Recent exposure to infectious diseases such as chickenpox. 
 
Age <2 years 
* Exclusion criteria for donors for matched unrelated products for the National Marrow Donor 
Program is done per NMDP protocol. 
 
3.4 Study Enrollment Procedures 
3.4.1 Consent Obtainment:  Allogeneic Recipients and Donors 
Informed consent will be obtained from all subjects during the first visit to the Clinical 
Center and before any screening procedures are conducted.  The Principal and/or 
Associate Investigators (PI/AI) will obtain the consent during a face-to-face interview.  
Commented [g9]: It may be useful to provide a link to this 
protocol or include criteria as an appendix to this protocol. 

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The PI and/or AI will counsel all participants.  The process of obtaining consent will be 
done with the presence of the PI, other study members i.e. Study Coordinator and/or 
Research Nurse, and a Case Manager. The Case Manager will serve as the impartial third 
part witness who attests to the information given to the patients as well as the validity of 
the signature obtained. The consent document for this study will be thoroughly reviewed, 
questions will be answered, and the consent form will then be signed in the presence of a 
witness. 
 
If a subject is a minor, the parent or legal guardian who signs the consent for the minor 
must be legally recognized as such.  Where deemed appropriate by the PI/AI and the 
child’s parent or legal guardian, the child will also be included in all discussions about 
the study and minor’s assent will be obtained.  The parent or legal guardian will sign on 
the designated line on the informed consent attesting to the fact that the child had given 
assent. 
 
During this study, all new information relating to risks and/or adverse events will be 
provided orally and/or in writing to all enrolled or prospective patient and donor 
participants and their parent/guardian.  Documentation will be provided to the IRB and if 
necessary, the informed consent document will be amended to reflect relevant 
information. 
 
3.4.2 Consent for Unrelated Donors  
Informed consent for donors in the National Marrow Donor Program is obtained per 
NMDP protocol. 
  
3.4.3 Participation of Children   
Congenital immunodeficient patients of all ages, especially children, suffer from 
debilitating and often lethal diseases that are incurable with conventional treatments. 
Currently, the only cure available for these disorders is bone marrow transplantation. This 
study aims to decrease the risk of transplantation related mortality, thus making more 
patients candidates for potentially curative therapy.  Bone marrow transplantation, 
however, poses greater than minimal risk with the prospect of direct benefit to pediatric 
participants (45CFR 46.102). As such, adequate provisions are made for soliciting the 
assent of the children and the permission of their parents or guardians (see Sections 3.4.2 
and 9.4). Children less than the age of 3 are excluded from participation as recipients on 
this protocol as the NIH does not have sufficient support to deal with possible 
complications related to transplant in pediatric patients less than 3.  Patients older than 65 
are excluded as well as it is known from standard transplantation that these patients have 
a higher risk of morbidity and mortality related to transplantation.  Given the 
experimental nature of this protocol, the risk benefit ratio is not warranted to include 
these patients at this time.  Further, the number of patients who would be eligible 
otherwise is exceedingly small. 
 

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4.0 STUDY IMPLEMENTATION 
4.1 Criteria for Withdrawal 
4.1.1 Patient’s (Recipient’s) Voluntary Withdrawal 
The patient (recipient) will be allowed to withdraw at any time, even after receiving the 
conditioning agents (as autologous recovery is expected) or after receiving the allograft, 
although withdrawal at these stages would be highly discouraged.  Once given the 
conditioning agents and/or allograft, if the patient (recipient) wishes to withdraw, he or 
she will have to remain within the care of the study physicians until either autologous or 
graft-mediated recovery is attained. 
 
Post – Infusion Obligations 
The patient (recipient) will be required to report to the clinic for follow-up assessments as 
specified in the study guidelines and Section 4.4, that is, weekly for the first 100 days, at 
4 and 6 months post-transplant, then every 6 months for the next year, then every 12 
months for the next 5 years post-transplant.  
 
4.1.2 Allogeneic Donor’s Voluntary Withdrawal 
Donors will be given ample opportunity to withdraw from the study prior to peripheral 
blood progenitor cells (PBPC)-collection by apheresis.  Donors also have the right, at any 
time, to elect not to participate in the research aspects of the study (donation of blood and 
bone marrow for non-routine tests). 
 
4.1.3 Involuntary Withdrawal of a Patient (Recipient)  
 If, after 2 G-CSF mobilization attempts on the donor at least 2 weeks apart, an 
inadequate cell number has been collected, the patient and donor will be 
withdrawn from the study, unless another donor is available.  
 Patients who fail to demonstrate donor T-cell engraftment will be taken off study 
treatment but will continue to be monitored by the study staff for 6 months post 
transplant for possible infectious complications related to the conditioning 
regimen.  After this period of follow up, patients will be referred back to their 
primary physician for further therapy. 
 Patients with disease relapse will be taken off study treatment but will continue 
to be monitored by the study staff for a minimum of 6 months post transplant for 
possible infectious complications related to the conditioning regimen.  The 
patient will then be approached to be consented for NIAID IRB Protocol 05-I-
0213 (Screening and Baseline Assessment of Patients with Abnormalities of 
Immune Function) if not already enrolled for alternative treatments or referred 
back to his/her referring physician, depending on what is considered to be in the 
best interest of the patient.  
 If this clinical trial is officially terminated.  This applies to the situation where 
the investigator, the sponsor or any regulatory agency for any reason terminates 
the entire study. 

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 If a subject becomes pregnant. Patients will be taken off study treatment and 
referred back to their primary physician for further therapy but will have contact 
follow-up by the study staff to document the outcome of the pregnancy. 
 If a patient develops a medical condition or circumstance where, in the opinion 
of the investigator, it is in the subject’s best interest to discontinue participation 
in the study. 
 
4.1.4 Involuntary Withdrawal of an Allogeneic Donor  
 If, after 2 G-CSF mobilization attempts at least 2 weeks apart on the donor, an 
inadequate cell number has been collected, the patient and donor will be 
withdrawn from the study, unless another donor is available. 
 If this clinical trial is officially terminated.  This applies to the situation where 
the investigator, the sponsor or any regulatory agency for any reason, terminates 
the entire study. 
 If a donor becomes pregnant. 
 If a donor develops a medical condition or circumstance where, in the opinion of 
the investigator, it is in the subject’s best interest to discontinue participation in 
the study. 
 
4.2 Study Method/Parameters 
4.2.1 Patient (Recipient) – Initial Screening 
The following will be done as part of the initial screen for all patients (recipient): 
o 
High resolution molecular HLA typing of patient and as many family members 
as possible to confirm complete matching of the donor 
o 
Dihydrorhodamine 123 (DHR) assay or other disease specific analysis 
o 
Serum ß-HCG for women of child-bearing potential 
o 
Complete medical history and physical examination, including weight. 
 
4.2.2 Patient (Recipient) – Baseline Testing and Evaluation 
o 
Coagulation screen, complete blood count (CBC) with differential 
o 
Chem 20 panel 
o 
Erythrocyte sedimentation rate (ESR) 
o 
Antibody screen for hepatitis B and C virus (HBV, HCV), human immuno-
deficiency virus (HIV), human T cell lymphotrophic virus type I and II (HTLV-
I/II), CMV, Epstein Barr virus (EBV), toxoplasma, and syphilis.  Consider 
purified protein derivative (PPD) test for patients from areas where tuberculosis 
is prevalent. 
o 
HLA antibody screen 

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o 
Lymphocyte  phenotype   
o 
T cell receptor Vß spectra type 
o 
Serum troponin-I 
o 
Complete lipid profile with triglycerides 
o 
Bone marrow aspirate 
o 
Collection of 24-hour urine for assessment of creatinine clearance 
o 
Chest radiograph, pulmonary function testing: forced vital capacity (FVC), 
forced expiratory volume in 1 second (FEV-1), diffusing capacity for the lungs 
measured using carbon monoxide (DLCO) 
o 
Sinus CT scans 
o 
Cardiac function: EKG, multi-acquisition gated (MUGA) scan and/or ECHO, 
24-hour Holter monitor 
o 
Nutritional assessment 
o 
Dental exam and clinical review 
o 
Social worker interview 
o 
Ophthalmology consultation 
o 
Interview with members of primary care team and visit to unit 
o 
Consent form signed 
o 
Durable power of attorney form completed 
 
4.2.3 Allogeneic Donor – Initial Screening 
o 
High resolution molecular HLA-typing of as many family members as 
necessary both to find a possible donor and to confirm HLA matching  
 
4.2.4 Allogeneic Donor Baseline – Testing and Evaluation 
o 
Confirm HLA identity of donor with patient 
o 
History and physical examination 
o 
CBC with differential, coagulation screen, Chem 20 panel 
o 
Lymphocyte phenotype   
o 
Hepatitis B, C, HIV, HTLV-I/II, CMV, EBV antibodies, rapid plasma reagin 
(RPR) 
o 
HLA antibody (Ab) screening 
o 
Fit to donate:  Orientation, visit to Department of Transfusion Medicine (DTM), 
inspection of veins to determine the need for a central line for apheresis 
o 
Consent to undergo G-CSF mobilization  (see below) 

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o 
Serum ß-HCG for women of child-bearing potential. 
 
4.2.5 NMDP Protocol 
Unrelated donors will be screened and evaluated according to NMDP guidelines. 
 
4.2.6 Cord Blood Tests 
For Cord Blood products, confirmatory HLA testing will be performed at the 
appropriate contract lab according to NMDP standard operating procedure (SOP).   
 
Samples will be collected and stored according to the NMDP follow up protocol. 
A donor blood sample will be obtained from the NMDP for lymphocyte 
phenotyping testing, which will be done here at NIH.  
 
 
4.3 Allogeneic Donor Intervention  
4.3.1 Mobilization and Collection of Allogeneic Peripheral Blood Progenitor Cells  
(see Appendix C) 
Starting at least 2 months before the anticipated transplant date, the eligible donor will 
receive granulocyte colony stimulating factor (G-CSF) at 10μg/kg/day subcutaneously for 
5-6 days.  
On the 5th day, the donor will be given a dose of G-CSF in the morning and then will be 
escorted down to the apheresis center in the DTM where a standard peripheral blood 
apheresis will be performed according to the DTM SOP.  The blood volume processed 
per apheresis procedure will be determined by DTM medical staff on the day of 
apheresis, based on peak CD34+ cell mobilization response to G-CSF and the CD34+ cell 
dose needed, based on the kilogram weight of the recipient.  This will range up to 35 
liters processed per day over 2 to 3 days, not to exceed a total of 75 liters over 3 days 
unless reviewed and approved by the medical director of the apheresis clinic.  In pediatric 
subjects, defined as less than 40 kg, a maximum of 8 blood volumes will be processed per 
day, for up to 2-3 days. 
The collections of peripheral blood progenitor cells (PBPC) will then be cryopreserved.  
Although the intent is to use the product within one year of collection, products may be 
used up to 10 years after collection and storage in accord with the Red Cross guidelines 
for maintenance and use of cryopreserved products.   If the two collections from the first 
mobilization combined are still inadequate, a repeat G-CSF mobilization (a minimum of 
2 weeks later at a higher dose of G-CSF will be done with the pooled products of the two 
mobilizations being given to the patient at the time of transplant.  
 
4.3.2 Allogeneic Peripheral Blood Progenitor Cell Transplant  
The target collection number for progenitor cells is  5 x 106 CD34+ cells/kg.  This 
product will be collected in advance and cryopreserved.  Donors will undergo apheresis 
for one or two days.  The minimum dose for a PBSC transplant will be  3.0 x 106 CD34+ 
cells/kg.  If this is not achieved in one apheresis procedure, then the donor will undergo a 

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second apheresis the following day.  If the combined product is still inadequate, the donor 
will be brought back after a minimum of two weeks in between collection and 
administration of G-CSF to be remobilized again using G-CSF at a dose of 16μg/kg.  
This will be added to the original collection.  If after two such attempts, an inadequate 
cell number has been collected, the patient and donor will be withdrawn from the study, 
unless another donor is available. 
 
4.3.3 Unrelated Donor Product Selection 
The product to be chosen for unrelated transplantation will follow the algorithm below: 
 
A)10/10 HLA match (Based on molecular high resolution typing) PBSCT 
 
If not available 
 
B) Best match cord blood product, minimum 4/6 HLA match with cell dose of 3 x 107 
nucleated cells/kg recipient weight. 
 
If the cell dose is insufficient, a second cord blood product will be used in combination 
(double cord) to ensure the minimum cell dose.  Again HLA matching will be a minimum 
of 4/6 HLA in relation to both the recipient and the first product. 
 
4.3.4 MUD Recipient Apheresis 
For those patients undergoing matched unrelated donor transplantation either with PBSC 
or cord blood, the patient will provide consent to undergoing NIAID IRB Protocol 94-I-
0073 for mobilization and apheresis.  Although the regimen is considered 
nonmyeloablative, given the higher risk of rejection of unrelated donor cells, CD34+ cells 
will be collected from the recipient.  This product will be stored as a back up in the case 
of graft failure or rejection and lack of autologous recovery.  Again, although the primary 
purpose of the collection is to use it within the peri-transplant period in the event of graft 
failure, products may be used up to 10 years after collection and storage as according to 
the Red Cross guidelines for maintenance and use of cryopreserved products. 
 
4.4 Patient (Recipient) Intervention 
4.4.1 Central Venous Line Placement 
A triple lumen Hickman catheter will be placed by an interventional radiologist or a 
surgeon if the patient does not already have a catheter in place.  Management of this 
access will be according to the SOP of the CC and nursing unit. 
 
4.4.2 Treatment Plan 
o 
All patients will be admitted to an inpatient unit for a minimum of 10 days prior 
to PBSC infusion.  The total anticipated number of inpatient days is 40. 
o 
Baseline studies will be performed as medically necessary to ensure no occult 
infection, which could necessitate delay of the transplant procedure.  These will 
consist of Chest CT, EKG, and erythrocyte sedimentation rate (ESR). 

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4.4.3 Group 1 -Preparative Regimen Schema (Daily Management) for Allogeneic 
Recipients 
Day -10 to Day -9  (10 to 9 days prior to cell infusion): 
 Patient will have serum troponin-I, ECHO, 24-hour Holter, Chest CT, EKG and 
daily ESR and CBC with differential.  
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -8 (8 days prior to cell infusion):  
 ESR and CBC with differential 
 Diphenhydramine (1-1.25mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15mg/kg orally (PO) followed by alemtuzumab 0.03 mg/kg 
in 100cc normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -7 (7 days prior to cell infusion): 
 ESR and CBC with differential 
 Diphenhydramine 1-1.25 mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15 mg/kg PO followed by alemtuzumab 0.1 mg/kg in 100cc 
normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -6,-5,-4 (6,5,4 days prior to cell infusion): 
 ESR and CBC with differential 
 Diphenhydramine 1-1.25 mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15 mg/kg PO followed by alemtuzumab 0.3 mg/kg in 100cc 
normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
                  Day -3 (3 days prior to cell infusion): 
 The patient will be administered clonazepam 0.6 mg/kg IV or equivalent 
medication (i.e., phenytoin).  
 30 minutes afterwards, the patient will receive IV busulfan at 5 mg/kg 
prepared in normal saline infused over 2 hours.  

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 Laboratory tests to be obtained are: ESR and CBC with differential. Drug 
(busulfan) levels will be obtained at the end of infusion, approximately 135, 
150, 180, 240, 300, and 360 minutes after starting the infusion of the drug. 
 The patient will be monitored with vital signs taken every 15 minutes for one 
hour, then every hour (q1h) until the completion of busulfan infusion, followed 
by approximately every 4 hours (q4h) while awake. (See also Section 11.6, 
administration of busulfan). Weight. 
 
Day -2 (2 day prior to cell infusion): 
 The patient will be administered clonazepam 0.6 mg/kg IV.  
 Thirty minutes afterwards, the patient will receive IV busulfan at 5 mg/kg 
prepared in normal saline given over 2 hours.   
 The patient will be monitored with vital signs taken every 15 minutes for one 
hour, then q1h until the completion of busulfan infusion, followed by 
approximately q4h while awake. (See also 11.7, administration of busulfan). 
Weight.   
 Laboratory tests to be obtained are:  ESR and CBC with differential.  
 
Day -1 (1 day prior to cell infusion): 
 No treatment  
    Laboratory tests to be obtained are:  ESR and CBC with differential. Weight 
 For adult patients, begin sirolimus 5 mg PO q4h for 3 doses, then 5 mg once a 
day (QD) to maintain trough levels between 10-20 ng/mL.  Patients will take 
sirolimus from Day -1 to Day 100 (minimum). 
 For pediatric patients, begin sirolimus 3 mg/m2 PO q4h for 3 doses, then 1 
mg/m2 once a day (QD) to maintain trough levels between 10-20 ng/mL. 
Pediatric patients will take sirolimus from Day -1 to Day 100 (minimum). 
 
Day 0 (cell infusion day): 
 The patient will be given the allogeneic peripheral blood allograft, which has 
been thawed and prepared following the SOP established in DTM.  
  The cells will be infused through patient’s central line as per CC policy and 
SOP on administration of blood products. 
 Vital signs will be taken within 15 minutes prior to the transfusion and then at 
approximately 15 minute intervals until the (last) bag is infused, followed twice 
by q1h vital signs, then approximately q4h while awake. Weight 
 Laboratory tests to be obtained are: ESR and CBC with differential. 
 
4.4.4 Group 2- Preparative Regimen Schema (Daily Management) for Matched 

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Unrelated and Cord Blood Recipients 
Day -11 to Day -9 (11 to 9 days prior to cell infusion): 
 Patient will have serum troponin-I, ECHO, 24-hour Holter, Chest CT, EKG and 
daily ESR and CBC with differential.  
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -8 (8 days prior to cell infusion):  
 ESR and CBC with differential 
 Diphenhydramine 1-1.25 mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15 mg/kg orally (PO) followed by alemtuzumab 0.03 
mg/kg in 100cc normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -7 (7 days prior to cell infusion): 
 ESR and CBC with differential 
 Diphenhydramine1-1.25 mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15 mg/kg PO followed by alemtuzumab 0.1 mg/kg in 100cc 
normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -6,-5,-4 (6,5,4 days prior to cell infusion): 
 ESR and CBC with differential 
 Diphenhydramine 1-1.25 mg/kg with maximum dose of 12.5 mg intravenously), 
acetaminophen 10-15 mg/kg PO followed by alemtuzumab 0.3 mg/kg in 100cc 
normal saline infused over 2 hours. 
 The patient will be monitored with vital signs with weight taken approximately 
every 8 hours while awake. 
 
Day -3 (3 days prior to cell infusion): 
 The patient will be administered clonazepam 0.6 mg/kg IV or equivalent 
medication (i.e., phenytoin).  
 30 minutes afterwards, the patient will receive IV busulfan at 2.5 mg/kg 
prepared in normal saline infused over 2 hours.  

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 Laboratory tests to be obtained are: ESR and CBC with differential. Drug 
(busulfan) levels will be obtained at the end of infusion, approximately 135, 
150, 180, 240, 300, and 360 minutes after starting the infusion of the drug. 
 The patient will be monitored with vital signs taken every 15 minutes for one  
hour, then every hour (q1h) until the completion of busulfan infusion, followed 
by approximately every 4 hours (q4h) while awake. (See also Section 11.7, 
administration of busulfan). Weight 
 
Day -2 (2 days prior to cell infusion): 
 The patient will be administered clonazepam 0.6 mg/kg IV.  
 Thirty minutes afterwards, the patient will receive IV busulfan at 2.5 mg/kg 
prepared in normal saline given over 2 hours.   
 The patient will be monitored with vital signs taken every 15 minutes for one 
hour, then q1h until the completion of busulfan infusion, followed by 
approximately q4h while awake. (See also Section 11.7, administration of 
busulfan). Weight  
 Laboratory tests to be obtained are:  ESR and CBC with differential.  
 
 
Day -1 (1 day prior to cell infusion): 
 The patient will be administered total body irradiation (TBI) of 300 cGy in 
two fractions of 1.5 cGy each in the Radiation Oncology Branch of the 
National Cancer Institute as follows:  
All patients will be treated with a linear accelerator using energies higher than 
4MV.  TBI will be delivered with lateral fields using extended SSD/SAD 
values of 200-500cm (depending on machine/vault size) and partial 
transmission lung blocks.  Patients will then receive AP/PA Mediastinal Boost 
fields using full blocks in the lung area.  Patients will be treated with TBI to a 
total dose of 300 cGy delivered in 150 cGy fractions bid, at least 6 hours apart 
(two total treatments in one day).  The mediastinal boost fields will be 
delivered at a dose of 75cGy per fraction for a total dose of 150cGy, also at 
least six hours apart.  Treatment planning will be performed to provide a mean 
lung dose of 150 cGy.  Occasionally, the total dose/technique of TBI may 
require modifications due to patient factors (unexpected or severe (grade 4-5) 
adverse events, serious medical illnesses not conducive to stable patient 
transfer, patient refusal, etc) or treatment factors (linear accelerator machine 
offline, etc.) Modifications to the radiation treatment will be at the discretion 
of the treating radiation oncologist and will be discussed with the Principal 
Investigator or Study Chairperson.  
 Laboratory tests to be obtained are:  ESR and CBC with differential.  

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 For adult patients, begin sirolimus 5 mg PO q4h for 3 doses, then 5 mg once a 
day (QD) to maintain trough levels between 10-20 ng/mL.  Patients will take 
sirolimus from Day -1 to Day 100 (minimum). 
 For pediatric patients, begin sirolimus 3mg/m2 PO q4h for 3 doses, then 1 
mg/m2 once a day (QD) to maintain trough levels between 10-20 ng/mL. 
Pediatric patients will take sirolimus from Day -1 to Day 100 (minimum). 
 
Day 0 (cell infusion day): 
 The patient will be given the unrelated donor peripheral blood allograft, which 
has been thawed (if cord blood) and prepared following the SOP established in 
DTM. Adult unrelated donor products will not be frozen and therefore will not 
require thawing. 
 The cells will be infused through patient’s central line as per CC policy and 
SOP on administration of blood products. 
 Vital signs will be taken within 15 minutes prior to the transfusion and then at 
approximately 15 minutes intervals until the (last) bag is infused, followed 
twice by q1h vital signs, then approximately q4h while awake. Weight 
 Laboratory tests to be obtained are: ESR and CBC with differential. 
 
4.4.5   Post Transplant Monitoring  (Day +1 to Discharge) 
The following will be obtained while the patient is an in-patient until discharged: 
 Once daily:  CBC with differential, Chem 20 panel, direct bilirubin, weight 
 Every 8 hours while awake: temperature, pulse, blood pressure, respiratory rate   
 Twice weekly:  reticulocytes, pre-albumin, and coagulation screen  
 Weekly:  CMV surveillance, serum cholesterol, triglycerides, and sirolimus 
levels 
 On Days +14, +30:  peripheral blood will be drawn to assess for donor-host 
chimerism in the lymphoid and myeloid lineages.  A sample of peripheral blood 
will also be obtained for chimerism analysis at the time of neutrophil recovery 
(i.e., absolute neutrophil count [ANC] > 500 cells/μL). On day +14 a 
lymphocyte phenotype will also be obtained. 
 
4.4.6 Hospital Discharge Criteria 
Patient will be discharged when the following criteria are fulfilled:   
 Patient afebrile, positive weight balance, no parenteral feeding required. 
 Neutrophil count greater than 1000 on 3 consecutive days. 
 Platelet transfusion requirement absent or manageable as an outpatient. 
 Patient or family able to care for Hickman catheter. 
Commented [ck10]: What will this consist of (i.e., PCR assay, 
antigenemia assay, cultures, etc?) 

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4.4.7 Discharge to Day 100 (Out-Patient) 
 At least weekly:  A complete physical exam, CBC with differential, DHR in 
appropriate patients, coagulation screen, Chem 20 panel, temperature, pulse, 
blood pressure, respiratory rate, weight, CMV antigenemia, and sirolimus levels 
to guide dosing, serum Immunoglobulin levels.   
 
 Monthly:  Serum cholesterol, triglycerides, pregnancy test will be done on all 
females of child-bearing potential monthly until 12 weeks after sirolimus is 
discontinued. 
 On Day +60 and Day +100:  Peripheral blood will be drawn to assess for donor-
recipient chimerism in the lymphoid and myeloid lineages. At day 100 a 
lymphocyte phenotype will also be determined. 
Ninety days (+/- 1 week) after the last dose Campath-1H:  ECHO, serum 
troponin-I, and 24 hour Holter monitor  
4.4.8 Beyond Day 100 
 At 6, 12, 18, 24, 36, 48 and 60 months:  CBC with differential, DHR in 
appropriate patients, Chem 20 panel, chest CT, chimerism analysis by PCR, and 
karyotype, lymphocyte phenotype, Vß spectra type, pulmonary function (12, 24 
months). Pregnancy test for females of child-bearing potential, serum 
cholesterol, triglycerides will be done at 6 months, Serum troponin levels and 
24 hour Holter monitor will done at the 6 month follow up visit. 
 At the 12 month evaluation:  Bone marrow aspirate samples will be obtained    
 Patients will be screened for CMV beyond Day 100 post transplant for a 
minimum of 6 months. 
 
4.4.9 Post Transplant Medical Management 
 Infection Prophylaxis  (See also Supportive Care Guidelines, available at 
http://intranettst2.cc.nih.gov/bmt/clinicalcare)  
 
Includes: PCP prophylaxis, HSV for appropriate patients, IVIG 
         Itraconazole or voriconazole fungal prophylaxis 
 Fever Regimen  (See Supportive Care Guidelines)  
 GvHD Prophylaxis (see Appendix A) 
 Sirolimus (Rapamune®) will be started on Day -1 with adult loading dose of 
5mg q4h for 3 doses, and then continued at 5 mg/day with a goal of trough 
levels being 10-20 ng/mL. For pediatric patients, sirolimus will be started on 
day-1 with the loading dose of 3 mg/m2 PO q4h for 3 doses, and then continued 
at 1 mg/m2 once a day (QD) to maintain trough levels between 10-20 ng/mL.  
Commented [mp11]: Consider including visit windows. 
Commented [mp12]: How often? Will this be weekly for 6 
months? 

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The sirolimus will be given for a minimum of 3 months to all patients; however, 
the total duration of administration will be determined by the presence or 
absence of GvHD and the level and kinetics of donor-recipient chimerism.  
 Transfusion Support (See Appendix B) 
 Nutrition  
Parenteral nutrition will be instituted if the patient’s daily caloric intake drops 
below 1000 Kcal/day.  
 
 
4.4.10 Management of Patient Complications 
The major complications are cytomegalovirus reactivation, acute and chronic GvHD, and 
relapse of the original disease.  Patients with these complications will be treated as 
follows: 
 CMV Reactivation: (See Supportive Care Guidelines) 
 Acute GvHD (See also Supportive Care Guidelines) 
Begin with standard dose cyclosporine (12 mg/kg given in 2 doses to maintain a 
level between 200-400 µg/L).   
Consider institution of extracorporeal photopheresis (ECP) in those patients 
willing to be so treated. 
 Chronic GvHD 
Sirolimus will be continued at 5 mg/day with a goal of trough levels being  
10-20 ng/mL (See Appendix A) with the duration dependent on the presence or 
absence of GvHD. 
Prednisone 20-60 mg or 1-5 mg/kg in pediatric patients daily according to 
severity. 
Change to alternate-day steroid along with sirolimus therapy when response is 
established. 
Non-responding patients may be treated with other standard of care therapies 
(such as cyclosporine, combination of psoralen and long-wave ultraviolet 
radiation [PUVA], thalidomide, mycophenolate, ECP, azathioprine, daclizumab, 
or infliximab) at the discretion of the attending physician. 
 Graft Rejection 
This transplant study uses a nonmyeloablative preparative regimen.  Therefore, 
autologous recovery is anticipated in patients who fail to engraft.  Patients who 
fail to demonstrate donor T-cell engraftment will be taken off study treatment, 
but will continue to be monitored by study staff for 6 months post-transplant, 
for possible infectious complications related to the conditioning regimen.  
Afterwards, they will be referred back to their primary physician for further 
therapy. 

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 Relapse of Original Disease 
Patients will be off study treatment and will be referred back to their primary 
physician to return to their standard treatment, after 6 months of monitoring for 
infectious complications. 
 
5.0 STUDY MODIFICATIONS  
Graft failures will be monitored throughout the study.  Specific need for preparative regimen 
modifications based on the incidence of graft failures will be made as discussed in Section 
4.4.10. 
 
 
6.0 ANALYSIS OF THE RESEARCH STUDY 
Specimens collected strictly for research purposes will not be read by a pathologist.  Samples 
will be ordered and tracked through the CRIS Research Screens.  Should a CRIS screen not be 
available, the NIH Form 2803-1 will be completed and will accompany the specimen and be 
filed in the medical record. 
 
 
6.1 Chimerism Studies 
We will use PCR analysis of microsatellites to identify the contribution of the donor PBPC to 
post-transplant hematopoiesis and to detect donor lymphocytes in the circulation (i.e., donor-
recipient chimerism). 
 
6.2 Bone Marrow Samples 
A volume (up to 25 mL) of bone marrow aspirate will be collected for research studies at the pre-
transplant, Day 100, and 6-month evaluation.  These will be used to help elucidate the 
contribution of the progenitor cells to the circulating component. 
 
6.3 Transthoracic Echocardiography 
Transthoracic echocardiography (ECHO) will be performed to assess baseline ejection fraction 
prior to first (i.e., Day -10 to Day -8) and at 90 days (+/- 1 week) after the last Campath-1H 
(alemtuzumab) infusion (i.e., Day -3). 
 
 
7.0 DATA AND SAFETY MONITORING  
Principal Investigator:  The safety of interventions and treatments associated with this study will 
be under continuous review by the investigative team.  Accrual, efficacy, and safety data will be 
monitored by the PI. The data generated during this study will be monitored by the Principal 
Investigators for safety and compliance with protocol-specified requirements. Close cooperation 
between the designated members of the Protocol Team will occur to evaluate and respond to 
individual adverse events in a timely manner.  
 
 
NIAID IRB:  Accrual and safety data will be reviewed annually by the IRB. A grade 4 SAE 
occurrence in any particular patient will necessitate the study being placed on voluntary hold by 

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the Principal Investigator until the safety data has undergone a review by the DSMB. The IRB 
will also be notified of the event per reporting requirements. In the event of an emergency 
outside the facilities of NIH, the patient’s personal physician will assess the situation and liaise 
with the PI accordingly, and transfer of care to NIH can be organized as clinically indicated. All 
privacy and confidentiality of all participants will be maintained according to NIH guidelines 
and policy. Prior to implementation of this study, the protocol and the proposed patient consent 
forms will be reviewed and approved by the properly constituted IRB operating according to the 
Title 45 of the Code of Federal Regulations, Part 46 (45 CFR 46) and 21 CFR 56.  The IRB will 
also approve all amendments to the protocol or informed consent document, and conduct 
continuing annual review so long as the study is open to accrual or follow up of subjects.  
 
NIAID Intramural Data and Safety Monitoring Board (DSMB):  While the NIAID Intramural 
Data and Safety Monitoring Board (DSMB) is required to monitor studies that use gene therapy 
methodology or involve multi-center studies presenting more than minimal risk to subjects or 
that generate randomized blinded data, there is provision that allows DSMB to monitor studies 
that pose more than minimal risk to its subjects.  
 
This study involves pediatric patients as young as 3 years who will undergo either an AlloBMT 
or MUD transplantation. Bone marrow transplantation carries a mortality rate as high as 40%. 
While this study aims to decrease the risk of transplant related mortality, 2 of the conditioning 
agents (Siromilus and Campath 1-H) are not approved by the FDA to be used in children <13 
years of age. Because this study poses more than a minimal risk to subjects, it falls under the 
NIAID DSMB policy on studies that may require DSMB monitoring.  
 
The NIAID DSMB will review the data and analysis plans of all intramural NIAID clinical 
studies that require DSMB oversight. The DSMB consists of experts in transplant related 
infectious diseases, biostatistics, and clinical trials. After the initial review, which occurs prior to 
opening the study to enrollment, the DSMB will review cumulative study data to evaluate safety, 
study conduct, and scientific validity and integrity of the trial on a semi-annual basis.  The 
DSMB members must be satisfied that the timeliness, completeness, and accuracy of the data 
submitted to them for review is sufficient for evaluation of the safety and welfare of study 
participants.  All cumulative safety data reports from the trial will be submitted to the Board 
within 14 business days prior to the review.  The DSMB will also assess the performance of 
overall study operations and any other relevant issues, as necessary. 
 
All serious adverse events (SAEs) will be included in these interim reviews.  SAEs that are 
unexpected and thought to be related to the experimental portion of the study will also be  
forwarded immediately to the DSMB via the DSMB Executive Secretary.  The DSMB may 
recommend early termination of the study for considerations of safety and efficacy. 
 
8.0 ADVERSE EVENTS 
8.1 Adverse Events Reporting Plan 
The NCI Common Terminology Criteria for Adverse Events (CTC version 3.0, 
http://ctep.cancer.gov/forms/CTCAEv3.pdf) will be use for scoring of adverse events from any 
of the research procedures.  
 

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An adverse event is defined as an untoward, undesired, unplanned, clinical event in the form of a 
sign, symptom, disease, or laboratory or physiological observation occurring in a subject 
participating in this clinical study regardless of causal relationship. A medical condition that is 
present when the subject enters the study is not defined as an adverse event unless this medical 
condition worsens after the subject has been entered into the study. Clinically significant 
laboratory abnormalities which require medical intervention (for example, abnormal X-rays, 
EKGs, etc.) that occur or worsen during the clinical study also are adverse events.   
 
A serious adverse event is any event occurring within the confines of the study that results in any 
of the following outcomes: 
 
Death 
 
A life-threatening adverse event-for the purpose of this study, this type of event is 
defined as any adverse event that places the subject at immediate risk of death from 
the event as it occurred. A life-threatening event does not include an event that, had 
it occurred in a more severe form, might have caused death, but as it actually 
occurred, did not create an immediate risk of death. 
 
New hospitalization or prolongation of existing hospitalization for the purpose of 
the study is for overnight admissions only. Emergency room and day or night  visits 
are not considered hospitalizations.  Any elective hospitalization for a preexisting 
condition that has not worsened does not constitute a serious adverse event. 
 
A persistent disability/abnormality is defined as a substantial disruption in a 
person’s ability to conduct normal life functions.  
 
Congenital anomaly/birth defect 
 
Additionally, important medical events that do not meet the above criteria may be considered  
serious adverse events when, based upon appropriate medical judgment, they may jeopardize the 
subject and may require medical or surgical intervention to prevent one of the outcomes listed in 
this definition. If there is any doubt whether the information constitutes a serious adverse event, 
the information will be treated as a serious adverse event for the purposes of this study.   
 
The relationship between an adverse event and the study procedures will be determined by the PI 
on the basis of his or her clinical judgment and the following definitions: 
 Definitely related:  An adverse event that follows a reasonable temporal sequence 
from the study procedures; follows a known response pattern to the study 
procedures; and, when appropriate to the protocol, is confirmed by improvement 
after stopping the study procedures and by reappearance of the reaction after repeat 
exposure to the study procedures; and cannot be reasonably explained by known 
characteristics of the subject’s clinical state or by other therapies. 
 Probably related:  An adverse event that follows a reasonable temporal sequence 
from the study procedures; follows a known response pattern to the study 
procedures; and, when appropriate to the protocol, is confirmed by improvement 
after stopping the study procedures; and cannot be reasonably explained by the 
known characteristics of the patient’s clinical state or by other exposures. 

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 Possibly related:  An adverse event that follows a reasonable temporal sequence 
from the study procedures and follows a known response pattern to the study 
procedures but could have been produced by the subject’s clinical state or by other 
therapies. 
 Unlikely Related: A potential relationship between study agent and the adverse 
event could exist (i.e., the possibility cannot be excluded), but the adverse event is 
most likely explained by causes other than the study agent (e.g., could readily 
have been produced by the subject’s clinical state or could have been due to 
environmental or other interventions) 
 Not related:  An adverse event for which sufficient information exists to indicate 
that the etiology is unrelated to the study procedures.  Two or more of the 
following variables apply: 
a. The adverse event does not follow a reasonable temporal sequence after 
the study procedures. 
b. The adverse event is readily explained by the patient’s clinical state or 
other therapies. 
c. Negative dechallenge—the adverse event does not abate upon cessation of 
the study procedures (assuming that it is reasonable to expect abatement of 
the adverse event within the observed interval). 
 
Serious adverse events that are not related to the study procedures may be considered to 
be related to the conduct of the protocol. 
 
8.1.1 The following are examples of situations that may be deemed significant and 
for which the Principal Investigator may institute a voluntary clinical hold: 
 A grade 4 SAE in any particular patient will necessitate placing the study on a 
voluntary hold by the Principal Investigator until the safety data has undergone a 
review by the DSMB. 
 A significant rate of development of GVHD in treated patients or failure to 
engraft in more than one patient.  The Principal Investigator may decide to place 
the study on a clinical hold for consideration of study amendment even if these 
events do not fulfill the criteria for stopping the study as defined in Section 10.0.  
 Any other criteria as determined by the Principal Investigator to warrant a 
clinical hold. 
 
 
8.2 Toxicity Criteria 
The severity of adverse events will be assessed according to the National Cancer Institute (NCI) 
Common 
Terminology 
Criteria 
Scale 
for 
Adverse 
Events 
(CTC 
version 
3.0, 
http://ctep.info.nih.gov/reporting/ctc.html) The following definitions will be used for toxicities 
that are not defined in the Common Toxicity Criteria Scale (please note that dose adjustment of 
the study drug is not applicable to this study): 

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1. Mild (Grade 1): The adverse event is noticeable to the patient but does not 
interfere with routine activity. The adverse event does not require discontinuing 
the study procedures.   
2. Moderate (Grade 2): The adverse event interferes with routine activity but 
responds to symptomatic therapy or rest. The adverse event may require 
modifying the study procedures but not discontinuing the study procedures. 
3. Severe (Grade 3): The adverse event significantly limits the subject’s ability to 
perform routine activities despite symptomatic therapy. In addition, the adverse 
event leads to discontinuing the study procedures. 
4. Life-threatening (Grade 4): The adverse event requires discontinuing the study 
procedures. The patient is at immediate risk of death. 
5. Death related to SAE (Grade 5) 
 
Abnormal biological or vital sign values that are considered clinically relevant by the 
investigator will be reported as an adverse event or as a serious adverse event. Certain 
information, while not necessarily meeting the definition of an adverse event, may nonetheless 
be of value for reporting.  
 
The severity of the AE will be recorded in the appropriate section of the AE Case Report form 
(CRF) and entered into a database for Institutional Review Board (IRB), Data and Safety 
Monitoring Board (DSMB) review. 
 
8.3 Recipients’ Adverse Events 
Adverse events used to evaluate the safety of this protocol regimen will be collected to include 
any unfavorable and unintended signs (including abnormal laboratory findings), symptoms or 
diseases (e.g., incidence of GvHD, graft failure, regimen-related toxicities, or infectious 
complications), which either occur during the study, having been absent at baseline or if present 
at baseline, appear to worsen and are determined to be possibly, probably or definitely related to 
this investigational treatment.   
 
8.3.1 AE for the Screening Period 
As part of the screening for enrollment into this study, we will conduct diagnostic tests 
that may lead to the discovery of conditions that may necessitate the stopping of 
enrollment into the trial.  If any of the following events occur or are discovered that have 
no relation to the screening interventions, we will document but will not be reporting to 
the IRB unless they meet the criteria of an SAE: 
 
Eastern Cooperative Oncology Group (ECOG) performance status of 3 or more. 
(See Supportive Care guidelines, available at 
http://intranettst2.cc.nih.gov/bmt/clinicalcare) 
 
Diffusion capacity of carbon monoxide (DLCO) < 60% predicted. 
 
Left ventricular ejection fraction: < 40%.  

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 
Transaminases > 5x upper limit of normal based on the patient’s clinical situation 
and at the discretion of the investigator. 
 
Psychiatric disorder or mental deficiency severe enough as to make compliance 
with the BMT treatment unlikely, and/or making informed consent impossible. 
 
Major anticipated illness or organ failure incompatible with survival from 
AlloPBSC transplant. 
 
Patient becomes pregnant or lactating 
 
HIV positive 
 
Uncontrolled seizure disorder 
8.3.2 Adverse Events for Enrolled Subjects 
The following expected outcomes will be documented but will not be reported to the IRB 
unless they meet the criteria of an SAE. 
 Transient cardiac arrhythmias 
 Transient cardiac insufficiency 
 Pulmonary insufficiency 
 Neutropenia and its complications 
 Thrombocytopenias and its complications 
 Anemia and its complications 
 Transfusion reactions 
 Treatable infections from bacteria, viruses, protozoa, and fungi 
 Late effects of transplant regimens including: cataracts, infertility, growth 
impairment, hypothyroidism, and dental caries. 
 Headache, insomnia, psychosis, mood changes, disorientation, seizures from 
metabolic imbalance. 
 Nausea, vomiting, diarrhea, mucositis, weight loss, dry mouth, hiccoughs, 
constipation. 
 Well characterized drug reactions – allergic manifestation, red man syndrome 
 Well characterized drug adverse effects from drugs routinely used in 
transplant recipients (e.g., preparative regimen, immunosuppressive drugs, 
and antimicrobials). 
 Common adverse effects of antiemetics, analgesics, anti-inflammatory agents, 
and known complications of steroid therapy. 
 Complications from intravenous catheters, thrombotic occlusion, infection, 
local reactions, cardiac arrhythmia.  
 

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The following expected outcomes will not be reported to the IRB at each occurrence 
unless they meet the criteria of an SAE.  The PI will incorporate these events into 
the protocol and consent document as appropriate.  They will be reported in 
summary form at the time of continuing review and at termination of the clinical 
study. 
 Acute GvHD 
 Chronic GvHD 
 Graft failure ⁄graft rejection 
 Veno-occlusive disease 
 Hemorrhagic cystitis 
 Regimen-related toxicity 
 CMV reactivation and disease 
 
8.4 Donors 
The following are expected outcomes for the Group 1 donors that will be listed in the 
protocol and informed consent but will not be reported to the IRB unless they meet the 
criteria of an SAE: 
 Common adverse effects of G-CSF administration (bone pain, fatigue, 
arthralgias, headache, insomnia, fever, worsening of pre-existing skin rashes, 
increases of alkaline phosphatase, lactate dehydrogenase and/or uric acid 
levels, elevated blood leukocyte count, or thrombocytopenia) 
 Hypotension during apheresis 
 
The following expected outcomes for the Group 1 donor would not be reported to the IRB 
at each occurrence unless they meet the criteria of an SAE.  The PI will incorporate these 
events into the protocol and consent document as appropriate.  They will be reported in 
summary form at the time of continuing review and at termination of the study. 
  
 Ischemic chest pain during G-CSF administration 
 Splenic enlargement 
 Cutaneous vasculitis 
 Bone pain, muscle aches, or headaches not controlled with non-narcotic 
analgesics 
 
8.5 Serious Adverse Events  
A serious adverse event (SAE) is defined as one of the following outcomes (in accordance with 
FDA 21 CFR 312.32 and the NIH intramural guidance for principal investigators on reporting 
adverse events): 
 Death from any cause 

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 Life threatening event, i.e., an event that places the subject, in the view of 
the investigator, at immediate risk of death from the event as it occurred 
 Any event that requires or prolongs in-patient hospitalization 
 Any event that results in persistent or significant disability/incapacity  
 Any congenital anomaly/birth defect diagnosed in a child of a subject who 
participated in this study 
 Other medically important events that in the opinion of the investigator 
may jeopardize the subject or may require intervention to prevent one of 
the other outcomes listed in the definition above 
 
8.6 Reporting of Serious Adverse Events to the Principal Investigator 
 
All SAEs will be reported to: 
 
 
Elizabeth Kang, M.D. 
 
 
Building 10-CRC, 6W- Room 63752 
 
 
Phone: 301-402-7567 
 
 
Email: Ekang@niaid.nih.gov  
 
8.7 Adverse Event Reporting Requirements to the NIAID IRB  
 Because risk in this study is primarily due to the experimental treatment defined in the research 
protocol for which efficacy and potential harms are untested and uncertain, the Principal 
Investigators will perform close monitoring of patient safety with prompt reporting of serious 
adverse events to the IRB. A grade 4 SAE in any particular patient will necessitate the study 
being placed on voluntary hold by the Principal Investigator until the safety data has undergone a 
review by the DSMB. The IRB will be notified of the event and the DSMB action. All reporting 
of such events will be done via email notification to the DSMB and IRB within 24 hours of our 
having knowledge of the details. A written report will be sent within 7 days of the email 
notification. 
 
Reporting of adverse events are outlined as follows: 
 
 All deaths will be reported in writing to the IRB within 7 days. 
 
Serious and unexpected adverse events: 
 Life-threatening events will be reported in writing to the IRB within 7 days of our 
having knowledge of the details 
 Non-life-threatening events will be reported in writing to the IRB within 15 days 
of our having a working knowledge of the details. 
 In-patient admission to the NIH or other hospital for infection or other 
inflammatory process likely related to transplantation will not be considered a 
serious adverse event for expedited reporting purposes, but will be noted in 
information provided at the time of the continuing review. Exception will be 
admission to an intensive care unit or transfer to an intensive care unit which will 

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be reported as a serious adverse event to the IRB within 15 days of our having a 
working knowledge of the details.  
The following types of adverse events will be reported to the IRB as part of the continuing 
review of the study: 
 Severe 
or 
medically 
significant 
adverse 
events 
that 
are 
possibly/ 
probably/definitely related to study procedures and require medical intervention. 
An example of such events may include mild to moderate allergic reaction (hives, 
headaches, muscle or joint pains), shortness of breath, fevers, visual changes, 
numbness or tingling in any part of the body. Mild expected adverse events are 
recorded in the medical record but are not reported in the continuing reviews. 
 Any other event or condition that in the judgment of the investigators represents a 
reportable event in this category. 
 
8.8 Reporting Serious Adverse Events to DSMB 
Reports of SAEs that are unexpected and thought to be related to the experimental portion of the 
study will be forwarded within 24 hours to the DSMB.  All SAEs will be included for review 
semiannually by the DSMB. If the serious adverse event is thought to be related to the 
experimental component of the study, accession to the protocol will be stopped until full 
discussions with the DSMB have been held.  
 
9.0 HUMAN SUBJECT PROTECTIONS 
9.1 Recruitment Plans and Procedures 
Recruitment will be made from patients or their family members who may already be 
participating or have participated in existing NIH studies and are being referred by their NIH 
physician for inclusion in this study.   
 
Participants will also be recruited outside the existing NIH patient population.  The Clinical 
Center Patient Recruitment and Public Liaison Office serves to provide protocol advertisement 
and contact information for both self-referring patients and physician referrals from outside NIH.  
The Clinical Center Volunteer Program website will provide the participants selected for this 
protocol will reflect the gender and ethnic diversity that is representative of the population. 
 
9.2 Institutional Review Board 
A copy of the protocol, proposed informed consent form, other written subject information, and 
any proposed advertising material will be submitted to the IRB for written approval.  In addition, 
the study will not proceed without prior approval by the IRB. 
 
The investigator must submit and, where necessary, obtain approval from the IRB, for all 
subsequent protocol amendments and changes to the informed consent document.  The 
investigator will notify the IRB of deviations from the protocol and serious adverse events.  The 
investigator will be responsible for obtaining IRB approval of the annual Continuing Review 
throughout the duration of the study. 
 

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9.3 Protocol Amendments and Study Termination 
No revisions to this protocol will be permitted without documented approval from the IRB that 
granted the original approval for the study.  In the event of a medical emergency, the 
investigators shall perform any medical procedures that are deemed medically appropriate.  
 
The Principal Investigators and NIAID-IRB reserve the right to terminate the study.  The 
investigator will notify the IRB in writing of the study’s completion or early termination. 
 
9.4 Informed Consent 
Before a subject may be enrolled in the study, it is the investigator’s responsibility to obtain 
written informed consent/assent from the subject, after adequate explanation of the aims, 
methods, anticipated benefits, and potential hazards of the study, and before any protocol-
specific procedures or study medications are administered.  All written information and other 
material to be used by subjects and investigative staff must use vocabulary and language that are 
clearly understood.  If the subject is a minor, the parent or legal guardian must sign the consent 
(See Section 3.4.1). 
The acquisition of informed consent will be documented in the subject’s medical records, as 
required by 21 CFR 312.62, and the informed consent form will be personally signed and dated 
by the subject and by the person who conducted the informed consent discussion.  The original 
signed informed consent form will be retained in the medical chart and a copy of the consent 
form will be provided to the subject. 
 
9.5 Privacy and Confidentiality Provisions 
The privacy and confidentiality of the participating subjects will be protected to the extent 
provided by federal, state, and local law.  Subject identifiers will be attached to clinical and 
laboratory data.  Data will be kept in laboratory or clinical sites and locked or password 
protected.  Medical records will be made available for review when required by the Food and 
Drug Administration, NIAID, or other authorized users, only under the guidelines set by the 
Federal Privacy Act.  Direct access includes examining, analyzing, verifying, and reproducing 
any records and reports that are important to the evaluation of the study.  The investigator is 
obligated to inform the subjects that the above named representatives will review their study-
related records without violating the confidentiality of the subjects.  Subjects will not be 
identified in any reports on this study. 
 
9.6 Policy Regarding Research-Related Injuries 
The Clinical Center will provide short-term medical care for any injury resulting from 
participation in this research.  In general, the National Institutes of Health, the Clinical Center, or 
the Federal Government will provide no long-term medical care or financial compensation for 
research-related injuries. 
 
9.7 Remuneration 
No remuneration will be provided to patients.  All medical treatments associated or necessitated 
by the protocol will be provided without charge to the patient, including medications taken as an 
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9.8 Hazards and Discomforts – Recipient 
9.8.1 Related to the Transplant 
The mortality from conventional BMT may be as high as 40%.  Although our data as 
well as that of others suggest a significant reduction in transplant-related mortality with 
nonmyeloablative “mini” PBSC transplantation, the procedure nevertheless carries 
significant risk.  It is therefore only appropriate to carry out this experimental procedure 
in the context of debilitating or life-threatening conditions and with full informed consent 
from the patient, donor, and immediate family.  The specific hazards of this study using a 
nonmyeloablative preparative regimen and high PBPC-content graft are graft rejection, 
graft versus host disease, and disease relapse.  The major discomforts are those of nausea, 
anorexia, diarrhea, fever and malaise, and intolerance of the isolation period.  Adverse 
effects of those drugs novel to nonmyeloablative transplantation are described in detail 
here. 
 
9.8.2 Related to Radiation 
Adverse effects of radiation have been well described. The most common include nausea 
and mucositis. Toxicities associated with TBI also include infections, cataracts, 
pulmonary insufficiency, liver toxicity and secondary late malignancies. [55, 56] 
Treatment-related mortality in series receiving comparable doses are rare. The risk of 
interstitial pneumonitis is of particular relevance given the use of busulfan with radiation; 
however, in order to reduce this risk, the lungs will be shielded and additionally, there 
will be one day of rest between the busulfan infusion and the administration of radiation.  
Importantly, the majority of the nonneoplastic effects were sub clinical and/or reversible. 
[57] Studies attempting to evaluate the risk induced by radiation alone suggest that there 
is a higher rate of solid tumors after radiation-based regimens.  Curtis et al. reported on 
19,229 patients and found a cumulative incidence rate of 2.2% at 10 years, and 6.7% at 
15 years, with higher doses of TBI associated with a higher risk of solid cancers. [58] 
However, the more important risk factor appears to be related to the level of 
immunosuppression as GVHD was also strongly linked to an increased risk of solid 
tumor development. In fact, in a study of 1036 patients, no relation could be found to 
radiation but the highest risk factor was felt to be the presence of chronic graft versus 
host disease, and long term treatment with cyclosporine. [59] Therefore the actual risk 
cannot be quantified for the low dose of 300cGy to be used in this trial; however, it is 
presumed to be minimal. [60-62]  
On this protocol, the total radiation dose will be 300cGy. In order to further reduce the 
adverse effects on nonlymphoid tissue, the radiation will be divided and given in two 
fractions.  Additionally, we will use lung shielding to minimize the pulmonary toxicity 
and in males use testicular shielding to minimize the risks of sterility secondary to 
radiation.  Should there be any graft rejection with this low dose of radiation, the IRB 
will be notified and the dose will be increased to 200cGy x2 with the same shielding 
used. 
 

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9.8.3 Related to Alemtuzumab (Campath-1H, Campath®) 
The safety and efficacy of alemtuzumab were evaluated in a multicenter, open-label, non-
comparative study in 93 patients with refractory B-cell chronic lymphocytic leukemia (B-
CLL) who had been previously treated with alkylating agents and had failed treatment 
with fludarabine. Adverse events are detailed below.  Previous treatment with alkylating 
agents and fludarabine may have contributed to both the range and severity of the adverse 
events observed. 
Infusion-related:  Adverse events resulted in discontinuation of alemtuzumab therapy in 
6% of the patients.  The most commonly reported infusion-related adverse events include 
rigors in 89% of patients, drug-related fever in 83%, nausea in 47%, vomiting in 33%, 
and hypotension in 15%.  Other frequently reported infusion-related events include rash 
in 30% of patients, fatigue in 22%, urticaria in 22%, dyspnea in 17%, pruritus in 14%, 
headache in 13%, and diarrhea in 13%.  Acute infusion-related events were most 
common during the first week of therapy.  Antihistamines, acetaminophen, antiemetics, 
meperidine, corticosteroids, and incremental dose-escalation were used to prevent or 
ameliorate infusion-related events. 
 
Infections:  In the earlier studies all patients were required to receive anti-herpes and anti-
PCP prophylaxis.  Forty (43%) of 93 patients experienced 59 infections (one or more 
infections per patient) during treatment or within 6 months of the last dose.  Of these, 34 
(37%) patients experienced 42 infections that were of Grade 3 or 4 severity; 11 infections 
(18%) were fatal.  Fifty-five percent of the Grade 3 or 4 infections occurred during 
treatment or within 30 days of last dose.  In addition, one or more episodes of febrile 
neutropenia (ANC ≤500 cells/L) were reported in 10% of patients.  The following types 
of infections were reported: Grade 3 or 4 sepsis in 12% of patients with 1 fatality, Grade 
3 or 4 pneumonia in 15% with 5 fatalities, and opportunistic infections in 17% with 4 
fatalities.  Candida infections were reported in 5% of patients; CMV infections in 8% 
(4% of Grade 3 or 4 severity);  Aspergillosis in 2% with fatal aspergillosis in 1%;  fatal 
mucormycosis in 2%;  fatal cryptococcal pneumonia in 1%;  Listeria monocytogenes 
meningitis in 1%;  disseminated herpes zoster in 1%;  Grade 3 herpes simplex in 2%;  
and Torulopsis pneumonia in 1%.  PCP pneumonia occurred in one (1%) patient who 
discontinued PCP prophylaxis.  In an earlier study, where anti-herpes and anti-PCP 
prophylaxis was optional, 37 (66%) patients had 47 infections while or after receiving 
Campath-1H therapy. 
 
Immunosuppression/Opportunistic 
Infections: 
 
Alemtuzumab 
induces 
profound 
lymphopenia.  Anti-infective prophylaxis is recommended upon initiation of therapy and 
for a minimum of 2 months following the last dose of alemtuzumab or until CD4+ counts 
are ≥200 cells/L.  The median time to recovery of CD4+ counts to 200/L was 2 
months; however, full recovery (to baseline) of CD4+ and CD8+ counts may take more 
than 12 months.  Because of the potential for GvHD in severely lymphopenic patients, 
irradiation of any blood products administered prior to recovery from lymphopenia is 
recommended. 
 
Hematologic: 
Commented [JPK13]: Please provide source citations for study 
results cited in this section. 

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 Pancytopenia/marrow 
hypoplasia: 
Alemtuzumab 
therapy 
was 
permanently 
discontinued in six (6%) patients due to pancytopenia/marrow hypoplasia, two (2%) 
of which were fatal. 
 Anemia:  Forty-four (47%) patients had one or more episodes of new onset National 
Cancer Institute-Common Toxicity Criteria (NCI-CTC) Grade 3 or 4 anemia.  Sixty-
two (67%) patients required red blood cell (RBC) transfusions.  In addition, 
erythropoietin use was reported in 19 (20%) patients.  Autoimmune hemolytic 
anemia secondary to alemtuzumab therapy was reported in 1% of patients.  A 
positive Coombs test without hemolysis was reported in 2% of patients. 
 Neutropenia:  Sixty-five (70%) patients had one or more episodes of NCI-CTC 
Grade 3 or 4 neutropenia.  Median duration of Grade 3 or 4 neutropenia was 28 days 
(range: 2 – 165 days). 
 Thrombocytopenia:  Forty-eight (52%) patients had one or more episodes of new 
onset Grade 3 or 4 thrombocytopenia.  Median duration of thrombocytopenia was 21 
days (range: 2 – 165 days).  Thirty-five (38%) patients required platelet transfusions 
for management of thrombocytopenia.  Autoimmune thrombocytopenia was reported 
in 2% of patients with one fatal case of alemtuzumab-related autoimmune 
thrombocytopenia. 
 Lymphopenia:  The median CD4+ count at 4 weeks after initiation of alemtuzumab 
therapy was 2 cells/L, at 2 months after discontinuation of alemtuzumab therapy, 
207 cells/L, and 6 months after discontinuation, 470 cells/L.  The pattern of 
change in median CD8+ lymphocyte counts was similar to that of CD4+ cells.  In 
some patients treated with alemtuzumab, CD4+ and CD8+ lymphocyte counts had 
not returned to baseline levels for ≥1 year post therapy. 
 
Cardiac:  The following events were reported in at least one patient treated on studies 
where Campath-1H was used as a single agent: cardiac failure, cyanosis, atrial 
fibrillation, cardiac arrest, ventricular arrhythmia, ventricular tachycardia, angina 
pectoris, coronary artery disorder, myocardial infarction, and pericarditis.  Some of these 
cardiac abnormalities may be irreversible.  For this reason, we will monitor subjects with 
an echocardiogram, a 24 hour Holter monitor and serum troponin levels before treatment 
begins, after the last dose of Campath-1H, and at the 6 month follow up visit.  We will 
also closely monitor subjects for cardiac symptomatology and ask them to immediately 
report any cardiac symptoms (palpitations, irregular pulse, difficulty in breathing, 
dizziness, swelling in the ankles, chest discomfort, or pain).  
 
9.8.4 Related to Sirolimus: 
The anticipated toxicities of sirolimus in this trial are those related to its 
immunosuppressive properties, such as an increased likelihood of infection, and mucous 
ulcers.  Other possible toxicities are listed here and include those reported with ≥ 3% and 
< 20% incidence in patients in any sirolimus treatment group in the two controlled 
clinical trials for the prevention of acute organ graft rejection: 
Commented [JPK14]: Please add source citations. 

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Body as a Whole:  abdomen enlarged, abscess, ascites, cellulitis, chills, face edema, flu 
syndrome, generalized edema, hernia, Herpes zoster infection, lymphocele, malaise, 
pelvic pain, peritonitis, sepsis; 
Cardiovascular System:  atrial fibrillation, congestive heart failure, hemorrhage, 
hypervolemia, 
hypotension, 
palpitation, 
peripheral 
vascular 
disorder, 
postural 
hypotension, syncope, tachycardia, thrombophlebitis, thrombosis, vasodilatation;  
Digestive System:  anorexia, dysphagia, eructation, esophagitis, flatulence, gastritis, 
gastroenteritis, gingivitis, gum hyperplasia, ileus, liver function tests abnormal, mouth 
ulceration, oral moniliasis, stomatitis;  
Endocrine 
System: 
Cushing's 
syndrome, 
diabetes 
mellitus, 
glycosuria, 
hypercholesterolemia, hyperlipidemia;  
Hemic 
and 
Lymphatic 
System: 
ecchymosis, 
leukocytosis, 
lymphadenopathy, 
polycythemia, thrombotic thrombocytopenic purpura (hemolytic-uremic syndrome); 
Metabolic and Nutritional:  acidosis, alkaline phosphatase increased, blood urea nitrogen 
(BUN) increased, creatine phosphokinase increased, dehydration, healing abnormal, 
hypercalcemia, hyperglycemia, hyperphosphatemia, hypocalcemia, hypoglycemia, 
hypomagnesemia, hyponatremia, lactic dehydrogenase increased, serum glutamic 
oxaloacetic transaminase (SGOT) increased, serum glutamic pyruvic transaminase 
(SGPT) increased, weight loss;  
Musculoskeletal System: arthrosis, bone necrosis, leg cramps, myalgia, osteoporosis, 
tetany;  
Nervous System: anxiety, confusion, depression, dizziness, emotional lability, 
hypertonia, hypesthesia, hypotonia, insomnia, neuropathy, paresthesia, somnolence;  
Respiratory System:  asthma, atelectasis, bronchitis, cough increased, epistaxis, hypoxia, 
lung edema, pleural effusion, pneumonia, rhinitis, sinusitis;  
Skin and Appendages:  fungal dermatitis, hirsutism, pruritus, skin hypertrophy, skin 
ulcer, sweating;  
Special Senses:  abnormal vision, cataract, conjunctivitis, deafness, ear pain, otitis media, 
tinnitus;  
Urogenital System: albuminuria, bladder pain, dysuria, hematuria, hydronephrosis, 
impotence, kidney pain, kidney tubular necrosis, nocturia, oliguria, pyelonephritis, 
pyuria, scrotal edema, testis disorder, toxic nephropathy, urinary frequency, urinary 
incontinence, urinary retention.  
 
Less frequently occurring adverse events included: mycobacterial infections, Epstein-
Barr virus infections, and pancreatitis.  
 
 

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9.8.5 Related to Busulfan 
Commonly listed adverse events include nausea and vomiting, hair loss, and seizure.  The 
risk of seizure will be minimized by the administration of a prophylactic anticonvulsant 
medication 30 minutes prior to busulfan treatment. 
 
Hematological Effects:  The most frequent serious consequence of treatment with 
busulfan at the recommended dose and schedule is profound myelosuppression occurring 
in all patients.  Severe granulocytopenia, thrombocytopenia, anemia, or any combination 
thereof may develop.  Frequent complete blood counts, including white blood cell 
differentials, and quantitative platelet counts should be monitored during treatment and 
until recovery is achieved.  Absolute neutrophil counts dropped below 500 cells/μL (i.e., 
<0.5x109/L) at a median of 4 days post transplant in 100% of patients treated in the 
busulfan clinical trial.  The absolute neutrophil count recovered at a median of 13 days 
following allogeneic transplantation when prophylactic G-CSF was used in the majority 
of patients.  Thrombocytopenia (< 25,000 cells/μL or requiring platelet transfusion) 
occurred at a median of 5-6 days in 98% of patients.  Anemia (hemoglobin <8.0g/dL) 
occurred in 69% of patients.  Antibiotic therapy and platelet and red blood cell support 
should be used when medically indicated. 
 
Pulmonary:  Bronchopulmonary dysplasia with pulmonary fibrosis is a rare but serious 
complication following chronic busulfan therapy.  The average onset of symptoms is 4 
years after therapy (range 4-10 years).  
 
Cardiac:  Cardiac tamponade has been reported in pediatric patients with thalassemia 
(8/400 or 2% in one series) who received high doses of oral busulfan and 
cyclophosphamide as the preparatory regimen for hematopoietic progenitor cell 
transplantation.  Of these 8 children, 6 died and 2 were saved by rapid pericardiocentesis.  
Abdominal pain and vomiting preceded the tamponade in most patients.  No patients 
treated in the busulfan injection clinical trials experienced cardiac tamponade. 
 
Neurological:  Seizures have been reported in patients receiving high dose oral busulfan 
at doses producing plasma drug levels similar to those achieved following the 
recommended dosage of busulfan.  Despite prophylactic therapy with phenytoin, one  
seizure was reported during an autologous transplantation clinical trial of Busulfex®.  
This episode occurred during the cyclophosphamide portion of the conditioning regimen, 
36 hours after the last busulfan dose.  Anti-convulsant prophylactic therapy should be 
initiated prior to busulfan treatment. 
 
Hepatic Effects: Current literature suggests that high busulfan area under the plasma 
concentration versus time curve (AUC) values (>1500μM/min) may be associated with 
an increased risk of developing hepatic veno-occlusive disease (HVOD).  Patients who 
have received prior radiation therapy, greater than or equal to 3 cycles of chemotherapy, 
or a prior progenitor cell transplant may be at an increased risk of developing HVOD 
with the recommended busulfan dose and regimen.  Based on clinical examination and 
laboratory findings, hepatic veno-occlusive disease was diagnosed in 8% (5/61) of 
patients treated with busulfan in the setting of allogeneic transplantation, was fatal in 2/5 
Commented [JPK15]: Please provide source citations for data 
presented in this section. 
Commented [JPK16]: Requires a reference citation. 

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cases (40%), and yielded an overall mortality from HVOD in the entire study population 
of 2/61 (3%).  Of the 5 patients diagnosed with HVOD, 3 were retrospectively found to 
meet the Jones’ criteria.  The incidence of HVOD reported in the literature from the 
randomized, controlled trials were 7.7% to 12%. 
 
Others:  Other reported adverse reactions include: headache (mild or moderate 64%, 
severe 5%), abdominal pain (mild or moderate 69%, severe 3%) asthenia (mild or 
moderate 49%, severe 2%), allergic reaction (mild or moderate 24%, severe 2%), 
injection site inflammation (mild or moderate 25%), injection site pain (mild or moderate 
15%), chest pain (mild or moderate 26%), back pain (mild or moderate 23%), myalgia 
(mild or moderate 16%), arthralgia (mild or moderate 13%), and ear disorder in 3%. 
 
Over dosage:  There is no known antidote to busulfan.  The principal toxic effects are 
bone marrow depression and pancytopenia but the central nervous system, liver, lungs, 
and gastrointestinal tract may be affected.  The hematologic status should be closely 
monitored and vigorous supportive measures instituted, if necessary.  Dialysis may be 
considered in the management of overdose, as there is 1 report of successful dialysis of 
busulfan.  Busulfan is metabolized by conjugation with glutathione; thus, administration 
of glutathione may be considered. 
 
 
9.9 Hazards and Discomforts – Donor 
9.9.1 Related to G-CSF 
The hazard to the donor is low.  The discomfort from G-CSF mobilization and apheresis 
for collection of blood stem cells are probably lower than those associated with marrow 
harvesting.  
G-CSF has been given to large numbers of normal donors without major adverse effects 
or long-term consequences.  The immediate adverse effects of G-CSF in 50-75% of 
recipients are bone pain, fatigue, insomnia, myalgia and headache.  These are usually 
mild and are self-limiting.  Reversible thrombocytopenia, with platelet counts falling to 
the region of 100,000/mm3 is frequent and to less than 60,000/mm3 after a third 
apheresis.  Two patients have been reported to experience non-fatal splenic rupture after 
more prolonged treatment with higher doses of G-CSF.  Of these 2 patients, 1 had 
concurrent mononucleosis, a second cause for splenic rupture.  Patients with ongoing 
ischemic heart disease have been reported to have angina seemingly temporally related to 
G-CSF administration and apheresis. 
 
9.9.2 Related to Central Line Placement 
It is estimated that about 50% of the donors will require intravenous central line 
placement to successfully complete apheresis.  Intravenous line placement in the femoral 
vein using a temporary double-lumen Arrow catheter carries a small risk of bleeding, 
bruising or pain and a very low risk of accidental injury to the adjacent artery and nerve.  
These risks are minimized by using only trained experienced staff for the procedure.  
 
Commented [JPK17]: Requires a reference citation. 

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9.9.3 Related to Apheresis 
Adverse reactions related to apheresis include hypotension resulting from transient blood 
volume loss and cutaneous paresthesia from the use of anticoagulant.  The former toxicity 
can be corrected by postural changes and volume replacement.  The latter is manageable 
with slowing the rate of anticoagulant infusion and/or providing calcium supplement.  In 
exceptional instances, the donor may be required to donate PBPC a third time or to give 
bone marrow.  Donation of PBPC on 3 successive days significantly increases the risk of 
thrombocytopenia (<100,000/μL).  However, thrombocytopenia is transient and unlikely 
to cause clinical sequelae.  There is no additional risk to the donor giving marrow after 
PBPC donation other than normally would be associated with bone marrow harvesting.  
 
9.10 Risks in Relation to Benefit 
9.10.1 For Adult Transplant Subjects 
Clinically the approach is ethically acceptable because we are targeting a patient group 
with a debilitating and often lethal hematological disease, incurable with conventional 
treatments other than allogeneic BMT.  The protocol aims to decrease the risk of 
transplant related mortality, thus making more patients candidates for potentially curative 
therapy. 
The research, therefore, involves more than a minor increase over minimal risk to 
subjects with the prospect of direct benefit (45 CFR 46.102) 
 
9.10.2 For Pediatric Donors 
The stem cell collection procedure is considered part of the research for the donors, 
namely the use of G-CSF in young children for mobilizing peripheral blood progenitor 
cells.  The risks associated with the stem cell collection procedure would be considered 
as risks of research for the pediatric donors. Therefore participation as a stem cell donor 
in this study is subject to the criteria set forth in 45 CFR 46, Subpart D. 
 
9.10.3 For Pediatric Donors Involved in Laboratory Research Studies   
The inclusion of children satisfies the criteria set forth in 45 CFR 46.404 as follows: 
 
(a) The research does not involve greater than minimal risk.  On this study, we will 
obtain blood specimens for research from pediatric donors that will be concurrent 
with clinically indicated sampling.  Therefore, there is no risk associated with 
sample collection for research because research will only be performed on material 
obtained during standard clinical intervention. 
 
Research specimens will be stored in Dr. Malech’s laboratory (LHD labs, Bldg 10 
CRC room 5-3750).  Samples will never be labeled with the child’s name.  Samples 
will be assigned a unique code known only to the principal investigator, which will 
serve as a link to the child’s clinical information collected as part of this research 
protocol.  No samples will be provided to investigators outside the branch.  
Therefore, confidentiality is protected. 

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Only those laboratory tests involving no greater than minimal risk will be 
conducted. Research will include genetic testing; therefore, there is genetic testing- 
associated risk.  
 
(b) Adequate provisions are made for soliciting the assent of the children and 
permission of their parents or guardians, as set forth in 45 CFR 46.408. 
 
Therefore, participation of pediatric donors in laboratory research during this study 
involves not greater than minimal risk (45 CFR 46.404). 
 
9.11 Informed consent 
The investigational nature and research objectives of this trial, as well as the procedures and their 
attendant risks and discomforts will all be carefully explained to the subject, and a signed 
informed consent document will be obtained prior to entry onto this study.  Drs. Kang or Malech 
will lead this discussion.  
 
If the subject is a minor, the parent who signs the consent for the minor must be a legally 
recognized parent or guardian.  Where deemed appropriate by the clinician, and the child's parent 
or guardian, the child will also be included in all discussions about the trial and a minor's assent 
will be obtained.  The parent or guardian will sign on the designated line on the informed consent 
document attesting to the fact that the child had given assent. 
 
If at any time during participation in the protocol new information becomes available relating to 
risks, adverse events, or toxicities, this information will be provided orally or in writing to all 
enrolled or prospective patient participants or parent/legal guardian.  Documentation will be 
provided to the IRB and if necessary the informed consent will be amended to reflect relevant 
information. 
 
 
10.0 
BIOSTATISTICAL CONSIDERATIONS 
This trial is designed to estimate the engraftment rate, which is anticipated to be about 80%.  The 
study has a sample size of 10 per group and this will allow us to estimate the engraftment rate 
with reasonable precision.  See table below:  
 
 
 
 
 
      N 
expected # engraftment 
assuming true rate is 80% 
95 % binomial exact CI 
     10 
           8 
.4439045    .9747893 
     15 
           12 
.5191089     .956688 
     25 
 
           20 
 
.5929626     .9316885 
 

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      N 
expected # engraftment 
assuming true rate is 70% 
95 % binomial exact CI 
     10 
7 
.3475471    .9332605 
     15 
11 
.4489968    .9221285 
     25 
 
18 
 
.5061232    .8792833 
 
 
10.1 
Stopping Rules 
 
Because this regimen is designed to decrease the risk of severe acute and chronic GvHD in 
patients with non-life threatening conditions, the study will be stopped and the design re-
evaluated after any death. We will also monitor the study for excessive graft rejection and 
excessive (acute and chronic) Grade III-IV GvHD, which we would consider a failure of the 
regimen. Therefore, regimen failure is a composite endpoint that means graft rejection, acute 
Grade III-IV GvHD, or chronic Grade III-IV GvHD.   Regimen failure will be evaluated for 
each patient during the 6 month period following the start of transplant.   We will stop the 
study if posterior probability that the regimen failure rate is greater than 30% exceeds 0.90. 
Stopping means that serious consideration will be given to modifying or terminating the 
protocol.     
 
 
11.0 
PHARMACEUTICALS 
11.1 Alemtuzumab (Campath-1H, Campath®) 
Generic name:  alemtuzumab  
Classification:  monoclonal antibody  
Action:  Monoclonal antibody directed against CD52 antigen, a surface glycoprotein expressed 
by lymphocytes  
Availability:  Commercial: Berlex Laboratories 
Dose:  30 mg IV 3  times a week after initial doses of 3mg and 10mg   (FDA approved dose for 
Chronic Lymphocytic Leukemia) 
Storage:  Stored at 2 to 8 degrees Celsius (36 to 46 degrees Fahrenheit) and protected from direct 
sunlight. Protect from freezing; discard if frozen. 
Stability:  Diluted solution for administration can be stored at room temperature (15 to 30 
degrees Celsius) or refrigerated, and should be used within 8 hours after dilution; protect solution 
from light. 
Adverse effects:  Nausea, vomiting, fever, chills, rigors, hypotension, rash, headaches, 
abdominal pain, myalgias, arthralgia, diarrhea, dyspnea, bronchospasm, angioedema, tumor lysis 
syndrome, infections, transient neutropenia and thrombocytopenia, pancytopenia, impaired 
sperm motility. 

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11.2  Daclizumab (Zenapax® ) 
General:  Zenapax® (Daclizumab) is an immunosuppressive, humanized immunoglobulin-G1 
monoclonal antibody.  It binds to the CD-25 or Tac subunit of human IL-2 receptors on T-
lymphocytes. The volume of distribution is approximately 6 liters and the systemic clearance is 
about 15 mL/hour. The elimination half-life is approximately 480 hours (20 days).  
 
Administration:  Daclizumab should be mixed with 50 mL of sterile 0.9% sodium chloride 
solution and administered via a peripheral or central vein over a 15 minute period. 
 
Adverse Drug Reactions:  Generally, daclizumab is a well tolerated agent. In clinical trials the 
most frequently reported adverse events were gastrointestinal disorders. Also reported are 
hypertension, hypotension, chest pain, tachycardia, edema, dyspnea, pulmonary edema, 
coughing, thrombosis, bleeding, and renal tubular necrosis.  
 
Dose:  In the treatment of GvHD: 1mg/kg IV on Days 1, 4, 8, 15, and 22.  
 
11.3 Human Recombinant Granulocyte Colony Stimulating Factor 
Generic:  G-CSF or Filgrastim (Amgen; Neupogen®) 
Classification:  glucoprotein 
Action:  Regulates the production of neutrophils from bone marrow progenitor cells and 
mobilizes primitive hematopoietic stem cells from the bone marrow into the circulation.    
Metabolism:  Absorption and clearance of G-CSF follow first order kinetics.  A positive linear 
correlation occurs between the parenteral dose and both the serum concentration and area under 
the concentration-time curves.  The elimination T1/2 is 3.5 hours. 
Administration:   G-CSF may be given, as a single daily dose, SC or IV over 30 min.  
Adverse effects:  Medullary bone pain, myalgia, headache, insomnia, chills, low grade fever, 
rash, injection site irritation, exacerbation of preexisting inflammatory conditions, splenomegaly, 
mild to moderate decreases in platelet number, and reversible elevations in uric acid, lactate 
dehydrogenase, and leukocyte alkaline phosphatase. 
 
G-CSF is a protein that is produced by normal human bone marrow cells.  G-CSF used in this 
study is produced by Escherichia coli with recombinant DNA technology.  The only differences 
between this product and natural G-CSF are an additional N-terminal methionine necessary for 
expression in E. coli and the fact that this product is unglycosylated.  G-CSF is approved by the 
US Food and Drug Administration for use in preventing chemotherapy-induced leukopenia.  
This drug has been used in cancer patients for prolonged periods of time without major toxicities.  
The extensive use of this drug in many patients has also proven its safety at a dose of 
10 mg/kg/day, an effective dose for up to 80% of subjects.  The most frequent adverse effect is 
bone pain originating from the stimulated proliferation of cell mass within the bone marrow.    
The pain is usually mild, can be alleviated most often by acetaminophen or non-steroidal 
analgesics, and will cease altogether once G-CSF is discontinued.  G-CSF administration is 
associated with increased inflammation in patients with autoimmune diseases or subjects with 
infections, but such patients are excluded from this study.    Leukocytosis is expected after 

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administration of G-CSF.  Other adverse reactions associated with the use of G-CSF as cited in 
the literature include: fatigue (50%), headache (30%), muscle aches (25%), insomnia (rare), 
fever (rare), transient but reversible increases of alkaline phosphatase, lactate dehydrogenase, 
and uric acid levels, exacerbation of preexisting skin rashes, and thrombocytopenia (mild, 
reversible). 
 
Recombinant G-CSF is available from the pharmacy at the National Heart, Lung and Blood 
Institute in colorless glass, single-use vials containing either 300mg in 1.0 mL vials or 480mg in 
1.6 mL vials (300 mg/mL).  It is formulated as a sterile, clear, colorless liquid in a 10 mM 
sodium acetate buffer at pH 4.0.  The quantitative composition (per mL) is: 
Recombinant G-CSF  
300 mg 
Acetate  
0.59 mg 
Mannitol  
50 mg 
Tween 80TM 
0.004% 
Sodium  
0.035 mg 
Water for injection (qs ad) to  
1.0 mL 
 
Storage: The intact vials of G-CSF should be stored under refrigeration (2 - 8°C). 
 
Stability: G-CSF in the intact vial is stable for 36 months when stored in a refrigerator at 2 - 8°C.  
A single brief exposure (up to 7 days) to elevated temperatures (< 37°C) does not affect the 
stability.  G-CSF should not be frozen, and vials which have been frozen should not be used. 
 
 
11.4 Infliximab (Remicade®) 
General:  Remicade® (Infliximab) is a chimeric monoclonal antibody directed against tumor 
necrosis factor-alpha (TNF-α).  TNF-α, a pro-inflammatory protein produced by immune cells, is 
believed to play a role in the pathogenesis of GvHD. Infliximab is a human/murine chimeric 
monoclonal antibody that binds specifically to both membrane-bound and soluble TNF-α.  
Administration:  Infliximab should be mixed with 250 mL of sterile 0.9% sodium chloride 
solution and administered via a peripheral or central vein over a period of not less than 2 hours. 
An in-line, sterile, non-pyrogenic, low-protein-binding filter with a pore size of 1.2um or less 
should be used for administration. 
Adverse Drug Reactions:  Infliximab is generally well tolerated. The most common adverse 
effect has been nausea. Infusion reactions that have been reported include chest pain, nausea, 
fever, facial flushing, headache, urticaria, dyspnea, and hypotension. Patients treated with 
infliximab are at an increased risk of developing respiratory infections. Other serious infections 
have also been reported including sepsis, reactivation of Hepatitis B, and disseminated 
tuberculosis. A tuberculin skin test is recommended prior to initiating therapy. Patients are at an 
increased risk of lymphoma (non-Hodgkin’s lymphoma and Hodgkin’s disease). Severe hepatic 
reactions, including acute liver failure, jaundice, hepatitis, and cholestasis have been reported 
rarely in post-marketing data.   
Dose:  In the treatment of GvHD: 10 mg/kg IV every week for a maximum of 4 doses. 
 
Commented [JPK18]: Is this information necessary here? 

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11.5 Sirolimus (Rapamune®) 
General:  Sirolimus is an immunosuppressant that is FDA approved for use in combination with 
cyclosporine and corticosteroids for prophylaxis of organ rejection after kidney transplantation. 
Administration:  Sirolimus is administered orally as either 1mg tablets or oral solution available 
in 1 mg/mL. 
Adverse Drug Reactions:  Common adverse effects attributed primarily to sirolimus include 
anemia, thrombocytopenia, hyperlipidemia, and hypertension. When used in combination with 
cyclosporine, careful monitoring of renal function is required because this combination has been 
associated with increases in serum creatinine. Co-administration of sirolimus with strong 
inhibitors or inducers of CYP3A4 and/or P-gp is not recommended. 
Dose:  Variable dosing. FDA approved adult dose for renal transplant rejection: loading dose of 
6 mg followed by a maintenance dose of 2 mg once daily. For patients 13 years or older who 
weigh less than 40 kg, the 1-time loading dose is 3 mg/m2 followed by a maintenance dose of 
1 mg/m2/day; the dose should be decreased by one-third for patients with hepatic impairment.  
Metabolism:  The drug is metabolized in the liver by cytochrome P450 3A enzymes and has an 
elimination half-life of about 60 hours. 
Vaccination: Use of live vaccines during treatment with sirolimus should be avoided as 
vaccination may be less effective. 
11.6 Busulfan 
Source: ESP Pharma 
Generic: busulfan 
Other: Busulfex® (IV formulation), Myleran® (PO formulation) 
Classification: Alkylating agent that prevents cell division by altering DNA  
Metabolism: Via the liver; sulfoxane, 3-hydroxysulfoxane, and other metabolites are formed and 
excreted in the urine. 
Formulation and Preparation 
Tetramethylene di(methanesulphonate); Butane-1,4-diol di(methanesulphonate).; supplied as an 
ampoule containing 60 mg/10 mL injection. 
 
Stability and Solubility 
Busulfan for injection should be refrigerated at 2 to 8°C. 
 
Incompatibilities and Drug Interactions 
Itraconazole decreases busulfan clearance.  Phenytoin increases busulfan clearance by 15%.   
 
Administration Procedure 
IV busulfan must be diluted prior to use with either 0.9% Sodium Chloride Injection, USP 
(normal saline) or 5% Dextrose Injection, USP (D5W). The diluent quantity should be 10 times 
the volume of busulfan injection, so that the final concentration is approximately 0.5 mg/mL. 

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Busulfan will be administered intravenously via a central venous catheter as a 2-hour infusion on 
2 consecutive days for a total dose of 10 mg/kg. Antiemetics of the 5-HT3 class should be 
administered prior to the first dose of busulfan and continued on a fixed schedule through 
busulfan administration. 
 
Clonazepam IV will be given daily for the 2 days of the infusion as anti-epileptic prophylaxis. 
 
Adverse effects: please see Section 9.8.5 for discussion on this. 
 
12.0 
BIOHAZARD CONTAINMENT 
As the transmission of HIV and other blood-borne pathogens can occur through contact with 
contaminated needles, blood, and blood products, appropriate blood and secretion precautions 
will be employed by all personnel in the drawing of blood and shipping and handling of all 
specimens for this study, as currently recommended by the Centers for Disease Control. 
 
All infectious specimens will be sent using the ISS-1 SAF-T-PAK mandated by the International 
Air Transport Association Dangerous Goods Regulations-Packing Instruction 602.   
Please refer to individual carrier guidelines, e.g., Fed Ex, Airborne, for specific instructions. 
 
13.0 
DATA COLLECTION, MANAGEMENT AND STORAGE  
13.1 Data and Safety Monitoring Plan 
Study data will be collected and maintained using the Crimson computerized system .    The 
Principal Investigator is responsible for assuring that the data collected is complete, accurate, and 
recorded in a timely manner.  Source documentation (the point of initial recording of a piece of 
data) should support the data collected on the case report form, and be signed and dated by the 
person recording and/or reviewing the data.  Source documents must include all recordings of 
observations or notations of clinical activities and all reports and records necessary for the 
evaluation and reconstruction of the clinical trial.  Source documents include, but are not limited 
to, the patient medical records, laboratory reports, ECG tracings, x-rays, radiologist reports, 
patient diaries, biopsy reports, ultrasound photographs, patient progress notes, pharmacy records, 
and any other similar reports or records of procedures performed during the subject’s 
participation in the protocol.  Data for Crimson will be collected during patient visits, phone calls 
with subjects and health care providers, patient diaries, and information abstracted from the 
patient’s medical record.  It is not acceptable for Crimson to be the only record of the patient’s 
participation in the study.  This is to ensure that anyone who would access the patient medical 
record has adequate knowledge that the patient is participating in a clinical trial.  
 
 
13.2 Study Monitoring 
The trial will be conducted in compliance with this protocol, International Conference on 
Harmonization (ICH) Guideline for Good Clinical Practices (GCP), and any applicable 
regulatory requirement(s).  Monitors under contract to the NIAID will visit the clinical research 
site to monitor all aspects of the study in accordance with the appropriate regulations.  The 
objectives of a monitoring visit will be:  
1) 
To verify the prompt reporting of all data points, including reporting SAEs, 
checking availability of signed informed consent 

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2) 
To compare individual subject records, data table pulls and the source 
documents (supporting data, laboratory specimen records and medical records 
to include physician progress notes, nurses’ notes, subjects’ hospital charts) 
3) 
To ensure protection of study subjects, compliance with the protocol, and 
accuracy and completeness of records.   
The monitors also will inspect the clinical site regulatory files to ensure that regulatory 
requirements (OHRP) and applicable guidelines (ICH-GCP) are being followed.  During the 
monitoring visits, the investigator (and/or designee) and other study personnel will be available 
to discuss the study progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms, data table 
pulls) and pertinent hospital or clinical records readily available for inspection by the local IRB, , 
the site monitors, and the NIAID staff for confirmation of the study data. 
 
The investigator is responsible for retaining all essential documents listed in the ICH Good 
Clinical Practice Guideline.  The essential documents should be maintained until at least 2 years 
after the last approval of a marketing application, or at least 2 years have elapsed since the 
formal discontinuation of clinical development of the investigational agent, or at least 3 years per 
NIH FWA, whichever is longest.  Storage of all trial-related documents will be such that 
confidentiality will be strictly maintained. 
 
 
14.0 
PLAN FOR USE AND STORAGE OF BIOLOGICAL SAMPLES  
Patients will be asked to provide 10 mL of blood so that genomic DNA can be isolated 
and stored for future genetic studies.  These studies will concern genes that are involved 
with regulation of inflammation, and innate or adaptive immune responses to infections.  
The tissue specimen blocks requested for this study, both paraffin-embedded and fresh-
frozen, will also be stored.  These specimens will be coded separately with a unique 
number and not by patient name.  A separate file will be maintained that will include the 
key for coupling the code number to a specific patient.  Both the specimens and the file 
will be stored in a locked room, in locked storage units for the purpose of maintaining 
confidentiality. Access to research samples will be limited using either a locked room or 
a locked freezer. Data will be kept in password-protected computers.  Only investigators 
or their designee(s) will have access to the samples and data.  We anticipate the storage 
of these samples for 10 years after the completion of the protocol. These specimens may 
be used in collaborative research, but the specimens will not be able to be linked with 
specific patients.  The research use of these stored specimens will be conducted in 
accordance with policy set by the NIH Office of Human Subjects Research and only with 
appropriate IRB approval.  Informed consent will be obtained for these purposes (see 
Consent Form). The Prinicipal investigator will notify the NIAID IRB under any of the 
following circumstances: 
a. The loss of key for coupling the code number to a specific patient  
b. An unauthorized entry into the locked room where samples are stored.  
c.  An unauthorized entry into computerized data 
 
 
 
 

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15.0 
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Watanabe, C., et al., Successful unrelated bone marrow transplantation for a patient with 
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11. 
Mancias, C., A.J. Infante, and N.R. Kamani, Matched unrelated donor bone marrow 
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12. 
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Tomizawa, D., et al., Allogeneic hematopoietic stem cell transplantation for seven 
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Weiden, P.L., et al., Antileukemic effect of chronic graft-versus-host disease: 
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15. 
Kolb, H.J., et al., Donor leukocyte transfusions for treatment of recurrent chronic 
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Porter, D.L., et al., Induction of graft-versus-host disease as immunotherapy for relapsed 
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Kolb, H.J., et al., Graft-versus-leukemia effect of donor lymphocyte transfusions in 
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18. 
Khouri, I.F., et al., Allogeneic blood or marrow transplantation for chronic lymphocytic 
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Kang, E.M., et al., Nonmyeloablative conditioning followed by transplantation of 
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20. 
Horwitz, M.E., et al., Treatment of Chronic Granulomatous Disease with 
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21. 
Grunebaum, E., et al., Bone marrow transplantation for severe combined immune 
deficiency. Jama, 2006. 295(5): p. 508-18. 
22. 
Gluckman, E., et al., Outcome of cord-blood transplantation from related and unrelated 
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23. 
Barker, J.N., et al., Rapid and complete donor chimerism in adult recipients of unrelated 
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24. 
Barker, J.N., et al., Transplantation of 2 partially HLA-matched umbilical cord blood 
units to enhance engraftment in adults with hematologic malignancy. Blood, 2005. 105(3): p. 
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25. 
Shulman, L.N., et al., Low-dose total body irradiation and G-CSF without hematopoietic 
stem cell support in the treatment of relapsed or refractory acute myelogenous leukemia (AML), 
or AML in second or subsequent remission. International Journal of Radiation 
Oncology*Biology*Physics, 1998. 42(5): p. 1113-1117. 
26. 
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engraftment in nonmyeloablated murine hosts. Exp Hematol, 2001. 29(6): p. 779-85. 
27. 
Kang, E., et al., In Vivo Persistence of Retrovirally Transduced Murine Long-Term 
Repopulating Cells Is Not Limited by Expression of Foreign Gene Products in the Fully or 
Minimally Myeloablated Setting. Hum Gene Ther, 2001. 12(13): p. 1663-72. 
28. 
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by nonmyeloablative conditioning. Hum Gene Ther, 1999. 10(11): p. 1783-90. 
29. 
Kang, E.M., et al., Persistent low-level engraftment of rhesus peripheral blood progenitor 
cells transduced with the fanconi anemia c gene after conditioning with low-dose irradiation. 
Mol Ther, 2001. 3(6): p. 911-9. 
30. 
Dufour, C., et al., Stem cell transplantation from HLA-matched related donor for 
Fanconi's anaemia: a retrospective review of the multicentric Italian experience on behalf of 
AIEOP-GITMO. Br J Haematol, 2001. 112(3): p. 796-805. 
31. 
Myburgh, J.A., et al., Transplantation tolerance in primates following total lymphoid 
irradiation and allogeneic bone marrow injection. II. Renal allografts. Transplantation, 1980. 
29(5): p. 405-8. 
32. 
Myburgh, J.A., et al., Total lymphoid irradiation in kidney and liver transplantation in the 
baboon: prolonged graft survival and alterations in T cell subsets with low cumulative dose 
regimens. J Immunol, 1984. 132(2): p. 1019-25. 

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33. 
Storb, R., et al., Stable mixed hematopoietic chimerism in dogs given donor antigen, 
CTLA4Ig, and 100 cGy total body irradiation before and pharmacologic immunosuppression 
after marrow transplant. Blood, 1999. 94(7): p. 2523-9. 
34. 
FEINSTEIN, L., et al., Nonmyeloablative Hematopoietic Cell Transplantation: Replacing 
High-Dose Cytotoxic Therapy by the Graft-versus-Tumor Effect. Ann NY Acad Sci, 2001. 
938(1): p. 328-339. 
35. 
Kang, E.M., et al., Busulfan pharmacokinetics, toxicity, and low-dose conditioning for 
autologous transplantation of genetically modified hematopoietic stem cells in the rhesus 
macaque model. Exp Hematol, 2006. 34(2): p. 132-9. 
36. 
Aiuti, A., et al., Correction of ADA-SCID by stem cell gene therapy combined with 
nonmyeloablative conditioning. Science, 2002. 296(5577): p. 2410-3. 
37. 
Dick, A.D., et al., Campath-1H therapy in refractory ocular inflammatory disease. Br J 
Ophthalmol, 2000. 84(1): p. 107-9. 
38. 
Coles, A.J., et al., Pulsed monoclonal antibody treatment and autoimmune thyroid disease 
in multiple sclerosis. Lancet, 1999. 354(9191): p. 1691-5. 
39. 
Isaacs, J.D., et al., Humanized anti-CD4 monoclonal antibody therapy of autoimmune 
and inflammatory disease. Clin Exp Immunol, 1997. 110(2): p. 158-66. 
40. 
Killick, S.B., et al., Sustained remission of severe resistant autoimmune neutropenia with 
Campath-1H. Br J Haematol, 1997. 97(2): p. 306-8. 
41. 
Pawson, R., et al., Treatment of T-cell prolymphocytic leukemia with human CD52 
antibody. J Clin Oncol, 1997. 15(7): p. 2667-72. 
42. 
Newman, D.K., et al., Prevention of immune-mediated corneal graft destruction with the 
anti- lymphocyte monoclonal antibody, CAMPATH-1H. Eye, 1995. 9(( Pt 5)): p. 564-9. 
43. 
Friend, P.J., et al., Reversal of allograft rejection using the monoclonal antibody, 
Campath- 1G. Transplant Proc, 1991. 23(4): p. 2253-4. 
44. 
Calne, R., et al., Campath IH allows low-dose cyclosporine monotherapy in 31 cadaveric 
renal allograft recipients. Transplantation, 1999. 68(10): p. 1613-6. 
45. 
Osterborg, A., et al., Phase II multicenter study of human CD52 antibody in previously 
treated chronic lymphocytic leukemia. European Study Group of CAMPATH-1H Treatment in 
Chronic Lymphocytic Leukemia. J Clin Oncol, 1997. 15(4): p. 1567-74. 
46. 
Osterborg, A., et al., Humanized CD52 monoclonal antibody Campath-1H as first-line 
treatment in chronic lymphocytic leukaemia. Br J Haematol, 1996. 93(1): p. 151-3. 
47. 
Bowen, A.L., et al., Subcutaneous CAMPATH-1H in fludarabine-resistant/relapsed 
chronic lymphocytic and B-prolymphocytic leukaemia. Br J Haematol, 1997. 96(3): p. 617-9. 
48. 
Kottaridis, P.D., et al., In vivo CAMPATH-1H prevents graft-versus-host disease 
following nonmyeloablative stem cell transplantation. Blood, 2000. 96(7): p. 2419-25. 
49. 
Hale, G., et al., CD52 antibodies for prevention of graft-versus-host disease and graft 
rejection following transplantation of allogeneic peripheral blood stem cells. Bone Marrow 
Transplant, 2000. 26(1): p. 69-76. 
50. 
Hale, G. and H. Waldmann, Recent results using CAMPATH-1 antibodies to control 
GVHD and graft rejection. Bone Marrow Transplant, 1996. 17(3): p. 305-8. 
51. 
Chakraverty, R., et al., Limiting transplantation-related mortality following unrelated 
donor stem cell transplantation by using a nonmyeloablative conditioning regimen. Blood, 2002. 
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52. 
Powell, J.D., C.G. Lerner, and R.H. Schwartz, Inhibition of cell cycle progression by 
rapamycin induces T cell clonal anergy even in the presence of costimulation. J Immunol, 1999. 
162(5): p. 2775-84. 
53. 
Kahan, B.D., Efficacy of sirolimus compared with azathioprine for reduction of acute 
renal allograft rejection: a randomised multicentre study. The Rapamune US Study Group. 
Lancet, 2000. 356(9225): p. 194-202. 
54. 
Groth, C.G., et al., Sirolimus (rapamycin)-based therapy in human renal transplantation: 
similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal 
Transplant Study Group. Transplantation, 1999. 67(7): p. 1036-42. 
55. 
Buchali, A., et al., Immediate toxicity during fractionated total body irradiation as 
conditioning for bone marrow transplantation. Radiother Oncol, 2000. 54(2): p. 157-62. 
56. 
Deeg, H.J., Acute and delayed toxicities of total body irradiation. Seattle Marrow 
Transplant Team. Int J Radiat Oncol Biol Phys, 1983. 9(12): p. 1933-9. 
57. 
Thomas, O., et al., Long-term complications of total body irradiation in adults. Int J 
Radiat Oncol Biol Phys, 2001. 49(1): p. 125-31. 
58. 
Curtis, R.E., et al., Solid cancers after bone marrow transplantation. N Engl J Med, 1997. 
336(13): p. 897-904. 
59. 
Kolb, H.J., et al., Malignant neoplasms in long-term survivors of bone marrow 
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Marrow Transplantation and the European Late Effect Project Group. Ann Intern Med, 1999. 
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60. 
Gerrard, G.E., et al., Toxicity and dosimetry of fractionated total body irradiation prior to 
allogeneic bone marrow transplantation using a straightforward radiotherapy technique. Clin 
Oncol, 1998. 10(6): p. 379-83. 
61. 
Bhatia, S., et al., Solid cancers after bone marrow transplantation. J Clin Oncol, 2001. 
19(2): p. 464-71. 
62. 
Deeg, H.J., et al., Malignancies after marrow transplantation for aplastic anemia and 
fanconi anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood, 1996. 87(1): 
p. 386-92. 
16.0 
 
 
 
 
 
 
 
 
 

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APPENDIX A: 
GvHD PROPHYLAXIS:  SIROLIMUS (RAPAMUNE®)  
 
Dose 
 
Sirolimus Day -1 to Day 100 in all patients.  Subjects will be advised not to take medication with 
grapefruit juice and not to take St. John’s wort while on medication.  Subjects must also be advised to 
limit exposure to sunlight and UV light due to an increased risk of skin cancer.  Women of child-bearing 
potential will be informed of the potential risks during pregnancy and that they should use effective 
contraception prior to initiation of drug.  
 
Levels 
 
Serum levels will be monitored as appropriate to maintain trough levels between 10-20 ng/mL. 
 
Mechanism of Action 
 
Sirolimus blocks T cell activation by inhibiting transport of cytoplasmic nuclear factor of activated T-
cells (NFAT) into the cell nucleus, thereby preventing IL-2 transcription. 
 
Adverse Drug Reactions:  (See Section 11.5) 
 
 

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APPENDIX B: 
TRANSFUSION 
OF 
RED 
CELLS 
TO 
RECIPIENTS 
OF 
ABO 
INCOMPATIBLE MARROW 
 
Major ABO incompatible recipient - donor 
 
Patient  
 
 
Donor  
 
 
Transfused RBC = patients  
O 
 
 
 
A , B or AB 
 
 
 
O 
A 
 
 
 
B  or AB 
 
 
 
A or O 
B 
 
 
 
A  or AB 
 
 
 
B or O 
 
Minor ABO incompatibility recipient - donor 
 
Patient  
 
 
Donor  
 
Transfused RBC = donor group 
A, B or AB 
 
 
O 
 
 
 
O 
AB 
 
 
 
B 
 
 
 
B or O 
AB  
 
 
 
A 
 
 
 
A or O 
 
TRANSFUSION OF PLATELETS TO RECIPIENTS OF ABO INCOMPATIBLE MARROW 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX C:   
 
 
IMMUNODEFICIENCY AlloBMT 
DONOR 
16.1.1 Schedule of Events 
 
Patient’s Name: ______________________                  MRN:__________________ 
 
 
Protocol Requirement Visit 1 
(Screen) 
Visit 2 
(Day 1) 
Visit 3 
(Day 2) 
Visit 4 
(Day 3) 
Visit 5  
(Day 4) 
Visit 6  
(Day 5) 
Visit 7 
(Day 6) 
DATE:  
 
 
 
 
 
 
CONSENT X 
 
 
 
 
 
 
H &P 
 (C=complete; D=directed) C 
 
 
 
 
 
 
CBC with diff 
X 
 
 
 
 
 
 
Chem 20  
X 
 
 
 
 
 
 
COAG 
Panel 
(PTT/PT/FIB/TT) 
X 
 
 
 
 
 
 
High resolution HLA 
typing 
X 
 
 
 
 
 
 
HLA Ab screening 
X 
 
 
 
 
 
 
HIV, Hep B, C antibody, 
HTLV I/II 
X 
 
 
 
 
 
 
EBV PCR 
X 
 
 
 
 
 
 
CMV antibodies 
X 
 
 
 
 
 
 
Serum Pregnancy test 
X 
 
 
 
 
 
 
RPR 
X 
 
 
 
 
 
 
Inspection of vessels by 
DTM to determine the 
need for CVC catheter for 
apheresis 
X 
 
 
 
 
 
 
LYMPH PHENO BMT 
X 
 
 
 
 
 
 
G-CSF injections 
 
X 
X 
X 
X 
X 
X* 
Apheresis 
for 
PBPC 
collection 
 
 
 
 
 
X 
X* 
OTHERS: 
 
 
 
 
 
 
 
 
X*- Day 6 of G-CSF injections followed by a second apheresis will only occur for patients whose 
product contains less than the minimum dose of CD34+ cells/kg after the first apheresis collection.  
 

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Note: 
1. All pediatric patients and adult patients requiring a CVC catheter for collection 
will be admitted as inpatients.  
2. The amount of blood that will be drawn from adult donors will not exceed 450 ml over any 
six- week period. For pediatric patients, we will not draw more than 3 ml/kg in a single blood 
withdrawal, and no more than 7 ml/kg will be drawn over any six-week period. 
 
Please see the excel spread sheet for the following appendices: 
 
Appendix D: Patient/Recipient AlloBMT INITIAL SCREENING 
 
Appendix E: Group 1: AlloBMT Recipient INPATIENT schedule of Event  
 
Appendix F: Group 2: MUD Recipient INPATIENT schedule of Events 
 
Appendix G: AlloBMT and MUD Recipient DISCHARGE TO DAY +100 
 
Appendix H: AlloBMT and MUD Recipient BEYOND DAY 100 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Commented [c20]: Note that these were not provided for 
review. 
Commented [JPK21]: Please convert each of these to MSWord 
documents and incorporate into protocol document. 

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APPENDIX I: Study Drug Information Sheets 
 

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