An Expanded Access Study Using the CliniMACS System to Offer Therapeutic Manipulated Grafts That Are CD34 Cell Enriched and T Cell Depleted for Allogeneic Stem Cell Recipients With Mismatched Related Donors or Borderline Organ Function
Phase: NA
Conditions: Malignant Diseases, Non-malignant Diseases
Sponsor: Rajni Agarwal

 
1 
 
 
AN EXPANDED ACCESS STUDY USING THE CLINIMACS® SYSTEM TO OFFER THERAPEUTIC MANIPULATED 
GRAFTS THAT ARE CD34 CELL ENRICHED AND T CELL DEPLETED FOR ALLOGENEIC STEM CELL RECIPIENTS 
WITH MISMATCHED OR MATCHED RELATED OR UNRELATED DONORS OR BORDERLINE ORGAN FUNCTION 
Division of Pediatrics: Stem Cell Transplantation Section 
 
 
Document History 
Notes 
Version 1 
Date: 3/17/2014 
Initial Submission 
Version 2 
Date: 05/01/2014 
Amendment 1 
Version 3 
Date: 06/01/2014 
Amendment 2 
Version 4 
Date 06/06/2014 
Amendment 3 
Version 5           Date 04/02/2018 
Amendment 4 
 
 
Principal Investigator: Rajni Agarwal, M.D. 
1000 Welch Road, 
 
Stanford, CA 94304. 
 
Phone: (650) 724-7173 
FAX: (650) 724-1164 
Rajnia@stanford.edu 
 
Stanford Co-PIs: 
Matthew Porteus, M.D., Ph.D., Kenneth Weinberg, M.D. 
 
Agnieszka Czechowicz MD, PhD 
 
 
Stem Cell Processing at Stanford: BMT Cellular Therapy Facility 
Stanford Health Care 
300 Pasteur Drive, 
 
Stanford, CA 94305 
T main: 
 
T lab:    
  
 
Biostatistician: 
 Ph.D. 
Health Research and Policy – Biostatistics at Stanford University Redwood Building, 
 
Stanford, California 94305-5405 
Phone: 
 
@stanford.edu 
 
 
IRB E-Protocol/IDE Number 
28663/IDE 
 
 

 
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TABLE OF CONTENTS 
Table of Contents 
1.0 OBJECTIVES ........................................................................................................................................................... 10 
1.1 Primary Objective .................................................................................................................................................. 10 
1.2 Secondary Objectives ............................................................................................................................................ 10 
2. BACKGROUND ......................................................................................................................................................... 11 
2.1 Previous Studies .................................................................................................................................................... 11 
2.3 Rationale ............................................................................................................................................................... 17 
2.4 Study Design .......................................................................................................................................................... 17 
3. PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES ..................................................................................... 18 
3.1 Inclusion Criteria ................................................................................................................................................... 18 
3.2 Exclusion Criteria: .................................................................................................................................................. 18 
3.3 
Informed Consent Process ............................................................................................................................... 18 
3.4 
Study Timeline ................................................................................................................................................. 19 
4. 
PATIENT CONDITIONING, TRANSPLANT, AND POST-TRANSPLANT CARE ............................................................... 19 
4.1 Patient Conditioning .............................................................................................................................................. 19 
4.2 Supportive Care Guidelines ................................................................................................................................... 19 
4.3 
Criteria for Removal from Study ....................................................................................................................... 19 
4.4 Duration of Participation in the Study and Follow-up. ............................................................................................ 19 
5 
DONOR SELECTION AND PROCEDURES ................................................................................................................ 19 
5.1 Mobilization Therapy for Peripheral Blood Leukapheresis ..................................................................................... 19 
5.2 Donor Follow-up .................................................................................................................................................... 20 
6 
INVESTIGATIONAL PROCEDURE INFORMATION ................................................................................................... 20 
6.1 Investigational Procedure ...................................................................................................................................... 20 
6.2 Release of product for transplant .......................................................................................................................... 20 
6.3 Agent Ordering ...................................................................................................................................................... 21 
6.4 Agent Accountability ............................................................................................................................................. 21 
7 
DOSE MODIFICATIONS ......................................................................................................................................... 21 
8 
ADVERSE EVENTS AND REPORTING PROCEDURES ................................................................................................ 21 
8.1 Potential Adverse Events ....................................................................................................................................... 21 
8.2 Adverse Event Reporting ....................................................................................................................................... 22 
9 
DATA FORMS AND SUBMISSION SCHEDULE ......................................................................................................... 24 
10 
MEASUREMENT ............................................................................................................................................... 24 
10.1 Primary and Secondary Outcome measures ......................................................................................................... 24 
10.2 Stopping Rules ..................................................................................................................................................... 24 
11 
REGULATORY CONSIDERATIONS ...................................................................................................................... 25 
11.1 Institutional Review of Protocol .......................................................................................................................... 25 
11.2 Data Management Plan ....................................................................................................................................... 25 
12 
STATISTICAL CONSIDERATIONS ........................................................................................................................ 25 
13 
REFERENCES ..................................................................................................................................................... 25 
APPENDIX A: Graft-Versus-Host Disease Grading ......................................................................................................... 31 
APPENDIX B: Current Criteria Used at Stanford University at the Adult and Pediatric Programs Stem Cell 
Transplantation Programs to establish Graft Failure .................................................................................................... 33 

 
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Protocol Synopsys: 
Principal Investigator at 
Stanford 
Rajni Agarwal, M.D. 
1000 Welch Road, Suite 
. Stanford, CA 94304. Mail Code: 
. 
Phone: (650) 724-7173. FAX: (650) 724-1164. rajnia@stanford.edu 
Funding Source 
None. 
Protocol Title 
An Expanded Access Study Using the CliniMACS® System to Offer Therapeutic 
Manipulated Grafts that are CD34+ Cell Enriched and T Cell Depleted for Allogeneic 
Stem Cell recipients with Matched or Mismatched Related Donors or with Borderline 
Organ Function 
Clinical Phase 
Phase II 
Background and 
Rationale 
Hypothesis: Transplantation of stem cells that have been CD34+ selected and T 
cell-depleted with the CliniMACS® device will prevent severe (grade III/IV) acute 
GvHD without the use of prophylactic post-transplant immunosuppression. The 
incidence of grade III/IV acute GVHD is predicted to be <10% after cell 
manipulation. 
Background: The CliniMACS® System (Device, Reagents, Tubing Sets, 
Instruments and PBS/EDTA Buffer) is manufactured and controlled under an ISO 
13485 and ISO 9001 certified quality system. In Europe, the CliniMACS System and 
Reagents are available as CE-marked medical devices for use in humans. Inthe 
USA, CliniMACS® System is available for use only under an approved 
Investigational New Drug (IND) application or Investigational Device Exemption 
(IDE). Its safety has been tested in multiple eIND and eIDE FDA submissions. As of 
2011, eighty four IDE protocols utilize the CliniMACS® CD34 Reagent to select 
CD34+ cells from heterogeneous hematologic cell populations. In 2011, Miltenyi 
Biotech HDE submitted CliniMACS® CD34 Reagent System as a Humanitarian 
Use Device (HUD) in its Registration Pathway in the U.S. Clinical data from BMT 
CTN 0303 Clinical – a phase II multi-center clinical trial sponsored by the BMT CTN 
(BB-IDE 11965) which enrolled 47 Acute Myeloid Leukemia (AML) patients after 
myeloablative therapy for HLA-matched allogeneic stem cell transplant recipients, 
without additional GvHD prophylaxis from October 2005 to December 
2008 -- supports “safety” and “probable benefit” arguments of the CliniMACS® 
System. In January, 2014, the CliniMACS CD34 Reagent System received FDA 
approval as a Humanitarian Use Device for processing hematopoietic progenitor 
cells collected by apheresis from an allogeneic, HLA-identical, sibling donor to 
obtain a CD34+ cell-enriched population for hematopoietic reconstitution following a 
myeloablative preparative regimen without the need for additional GvHD prophylaxis 
in acute myeloid leukemia. (44) 
Rationale: Hematopoietic stem cell transplantation (HSCT) is now recognized as an 
effective form of therapy for an increasing number of malignant and non- malignant 
disorders. Therefore, extending the donor pool to alternative source of 
hematopoietic stem cells (HSCs) that is capable of reconstituting hematopoiesis 
after intensive myeloablative therapy is an important goal in this field. The preferred 
donor for an allogeneic HSCT is an HLA identical sibling. However, less than 25% of 
patients requiring a transplant will have such a donor. The purpose of this protocol 
is to provide access to the CliniMACS® System to patients who only have donors 
with a significant HLA-mismatched compatibility (alternative donors) or have a 
matched related donor but cannot receive post immunosuppressive therapy due to 
organ function. By manipulating the product graft through this platform, we can offer 
a CD34 enriched and T-cell depleted graft that addresses the main morbidity and 
mortality issue for this type of hematopoietic stem cell transplant (HSCT): 
overwhelming (grade III/IV) graft-versus-host disease (GvHD). The cell manipulation 
provides cellular therapeutic GvHD prophylaxis, therefore 

 
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eliminating the need of post-HSCT immunosuppressant therapies making this 
process also a viable alternative to deliver a life-saving HSCT for patients who do 
not have a matched related donor and for those with borderline organ function -- 
who might not endure the often-toxic side-effects of traditional post-HSCT 
immunosuppressive therapy. 
Objectives 
This is a prospective study to provide access to an investigational device and 
reagents used for progenitor cell sorting of a stem cell transplant donor graft to 
determine the ability of CD34+ cell selection and T Cell depletion (using the 
CliniMACS® device as the sole GvHD prophylaxis) to prevent severe (grade III- 
IV) acute GvHD in recipients of mismatched related or unrelated donor 
hematopoietic stem cell transplants or with borderline organ function -- who might 
not endure the often toxic side-effects of traditional post-HSCT immunosuppressive 
therapy. 
Primary Endpoint(s) 
The primary endpoint of the study is the incidence of severe (grade III/IV) acute 
GvHD occurring by Day +100 after transplant. 
Secondary Endpoint(s) 
Outcomes measures for the secondary endpoints will include: 
Engraftment: The incidence of primary graft failure and graft rejection will be 
determined. 
Chimerism at Day 100: The percentage of donor cells will be reported for all 
evaluable (without disease progression) patients. 
Immune recovery: The time to CD4 count >100 and > 200 and PHA ≥ 10% and 30% 
will be calculated. 
Severe toxicities: The incidence of severe toxicities will be determined. This will 
include grade 3/4 stem cell product infusion-related toxicity. 
Post-transplant infections (bacterial, viral and fungal): Post-transplant infections will 
be described by incidence and type. 
Post-transplant lymphoproliferative disease (PTLD): The incidence of PTLD will be 
calculated. 
Transplant-related mortality (TRM): The incidence of TRM will be calculated at Day 
100 after transplant and at one year. 
Disease-free survival. 
Overall survival: Overall survival will be estimated using the Kaplan-Meier method. 
Device (CliniMACS®) performance parameters: The parameters will be 
summarized for all products that are processed and median and ranges will be 
determined. 
Study Design 
Participants will be enrolled with alternative (mismatched/haplocompatible) 
related/unrelated donors or matched related/unrelated donors for an initial HSCT.  
Study Centers 
This is a Stanford University, Principle Investigator (PI) initiated protocol conducted 
at LPCH and Stanford Hospital and Clinics. 
Duration of Study per 
Subject 
Individual participants will be followed for outcomes related to the experimental 
device including acute and chronic GvHD and engraftment for a minimum of one 
year. Subsequent follow-up care will follow the standard for all transplant patients. 
Subject Population 
Pediatric and adult patients up to 35 years old, of any gender, and ethnic 
background, with malignant and/or non-malignant diagnosis that are treatable 
though allogeneic stem cell transplantation.  

 
5 
Eligibility criteria 
Inclusion Criteria: 
 
Participants greater than one month of age. 
 
Participant has a disorder that can be treated with a hematopoietic stem cell 
transplant 
Participant’s is eligible for an allogenic HSCT as per current institutional SOP 
and FACT standards 
 
Participant must have a related or unrelated matched or mismatched-
related donor who is: 
o 
Able to receive granulocyte colony-stimulating factor (G-CSF) and 
undergo apheresis either through placement of catheters in 
antecubital veins or a temporary central venous catheter OR agrees 
on a bone marrow harvest; 
o 
Healthy as per donor selection screening (following current SOP 
based on standards of foundation for accreditation of cellular 
therapy and stem cell transplantation – FACT); 
o 
Willing to participate and sign consent. 
 
Participant or Legal Authorized Representative is able to sign informed 
consent (and signed assent, if applicable) for transplant. 
Exclusion Criteria: 
 
Participant does not qualify for an allogeneic transplant due to medical 
screening, underlying disease, or lack of alternative donors. 
 
Any condition that compromises compliance with the procedures of this 
protocol, as judged by the principal investigator. 
Discontinuation Criteria 
(Safety Considerations) 
Principal Investigator will evaluate and report all grade 3 or 4 adverse reactions that 
are unexpected and possibly related to the investigation in process and are not 
expected TRM as per protocol definition. If it becomes clear that the study treatment 
is less effective than other available treatments or the toxicity is unacceptable we 
will follow the stopping rules. 
Stopping rule for primary graft failure: The first 30 enrolled patients (related and 
unrelated) will be evaluated for the stopping rule for primary graft failure. The trial is 
stopped if there are ≥ b graft failures out of k resolved patients. Only points where 
stopping is possible are listed. 
The stopping rule for graft failure yields the probability of stopping the trial of 0.05 if 
the rate of graft failure is 0.1. The probability of stopping the trial is 0.41 if the graft 
failure rate is 0.2, 0.82 if the graft failure rate is 0.3, and 0.96 if the graft failure rate 
is 0.4. These probabilities were calculated based on the binomial distribution. 
If the study reaches a stopping boundary, the study will be suspended. Proper use 
of the stopping rule table will be ensured by the Study Investigator. 
Stopping rule for Grade ≥ IV toxicity: If any patient develops grade ≥ IV acute 
GvHD, grade ≥ IV infection, or any unexpected grade ≥ IV toxicity by 4 weeks post-
transplant, the protocol will be halted and the processing re-evaluated before 
 
k 2 4 5 6 8 9 10 12 13 14 15 16 18 19 20 21 23 24 25 26 27 28 30
b 2 3 3 3 4 4 4 5 5 5 5 5 6 6 6 6 7 7 7 7 7 7 8 

 
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proceeding. 
Conditions for individual patient termination: 
Disease progression 
Need for exclusionary concurrent treatment (per PI discretion) 
Withdrawal of informed consent 
Protocol non-compliance 
The PI, through the reports from the Stem Cell Laboratory, will also monitor system 
performance based on: 
Cellular composition of final product 
Yield (% recovery) of CD34+ cells from the starting product 
CD3+ cell reduction 
Sterility 
Purity 
Sample Size 
The study will enroll 30 transplant recipients at Stanford. 
Statistical Methodology 
Statistical analysis results will be reported using summary tables, figures, and data 
listings. Continuous variables will be summarized using the mean, standard 
deviation, median, minimum, and maximum. Categorical variables will be 
summarized by numbers and percentages of subjects in corresponding categories. 
All raw data obtained from the case report form (CRF) and any derived data will be 
summarized and included in data listings. Missing data will not be imputed. 
 
 
 

 
7 
 
 
 
 
List of Abbreviations and Definition of Terms 
List of Abbreviations 
AE 
Adverse event 
AdV 
Adenovirus 
aGvHD 
Acute Graft-versus-Host Disease 
ALL 
Acute Lymphoblastic Leukemia 
Allo 
Allogeneic 
AML 
Acute Myelogenous Leukemia 
ANC 
Absolute Neutrophil Count 
BM 
Bone marrow 
BM bx. 
Bone marrow biopsy 
BMI 
Body mass index 
BMT 
Bone Marrow Transplant 
BP 
Blood pressure 
BSA 
Body surface area 
oC 
Degrees Celsius 
CB 
Cord blood 
CBC 
Complete blood count 
CD 
Cluster of Differentiation 
CFR 
Code of Federal Regulations 
cGvHD 
Chronic Graft-versus-Host Disease 
CIBMTR 
Center for International Blood and Marrow Transplant Research 
CML 
Chronic Myelogenous Leukemia 
CMV 
Cytomegalovirus 
CNS 
Central nervous system 
Co-PI(s) 
Co-Investigator(s) 
CRF 
Case report/record form 
CR 
Complete response 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTN 
Clinical Trials Network 
DFS 
Disease Free Survival 
DLI 
Donor Leukocyte Infusion 
DLT 
Dose Limiting Toxicity 
DSMB 
Data Safety Monitoring Board 
DSMC 
Data and Safety Monitoring Committee 
EBV 
Epstein Barr Virus 
EC 
Ethics Committee 
ECG 
Electrocardiogram 
Echo/ECCO 
Echocardiogram 

 
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EFS 
Event-free survival 
EIDE 
Emergency investigational device exemption 
EIND 
Emergency Investigational new drug 
F 
Fahrenheit 
FA 
Fanconi Anemia 
FACT 
Foundation for the Accreditation of Cellular Therapy 
FDA 
Food and Drug Administration 
GCP 
Good clinical practice 
G-CSF 
Human granulocyte-colony stimulating factor 
GI 
Gastrointestinal 
GFR 
Glomerular filtration rate 
GvHD 
Graft-versus-Host Disease 
HBV 
Hepatitis B Virus 
HCG 
Human chorionic gonadotropin 
HCV 
Hepatitis C Virus 
Hgb 
Hemoglobin 
HIV 
Human Immunodeficiency Virus 
HHV 
Human Herpes Virus 
HLA 
Human leukocyte antigen 
HPF 
High-power field 
HR 
Heart rate 
HSCs 
Hematopoietic stem cells 
HSCT 
Hematopoietic Stem Cell Transplant 
HSV 
Herpes Simplex Virus 
HTN 
Hypertensions 
HUD 
Humanitarian Use Device 
IB 
Investigator Brochure 
IDE 
Investigational Device Exemption 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
IV 
Intravenous 
LAR 
Legal authorized representative 
LLN 
Lower limit of normal 
LN2 
Liquid Nitrogen 
mAb 
Monoconal antibody 
MDS 
Myelodysplastic Syndrome 
MRD 
Matched related donor 
NCI 
National Cancer Institute 
NCI CTCAE 
National Cancer Institute common terminology criteria for adverse events 
NHL 
Non-Hodgkin’s Lymphoma 

 
9 
NIH 
National Institutes of Health 
NK 
Natural killer 
NMDP 
National Marrow Donor Program 
OS 
Overall survival 
PBSC 
Peripheral blood stem cells 
PCR 
Polymerase chain reaction 
PD 
Progressive diseased 
Peds 
Pediatric patients 
PFS 
Progression free survival 
PI 
Principal investigator 
PLT 
Platelet 
PMRD 
Partially matched donor 
PPA 
Per protocol analysis 
PR 
Partial response 
PTLD 
Post-transplant lymphoproliferative disorder 
RR 
Response rate 
SAE 
Serious adverse event 
SCCI 
Stanford Center for Clinical Informatics 
SD 
Stable disease 
SCT 
Stem cell transplant 
SCTT 
Stanford Blood and Marrow Cellular Therapeutics & Transplantation Laboratory 
SOP 
Standard Operating Procedure 
SRC 
Scientific Review Committee 
T. bili 
Total bilirubin 
TBI 
Total body irradiation 
TRM 
Transplant-related mortality 
TTP 
Time to progression 
UCB 
Umbilical Cord Blood 
ULN 
Upper limit of normal 
UNK 
Unknown 
URD 
Unrelated donor 
VZV 
Varicella Zoster Virus 
WBC 
White blood cell 
WHO 
World Health Organization 

 
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1.0 OBJECTIVES 
A major issue in alternative donor (mismatched related and unrelated donor) transplantation is the development 
of graft-versus-host disease (GvHD). For those who cannot receive post-transplant immunosuppressive therapy, 
any form of graft versus host disease, even with a matched related or unrelated donor can also be a complication. 
Several clinical trials have shown that the use of T cell-depleted peripheral blood stem cells (PBSC) reduces 
GvHD in hematopoietic stem cell transplants (HSCT). By using a purified stem cell graft, there is a reduced risk of 
post-transplant graft versus host disease in patients who have a mismatched donor or cannot receive post-
transplant immunosuppression. 
 
This is a prospective study to provide access to an investigational device and reagents used for progenitor cell 
sorting. The hypothesis is that the CliniMACS® System will allow for a manipulated graft – with augmented 
CD34+ cells and T cell-depleted numbers --- in a ratio that will prevent severe (grade III/IV) acute GvHD (as 
defined on appendix C) without the use of prophylactic post-transplant immunosuppression. The incidence of 
grade III/IV acute GVHD is predicted to be lesser than 10% based on similar ongoing trials (17).  
 
The objective is to manipulate (alter the ratio) specific cells in the donor graft to determine the ability of 
augmented CD34+ cells and T cell-depleted products to prevent severe (grade III/IV) acute GvHD in recipients of 
mismatched related donor hematopoietic stem cell transplants as well as to candidates of allogeneic (allo) 
transplant with borderline organ function -- who might not endure the often toxic side- effects of traditional post-
HSCT immunosuppressive therapy. 
 
The patients will receive conditioning therapy based on their diagnosis and per instructional guidelines for treating 
the disease. The transplant recipients will be followed for one year post- transplant for the development of GvHD, 
engraftment, post-transplant infections, disease relapse, and overall survival. 
 
1.1 Primary Objective 
To determine the ability of CD34+ cell selection using the CliniMACS® device as the sole GvHD prophylaxis to 
prevent severe (grade III-IV) acute GVHD in recipients of alternative donors hematopoietic stem cell transplants 
and/or potential matched-related allogeneic SCT recipients with borderline organ function (who might not tolerate 
traditional post-HSCT GvHD prophylaxis without increased morbidity). 
 
 
1.2 Secondary Objectives 
Assess the ability of this approach to serve as a platform for strategies to accelerate post- transplant 
immunological recovery. 
 
Evaluate chimerism at day +100 (from the date of transplant graft infusion), and the rate of engraftment in 
recipients of CD34+ cell selected, T cell-depleted transplants from mismatched related donors. 
 
Evaluate the rates of post-transplant occurrences, including: 
a) Graft rejection and graft failure 
b) Immune recovery 
c) Transplant-related severe toxicities 
d) Post-transplant infections 
e) CMV infection and disease 
f) 
EBV-related post-transplant lymphoproliferative disorder (PTLD) 
g) Relapse of malignant-diagnosis 
h) Transplant-related mortality 
i) 
Disease-free survival (DFS) and overall survival (OS) 
 
Monitor device performance: 
a) Cellular composition of final product 
b) Yield of CD34+ cells 
c) CD3+ cell depletion 
d) Sterility 
e) Purity 

 
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2. BACKGROUND 
2.1 Previous Studies 
Hematopoietic stem cell transplantation (HSCT) is now recognized as an effective form of therapy for an 
increasing number of malignant and non-malignant disorders. Therefore, extending the donor pool to alternative 
sources of hematopoietic stem cells (HSCs) that are capable of reconstituting hematopoiesis after intensive 
myeloablative therapy is an important goal in this field. The preferred donor for an allogeneic HSCT is an HLA 
identical sibling. However, less than 25% of patients requiring a transplant will have such a donor. Alternative 
stem cell sources include volunteer matched unrelated donors (URD) and unrelated donor cord blood (CB).(1) 
For patients lacking either a matched sibling donor or a suitable unrelated donor in a timely fashion, mismatched 
related donors could be a potential option. 
 
To overcome the lack of suitable donors, transplant centers around the world started using mismatched 
(haplocompatible or sharing only one of two haplotypes) related donors. In the past, the major challenges have 
been engraftment, graft-versus-host disease (GvHD), and an increased incidence of infection and disease 
relapse. 
 
Outcomes have improved markedly in recent years with the availability of cell selection devices that allow the 
administration of a large number of stem cells with a low dose of T cells and the development of more intensely 
immunosuppressive conditioning regimens. In an effort to reduce the high risk of fatal GvHD associated with 
these mismatched donors, the stem cells need to be processed to significantly reduce the number of donor T 
cells present in the graft. CD34 is a receptor that is expressed on early hematopoietic stem cells (HSC). 
Monoclonal antibodies are available that efficiently bind to the CD34 antigen. Studies have shown that positive 
selection of CD34+ marrow or blood cells results in a significant (> 4 log) depletion of T cells from the preparation 
(8-10). This approach is less time-consuming and may be more efficient than earlier approaches because it 
specifically targets the HSC. A cell separation system for clinical use is available for evaluation in the US after 
extensive use in Europe (11,12). The Miltenyi Biotec Inc. CliniMACS® CD34+ Reagent System has the 
advantages of the best T cell depletion efficiency achievable and a very high efficiency of CD34+ cell recovery so 
that fewer apheresis are necessary. The CliniMACS® device uses a sterile, closed magnetic sorting system to 
isolate CD34+ stem cells.     
 
The bone marrow or peripheral blood stem cell product obtained from the donor is incubated with a murine anti-
CD34 monoclonal antibody conjugated to small super- paramagnetic beads composed of iron dextran 
(commercially available to treat iron deficiency). The murine antibody has been used in clinical trials in humans. 
The dose administered to the transplant after processing is 100 times lower than therapeutic levels. Following 
incubation, unbound reagent is removed by washing the cell product and the product loaded onto the 
CliniMACS®. The cells are passed through a strong magnetic field applied to the CliniMACS® column and the 
CD34+ cells retained on the column while other cells (T, B, and NK cells, monocytes, and neutrophils) pass 
through into a waste bag. The magnet is withdrawn and the CD34+ cells eluted from the demagnetized column 
into a blood transfer pack for final formulation prior to release for infusion. 
 
It has been well established that allogeneic peripheral blood stem cells (PBSC) recruited into the blood by 
administering G-CSF can successfully and durably engraft in both matched (13,14) or mismatched (15,16) 
transplant settings. PBSC recruited with cytokines generally engraft earlier and contain a larger number of HSC 
compared to marrow HSC (14,15). 
 
Much of the work that has been done with this approach has been reported by a group in Perugia, Italy. They 
used the CliniMACS® device to lower the T cell content of the donor PBSC to a median of 1 x 104/kg (range 0.04 
– 3 x 104/kg). Ninety-one percent of patients had primary engraftment. Six of seven patients who did not have 
primary engraftment were successfully engrafted after a second transplant, making the overall engraftment rate 
99%. Grade III-IV acute GvHD occurred in 2% of patients. The transplant-related mortality was 37%, with the 
majority of deaths due to infection (bacterial, viral, and fungal). Relapse occurred in 16% of 66 patients who were 
in remission at the time of transplant. The two-year EFS for patients in remission was 48% for AML and 46% for 
ALL (17). 
 
 
The results of haplocompatible transplantation in children have been encouraging. Handgretinger reported a 46% 

 
12
two years EFS in patients with ALL in CR1-3 (18) Ortin et al. reported 75% EFS of patients with ALL in CR2/3 
with median follow-up of 18 months (range 6-29) (19). The international experience was reviewed at a conference 
in Naples in 2004. Lang updated the Tubingen, Germany experience when he reported a 44% 2 years EFS for 21 
children with ALL in CR1-3 (20) 
 
Advantages of haplocompatible transplantation are rapid engraftment and the very low incidence of severe acute 
GVHD. Ortin et al. reported a median time to ANC > 500 and to platelet count > 50,000 of 12 and 20 days, 
respectively, with the incidence of grade III/IV acute GvHD being 5% (19). Lang reported a 1% incidence of grade 
III/IV acute GvHD (21). 
 
In a retrospective review of the experience of 3 investigators in the U.S. (Cowan, Gilman, Sleight) with alternative 
(haplocompatible) donor transplant using CD34+ cell selection for T cell depletion, 13/18 (72%) were surviving 
with follow-up of 7 months – 7 years (median 31 months)(22). The median patient age was 8 years (range 1-20). 
Patient with malignancy (n=13) included: AML - CR1 (primary induction failure, failed cord blood transplant) [1], 
CR2 [3]; MDS - RA/RARS [2], RAEB [2] AM  (and Fanconi anemia, FA) [1]; CML - CP2 [1]; ALL - CR3 [2]; NHL - 
CR2 [1]. Patients with non-malignant (n=5) disease included severe aplastic anemia (1 with prior bone marrow 
transplant three years earlier) [4] and Wiskott-Aldrich syndrome [1]. 
 
Fourteen donors were a 3/6 HLA match and 5 were a 4/6 match (one patient had two transplants using different 
donors). A CD34-positive selection device – Miltenyi CliniMACS® (15), Isolex (3) – was used to select stem cells 
and deplete T lymphocytes. Patients received a median of 18 x 106 CD34+ (stem) cells/kg (range 6-28) and 3 x 
104 CD3+ (T) cells/kg (range 0.3-11). 
 
Sustained primary engraftment occurred in 15/18 (83%) patients. Primary graft failure occurred in one patient. 
Two patients had immunological rejection following HHV-6 reactivation. They both engrafted after a second 
transplant; therefore the overall engraftment rate was 94%. The median time to an ANC >0.5 x 109/L was 12 days 
(range 9-21). Platelet recovery occurred in 16/18 at a median of 17 days (range 9-22). Grade II acute GvHD was 
seen in 4/17 patients (24%). Grade III-IV acute GvHD was seen in 1 pt. (6%) with overlap syndrome (acute + 
chronic GvHD) associated with HHV-6 reactivation. Nine patients received DLI and/or stem cell boosts (boosts for 
graft rejection); 4 had grade II GvHD (3/4 had a history of acute GvHD) and none had grade III-IV GvHD. After 
DLI and/or stem cell boost, two patients developed extensive chronic GvHD and one developed overlap 
syndrome. The day 100 mortality and one year transplant-related mortality were 11% and 19%, respectively. Four 
patients (of 13 at risk, 31%) have relapsed; 1 patient with cytogenetic relapse is in CR > 1 year later. The 2 year 
predicted survival is 64% (60% for 13 patients with malignant disease and 75% for 5 patients with non- malignant 
disease). 
 
Infections were common. All patients were at risk for CMV reactivation. Seven patients (39%) reactivated CMV. 
All cases were responsive to anti-viral therapy and/or DLI. No CMV disease was seen. Seven patients had 
adenovirus reactivation and six had HHV-6 reactivation. EBV reactivation occurred in 5/18 (28%) patients, three 
of whom manifested signs of post-transplant lymphoproliferative disorder. 
Patients received a median of 3 x 104 CD3+ cells/kg at the time of transplant. Some patients received additional 
donor T cells (DLI) for viral reactivation. At three months post-transplant, only 4 of the 15 evaluable patients had a 
CD4 count > 100. By nine months post-transplant, 10 of the 13 evaluable patients had a CD4 count > 200. 
 
There is an ongoing study at the University of California, San Francisco Medical Center of a prospective trial of 
CD34+ selected (with the CliniMACS® device) PBSC from mismatched related donors (protocol CC# 01151). 
Patients received a conditioning regimen including TBI 1200 cGy, thiotepa, fludarabine, and rabbit-ATG (3.5 
mg/kg). Patients received a fixed T cell dose of 3 x 104/kg at the time of transplant. Seventeen evaluable patients 
have been reported with the following diagnoses: ALL (3), AML (6), biphenotypic leukemia (1), CML (1), MDS (1), 
aplastic anemia (2), congenital amegakaryocytic thrombocytopenia (1) combined immunodeficiency (1), and 
hemophagocytic lymphohistiocytosis (1). 
 
Twelve of the 17 (65%) were alive and well as of 2010, with follow-up ranging from 3 months to 6.5 years (median 
follow-up 2.5 years). Survivors include 7/12 (58%) with malignant disease and 5/5 (100%) with non-malignant 
disease. One patient died at 7 months after transplant due to parainfluenza and Paecilomyces infections and

 
13 
another died at 23 months after transplant due to disseminated Mucor infection, a fungus from the class of 
Zygomycetes. There was no severe (grade III/IV) acute GvHD. Only 3/17 (18%) of patients achieved > 100 CD4+ T 
cells/uL by 100 days after transplant and 3/4 of these patients either had received donor T cell infusion for serious 
viral infections prior to 100 days or had GvHD (in which case the T cells probably represented those causing the 
GvHD and not providing protection against infection). 
 
A variety of genetic diseases of lymphohematopoiesis have been effectively treated and either cured or ameliorated 
by allogeneic HSCT from an HLA-matched histocompatible healthy sibling [45, 46]. The conditioning regimen of 
myeloablative high dose Busulfan along with immunoablative high-dose cyclophosphamide (BU- CY) ± anti-
thymocyte globulin (ATG) was developed in the 1980’s after important clinical studies were conducted to better 
understand the prerequisites for replacement of hematopoietic stem cells (HSC) carrying deleterious mutations with 
those derived from matched healthy donors [46]. These studies established that, in the setting of histocompatible 
transplant, ablation of host hematopoietic stem cells with high-dose BU (16 mg/kg, 600 mg/m2, or 640 mg/m2) and 
of host immunity with CY ± ATG allowed durable engraftment of donor HSCs. 
 
Since most patients lack a histocompatible sibling donor, a major challenge in the treatment of the genetic 
lymphohematological disorders is the development of HSCT regimens that result in high levels of engraftment while 
avoiding regimen-related toxicity and GVHD. Higher risks of graft rejection and GVHD with the use of alternative 
donors, including unrelated donors (URD), mismatched related donors (mMRD), umbilical cord blood units, and 
haplo-identical donors, makes these transplants high risk and are associated with high rates of TRM as well. 
Consequently, the results of HSCT for patients with genetic disorders, using alternative donors has been both limited 
and suboptimal [47-54]. 
 
Given the especially increased risk of rejection with haploidentical transplants in patients with non-malignant 
disorders [52,54] , we are proposing the use of additional immunosuppression to the standard back-bone of 
Busulfan/cyclophosphamide/anti- thymocyte globulin, by adding Fludarabine. 
 
Miltenyi supported a Phase II multi-center clinical trial sponsored by the BMT CTN (BB-IDE 11965) which enrolled 
47 AML patients from October 2005-December 2008 Evaluated the use of the CliniMACS® CD34 Reagent System 
for selecting CD34+ cells from HLA-matched related donors for allogeneic stem cell transplantation after 
myeloablative therapy in patients with Acute Myeloid Leukemia (AML) in 1st or 2nd CR, without additional GvHD 
prophylaxis 
 

 
14
 
 
 
Post processing outcomes in all centers were able to process grafts that met the study criteria. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CD34+ Target Dose 
 
 
All patients received the minimum CD34+ dose (> 2.0 x 106 cells/kg) 
 
84.1% of patients received > 5 x 106 CD34+ cells/kg 
 
 
 
 
 
 
 
 
 
 
 

 
15
 
Final Cellular Product Summary 
 
 
All Gram stains/14 day cultures were negative 
 
All endotoxin < 5.0 EU/kg 
 
No significant infusion related toxicities observed 
 
Primary Endpoint 6 Month Disease-Free Survival of >75% 
The BMT CTN 0303 conclusions was that HCT following myeloablative preparative regimen for patients with AML in CR1 
or CR2 can be performed in a multicenter setting using the CliniMACS® CD34 Reagent System without additional post- 
transplant pharmacologic GvHD prophylaxis 
All 1º and most 2º endpoints were met, demonstrating: 
 
81.8% Disease-Free Survival 6 months post TX 
 
No primary graft failure; consistent neutrophil and platelet engraftment 
 
Acute GvHD grades II-III <23%. No Grade IV aGvHD 
 
Chronic GvHD at 2 years 19%; extensive 6.8% 
 
TRM <20% at 2 years 
 
Overall risk of relapse was low at 23.7% at 2 years 
 
The CliniMACS® CD34 Reagent System consistently produced a graft with > 2 x 106 CD34+ cells/kg and < 1 x 105 CD3+ 
cells/kg with no reported device related toxicities. 
 
In January, 2014, the CliniMACS® CD34 Reagent System received FDA approval as a Humanitarian Use Device for 
processing hematopoietic progenitor cells collected by apheresis from an allogeneic, HLA-identical , sibling donor to 
obtain a CD34+ cell-enriched population for hematopoietic reconstitution following a myeloablative preparative regimen 
without the need for additional GvHD prophylaxis in acute myeloid leukemia.(44)  
 
In summary, the use of CD34+ selected PBSC and manipulated bone marrow grafts without post-transplant GvHD 
prophylaxis for children was associated with rapid engraftment, low 100-day mortality, very low incidence of severe 
GvHD, and excellent survival. The overall survival compares favorably with matched-related and URD HSCT. Immune 
reconstitution was slow and post-transplant infections contributed to morbidity and mortality. 
2.2 Study Device Information 
Miltenyi Biotec, Inc. 
Brand: CliniMACS® CD34 Reagent System Information 

 
16
 
The CliniMACS® CD34 Reagent System is a medical device that is used in vitro to select and enrich specific cell 
populations. 
 
The CliniMACS® CD34 Reagent System is comprised of four primary components: 
 
CliniMACS® CD34 Reagent: a sterile monoclonal antibody reagent specific for CD34+ cells. 
 
CliniMACS® plus Instrument: a software controlled instrument that processes the blood sample (cell 
product). 
 
CliniMACS® Tubing Sets: single-use, sterile, disposable tubing sets with two proprietary cell selection 
columns (CliniMACS® Tubing Set and CliniMACS® Tubing Set LS) 
 
CliniMACS® PBS/EDTA Buffer: a sterile, isotonic phosphate-buffered, 1 mM EDTA, saline solution, used 
as external wash and transport fluid for the in vitro preparation of blood cells 
 
Distributor: 
Corporate Headquarters Miltenyi Biotec Inc. 
12740 Earhart Avenue 
Auburn, CA 95602 
 
Manufacturer: 
Miltenyi Biotec GmbH, Clinical Products Friedrich-Ebert Strasse Technologiepark H-13 
D51429 
Bergisch Gladbach, Germany 
 
Manufacturer Contact: Miltenyi Biotec Inc., Suite 305 120 Presidential Way 
Woburn, MA 01801 
Phone: (781) 782-1910 
Fax: (781) 782-1920 
 
CliniMACS® Tubing Sets (Standard and Large Scale) Standard: Up to 0.6 x 109 CD34+ cells from up to 60 x 109 
Cells 
 

 
17 
Large Scale: 0.6-1.2 x 109 CD34+ cells from 60-120 x 109 Cells 
 
 
CliniMACS® PBS/EDTA Buffer 
 
2.3 Rationale 
Cell dose is the most important factor affecting clinical outcomes in adult patients undergoing transplantation. 
Transplantation with a cell dose of less than 2.5 x 107 nucleated cells per kilogram or a CD34 dose of less than 1.7 x 
105 per kilogram is associated with higher rates of non-engraftment, non-relapse mortality and lower survival (14). 
Alternative donors can provide ideal cell dose grafts and have the advantage of being rapidly available, but the 
higher probabilities of severe acute and extensive chronic GvHD due to HLA-mismatched is a significant source of 
morbidity and mortality. This study provides access to a model that potentially benefits from alternative NMDP and 
related donor pools to test the safety and efficacy of manipulated grafts using the CliniMACS® System. The aim is to 
ensure robust cell dose grafts with the best available donor for allogeneic HSCT recipients while providing a method 
for T cell depletion through CD34+ selection as the single form of GvHD prophylaxis. Currently, there is not a FDA-
approved method for this process in the United States. 
 
The outcomes in relatively small studies for children receiving unrelated donor transplants using the CliniMACS® 
have been comparable to or better than those receiving T cell depleted transplants with post-transplant 
immunosuppression (21, 25). This study will enroll up to 30 transplant recipients at Stanford. As the recommended 
prescription of allogeneic transplantation for different underlying diseases grows, enrollment on this protocol is 
expected to gradually increase in relation to the overall number of transplants performed in our center. 
 
2.4 Study Design 
This is a PI-initiated, single institution, open (no masking is used), prospective study to provide access to allogeneic 
graft manipulation through CliniMACS® System for qualifying candidates. 
 
Currently, no FDA-approved method for T cell depletion exists. Recent experience with the CliniMACS® device has 
produced results with a 70-75% survival in children, many of whom were high-risk patients (19, 22, 36). 

 
18 
 
Patients will be enrolled with alternative (mismatched/haplocompatible) donors for an initial transplant. 
 
The results with mismatched related or unrelated donor transplants so far justify expanding this approach to related 
donor recipients who have contraindications to post-transplant immunosuppression due to borderline organ function. 
Matched-related or unrelated donor recipients with borderline organ function will be allowed to enroll.  
 
It is anticipated that the use of the CliniMACS® device will result in a very low risk of GvHD without the need for post-
 
transplant immunosuppression. 
 
This protocol will allow the use of patient-specific conditioning regimens that are available through open IRB studies 
in our institution or through open treatment plans from either the pediatric or adult stem cell transplant groups at 
Stanford University. 
 
The follow-up schedule and affiliated data collection forms for the allogeneic recipients in this study are the standard 
CIBMTR data forms and procedures. 
All recipients enrolled in the study and will be assessed for: neutrophil and platelet engraftment, graft rejection, 
serious infusion reaction, transmission of infection from HSCs infusion, EFS, TRM, acute and chronic GvHD, HSCT-
related toxicities. 
 
3. PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES 
Refer to the Participant Eligibility Checklist in Appendix A. 
 
3.1 Inclusion Criteria 
 Participants greater than one month of age. 
 Participant has a disorder that can be treated with a hematopoietic stem cell transplant 
 Participant’s is eligible for an allogenic HSCT as per current institutional SOP and FACT standards. 
 Participant must have a related or unrelated matched or mismatched-related donor who is: 
 Able to receive granulocyte colony-stimulating factor (G-CSF) and undergo apheresis either through 
placement of catheters in antecubital veins or a temporary central venous catheter OR agrees on a bone 
marrow harvest; 
 Healthy as per donor selection screening (following current SOP based on standards of foundation for 
accreditation of cellular therapy and stem cell transplantation – FACT); 
 Willing to participate and sign consent. 
 Participant or Legal Authorized Representative is able to sign informed consent (and signed assent, if 
applicable) for transplant. 
 
3.2 Exclusion Criteria: 
 
Participant does not qualify for an allogeneic transplant due to medical screening, underlying disease, or 
lack of alternative donors. 
 
Any condition that compromises compliance with the procedures of this protocol, as judged by the principal 
investigator. 
 
3.3  Informed Consent Process 
All participants will be provided a consent form describing the experimental nature of the study with sufficient 
information for participants to make an informed decision regarding their participation. 
 
The attending physician discusses the rationale, logistics, risks, and benefits of the study with the patient. Alternative 
therapies are discussed. The subject's bill of rights is reviewed. The recipient is given a copy of the consent form to 
take home and read. Then a meeting is scheduled for a consent conference to review the rationale, logistics, 
treatment plan, risks, benefits and consent form. The subject's bill of rights is reviewed with an emphasis placed on 
voluntary participation. Patients are given time to ask questions and do not need to sign the consent form until they 
are ready to proceed. In general, there is one hour devoted to the consent discussion with a clinical nurse specialist 
or research nurse educator.  The attending physician is available to discuss the study with the patient at any time. 

 
19 
For pediatric patients, all efforts will be made to have both parents sign. If one parent is unable to sign, the reason 
will be documented in the consent note that is part of the patient permanent electronic medical records. 
 
During the consent teaching, the physician, clinical nurse specialist or research nurse frequently asks questions of 
the subject to assess their understanding and a consent note is written documenting the patients understanding. For 
patients that are not fluent in English, the consent procedure is performed with an interpreter present (or via 
telephone), or a signer for those that are hearing impaired. Consents are translated into Spanish as needed. 
Patients are evaluated by a physician, nurse coordinator, clinical research nurse or clinical nurse specialist and 
social worker. Any member of the team can request a psychiatric evaluation if a concern of competency to provide 
consent is raised 
 
3.4  Study Timeline 
There will be no primary completion timeline to this study since the goal isto care for all HSCT recipients that need 
manipulated transplant graft. Subjects will continue to be followed on study until death or per institutional guidelines, 
whichever is longer. 
 
4. PATIENT CONDITIONING, TRANSPLANT, AND POST-TRANSPLANT CARE 
4.1 Patient Conditioning 
Patient conditioning will be determined by disease and institutional guidelines for treatment. 
 
4.2 Supportive Care Guidelines 
Standard supportive care guidelines will be followed as per institutional standards and may vary with the condition or 
problems of the individual patients. 
 
4.3 Criteria for Removal from Study 
Conditions for individual patient termination: 
 
Disease progression 
 
Need for exclusionary concurrent treatment (per PI discretion) 
 
Withdrawal of informed consent 
 
Protocol non-compliance 
 
The Principal Investigator may terminate the study for any of the following reasons: 
 
Significant toxicities related to the product graft 
 
If it becomes clear that the study treatment is less effective than other available treatments. 
 
4.4 Duration of Participation in the Study and Follow-up. 
Patients will remain in the hospital until there is evidence of engraftment of donor cells as well as recovery from the 
side effects of transplant conditioning. When the patient has engrafted and meets institutional hospital discharge 
criteria, they will be discharged from the inpatient facility. Patients will be followed as outpatients at regular intervals 
in the Bass Center Outpatient Center in the Stem Cell Transplant Clinic. Duration of participation and follow up in 
study will conclude at day 100 post-transplant.  
 
 
5 DONOR SELECTION AND PROCEDURES 
Donors will undergo standard of care procedures for mobilization and leukapheresis. They will be asked to sign 
consents per standard of care procedures. There will be no research procedures for the donor. 
 
5.1 Mobilization Therapy for Peripheral Blood Leukapheresis 
Following screening and enrollment, the donor will receive mobilization therapy per institutional guidelines with daily 
G-CSF. Recommended dose of G-CSF is 16 mg/kg/day subcutaneously (rounded off to the nearest vial size of 
either 300 or 480 µg). Donors will receive G-CSF once a day for 5 days, with the leukapheresis occurring on day 5. If 
needed, an additional dose of G-CSF and leukapheresis procedure may occur on day 6 if needed to meet cell dose 
requirements. Plerixafor (mozibil) may also be given per institutional standards to increase peripheral blood stem 
cells prior to leukapheresis procedure.  

 
20 
 
Target post-selection cell dose is 20 x 106 cells/kg with a CD3 cell dose of less than or equal to 3 x 104 cells/kg.   
 
 
5.2 Donor Follow-up 
Donors will be followed per institution guidelines following FACT standards of care. 
 
6 INVESTIGATIONAL PROCEDURE INFORMATION 
6.1 Investigational Procedure 
CD34+ Selection 
CD34+ cell selection will be performed according to procedures given in the CliniMACS Users Operating Manual 
and institution SOPs. The processing will be performed at the Stanford BMT Cellular Therapeutics & Transplantation 
Laboratory at the Stanford University Medical Center. 
 
The target cell doses will be ≥ 20 x 106 CD34+ cells/kg. A minimum dose of ≥ 5 x 106 CD34+ cells/kg will be 
acceptable. The target T cell dose will be ≤ 3 x104 CD3+ cells/kg. 
 
The stem cells will be infused via central venous line into the recipient. 
 
If > 20 x 106 CD34+ cells/kg are available, then 20 x 106/kg will be infused and the remainder will be cryopreserved 
in aliquots of 5 x 106 CD34+ cells/kg. 
 
Additional cell doses may be cryopreserved for future use. 
6.2 Release of product for transplant 
Sterility (routine USP culture for bacteria and fungi), endotoxin testing, gram stain, and viability studies will be 
performed in addition to the cell immunophenotyping. We already have experience with all of these procedures in 
other protocols at this institution (A Feasibility Trial of Post-Transplant Infusion of Allogeneic Regulatory T Cells 
Simultaneously with Allogeneic Conventional T Cells in Patients with Hematologic Malignancies undergoing 
Allogeneic Myeloablative Hematopoietic Cell Transplantation from Haploidentical Related Donors) and through 
pediatric stem cell transplant eIND and eIDEs submitted from this institution. 
 
Viability, gram stain and endotoxin testing will be done prior to the release of the product for infusion. If the viability is 
≥70%, the Gram stain is negative, and the CD34+ cell and CD3+ cell doses meet the criteria, the product will be 
released for infusion (target cell doses will be ≥ 20 x 106 CD34+ cells/kg. The target T cell dose will be ≤ 3 x104 
CD3+ cells/kg.) 
 
If the culture becomes positive, appropriate antibiotics, per institutional protocols, will be administered to the 
recipient. The donor will undergo a clinical evaluation for infection and the reagents (including the aliquoted 
reagents) and procedures will be tested and reviewed to try to identify the source. If the gram stain is positive, the 
cells will be cryopreserved until the culture results are back, and the donor will undergo another leukapheresis 
providing he or she has no clinical evidence of infection. 
 
All donor apheresis collections will be assessed as follows: 
 
Cell count by impedance counter (Coulter Z1) 
 
Viability by Propidium Iodide (PI) exclusion (flow cytometric assay) with target viability ≥70%, 
 
Sterility in accordance with 21 CFR 610.12 using Fluid Thioglycollate medium and Tryptic Soy Broth 14-day 
cultures 
 
Phenotypic analysis by flow cytometry for cell surface expression of CD34 and CD3 
 
Final Products for infusion, including CD34 selected cells and CD3 fractions will be assessed prior to cryopreservation 
as follows: 
a) Cell count by hemacytometer as per SCTT current SOP 
b) Viability by Propidium Iodide (PI) exclusion (flow cytometric assay) 
c) Sterility in accordance with 21 CFR 610.12 using FTM and TSB 14-day cultures as per current SCTT current 
SOP 
d) Phenotypic analysis by flow cytometry for cell surface expression of CD34 and CD3 

 
21 
e) Endotoxin testing post-selection on each graft product as per SCTT current SOP 
 
6.3 Agent Ordering 
MACS Cell Separation Reagents are available through MACS and CliniMACS® Products 
 
Miltenyi Biotec Inc. 
2303 Lindbergh Street Auburn, CA, 95602, USA Phone: +1 530 888 8871 
TOLL FREE: 800 FOR MACS 
Fax: +1 530 888 8925 
Email: macs@miltenyibiotec.com 
 
 
CliniMACS® plus Instruments are installed by qualified Miltenyi personnel 
 
Installation and Operational Qualification (IQ/OQ) is performed at time of installation 
 
All subsequent servicing and Preventative Maintenance are performed by qualified Miltenyi personnel 
 
Emergency Hotline Support 
 
6.4 Agent Accountability 
All quality validation will be checked at the Stanford BMT Cellular Therapeutics & Transplantation Laboratory at 
Stanford University Medical Center. The SCTT also has SOPs for cell sorting of PBSC and BM grafts to ensure 
consistent efficacy of the process. Cross reference letters are issued by the vendor, as per FDA guidelines, to notify 
dispensation of all supplies needed for the cell sorting. 
All Reagents and materials are accounted for as per an approved Investigational New Drug (IND) application or 
Investigational Device Exemption (IDE) FDA guideline. 
 
7 DOSE MODIFICATIONS 
None anticipated. These are part of transplant issues but not specific to the use of the investigational device 
(CliniMACS® System). 
 
8 ADVERSE EVENTS AND REPORTING PROCEDURES 
8.1 Potential Adverse Events 
Risk of contamination of the cell preparation with biologic or other foreign material. The sterility of system 
components that contact the cell sample and the detailed processing steps are designed to minimize potential 
contamination. 
 
Significant animal and human studies have been done using these super-paramagnetic beads which are small in 
size (~50 nm in diameter) and are composed of iron oxide and dextran conjugated to murine monoclonal antibodies. 
These magnetic particles form a stable colloidal suspension and do not precipitate or aggregate in magnetic fields. 
The concentration of the conjugate is equivalent to 22 µg of antibody protein per ml of reagent, 800 µg/ml of dextran 
and 800 µg/ml of iron. Detailed toxicity studies have been undertaken to assess the safety of the antibody reagent 
when delivered to monkeys and rabbits in dosages significantly greater than the projected maximum dosage 
anticipated in clinical use (CliniMACS® Investigator brochure). There have been more than 100 separations for 
clinical use of the CliniMACS® system. 
 
Reaction to CliniMACS® reagent - (murine monoclonal antibody is conjugated to an iron- dextran moiety. The iron 
dextran exposure from a single CliniMACS® separation is ~0.5 mg and less than 1 mg dextran. 
 
The other reagent is the murine monoclonal antibody in which there is a risk of an anaphylactic reaction. The anti-
CD34 monoclonal antibody, AC101 has been tested for safety in conformance with US standards. Systemic 
reactions appear related to the dose and rapidity of administration. Therapeutic levels (for cancer therapy or 
prevention of graft rejection) of mAb appear to be in the range of 2.5-5 mg/ml. The most commonly reported side 
effects have been myalgia, arthralgia, and flu-like symptoms. The CliniMACS® system results in the administration of 
a maximum of <15µg of antibody, 100x lower than therapeutic levels. Furthermore, studies have shown that the 
levels of antibody used in the CliniMACS® system do not induce complement activation in vitro. 
 

 
22 
8.2 Adverse Event Reporting 
Recipients of allogeneic hematopoietic cell transplantation face significant risks. The risk of treatment related 
morbidity and mortality following allogeneic transplant is related to the underlying disease, disease status at the time 
of transplant, prior therapies and other coexisting health problems. Significant risks include: 
 
Infection. Risks include bone marrow suppression, prolonged hospitalization, invasive procedures, prior 
exposure to micro-organisms and immunosuppressive medications for the prevention and treatment of graft 
versus host disease. Infections may be caused by bacteria, viruses, protozoa or fungus. 
 
Graft versus host disease. Acute GvHD can range from a mild and treatable problem to a life threatening 
complication. Chronic GvHD can vary from a mild to an extensive disorder affecting almost any body tissue. 
Chronic GvHD can negatively affect functional status and quality of life. 
 
Graft failure. Although rare, graft failure is a fatal complication. 
 
Infertility in both genders and premature menopause in women at risk. 
 
Relapse. All patients face a risk of relapse. The risk is highest for those patients with active or resistant 
disease at the time of transplantation. 
 
Definitions used for Reporting 
Adverse Event: Any untoward medical occurrence in a patient or clinical investigation subject administered 
a pharmaceutical product, medical treatment or procedure and which does not necessarily have to have a 
causal relationship with this treatment. An adverse event can therefore be any unfavorable and unintended 
sign (including an abnormal laboratory finding), a symptom, or disease temporally associated with the use of 
a medicinal product, medical treatment or procedure whether or not considered related to medicinal product 
or treatment. 
 
Life Threatening Adverse Event: Any adverse event that, in the view of the investigator, places the patient 
at immediate risk of death from the reaction. 
 
Unexpected Adverse Event: An adverse event, the nature or severity of which is not consistent with the 
applicable product information (Investigator’s Brochure, product insert). For studies that do not involve 
investigational products or devices, an unexpected adverse event is an adverse event that is not described 
in the transplant medical literature or consent form. 
 
Serious Adverse Event (SAE): Any adverse event occurring that results in any of the following outcomes: 
death, a life threatening adverse event, a persistent or significant disability/incapacity, a congenital anomaly, 
requires intervention to prevent permanent impairment or damage. Unless the Principal Investigator is using 
an investigational agent, s/he is not bound by the definition in Title 22 CFR. Stanford IRB definition(s) and 
requirements apply when applicable, Unanticipated Problems Involving Risks to Participants or Others (UPs) 
and Unanticipated Adverse Device Effect (UAD)  
 
All grades 3-5 organ-related adverse events not due to the primary malignancy or pre-existing condition will be 
collected from day 0 through resolution of the SAE. Grade 4 infusion reactions will be reported expeditiously and as 
a separate listing in the FDA Annual Report. 
 
Distinction between Serious and Severe Adverse Events: The term severe is used to describe the intensity 
(severity) of a specific event, for example mild, moderate or severe. The event itself however, may be of relatively 
minor medical significance, for example a severe headache. This is not the same as serious, which is based on the 
patient/event outcome and is usually associated with events that pose a threat to the patient’s life or functioning. 
Seriousness, not severity, serves as a guide for defining regulatory obligations. 
 
All Adverse events will be graded using the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.3 found on the following website: 
 
http://ctep.cancer.gov/reporting/ctc.html 
 
The Principal Investigator (PI) or designee will assess each Adverse Event (AE) to determine whether it is expected 
or unexpected according to the Informed Consent, Protocol Document, or Investigator’s Brochure; and its 

 
23 
relationship to the investigational product.  
 
For the purpose of this study, only AE’s found to be directly related to a study-mandated intervention will be 
captured.  The following adverse events, if felt to be related to study treatment, will be captured, but not reported as 
SAEs:  
 
Hospitalization, prolongation of hospitalization, or escalation of care due to study-mandated intervention  
 
Disease Relapse 
 
Any problems with CliniMACS device during cell separation or inability to achieve CD34+ cell and CD3+ 
cell target doses 
 
Any positive culture of HSCT product 
 
Failure to engraft or graft rejection  
 
All Serious Adverse Events (SAEs) will be tracked until resolution. SAEs will be collected from the time of stem cell 
infusion until 30 days post cell infusion.  SAEs will be reported to the IRB if they meet the definition of expedited 
reporting per institutional regulations. All AE’s and SAE will be reported at continuing renewal.  
 
Reports (copy of faxed MedWatch) will also be sent to Miltenyi Biotec Incorporated, to the director of Regulatory 
Affairs and Safety Officer at (781) 782-1920. 
 
Serious Adverse events will be reported on the MedWatch form 3500a when it meets the definition of expedited FDA 
reporting. All deaths will be examined to determine if they are related to the use of the CliniMACS device or infusion 
of manipulated product. If the death is determined to be related, the event leading to the death must be reported 
within 24 hours of the Investigator’s awareness of the event. 
 
The following SAEs require expedited reporting (as soon as possible but within at least 7 calendar days of the 
investigator learning of the event): 
 
Deaths: All deaths up to 100 days post-transplant. This includes deaths from the common and expected grade 3-4 
toxicities noted below. Deaths that occur outside of Stanford will be reported within 5 working days of knowledge of 
the death. It must be noted that obtaining detailed information on the cause and circumstances of a death occurring 
at another institution can be difficult. Excludes deaths related to relapse of underlying disease, which will reported in 
the annual report. 
 
All serious and unexpected toxicities defined as those toxicities not identified in the transplant literature or in the 
consent form and that are felt to be related to protocol-mandated procedures. 
 
The following will not be reported as SAEs: 
 
 Hospitalizations: Approximately 50% of allogeneic transplant recipients will be readmitted to the hospital. The 
most common indications for readmission of an allogeneic HCT recipient are fever, failure to maintain nutritional 
status and graft versus host disease. Only hospitalizations felt to be related to study mandated interventions 
should be reported as SAE’s.  
 Relapse of disease: Disease relapse unfortunately remains a significant problem following both autologous and 
allogeneic transplantation. The risk of relapse is influenced by both patient and disease variables. The risk of 
relapse following allogeneic transplant is extremely dependent on the disease being treated but ranges from 
10% (for patients with severe aplastic anemia) to 80% (for patients with refractory acute leukemia).  
 Common and expected grade 3 - 4 toxicities of HSCT that are well described in the transplant literature, the 
product inserts or stated in the consent form and do not result in death. This includes but is not limited to 
neutropenia, thrombocytopenia, anemia, thrombotic microangiopathy, bleeding requiring transfusions, edema, 
hypertension, hypotension, gastritis, mucositis, nausea, vomiting, diarrhea, hematuria, central venous catheter 
infections, febrile episodes, sepsis, mental status changes, insomnia, mood alterations, seizures, tremor, pain, 
hypoxia, pleural effusion, pneumonitis, incontinence, infertility, laboratory abnormalities, veno-occlusive disease, 
graft failure, cardiac arrhythmias and graft versus host disease. 
 
Secondary Malignancies. The occurrence of secondary malignancies and associated mortality is a known risk 
of cancer therapies. The occurrence of secondary malignancies will be reported in the annual report. 

 
24 
 
9 
DATA FORMS AND SUBMISSION SCHEDULE 
Primary internal data monitoring will be performed by the Principal Investigator or designee. The PI will review data 
to assure the validity of data, as well as the safety of the subjects. The PI will also monitor the progress of the trial. 
The PI is responsible for maintaining the clinical protocol, reporting adverse events, assuring that consent is 
obtained and documented, reporting of unexpected outcomes, and reporting the status of the trial in the continuing 
renewal report submitted annually to the IRB and SRC. The PI may designate any or all of these tasks to qualified 
and trained staff. 
 
Patient enrollment and toxicity/reporting will also be entered by the data-manager, coordinator, or designee into the 
CIBMTR and on the Program’s REDCap database by the research staff. 
 
10 MEASUREMENT 
10.1 Primary and Secondary Outcome measures 
The primary outcome will be severe (grade III/IV) acute graft-versus-host disease. 
 
Secondary outcomes will be engraftment date (ANC>500 for 3 consecutive days, and 
>80% donor cells in blood), graft failure and graft rejection, immune recovery, infection, CMV infection and disease, 
EBV-related PTLD transplant-related toxicity and mortality, transplant-related mortality, grade 3/4 stem cell product 
infusion-related toxicity, relapse, DFS, OS. 
 
10.2 Stopping Rules 
 
The study includes stopping rules based on the incidence of graft failure (or graft rejection), and acute GVHD. 
 
Stopping rule for primary graft failure: The first 30 enrolled patients (related and unrelated) will be evaluated for the 
stopping rule for primary graft failure. The trial is stopped if there are ≥ b graft failures out of k resolved patients. Only 
points where stopping is possible are listed. 
 
k 
2 4 5 6 8 9 10 12 13 14 15 16 18 19 20 21 23 24 25 26 27 28 30 
b 
2 3 3 3 4 4 4 
5 
5 
5 
5 
5 
6 
6 
6 
6 
7 
7 
7 
7 
7 
7 
8 

 
25 
The stopping rule for graft failure yields the probability of stopping the trial of 0.05 if the rate of graft failure is 0.1. 
The probability of stopping the trial is 0.41 if the graft failure rate is 0.2, 0.82 if the graft failure rate is 0.3, and 0.96 if 
the graft failure rate is 0.4. 
 
These probabilities were calculated based on the binomial distribution. The stopping rule was generated as 
described by Ivanova et al. (40). 
 
 
If the study reaches a stopping boundary, the study will be suspended. At this point it may be terminated by the PI or 
submitted to the safety monitor with a description of the failures to date and a rationale for why the study should be 
continued. Proper use of the stopping rule table will be ensured by the Study Investigator. 
 
Stopping rule for Grade ≥ IV toxicity: If any patient develops grade ≥ IV acute GvHD, grade ≥ IV infection, or any 
unexpected grade ≥ IV toxicity by 4 weeks post-transplant, the protocol will be halted and the processing re-
evaluated before proceeding. 
 
11 REGULATORY CONSIDERATIONS 
11.1 Institutional Review of Protocol 
The protocol, the proposed informed consent and all forms of participant information related to the study will be 
reviewed and approved by the Stanford IRB and other regulatory groups as necessary and required. Any changes 
made to the protocol will be submitted as a modification and will be approved by the IRB prior to implementation. 
The Protocol Director will disseminate the protocol amendment information to all participating investigators. 
 
11.2 Data Management Plan 
The PI, or his/her designee, will prepare and maintain adequate and accurate participant case histories with 
observations and data pertinent to the study. Study specific Case Report Forms (CRFs) will document treatment 
outcomes for data analysis. Case report forms will be developed using the REDCap database system and will be 
maintained by the SCT clinical research nurse and/or program’s CRA. 
 
12 STATISTICAL CONSIDERATIONS 
Interim descriptive analyses will be performed annually. As the eligibility for this protocol is very broad, analyses will 
be done separately for several pre-specified subgroups. 
 
First, analyses will be conducted separately for patients transplanted for malignant diseases and those transplanted 
for non-malignant diseases. They will also be analyzed separately for transplants using mismatched related versus 
alternative donors. Finally, analyses will be conducted separately for primary vs. secondary allogeneic transplants. 
Further separation of the study population such as adults vs. pediatrics and myeloablative vs. non-myeloablative 
conditioning regimens will be considered depending on the patient numbers. In particular, neutrophil and platelet 
engraftment will only be analyzed using transplants with myeloablative conditioning regimens. 
 
Baseline characteristics will be described using frequencies/percent or median/range as appropriate. 
 
The study will enroll 30 transplant recipients at Stanford. As the recommended prescription of allogeneic 
transplantation for different underlying diseases grows, enrollment on this protocol is expected to gradually increase 
in relation to the overall number of transplants performed in our center. 
 
 
 
 
 
 
 
 
 
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26 
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27 
 
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(22) Sleight B, Cowan M, Horn B, Jaroscak J, McGuirk J, Gilman A. Selected haplocompatible donor stem cell 
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(23) Ruggeri L, Capanni M, Urbani E et al. Effectiveness of donor natural killer cell alloreactivity in mismatched 
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(30) Dvorak CC, Gilman AL, Horn B, Cowan MJ. Primary graft failure after umbilical cord blood transplant rescued 
by parental haplocompatible stem cell transplantation. J Pediatr Hematol Oncol. 2009 Apr;31(4):300-3. 
 
(31) Lacerda JF, Martins C, Carmo JA et al. Haploidentical stem cell transplantation with purified CD34 cells after a  
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28 
radiation. Bone Marrow Transplant 2003 March;31(6):437-40. 
 
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29 
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31
APPENDIX A: Graft-Versus-Host Disease Grading 
 
The modified Keystone criteria will be used for acute GvHD scoring for both pediatric and adult participants. 
See tables below. 
If the criteria are not met but clinical signs and symptoms are consistent with GvHD and systemic therapy was 
begun, then the diagnosis of acute GvHD can be made. The staging of acute GvHD in the affected organ will 
be determined by the scoring team based on the severity of symptoms using the NIH Consensus Organ 
Scoring System of chronic GvHD, as a guide. 
The onset date for acute GvHD will typically be the date when the systemic immunosuppression is added or 
escalated. 
When acute GvHD progresses directly to chronic GVHD and a definite onset date cannot be determined, 
d+100 will be used for the onset date of chronic GvHD. 
The diagnosis of acute or chronic GvHD will typically be based on clinical signs and symptoms and are not 
based on the time of symptoms after transplant or donor lymphocyte infusion (DLI). 
The NIH Consensus recommendations will be used to establish the diagnosis of chronic GVHD: 
 
Distinction from acute GvHD; 
At least one diagnostic sign OR at least one distinctive sign confirmed by biopsy or other tests; 
Exclusion of other diagnosis. 
Chronic GvHD stated as suspected, presumed, and possible or not meeting the NIH consensus criteria be 
reviewed at the team’s weekly patient meetings. 
 
Signs and Symptoms described as: 
Chronic GvHD? 
Inactive or Quiescent 
No 
Irreversible symptoms present 
Yes 
No symptoms, patient on GvHD therapy No 
 
 
Article I. 
Acute GvHD Grading 
 
GradeSkin 
Liver 
Gut 
0 
None 
None 
None 
1 
Stages 1 - 
2 
None 
None 
2 
Stage 3, or Stage 1, or 
Stage 1 
3 
Stages 2 - 3, 
or 
Stages 2 - 
4 
4* 
Stage 4, or Stage 4 
 
 
 Grade 4 may also include lesser organ involvement but with an extreme decrease in performance 
status. 

 
32
Article II. 
Acute GvHD Staging 
 
Stage* Skin† 
Gut (adults)§¶ 
Gut (children) §¶ 
Liver‡ 
0 
No evidence of 
GvHD 
< 500 mL/d diarrhea 
< 10 mL/kg/d diarrhea 
T.bili < 2.0 
mg/dL 
1 
< 25% body surface 
area involved 
500 - 999 mL/d diarrhea, or 
persistent nausea with 
histologic evidence 
10 - 15 mL/kg/d diarrhea, or 
persistent nausea with 
histologic evidence 
T.bili 2.0 - 3.0 
mg/dL 
2 
25% - 50% 
1000 - 1499 mL/d diarrhea 
16 - 20 mL/kg/d diarrhea 
T.bili 3.1 - 6.0 
mg/dL 
3 
> 50% 
>/= 1500 mL/d diarrhea 
21 - 25 mL/kg/d diarrhea 
T.bili 6.1 - 15.0 
mg/dL 
4 
With bullous 
formation 
Severe abdominal pain 
with/without ileus 
> 26 mL/kg/d diarrhea 
T.bili > 15.0 
mg/dL 
 
 
 Criteria for grading minimum degree of organ involvement required conferring that grade. 
† Use "rule of nines" or burn chart to determine extent of rash. 
Range given as total bilirubin level. 
§ Volume of diarrhea applies to adults. For pediatric patients, the volume of diarrhea should be based 
on body surface area. 
¶ Persistent nausea with histologic evidence of GVHD in the stomach or the duodenum. 
 
Article III. 
Chronic Graft-versus-Host Disease Grading 
 
ONSET: 
 
De Novo – no prior aGVHD 
 
Interrupted – prior aGvHD resolved before onset of cGvHD 
 
Progressive – prior aGvHD did not resolve before onset of cGvHD 
 
GRADE: 
Limited – localized skin involvement and/or hepatic dysfunction due to cGvHD Extensive – one or more of 
the following: 
 
generalized skin involvement; or 
 
liver histology showing chronic aggressive hepatitis, bridging necrosis or cirrhosis; or 
 
involvement of eye: Schirmer test with < 5 mm wetting; or 
 
involvement of minor salivary glands or oral mucosa demonstrated on labial biopsy; or 
 
involvement of any other target organ 
 
SEVERITY: 
 Mild – signs and symptoms of cGvHD do not interfere substantially with function 
and do not progress once appropriately t 
treated with local therapy or standard 
systemic therapy (corticosteroids and/or cyclosporine or FK 506) 
 Moderate – signs and symptoms of cGvHD interfere somewhat with function despite appropriate 
therapy or are progressive through first line systemic therapy (corticosteroids and/or cyclosporine or 
FK 506) 
 Severe – signs and symptoms of cGvHD limit function substantially despite appropriate therapy or 
are progressive through second line therapy 
 
 

 
33 
APPENDIX B: Current Criteria Used at Stanford University at the Adult and Pediatric 
Programs Stem Cell Transplantation Programs to establish Graft Failure