An Expanded Access Study of the Feasibility of Using the CliniMACS® Device for CD34+ Cell Selection and T Cell Depletion for Graft-versus-Host Disease Prophylaxis in Alternative Donor Stem Cell Transplant Recipients
Conditions: Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Chronic Myeloid Leukemia, Myelodysplastic Syndrome, Lymphomas, Bone Marrow Failure, Hemoglobinopathy, Immune Deficiency, Osteopetrosis, Cytopenias, Leukocyte Disorders, Anemia Due to Intrinsic Red Cell Abnormality
Sponsor: Christopher Dvorak

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 1 of 53 
 
 
`An Expanded Access Study of the Feasibility of Using the CliniMACS® Device for CD34
+
 
Cell Selection and T Cell Depletion for Graft-versus-Host Disease Prophylaxis in 
Alternative Donor Stem Cell Transplant Recipients 
 
 
Protocol Number: 10082 
Version Number: 6.0 
Version Date: 01 Jan. 2018 
 
Study device:  
CliniMacs® BB-IDE-#8817 
IND: 14442 
 
 
Principal Investigator 
Morton J Cowan, MD 
UCSF Benioff Children’s Hospital 
550 16th Street, 4th Floor,  
Mailstop 0434 
San Francisco, CA 94143-1278 
Phone: (415) 476 – 2188 
Fax:   (415) 502 – 4867 
E-mail: mcowan@peds.ucsf.edu 
 
 
Co-Investigators 
Christopher Dvorak, MD 
Kristin Shimano, MD 
          Jeffrey Wolf, MD 
     Robert Goldsby, MD 
       James Huang, MD 
          Neil Shah, MD 
         Gauri Sunkersett, DO 
Justin Wahlstrom, MD 
   Thomas Martin, MD 
     
             
 
 
  Alexis Melton, MD 
 
 
Statistician 
Vivian Weinberg, PhD 
 
 
Clinical Research Coordinator 
Jueleah Expose’-Spencer 
 
Revision History 
                                                    Version 6.0     01 Jan. 2018 
                                                    Version 5.0     01 Sept. 2016 
                                                    Version 4.0     01 June 15 
                                                    Version 3.0     11 April 2012 
Version 2.0     12 August 2011 
Version 1.0 
12 August 2010 
                                                    Version 0.3       21 June 2010 
                                                    Version 0.2       07 June 2010 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 2 of 53 
 
 
                   
     Participating Institutions/ Investigators Table 
 
Principal Investigator:  Morton Cowan, M.D.  
Contact: Morton Cowan, M.D. 
Institution: University of California San Francisco, Benioff Children’s Hospital 
Address:  550 16th Street, 4th Floor, Mailstop 0434, San Francisco, CA 94143 
Phone: 
 415-476-2188 
 
 
 
 
 
 
 
Fax: 415-502-4867 
Email: mcowan@peds.ucsf.edu 
Sub-Investigators: Christopher Dvorak, MD;; Justin Wahlstrom, MD; Robert Goldsby, MD;  Kristin Shimano, MD; James Huang, 
MD; Jeffrey Wolf, MD; Neil Shah, MD; Thomas Martin, MD, Alexis Melton, MD 
 Principal Investigator: Carrie L. Kitko, M.D.  
 
 Institution: Vanderbilt University-Ingram Cancer Center  
Contact: Laura Currie, R.N, B.S.N Clinical Trials Manager 
Address: Clinical Trials Shared Resources, 2141 Blakemore Ave., Nashville, TN 37212 
Phone: 615-936-3877               
 Fax: 615-936--5850 
Email: laura.currie@vandervilt.edu 
Sub-Investigators: Richard Ho, M.D   James Connelly, M.D. 
_____________________________________________________________________________ 
Principal Investigator: 
 
 
 
Institution:  
Contact: 
Address: 
Phone: 
 
 
Fax: 
 
 
 
Email: 
Sub-Investigators: 
Principal Investigator: 
 
 
 
 Institution:   
Contact: 
Address: 
Phone:                
 
 Fax: 
 
 
 
Email: 
______________________________________________________________________________________________________ 
Principal Investigator: 
 
 
 
 Institution:  
Contact: Laura Currie,  
Address:  
Phone: 
 
 
 Fax: 
 
 
Email:  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 3 of 53 
 
Sub-Investigators:  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 4 of 53 
 
TABLE OF CONTENTS  
 
PARTICIPATING SITES ............................................................................................................ 2 
SCHEMA…………………………………………………………………………………………………..6 
 
 
 
 
 
 
 
 
 
             
 
LIST OF ABBREVIATIONS ........................................................................................................ 8 
SHORT ABSTRACT .................................................................................................................. 9 
 
1.0  GOALS AND OBJECTIVES .............................................................................................. 9 
1.1  Primary objective ...................................................................................................... 9 
1.2  Secondary objectives ................................................................................................ 9 
 
2.0  BACKGROUND AND RATIONALE ..................................................................................10 
2.1  Stem cell transplantation..........................................................................................10 
2.2  Mismatched (haplocompatible) related donor stem cell transplant ...........................11 
2.3  NK alloreactivity .......................................................................................................13 
2.4  Preliminary data .......................................................................................................14 
2.5 Immune reconstitution .............................................................................................14 
2.6  Rationale for including alternative conditioning regimens .........................................15 
 
3.0  STUDY DESIGN AND ELIGIBILITY CRITERIA ................................................................15 
3.1  Study design ............................................................................................................15 
3.2  Recipient inclusion criteria .......................................................................................16 
3.3  Recipient exclusion criteria ......................................................................................17 
3.4  Donor selection criteria………………………………………………………………...17-17 
3.5  Patient recruitment ...................................................................................................18 
3.6  Patient registration ...................................................................................................18 
 
4.0  INVESTIGATIONAL TREATMENT PLAN.........................................................................19 
4.1  Dose .......................................................................................................................19 
4.2  Duration of Therapy ................................................................................................18 
 
 
5.0  DOSE MODIFICATIONS AND TOXICITIES .....................................................................19 
5.1  Recipient toxicity from the conditioning regimen ......................................................19 
5.1.1 Thiotepa .......................................................................................................19 
5.1.2 Fludarabine ..................................................................................................20 
5.1.3 Total body irradiation (TBI) ...........................................................................20 
5.1.4 Rabbit anti-thymocyte globulin (rATG) ..........................................................20 
5.1.5 Melphalan ....................................................................................................20 
5.1.6 Cyclophosphamide .......................................................................................20 
5.1.7 Busulfan .......................................................................................................20 
5.1.8 Infection and bleeding ..................................................................................20 
5.1.9 Mortality .......................................................................................................20 
5.1.10 Secondary malignancy .................................................................................21 
5.1.11  Decreased IQ ...............................................................................................21 
5.1.12 Short Stature ................................................................................................21 
 
 
 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 5 of 53 
 
5.2  Recipient Toxicity from Transplant ...........................................................................21 
5.2.1 Failure of engraftment ..................................................................................21 
5.2.2 Delayed T-cell reconstitution and increased infections .................................21 
5.2.3 Increased risk of leukemic relapse ...............................................................21 
                 5.2.4   Graft vs. Host Disease……………………………………………………………..20 
         5.3   Toxicity from Miltenyi Biotec Inc. CliniMACS® Reagent System Processing………..21  
5.4  Donor  PBSC toxicity ...............................................................................................23 
5.4.1 G-CSF ..........................................................................................................23 
5.4.2  Plerixafor ......................................................................................................22 
5.4.3  Apheresis .....................................................................................................23 
 
6.0  DEVICE INFORMATION ..................................................................................................24 
6.1  CD34+  cell processing ............................................................................................24 
6.2  Release of product for transplant .............................................................................25 
 
7.0  SCHEDULE OF ASSESSMENTS ....................................................................................25 
7.1 Screening assessments ...........................................................................................25 
7.1.1 Recipient pre-transplant evaluation (within 30 days of admission): ...............25 
7.1.2  Donor pre-apheresis evaluation (must be done within 30 days of donation) 
 
(related donors only): ...................................................................................26 
7.2  Therapy ..................................................................... Error! Bookmark not defined. 
7.2.1  Recipient cytoreductive regimen - this will be patient-specific. ......................26 
7.2.2  GVHD prophylaxis and therapy ....................................................................27 
7.2.3 Infection prophylaxis and pre-emptive therapy .............................................27 
7.2.4  Supportive care ............................................................................................29 
7.3  Mobilization and collection of donor peripheral blood stem cells ..............................29 
7.4  Treatment of graft failure..........................................................................................30 
7.5  Therapeutic donor lymphocyte infusion (DLI) ...........................................................30 
7.5.1 Collection of therapeutic donor lymphocytes: ...............................................30 
7.5.2  DLI Dose ......................................................................................................31 
7.6  Follow-up assessments ...........................................................................................31 
 
8.0  CRITERIA FOR TERMINATION ......................................................................................31 
8.1  Conditions for terminating the study .........................................................................31 
8.2  Conditions for individual patient termination .............................................................31 
 
9.0  STATISTICAL CONSIDERATIONS ..................................................................................31 
9.1 Hypotheses..............................................................................................................31 
9.2  Sample Size ............................................................................................................31 
9.3 Endpoint definitions .................................................................................................32 
9.4  Plan of analysis .......................................................................................................33 
9.5 Stopping rules ..........................................................................................................34 
9.6  Accrual ....................................................................................................................35 
9.7  Estimated duration of study .....................................................................................35 
9.8  Replacement policy .................................................................................................35 
 
10.0 CRITERIA FOR EVALUATION .........................................................................................35 
10.1  Monitored outcomes ................................................................................................35 
 
11.0 DATA AND SAFETY MONITORING PLAN ......................................................................36 
11.1  Oversight and monitoring plan .................................................................................36 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 6 of 53 
 
11.2  Monitoring and reporting guidelines .........................................................................36 
11.3 Review and oversight requirements .........................................................................36 
11.3.1 Adverse event definition ...............................................................................36 
11.3.2 Adverse event reporting ...............................................................................37 
11.3.3 Serious adverse events definition .................................................................37 
11.3.4  Special AE documentation guidelines for this protocol .................................37 
11.3.5 Adverse event reporting procedures.............................................................38 
11.3.6 Review of adverse event rates .....................................................................39 
 
12.0 ETHICAL ASPECTS ........................................................................................................39 
12.1 Regulatory considerations .......................................................................................39 
12.2 Institutional Review Board .......................................................................................39 
 
13.0  DATA FORMS AND SUBMISSION SCHEDULE ..............................................................39 
 
APPENDICES 
 .....................................................................................................................41 
APPENDIX I – Alternative conditioning regimens: ............................................................41 
APPENDIX II – DONOR (pre-administration through collection) .......................................43 
APPENDIX III – RECIPIENT (pre-administration through transplant) ...............................44 
APPENDIX IV – RECIPIENT (follow-up schedule) ...........................................................45 
APPENDIX V – Acute GVHD staging and grading for children** ......................................46 
APPENDIX VI - Chronic GVHD staging and grading for children** ...................................47 
APPENDIX VII - Device information .................................................................................48 
APPENDIX VIII - SOP #206.04 Recipient Selection and Treatment Regimen for Pediatric 
Bone Marrow Transplant (PBMT). ....................................................................................49 
APPENDIX IX - SOP #206.04 Donor and Donor Source Selection for Pediatric Bone 
Marrow Transplant (PBMT). .............................................................................................49 
 
REFERENCES .................................................................................................................50 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 7 of 53 
 
SCHEMA 
 
Patient population: Patients with a malignant or non-malignant disease who can benefit from 
alternative stem cell transplantation, who lack a healthy HLA-identical related donor at least one 
year of age, and who have a mismatched related (≥ 4/8 and ≤ 7/8) or unrelated (6/8 or 7/8) 
donor able to receive G-CSF and undergo apheresis. 
 
Study design: 
 
SCHEMA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
¶ 
 
 
 
  
 
 
 
 
RECIPIENT 
Eligible for BMT with an eligible 
mismatched donor (related or unrelated)  
DONOR 
Apheresis  
DONOR 
Eligible for apheresis 
RECIPIENT 
Admitted to BMT unit to begin conditioning 
therapy  
RECIPIENT  
Response outcome measured by: 
engraftment at 4 weeks that is sustained 
through day 100. 
RECIPIENT 
PBSC transplant 
CD 34+ processing with 
CliniMACS® system  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 8 of 53 
 
LIST OF ABBREVIATIONS 
 
AE 
 
adverse event 
ALL 
 
acute lymphocytic leukemia 
AML 
 
acute myelocytic leukemia 
ANC 
 
absolute neutrophil count 
APC 
 
antigen presenting cells 
ASCO  
American Society for Clinical Oncology 
BMT 
 
bone marrow transplant 
BSA 
 
body surface area 
CB 
 
cord blood 
CHR 
 
Committee on Human Research (UCSF IRB) 
CIBMTR 
Center for International Blood and Marrow Transplant Research 
CML 
 
chronic myelogenous leukemia 
CMV 
 
cytometolovirus 
CR 
 
complete response 
CRF 
 
case report form 
CTCAE  
Common Terminology Criteria for Adverse Events 
DFS 
 
disease-free survival 
DLI 
 
donor lymphocyte infusion 
DLT 
 
dose limiting toxicity 
EBV 
 
Epstein - Barr virus 
EFS 
 
event-free survival 
FA 
 
Fanconi Anemia 
FDA 
 
Food and Drug Administration 
FHCRC 
Fred Hutchinson Cancer Research Center 
GCP 
 
Good Clinical Practice 
G-CSF  
human granulocyte-colony stimulating factor 
GVHD  
graft versus host disease 
HLA 
 
human leukocyte antigen 
HSC 
 
hematopoietic stem cell 
ICH 
 
International Conference on Harmonization 
IDE 
 
investigational device exemption 
IEC 
 
Independent Ethics Committee 
IRB 
 
institutional review board 
JMML  
juvenile myelomonocytic leukemia 
KIR 
 
killer cell immunoglobulin-like receptor 
MDS 
 
myelodysplastic syndrome 
MRI 
 
magnetic resonance imaging 
MUD 
 
matched unrelated donor 
NMDP  
National Marrow Donor Program 
OS 
 
overall survival 
PBSC  
peripheral blood stem cells 
PCR 
 
polymerase chain reaction 
PI 
 
Principal Investigator 
PTLD  
post-transplant lymphoproliferative disorder 
PRC 
 
Protocol Review Committee, UCSF HDFCCC 
rATG 
 
polyclonal rabbit anti-thymocyte globulin  
SAE 
 
serious adverse event 
SOP 
 
Standard Operating Procedure  
 
 
TBI 
 
total body irradiation 
TMP/SMX 
Trimethoprim-sulfamethoxazole 
TRM 
 
Transplant related mortality 
  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 9 of 53 
 
 
SHORT ABSTRACT  
  
A major issue in alternative donor (mismatched related and unrelated donor transplantation is 
the development of graft-versus-host disease (GVHD).  Several clinical trials have shown that 
the use of T-cell depleted peripheral blood stem cells (PBSC) reduces GVHD in alternative 
donor transplants.  The purpose of this study is to determine the ability of CD34 positive 
selection and T cell depletion using the CliniMACS® Device as the only GVHD prophylaxis to 
prevent severe acute GVHD in recipients of an alternative donor PBSC transplant.  Mismatched 
related donors will match 4, 5, 6 or 7 out of 8 HLA antigens (haplocompatible) and unrelated 
donors will match 6 out of 8, or 7 out of 8 HLA antigens with the transplant recipient.   
The patients will receive conditioning therapy based on their diagnosis which may include 
chemotherapy, anti-thymocyte globulin (ATG), +/- total body irradiation (TBI).  The transplant 
recipients will be followed for 2 years post-transplant for the development of GVHD, 
engraftment, post-transplant infections, disease relapse, and overall survival.   
   
 
1.0  
GOALS AND OBJECTIVES  
 
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 donor 
(mismatched related donor and unrelated donor) hematopoietic stem cell transplants.  
  
 
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, and the rate of engraftment in recipients of CD34+ cell 
selected, T cell-depleted transplants from alternative 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) Leukemia relapse  
h) Transplant-related mortality 
i) Disease-free survival (DFS) and overall survival (OS)  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 10 of 53 
 
Monitor device performance:  
a) Purity of selected product  
b) Yield of CD34+ cells  
c) CD3+ cell depletion  
d) Sterility  
 
 
2.0  
BACKGROUND AND RATIONALE  
 
2.1  
Stem cell transplantation  
 
Stem cell transplantation (SCT) can cure some children with marrow failure syndromes, 
inherited immunodeficiencies, myelodysplastic syndrome (MDS), acute lymphocytic leukemia 
(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), lymphoma, 
white blood cell disorders (chronic granulomatous disease, osteopetrosis), and red blood cell 
disorders (sickle cell disease, thalassemia). The preferred donor for transplantation is an HLA 
identical sibling. However, less than 20% of patients requiring a transplant will have such a 
donor. Alternative stem cell sources include volunteer matched unrelated donors (MUD) and 
unrelated donor cord blood (CB). Approximately 90% of Caucasians and 60% of African-
Americans will have a 5/6 or 6/6-matched unrelated marrow donor. Approximately 80% of 
Caucasians and 40% of African-Americans will have a 5/6 or 6/6-matched unrelated cord blood 
donor. Most patients will have a 4/6-matched cord blood available (1).  
  
The largest experience using unrelated donors is with unrelated donor bone marrow transplants. 
Single center data for children include a report from Fred Hutchinson Cancer Research Center 
(FHCRC) that described 47% 2 yr event-free survival (EFS) for ALL in CR1/2 and 46% for AML 
in CR1/2 (2). The incidence of severe, life-threatening (grade III-IV) acute GVHD was 37% and 
62% of HLA-matched and mismatched recipients, respectively. Data from the Center for 
International Blood and Marrow Transplant Research (CIBMTR) from 1996-2001 shows a 50% 
2 yr EFS for ALL in CR1 and 42% 2 yr EFS for ALL in CR2+. This compares to CIBMTR data for 
matched sibling transplants over the same time period that shows a 70% 2 yr EFS for ALL in 
CR1 and 60% 2 yr EFS for ALL in CR2+ (3). 
 
For unrelated donor cord blood transplants, the University of Minnesota reported a 55% 2 yr 
EFS for ALL in CR1, 32% 2 yr EFS for ALL in CR2+, and 33% for AML in CR2+. The incidence 
of grade III-IV acute GVHD was 11%. The use of a 4/6-matched donor was associated with a 
relative risk of 2.4 for death compared to a 5/6 or 6/6-matched donor (p=0.01) (4). A larger 
review (562 patients) by Rubinstein et al. reported a 23% incidence of grade III-IV acute GVHD. 
The cumulative incidence of transplant-related events increased as the number of HLA 
disparities increased (5).  
  
A comparison of MUD and CB donor sources in 541 children transplanted for acute leukemia 
showed a 2 year event-free survival of 43% for unmanipulated MUD transplant, 37% for T cell-
depleted MUD transplant, and 31% for cord blood transplant. The incidence of grade III-IV acute 
GVHD was 29%, 8%, and 21% in the respective groups (6). A recent retrospective review of 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 11 of 53 
 
MUD and CB donor transplant for 503 children with leukemia transplanted in the US showed the 
following results (7):  
 
a) 2 yr EFS ~ 40% after matched and mismatched unrelated donor bone marrow 
transplant,  
b) 2 yr EFS ~ 40% after 4/6 matched unrelated donor cord blood, ~ 45% after 5/6, ~ 65% 
after 6/6 matched (there were only 35 patients with a 6/6 matched donor),  
c) Severe (Grade III/IV) acute GVHD 18% and 32% after matched and mismatched 
unrelated donor bone marrow transplant, respectively,  
d) Severe (Grade III/IV) acute GVHD 27% after 4/6 matched unrelated donor cord blood, 
20% after 5/6, 9% after 6/6 matched,  
e) Relapse 39% and 31% after matched and mismatched unrelated donor bone marrow 
transplant, respectively,  
f) Relapse 19% after 4/6 matched unrelated donor cord blood, 27% after 5/6, 31% after 6/6 
matched.  
 
2.2  
Mismatched (haplocompatible) related donor stem cell transplant       
 
Another donor source that has been reported is a mismatched (haplocompatible or sharing only 
one of two haplotypes) related donor. In the past, the major challenges have been engraftment, 
graft-versus-host disease (GVHD), and an increased incidence of infection and 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 order 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, and 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 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 100x lower than therapeutic levels. Following incubation, the 
product passes through a strong magnet. The CD34+ cells remain at the level of the magnet 
and other cells (T, B, and NK cells, monocytes, and neutrophils) pass through into a waste bag. 
The magnet is then turned off and the CD34+ cells are released into a bag containing the final 
product.   

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 12 of 53 
 
 In addition, 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 the 
matched (13,14) or mismatched (15,16) relative. PBSC recruited with cytokines 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 a conditioning regimen of total body irradiation (TBI), thiotepa, 
fludarabine, and rabbit ATG for 101 patients. 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 2 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% 2 yr 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 mo. (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 yr. 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 mo. – 7 yrs. (median 31 mo.)(22). The 
median pt age was 8 yrs. (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] AML (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 BMT 3 yrs 
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. The conditioning regimen was TBI 12-14 Gy in 6 fractions, thiotepa, 
fludarabine, and ATG. Fractionated TBI was replaced with single fraction TBI (2 pts.) or 
melphalan (3 pts) as clinically indicated.  
 
Cyclophosphamide was used instead of thiotepa for one pt with FA. No post-transplant graft-
versus-host disease (GVHD) prophylaxis such as Tacrolimus or methotrexate was given. 
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).   

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 13 of 53 
 
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). Primary (occurring after SCT and prior to donor 
lymphocyte infusion [DLI]) 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 1 year transplant-related mortality 
were 11% and 19%, respectively.  Four patients (of 13 at risk, 31%) have relapsed; 1 pt with 
cytogenetic relapse is in CR > 1 year later. The 2 yr 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 6 had HHV-6 reactivation. EBV 
reactivation occurred in 5/18 (28%) patients, 3 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 3 
months post-transplant, only 4 of the 15 evaluable patients had a CD4 count > 100. By 9 
months post-transplant, 10 of the 13 evaluable patients had a CD4 count > 200.  
  
In summary, the use of megadose CD34+ selected PBSC without post-transplant GVHD 
prophylaxis for children was associated with rapid engraftment, a low 100-day mortality, a very 
low incidence of severe GVHD, and excellent survival. The overall survival compares favorably 
with MSD and MUD HSCT. Immune reconstitution was slow and post-transplant infections 
contributed to morbidity and mortality.  
  
2.3  
NK alloreactivity  
 
The risk of relapse with haplocompatible transplantation may be reduced by utilizing both donor 
NK cells for a graft-versus-leukemia effect and donor lymphocyte infusions (DLI) to accelerate T 
cell reconstitution. NK alloreactivity has been shown to be present for myeloid leukemias and for 
lymphoid leukemias (23, 24). Alloreactivity can be determined by HLA-C typing with even better 
results with the addition of NK receptor typing (23, 24). In addition to HLA compatibility, the 
choice of the optimal donor source may depend on the age and size of the recipient, the type of 
leukemia, disease status, pre-transplant organ dysfunction, and pre-transplant infections.  
  
There is limited experience with unrelated donor transplants for children following CD34+ cell 
selection and T cell depletion with the CliniMACS device (25). In 30 patients with leukemia, 
primary engraftment was observed in 84% with the remainder engrafted after a second 
transplant. Grade III-IV acute GVHD occurred in 7% of patients and followed HHV-6 infection in 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 14 of 53 
 
both patients. The 2 year survival was 44% for patients in remission at the time of transplant 
(25). The same approach was used for unrelated donor transplantation for 14 children with non-
malignant disease. Observed overall survival was 100% (follow-up 1-7 years) and no grade III-
IV acute GVHD occurred (21).   
  
In a large prospective study of T cell depletion versus immunosuppressive drugs for GVHD 
prophylaxis for adults undergoing matched unrelated donor (MUD) transplant, there was no 
difference in 3 year EFS, 27 vs 34%, respectively (26). However, the T cell depletion used for 
this trial was much less intense than that achieved with the CliniMACS® device, as 
demonstrated by an 18% incidence of grade III-IV acute GVHD (37% in the non-T cell-depleted 
arm) (26). Post-transplant infections including CMV and aspergillus were more problematic after 
T cell depletion (27). Interestingly, the outcome for recipients of T cell-depleted bone marrow 
who did not have a fungal infection was better than that for patients receiving T replete bone 
marrow. This suggests that successful prevention of post-transplant infection by hastening 
immune recovery may result in a superior outcome for T cell-depleted transplants.  
  
2.4  
Preliminary data  
 
We have an ongoing study at UCSF 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 enrolled with the following diagnoses: ALL (3), AML 
(6), bilineage 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%) are alive and well, 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. Of note is that 9/11 patients treated after the last protocol 
amendment are alive and well, including 5/7 with malignant disease. Both deaths were due to 
late infection. One patient died at 7 months after transplant due to Para influenza and 
Paecilomyces infections and the other died at 23 months after transplant due to disseminated 
Mucor infection. 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).    
  
2.5 
Immune reconstitution  
 
A major challenge of previous approaches to haplocompatible donor transplantation is the 
prolonged immunodeficiency that follows transplant (28). This results in viral and fungal 
infections and is the primary cause of transplant-related mortality (29). As part of this study, 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 15 of 53 
 
patients who have engrafted without evidence of GVHD will be eligible to receive donor 
lymphocyte infusions if clinically indicated (See section 7.5) 
  
2.6  
Rationale for including alternative conditioning regimens  
 
Patients who are not eligible for the predecessor ongoing study described above include those 
who need an individualized conditioning regimen (ie. contraindication to the total body irradiation 
(TBI), fludarabine, thiotepa, and ATG regimen) or because of diseases/conditions not allowed in 
the eligibility criteria (e.g. non-Hodgkin’s lymphoma, poor lung function) or who do not have a 
related haploidentical donor available or who have failed a prior transplant from a related or 
unrelated donor, e.g. unrelated cord blood transplant and who need a rescue transplant using 
an alternative donor. Examples of such patients that we have transplanted include (1) patient 
with AML who failed to engraft after a cord blood transplant and had invasive fungal infection – 
alive and well 4 years after transplant, and (2) patient with rare immunodeficiency – NEMO 
syndrome – who had significant lung dysfunction and was on oxygen at the time of transplant – 
alive and well 3 years after transplant, and (3) a patient with CD40 ligand deficiency who 
rejected a mismatched unrelated donor cord blood transplant and was successfully 
reconstituted with a paternal CD34+ T cell depleted haplocompatible transplant (30).   
  
Recent studies using the CliniMACS® device, including our own, used a CD3+ cell (T cell) dose 
of < 3 x 104/kg. Conditioning regimens include the TBI-containing regimen used by the Perugia 
group and in our study as well as non-TBI containing regimens as indicated by the patient’s 
disease and clinical condition. The substitution of Melphalan for TBI has been reported by 
several groups (22, 31, and 32). Regimens have been developed for patients in poor clinical 
condition (32, 33) or with DNA repair syndromes (34,35). Because this protocol is designed to 
look at the ability of the CliniMACS® device to prevent GVHD, the conditioning regimen used 
will be chosen based on the patient’s disease and clinical condition.  
  
 
3.0  
STUDY DESIGN AND ELIGIBILITY CRITERIA  
 
 3.1  
Study design  
 
Patients will be enrolled with alternative (mismatched/haplocompatible) related donors or 
unrelated donors either for an initial transplant or as a rescue following rejection of a previous 
graft or relapse following a previous transplant. For patients with mismatched related donors, 
the majority of clinical experience has been with a T cell-depleted PBSC product. Currently, no 
FDA-approved method for T cell depletion exists. Recent experience with the CliniMACS® 
device has produced excellent results with a 70-75% survival in children, many of whom were 
high risk patients (19, 22, and 36).   
  
Patients that receive transplants from unrelated donors usually receive stem cells that are not T 
cell-depleted. However, this is associated with a high risk of GVHD. The excellent results with 
mismatched related donor transplants justify expanding this approach to unrelated donor 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 16 of 53 
 
transplant recipients if the HLA mismatch is sufficiently great. 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. 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 replete transplants with post-transplant immunosuppression (21,25).  
  
This protocol will allow the use of patient-specific conditioning regimens. Some patients have 
contraindications to certain components of the conditioning regimen used for our ongoing study 
under BB-IND 8817 (CC# 01151). An example is a patient with pre-existing organ dysfunction 
that would be better served by the use of a reduced intensity conditioning regimen. Another 
example is a patient for who total body irradiation is contraindicated due to very young age or 
prior radiation therapy.  Finally, patients who would be otherwise eligible for the predecessor 
study but who do not have an eligible related donor or a closely matched unrelated donor would 
be eligible for this study. The target CD3+ T cell dose that will be given will be 3 x 104/kg. The 
UCSF 01151 protocol uses a dose of 3 x 104/kg. The T cell dose in the graft is usually < 1 x 
104/kg after processing and T cells are added to the product.  
  
3.2  
Recipient inclusion criteria  
 
 
Patient must have a malignant or non-malignant disease that can benefit from alternative 
stem cell transplantation according to standard practice guidelines for including patients 
for transplant as outlined in UCSF Pediatric BMT Standard Operating Procedure (SOP) 
#206.04 Recipient Selection and Treatment Regimen for Pediatric Bone Marrow 
Transplant (PBMT). Examples include acute and chronic leukemias, myelodysplastic 
syndrome, lymphoma, severe acquired and congenital cytopenias, white and red blood 
cell abnormalities, inborn errors of metabolism and immunodeficiencies.  Patient with 
Fanconi's Anemia will be eligible regardless of match with donor. 
 
Patients with acute leukemia (AML excepted) or lymphoma must be in remission at the 
time of transplant.  
 
 
Patients must lack a healthy and willing HLA-identical related donor.  
 
 
If recipient is female and of child-bearing age, negative pregnancy test.  
 
Patient must either:  
o A) Have a mismatched related or an unrelated donor,  
 
Mismatched related or unrelated donors must be: 
o Able to receive G-CSF +/- Plerixafor and undergo apheresis either 
through placement of catheters in antecubital veins or a temporary 
central venous catheter 
o Able to undergo Bone Marrow Harvest 
o Healthy and willing 
o See Donor selection criteria below 
o B) Be suitable for an autologous gene-modified transplant: 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 17 of 53 
 
o using either bone marrow  or cytokine-mobilized peripheral blood stem cells 
(PBSC)  
 
o Patient or authorized guardian must sign informed consent for this study.  
 
3.3  
Recipient exclusion criteria  
 
 
Patient with an anticipated life expectancy of < 1 month  
 
 
Active infectious hepatitis or CMV disease (organ involvement)  
 
 
HIV or HTLV-I/II infection  
 
 
Cardiac ejection fraction < 45%; can be lower if patient is not in clinical cardiac failure 
and a reduced intensity conditioning regimen is used.  
 
 
Creatinine clearance <60 ml/min/1.72 m2; can be lower if a reduced intensity 
conditioning regimen is used.  
 
 
Pulmonary diffusion capacity (adjusted for Hgb), FEV1, or FVC <60% of predicted or O2 
sat > 94% on room air if unable to perform PFTs; can be lower if a reduced intensity 
conditioning regimen is used.  
 
 
Serum ALT > 5 x upper limit of normal (can be up to 10x upper limit of normal if a 
reduced intensity conditioning regimen is used) or bilirubin > 2.  
 
 
Performance score (Lansky/Karnofsky) < 50  
 
 
Any condition that compromises compliance with the procedures of this protocol, as 
judged by the principal investigator.  
 
3.4  
Donor selection criteria:  
Must have a complete medical history, physical and screening for infectious diseases 
that are acceptable for donation.  See Section 7.1.2 for details of evaluation.   
Donors must be willing to sign informed consent for this study. If donor is <18 years of 
age, donor must be willing to give assent and parents are willing to sign informed 
consent.  
o 3.4.1 Related Donors 
o Sibling, half-sibling, parent, cousin, aunt, uncle or grandparent will all be 
considered eligible.  
o HLA antigen genotypic match ≥4/8 and ≤7/8 (haplocompatible).  
o If donor is female and of child-bearing age, negative pregnancy test if GCSF is to 
be administered. 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 18 of 53 
 
 
o Criteria to consider when choosing among related haplo donors are:  
o CMV positive donor is preferred over other factors. CMV negative donor can be 
used ONLY if recipient is CMV negative. 
o HLA disparity i.e. 2 Ag mismatch preferred over 3 Ag mismatch; Drβ1 match 
preferred over class I match; HLA-C matched preferred over A and B match.  
o KIR mismatch in GVH direction is preferred for patients with malignant disorders  
o ABO compatibility  
o Absence of anti-HLA antibodies in recipient directed against donor antigens.  
 
 
3.4.2 Unrelated Donors 
 
o 6/8 or 7/8 HLA antigens match (if two mismatches, they must be at different loci).  
o Absence of anti-HLA antibodies in recipient directed against donor antigens.  
o If donor is female and of child-bearing age, negative pregnancy test if GCSF is to 
be administered. 
o Must follow NMDP/Be The Match Algorithm Guidelines for Unrelated Donors 
below:  
 
NMDP: For unrelated donors: Per New Algorithm, Jan. 14, 2016, 
effective immediately, NMDP/Be The Match is adopting a revised 
algorithm for determining if a donor is a research subject on their 
recipient’s research protocol: The NMDP will inform the donor of the 
activities and the use of donor’s apheresis product to be used in this 
study and obtain written consent from the donor. Transplant centers are 
sent documentation of the donor consent to participate in the research 
support activities. (The revised algorithm can be found on the “Be the 
Match Clinical Network”) 
 
3.4.3 Autologous Donors 
 
o If donor is female and of child-bearing age, negative pregnancy test if GCSF is to 
be administered. 
o Age ≥2 months for autologous bone marrow collection and CD34 selection (Gene 
Therapy Protocol, CHR# 16-18983). 
 
 3.5  
Patient recruitment  
 
Candidates are referred to study investigators from pediatric hematology/oncology specialists 
and immunologists.  Patients and parents will be seen in the pediatric BMT outpatient clinic or 
as an inpatient where consent will be presented.   
  
3.6  
Patient registration  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 19 of 53 
 
The patients will be registered by completion of eligibility forms and data entry into the UCSF 
Helen Diller Family Comprehensive Cancer Center Clinical Trial Management System (CTMS) 
and each patient will be assigned a unique subject study ID.   
 
 
4.0  
INVESTIGATIONAL TREATMENT PLAN   
 
4.1 
 Dose  
 
The plan is to use the CliniMACS® device to prevent severe (grade III/IV) acute GVHD without 
the need for post-transplant immunosuppressive medications. The target dose of CD34+ cells is 
≥ 20x106/kg, but a minimum of ≥2x106/kg is required. If the yield is <2x106/kg, we will attempt to 
obtain more donor cells (see Section 7.3). The target dose of CD3+ cells is 3x104/kg.  The 
ClinicMACS® CD34+ Reagent System will be used for donor CD34+ cell selection. If we end up 
infusing <5x10^6 CD34/kg we will initiate a search for a back-up donor in the event the graft 
fails or is rejected and a second transplant is necessary. Target dosing for autologous gene 
therapy is defined in the Gene Therapy Protocol, LVXSCID-ND: “A pilot feasibility study of gene 
transfer for x-linked Severe Combined Immunodeficiency in newly diagnosed infants using a 
self-inactivating lentiviral vector to transduce autologous CD34+ hematopoietic cells  
(CHR # 16-18983)”. 
 
4.2  
Duration of therapy    
 
Patients will be followed for outcomes related to the device including acute and chronic GVHD 
and engraftment for 2 years. Subsequent follow-up care is standard for all transplant patients, 
and additional data related to secondary endpoints will be collected as available (see Appendix 
IV).  
 
5.0  
DOSE MODIFICATIONS AND TOXICITIES  
 
These are part of related transplant-issues but not specific to the use of the investigational 
device (CliniMACS® device).  
  
5.1  
Recipient toxicity from the conditioning regimen [MC1] 
 
5.1.1 Thiotepa 
Risks of thiotepa include alopecia, mucositis, and hepatic toxicity. There is also cutaneous 
toxicity which can result in erythema and breakdown of the skin especially in the neck, 
axilla, and inguinal and perianal areas. This can be painful and can become secondarily 
infected. Significant skin toxicity can be prevented by frequent baths or showers when the 
drug is given. In combination with fludarabine and TBI the risk of fatal hepatic toxicity or 
fatal pulmonary toxicity is <5%. There has also been a multiorgan failure syndrome 
reported in association with thiotepa containing regimens. The incidence is low (<5%) and 
was seen when doses greater than 10 mg/kg were used.  
 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 20 of 53 
 
5.1.2 Fludarabine 
The primary toxicity of fludarabine in the doses used has been severe T and B cell 
immunodeficiency. It has also been associated with the development of immune mediated 
hemolytic anemias, unlikely to occur as part of an ablative conditioning regimen for BMT. 
Other side effects include nausea, vomiting and neurotoxicity (seizures, confusion). 
Neurotoxicity is very unlikely at the doses used on this study.  
  
 
 
5.1.3 Total body irradiation (TBI) 
The total TBI dose is 1200 cGy given in 6 fractions. All fractions will use 50% shielding of 
the lungs. Potential acute toxicities associated with the use of TBI include nausea, vomiting, 
and parotitis. Sub-acute complications or side effects of TBI include hair loss, mucositis, 
diarrhea, veno-occlusive disease, leukoencephalopathy and interstitial pneumonitis. Chronic 
complications include cataracts in about 20% of cases, delayed sexual development, short 
stature, and sterility in >95% of cases, and thyroid dysfunction in 5-10% of cases.  
 
 5.1.4 Rabbit anti-thymocyte globulin (rATG) 
The most important risk associated with rATG is the development of a severe allergic 
reaction (i.e. anaphylaxis) which is rare. More common side effects of rabbit ATG include 
fever and rash.  
 
5.1.5 Melphalan 
Common side effects of melphalan include nausea, vomiting, low blood counts, mucositis, 
sterility, and hair loss. Much less common side effects include liver and lung damage.  
 
 5.1.6 Cyclophosphamide 
Common side effects of cyclophosphamide include nausea, vomiting, low blood counts, 
sterility, and hair loss. Less common side effects include water retention, damage to the 
lining of the bladder leading to blood in the urine, and liver damage. Rare side effects 
include damage to the lungs and heart and secondary cancer (leukemia and lymphoma).  
 
5.1.7 Busulfan 
Common side effects of busulfan include nausea, vomiting, low blood counts, mucositis, 
sterility, temporary darkening of the skin, and hair loss. Less common side effects include 
liver damage. Rare side effects include lung injury and seizures (the latter are rare when 
prophylactic anti-convulsants are used).  
 
    5.1.8 Infection and bleeding 
      In general, due to the 1-2 weeks of neutropenia, thrombocytopenia and mucositis, there is a     
significantly increased risk of infection and bleeding. The mortality associated with these 
complications is generally <5%.  
 
    5.1.9 Mortality 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 21 of 53 
 
 The reported transplant related mortality (organ failure, infection, bleeding) is ~30% for   
patients with high-risk malignancies (25, 31, 32). In a limited number of children with non-
malignant diseases the TRM was lower (10%) but until more patients in this category are 
evaluated, we will assume it is as high as 30%. For patients undergoing a transplant who have 
already failed a prior transplant (e.g. graft rejection post umbilical cord blood transplant) the 
TRM could be as high as 50%. 
 
 
5.1.10 Secondary malignancy 
There is also an overall reported risk of ~2-10% of a malignancy occurring up to 15 years post-
transplant. The majority of these are lymphoproliferative disease related to EBV infection. 
Because these patients will not receive cyclosporine prophylaxis or post-transplant ATG, and 
because the processing procedure removes almost all B cells from the donor graft (the source 
of EBV in these circumstances), this risk is likely to be lower.   
 
5.1.11 Decreased IQ 
There is about a 30% risk of a decreased IQ (7 points) at 1 year post transplant (37). This 
occurs regardless of the conditioning regimen and by 3 years there appears to be some 
recovery of IQ points. Of the remaining patients, 1/3 has no change in their IQ and 1/3 increases 
their IQ by about 7 points.  
 
5.1.12 Short stature 
The risk of short stature post-transplant is related to the type of conditioning. With TBI the risk is 
highest (25%) while with busulfan containing regimens it is ~10%.  
 
 5.2  Recipient Toxicity from Transplant  
 
5.2.1 Failure of engraftment 
Published reports using 1-10x106 CD34+ cells/kg haplocompatible stem cell enriched T cell 
depleted PBSC with TBI-containing regimens have initial engraftment rates of >80% although 
with second transplants the engraftment rate has been >90%. We believe that the combination 
of thiotepa, fludarabine, ATG and TBI plus the large number of CD34+ cells in the graft should 
result in a primary engraftment rate of at least 80%.  
 
5.2.2 Delayed T-cell reconstitution and increased infections 
The chance of delayed T-cell reconstitution and increased infections is likely. In both children 
and adults who have been heavily pretreated it has taken as long as a year for T cell immunity 
(i.e. CD4+ cells > 200) to recover. The most commonly reported cause of transplant-related 
death with this kind of transplant is infection.  
 
5.2.3 Increased risk of leukemic relapse 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 22 of 53 
 
The extent of increased risk of leukemic relapse with PBSC is unknown. The studies of CD34+ 
PBSC haplocompatible transplants that have been reported to date do not demonstrate a higher 
than expected relapse rate.   
  
5.2.4 Graft vs. Host Disease 
In other studies in which <7x104 CD3+ cells/kg of haplocompatible PBSC are infused the 
incidence of grade III-IV acute GVHD has been <5%.  In our current study using infusions up to 
6x104 CD3/kg we have seen an incidence of ~30% grade I-II acute GVHD and ~5% chronic 
GVHD. There have been no deaths due to GVHD. 
 
 
5.3  
Toxicity from Miltenyi Biotec Inc. CliniMACS® reagent system processing  
 
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.  
Paramagnetic microspheres - 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 300 separations 
for clinical use of the CliniMACS® system.  
 
 Reaction to CliniMACS® reagent - (murine monoclonal antibody conjugated to an iron-dextran 
moiety). Iron dextran is commercially available as a sterile solution of iron dextran complex for 
the treatment of severe iron-deficient syndromes. It contains 5% iron and 20% dextran, and its 
safety profile has been well characterized. Iron dextran solution contains 50 mg/ml of elemental 
iron, most of which is present in the ferric state. A total dose of iron-dextran for the average 70 
kg person is calculated to be approximately 2 gm over several days (single dose of 100 mg). 
The iron dextran exposure from a singe CliniMACS® separation is ~0.5 mg and less than 1 mg 
dextran, 100x lower than a single dose and 1000x lower than a total dose.  
 
CD34+ monoclonal antibody -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 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 23 of 53 
 
levels. Furthermore, studies have shown that the levels of antibody used in the CliniMACS® 
system do not induce complement activation in vitro.  
  
5.4  
Donor PBSC toxicity  
 
The toxicities listed below are of concern for all donors and not specific to this protocol or the 
use of the investigational device (CliniMACS® device).  
 
5.4.1 G-CSF 
For the recruitment of PBSC, G-CSF is known to cause bone pain in most patients as well 
as other symptoms including headache, bone discomfort or ache, ankle swelling or fluid 
retention. These symptoms are dose related and generally controlled with analgesics such 
as Tylenol or ibuprofen (17-19). There is at least a theoretical risk of inducing a malignancy 
with G-CSF although the extensive experience to date with normal donors does not indicate 
that this will be a problem. Patients with aplastic anemia or Kostmann’s syndrome who have 
been chronically treated with G-CSF do not have any higher incidence of malignancy than is 
normally found with these disorders. Since G-CSF could theoretically interfere with 
embryogenesis, its use is contraindicated in pregnant women. 
 
 
5.4.2 Plerixafor 
Plerixafor has been used safely in a large number of patients and a limited number of 
healthy volunteers. In patients plerixafor was given in combination with G-CSF, the use of 
Plerixafor added to GCSF with GCSF dosing changed to 10 mg/kg/day x4 would be 
permitted as an alternative to the use of GCSFx8 or GCSFx4 without Plerixafor.  
The most frequently occurring AEs (≥10%) were diarrhea, nausea, bone pain, fatigue, 
injection site reaction, headache, paraesthesia, back pain, hypokalemia, arthralgia, catheter 
site pain, vomiting, and dizziness. Also flatulence and insomnia (≥5%) have been rarely 
reported. The majority of patients experienced AEs that were mild or moderate and resolve 
within 24 hours. Systemic reactions (including urticaria, periorbital swelling, dyspnea, or 
hypoxia) and vasovagal reactions, orthostatic hypotension and/or syncope were seen in less 
than 1% of patients. The first studies with plerixafor in healthy volunteers and patients date 
from 2003 so follow-up is >10 years. 
 
5.4.3 Apheresis 
Apheresis has been associated with decreased platelets and temporary hypotension as well 
as hypocalcemia and some risk of bleeding because of anti-coagulation. There is also pain 
from the insertion of needles into the antecubital veins and the discomfort of a central line if 
necessary. All of these are reversible and have been tolerated in previously reported 
studies. There is the small risk (<1%) that a healthy donor might require a platelet 
transfusion. There is also the risk that peripheral venous access might be inadequate and 
placement of a temporary central line under local anesthesia will be necessary (17,18). The 
risks of inserting a central line include bleeding, infection and/or pneumothorax. There are 
abnormalities in the circulating white cell populations (T cells and stem cells) that occur after 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 24 of 53 
 
apheresis in normal donors. Most of these appear to resolve (i.e. numbers return to normal) 
within the first 3 months post donation, but there may be abnormalities or long-term side 
effects that at this time have not been identified.  
 
6.0  
DEVICE INFORMATION   
 
 (Milteny Biotec, Inc. CD34+ ClinicMACS® reagent system information)  
 
6.1  
CD34+ cell processing  
 
The collection will be stem cell enriched and T-cell depleted. The CD34+ cells are positively 
selected using the CliniMACS® System.  
  
The pheresis product may be stored overnight at 4oC and at a concentration < 200 x 106 cells/ml 
and processed the following morning. Products may be pooled (i.e. first and second collection 
pooled and third and fourth collection pooled) for processing and cell selection, or the 
collections may be processed and CD34+cells selected on each day.  The determination of 
whether to store product overnight or select daily will be individualized per patient and be based 
on patient cell counts and CD34+ column cell capacity.  CD34+ cell selection will be performed 
using the Miltenyi CliniMACS® system. The processing will be performed at University of 
California-San Francisco.    
  
The target cell doses will be ≥ 20x106 CD34+ cells/kg . A dose of ≥10x106 CD34+ cells/kg will 
be acceptable. The target T cell dose will be 3x104 CD3+ cells/kg.   
 
In the unlikely event that the CD3+ cell count is too high in order to achieve the minimum 
acceptable CD34+ cell dose, the patient can be given long course methotrexate or a course of 
cellcept along with a calcineurin inhibitor.  
 
The stem cells will be injected intravenously into the recipient. Prophylactic Cefazolin* will be 
given for 24 hours beginning just prior to the infusion.  
 
*Patients with penicillin allergy will be excluded from Cefazolin administration 
 
If >35x106 CD34+ cells/kg are available, then 20x106/kg will be infused and the remainder will 
be cryopreserved. Otherwise, the full dose up to 35x106/kg will be infused.  
  
Five aliquots of the negative fraction (non CD34+ selected cells) containing the CD3+ cells will 
be made and cryopreserved for future DLI. Each aliquot will contain a minimum of 10x104 CD3+ 
cells/kg body weight of the recipient.  
 
Processing of samples received from participating centers:  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 25 of 53 
 
Fresh harvested Hematopoietic progenitor cells (HPC) product are shipped to UCSF and then 
processed and cryopreserved and shipped back to the participating center for thawing and 
infusion. 
 
 6.2  
Release of product for transplant  
 
The studies that will be done in addition to the cell immunophenotyping will be sterility (routine 
USP culture for bacteria and fungi), endotoxin testing, gram stain, and viability. We already have 
experience with all of these procedures in other protocols, specifically, BB-IND 8817.  
  
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.   
 If the culture becomes positive, appropriate antibiotics will be started. 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.  
  
 
7.0  
SCHEDULE OF ASSESSMENTS   
 
 (See Appendices II-III for Assessment Tables)  
 
7.1 Screening assessments  
 
7.1.1 Recipient pre-transplant evaluation (within 30 days of admission):  
 
 
Pulmonary function tests if patient age allows (usually > 5 years of age); if not, the 
patient will have pulse oximetry on room air.  
 
Echocardiogram and ECG  
 
CXR or chest CT scan (if prior history of lung disease) 
 
Creatinine clearance by Schwartz formula, urine collection, or GFR scan  
 
Testing for syphilis, CMV, HIV, EBV, HSV, VZV, Hepatitis B & C, HTLV I/II, and 
toxoplasmosis  
 
ABO typing and antibody screen  
 
Liver function tests, electrolytes, BUN, creatinine, CBC, differential.  
 
Marrow/Lumbar Puncture (LP) within 2 weeks of admission for patients with 
MDS/leukemia  
 
HLA typing  
 
Complete history and physical exam  
 
Evaluation for anti-HLA antibodies against donor antigens  
 
Pregnancy test if female and of childbearing age.  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 26 of 53 
 
 
7.1.2 Donor pre-apheresis evaluation (must be done within 30 days of donation) 
(related donors only):  
 
Complete history will include:  
 
Surgical/anesthesia history  
 
Review of systems  
 
History of inherited conditions and chronic illness  
 
History of hematological problems and immunological disorders  
 
History of cancer  
 
Donor health questionnaire which includes transfusion, vaccination and travel history  
 
List of current medications and allergies.  
 
Screen for evidence of syphilis, CMV, HIV, EBV, Hepatitis B & C, HTLVI/II, West Nile 
virus, Chagas’ disease, and toxoplasmosis  
 
ABO typing and antibody screen  
 
Liver function tests, electrolytes, BUN, creatinine, CBC, diff, PT, PTT  
 
HLA typing  
 
ECG  
 
Chest x-ray    
 
Pregnancy test if female and of child-bearing age  
 
Donor eligibility will be determined in accordance with 21 CFR 1271.45-.90 and the UCSF 
BMT Program Hematopoietic Progenitor Cell Donor and Recipient Evaluation Guidelines.  
 
Unrelated donor product will be received via the National Marrow Donor Program (NMDP). 
Donors will have been evaluated per NMDP standard protocols.  
 
7.2 Therapy  
 
7.2.1 Recipient cytoreductive regimen - this will be patient-specific.  
The standard regimen for leukemia will be:  
 
Total Body Irradiation (TBI) followed by chemotherapy (see table below):  
Day -9 
200 cGy TBI x 2 
Day -8 
200 cGy TBI x 2 
Day -7 
200 cGy TBI x 2 
Day -6 
Fludarabine 40 mg/m2 (1.33 mg/kg if ≤ 12 kg body weight) 
Thiotepa 10 mg/kg/day divided into 2 doses 12 hours apart 
Day -5 
Fludarabine 40 mg/m2;  
Day -4 
Fludarabine 40 mg/m2;  
Day -3 
Fludarabine 40 mg/m2; Rabbit ATG 0.5 mg/kg 
Day -2 
Fludarabine 40 mg/m2; Rabbit ATG 1.5 mg/kg 
Day -1 
Rabbit ATG 1.5 mg/kg 
Day 0 
Transplant 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 27 of 53 
 
 
Example of other conditioning regimens include (see Appendix I for details):  
 
Substitution of melphalan 140 mg/m2 for TBI especially for non-malignant, non SCID 
diseases. 
 
Substitution of busulfan and melphalan for TBI and thiotepa  
 
Substitution of cyclophosphamide for thiotepa in patients with Fanconi anemia  
These modifications will be based upon published conditioning regimens (22,31-35).  
 
The autologous gene therapy conditioning uses targeted busulfan at a low dose 
exposure that is specified in the gene therapy protocol. 
  
Patients with severe combined immunodeficiency and lack of NK cells or function and no 
GVHD due to maternal engraftment may be transplanted without conditioning.  
  
Total dose of the TBI (1200 cGy) will be delivered in 200 cGy fractions separated by at least 
6 hours. The patient will be treated with AP and PA fields and lungs will be partially blocked 
such that the dose to the lungs will be 600cGy (50% clinical shielding of lungs). For 
patients with MDS/leukemia, there will be an extra boost of radiation to the ribs). 
 Thiotepa will be given IV on day –6 at a dose of 5 mg/kg every 12 hrs x 2. Each infusion will 
be given over 4 hrs. Very careful attention to skin care during thiotepa treatment is 
necessary.  The patient should shower (or bath for small children) at least three times daily 
during administration and for 24 hrs after the last dose of thiotepa.    
  
Fludarabine will be administered each day, from–6 to–2, at a dose of 40 mg/m2 as a 30 
minute infusion.  The fludarabine dose will be reduced by 20% for patients with creatinine 
clearance ≤70 ml/min/1.73m2.  
  
Rabbit ATG will be infused each day, from –3to –1 as 10 hour infusion.  
  
7.2.2  
GVHD prophylaxis and therapy  
 
There will be no post-transplant GVHD prophylaxis given unless the CD3+ cell dose is too 
high (see Section 6.1).  
 
Initial treatment of GVHD will be prednisone or methylprednisolone 1-2 mg/kg/d. A rapid 
taper can be used if there is a quick and complete response. Additional therapy may be 
added if needed.  
 
7.2.3 Infection prophylaxis and pre-emptive therapy  
 
The infection prophylaxis listed below can be modified by the principal investigator if 
clinically necessary (this includes changing drugs and time period as clinically relevant).     
 
Herpes viruses prophylaxis:  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 28 of 53 
 
a) acyclovir 250 mg/m2/dose IV q8h until discharge then PO per standard supportive care 
guidelines until the CD4>200 and PHA response ≥30% of lower normal control.  
 
CMV:  
All recipients will have CMV testing by PCR once per week until day +100 after transplant 
and then q2 weeks until CD4>200/uL. Monitoring should be more frequent in patients who 
have prolonged CMV reactivation after transplant. If the PCR test becomes positive, 
ganciclovir will be started at an induction dose of 5 mg/kg IV q12 x 7-14 days, then a 
maintenance dose of 5 mg/kg/day 5-7 days per week until PCR is negative x 2 weeks 
(assuming the patient remains asymptomatic). Valganciclovir can be used as prophylaxis 
following ganciclovir therapy if CMV reactivation occurred when the patient was on acyclovir 
prophylaxis. Alternatively, patients may be treated with foscarnet induction and 
maintenance. If CMV PCR does not clear with ganciclovir therapy or if drug resistance 
develops, DLI should be considered. Patient may also be eligible for CMV-specific cytotoxic 
T cell study. 
 
EBV: 
EBV by PCR will be obtained weekly beginning at 1 month post SCT and continuing until 
day +100 after SCT then q2wks until CD4>200. Monitoring should be more frequent in 
patients who have EBV reactivation after transplant. If the EBV viral load is > 1000 copies/ml 
or if any level of positivity is present with fever and/or other clinical findings such as 
adenopathy, rituximab 375 mg/m2 IV weekly x 2-4 will be given. CT scan of head, neck, 
chest, abdomen, and pelvis should be considered to evaluate for adenopathy. A therapeutic 
donor lymphocyte infusion can also be considered.  
 
HHV-6:  
HHV-6 PCR will be obtained weekly until day +60 after transplant and then q2wks until day 
+100.  
 
Adenovirus:  
Adenovirus PCR will be obtained on patients with fevers and negative bacterial/fungal blood 
cultures. For adenovirus reactivation (positive PCR), cidofovir will be given. This will be 
given as 5 mg/kg weekly or 1 mg/kg three days per week until the PCR is negative.  
 
 
Pneumocystis carinii prophylaxis: 
Trimethoprim/sulfamethoxazole 5 mg/kg/day of TMP component divided BID for 3 
consecutive days per week. If TMP/SMX is not tolerated, alternative prophylaxis will be 
given per institutional policy.  
 
Fungal prophylaxis:  
Fluconozole 3 mg/kg (max 200 mg) PO (or IV if PO not tolerated) qd will be started on Day 
+1 and continued until CD4 > 200 and PHA ≥30% lower limit of normal control.  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 29 of 53 
 
Bacterial prophylaxis:  
None  
 
IVIG:  
Gammaglobulin will be administered at a dose of 200 mg/kg every 2 weeks while 
hospitalized and then 400 mg/kg every 4 weeks until IgM ≥ 50 AND isohemaglutinin titers 
are ≥1:8.  
  
7.2.4  
Supportive care  
 
No post-transplant G-CSF unless the ANC is <500 on day +14; G-CSF may be started as 
early as Day +6 in patients with active fungal infections at the discretion of the treating 
physician.  
 
All transfusions (except the stem cell product) will be irradiated and leukodepleted. In 
addition, CMV negative PRBC will be used.  
 
Direct and indirect Coombs test will be checked q 2 weeks beginning at 8 weeks post BMT 
until the CD4>200 and PHA≥30% of the lower limit of normal control because of the risk of 
autoimmune hemolytic anemia.  
 
 7.3  
Mobilization and collection of donor peripheral blood stem cells  
 
The donor will receive 4 days of granulocyte colony stimulating factor (G-CSF, Neupogen®) 
administered subcutaneously (5 µg/kg/dose) twice a day. The total daily dose may be reduced 
to 10 mcg/kg and the dose may be given once a day for an unrelated donor if the recommended 
dosing is not possible.  When determined to be clinically necessary by the treatment team, 
Plerixafor 240 mcg/kg will be administered subcutaneously approximately 9 hours prior to 
planned collection. This will generally be required for donors of patients > 15 kg, where 
historically a 2nd day collection has almost always been required. The donor will undergo an 
outpatient apheresis using antecubital veins for venous access if possible. If the peripheral 
venous access is inadequate, a temporary central line will be placed as an outpatient. The 
morning dose of G-CSF will be omitted on the day of collection if apheresis will take place in the 
morning. G-CSF will be continued until the completion of apheresis. If the donor’s white blood 
count determined on the 4th day and subsequent days of G-CSF (for patients receiving 5 
mcg/kg/dose q12) is greater than 60 x 109 cells/L, the G-CSF dose on the following day should 
be decreased to 3ug/kg/day.    
  
For the apheresis, 5-20 x 1010 mononuclear cells (depending on the recipient’s body weight and 
efficacy of CD34+ cell mobilization in the donor) will be collected which may require 1-3 
apheresis procedures. Additional days of collection will require additional doses of  G-CSF and 
possibly Plerixafor. Each pheresis procedure will process 5 blood volumes (max 25 L) and will 
be performed in accordance with the UCSF BMT Program Pediatric PBSC Mobilization and 
Collection Guidelines.  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 30 of 53 
 
In the unlikely event the yield of CD34+ cells is too low or the product is not deemed acceptable 
for infusion, a related donor may be mobilized again (probably using Plerixafor (AMD3100) or 
another relative may be asked to act as an alternative donor. Unrelated donors may be asked 
for a second donation or another unrelated donor will be sought. It is our standard policy with 
unrelated donor transplants to identify an alternative if the first donor doesn’t engraft. We would 
do the same for this protocol. Each occurrence of a transplant procedure will use cellular 
product from only one donor.  
  
7.4  
Treatment of graft failure  
 
If at 4 weeks post-transplant there is no evidence of engraftment based upon blood counts, 
bone marrow examination and chimerism assays (<5% donor cells), infusion of a second PBSC 
transplant will be offered using the same or another donor (the other parent, sibling, or unrelated 
donor). Additional chemotherapy and immunosuppression will be given prior to the second 
transplant as needed. If pancytopenia occurs at a later time point, the same approach may be 
used.   Post-transplant endpoints for the second transplant will not be included in study 
analyses. 
 
 7.5  
Therapeutic donor lymphocyte infusion (DLI)  
 
Indication:  
 
Mixed Chimerism (< 80% donor) or increasing recipient chimerism  
 
Relapse  
 
EBV-related PTLD  
 
Viral infections that progress despite treatment with anti-virals 
 
In adequate T cell reconstitution 
o CD4<100 on day +100 
o PHA<10% of normal control level on day +100  
 
7.5.1 Collection of therapeutic donor lymphocytes:  
DLI will usually be obtained from the cryopreserved negative fraction (T cell-containing) of 
the CD34+ cell selected G-CSF-mobilized PBSC.   
 
T cells from the G-CSF-mobilized PBSC may be less alloreactive than T cells collected from 
blood when G-CSF is not present. G-CSF increases Th2 cells that decrease IL-12 production 
and delays immune recovery. At the discretion of the treating physician, a new collection can be 
performed to provide DLI depending on the clinical situation (such as leukemic relapse).  For 
larger recipients, the stem cell donor will undergo lymphocytopheresis to collect CD3+ cells in 
aliquots of 5 X 104/kg, 1 X 105/kg, 5 X105/kg, and 106/kg. For small children requiring small 
doses, donors may give whole blood if ABO compatibility permits the use of whole blood as a 
source of DLI.  All additional CD3 collections, whether from a blood draw or an apheresis will be 
GCSF mobilized comparable to the initial mobilization for the transplant. 
 
DLI will not be given if the patient has active GVHD and/or is on immunosuppressive 
therapy for previously active GVHD.  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 31 of 53 
 
  
7.5.2  
DLI Dose 
The dose of DLI (3x104 – 1x105 CD3/kg) will be determined by the treating physician based 
on the clinical situation.  
 
7.6  
Follow-up assessments  
 
Follow-up studies which are standard for all transplant patients will be performed according to 
BMT guidelines and include assessment of engraftment, marrow function, immune status, liver 
and kidney function, pulmonary function tests, and echocardiogram. (See Appendix IV) 
 
 
8.0 
 CRITERIA FOR TERMINATION   
 
8.1  
Conditions for terminating the study  
 
The Principal Investigator may terminate the study for any of the following reasons:    
 
Significant toxicities  
 
If it becomes clear that the study treatment is less effective than other available 
treatments.  
  
8.2  
Conditions for individual patient termination  
 
The Principal Investigator may terminate the participation of an individual patient for any of the 
following reasons:  
 
Disease progression  
 
Need for exclusionary concurrent treatment  
 
Withdrawal of informed consent  
 
Protocol non-compliance  
 
Lost to follow-up  
  
 
9.0  
STATISTICAL CONSIDERATIONS    
 
9.1 
 Hypotheses  
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%. This has been our experience in 18 reported patients (22) and currently 
25 treated recipients of haplocompatible donor transplants.  
  
9.2  
Sample Size 
Patients will be enrolled into one of two cohorts, haplocompatible related donor (n=60) and 
unrelated donor (n=20). The study objective is to determine the ability of CD34+ selection using 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 32 of 53 
 
the CliniMACS® device as the sole GVHD prophylaxis to prevent severe (grade III-IV) acute 
GVHD by Day +100 after transplant in recipients of alternative donor stem cell transplants. It is 
anticipated that the incidence of GVHD will not be affected by whether or not patient has had a 
previous transplant. Based on the literature and our experience, graft rejection is expected to 
occur in < 10% of patients. Sixty patients with related donors will allow estimating the proportion 
of severe acute GVHD with the exact 95% confidence interval with a maximum total width of 
0.168 if the observed rate of severe acute GVHD does not exceed 10%. If a maximum 
occurrence of 6 severe acute GVHD events is observed in 60 patients, the exact 95% 
confidence interval is (0.04, 0.21). For 20 patients with unrelated donors the maximum width of 
the exact 95% confidence interval for a 10% maximum occurrence of GVHD is 0.305. A very 
low incidence of severe GVHD is predicted based on a reported incidence of 2% in a large 
study of adults (17) and a 0% incidence in a large study in children (21) and our experience 
(22).  
 
The maximum total width of the exact 95% confidence interval is displayed below for an 
observed proportion of severe acute GVHD not exceeding 10% based upon the total number of 
evaluable patients: 
 
Related (n=60) 
Unrelated (n=20) 
# Evaluable Pts.    Total Width for 
the Exact 95% CI 
# Evaluable Pts.    Total Width for 
the Exact 95% CI 
60 
0.168 
20 
0.305 
29 
0.159 
19 
0.259 
28 
0.161 
18 
0.272 
27 
0.164 
17 
0.286 
26 
0.166 
16 
0.300 
25 
0.169 
15 
0.317 
 
9.3 
 Endpoint definitions  
 
1. GVHD: Grading of GVHD will be according to Appendix V and VI  
 
2. Engraftment: Primary graft failure (lack of engraftment) will be the lack of recovery of 
ANC to > 500 by Day +28 after transplant in the absence of immunological graft 
rejection. This endpoint is intended to monitor graft quality after manipulation.  
 
3. Graft rejection:  
 
a. Initial evidence for marrow recovery and engraftment with subsequent 
pancytopenia without another cause (i.e. Infection and/or drug therapy)  
 
b. Decrease in chimerism greater than 50% from highest level achieved  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 33 of 53 
 
4. Chimerism at Day 100: Chimerism (percentage of donor cells) will be assessed at Day 
100 post-transplant in evaluable (without disease progression) patients.  
 
5. Immune recovery: Immune recovery will be assessed by the time to CD4>100 and 
CD4>200 and the PHA to ≥10 and 30% of lower limit of normal.  
 
6. Severe toxicity: Severe toxicity will include Grade 3 unexpected (for transplant recipient) 
toxicity or any non-hematological grade 4 toxicity.  
 
7. Post-transplant infections: Infections that occur following the initiation of the conditioning 
regimen.  
 
8. CMV infection and disease: CMV infection will be reactivation detected by CMV PCR on 
plasma and CMV disease will be evidence of organ involvement (eg. CMV pneumonia, 
enteritis).  
 
9. Post-transplant lymphoproliferative disease (PTLD): PTLD will include patients that have 
clinical PTLD and those with EBV viral load > 1000 copies/mL.  
 
10. Transplant-related mortality (TRM): TRM will include death due to regimen-related 
toxicity or GVHD (usually all causes other than disease relapse or when infection is 
present at enrollment).  
 
11. Disease free survival (DFS) and overall survival (OS):  Disease-free survival will be 
survival without relapse, including molecular, cytogenetic, and morphological relapse. 
DFS and OS at one and two years will be determined from the day of transplant. DFS 
and OS at 5 years after transplant, pending availability of clinical follow-up data, will be 
determined from the day of transplant. 
 
12. Device (CliniMACS®) performance parameters: The parameters will include the purity of 
the CD34+ selected stem cell product, yield of CD34+ cells after selection, degree of 
CD3+ cell depletion after selection, and viability and sterility of the stem cell product after 
selection.  
 
9.4  
Plan of analysis 
  
The primary endpoint of the study is the incidence of severe (grade III/IV) acute GVHD 
occurring by Day +100 after transplant. This will be assessed for each cohort separately (related 
and unrelated donor). In our experience, almost all patients who develop acute GVHD after this 
type of transplant do so by Day +30.  
  
The following variables will be assessed as secondary endpoints with results reported for each 
study cohort. Frequencies of secondary endpoints will be summarized by proportions with 95% 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 34 of 53 
 
confidence intervals. Percentages will be summarized by mean values with 95% confidence 
intervals. Durations will be measured from the date of transplant:   
 
a) Engraftment: The incidence of primary graft failure and graft rejection will be determined.  
 
b) Chimerism at Day 100: The percentage of donor cells will be reported for all evaluable 
(without disease progression) patients.   
 
c) Immune recovery: The time to CD4 count >100 and > 200 and PHA≥10% and 30% will 
be calculated.   
 
d) Severe toxicities: The incidence of severe toxicities will be determined. This will include 
grade 3/4 stem cell product infusion-related toxicity.  
 
e) Post-transplant infections: Post-transplant infections will be described by incidence and 
type.  
 
f) CMV infection and disease: The incidence of each will be calculated.  
 
g) Post-transplant lymphoproliferative disease (PTLD): The incidence of PTLD will be 
calculated.  
 
h) Transplant-related mortality (TRM): The incidence of TRM will be calculated at Day 100 
after transplant and long term.  
 
i) Disease-free survival: The method of Craddock et al. (38) will be used to estimate DFS. 
The definition of DFS will parallel that of leukemia-free survival proposed by Craddock.   
 
j) Overall survival: Overall survival will be estimated using the Kaplan-Meier method (38).  
 
k) Device (CliniMACS®) performance parameters: The parameters will be summarized for 
all products that are processed and median and ranges will be determined.  
  
The endpoints will be monitored at least annually. The frequency will depend on the number 
of patients enrolled.   
  
9.5 
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 ≥ bk 
graft failures out of k resolved patients. Only points where stopping is possible are listed.  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 35 of 53 
 
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 
 
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.  
 
9.6  
Accrual  
 
Patients will be recruited by the PI and co-investigators. A total of 80 patients will be enrolled 
into the study: 30 patients into an alternative related donor cohort and 20 patients into an 
unrelated donor cohort.     
 
9.7  
Estimated duration of study    
 
The study will require 5 years for accrual and 2-5 additional years for follow-up studies. In 
addition, DFS and OS will be further analyzed using available data from standard clinical follow-
up at 3-years, 4-years, and 5-years post-transplant.  
 
9.8  
Replacement policy  
 
Enrolled patients who fail to complete the study will not be replaced. If a patient dies prior to 
evaluation for engraftment, the patient will not be included in analysis of the primary outcome 
(see Section 9.2).  
  
10.0  CRITERIA FOR EVALUATION  
 
10.1  Monitored outcomes    
 
The primary outcome will be severe (grade III/IV) acute graft-versus-host disease.    
 
Secondary outcomes will be engraftment (ANC>500 and >80% donor cells in blood), graft 
failure and graft rejection, immune recovery, infection, CMV infection and disease, EBV-related 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 36 of 53 
 
PTLD transplant-related toxicity and mortality, transplant-related mortality, grade 3/4 stem cell 
product infusion-related toxicity, relapse, DFS, OS.  
  
If a second PBSC transplant is offered as described in Section 7.4, post-second-transplant 
outcomes will not be included in study analyses. 
 
 
11.0 DATA AND SAFETY MONITORING PLAN   
 
11.1  Oversight and monitoring plan  
 
The UCSF-CCC Data Safety Monitoring Committee (DSMC) is responsible for monitoring data 
quality and patient safety for all UCSF-CCC institutional clinical studies. 
A summary of DSMC activities for this study includes: 
 
Review of subject data  
 
Review of all serious adverse events 
 
Minimum of a yearly audit 
 
Data Safety Monitoring Committee Contacts: 
 
DSMC Chair:   
Alan Venook, MD 
Phone: 
 
(415) 353-2745 
Email: 
 
venook@cc.ucsf.edu 
Box:  
 
1705 
 
DSMC Monitors: 
Box: 1297 
 
 11.2  Monitoring and reporting guidelines  
 
Investigators will conduct continuous review of data and patient safety at weekly study group or 
site committee meetings where the results of each patient’s treatment are discussed.  The 
discussion will be documented in the minutes and made available to the DSMC as requested.  
The discussion will include number of patients, significant toxicities as described in the protocol,  
and observed responses.  All grade 3-5 unexpected adverse events (AE) and serious adverse 
events (SAE) related to study participation will be reported to the FDA, device manufacturer, 
CHR, DSMC and Miltenyi Biotec, Inc.     
 
11.3 Review and oversight requirements  
 
11.3.1 Adverse event definition  
A clinical adverse event is any unfavorable or unintended sign, symptom or disease 
temporally associated with the use of an investigational product occurring in a research 
patient during treatment or post-treatment follow-up period, regardless of causality 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 37 of 53 
 
assessment.  This includes adverse clinical or laboratory findings, intercurrent illness, or an 
exacerbation or progression of a disease/condition present at baseline. 
An unexpected adverse event is any adverse experience where the specificity or severity of 
which is not consistent with the current investigator brochure; or, if an investigator brochure 
is not required or available, the specificity or severity of which is not consistent with the risk 
information described in the general investigational plan or elsewhere in the current 
application.  
 
11.3.2 Adverse event reporting    
Adverse events will be graded using the NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 5.0 found on the following website: 
http://ctep.cancer.gov/reporting/ctc.html  
  
Laboratory test value abnormalities will not be recorded as adverse events unless they are 
designated as serious, require treatment, or cause premature withdrawal (see Serious 
Adverse Events definition below).  
 
Specifications to the grading of acute and chronic GVHD are given in Appendix V and VI.   
 
Causality will be rated as definitely, probably, possibly, or unlikely related, or unrelated to 
the CD34+ CliniMACS® collection. The Principal Investigator is responsible for making an 
assessment of whether or not it is reasonable to suspect a causal relationship between the 
adverse event and the investigational product.  
 
11.3.3 Serious adverse events definition  
A serious adverse experience (SAE) is any adverse experience occurring at in a study 
patient that results in any of the following outcomes:  
1. Death  
2. Life-threatening (places the subject, in the view of the investigator, at immediate risk 
of death from the reaction as it occurred)  
3. Inpatient hospitalization or prolongs existing hospitalization  
4. Persistent or significant disability/incapacity (a substantial disruption of a person’s 
ability to conduct normal life functions)  
5. Birth defect/congenital anomaly  
6. Any important medical event that may not result in prior listed outcomes but, based 
upon appropriate medical judgment, may jeopardize the subject, and may require 
medical and surgical intervention to prevent one of the prior listed outcomes.  
 
11.3.4  
Special AE documentation guidelines for this protocol  
 
The following AEs will be recorded in the UCSF Helen Diller Family Comprehensive Cancer 
Center CTMS database: 
1. Any evidence of grade III/IV acute or extensive chronic GVHD using protocol appendices 
V and VI  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 38 of 53 
 
2. Toxicities of any organ Grade 3, 4, 5 using CTCAE version 4.0  
3. Hospitalization  
4. Relapse  
5. Any change in conditioning regimen outside of planned  
6. Any problems with CliniMACS device during cell separation or inability to achieve CD34+ 
cell and CD3+ cell target doses.  
7. Failure to engraft or graft rejection  
 
11.3.5 
 Adverse event reporting procedures  
 
Adverse Events (AEs) will be recorded on the OnCore eResearch database, all grade 3-5 
expected and unexpected AEs will be recorded and updated at each visit.    
 
Serious Adverse Event Reporting  
 
Serious Adverse Event reporting will be in accordance with the UCSF- Committee on 
Human Research Regulations and Code of Federal Regulation Title 21 Volume 5 Part 
312.32.  
 
UCSF CHR website for guidance in reporting serious adverse events 
http://www.research.ucsf.edu/chr/Guide/Adverse_Events_Guidelines.pdf 
 
FDA website for guidance in reporting serious adverse events  
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr+312.32 
  
MedWatch forms and information:  
http://www.fda/gov/medwatch/getforms.html 
  
CliniMACS® CD34+ Reagent System:  
Fax MedWatch form directly to the Safety Officer, Miltenyi Biotec Inc. (781-782-1920)  
  
Serious Adverse events will be reported on the MedWatch form when it meets the definition 
of expedited FDA reporting.   
  
If the SAE is death and determined to be possibly, probably, or definitely related to 
the investigational product or any research related procedure, the event must be 
reported via phone or in person to the UCSF DSMC chair or his designee within 24 
hours of the Principal Investigator’s awareness of the event. Written documentation 
of the verbal communication will be sent via e-mail to the DSMC Chair or his designee 
by the reporting investigator; a copy of the e-mail must be sent to the DSMC 
Administrator and DSMC Coordinator.    
  
All subjects/patients with serious adverse experiences must be followed up for outcome. 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 39 of 53 
 
11.3.6 
Review of adverse event rates  
 
If the study has an increase of unexpected or expected Adverse Events grade 3 or 4 above 
the rate reported in the protocol or investigational brochure or device documentation, this 
will be reported to the DSMC at the time of identifying the increased rate, and each quarterly 
report will indicate if the AE incidence is within the expected range.   If at any time the 
Investigator stops enrollment or stops the study due to safety issues, the DSMC Chair and 
Administrator must be notified within 24 business hours via email. The DSMC must receive 
a formal letter within 10 business days and the CHR must be notified.  
 
 
12.0 ETHICAL ASPECTS   
 
12.1  Regulatory considerations  
 
This study will be reviewed by the UCSF Comprehensive Cancer Center Protocol Review 
Committee, in addition to the UCSF Committee on Human Research (IRB, see below). In 
addition, the study protocol will be filed with the FDA as a new study under BB-IDE#8817, in 
addition to current study CC#01151.  As per FDA regulations, (21 CFR 812.150), annual reports 
will be submitted to the FDA within 60 days of the anniversary date that the IDE went into effect.  
 
 12.2 Institutional Review Board  
 
This protocol and the informed consent will be approved by the UCSF CHR (IRB).  The Principal 
Investigator is responsible for keeping the CHR advised of the progress of the study and of any 
changes made in the protocol prior to implementation.  The Principal Investigator will also keep 
the CHR informed of any significant adverse reactions, and any protocol exceptions or 
deviations.  Records of all study review and approval documents must be kept on file by the 
Principal Investigator and are subject to FDA inspection during or after completion of the study.  
The CHR will receive notification of the termination of the study.  
  
13.0  DATA FORMS AND SUBMISSION SCHEDULE   
 
Forms will be completed and documented for each patient. A research chart with completed 
paper case report forms and supporting documentation of reportable AEs and SAEs for all 
enrolled patients will be maintained in a locked cabinet in the BMT office.  Patient enrollment 
information and toxicity/reporting information will also be entered by the coordinator into the 
CTMS database.  
 
FORMS 
COMPLETION SCHEDULE 
Enrollment forms: 
 
Recipient Eligibility Checklist 
 
Donor Eligibility Checklist 
 
Recipient Baseline Information 
After the subject and donor have given informed 
consent and have been screened, and prior to 
recipient beginning conditioning therapy or donor 
receiving G-CSF prior to apheresis. 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 40 of 53 
 
 
Signed Consents 
Treatment forms: 
 
Transplant Data Form 
 
Cell Processing Report 
Completed transplant form one week after 
conditioning treatment. Within one week of the 
donor cell processing complete processing form. 
Follow-up forms: 
 
Acute GVHD Form 
 
Post-transplant Assessment Form 
Starting at 4 weeks after transplant, following 
protocol-specified time points (see Appendix IV) 
AE/SAE reporting: 
 
Study AE/SAE Form 
 
CHR AE/SAE reporting 
 
FDA Med Watch 3500a 
See section 11.3 for reportable AEs and SAEs 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 41 of 53 
 
APPENDICES  
 
APPENDIX I – Alternative conditioning regimens:  
 
Chemotherapy Alone Regimen (for patients who have a contraindication to TBI or non-
malignant disease (22).  
 
1. On day –7, administer melphalan 140mg/m2 IV over 30 minutes x 1 dose.    
 
2. On day –6, thiotepa 5mg/kg/dose will be administered intravenously in two doses (each 
dose of 4 hours) for a total dose of 10mg/kg/day.  
 
3. On days –6 through –2, fludarabine 40 mg/m2/d (infuse over 30 minutes) will be 
administered in the early morning hours on each of the five days (there will be at least 24 
hrs between the last dose of fludarabine and  the stem cell infusion).  Fludarabine dose 
will be reduced by 20% for creatinine clearance of < 70 ml/min/1.73m2.  
 
4. Rabbit ATG (thymoglobulin) 2.5 mg/kg on days –3 to day –1 infused over 6 hours.  
 
Reduced Intensity Regimen (for patients with decreased organ function) (31,32)  
 
1. On day -7 through day -3, fludarabine 30 mg/m2/d (infused over 30 minutes) will be 
administered in the early morning hours on each of the five days.  Fludarabine dose will 
be reduced by 20% for creatinine clearance of 30-70 ml/min/1.73m2.  
 
2. On day –3, thiotepa 5mg/kg/dose will be administered intravenously in two doses (each 
dose of 4 hours) for a total dose of 10mg/kg/day.  
 
3. On days –2 and –1, melphalan 60 mg/m2/d (infuse over 30 minutes) will be administered 
in the morning (there will be at least 24 hrs between the last dose of melphalan and  the 
stem cell infusion).  
 
4. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -7 infused over 4 hrs and then 2.5 mg/kg 
for four daily doses from day –6 to day –3 infused over 6 hours.  
 
 Regimen for chromosome breakage disorders like Fanconi anemia with aplasia (34)  
 
1. On day -7, 300 cGy TBI with thymic shielding per protocol. 
 
2. On day -6 to day -3, fludarabine 35 mg/m^2/day IV over 1 hour. Fludarabine dose will be 
reduced for decreased creatinine clear and and for children under 1 year of age per 
protocol. 
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 42 of 53 
 
3. Day -6 to day -3, cyclophosphamide 10mg/kg/day over 2 hours with mesna 
administration per protocol. 
 
4. On days -6 to -2, equine ATG 30mg/kg/day IV over 4 hours. 
  
 Regimen for chromosome breakage disorders like Fanconi anemia with MDS/AML (35)  
 
1. On day -7, a 450 cGy single dose of total body irradiation is delivered.  
 
2. On day -5 through day -2, cyclophosphamide 10mg/kg/dose will be administered 
intravenously (each dose to run over 1 hour) on each of the four days.  
 
3. On day -6 through day -2, fludarabine 30 mg/m2/d  (infused over 30 minutes) will be 
administered in the early morning hours on each of the five days (there will be at least 24 
hrs between the last dose of fludarabine and  the stem cell infusion).  Fludarabine dose 
will be reduced by 20% for creatinine clearance of < 70 ml/min/1.73m2. 
  
4. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -6 infused over 4 hrs and then 2.5 mg/kg 
for four daily doses from day -5 to day -2 infused over 6 hours 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 43 of 53 
 
Appendix II – DONOR (pre-administration through collection) 
 
Assessments/Tests 
w/in 30 days Day 1 Day 2 Day 3 
Day 4 Day 5 Day 6 
History & Physical 
X 
 
 
 
 
 
 
Serological Testing1 
X 
 
 
 
 
 
 
ABO Typing 
X 
 
 
 
 
 
 
LFTs, CBC/diff, platelet count, 
creatinine, CMP, Mg, Ca 
X 
 
 
 
 
 
 
ECG 
X 
 
 
 
 
 
 
CXR 
X 
 
 
 
 
 
 
HLA Typing 
X 
 
 
 
 
 
 
Pregnancy test2 
X 
 
 
 
 
 
 
G-CSF3 
 
X 
X 
X 
X 
 
X4 
Apheresis 
 
 
 
 
 
X 
X4 
CD34+ Selection 
 
 
 
 
 
 
X 
1 Serological testing for CMV,syphilis, HIV including p24, EBV, HSV, Hepatitis B C (must be done or repeated within 
30 days of donation), HTLV I/II.  
2 If female of child-bearing age.  
3 The donor will receive 4 days of G-CSF (Neupogen) administered subcutaneously (8 g/kg/dose) twice a day. On 
day 5 donor will undergo apheresis.   
4 Additional days (Day 5, 6, 7) of G-CSF may be given if the donor does not mobilize enough stem cells.  Then 
apheresis will occur on Day 8.    
  
  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 44 of 53 
 
APPENDIX III – RECIPIENT (pre-administration through transplant) 
 
Time Period 
→ 
Pre-
administration 
Cytoreductive Regimen 
w/in 30 
days 
w/in 2 
weeks 
Day 
-9 
Day 
-8 
Day 
-7 
Day 
-6 
Day 
-5 
Day 
-4 
Day 
-3 
Day 
-2 
Day 
-1 
Transplant 
(Day 0) 
PFTs 
X 
 
 
 
 
 
 
 
 
 
 
 
ECHO & ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
Creatinine 
clearance 
X 
 
 
 
 
 
 
 
 
 
 
 
Infectious disease 
testing1 
X 
 
 
 
 
 
 
 
 
 
 
 
ABO typing 
X 
 
 
 
 
 
 
 
 
 
 
 
Anti-HLA 
Antibodies 
X 
 
 
 
 
 
 
 
 
 
 
 
LFTs 
X 
 
 
 
 
 
 
 
 
 
 
 
Creatinine 
X 
 
 
 
 
 
 
 
 
 
 
 
Marrow/LP2 
 
X 
 
 
 
 
 
 
 
 
 
 
HLA Typing 
X 
 
 
 
 
 
 
 
 
 
 
 
History & Physical 
X 
 
 
 
 
 
 
 
 
 
 
 
CXR or CT (chest) 
X 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test3 
X 
 
 
 
 
 
 
 
 
 
 
 
TBI 
 
 
X 
X 
X 
 
 
 
 
 
 
 
Fludarabine4 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
Thiotepa 
 
 
 
 
 
X 
 
 
 
 
 
 
rATG 
(Thymoglobulin®)4 
 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
X 
 
Transplant 
 
 
 
 
 
 
 
 
 
 
 
X 
1Serological Testing for CMV, HIV (including p24), EBV, HSV, Hepatitis B, C, HTLV I/II. 
2Within 2 weeks of admission for patients with leukemia/MDS. 
3If female of childbearing age. 
4Alternative conditioning regimen may be used based on disease diagnosis as stated in   
Appendix I 
  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 45 of 53 
 
APPENDIX IV – RECIPIENT (follow-up schedule) 
 
 
Weeks Post-BMT 
Months Post-BMT 
Time Point 
0 1 
2 
3 
4 
8 
12 6 A 9 A 
12 B 15 A 18 A 21 A 24  
36 
48 
60 
Engraftment 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lymphocyte 
Phenotyping 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Mitogen 
StudiesC 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
CoombsD 
 
 
 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
Kidney function 
 
 
 
 
 
 
X 
X 
X 
X 
 
X 
 
X 
 
 
 
Liver function 
 
 
 
 
 
 
X 
X 
X 
X 
 
X 
 
X 
 
 
 
PFTs 
 
 
 
 
 
 
 
 
 
X 
 
 
 
X 
X 
X 
X 
ECHO 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
A Recommended follow-up timepoints per standard post-transplant care 
 
B Patients will be followed for outcomes related to the device including acute and chronic GVHD 
and engraftment at study-specified timepoints through Day +100 (Week 12). Subsequent follow-
up care is standard for all transplant patients, and data will be collected for OS, DFS, and other 
secondary endpoints through 2 years post-transplant to fulfill study objectives, and up to 5 years 
if standard follow-up data are available.   
 
C Direct and indirect Coombs test will be checked q 2 weeks beginning at 8 weeks post BMT 
until the CD4>200 and PHA≥30% of the lower limit of normal control because of the risk of 
autoimmune hemolytic anemia.  
 
D Mitogen studies can be discontinued once results are normal at physician discretion. 
 
  
  
  
  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 46 of 53 
 
APPENDIX V – Acute GVHD staging and grading for children** 
  
ORGAN 
STAGE 
DESCRIPTION 
SKIN 
1 
Maculopapular rash < 25% of 
BSA 
 
2 
25 – 50% of BSA 
 
3 
Generalized erythroderma 
 
4 
Desquamation and bullae 
 
LIVER 
1 
Bilirubin 2 – 3 mg/dL 
 
2 
Bilirubin 3.1 – 6 mg/dL 
 
3 
Bilirubin 6.1 – 15 mg/dL 
 
4 
Bilirubin > 15 mg/dL 
 
GUT 
1 
Diarrhea > 500 – 1000 ml/day  
(> 280 mL/m2 – 556 mL/m2/day) 
OR persistent UGI symptoms 
 
2 
Diarrhea > 1000 – 1500 ml/day  
(> 556 mL/m2 – 833 mL/m2/day) 
 
3 
Diarrhea > 1500 ml/day  
 (> 833 mL/m2/day) 
 
4 
Severe abdominal pain or ileus 
 
GRADE 
Skin 
Liver                    Gut 
I 
1-2 
   0                          0 
II 
3     and/or       1            and/or        1 
III 
-- 
 2-3          and/or      2-3 
IV 
4     and/or       4            and/or        4 
** Adapted from Przepiorka and Jacobsohn articles (41, 42).  
  
  
  
 

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 47 of 53 
 
APPENDIX VI - Chronic GVHD staging and grading for children**  
 
 LIMITED CHRONIC GVHD  
 
1. Localized skin involvement and/or  
 
2. Hepatic dysfunction due to chronic GVHD  
  
 
EXTENSIVE CHRONIC GVHD  
 
Either:  
 
1. Generalized skin involvement, or  
 
 
2. Localized skin involvement and/or hepatic dysfunction due to chronic GVHD  
 
  
 
Plus:  
 
a) Liver histology showing chronic aggressive hepatitis, bridging necrosis, or cirrhosis, or  
 
 
 
b) eye involvement (Schirmer test with < 5mm wetting), or  
 
 
 
c) involvement of minor salivary glands or oral mucosa demonstrated on labial biopsy, or  
 
 
d) involvement of any other target organ  
  
3.  Involvement of two target organs  
 ** Adapted from Glucksberg (43).  
  
  
  
  
  
  

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 48 of 53 
 
APPENDIX VII - Device information   
  
Brand: CliniMACS® CD34 Reagent System Information  
The CliniMACS® CD34 Reagent System is a medical device that is used in vitro to select and 
enrich specific cell populations. When using the CD34 Reagent, the system selects CD34+ cells 
from heterogeneous hematological  
  
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   
 
 University of California, San Francisco CliniMACS® cell sorter Device information:  
1) Serial number: 000288  
2) Catalogue number: 15101  
3) Part number:  44085  
  
  
Distributor:                             
 
 
Manufacturer:  
Corporate Headquarters     
 
 
 
Miltenyi Biotec GmbH, Clinical Products  
Miltenyi Biotec Inc.      
 
 
 
Friedrich-Ebert Strasse  
12740 Earhart Avenue    
 
 
 
Technologiepark H-13  
Auburn, CA 95602      
 
 
 
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 Protocol  v6.0       01-Jan-18                                                   Page 49 of 53 
 
APPENDIX VIII - SOP #206.04 Recipient Selection and Treatment Regimen for Pediatric 
Bone Marrow Transplant (PBMT). 
 
---Attached (7 pages)--- 
 
 
 
APPENDIX IX - SOP #206.04 Donor and Donor Source Selection for Pediatric Bone 
Marrow Transplant (PBMT). 
 
---Attached (11 pages)---

CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 50 of 53 
 
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CliniMACS Protocol  v6.0       01-Jan-18                                                   Page 51 of 53 
 
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