Conditions: Non-Malignant Hematologic Disorders
Keywords: T-cell B-cell CliniMACS System Melphalan Thiotepa Clofarabine Fludarabine 17-596
Allocation: NON_RANDOMIZED | Intervention model: PARALLEL
Enrollment: 1
Healthy volunteers: no

Brief summary:
The purpose of this study is to find out if removing a specific type of white blood cell (called alpha beta T-cell) that help make up the transplant donor's stem cells can improve results of blood stem cell transplant for the participant's disease.

Detailed description:

Arms:
arm 1: Melphalan 70 mg/m2/day x 2, Thiotepa 7.5 mg/kg/day x 2 and Clofarabine 20-30 mg/m2/day x 5. Patients will also receive rabbit anti-thymocyte globulin at 2.5 mg/kg/day x 3 doses prior to the start of conditioning. arm 2: Melphalan 70 mg/m2/day x 2, Thiotepa 7.5 mg/kg/day x 2 and Fludarabine 30 mg/m2/day x 5. Patients will also receive rabbit anti-thymocyte globulin at 2.5 mg/kg/day x 3 doses prior to the start of conditioning.

Interventions: intervention 1: Melphalan intervention 2: Thiotepa intervention 3: Clofarabine intervention 4: Fludarabine intervention 5: Anti-Thymocyte Globulin (Rabbit) (Thymoglobulin®) intervention 6: CliniMACS reagents
Intervention descriptions: intervention 1: Melphalan 70 mg/m2/day x 2 intervention 2: Thiotepa 7.5 mg/kg/day x 2 intervention 3: Clofarabine 20-30 mg/m2/day x 5 intervention 4: Fludarabine 30 mg/m2/day x 5 intervention 5: antithymocyte globulin (ATG) (rabbit ATG 2.5 mg/kg/day x 3 or equine ATG 15 mg/kg/day x 3 (or 40 mg/kg/day x 1 equine ATG if rabbit ATG is not tolerated) during pre-transplant conditioning to deplete host T-cells that could hamper engraftment. intervention 6: Products will then undergo TCR-αβ+ and CD-19 depletion using the CliniMACS.

Protocol document:
Prot_SAP_000.pdf: 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 1 of 55 
 
 
 
 
 
 
 
A Phase II Trial of Alpha Beta T-cell and CD19 B-cell depleted Peripheral Blood Stem Cell 
Transplantation using the CliniMACS System for patients with Non-Malignant Hematologic 
Disorders from matched or mismatched, related or unrelated donors 
 
PROTOCOL FACE PAGE FOR 
MSK THERAPEUTIC/DIAGNOSTIC  PROTOCOL 
 
 
Principal Investigator/Department: 
Maria Cancio, M.D. 
Pediatrics 
Co-Principal  Investigator(s)/Department: 
Farid Boulad, M.D. 
Miguel Angel Perales, M.D. 
Scott Avecilla, M.D., Ph.D. 
Pediatrics 
Medicine 
Laboratory Medicine 
Investigator(s)/Department: 
Kevin Curran, M.D. 
Jaap-Jap Boelens, M.D., Ph.D. 
Nancy Kernan, MD 
Susan Prockop, M.D. 
Andrew Kung, M.D., Ph.D. 
Andromachi Scaradavou, M.D. 
Richard O'Reilly, M.D. 
Barbara Spitzer, M.D. 
Juliet Barker, M.B.B.S. 
David J. Chung, M.D., Ph.D. 
Sergio Giralt, M.D. 
Boglarka Gyurkocza, M.D, 
Katharine Hsu, M.D., Ph.D. 
Ann Jakubowski, M.D., Ph.D. 
Parastoo Dahi, M.D. 
Heather Landau, M.D. 
Esperanza Papadopoulos, M.D. 
Doris Ponce, M.D. 
Craig Sauter, M.D. 
Brian Shaffer, M.D. 
Roni Tamari, M.D. 
Marcel R.M. van den Brink, M.D., Ph.D. 
James Young, M.D 
Alan M. Hanash, M.D. 
Michael Scordo, M.D. 
Christina Cho, M.D. 
Gunjan Shah, M.D. 
Cheryl Goss, M.D. 
Audrey Mauguen, Ph.D. 
Victoria Szenes, NP 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Pediatrics 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Medicine 
Laboratory Medicine 
Epidemiology and 
Biostatistics 
Pediatrics 


Page 2 of 55 
 
 
 
 
 
 
 
 
 
atrics 
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Memorial Sloan Kettering Cancer 
Center IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Melissa Varda, RN 
Pedi 
Jennifer Bieler, RN 
Nurs 
Adlynn Parado, RN 
Nurs 
Elaina Preston, MSHS, PA-C 
Medi 
Shani Irby, NP 
Nurs 
Nicole LeStrange, NP 
Nurs 
Tara Doga, NP 
Nurs 
Sheila Kenny, NP 
Nurs 
Chelsea Mintz, NP 
Nurs 
Emily Patterson, NP 
Nurs 
Courtney McElrath, NP 
Nurs 
Jason Batalha, NP 
Nurs 
Marci Andrejko, NP 
Nurs 
Stefanie Kelly, NP 
Nurs 
Melissa Cortes, NP 
Nurs 
Natasia Rodriguez, RN 
Nurs 
Caroline Carino, RN 
Nurs 
Abigail Cohen, NP 
Nurs 
Carter Hibbs, NP 
Nurs 
Meighan Palazzo, NP 
Nurs 
Megan Scott, NP 
Nurs 
Allison Tucker, NP 
Nurs 
Naomi Cazeau, NP 
Nurs 
Christina Kiss, NP 
Nurs 
Consenting  Professional(s)/Department: 
Maria Cancio, M.D. 
Pediatrics 
Jaap-Jap Boelens, M.D., Ph.D. 
Pediatrics 
Farid Boulad, M.D. 
Pediatrics 
Kevin Curran, M.D. 
Pediatrics 
Nancy Kernan, MD 
Pediatrics 
Andrew Kung, M.D., Ph.D. 
Pediatrics 
Richard O'Reilly, M.D. 
Pediatrics 
Susan Prockop, M.D. 
Pediatrics 
Andromachi Scaradavou, M.D. 
Pediatrics 
Barbara Spitzer, M.D. 
Medicine 
Juliet Barker, M.B.B.S. 
Medicine 
David J. Chung, M.D., Ph.D. 
Medicine 
Sergio Giralt, M.D. 
Medicine 
Boglarka Gyurkocza, M.D, 
Medicine 
Katharine Hsu, M.D., Ph.D. 
Medicine 
Ann Jakubowski, M.D., Ph.D. 
Medicine 
Robert Jenq, M.D. 
Medicine 
Parastoo Dahi, M.D. 
Medicine 
Heather Landau, M.D. 
Medicine 
Esperanza Papadopoulos, M.D 
Medicine 
Miguel-Angel  Perales, M.D. 
Medicine 
Doris Ponce, M.D. 
Medicine 


Page 3 of 55 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
 
 
 
 
 
Craig Sauter, M.D. 
Medicine 
Brian Shaffer, M.D. 
Medicine 
Roni Tamari, M.D. 
Medicine 
Marcel R.M. van den Brink, M.D., Ph.D 
Medicine 
James Young, M.D 
Medicine 
Alan M. Hanash, M.D. 
Medicine 
Michael Scordo, M.D. 
Medicine 
Christina Cho, M.D. 
Medicine 
Gunjan Shah, M.D. 
Medicine 
 
 
Please Note: A Consenting Professional must have completed the mandatory Human 
Subjects Education and Certification Program. 
 
 
 
 
 
Memorial Sloan Kettering Cancer Center 
1275 York Avenue 
New York, New York 10065 
OneMSK  Sites 
Manhattan 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 4 of 55 
 
 
 
Table of Contents 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA ....................................................................... 4 
2.0 
OBJECTIVES AND SCIENTIFIC AIMS ................................................................................ 6 
3.0 
BACKGROUND AND RATIONALE ..................................................................................... 6 
4.0 
OVERVIEW OF STUDY DESIGN/INTERVENTION ................................................................. 11 
4.1 
Design................................................................................................................................. 11 
4.2 
Intervention .............................................................................................................................. 11 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS ........................................................................... 12 
6.0 
CRITERIA FOR SUBJECT ELIGIBILITY ............................................................................... 17 
6.1 
Subject Inclusion Criteria ..................................................................................................... 17 
6.2 
Subject Exclusion Criteria ................................................................................................... 19 
7.0 
RECRUITMENT PLAN .................................................................................................................... 20 
8.0 
PRETREATMENT EVALUATION ...................................................................................... 20 
9.0 
TREATMENT/INTERVENTION PLAN ................................................................................ 21 
10.0 
EVALUATION DURING TREATMENT/INTERVENTION .................................................... 29 
11.0 
TOXICITIES/SIDE EFFECTS .................................................................................................... 31 
12.0 
CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT ......................... 40 
13.0 
CRITERIA FOR REMOVAL FROM STUDY ....................................................................... 42 
14.0 
BIOSTATISTICS ............................................................................................................... 42 
15.0 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 
44 
15.1 
Research Participant Registration ....................................................................................... 44 
15.2 
Randomization .................................................................................................................... 44 
16.0 
DAT A M ANAGEMENT ISSUES ........................................................................................ 44 
16.1 
Quality Assurance............................................................................................................... 45 
16.2 
Data and Safety Monitoring .................................................................................................... 45 
17.0 
PROTECTION OF HUMAN SUBJECTS ............................................................................ 46 
17.1 
Privacy ................................................................................................................................ 47 
17.2 
Serious Adverse Event (SAE) Reporting ................................................................................ 47 
18.0 
INFORMED CONSENT PROCEDURES................................................................................... 48 
19.0 
REFERENCES ....................................................................................................................... 49 
20.1 
APPENDICES .................................................................................................................. 54 
• 
1.0 
PROTOCOL SUMMARY AND/OR SCHEMA 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 5 of 55 
 
 
 
This is a Phase II trial for the use of 
- 
Two specific chemotherapy-only cytoreductive regimens including 
Melphalan Thiotepa + [Clofarabine or  Fludarabine], followed by 
- 
GCSF-mobilized peripheral blood stem cell (PBSC) grafts from HLA-matched or HLA- 
mismatched, related or unrelated donors 
- 
Using a novel T-cell depletion method with TCR αβ T-cell depletion and CD19 B-cell 
depletion using the Miltenyi CliniMACS device 
- 
In patients with non-malignant hematologic disorders 
 
The primary objective of the trial is to assess two-year overall survival. 
 
The secondary objectives of the trial are to assess: 
 
1. Neutrophil  engraftment following  T- and B- cell depleted  grafts in patients  with non- 
malignant hematologic disorders using a chemotherapy-only cytoreduction. 
2. The rate of grade III-IV  acute and chronic GvHD  following  this novel  T- and B- cell 
depleted approach. 
3. Graft composition CD34, T-α/β, T-γδ, B, and NK cells post T-cell and B-cell depletion. 
4. The immune reconstitution following α/β T-cell depleted stem cell transplant as measured 
at approximately 1, 2, 3, 6, and 12 months. 
5. The rate and specifics of the immune reconstitution post transplant compared to those 
achieved in CD34 positively selected grafts as part of protocol 10-050. 
6. The CD34+ cell dose using this negative selection method with TCR alpha beta T-cell 
and CD19 B-cell depletion method and compare to the cell dose achieved using a 
positive selection method in CD34 positive grafts as part of protocol 10-050. 
7. The   incidence  of  post-transplant   infectious   complications,  including   CMV,   EBV, 
adenovirus and HHV6  (viremia, organ involvement and infection). 
8. The incidence of transplant-related mortality and of disease free survival post transplant. 
 
For this trial, patients will be assigned to receive one of two conditioning regimens, based 
on their disease, disease severity, organ status 
and history of red blood cell 
alloimmunization. Both regimens have demonstrated safety and efficacy in facilitating 
engraftment of CD34+ selected allografts in MSKCC protocol 10-050. 
 
The two conditioning regimens are: 
 
Regimen A:  Melphalan/Thiotepa/Clofarabine 
Melphalan 70 mg/m2/day x 2, Thiotepa 7.5 mg/kg/day x 2 and Clofarabine 20-30 mg/m2/day 
x 5. 
 
Regimen B: Melphalan/Thiotepa/  Fludarabine 
Melphalan 70 mg/m2/day x 2, Thiotepa 7.5 mg/kg/day x 2 and Fludarabine 30 mg/m2/day 
x 5. 
 
Patients will also receive rabbit anti-thymocyte globulin at 2.5 mg/kg/day x 3 doses prior to 
the start of conditioning. If a severe adverse reaction to rabbit ATG occurs, the patient will 
receive horse ATG for subsequent doses. If hATG is not tolerated, alemtuzumab will be 
requested through the Campath Distribution Program. 
 
On day -1, patients will receive rituximab 200mg/m2 for in vivo donor and recipient B-cell 
depletion to reduce the risk of Epstein-Barr virus (EBV)-related posttransplantation 
lymphoproliferative  disease (PTLD). 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 6 of 55 
 
 
 
 
Approximately twenty-four to forty-eight hours after completion of cytoreduction, patients 
will receive an αβ T-cell and CD-19 depleted, GCSF-mobilized PBSC transplant. 
 
Donors for patients enrolled on this protocol will be 
HLA matched related 
HLA matched unrelated 
HLA mismatched related 
HLA mismatched unrelated 
 
Stratification will be by donor. The sample size is as follows: 
Donor Type 1 (Related or Unrelated ≥ 7/8 HLA-antigen matched): 30 patients 
Donor Type 2 (Related donors 4-6/8 HLA-antigen matched): 30 patients 
 
 
2.1 
OBJECTIVES AND SCIENTIFIC AIMS 
 
Primary 
The primary objective of the trial is to assess: Two-year overall survival. 
 
Secondary: 
The secondary objectives of the trial are to assess: 
 
1. Neutrophil  engraftment  following  T-  and  B-  cell  depleted  grafts  in  patients  with  non- 
malignant hematologic or immune disorders using a chemotherapy-only cytoreduction. 
2. The rate of grade III-IV  acute and chronic GvHD following this novel T- and B- cell depleted 
approach. 
3. Graft composition CD34, T-α/β, T-γδ, B, and NK cells post T-cell and B-cell depletion. 
4. The immune reconstitution following α/β T-cell depleted stem cell transplant as measured 
at approximately 1, 2, 3, 6, and 12 months. 
5. The rate and specifics of the immune reconstitution post transplant compared to those 
achieved in CD34 positively selected grafts as part of protocol 10-050. 
6. The CD34+ cell dose using this Negative Selection method with TCR alpha beta T-cell 
and CD19 B-cell depletion method and compare to the cell dose achieved  using a 
Positive Selection method in CD34 positive grafts as part of protocol 10-050. 
7. The incidence of post-transplant infectious complications, including CMV, EBV, adenovirus 
and HHV6 (viremia, organ involvement and infection). 
8. The incidence of transplant-related mortality and of disease free survival post transplant. 
 
 
3.0 
BACKGROUND AND RATIONALE 
 
Allogeneic hematopoietic cell transplantation (alloHCT) has long been recognized as a cure 
for many life threatening non-malignant hematologic and immunologic disorders such as 
sickle cell disease, thalassemia major, inherited and acquired bone marrow failure 
syndromes, and immunodeficiency disorders. While HCT offers the only curative option for 
patients resistant to standard therapies, it may be associated with acute and chronic 
morbidity and mortality secondary to both the cytoreductive regimens and transplant related 
complications, primarily GVHD. AlloHCT from donors other than HLA-matched siblings has 
been performed primarily using unmodified grafts, while T-cell depleted transplants have 
traditionally required conditioning regimens that include total body irradiation (TBI), 
especially for recipients of related mismatched or unrelated donors, due to the higher risk 
of graft rejection in multiply transfused patients. However, this approach leads to a high risk 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 7 of 55 
 
 
 
for GVHD and to the late effects of TBI. In patients  with non-malignant hematological 
disorders, reducing toxicity of cytoreduction using chemotherapy only and reducing the 
chance of GVHD by using T-cell depletion are fundamental in reducing acute and late 
complications. 
 
Only 25% of patients who are candidates for allogeneic transplantation have matched 
related donors. In addition, for patients with genetic disorders, potential matched related 
donors may not be suitable as they may be affected by the genetic disease. For patients 
lacking a matched related donor, the likelihood of finding an unrelated donor depends on 
the frequency of the patient’s HLA haplotype as determined by racial and ethnic 
background. 
 
Based on recent data from our institution, 20% of patients had related stem cell donors, 
while 80% of patients required transplants from unrelated or mismatched related donors. 
Alternative donor transplantation, using haploidentical related donors is being increasingly 
used for many individuals, particularly those belonging to minority groups. However, 
advances in alternative donor transplantation have been hindered by GVHD and graft 
rejection, mediated by donor and recipient alloreactive T-cell response, respectively.(1) 
 
The risk of acute GVHD following allogeneic from HLA-matched siblings is 20-60% despite 
the use of immunosuppressive agents like cyclosporine A (CSA), tacrolimus (FK506), 
methotrexate (MTX), antithymocyte globulin (ATG) and corticosteroids, alone or in 
combination (2-5). Grades II-IV acute GVHD is associated with an increased risk of 
transplant-related mortality (21, 22). Mortality rates among patients who develop GVHD can 
be as high as 75% when that disease is unresponsive to therapy (22). Higher rates of acute 
and chronic GVHD are observed following matched unrelated (6) and unmanipulated 
haploidentical transplants (>30% and >70% respectively) (7, 8). 
 
Over the past twenty years, several techniques for T-cell depletion (TCD) of donor grafts 
have been introduced and evaluated for their capacity to prevent acute and chronic GVHD. 
These include techniques involving physical adsorption of T-cells to protein ligands such as 
lectins, elutriation, and immunoadsorption or immuno deletion with T-cell or lymphocyte- 
specific monoclonal antibodies (9-13). Comparative analyses have demonstrated that these 
techniques vary widely in their capacity to deplete T-cells (1.5-3.5 log10 depletion) (14). The 
relationship between the T-cell dose transplanted and the risk of acute GVHD is complex 
and varies depending upon the degree of MHC-compatibility, graft source (bone marrow 
versus peripheral blood), type of GVHD prophylaxis (pharmacological versus T-cell 
depletion), method of T-cell depletion, conditioning regimen, use of in vivo depleting 
antibodies (15-18), as well as patient and donor characteristics including underlying 
disease, age, gender and parity (19-21). Our previous studies in more than 200 patients 
suggested that the threshold dose of T-cells required to induce grade II-IV acute GVHD after 
HLA-matched sibling transplants is 105 clonable T-cells/kg (31). A 2.8-3.0 log10 reduction in 
marrow grafts or a 4.0-4.5 log10 reduction in peripheral blood stem cell (PBSC) grafts is 
required if this threshold is not to be exceeded. This likely explains the variable reductions 
in GVHD and inconsistent reductions in chronic GVHD observed with several techniques 
despite the concomitant use of CSA and MTX prophylaxis. 
 
Two methods of T-cell depletion are effective in reducing or preventing acute GVHD in both 
HLA-matched and HLA-disparate transplant recipients without co-administration of CSA 
and/or MTX. The first is: T-cell depletion of a marrow allograft by soybean lectin 
agglutination and E-rosette depletion (SBA), an approach reported by Memorial Sloan- 
Kettering Cancer Center (MSKCC) (9, 22) and the University of Perugia (23). This method 
is associated with a 0-5% incidence of acute GVHD in HLA-matched transplant recipients 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 8 of 55 
 
 
 
and a 0-8% incidence in HLA haplotype transplant recipients. The second is: depletion of T- 
cells by positive selection of CD34+ hematopoietic cells from GCSF mobilized peripheral 
blood and transplantation of these CD34+ cell-enriched, T-cell depleted cell fractions (24, 
25). 
 
In the CliniMACS system (Miltenyi Biotec) system, stem cells (GCSF mobilized PBSC) are 
introduced into a closed device, which treats the cells with anti CD34 MoAB coated 
paramagnetic beads. The CD34+ progenitor cells are then separated from other cells by 
passage through an electromagnetic field, and then washed and eluted. In the large Perugia 
series, recipients of HLA haplotype disparate CD34+ PBSC isolated on the CliniMACS 
device had only a 10% probability of developing acute or chronic GVHD (26). In a 
multicenter trial conducted by the BMT Clinical Trials Network under an FDA IND, for which 
Dr. Richard O’Reilly was principal investigator, this method yielded a progenitor-enriched 
cell fraction that provided doses of progenitor cells ranging from 2.4 – 31.3 (med 7.9) x 106 
CD34+ cells kg and T-cell doses of 1.1-84.9 (median 6.6) x 103 CD3+ cells/kg. HLA matched 
related HCT fractionated by this approach were administered after cytoreduction with our 
protocol of hyperfractionated TBI, thiotepa and cyclophosphamide in patients with 
hematologic malignant disorders. These transplants provided consistent engraftment (1 
late graft failure in 44 patients transplanted), and were associated with incidences of 20.5% 
grade 2-4 acute GVHD and 7.6% extensive chronic GVHD. 
 
We retrospectively analyzed data on 18 patients affected by non-malignant hematologic 
disorders lacking an HLA-identical sibling donor who underwent an alternative donor 
allogeneic HCT at our institution between 2005 and 2013. Of those, 50% had received 
immunosuppressive treatments, 72% had a history of infections, and 56% were transfusion- 
dependent at the time of transplant. Cytoreduction included a combination of three of five 
agents: fludarabine, melphalan, thiotepa, busulfan and cyclophosphamide. Grafts were T- 
cell depleted. All evaluable patients engrafted; one developed secondary graft rejection. 
Five patients died of transplant complications. The cumulative incidence of GvHD was 6%. 
No patient had recurrence of their non-malignant disease. 5-year overall survival was 77%. 
Age at transplant <6 years was strongly associated with better survival. Based on these 
results, transplant with chemotherapy-only cytoreductive regimens and T-cell depleted stem 
cell transplants could be recommended for patients with high risk non-malignant 
hematologic disorders (NMHD) especially at a younger age. (27) Although extensive T- 
lymphocyte depletion from the grafts lowers the rate of GVHD, other immune cells, which 
may be beneficial to engraftment and innate immunity, are also removed from the graft 
during CD34 selection, thus contribute to risk of graft rejection and a delayed immune 
reconstitution (28). 
 
In the era of alternative donor transplantation with T-cell depleted grafts, graft rejection is a 
major obstacle following allogeneic HCT. A major cause of graft failure is the recipient’s 
immune response against donor immuno hematopoietic cells. The risk of rejection or graft 
failure is higher in HLA-mismatched or unrelated donor grafts, T-cell depleted transplants 
and major ABO blood group incompatibility (29, 30). In addition, multiply transfused patients 
with non-malignant hematologic disorders such as sickle cell disease, thalassemia major 
and severe aplastic anemia, have an increased risk of graft rejection (5-60%), since their 
memory T-cells recognize major or minor HLA antigens on donor grafts(31, 32). 
 
To tackle the barrier to engraftment, the use of megadose of GCSF-mobilized peripheral 
blood stem cells was first reported by the group of Perugia with rate of engraftment over 
90% and the incidence of both grade II-IV acute and chronic GVHD less than 10% (33). In 
addition, aggressive inhibition of the host immune system using either myeloablative or 
immunoablative drugs as a conditioning  regimen was used to augment the likelihood  of 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 9 of 55 
 
 
 
engraftment. For instance, the addition of hydroxyurea, azathioprine and fludarabine to 
busulfan/cyclophosphamide based regimen reduced the risk of rejection from 30% to 8% in 
high risk thalassemia major patients receiving HLA matched related stem cell transplant (34, 
35). 
 
Recently, several studies in T-cell depleted grafts have shown graft failure rates ranging 
from 0 to 17% (36-40) in haploidentical HCT by using fludarabine/clofarabine, thiotepa and 
melphalan or fludarabine-based regimens. Whereas all patients receiving matched related 
or unrelated donor stem cells after fludarabine, busulfan plus ATG conditioning achieved 
primary engraftment (41-43). 
 
Despite downward trends in GVHD achieved by effective reduction of T cells in the graft , 
improved engraftment using a megadose of stem cells after GCSF mobilization, and 
aggressive inhibition of the host immunesystem by immunoablative conditioning, transplant 
related mortality rate is still high, ranging between 5-37% (44-47) in pediatric studies. This 
unacceptably high mortality is attributed to delayed immune reconstitution. (24) 
 
However, there is significant evidence of lower rate of mortality secondary to infection in 
patients achieving faster T- and B-cell lymphocyte counts compared with those did not 
during the first 6 months or even during the whole first year post SCT (48). 
 
It is known that, γδ T cells (also termed “innate-like” T cells) constitute 1-10% of peripheral 
blood lymphocytes and have several functions, including response against intracellular and 
extracellular pathogens. These cells can recognize their targets in an MHC-independent 
manner through activating receptors NKG2D, TLRs, DNAM-1 and exhibit a pre-activate 
phenotype allowing rapid cytokine production suchas IFN- γ and TNF-α and strong cytotoxic 
response upon activation (36). In contrast, αβ T cells are restricted by MHC molecules, 
which lead to increased risk of GVHD (37). Hence, a new technique of graft manipulation to 
hasten immune recovery relies on removal of alpha beta T lymphocytes by using a 
biotinylated anti-TcR αβ antibody followed by an anti-biotin antibody conjugated to magnetic 
microbeads.  This approach preserves γδ+ T cells, natural killer (NK) cells and other cells 
in the graft. In addition to TCRab depletion, CD19+ B-lymphocyte depletion is employed for 
prevention of post-transplant EBV-associated lymphoproliferative disease (49). Over the 
past several years, this approach has been widely and successfully used in hematopoietic 
stem cell transplantation for both hematological malignant and nonmalignant disorders. 
 
The outcomeof the first clinical experience in pediatrics at the Children’s University Hospital 
Tubingen with TcRαβ/CD19 depleted haploidentical transplantation with melphalan, 
thiotepa, fludarabine or clofarabine preparative regimens, showed a rapid  donor 
engraftment and rapid T-cell immune reconstitution at day +28 post-transplant without 
transplant related mortality (50). Similarly, data published by Bertaina et al. shows sustained 
engraftment of donor hematopoiesis without GVHD occurrence in most patients and 2-year 
probability of disease-free survival 91.1%.(38) Several series (see appended tables) 
demonstrated that this method has superior outcomes with accelerated immune recovery in 
natural killer (NK)-, B- and T-cell subsets when be compared with positive CD34+ selected 
stem cells transplant. (38, 49-51) 
 
The recovery of CD 56+ NK cells, the first lymphoid cells to emerge following HCT (48), was 
detectable in the first week post HCT and significantly increased at day +14 when compared 
to a historical control. In addition, B cell recovery started at day +30 and normalized at day 
+127. At day +30, TcRαβ/CD19 depleted recipients had significantly higher CD3+ and 
CD4+ cell numbers than control group (49, 51). For subsets of T-cells, γδ+ T cells expanded 
faster than TcRαβ lymphocytes in the first 30 days post HCT; however, αβ T-cells were 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
Page 10 of 55 
 
 
 
predominant by day +90 (40). Similarly, Bertaina et al have reported the results of 
haploidentical related HCT in nonmalignant disorders fractionated by this approach 
administered after cytoreduction with a fludarabine base regimen.  In their experience, γδ+ 
T cells recovered speedily followed by gradual increase of αβ T lymphocytes in a year, with 
9.3% incidence of TRM (38). 
 
Herein we propose to evaluate the potential of HCT grafts selectively depleted of αβ+ T- 
cells and CD19+ B-cells by the CliniMACS system, when administered after each of two 
cytoreductive regimens to maximize sustained engraftment among recipients at high risk of 
graft rejection, particularly those with history of multiple transfusions, prevent or abrogate 
acute and chronic forms of GVHD and to accelerate immune reconstitution to reduce 
infection related morbidity and decrease treatment related mortality. 
 
Published studies using TCR- α/β depletion include an additional depletion of CD19+ B- 
lymphocytes prior to allograft infusion. The purpose of this additional step is to reduce the 
reservoir for EBV from the donor allograft and in turn minimize the risk for donor derived 
EBV- post transplant lymphoproliferative disorder (PTLD). Rituximab 200 mg/m2 has also 
been used for in vivo donor and recipient B-cell depletion to reduce as muchas possible the 
risk of Epstein-Barr virus (EBV)-related posttransplantation lymphoproliferative disease 
(PTLD) (37). Infusion of low dose peritransplant rituximab has demonstrated efficacy to 
reduce EBV viremia and PTLD without impacting long term immune reconstitution (52). 
 
At our Center, we have used the two regimens melphalan/thiotepa/clofarabine and 
melphalan/thiotepa/fludarabine followed by CD34 selected grafts in patients receiving HCT 
from alternative donors. Two patients rejected their grafts following melphalan thiotepa 
clofarabine, while 4 patients suffered recovery of host chimerism post transplant. Moreover, 
several reports of TCR- α/β and CD19 depleted transplants(38, 53) have described a rate 
of primary graft failure approaching 17%. In our CD34+ protocol and in the alpha beta 
negative reports, conditioning included thiotepa 10 mg/kg divided in two doses. We have 
decided to increase the dose of thiotepa for our new protocol from 10 mg/Kg to 15 mg/Kg 
for increased immunosuppression and myelosuppression and for the improvement of 
engraftment  and chimerism. 
 
The combination of melphalan 70 mg/m2/day x 2, thiotepa 5 mg/kg/day x 2 (or 10 mg/kg/day 
x 1) and clofarabine 20-30 mg/m2/day x 5 has been successfully used to treat 64 patients 
at our center with unmodified and T-cell depleted grafts from HLA-matched, mismatched 
related, matched unrelated and mismatched unrelated donors.  Engraftment occurred in 59 
of 61 evaluable patients. Grade II-IV acute GvHD occurred in 4/20 (20%) evaluable patients 
in the TCD group. With a median follow up of 15.4 months for the TCD group, the overall 
survival and disease free survival rates were 64.1% and 60.7% (54). This cytoreductive 
regimen has shown promise as radiation sparing conditioning for patients with acute 
leukemias, including in the relapsed setting (55). 
 
The second combination of melphalan 70 mg/m2/day x 2, thiotepa 5 mg/kg/day x 2 (or 10 
mg/kg/day x 1) and fludarabine 30mg/m2/day x 5, was employed as preparative conditioning 
for seven CD34+ T cell depleted Isolex transplants and, under single patient use INDs, 3 T 
cell depleted CD34+ (CliniMACS) PBSC transplants to treat 10 patients: refractory leukemia 
(n=2), aplastic anemia, (n=3), PNH or refractory autoimmune cytopenia (n=2), lethal 
congenital immune deficiencies (n=3). The regimen induced only moderate GI toxicity. 
However, in each of 9 evaluable patients, we observed rapid reconstitution with full donor 
chimerism and without GVHD. This regimen is proposed for use in (1) patients heavily 
sensitized by transfusions for whom, melphalan and clofarabine alone may not be adequate 
to ensure engraftment, (2) patients who, because of prior hepatic and/or pulmonary injury 


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are at high risk of severe and life threatening toxicity (3) patients with life threatening 
nonmalignant acquired and genetic disorders of hematopoiesis and immunity (27). 
 
Summary of Rationale for the Proposed Study: 
 
In the current study we propose to conduct HLA matched or mismatched unrelated and 
mismatched related donor allotransplant using TCR-α/β+ cell depletion based GVHD 
prophylaxis. The TCR-α/β+ cell depletion platform has validated results at other centers 
using HLA matched donors and in related HLA-haploidentical donors with an acceptable 
rate of acute GVHD and excellent engraftment and long-term disease control (37). This 
platform represents a logical extension of the CD34+ selection based GVHD prophylaxis for 
mismatched unrelated donors in that it allows for innate effector cells such as NK and TCR- 
γδ+ T-cells to participate in pathogen control early after allo HCT. The proposed study 
addresses a shortcoming in the use of CD34+ selection for HLA mismatched donors in that 
diminished immune reconstitution is a major contributor to early transplant related mortality. 
Immune reconstitution in participants of the proposed study will be compared to those 
undergoing HLA mismatched allotransplant with both unmodified and CD34+ selected 
grafts. 
 
 
 
 
 
 
 
 
4.0 
OVERVIEW OF STUDY DESIGN/INTERVENTION 
 
o 4.1 
Design 
 
This phase 2 trial is designed to assess the efficacy of αβ T-cell and B-cell depleted 
peripheral blood stem cell transplants for patients with non-malignant hematologic 
disorders. The study population is divided into two groups defined by donor type: 1) donors 
who are ≥ 7/8 HLA-antigen matched and 2) related donors who are 4-6/8 HLA-antigen 
matched. A maximum of 30 patients in each group is planned for accrual onto the study. 
The primary endpoint of the study is two-year overall survival. It is anticipated that accrual 
will last three years and each patient will be followed for a minimum of two years. The data 
analysis and data monitoring will be conducted separately in each donor group. 
4.2 
Intervention 
 
Patients with non-malignant hematological disorders who fulfill eligibility requirements and 
consent to treatment, will receive one of two conditioning regimens, based on their disease 
and clinical parameters including age, prior treatment history, or the presence of 
comorbidities that could increase risk of graft failure or severe toxicity or in the post 
transplant period. 
 
The two cytoreduction regimens to be evaluated are: 


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A. Melphalan/Thiotepa/Clofarabine 
a. Melphalan (70 mg/m2/day x 2) 
b. Thiotepa (7.5 mg/kg/day x 2 or 15 mg/kg/day x1) 
c. Clofarabine (20-30 mg/m2/ day x 5) 
 
B. Melphalan/Thiotepa/Fludarabine 
 
a. Melphalan (70 mg/m2/day x 2) 
b. Thiotepa (7.5 mg/kg/day x 2 or 15 mg/kg/day x1) 
c. Fludarabine (30 mg/m2/ day x 5 ) 
ATG will be given to all transplant recipients. 
ATG dosing will be as follows: antithymocyte globulin (ATG) rabbit ATG 2.5 mg/kg/day x 3 
or equine ATG 15 mg/kg/day x 3 (or 40 mg/kg/day x 1 equine ATG if rabbit ATG is not 
tolerated) during pre-transplant conditioning to deplete host T-cells that could hamper 
engraftment. 
 
Donors will be evaluated according to standard National Marrow Donor Program criteria. 
Donors will donate a standard, G-CSF mobilized peripheral blood hematopoietic progenitor 
cell (HPC (A)) product that will subsequently be transferred to the MSKCC Cell Therapy 
Laboratory for processing. HPC (A) products will then undergo TCR-αβ+ and CD-19 
depletion using the CliniMACS reagents according to the manufacturer’s guidelines. Excess 
starting product(s) not undergoing TCR-αβ+ and CD-19 depletion may be processed for 
CD34 enrichment when applicable. 
Following preparative cytoreduction, all patients will receive HPC (A) depleted of α/β T-cells 
and CD-19 B-cells. The targeted dose of progenitor cells is > 5 x 106 CD34+ cells/kg; the 
targeted dose of relevant T-cells TCR-alpha beta limited to < 10 x 104 CD3+ cells/kg for 
matched related donor and < 1.0 x 104 CD3+ cells/kg for alternative donor; and TCR- gamma 
delta >1.0 x 106. 
Following transplantation, the patients will receive transfusions and supportive care 
according to the guideline of the Transplant Service in Pediatrics and Medicine. These 
guidelines will also be invoked for prophylaxis and treatment of infectious complications. 
 
The patients will then be evaluated sequentially for toxicities, engraftment, acute and/or 
chronic GVHD, the kinetics and quality of hematopoietic and immune reconstitution, and 
both relapse-free survival and overall survival. 
 
5.0 
THERAPEUTIC/DIAGNOSTIC AGENTS 
 
5.1 Thiotepa, melphalan, fludarabine and clofarabine are standard cytoreductive agents 
that will be employed in the two regimens as detailed in the treatment plan. ATG will be 
administered for further immunoablation. 
 
 
5.1.2. Thiotepa (Thioplex® ) 


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Formulation: 15 mg vial lyophilized powder; must be diluted prior to infusion. 
 
Reconstitution: Add 1.5 mL of sterile water for injection (SWFI) to 15mg vial to 
yield 10mg/mL.  Solutions which are grossly opaque or contain a precipitate, 
should not be used.  In order to eliminate haze, solutions should be filtered through 
a 0.22-micron filter prior to administration. 
 
Storage and Stability: 
1. Store vials in refrigerator and protect from light. 
2. Reconstituted solutions are stable for 24 hours under refrigeration and room 
temperature. 
3. Reconstituted solutions further diluted in NS (0.9% sodium chloride) at 
concentrations of 1mg/mL, 2mg/mL and 5 mg/mL are stable for up to 24 hours 
at room temperature and 48 hours under refrigeration. 
4. Diluted solutions of 0.5 mg/mL in NS should be used immediately after 
preparation. 
 
Preparation: 
 
1. Standard IV fluid: NS 
2. Final concentration range up to: 5mg/mL. 
 
Clinical Considerations: 
 
Hydration: NA 
 
Emetic Potential: High 
 
Incompatibilities: Cisplatin, filgrastim (G-CSF), vinorelbine. 
 
 
5.1.3 Melphalan (Evomela®) 
 
a. Source and Pharmacology:  Supplier: Spectrum Pharmaceuticals Inc,. A 
derivative of nitrogen mustard, an analog of mustard gas.  It is a polyfunctional 
alkylating agent that causes miscoding, cross-linkage of DNA, and single-strand 
breakage of DNA.  It inhibits cellular glycolysis, respiration, and protein 
synthesis.  It is cell cycle-phase non-specific. 
 
b. Formulation and Stability: A lyophilized powder of 50 mg melphalan and 20 
mg povidone per vial.  Also provided is 10 mL of sterile diluent for use in 
reconstituting the product and a 0.45 micron filter. 
 
c. Solution Preparation: 50 mg/vial: Reconstitute by rapidly injecting 8.6 mL of 
the supplied diluent into the vial to yield a final concentration of 5 mg/mL. Shake 
vigorously until the solution is clear. 


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d. Storage and Stability:  The intact solution should be stored at room 
temperature (15-30°C) protected from light.  Do not refrigerate reconstituted 
product. Stability of prepared infusions varies by final concentration. (See MSK 
Pediatric Chemotherapy Guidelines). 
 
Concentration of Solution (mg/mL) 
Duration of Stability at Room 
Temperature (hours) 
0.45 
4 
1 
7 
2 
11 
5 
25 
 
 
e. Administration:   Intravenous, over 30 minutes. 
 
 
 
5.1.4 
  Fludarabine (FLUDARA® ) 
 
a. Source and Pharmacology: Supplier: Berlex Laboratories, Inc. FLUDARA 
FOR INJECTION contains fludarabine phosphate, a fluorinated nucleotide 
analog of the antiviral agent vidarabine, 9-ß-D-arabinofuranosyladenine (ara-A) 
that is relatively resistant to deamination by adenosine deaminase. The 
chemical name for fludarabine phosphate is 9H-Purin-6-amine, 2-fluoro-9-(5-O- 
phosphono-ß-D-arabinofuranosyl). Fludarabine  phosphate is rapidly 
dephosphorylated to 2-fluoro-ara-A and then phosphorylated intracellularly by 
deoxycytidine kinase to the active triphosphate, 2-fluoro-ara-ATP. This 
metabolite appears to act by inhibiting DNA polymerase alpha, ribonucleotide 
reductase and DNA primase, thus inhibiting DNA synthesis. The mechanism of 
action of this antimetabolite is not completely characterized and may be multi- 
faceted. 
 
b. Formulation and Stability: Each vial of sterile lyophilized solid cake contains 
50 mg of the active ingredient fludarabine phosphate, 50 mg of mannitol, and 
sodium hydroxide to adjust pH to 7.7. The pH range for the final product is 7.2- 
8.2. Reconstitution with 2 mL of SWFI results in a solution containing 25 mg/mL 
of fludarabine phosphate intended for intravenous administration. FLUDARA 
FOR INJECTION is supplied in a clear glass single dose vial (6 mL capacity) 
and packaged in a single dose vial carton in a shelf pack of five 
 
c. Solution Preparation: FLUDARA should be prepared for parenteral use by 
aseptically adding SWFI. When reconstituted with 2 mL of SWFI, the solid cake 
should fully dissolve in 15 seconds or less; each mL of the resulting solution  
will contain 25 mg of fludarabine phosphate, 25 mg of mannitol, and sodium 
hydroxide to adjust the pH to 7.7. The pH range for the final product is 7.2-8.2. 
In clinical studies, the product has been diluted in 100 cc or 125 cc of D5W (5% 
dextrose in water)  or NS. 


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d. Storage and Stability: FLUDARA is supplied as a white, lyophilized solid 
cake. Each vial contains 50 mg of fludarabine phosphate, 50 mg of mannitol 
and sodium hydroxide to adjust pH to 7.7. The pH range for the final product is 
7.2-8.2. Store under refrigeration, between 2°-8° C (36°-46° F). Infusions 
prepared in D5W or NS are stable for up to 16 days at 25°C. 
 
e. Administration: Intravenous, over thirty minutes. 
 
 
5.1.5 C lofar abine (C lolar ™)  
a. Formulation and Stability: 
 
Clofarabine is formulated at a concentration of 1 mg/mL in sodium chloride (9 
mg/mL). Clofarabine is supplied in a 1 mg/mL, 20 mL vial. The pH range of the 
solution is 4.0 to 7.0. The solution is clear with color ranging from colorless to yellow 
and is free from visible particulate matter. Genzyme will be supplying the drug for 
this study. 
 
Vials containing undiluted Clofarabine for injection should be stored at controlled 
room temperature (15 to 30°C). Shelf-life studies of intact vials are currently ongoing. 
Clofarabine for injection should be filtered through a sterile 0.2 µm syringe filter and 
then further diluted with D5W or NS prior to IV infusion. The resulting admixture may 
be stored at room temperature, but must be used within 24 hours of preparation. 
 
b. Administration: 
Clofarabine will be administered by IV infusion over 2 hours daily for 5 consecutive 
days. To prevent drug incompatibilities, no other medications should be 
administered simultaneously through the same IV line. 
 
5.2 Anti-Thymocyte Globulin (Rabbit) (Thymoglobulin®) 
 
a. Source and pharmacology: Supplier: Sangstat, The Transplant Company®. 
Thymoglobulin® [Anti-thymocyte Globulin (Rabbit)] is a purified, pasteurized, gamma 
immune globulin, obtained by immunization of rabbits with human thymocytes. This 
immunosuppressive product contains cytotoxic antibodies directed against antigens 
expressed on human T-lymphocytes. 
 
b. Formulation and stability: Thymoglobulin is a sterile, freeze-dried product for 
intravenous administration after reconstitution with SWFI. Each package contains 
two 7 mL vials: Vial 1: Freeze-Dried Thymoglobulin Formulation Active ingredient: 
Anti-thymocyte Globulin (Rabbit) 25 mg - Inactive ingredients: Glycine (50 mg), 
mannitol (50 mg), sodium chloride (10 mg); Vial 2: Diluent SWFI 5 mL. The 
reconstituted preparation contains approximately 5 mg/mL of Thymoglobulin, of 
which >90% is rabbit gamma immune globulin (IgG). The reconstituted solution has 
a pH of 7.0± 0.4. Human red blood cells are used in the manufacturing process to 
deplete cross-reactive antibodies to non-T-cell antigens. The manufacturing process 


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is validated to remove or inactivate potential exogenous viruses. All human red blood 
cells are from US registered or FDA licensed blood banks. A viral inactivation step 
(pasteurization, i.e., heat treatment of active ingredient at 60°C/10 hr) is performed 
for each lot. Each Thymoglobulin lot is released following potency testing 
(lymphocytotoxicity and E-rosette inhibition assays), and cross-reactive antibody 
testing (hemagglutination, platelet agglutination, anti-human serum protein antibody, 
antiglomerular basement membrane antibody, and fibroblast toxicity assays on 
every 5th lot). 
c. Solution preparation: Each reconstituted vial contains 25 mg or 5 mg/mL of 
Thymoglobulin. Transfer the contents of the calculated number of Thymoglobulin 
vials into the bag of infusion solution (saline or dextrose). Recommended volume: 
per one vial of Thymoglobulin use 50 mL of infusion solution (total volume usually 
between 50 to 500 mL). Mix the solution by inverting the bag gently only once or 
twice. 
d. Storage and stability: Store in refrigerator between +2° C to +8° C (36° F to 46° 
F). Protect from light. Do not freeze. Do not use after the expiration date indicated 
on the label. Reconstituted vials of Thymoglobulin should be used within 4 hours. 
Infusion solutions of Thymoglobulin must be used immediately. Any unused drug 
remaining after infusion must be discarded. 
 
e. Administration:  Infuse through a 0.22-micron filter. 
 
5.4 Rituximab (Rituxan ®) 
 
a. Source and pharmacology: Rituximab is a monoclonal antibody directed 
against the CD20 antigen on the surface of B-lymphocytes. CD20 regulates cell 
cycle initiation; and, possibly, functions as a calcium channel. Rituximab binds 
to the antigen on the cell surface, activating complement-dependent B-cell 
cytotoxicity; and to human Fc receptors, mediating cell killing through an 
antibody-dependent cellular toxicity. B-cells are believed to play a role in the 
development and progression of rheumatoid arthritis. Signs and symptoms of RA 
are reduced by targeting B-cells and the progression of structural damage is 
delayed. 
 
b. Formulation and stability: Store intact vials at 2°C to 8°C (36°F to 46°F); do 
not freeze. Do not shake. Protect vials from direct sunlight. Solutions for infusion 
in NS or D5W are stable at 2°C to 8°C (36°F to 46°F) for 24 hours and at room 
temperature for an additional 24 hours  (although because there is no 
preservative, the manufacturer recommends storing refrigerated). 
 
 
c. Solution preparation: Solution, Intravenous [preservative free]: Rituxan: 10 
mg/mL (10 mL, 50 mL) [contains polysorbate 80]. Rituximab for injection should 
be diluted in normal saline to a concentration of 1-4 mg/mL. 


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d. Storage and stability: Store vials under refrigeration once diluted, protect from 
sunlight. Diluted rituximab is stable for 24 hours refrigerated and 12 hours at 
room temperature. 
 
 
e. Administration: Withdraw necessary amount of rituximab and dilute to a final 
concentration of 1 to 4 mg/mL with NS or D5W. Gently invert the bag to mix the 
solution. Do not shake. Do not mix or dilute with other medications. Compatible 
in polyvinyl chloride (PVC) and polyethylene bags. Anticipate infusion reactions, 
particularly in individuals who have not previously received rituximab infusion. 
Infusion schedule will be adjusted according to standard guidelines. Emetogenic 
potential is low. Patients should receive acetaminophen 10-15 mg/kg/dose PO 
(max 650 mg/dose) and diphenhydramine 0.5-1 mg/kg/dose PO or IV (max 50 
mg/dose) 30-60 minutes prior to infusion. 
 
 
6.1 
CRITERIA FOR SUBJECT ELIGIBILITY 
 
6.2 
Subject Inclusion Criteria 
 
6.2.1 Lethal disorders of Hematopoiesis correctable by transplant for which α/β 
T-cell and CD-19 depleted allogeneic hematopoietic stem cell transplantation is 
indicated including: 
a) Hemoglobinopathies: 
 
• 
Sickle cell disease (HbSS, HbSC, HbSB0 thalassemia, HbSB+, HbSD, HbSE) 
with at least one of the following criteria (Walters et al): 
1. Cerebrovascular accident lasting longer than 24 hours 
2. Impaired neuropsychological function with abnormal brain MRI/MRA 
3. Recurrent hospitalizations (>2 episodes/year over several years) or 
exchange transfusions for acute chest syndrome 
4. Recurrent priapism 
5. Stage I or II sickle chronic lung disease 
6. Sickle cell nephropathy (moderate to severe proteinuria or glomerular 
filtration rate 30-50% of predicted normal value for age) 
7. Bilateral proliferative retinopathy with major visual impairment in at least 
one eye 
8. Osteonecrosis of multiple joints 
9. Red cell alloimmunization during chronic transfusion therapy 
 
• 
Thalassemia major with at least one of the following criteria: 
1. Age <16 years 
2. Available  HLA-identical sibling 
3. Red blood cell transfusion dependency 
4. Lucarelli class 1 or 2 risk status (i.e. with only 0-2 of the following factors: 
hepatomegaly, portal fibrosis, or poor response to chelation therapy) 


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5. Recurrence of disease after previous stem cell transplant 
 
 
b) Bone Marrow Failure Syndromes: 
• Aplastic anemia refractory to immunosuppressive therapy 
• Diamond Blackfan Anemia refractory to conventional therapy 
• Shwachman-Diamond Syndrome 
• Severe  Congenital Neutropenia 
• Congenital Amegakaryocytic  Thrombocytopenia 
• Thrombocytopenia Absent Radii syndrome 
• Other marrow failure disorders not otherwise specified 
 
c) Autoimmune  cytopenias refractory to all conventional treatments 
• Autoimmune hemolytic anemia 
• Immune thrombocytopenia 
• Evan’s syndrome 
• Pure red cell aplasia 
 
d) Histiocytic disorders: 
• Hemophagocytic  lymphohistiocytosis 
• High risk, recurrent or refractory Langerhans cell histiocytosis 
• Secondary HLH 
 
 
 
6.2.2 
Subject Inclusion 
• 
Recipient’s age birth to < 70 years old 
• 
Patients must have adequate organ function measured by: 
 
Cardiac:  asymptomatic or if symptomatic then LVEF at rest must be > 50% and 
must improve with exercise. 
Hepatic:  < 3x ULN AST and < 1.5 mg/dl total serum bilirubin, unless there is 
congenital benign hyperbilirubinemia or if the hyperbilirubinemia is directly caused 
by the disease in which the patient is receiving a transplant for. Patients with 
higher bilirubin levels due to causes other than active liver disease are also eligible 
with PI approval e.g. patients with PNH, Gilbert’s disease or other hemolytic 
disorders. 
Pulmonary:  asymptomatic or if symptomatic, DLCO > 50% of predicted (corrected 
for hemoglobin). 
Renal:  serum creatinine <1.5x normal for age. If serum creatinine is outside the 
normal range, then CrCl > 70 mL/min/1.73m2 (calculated or estimated) or GFR 
(mL/min/1.72m2) >30% of predicted normal for age. 


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Normal GFR in Children and Young Adults 
 
Age 
Mean GFR +- SD 
(mL/min/1.73 m2) 
1 week 
40.6 + / - 14.8 
2-8 weeks 
65.8 + / - 24.8 
>8 weeks 
95.7 + / - 21.7 
2-12 years 
133.0 + / - 27.0 
13-21 years (males) 
140.0 + / - 30.0 
13-21 years (females) 
126.0 + / - 22.0 
 
Abbrev iations: GFR, glomerular f iltration rate; SD, standard dev iation 
Greater than 2 y ears old: Normal GFR is 100 mL/ min. 
Inf ants: GFR must be corrected f or body surf ace area 
 
Each patient must be willing to participate as a research subject and must sign an informed 
consent form. 
 
6.1.3. Donor Inclusion Criteria 
• Each donor must meet criteria outlined by institutional guidelines and be 
medically eligible to donate according to NMDP (or equivalent donor 
search organization) criteria including testing for antibodies to Human T- 
Lymphotrophic Virus Types I & II (Anti-HTLV-I/II)  and screening for West 
Nile Virus, Creutzfeldt–Jakob disease and Zika. 
• 
Pediatric donors should weigh ≥ 25.0 kg, have adequate peripheral venous  
catheter access for leukapheresis or must agree to placement of a central catheter. 
• 
Donor should be healthy and agree to receive G-CSF followed by donation of 
peripheral blood stem cells. 
• 
Donors must agree to anesthesia and marrow donation (in cases of inadequate 
PBSC collection). 
• 
Related or unrelated donors who are 7/8 or 8/8 HLA-antigen matched for 
haplotypes A, B, C, DRB1 OR 
• 
Related donors who are 4-6/8 HLA-antigen  matched. 
 
6.1.4 
Donor Exclusion Criteria 
Donors who are seropositive for HIV-I/II  or HTLV-I/II  and female patients who 
are pregnant or breastfeeding will not be eligible for this study. 
 
6.2 
Subject Exclusion Criteria 
 
• 
Inherited DNA repair deficiency: 
Fanconi Anemia 
Dyskeratosis Congenita 
These are presently undergoing transplantation based on a multi-center protocol 


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• 
Inherited metabolic disorders: 
Hurler Syndrome 
Sly syndrome (MPSVIII) 
α-Mannosidosis 
X- ALD 
Osteopetrosis 
• 
Stage III-IV  sickle chronic lung disease 
• 
Patients with Thalassemia Major with Pesaro risk score >II 
• 
Female patients who are pregnant or breast-feeding 
• 
Active viral, bacterial or fungal infection 
• 
Patient seropositive for HIV-I/II;  HTLV-I/II 
• 
Karnofsky (adult)/Lansky (pediatric) < 70% 
 
 
7.1 
RECRUITMENT PLAN 
 
Patients who fulfill the eligibility criteria as listed in Section 6.0 will be recruited for this study 
by an attending physician of the Allogeneic BMT services in Medicine or Pediatrics. This 
protocol will take due notice of NIH/ADAMHA policies concerning inclusion of women and 
minorities in clinical research populations. We anticipate that this study will preferentially 
benefit persons of racial or ethnic minority populations as these individuals are less likely to 
have a HLA matched allo HCT donor. Otherwise, we expect that the study population will 
be fully representative of the range of patients referred for transplant without exclusion as 
to age or gender with the exception that pregnant women are excluded from participation in 
this study. 
 
 
8.0     PRETREATMENT EVALUATION 
 
The patient will receive an extensive medical evaluation within approximately 45 days prior 
to starting preparatory cytoreduction. This evaluation may include the below tests: 
• 
Complete physical exam and medical history 
• 
Complete Blood Count (CBC), coagulation studies (PT/aPTT/INR), Blood Type (ABO) 
and screen, serum chemistries including BUN,  creatinine, electrolytes, glucose, total 
protein, albumin, liver function tests (AST, ALT, bilirubin, alkaline phosphatase) 
• 
Pregnancy test for women of childbearing potential 
• 
Infectious  disease  markers  will  be  tested  per  FDA,  AABB,  NY  State,  and  FACT 
standards. 
• 
Urinalysis 
• 
Electrocardiogram, echocardiogram 
• 
Samples of bone marrow and/or peripheral blood cells will be obtained to define 
donor/host genetic differences and to determine engraftment of donor cells (may be 
completed outside of 45-day window) 


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• 
Donor specific class 1 and class 2 anti-HLA antibody titers for mismatched donors 
• 
Pre-transplant research sample 
 
When indicated 
• 
Dental evaluation 
• 
Disease Evaluation: Hemoglobin electrophoresis, bone marrow aspirate and/or biopsy 
• 
Assessment of iron overload (ferritin, liver and/or heart MRI) 
• 
Pulmonary function test for patients older than 7 years 
• 
Chest X-ray and/or other types of scans (MRI, CT scan and PET scan) 
• 
Transcranial Doppler Evaluation (TCD) 
• 
Neuropsychological testing 
 
 
9.1 
TREATMENT/INTERVENTION PLAN 
 
9.2 Selection of Cytoreduction Regimen 
 
Patients eligible for this protocol include individuals with non-malignant disorders of 
hematopoiesis or immunity who fulfill eligibility requirements and consent to treatment. 
Stratification of patients to one of the two disease-targeted cytoreductive regimens will be 
based on the patient’s disease as well as clinical parameters that could increaserisk of graft 
failure or severe toxicity in the post transplant period. Examples of disease indications for 
each of the two cytoreductive regimen are summarized below. The basis for selection of a 
specific regimen can be briefly described. 
Regimen A, which combines clofarabine, melphalan and thiotepa has been tested with 
unmodified HLA-compatible transplants (IRB 06-125) and has shown particular promise in 
the treatment of hematologic malignancies. (54, 55) This regimen has been successfu llly 
used for some non-malignant disorders in MSKCC 10-050 Arm C. 
Regimen B, which combines fludarabine, melphalan and thiotepa has been employed 
successfully for cytoreduction of heavily transfused patients with congenital acquired 
cytopenias, aplastic anemia, genetic immune deficiencies, and acquired auto immune 
disorders resulting in severe autoimmune cytopenias and lymphoproliferative disease and 
refractory leukemia. The immunosuppressive activity of thiotepa and fludarabine, coupled 
with the myeloablation induced by melphalan and thiotepa, has generated consistent full 
engraftment with only moderate gastrointestinal toxicities. This regimen has been 
successfully used in MSKCC 10-050 Arm D. 
9.3 Admission  for Transplantation 
 
Patients will be treated as inpatient on the Memorial Hospital Allogeneic Transplant 
Services in Pediatrics or Medicine. All orders will be administered upon admission as per 
institutional standard of practice. 
9.4 Cytoreduction Regimens in Preparation for Transplantation 
 
Please note: prior to transplant, an additional day of rest may be added to the treatment 
schedule, due to unforeseen scheduling issues. 


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9.3.3 Regimen A: Clofarabine, Thiotepa, Melphalan 
9.3.3a Clofarabine 
Clofarabine will be administered via approximately a 2-hour intravenous infusion at a dose 
of  20-30 mg/m2/day for 5 doses. Doses of 30 mg/m2/day are better tolerated by pediatric 
patients than adults. Patients ≤ 18 years of age deemed suitable may receive clofarabine 
at 30mg/m2/day with PI approval. 
The timing of conditioning may be lengthened by 1-2 days based if deemed clinically 
necessary because of elevation of liver enzymes, and approved by the PI. 
9.3.3b Thiotepa 
 
Thiotepa will be administered via approximately a 4-hour intravenous infusion at a dose of 
7.5 mg/kg/day over approximately 4 hr daily x 2 days (Day-5 and Day -4). Thiotepa may 
also be administered at 15 mg/kg/day for 1 dose (day –4) depending on scheduling of 
transplant harvests. In select cases in which the peripheral blood stem cells must be 
harvested a day later than requested due to a scheduling issue with the donor or Stem 
Cell Processing Laboratory, there may be a day of rest between the last day of clofarabine 
and the first day of thiotepa. Thiotepa dosing will be adjusted if patient is > 125% of ideal 
body weight and will be calculated based on adjusted ideal body weight, as per MSKCC 
standard of care guidelines. 
 
 
9.3.3c Melphalan 
 
Melphalan will be administered via approximately a 30 minutes infusion at a dose of 70 
mg/m2/day for 2 doses. Melphalan dosing will be adjusted if patient is > 125% of ideal 
body weight and will be calculated based on adjusted ideal body weight, as per MSKCC 
standard of care guidelines. 
 
Dosing adjustments for patients ≤ 3 years will be made on case by case basis. 
 
 
9.3.3d Rabbit Anti-thymocyte  Globulin* (Thymoglobulin®) and Methylprednisolone 
(MPD) 
Rabbit ATG (Thymoglobulin®) will be administered in the pre-transplant period for all 
research participants at 2.5 mg/kg/day x 3 days. If the patient has a history of allergy or 
intolerance to rabbit ATG, equine antithymocyte globulin at a dose of 15 mg/kg x 3 or 40 
mg/kg x 1 may be used. If severe reaction is encountered after the first dose of ATG, the 
second dose can be delayed until day +5.  Methylprednisolone will be given at 1 
mg/kg/day x 3 days with the ATG administration and will be discontinued thereafter. 
Additional medications to prevent or treat reactions will be administered as indicated 
according to institutional guidelines. If the patient is receiving a second transplant from the 
same donor, ATG administration will be at the discretion of the physician. ATG dosing will 


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Approval date: 02-Oct-2019 
Page 23 of 55 
 
 
 
be adjusted if patient is > 125% of ideal body weight and will be calculated based on 
adjusted ideal body weight, as per MSKCC standard of care guidelines. 
 
9.3.3 e Rituximab 
 
Rituximab will be administered based on actual weight according to institutional protocols 
to minimize infusion reaction. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Approximate  Schema for Regimen A 
 
EXAMPLE OF ROAD MAP OF PREPARATION FOR TRANSPLANT 
 
Monday 
Tuesday 
Wednesday 
Thursday 
Friday 
Saturday 
Sunday 
-12 
Rabbit ATG 
2.5 mg/Kg/day 
IV over 12 hrs 
-11 
Rabbit ATG 
2.5 mg/Kg/day 
IV over 12 hrs 
-10 
Rabbit ATG 
2.5 mg/Kg/day 
IV over 12 hrs 
-9 
-8 
-7 
-6 
-5 
-4 
-3 
Clofarabine 
Clofarabine 
Clofarabine 
Clofarabine 
Clofarabine 
Thiotepa 
Melphalan 
20-30 
20-30 
20-30 
20-30 
20-30 
7.5 mg/Kg/dose 
70 mg/m2/dose 
mg/m2/dose 
mg/m2/dose 
mg/m2/dose 
mg/m2/dose 
mg/m2/dose 
IV over 4 hours 
IV over 30 min 
IV over 2 hours 
IV over 2 hours 
IV over 2 hours 
IV over 2 hours 
IV over 2 hours 
Thiotepa 
7.5 mg/Kg/dose 
IV over 4 hours 
-2 
Melphalan 
70 mg/m2/dose 
IV over 30 min 
Start 
Tacrolimus 
-1 
Rituximab 
200 mg/m2 
0 
TCR αβ - 
CD19- 
Stem cell 
transplant 
+1 
+2 
+3 
+4 


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(Haplo) 
+5 
+6 
+7 
Start G-CSF 
 
 
9.3.4 Regimen B: Fludarabine, Thiotepa, Melphalan 
9.3.4a Fludarabine 
Fludarabine will be administered via approximately a 30-minute infusion at a dose of 30 
mg/m2/day for 5 days. Fludarabine may be adjusted in the case of renal toxicity. 
9.3.4b Melphalan 
 
Melphalan will be administered via approximately a 30-minute infusion at a dose of 70 
mg/m2/day for 2 doses. Melphalan dosing will be adjusted if patient is > 125% of ideal 
body weight and will be calculated based on adjusted ideal body weight, as per MSKCC 
standard of care guidelines. 
9.3.4c Thiotepa 
 
Thiotepa will be administered via approximately a 4 hour intravenous infusion at a dose of 
7.5 mg/kg/day IV over approximately 4 hr daily x 2 days. Thiotepa may also be 
administered at 15 mg/kg/day for 1 dose depending on scheduling of transplant harvests. 
Thiotepa dosing will be adjusted if patient is > 125% of ideal body weight and will be 
calculated based on adjusted ideal body weight, as per MSKCC standard of care 
guidelines. 
In select cases in which the peripheral blood stem cells must be harvested a day later than 
requested due to a scheduling issue with the donor or Stem Cell Processing Laboratory, 
there may be a day of rest between the last day of melphalan and the first day of 
fludarabine and thiotepa. 
9.3.4d Rabbit Anti-thymocyte  Globulin (Thymoglobulin®) and Methylprednisolone 
(MPD) 
Rabbit ATG will be administered in the pre-transplant period for all research participants  
on this arm. Research participants will receive rabbit ATG (Thym oglobulin®) at 2.5 
mg/Kg/day x 3 days. If the patient has a history of allergy or intolerance to rabbit ATG, 
equine antithymocyte globulin at a dose of 15 mg/kg x 3 or 40 mg/kg x 1 may be used. If 
severe reaction is encountered after the first dose of ATG, the second dose can be  
delayed until day +5.  Methylprednisolone  will be given at 1 mg/Kg/day x 3 days with the 
ATG administration and will be discontinued thereafter. If patient is receiving a second 
transplant from the same donor, ATG administration will be at the discretion of the 
physician. ATG dosing will be adjusted if patient is > 125% of ideal body weight and will be 


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Approval date: 02-Oct-2019 
Page 25 of 55 
 
 
 
calculated based on adjusted ideal body weight, as per MSKCC standard of care 
guidelines. 
 
 
9.3.4e Rituximab 
 
Rituximab will be administered based on actual weight according to institutional protocols 
to minimize infusion reaction. 
Approximate  Schema for Regimen B 
 
EXAMPLE OF ROAD MAP OF PREPARATION FOR TRANSPLANT 
 
MONDAY 
TUESDAY 
WEDNESDAY 
THURSDAY 
FRIDAY 
SATURDAY 
SUNDAY 
-11 
Rabbit ATG 
2.5 mg/Kg/d 
-10 
Rabbit ATG 
2.5 mg/Kg/d 
-9 
Rabbit ATG 
2.5 mg/Kg/d 
-8 
Melphalan 
70 mg/m2/day 
IV X 1 
-7 
Melphalan 
70 mg/m2/day 
IV X 1 
-6 
Fludarabine 
30 
mg/m2/day 
IV X 1 
Thiotepa 
7.5 mg/kg/day 
IV X 1 
-5 
Fludarabine 
30 mg/m2/day 
IV X 1 
Thiotepa 
7.5 mg/kg/day 
IV X 1 
-4 
Fludarabine 
30mg/m2/day 
IV X 1 
-3 
Fludarabine 
30 mg/m2/day 
IV X 1 
-2 
Fludarabine 
30 mg/m2/day 
IV X 1 
Start 
Tacrolimus 
(Haplo) 
-1 
Rituximab 
200 mg/m2 
0 
TCR αβ - 
CD19- 
Stem cell 
transplant 
+7 
Start G-CSF 
 
 
 
 
9.4 Prophylaxis against acute graft-versus-host disease 
 
Tacrolimus or Mycophenolate mofetil (MMF) will be used as GVHD prophylaxis for 
haploidentical grafts. The choice of agent will be based on clinical status. 
Prophylaxis will be started on day -2, tapered from day +30 to +60 and discontinued. 
 
1. Tacrolimus 
Tacrolimus will be started day –2 at an approximate dose of 0.03 mg/kg/24 hours as a 
continuous infusion. The dose of tacrolimus will be adjusted to achieve a level between 5 
and 15 ng/mL. Once oral medications are tolerated, patients may be converted to oral 
tacrolimus at 3-4 times the current IV dose divided every 8 to 12 hours. 
In case of major toxicity, such as CNS symptomatology, tacrolimus may be discontinued 
earlier and substituted with other immunosuppressive medications as per clinical care 
guidelines. 
 
The immunosuppressive regimens on this protocol may conform to current practice where 
additional agents may be introduced in addition to Tacrolimus. 


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2. Mycophenolate  mofetil  (MMF) 
 
Prophylaxis Dosing Starting on Day -2: 
Recipient > 12 years and < 50 kg 
15 mg/kg q 8 hours 
Recipient > 12 years and ≥ 50 kg 
15 mg/kg q 8 hours (Min 1 g q 8 hours. Max 1.5 g q 8 hours) 
Recipient ≤ 12 years 
30 mg/kg IV q 8 hours (Max dose 1.5g q 8 hours) 
 
In preparation for discharge, switchto oral route (CellCept or generic mycophenolate mofetil 
and same dosing as IV) and round to tablet size. 
• 
No dose adjustments for renal or liver disease are needed routinely unless severe organ 
dysfunction. Measurements of serum blood levels of MMF should be done routinely as per 
current MSKCC guidelines to guide assessment of potential MMF toxicity in the event of 
unexpected myelosuppression. If patient > +28 days and slow engraftment, consideration 
can be made to reduce dosing to q12 after discussion with PI or co-PI. See MSKCC 
guidelines for further information about MMF  levels. 
 
For patients with active GvHD, tacrolimus or MMF may be continued for longer periods of 
time according to clinical care guidelines. 
 
9.5. Stem Cell Transplantation   
 
 
9.5.1 
PBSCT 
 
Donor peripheral blood progenitor cells: stimulation, harvesting, isolation and T-cell 
depletion. For related donors, GCSF will be administered according to MSKCC standard 
of care. For unrelated donors, the G-CSF will be administered and the leukapheresis 
obtained according to the National Marrow Donor Program protocol and IND. Mobilization 
with Plerixafor will be considered in selected cases. 
 
Donor bone marrow cells: Harvesting, Isolation and T-cell depletion 
 
In cases of failed peripheral stem cell donor mobilization, bone marrow harvesting will be 
performed. Under general anesthesia, and after appropriate surgical preparation, bone 
marrow will be harvested by repeated aspiration from the iliac crests bilaterally into 
heparinized syringes, and pooled in heparinized saline, per FACT approved institutional 
guidelines.  After filtration to remove boney spicules, and marrow harvest, collected in a 
sealed sterile transfusion bag, is transported to the laboratory for processing and selection 
of CD34+ cells on the CliniMACS device. 
 
Processing prior to selection of CD34+ progenitor cells on the CliniMACS device, consists 
of initial measurements of total volume and cell count, acquisition of samples required for 
immunocytometry and cultures, and thereafter, depletion of red cells by hetastarch 
sedimentation, collection of the leukocyte rich plasma and washing, according to the 
laboratory’s standard operating procedures. 
 
Following this processing, the cells are resuspended in CliniMACS PBS/EDTA buffer 
supplemented with sterile human intravenous immune globulin at a concentration of 1.5 
mg IVIG/mL,  and thereafter incubated with the antiCD34 antibody coated paramagnetic 


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beads, and then separated on the CliniMACS device, as detailed for selection of CD34+ 
progenitors from GCSF-mobilized PBSCT. 
 
9.5.2 Isolation of CD34+ TCR α/β T-cell and CD-19 Depleted hematopoietic 
progenitor cells with the CliniMACS TM System,  Miltenyi Biotec. 
 
a. Mobilization of Donor 
Allogeneic donors will be mobilized per institutional standards. 
 
b. Donor Stem Cell Apheresis 
 
Donor stem cell apheresis, HPC(A) will be performed per institutional standards.  The 
donor shall not undergo more than 2 days of collection unless extenuating circumstances 
permit for an additional day (total 3 days). 
c. TCRαβ+ T-Cell Reduction using the Miltenyi CliniMACS® 
 
 
 
Reduction of TCRαβ T-cells and CD19+ B-cells from the donor HPC(A) product will be 
performed according to procedures given in the CliniMACS® Users Operating Manual and 
institutional Standard Operating Procedures (SOP) implemented in the Cell Therapy 
Laboratory (see Appendix for SOP). 
The total nucleated cell count, CD34+ cell count, TCRαβ+ CD3+ T-cell count, and CD19+ 
cell count will be analyzed pre- and post-selection and recorded. 
Day 1: TCRαβ + CD19 reduction and Day 2: CD34 selection 
 
A single TCRαβ+ T-cell and CD19+ B cell reduction procedure will be performed for 
donors to achieve a target of CD34+ cells/kg cell dose for the final product ≥ 5 x 106 and 
CD3+ TCRαβ+ cell count ≤ 1 x 104 cells/kg recipient total body weight for mismatched 
donors. An additional CD34+ cell selection may be performed to achieve the target CD34+ 


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cell dose, if required.  If there are less than a total of 2 x 106  CD34+ cells/kg recipient body 
weight in the combined allografts, the laboratory will contact the investigator.  If required, 
CD34+ cell selection on additional HPC(A) will be performed according to procedures  
given in the CliniMACS® Users Operating Manual and approved institutional SOP. Testing 
of the product will be conducted in a CLIA certified laboratory as described in the CMC 
section. If needed, the order of cell selection may be altered in order to accommodate 
resource limitations. 
 
The TCRαβ graft is depleted of potentially allo-reactive T cells (TCR a-b positive cells). 
Because all other cells are retained (CD34, TCR gamma-delta, NK cells etc) the size of 
the graft is similar to an unmodified PBPC graft, and therefore it is usually dispensed in a 
bag. 
 
The CD34 selected graft only retains the CD34 cell fraction, which is relatively rare and 
therefore the volume of the product is much smaller and as such the product is dispensed 
in a syringe. 
 
If both products are available simultaneously to infuse, the recommendation is to first 
infuse the CD34 selected graft due to its small volume and nearly absent reaction rate. 
Once the CD34 selected graft has been infused, then the bag of the TCRαβdepleted graft 
may be administered as per SOP for HPCs in the bag format. There is no necessity for a 
delay between infusions. 
 
Pre-medications and fluids should be administer per standard BMT practice guidelines for 
CD34 selected products. 
d. Analysis  of Allograft 
 
Samples will be taken from each leukapheresis product after collection and before 
processing and on each fraction of cells after processing on a single CliniMACS® tubing 
set.  Pre- and post-selected target cells (CD34+ and CD3+) will be characterized as 
follows: 
• 
Gram stain (performed locally) post selection 
• 
Total nucleated cell (TNC) count pre- and post-selection 
• 
Endotoxin testing pre-release (performed locally) 
• 
Flow cytometric analysis for CD34+ cells and for CD3+ cell pre- and post-selection 
performed locally using validated SOP 
• 
Flow cytometric analysis of monocytes, B-cells, NK cells in the cell-selected 
products 
• 
Viability testing (7-AAD method) pre- and post-selection 
 
Criteria for release of final graft product for infusion. 
• 
Viability ≥ 70% after selection 
• 
Negative Gram stain 
• 
Cumulative CD34+ cell content ≥ 2.0 x 106  CD34+ cells/kg 
• 
Cumulative T cell content ≤ 10 x 104 TCRαβ+CD3+ cells/kg; related matched donor 


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• 
Cumulative T cell content ≤ 1x 104  TCRαβ+CD3+ cells/kg; alternative, mismatched 
donor 
 
9.5.3 Transplantation of the T-cell depleted stem cells. 
 
The CD34+T-cell depleted peripheral blood progenitor cells will be suspended in a volume 
of approximately 20-50 mL will be infused intravenously over approximately 15 minutes 
with monitoring of vital signs. 
9.6. 
Supportive Care 
 
a. Prophylaxis against infections 
 
Standard of care guidelines will be followed for prophylaxis against post transplant 
infections by opportunistic organisms, including Pneumocystis jiroveci, fungal organisms, 
DNA herpes viruses and more specifically CMV. 
b. Prophylaxis against menorrhagia 
 
All post-pubertal females will receive prophylaxis against menorrhagia according to our 
standard of care guidelines. 
 
c. Transfusions 
 
Following initiation of the pre-transplant cytoreduction, all blood products for transfusion, 
with the exception of the peripheral blood stem cell, will be irradiated to 3,000 cGy to 
inactivate lymphocytes capable of initiating lethal GvHD. Blood products are irradiated in 
the blood bank, using a cesium gamma emitter. Platelets will be administered for clinical 
evidence of active hemorrhage. Platelets will be administered at the discretion of the 
treating attending and as per standard of care. 
 
 
 
 
d. 
Nutritional support 
 
Nutritional status will be carefully monitored by the physician, and high-calorie parenteral 
alimentation will be introduced as needed. Vitamin supplements will be as clinically 
indicated. 
 
 
10.1 EVALUATION DURING TREATMENT/INTERVENTION 
 
10.1 Post-transplant evaluation 
 
The chart below shows the approximate dates for tests and procedures performed after 
transplant. Certain tests may be held or repeated at the discretion of the treating physician 
if deemed in the best clinical interest of the patient. Additional testing may be performed as 
is clinically indicated. Scheduled evaluations on day +30 and +100 may occur ±7 days from 


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Page 30 of 55 
 
 
 
the schedule time point.  Long term follow  up testing may be done  ±21 days  from the 
scheduled time point. 
Post-transplant GVHD assessments will be performed as per institutional standards. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1. May be done more frequently  if clinically indicated 
2. PBL phenotyping  should be performed  until CD34 counts is >200 or if patient is being immunized 
3. Should be performed  until normal or reached plateau 
4. CMV testing if patient or donor CMV positive only. Testing more or less frequently  as clinically 
indicated 
Activity 
Transplant  to 
Discharge 
DISCHARGE 
TO DAY 100 
Day 100 
6 Months 
12 months 
24 
Months 
Blood counts and 
chemistry 
(CBC, CMP) 
CBC: Daily 
CMP: 2x a week 
CBC/CMP: 
Weekly, 2x a 
week, or every 
2-3 weeks 
Every 2-4 weeks 
X 
Lineage Specific 
chimerism 
including T-cells 1 
NA 
Day +30 
Day +60 
X 
X 
X 
X 
Peripheral blood 
lymphocyte (PBL) 
phenotyping2 
NA 
Day +30 
Day +60 
X 
X 
X 
X 
In vitro response 
of PBL to 
standard panel of 
mitogens 2,3 
NA 
NA 
X 
X 
X 
X 
Viral PCR: CMV, 
EBV, ADV4 
Twice weekly4 
As clinically 
indicated 
Twice weekly4 
As clinically 
indicated 
Weekly4 
As clinically 
indicated 
Every 2 
Every two 
weeks 4 
weeks 4 
If clinically 
If clinically 
indicated 
indicated 
Research bloods 
Once 
Day +30 
Day +60 
X 
X 
X 
X 


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IRB Number: 17-596 A(2) 
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For patients weighing ≥10 kg, research samples will consist of three 10 mL solution heparin tubes 
of blood on the days described above. If a patient is at risk for exceeding maximum allowable 
blood draw limits (per Seattle Children's Hospital Guideline for Maximum Blood Volumes), 
clinically laboratory assessments will be prioritized over research samples. 
 
11.0 
TOXICITIES/SIDE EFFECTS 
 
Patients recruited to this transplantation trial are individuals who are either referred by physicians 
or self-referred for peripheral blood stem cell transplantation as a potentially curative treatment for 
their life-threatening disorders. Prior to consideration for transplant, all patients undergo a series 
of 1-3 hour consultations discussing the risks and potential benefits of an allogeneic stem cell 
transplantation and the different procedures which will be a normal part of the transplant course. 
The risks and potential benefits of the transplant procedure, as well as the participation in any 
given research, experimental, or therapeutic protocol are also discussed. 
In addition to tracking patients for survival and graft failure, all participants will also be monitored 
for post-transplant toxicities. Toxicities will be graded according to NCI CTCAE version 5.0 at the 
time points outlined in Section 10.1. All grade 3-4 adverse events will be captured and assessed 
for attribution to protocol treatment. Additional toxicities will be reviewed and graded/attributed at 
the discretion of the PI. Toxicities which are attributable to underlying disease and/or grade 1-2 
expected toxicities from the transplant will not be tracked. 
The risks of short-term treatment with G-CSF are likely negligible. However, administration of 
GCSF is frequently associated with low grade fever and low back pain which usually resolves 
within one day following cessation of GCSF treatment. Furthermore, there has now been one 
recorded patient who developed acute splenomegaly and splenic rupture in response to high  
dose GCSF. The bone pain may require treatment with analgesics. The risks of a leukapheresis 
are negligible, involving  an occasional vasovagal response to venipuncture and the minimal 
hemodynamic alterations associated with single unit phlebotomies. To protect against these risks, 
leukapheresis are conducted in the Blood Bank Donor Room with full medical and nursing 
supervision and support systems to address adverse events. 
 
11.1.1  
 
11.1.2 Thiotepa 
 
 
 
 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving Thiotepa, more than 20 and up to 100 may have: 
• Nausea, vomiting 
• Pain 
• Loss of appetite 
• Tiredness 
• Hair loss 
• Rash 


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11.1.5 Melphalan 
 
 
 
 
 
 
 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Melphalan,  from 4 to 20 may have: 
• Anemia,  kidney problems which may cause tiredness, bruising,  swelling,  or may require 
dialysis  or require blood transfusions 
• Infection,  especially when white blood cell count is low 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving Melphalan,  more than 20 and up to 100 may have: 
• Sores in mouth which may cause difficulty swallowing 
RARE, AND SERIOUS 
In 100 people receiving Thiotepa, 3 or fewer may have: 
• Allergic  reaction which may cause rash, low blood pressure, wheezing,  shortness of breath, 
swelling of the face or throat 
• Cancer of bone marrow caused by chemotherapy 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Thiotepa, from 4 to 20 may have: 
• Infection,  especially when white blood cell count is low 
• Anemia  which may require transfusion 
• Bruising,  bleeding 
• Damage to the bone marrow (irreversible)  which may cause infection,  bleeding,  may 
require transfusions 
• Absence of menstrual  period 
• Loss or absence of sperm 
• Blurred vision 
• Blood in urine 
• Swelling of feet or lower legs 


Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
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11.1.6 Fludarabine 
 
 
 
 
 
 
 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Fludarabine,  from 4 to 20 may have: 
• Anemia,  kidney problems which may cause tiredness, bruising,  or swelling 
• Nausea, chills 
• Feeling of "pins  and needles" in arms and legs 
• Damage to organs (brain, lungs, others) which may cause tiredness, changes in thinking or 
shortness of breath 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving Fludarabine,  more than 20 and up to 100 may have: 
• Infection,  especially when white blood cell count is low 
• Vomiting,  loss of appetite 
• Tiredness, fever 
• Pain 
• Bruising,  bleeding 
• Cough 
• Increased risk of unusual infections  lasting more than 6 months 
RARE, AND SERIOUS 
In 100 people receiving Melphalan,  3 or fewer may have: 
• Heart stops beating 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Melphalan,  from 4 to 20 may have: 
• Bruising,  bleeding 
• Allergic  reaction which may cause rash, low blood pressure, wheezing,  shortness of breath, 
swelling of the face or throat 
• Hepatitis  which may cause yellow  eyes and skin, tiredness 
• A new cancer resulting from treatment  of a prior cancer 
• Cancer of bone marrow caused by chemotherapy 
• Kidney damage which may cause swelling,  may require dialysis 
• Scarring of the lungs  which may cause shortness of breath 


Page 34 of 55 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
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OCCASIONAL, SOME MAY BE SERIOUS 
 
 
In 100 people receiving Fludarabine,  from 4 to 20 may have: 
• Confusion 
 
 
 
 
 
 
 
 
 
 
 
 
11.1.7 Clofarabine 
 
 
 
 
 
 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Clofarabine,  from 4 to 20 may have: 
• Severe blood Infection 
• Kidney damage which may cause swelling,  may require dialysis 
• Stroke which may cause paralysis,  weakness 
• Severe skin rash with blisters  and can involve  inside of mouth and other parts of the body 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving Clofarabine,  more than 20 and up to 100 may have: 
• Anemia  which may cause tiredness, or may require blood transfusions 
• Abnormal heartbeat 
• High blood pressure 
• Diarrhea, nausea, vomiting,  chills 
• Tiredness 
• Fever 
• Bruising,  bleeding 
• Infection,  especially when white blood cell count is low 
• Loss of appetite 
• Pain 
• Headache 
 • Worry 
• Nose bleed 
• Itching,  rash 
• Low blood pressure which may cause feeling faint 
• Damage to the liver 
RARE, AND SERIOUS 
In 100 people receiving Fludarabine,  3 or fewer may have: 
• Kidney damage which may require dialysis 


Page 35 of 55 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
 
 
 
 
 
 
 
 
 
 
11.1.8 Rabbit Antithymocyte   Globulin (Thymoglobulin) 
 
The ATG to be used in this trial is a purified preparation of rabbit gamma globulin containing 
high concentrations of antibodies against human lymphocytes. The preparation may contain 
low levels of antibody that cross-react with human platelets, white cells or red cells. The 
potential side effects of ATG are: 
 
 
 
 
 
 
RARE, AND SERIOUS 
In 100 people receiving  ATG  3 or fewer may have: 
• Severe, possibly fatal, allergic  reaction 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving  ATG more than 20 and up to 100 may have: 
• Skin rash and itching 
• Fever and chills 
• Lowered blood pressure 
• Decreased ability to form blood clots; this may lead to difficulty in stopping bleeding,  easy 
bruising and blood in urine or stool. 
• Serum sickness- causes severe skin rashes, mouth and vaginal sore, pain and swelling in the 
joints or kidney damage. Serum sickness is treatable with antihistamines  and steroids 
• Increased chance of infection 
RARE, AND SERIOUS 
In 100 people receiving Clofarabine,  3 or fewer may have: 
• Damage to the bone marrow (irreversible)  which may cause infection,  bleeding,  may 
require blood transfusions 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving Clofarabine,  from 4 to 20 may have: 
• Fluid in the organs which may cause low blood pressure, shortness of breath, swelling of 
ankles 
• Flushing 


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11.1.9 Rituximab 
 
 
 
 
 
OCCASIONAL, SOME MAY BE SERIOUS 
 
In 100 people receiving  Rituximab,  from 4 to 20 may have: 
 
• Bruising,  bleeding 
• Abnormal heartbeat 
• Heart attack or heart failure  which may cause shortness of breath, swelling of ankles, and 
tiredness 
• Sores in eye 
• A tear or a hole in the bowels that may require surgery 
• Diarrhea, vomiting 
• Pain 
• Swelling of the body 
• Hepatitis,  or liver damage which may cause yellow  eyes and skin 
• Dizziness,  headache 
• Kidney damage which may require dialysis 
• Cough 
• Scarring of the lungs 
• Stuffy nose 
• Blockage of internal organs which may cause shortness of breath, wheezing,  vomiting 
• Increased sweating 
• Itching,  rash, blisters on the skin 
• Severe skin rash with blisters  and peeling which can involve  mouth and other parts of the 
body 
• Low blood pressure which may cause feeling faint 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving  Rituximab,  more than 20 and up to 100 may have: 
• Nausea 
• Chills,  fever 
• Reaction during or following infusion of the drug 
• Infection,  especially when white blood cell count is low 
• Anemia  which may require  blood transfusions 
• Numbness and tingling of the arms and legs 
• Tiredness 


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11.2 Toxicities/Side  effects  of  the   transplant  and   the  combined   effects  of  the 
conditioning regimen and transplant 
 
In addition to tracking patients for survival and graft failure, all participants may also be monitored 
for post-transplant toxicities. 
 
Grade 3-4 toxicities which occur within 30 days following a transplant AND are also possibly, 
probably, or definitely attributable to the CliniMACS device will be graded and attributed. Additional 
toxicities will be reviewed and graded/attributed at the discretion of the PI. 
 
Toxicities which are attributable to underlying disease and/or grade 1-2 expected toxicities from the 
transplant will not be tracked. 
 
The grading for monitoring transplant related toxicities will be based on the CTCAE 4.0 (57). 
 
11.2.1 
Potential Sensitization to Murine Proteins 
 
Mouse protein antibodies are used in the CliniMACS processing procedures. If the recipient has a 
pre-existing allergy, he or she may be at risk for allergic reactions during infusion of the processed 
cells, although the residual amount of murine protein in the final product is very low (estimated 
maximum dose for a 50 kg patient would be 30 µg). To date, no allergic reactions are reported in 
patients receiving cells processed with the CliniMACS System. Epinephrine and antihistamines will 
be available at the recipient's bedside during the PBSC infusion. 
 
 
11.2.2 
PBSC Infusion 
 
Symptoms may include changes in heart rate and/or rhythm, changes in blood pressure, fever, 
chills, sweats, nausea, vomiting, diarrhea, abdominal cramping, hemoglobinuria, acute renal failure, 
allergic reactions, respiratory dysfunction, or headache. 
 
11.2.3 
Infections 
 
Transplantation puts the patient at higher risk for bacterial, viral, or fungal infections, which are 
potentially life threatening.  These risks are potentially higher with TCD transplants.  Prophylaxis 
will be initiated and patients will be closely monitored for signs of infections and will receive early 
and appropriate treatment. Low B and T-cell count for an additional 3-12 months after 
transplantation increases the risk for certain opportunistic infections including pneumocystis jiroveci 
pneumonia, cytomegalovirus, and others. Medications are given to reduce the chance of infections. 
Patients  will  receive treatment if  they do  get an  infection and  most infections  can be treated 
RARE, AND SERIOUS 
In 100 people receiving  Rituximab,  3 or fewer may have: 
• Damage to the brain caused by a virus which may result in tiredness,  weakness, changes in 
thinking,  and disability.   This is called progressive  multifocal leukoencephalopathy (PML). 
• Heart stops beating 


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successfully with antibiotics. Patients will stay in the hospital longer or be readmitted if found to 
have an infection. Patients are watched closely for bleeding and given platelet transfusions to 
prevent serious bleeding, but minor bleeding may occur. 
 
 
11.2.4 
Microbial Contamination of PBSC 
 
There is a potential that processing the leukapheresis product will inadvertently introduce 
microorganisms that could cause infection in the recipient after the cells are infused. All precautions 
to maintain sterility will be taken. Cultures of the leukapheresis product and the selected product 
will be obtained to monitor for contamination. 
 
11.2.5 
Graft Failure / Poor Marrow Function 
 
T cell depletion of donor cells is associated with an increased incidence of graft failure in allogeneic 
transplant recipients. After allogeneic transplantation, the recipient's marrow function may be poor 
and leukopenia, anemia, or thrombocytopenia may result from many causes including  graft 
rejection induced by surviving host immune T-cells, or ongoing suppression of engrafted donor 
blood-forming cells by GVHD,  infection or marrow suppression or immunosuppressive drugs and 
other medications. Graft failure may result in death if not reversed. In patients with immune rejection 
second transplants can be administered with immunosuppressive therapy, including non- 
myeloablative conditioning regimens. For patients who are engrafted with donor cells but have 
severe cytopenia affecting one or more blood cell lineages, secondary transplants of CliniMACS 
fractionated CD34+ T-cell depleted PBSCs may be administered to booster and replenish donor 
hematopoietic cells without conditioning or after treatment with anti- thymocyte globulin. 
 
11.2.6 
Graft-versus-host Disease 
 
Acute or chronic GVHD may develop after allogeneic transplantation that can be disabling and 
can lead to death.  GVHD is thought to be initiated by T cells contained in the PBSC graft.  CD34+ 
selection and CD3+ depletion reduces the number of T cells in the PBSC but GVHD can occur 
after TCD transplants. Acute and/or chronic GVHD will be treated with immunosuppressive drugs 
as per the transplant service guidelines. 
 
Acute GVHD usually occurs in the first 3 months or as immunesuppressive medications are tapered 
and may cause: skin rashes, nausea, vomiting, diarrhea, hepatitis, increased risk of infection, 
ulceration of the surfaces of the oral cavity, esophagus, and intestines, and suppressed or delayed 
recovery of the hematopoietic and immune system. 
 
Chronic GVHD can occur any time after the first 3 months and is manifested to varying degrees by 
scleroderma-like changes of the skin, cirrhosis of the liver, sclerosis of lacrimal and salivary ducts, 
chronic inflammation and scarring of the gastrointestinal tract with consequent malabsorption and 
diarrhea, chronic bronchitis, and suppression of the immune system. This can be treated with 
standard or protocol-based experimental immunosuppression, but may be refractory. 
 
Severe GVHD: Rarely, GVHD can be severe or deadly. Severe acute GVHD could involve a severe 
skin rash like a burn, severe vomiting and/or diarrhea, liver failure and infections or bleeding. Severe 
acute GVHD will be treated with intense immunosuppressive therapy according to standard clinical 
practice or other experimental protocol. Severe chronic GVHD could involve similar symptoms but 
may produce other symptoms such as severe skin changes, severe dry eyes and weight loss. 


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Steroids, as treatment for GVHD: inability to sleep, high blood sugar, puffiness of the face, changes 
in the skin, high blood pressure, increased risk of infection, weight gain, reduced growth in children, 
thinning of the bones. 
 
 
11.2.7 
Veno-occlusive Disease (VOD) of the Liver 
 
VOD is a manifestation of damage to the liver by the conditioning regimen that usually develops 
within two weeks after allogeneic transplant and is characterized by at least two of the following: 
• 
Hyperbilirubinemia (total bilirubin > 2 mg/dL) 
• 
Hepatomegaly or right upper quadrant pain, or 
• 
Rapid weight gain (> 5% above baseline) 
 
Recipients developing VOD will be monitored closely and will receive appropriate supportive care 
and careful fluid management.  TCD is not expected to affect the risk of VOD. 
11.2.8 End Organ Damage 
 
End organ damage of all or any of the major organs, including the brain, may occur as a result of 
cumulative toxicity from anti-neoplastic therapy, reactions to other drugs, and as a result of 
destructive processes (e.g., infection, GVHD, etc.) and may have a fatal outcome.  Toxicities may 
occur in any individual patient due to multiple events and cumulative effects that may involve any 
and all organs, including the brain.  Brain damage can result in severe loss of cognitive or 
neurologic function.  Data from previous studies do not suggest that the risk of end organ damage 
is appreciably affected by TCD or the preparative regimens to be used in this study. 
11.2.9 Lymphoproliferative Syndrome 
 
Recipients of TCD allogeneic grafts have an increased risk of developing lymphoproliferative 
syndromes caused by EBV infection (56, 57). This syndrome should be included in the differential 
diagnosis of recipients with unexplained  symptoms such as fever, diarrhea, hepatomegaly or 
lymphadenopathy.  Biopsy evaluation is required to make the diagnosis.  EBV PTLD may rapidly 
progress and can be fatal if not treated.  Management of suspected EBV PTLD should be 
discussed with one of the Protocol P.I.s. EBV PTLD can be treated with rituximab and/or infusion 
of 106  T cells/kg from the donor.  Rituximab has been shown to induce regression in 50 - 70% of 
cases (58).  However, Rituximab does not enter the CNS and is not effective in treating CNS 
disease.  Donor lymphocyte infusions may induce regression in > 90% of cases of EBV PTLD and 
are effective in CNS disease but may cause GVHD (59). EBV-immune cells are experimental, but 
can also induce regression of EBV PTLD without risk of graft vs host disease (60, 61). 
11.2.11 Serious Bleeding 
 
Serious bleeding may result from low platelet counts and /or injury to tissues from treatment. This 
can happen despite platelet transfusions. Bleeding is rarely fatal. 
 
11.2.12 Risk of secondary cancer 


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Secondary cancers may occur after chemotherapy. The risk of developing a secondary cancer of 
the skin, cervix, etc., which has been seen in other transplant studies is less than 5%. 
 
11.2.13 Death 
 
There is an approximately 5-10% risk of treatment related mortality within the first month of 
transplant due to the risk of severe regimen related toxicity, hemorrhage, opportunistic infection, 
or other complications. It is not expected that the regimens to be used in this protocol will 
increase this risk. 
12.0 
CRITERIA FOR THERAPEUTIC RESPONSE/OUTCOME ASSESSMENT 
 
Definition of events in the post-transplant course important for analysis and treatment 
 
12.1 
Engraftment and Chimerism 
 
Engraftment will be documented by analysis of blood cells, T cells and bone marrow cells for 
chimerism by standard cytogenetic studies at about 1 month, 100 days, 6 months, 12 months, 
and 24 months post-transplant or as needed thereafter. 
 
12.2 
Graft failure or rejection 
 
Primary non-engraftment is diagnosed when the patient fails to achieve an ANC  500/l at any 
time in the first 28 days post-transplant. If host T-cells capable of specifically inhibiting donor 
hematopoietic progenitor growth in vitro are concurrently detected during graft failure, a 
presumptive diagnosis of immune mediated rejection is made (62, 63). If (1) after achievement of 
an ANC  500/mm3, the ANC declines to < 500/mm3 for more than 3 consecutive days in the 
absence of relapse, or, (2) there is absence of donor cells in the marrow and/or blood as 
demonstrated by chimerism assay in the absence of relapse, a diagnosis of secondary graft 
failure is made. 
Patients with evidence of graft failure may have additional studies drawn to ascertain cause and 
define relevant histo incompatibilities.  These analyses may include (1) Evaluation of bone 
marrow aspirates and biopsies, when indicated, (2) Culture and/or molecular analyses of marrow 
and blood for viral pathogens potentially causing graft failure including CMV, HHV6 and 
parvovirus B19, (3) Immunophenotypic and genetic analysis of circulating T-cells and NK cells to 
ascertain their origin and potential function, (4) Analysis of the functional activity of residual 
circulating lymphocytes to determine whether and to what degree they exhibit cytotoxic or cyto- 
inhibitory activity against donor host or third party PHA-stimulated blasts or clonogenic 
hematopoietic progenitor cells. If donor-specific reactivity is identified, attempts will be made to 
identify targeted specificities (HLA or minor alloantigens) whenever possible. 
Patients who suffer graft failure may be considered for a secondary transplant. The need for 
additional immunosuppression or treatment for viral infection prior to the secondary transplant will 
be determined by the results obtained from chimeric and viral studies. 
 
12.3. 
Graft-versus-host disease 
 
Standard BMT-CTN and IBMTR systems clinical criteria as defined by Rowlings, et al (64) will be 
used to establish and grade acute GvHD. 


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To determine the severity of acute GvHD, data will be collected approximately weekly to 
characterize the severity of symptoms and signs caused by GvHD and to evaluate possible 
confounding factors. Real time data collection will include descriptive characteristics of rash and 
estimated body surface area involved, extent of dermal/epidermal separation, identification of 
concomitant causes of increased bilirubin other than GvHD, presence or absence of nausea, 
vomiting or anorexia persistent after engraftment, peak diarrhea volume with annotations 
concerning the presence after engraftment, peak diarrhea volume with annotations concerning 
the presence or absence of urinary mixing and estimates of true diarrhea volume, presence or 
absence of abdominal cramps, presence or absence of frank stool blood or melena, concomitant 
causes of GI symptoms other than GvHD, biopsy results, identification of any agents used for 
treatment and autopsy results. 
Patients will be observed for acute and/or chronic GvHD as long as they have not received donor 
derived leukocytes infusion (DLI). If at any time, a patient receives DLI, that time will represent the 
end-time for evaluation of GvHD. Graft-versus-host disease occurring after DLI infusions will be 
analyzed separately. 
Patients with moderate to severe acute GvHD (grade II-IV)  will be treated in standard fashion with 
high-dose I.V. methylprednisolone (2-20 mg/kg/day) or in combination with other 
immunosuppressants as per ongoing trials on GvHD and at the discretion of the treating  
attending. Patients failing to respond to steroids will be considered for treatment with experimental 
treatments available at the time of diagnosis of GvHD. 
Chronic GvHD will be diagnosed and graded according to the NIH Consensus development 
project on criteria for clinical trials in chronic GVHD (65) treated with standard or experimental 
immunosuppressive therapy. Treatment will consist of corticosteroids, cyclosporin A or 
azathioprine, or combinations of these agents. Other novel treatments could be used if available, 
i.e. thalidomide and psoralen/ultraviolet  A phototherapy (PUVA). 
 
12.4. 
Transplant related mortality 
 
Transplant-related mortality (TRM) includes fatal complications resulting from the allogenic 
transplant and/ or treatment regimens such as graft failure, GvHD, hemorrhage, and infections. 
12.5. 
Infections 
 
The occurrence of life-threatening opportunistic infections will be evaluated according to the 
criteria established by BMT CTN and will correlate this with the level of immune recovery. The 
infection-related mortality will be also determined. Patients will be considered to have died from 
infection if death is attributed to a recent severe infection and/or infection was identified at 
autopsy. Patients with relapsed disease or GVHD before death will be excluded from the above 
definition, even if an infection was the final cause of death. 
 
12.6. 
Cellular Immunological reconstitution 
Immune recovery after transplantation with a αβ T-cell/CD-19 lymphocyte depleted graft will be 
evaluated by phenotypic and functional studies. B cell, T-cell subsets and NK cell analysis will be 
performed at baseline, weekly until day 100 or discharge, and monthly thereafter. Phenotypic 
analysis will be done by flow cytometry; analysis will be performed on mononuclear cells labeled 


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with mAb panels to allow identification of the main T cell subsets. Functional studies using T cell 
subsets shortly after collection and after in vitro expansion. 
12.7 Disease recurrence 
 
Disease recurrence would be considered if engraftment of donor hemopoietic cells have not been 
achieved and function or phenotypic abnormalities of hemopoietic or lymphoid lineages affected, 
occur. 
12.7 
Disease-free Survival 
 
DFS is defined as the minimum time interval of times to relapse/recurrence, to death or to the last 
follow-up, from the time of transplant. 
12.8 
Overall Survival 
 
Overall survival is defined as time from transplant to death or last follow-up. 
 
12.9 
CD34+ and CD3+ Cell Doses 
 
Total CD34+ and CD3+ cell doses will be calculated based on results of flow cytometric analysis. 
 
 
13.1 
CRITERIAFOR REMOVAL FROM STUDY 
 
Research participants may be removed from the study if requested by the research participant. 
Management will depend on where they are in their treatment course. Such research participants 
will receive appropriate supportive care. Patients may also be removed from the study at any 
point deemed appropriate by the principle investigator. 
 
Patients may be removed from study, and not followed further, if one or more of the following 
events occur: 
 
• 
Significant noncompliance on the part of the patient 
• 
Refusal of the patient to continue treatment or observations 
• 
Graft failure 
• 
Decision by the investigator that termination is in the patient’s best medical interest 
• 
Unrelated medical illness or complication 
• 
Lost to follow-up 
 
14.0 
BIOSTATISTICS 
 
This phase 2 trial is designed to assess the efficacy of αβ T-cell and B-cell depleted 
peripheral blood stem cell transplants for patients with non-malignant hematologic or 
immune disorders. The study population is divided into two groups defined by donor type: 
1) donors who are ≥ 7/8 HLA-antigen matched and 2) related donors who are 4-6/8 HLA- 
antigen matched. A maximum of 30 patients in each group is planned for accrual onto the 
study.  The primary endpoint of the study is two-year overall survival. It is anticipated that 
accrual will last three years and each patient will be followed for a minimum of two years  
if he/she has not failed. The data analysis and data monitoring will be conducted 
separately in each donor group. It is anticipated that accrual will last three years and each 


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patient will be followed for a minimum of two years if he/she has not failed. Patients lost to 
follow-up prior to the two-year endpoint will be counted as a failure for the purpose of the 
primary endpoint evaluation. The data analysis and data monitoring will be conducted 
separately in each donor group. 
 
For patients with donor type 1, a two-year overall survival (OS) rate greater than 0.75 would be 
considered a success. A single stage design that differentiates between two-year OS population 
rates of 0.75 and 0.92 will be used to assess treatment efficacy. At the conclusion of the study, if at 
least 26/30 patients survived for at least two years, then the treatment will be declared a success. 
The probability of declaring the treatment a success is 0.10 when the two-year OS rate in the 
population is 0.75 and increases to 0.91 when the two-year OS rate is 0.92. 
 
For patients with donor type 2, a two-year OS rate greater than 0.65 would be considered a success. 
A single stage design that differentiates between two-year OS population rates of 0.65 and 0.87 
will be used to assess treatment efficacy. At the conclusion of the study, if at least 24/30 patients 
survived for at least two years, then the treatment will be declared a success. The probability of 
declaring the treatment a success is 0.06 when the two-year OS rate in the population is 0.65 and 
increases to 0.91 when the two-year OS rate is 0.87. 
 
In order to reduce patient risk, the study design includes early termination in the event of excessive 
graft failure, acute graft versus host disease, or infection-related mortality during the accrual period. 
The stopping rules for excessive failure and the corresponding power calculations are derived 
separately for the two donor groups and are given in the tables below. The boundaries are based 
on a maximum accrual of 30 patients. In the event that the stopping boundary is cros sed for one 
donor group, the study will continue accrual in the other donor group. The calculations in the table 
are based on marginal binomial probabilities. 
 
 
 
 
 
 
 
 
Failure type 
Donor type 1 
# of failures needed 
Failure rate 
Probability boundary 
to stop the study 
in the population 
is crossed 
Graft failure 
2 in the first 26 patients 
3 within 30 patients 
0.02 
0.10 
0.15 
0.93 
Acute Graft 
Versus Host 
Disease 
(Grades 3-4) 
2 in the first 10 patients 
3 in the first 20 patients 
4 within 30 patients 
0.04 
0.09 
0.20 
0.91 
Infection-related 
Mortality 
2 in the first 26 patients 
3 within 30 patients 
0.02 
0.10 
0.15 
0.93 
 
 
Donor type 2 
 
# of failures needed 
Failure rate 
Probability boundary 
to stop the study 
in the population 
is crossed 
 
Failure type 
Graft failure 
2 in the first 26 patients 
0.02 
0.10 


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3 within 30 patients 
0.15 
0.93 
Acute Graft 
Versus Host 
Disease 
(Grades 3-4) 
3 in the first 10 patients 
4 in the first 20 patients 
5 within 30 patients 
0.07 
0.08 
0.25 
0.92 
Infection-related 
Mortality 
2 in the first 10 patients 
3 in the first 20 patients 
4 within 30 patients 
0.04 
0.09 
0.20 
0.91 
 
 
At the conclusion of the study, Kaplan-Meier estimates of overall and disease-free survival will be 
computed. The cumulative incidence function will be used to evaluate the cause-specific probability 
of graft failure, acute and chronic GvHD, infectious complications, and transplant-related mortality.  
At the outset of transplant procedure, the immune reconstitution parameters will be recorded and 
summarized over all patients. Over time post-transplant, nonparametric curve smoothing methods 
will be used to estimate the population  trajectory of the immunologic recovery parameters. Linear 
mixed models  will be employed  to compare the  recovery rates  to patients  who received CD34 
positively selected grafts as part of protocol 10-050. The Wilcoxon rank sum test will be used to 
compare the CD34+ cell doses for patients transplanted on this protocol to the cell doses of patients 
with non-malignant disorders who received CD34 positively selected grafts. 
 
15.1 
RESEARCH PARTICIPANT REGISTRATION AND RANDOMIZATION PROCEDURES 
 
15.2 
Research Participant Registration 
 
Confirm eligibility as defined in the section entitled Criteria for Subject Eligibility. 
 
Obtain informed consent, by following procedures defined in section entitled Informed 
Consent Procedures. 
During the registration process registering individuals will be required to complete a 
protocol specific Eligibility Checklist.  The individual signing the Eligibility Checklist is 
confirming is confirming whether or not the participant is eligible to enroll in the study. 
Study staff are responsible for ensuring that all institutional requirements necessary to 
enroll a participant to the study have been completed. See related Clinical Research 
Policy and Procedure #401 (Protocol Participant Registration). 
 
 
15.3 
Randomization 
 
This research study does not require a randomization. 
 
16.1 
DATA MANAGEMENT ISSUES 
 
A Clinical Research Coordinator (CRC)will be assigned to the study and will be 
responsible for both pediatric and adult accruals. The responsibilities of the CRC and 
principal investigator include project compliance, data collection, abstraction and entry, 
data reporting, regulatory monitoring, problem resolution and prioritization, and coordinate 
the activities of the protocol study team.  The data collected for this study will be entered 


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into The Clinical Research Data Base (CRDB), a secure database.  Source documentation 
will be available to support the computerized patient record. 
16.2 
Quality Assurance 
 
Weekly registration reports will be generated to monitor patient accruals and 
completeness of registration data.  Routine data quality reports will be generated to 
assess missing data and inconsistencies.  Accrual rates and extent and accuracy of 
evaluations and follow-up will be monitored periodically throughout the study period and 
potential problems will be brought to the attention of the study team for discussion and 
action. 
Random-sample data quality and protocol compliance audits will be conducted by the 
study team, at a minimum of two times per year, more frequently if indicated. 
16.3 
Data and Safety Monitoring 
 
The Data and Safety Monitoring (DSM) Plans at Memorial Sloan-Kettering Cancer Center 
were approved by the National Cancer Institute in September 2001.  The plans address  
the new policies set forth by the NCI in the document entitled “Policy of the National 
Cancer Institute for Data and Safety Monitoring of Clinical Trials” which can be found at: 
http://cancertrials.nci.nih.gov/researchers/dsm/index.html.    The DSM Plans at MSKCC 
were established and are monitored by the Clinical Research Administration.  The MSKCC 
Data and Safety Monitoring Plans can be found on the MSKCC Intranet at: 
http://mskweb2.mskcc.org/irb/index.htm 
There are several different mechanisms by which clinical trials are monitored for data, 
safety and quality.  There are institutional processes in place for quality assurance (e.g., 
protocol monitoring, compliance and data verification audits, therapeutic response, and 
staff education on clinical research QA) and departmental proc edures for quality control, 
plus there are two institutional committees that are responsible for monitoring the activities 
of our clinical trials programs.  The committees: Data and Safety Monitoring Committee 
(DSMC) for Phase I and II clinical trials, and the Data and Safety Monitoring Board 
(DSMB) for Phase III clinical trials, report to the Center’s Research Council and  
Institutional Review Board. 
During the protocol development and review process, each protocol will be assessed for 
its level of risk and degree of monitoring required.  Every type of protocol (e.g., NIH 
sponsored, in-house sponsored, industrial sponsored, NCI cooperative group, etc.) Will be 
addressed and the monitoring procedures will be established at the time of protocol 
activation. 


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17.1 
PROTECTION OF HUMAN SUBJECTS 
 
The risks associated with a T-cell depleted transplant after cytoreduction by any of the two 
conditioning regimens under study are those associated with the toxicities of the conditioning 
regimens, as detailed in Section 11.0, as well as the risks of an allogeneic transplant, 
particularly graft failure, or graft vs. host disease, as also detailed in Section 11.0. 
To protect against the toxicities of the cytoreductive regimens, the patient will be transplanted 
in a single room, HEPA filtered environment. Organ toxicities such as mucositis, enteritis and 
hepatic dysfunction as well as infectious complications will be treated by standard  
procedures developed for transplantation to support our patients.  Blood and platelet counts 
will be supported by transfusion.  Graft failure might necessitate a second transplant, after 
additional conditioning.  Approaches to the diagnosis and treatment of graft failure that  
secure consistent engraftment have been developed by the Transplantation Services. 
Similarly, advanced treatments will be instituted in the event the patient develops graft vs. 
host disease. 
Despite a transplant, the patient’s disease may recur.  In this case, standard and/or 
experimental therapies, such as phase I/II drugs, antibodies or cell therapies, will be 
available to the patient for consideration as treatment options. 
Benefits: 
 
A transplant is administered with curative intent.  The approaches being evaluated may 
achieve this goal and may also be effective in preventing acute and chronic graft vs. host 
disease. 
The results of this study will also define risks and benefits of T-cell depleted grafts 
fractionated on the CliniMACS device, when administered after each of the cytoreduction 
regimen proposed for study. This may greatly accelerate further development of 
transplantation approaches employing this approach to T-cell depletion. 
Consent Process: Participation in this study is voluntary. All patients will be required to sign a 
statement of informed consent which must conform to MSKCC IRB guidelines. 
Alternatives:  Enrollment in this study is voluntary. Alternative treatment options will be 
presented to the patient prior to taking part in this study. Alternative treatment options may 
include getting a transplant from a volunteer unrelated donor, if one is available; getting 
treatment with transplant without being on a study; taking part in another study; or getting no 
treatment. 
Costs: The patient’s health plan/insurance company will need to pay for all of the costs of 
treatment in this study. The patient will be responsible for the costs of standard medical care, 
all hospitalizations and any transplant complications. Pre-authorization for the transplant will 
be cleared with the health plan/insurance company prior to admission. Patients will not be 
paid for taking part in this study. Research tests will be done at no cost to the patient. 


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Confidentiality: Every effort will be made to maintain patient confidentiality. Research and 
hospital records are confidential. 
17.2 
Privacy 
 
MSK’s Privacy Office may allow the use and disclosure of protected health information 
pursuant to a completed and signed Research Authorization form. The use and disclosure of 
protected health information will be limited to the individuals described in the Research 
Authorization form. A Research Authorization form must be completed by the Principal 
Investigator and approved by the IRB and Privacy Board (IRB/PB). 
The consent indicates that individualized  de identified information collected for the purposes 
of this study may be shared with other qualified researchers. Only researchers who have 
received approval from MSK will be allowed to access this information which will not include 
protected health information, such as the participant’s name, except for dates. It is also 
stated in the Research Authorization that their research data may be shared with other 
qualified  researchers. 
17.3 
Serious Adverse Event (SAE) Reporting 
 
An adverse event is considered serious if it results in ANY of the following outcomes: 
 
• 
Death 
• 
A life-threatening adverse event 
• 
An adverse event that results in inpatient hospitalization or prolongation of existing 
hospitalization 
• 
A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
A congenital anomaly/birth defect 
• 
Important Medical Events (IME) that may not result in death, be life threatening, or 
require hospitalization may be considered serious when, based upon medical judgment, 
they may jeopardize the patient or subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition 
 
Note: Hospital admission for a planned procedure/disease treatment is not considered an 
SAE. 
 
SAE reporting is required as soon as the participant signs consent. SAE reporting is 
required for 30-days after the participant’s last investigational treatment or intervention. Any 
events that occur after the 30-day period and that are at least possibly related to protocol 
treatment must be reported. 
 
If an SAE requires submission to the IRB office per IRB SOP RR-408 ‘Reporting of Serious 
Adverse Events’, the SAE report must be sent to the IRB within 5 calendar days of the event. 
The IRB requires a SAE report be submitted electronically to the SAE Office as follows: 


Page 48 of 55 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
 
 
 
All SAEs must be submitted in PIMS. If an SAE requires submission to the Human Research 
Protection Program (HRPP) office per IRB SOP RR-408 ‘Reporting of Serious Adverse 
Events’, the SAE report must be submitted within 5 calendar days of the event.  All other 
SAEs must be submitted within 30 calendar days of the event. 
 
The report should contain the following information: 
 
• 
The date the adverse event occurred 
• 
The adverse event 
• 
The grade of the event 
• 
Relationship of the adverse event to the treatment(s) 
• 
If the AE was expected 
• 
Detailed text that includes the following 
o An explanation of how the AE was handled 
o A description of the participant’s condition 
o Indication if the participant remains on the study 
• 
If an amendment will need to be made to the protocol and/or consent form 
• 
If the SAE is an Unanticipated Problem 
 
 
 
The SAE report should be completed as per above instructions. If appropriate, the report will 
be forwarded to the FDA by the IND Office 
 
 
 
18.0 
INFORMED CONSENT PROCEDURES 
 
Before protocol-specified procedures are carried out, consenting professionals will explain  
full details of the protocol and study procedures as well as the risks involved to participants 
prior to their inclusion in the study. Participants will also be informed that they are free to 
withdraw from the study at any time. All participants must sign an IRB/PB-approved consent 
form indicating their consent to participate. This consent form meets the requirements of the 
Code of Federal Regulations and the Institutional Review Board/Privacy Board of this Center. 
 
The consent form will include the following: 
 
1. The nature and objectives, potential risks and benefits of the intended study. 
2. The length of study and the likely follow-up required. 
3. Alternatives to the proposed study (This will include available standard and 
investigational therapies; additionally, patients will be offered an option of supportive 
care). 
4. The name of the investigator(s) responsible for the protocol. 
5. The right of the participant to accept or refuse study interventions/interactions and to 
withdraw from participation at any time. 
Before any protocol-specific procedures can be carried out, the consenting professional will 
fully explain the aspects of patient privacy concerning research specific information.  In 


Page 49 of 55 
Memorial Sloan Kettering Cancer Center 
IRB Number: 17-596 A(2) 
Approval date: 02-Oct-2019 
 
 
 
addition to signing the IRB Informed Consent, all patients must agree to the Research 
Authorization component of the informed consent form. 
Each participant and consenting professional will sign the consent form. The participant must 
receive a copy of the signed informed consent form. 
 
 
 
 
19.0 
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Memorial Sloan Kettering Cancer Center 
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27. 
Mussetti A, Kernan NA, Prockop SE, Scaradavou A, Lehrman R, Ruggiero JM, et al. 
Allogeneic  hematopoietic stem cell transplantation for nonmalignant hematologic disorders using 
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28. 
Booth C, Veys P. T cell depletion in paediatric stem cell transplantation. Clinical and 
experimental immunology. 2013;172(2):139-47. 
29. 
Jakubowski AA, Small TN, Kernan NA, Castro-Malaspina H, Collins N, Koehne G, et al. T 
cell-depleted unrelated donor stem cell transplantation provides favorable disease-free survival for 


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Memorial Sloan Kettering Cancer Center 
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Approval date: 02-Oct-2019 
 
 
 
adults with hematologic malignancies. Biology of blood and marrow transplantation : journal of the 
American Society for Blood and Marrow Transplantation. 2011;17(9):1335-42. 
30. 
Mattsson J, Ringden O, Storb R. Graft failure after allogeneic hematopoietic cell 
transplantation. Biology of blood and marrow transplantation : journal of the American Society for 
Blood and Marrow Transplantation. 2008;14(1 Suppl 1):165-70. 
31. 
Champlin RE, Horowitz MM, van Bekkum DW, Camitta BM, Elfenbein GE, Gale RP, et al. 
Graft failure following bone marrow transplantation for severe aplastic anemia: risk factors and 
treatment results. Blood. 1989;73(2):606-13. 
32. 
Storb R, Prentice RL, Thomas ED, Appelbaum FR, Deeg HJ, Doney K, et al. Factors 
associated with graft rejection after HLA-identical marrow transplantation for aplastic anaemia. 
British journal of haematology. 1983;55(4):573-85. 
33. 
Aversa F, Tabilio A, Terenzi A, Velardi A, Falzetti F, Giannoni C, et al. Successful 
engraftment of T-cell-depleted haploidentical "three-loci" incompatible transplants in leukemia 
patients by addition of recombinant human granulocyte colony-stimulating factor-mobilized 
peripheral blood progenitor cells to bone marrow inoculum. Blood. 1994;84(11):3948-55. 
34. 
Tolar J, Deeg HJ, Arai S, Horwitz M, Antin JH, McCarty JM, et al. Fludarabine-based 
conditioning for marrow transplantation from unrelated donors in severe aplastic anemia: early 
results of a cyclophosphamide dose deescalation study show life-threatening adverse events at 
predefined cyclophosphamide dose levels. Biology of blood and marrow transplantation : journal of 
the American Society for Blood and Marrow Transplantation. 2012;18(7):1007-11. 
35. 
Sodani P, Gaziev D, Polchi P, Erer B, Giardini C, Angelucci E, et al. New approach for bone 
marrow transplantation in patients with class 3 thalassemia aged younger than 17 years. Blood. 
2004;104(4):1201-3. 
36. 
Locatelli F, Bauquet A, Palumbo G, Moretta F, Bertaina A. Negative depletion of alpha/beta+ 
T cells and of CD19+ B lymphocytes: a novel frontier to optimize the effect of innate immunity in 
HLA-mismatched hematopoietic stem cell transplantation. Immunology letters. 2013;155(1-2):21-3. 
37. 
Kharya G, Nademi Z, Leahy TR, Dunn J, Barge D, Schulz A, et al. Haploidentical T-cell alpha 
beta receptor and CD19-depleted stem cell transplant for Wiskott-Aldrich syndrome. The Journal of 
allergy and clinical immunology. 2014;134(5):1199-201. 
38. 
Bertaina A, Merli P, Rutella S, Pagliara D, Bernardo ME, Masetti R, et al. HLA-haploidentical 
stem cell transplantation after removal of alphabeta+ T and B cells in children with nonmalignant 
disorders. Blood. 2014;124(5):822-6. 
39. 
Merli P, Li Pira G, Bertaina V, Sinibaldi M, Lucarelli B, Brescia LP, et al. Immune 
Reconstitution after Adoptive Infusion of BPX501 Cells (donor T cells transduced with iC9 suicide 
gene) in Children Given Alpha/Beta T-Cell Depleted HLA-Haploidentical  Hematopoietic Stem Cell 
Transplantation (haplo-HSCT): Preliminary Phenotypic and Functional Results of a Phase I-II Trial. 
Blood.  2015;126(23):3093-. 
40. 
Lang P, Feuchtinger T, Teltschik H-M, Schumm M, Schlegel P, Pfeiffer M, et al. 
Transplantation Of TcRαβ/CD19 Depleted Stem Cells From Haploidentical  Donors In Children: 
Current Results. Blood. 2013;122(21):692-. 
41. 
Witte Md, der Wagen Lv, Slaper-Cortenbach I, Kuball J. Alpha/Beta T-cell Depleted 
Allogeneic Stem Cell Transplantation from Matched Related and Unrelated Donor Grafts in Patients 
with Poor Risk Leukemia. Biology of Blood and Marrow Transplantation.21(2):S277-S8. 
42. 
van der Wagen L, te Boome LC, Nijhof I, Schoordijk M, Buter D, Meijer E, et al. Treatment of 
Chronic Graft Versus Host Disease with a Combination of B-Cell Depletion and Tyrosine Kinase 
Inhibition.  Blood. 2015;126(23):1939-. 
43. 
Bertaina A, Lucarelli B, Masetti R, Merli P, Rondelli R, Li Pira G, et al. Children with Acute 
Leukemia Given Hematopoietic Stem Cell Transplantation (HSCT) from an HLA-Compatible Sibling, 
or an Unrelated Donor (UD) or an HLA-Haploidentical  Relative after Alpha/Beta T-Cell Depletion 
Have a Comparable Outcome. Blood. 2015;126(23):195-. 
44. 
Ortin M, Raj R, Kinning E, Williams M, Darbyshire PJ. Partially matched related donor 
peripheral blood progenitor cell transplantation in paediatric patients adding fludarabine and anti- 
lymphocyte gamma-globulin. Bone marrow transplantation.  2002;30(6):359-66. 


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45. 
Klingebiel T, Cornish J, Labopin M, Locatelli F, Darbyshire P, Handgretinger R, et al. Results 
and factors influencing outcome after fully haploidentical hematopoietic stem cell transplantation in 
children with very high-risk acute lymphoblastic leukemia: impact of center size: an analysis on 
behalf of the Acute Leukemia and Pediatric Disease Working Parties of the European Blood and 
Marrow Transplant group. Blood. 2010;115(17):3437-46. 
46. 
Lang P, Greil J, Bader P, Handgretinger R, Klingebiel T, Schumm M, et al. Long-term 
outcome after haploidentical stem cell transplantation in children. Blood cells, molecules & diseases. 
2004;33(3):281-7. 
47. 
Marks DI, Khattry N, Cummins M, Goulden N, Green A, Harvey J, et al. Haploidentical stem 
cell transplantation for children with acute leukaemia. British journal of haematology. 
2006;134(2):196-201. 
48. 
Koehl U, Bochennek K, Zimmermann SY, Lehrnbecher T, Sorensen J, Esser R, et al. 
Immune recovery in children undergoing allogeneic stem cell transplantation: absolute CD8+ CD3+ 
count reconstitution is associated with survival. Bone marrow transplantation. 2007;39(5):269-78. 
49. 
Lang P, Feuchtinger T, Teltschik HM, Schwinger W, Schlegel P, Pfeiffer M, et al. Improved 
immune recovery after transplantation of TCRalphabeta/CD19-depleted allografts from 
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Handgretinger R, Lang P, Feuchtinger TF, Schumm M, Teltschik H-M, Schulz AS, et al. 
Transplantation of TcRαβ/CD19 Depleted Stem Cells From Haploidentical Donors: Robust 
Engraftment and Rapid Immune Reconstitution In Children with High Risk Leukemia. Blood. 
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Oevermann L, Lang P, Feuchtinger T, Schumm M, Teltschik HM, Schlegel P, et al. Immune 
reconstitution and strategies for rebuilding the immune system after haploidentical stem cell 
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Dominietto A, Tedone E, Soracco M, Bruno B, Raiola AM, Van Lint MT, et al. In vivo B-cell 
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Lang PJ, Schlegel PG, Meisel R, Schulz AS, Greil J, Bader P, et al. TCR-Alpha/Beta and 
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Papadopoulos EB, Ladanyi M, Emanuel D, Mackinnon S, Boulad F, Carabasi MH, et al. 
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Kernan NA, Bordignon C, Heller G, Cunningham I, Castro-Malaspina H, Shank B, et al. Graft 
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Bordignon C, Keever CA, Small TN, Flomenberg N, Dupont B, O'Reilly RJ, et al. Graft failure 
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Rowlings PA, Przepiorka D, Klein JP, Gale RP, Passweg JR, Henslee-Downey PJ, et al. 
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Jagasia MH, Greinix HT, Arora M, Williams KM, Wolff D, Cowen EW, et al. National Institutes 
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2015;21(3):389-401  e1. 


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20.0 
APPENDICES 
 
Table I: Alpha beta T -Cell Depleted stem cell transplants for Non-Malignant Disorders (+/- 
malignant) 
 
CD19 depleted 
Donor 
N 
ATG 
Conditioning 
Intensity 
GvHD PPx 
Rituximab 
Engraftment/Graft 
failure 
GVHD a/c 
Infection 
TRM 
OS 
Karakukcu 
M 
EBMT Abs 
2016 
Both 
(NMHD13) 
Ped 
No 
Haplo 
34 
ATG 
Flu/Mel/Thio 
RIC 
MMF if αβ 
>2.5x104/k 
g 
(50% of 
all) 
Yes 
11% 
D-1 
Engraft 
(D+12) 
3% graft 
failure 
20% (II 
skin) 
12-15% 
(III-IV 
GI/liver) 
80% CMV react 
15% of CMV react 
had EBV react 
14.7% 
(GvHD, 
sepsis, 
relapsed)
64.7% 
Yadav SP 
EBMT Abs 
2016 
NMHD 
(FA, WAS, 
Thal in 
Ped/adult) 
Yes 
Haplo 
4 
(+2 req 
2nd graft) 
ATG- 
T(2/4) 
TBI/Flu/Cy +/- thio – 
failed x 2 pts. 
Campath/Flu/Cy 
Campath/Flu/Treo/Thio 
( doses not specified) 
RIC 
No 
No 
2/4 failed 50% (II) 
No 
chronic 
75% CMV react, 
25% TMA 
25% JC enceph 
25% BK encep & 
emorrhagic cystiti
25% 
(BK ) 
75% 
Mainardi C 
Letter to 
editor BMT
2016 
(Hurler) 
Yes 
MUD 
1 
ATG (? 
Dose) 
Flu/Treo/Mel 
RIC 
MMF <2M 
No 
Engraft 
on 
D+14 
“ mild GI” CMV reactivation 
0 
Lang P 
BMT 2015 
Both (5 
NMHD) 
Ped 
Yes 
Haplo 
41 
ATG-F 15 
mg/kg (1- 
4-5-5 on D- 
12 to -9) or 
OKT-3 
(pre 2012) 
Flu 40 mg/m2/day or 
Clo 50 mg/m2/day (D-8
to -5) 
Mel 70 mg/m2/day (D-3
to -2) 
Thio 10 mg/kg D-4 
MAC/RI 
C 
MMF until 
D+30 
No 
88% 
Engraft 
(+D10) 
Rej 12% 
76% (0-I) 
10%(II) 
15%(III or 
IV) 
Chronic: 
limited 
- 
(focuses on immune
reconstitution) 
48% died
(17/41 
died of 
relapse) 
51.2% 
(relapsed) 


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*peak level 
of ATG 
18%; 
extensive 
9% 
Airoldi I 
Blood 2015
Both 
(12NMHD) 
Ped 
Yes 
Haplo 
27 
r-ATG (D- 
5 to -3) 
Treo/Flu +/-Thio 
Treo/Thio/Mel 
TBI/Cy/Flu 
Bu/Thio/Flu 
Bu/Cy/Mel 
MA 
No 
Yes 
D-1 
- 
- 
CMV reac 55% 
1 from 
Adeno 
81% (all) 
100% in 
NMHD 
Tumino M 
Letter to 
BMT 2014 
(SAA in 
ped) 
Yes 
Haplo 
1 
ATG-T 
Thio 10 mg/kg, Cy 200 
mg/kg, 
TLI 7.5Gy 
MA 
CSA if αβ 
>2.5x104 
No 
Engraft 
D+14 
No 
CMV reactivation 
Invasive 
spergillosis (lung 
No 
Kharya G 
Allergy and
Clinical 
Immunol 
2014 
(WAS) 
Yes 
Haplo 
1 
ATG- 
F(15mg/Kg 
) 
Bu (AUC 80 mg/L h), 
Flu 160/m2, 
Thio 15mg/kg 
RIC 
CSA+MM 
F 
No 
Engraft 
D+15 
- 
CMV reacti 
No 
100% 
Bertaina 
Blood 2014 
NMHD Ped 
Yes 
Haplo 
(KIR-B 
preferred) 
23 
ATG-F 4 
mg/kg/day 
(D-5 to -3) 
Bu/Thio/Flu 
Treo/Thio/Flu 
Treo/Flu (SCID) 
Flu/Cy+/-TBI  (single 
dose at 200 cGy) (SAA 
or FA) 
RIC 
No 
Yes 
D-1 
200 
mg/m2 
82% 
engraft 
(D+13) 
20% 
graft 
failure 
13% (I-II) 
no cGvHD
CMV+Adeno=38% 
9.3% 
91.3%