TCRαβ-depleted Progenitor Cell Graft With Additional Memory T-cell DLI, Plus Selected Use of Blinatumomab, in Naive T-cell Depleted Haploidentical Donor Hematopoietc Cell Transplantation for Hematologic Malignancies
Phase: PHASE2
Conditions: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Myelodysplastic Syndromes (MDS), NK-Cell Leukemia, Hodgkin Lymphoma, Non Hodgkin Lymphoma (NHL), Juvenile Myelomonocytic Leukemia (JMML), Chronic Myeloid Leukemia (CML)
Sponsor: St. Jude Children's Research Hospital

Official Study Title: TCRαβ-DEPLETED PROGENITOR CELL GRAFT WITH
ADDITIONAL MEMORY T-CELL DLI, PLUS SELECTED USE OF BLINATUMOMAB,
IN NAIVE T-CELL DEPLETED HAPLOIDENTICAL DONOR HEMATOPOIETC CELL
TRANSPLANTATION FOR HEMATOLOGIC MALIGNANCIES
CTG Number: NCT03849651
Document Date: 03/01/2024

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
St. Jude
HAP2HCT
Initial version dated: 03/28/2018 (IRB Approved: 01/09/2019) Activated: 1/17/2019
Amendment 1.0: 2/05/2020 (IRB Approved: 3/10/2020) Activated: 04/23/2020
Letter of Amendment 1.0: 03/24/2020 (IRB approved: 04/02/2020)
Amendment 2.0: 03/30/2021 (IRB Approved: 05/11/2021) Activated: superseded
Revision 2.1: 06/18/2021 (IRB Approved: 07/21/2021) Activated: 09/21/2021
Amendment 3.0 dated: 07/25/2022 (IRB Approved: 01/06/2023) Activated: 03/08/2023
Revision 3.1 dated: 04/07/2023 (IRB Approved: 04/19/2023 ) Activated: 04/21/2023
Amendment 4.0 dated: 08/07/2023 (IRB Approved: 10/10/2023) Activated: 12/15/2023
Amendment 5.0 dated: 03/01/2024 (IRB Approved: ) Activated:
TCRαβ-DEPLETED PROGENITOR CELL GRAFT WITH ADDITIONAL MEMORY T-
CELL DLI, PLUS SELECTED USE OF BLINATUMOMAB, IN NAÏVE T-CELL
DEPLETED HAPLOIDENTICAL DONOR HEMATOPOIEITC CELL
TRANSPLANTATION FOR HEMATOLOGIC MALIGNANCIES
IDE #16971
Principal Investigator
Brandon M. Triplett, MD1
Sub-Investigators
Stephen Gottschalk, MD1
Subodh Selukar, PhD 2
Ewelina Mamcarz, MD1
Akshay Sharma, MD1
Ashok Srinivasan, MD1
Salem Akel, PhD3
Renee Madden, MD1
Paul Thomas, PhD4
Amr Qudeimat, MD1
Ben Youngblood, PhD4
Aimee Talleur, MD1
Kristine Crews, PharmD5
Ali Suliman, MD1
Diana Wu, PharmD5
Swati Naik, MD1
Paulina Velasquez, MD1
Esther Obeng, MD, PhD6
Rebecca Epperly, MD1
Caitlin Zebley, MD, PhD1
Senthil Bhoopalan, MBBS, PhD1
Department of Bone Marrow Transplantation and Cellular Therapy1
Department of Biostatistics2
Human Applications Lab3
Department of Immunology4
Department of Pharmaceutical Sciences5
Department of Oncology6
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
(901) 595-3300
This is a confidential research document. No information may be extracted without permission of the principal investigator.

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
TABLE OF CONTENTS
Study Summary
1.0
OBJECTIVES…………………………………….…………………………
1
1.1
Primary Objectives………………………………………………..………………..…
1
1.2
Secondary Objectives……………………………………………….………………….
1
1.3
Exploratory Objectives…………………………………………….…………………..
1
2.0
BACKGROUND AND RATIONALE……………..…………………………………
2
2.1
Overview………………………………………………………………………………..
2
2.2
Haploidentical HCT……………………………………………………………………..
2
2.3
Rationale for Immunomagnetic T Cell Depletion of Hematopoietic Progenitor Cell
Graft…………………………………………………………………………………….
4
2.4
Rationale for CD45RA-depleted DLI………………………………………………….
5
2.5
Rationale for the Reduced Intensity Regimen………………………………………….
9
2.6
Rationale for Blinatumomab………………………………………………………..…..
11
2.7
Minimal Residual Disease………………………………………………………………
13
2.8
Rationale for the Present Study…………………………………………………………
13
2.9
Rationale for Early DLI Cohort in Amenment 5……………………………………….
14
3.0
RESEARCH PARTICIPANT ELIGIBILITY CRITERIA AND STUDY
ENROLLMENT……………………………………………………………………....
15
3.1
Inclusion Criteria for Transplant Recipient..…………………………………………..
15
3.2
Inclusion Criteria for Haploidentical Donor…..………………………………………
17
3.3
Research Participant Recruitment and Screening……………………………………..
17
3.4
Enrollment on Study at St. Jude……………………………………………………….
18
4.0
TREATMENT PLAN…………………………………………………………………
18
4.1
Preparative Regimen…………………………………………………………………..
18
4.2
Blinatumomab Administrations……………………………………………………….
20
4.3
Donor Selection………………………………………………………………………..
22
4.4
Donor Mobilization and Graft Collections …………………………………………….
23
4.5
Progenitor Cell Graft Preparation………..……………………………………………
24
4.6
Additional Progenitor Cell Graft Administration ……………………………………..
25
4.7
Donor Lymphocyte Infusions………………………………………………………….
25
4.8
Quality Assurance of Cellular Products ………………………………………………..
29

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
5.0
DRUG/DEVICE/BIOLOGIC AGENT INFORMATION………….………………….
29
5.1
Medications.…………………….…………………………………..………………..…
29
5.2
CliniMACSTM System…………………………….…..………………….…………….
36
6.0
REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS……………………..
37
6.1
Schedule of Evaluations………………………… …………………………………….
37
6.2
Long-term Follow-up Evaluations………………...…………………………………..
37
6.3
Evaluation for Chimerism and Engraftment …………………………………………..
37
6.4
Evaluation for Immune Reconstitution …………………………………….………….
38
6.5
Evaluations for ATG pharmacokinetics testing (optional) ……...…………………….
38
6.6
Minimal Residual Disease Evaluations…………..……………………………………
38
6.7
Research Tests on Haploidentical Donor………………………………………………
39
7.0
EVALUATION CRITERIA………...………………………………………………….
39
7.1
Toxicity Evaluation Criteria…………………………………………………………..
39
7.2
Hematologic Recovery Criteria……………………………………………………….
39
7.3
Graft failure Criteria…………………………………………………………………..
39
8.0
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY
CRITERIA………………………………………………………………………………
40
8.1
Recipient Criteria……………………………………………………………………..
40
8.2
Off therapy Criteria……………………………………………………………………..
40
8.3
Donor Criteria…………………………………………………………………………..
40
9.0
SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS………………..
41
9.1
Reporting Adverse Experiences and Deaths to St. Jude IRB………………………..
41
9.2
Reporting to St. Jude Institutional Biosafety Committee ………………………………
42
9.3
Reporting to FDA………………………………………………………………………
43
9.4
Reporting to St. Jude Office of Regulatory Affairs …………………………………..
43
9.5
Continuing Review Reports……………………………………………………………
43
9.6
Reporting to Miltenyi Biotec…………………………………………………………
44
9.7
Reporting to the CIBMTR……………………………………………………………..
44
9.8
Data and Safety Monitoring Board ……………………………………………………
44

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
10.0
DATA COLLECTION, STUDY MONITORING, AND CONFIDENTIALITY………
45
10.1
Data Collection and Submission……………………………………….……..……….
45
10.2
Study Monitoring…………………………..……………………………….………….
45
10.3
Confidentiality………………………………………………………….………………
45
10.4
Genomic Data Sharing………………………………………………………………….
45
11.0
STATISTICAL CONSIDERATIONS……………..……………..……….……………
46
11.1
Statistical Design and Analysis for the Primary Objectives and Stopping Rules ….…
46
11.2
Statistical Analysis for Secondary Objectives …………………..…………………….
51
11.3
Analysis for Exploratory Objectives …………………………………………………..
52
11.4
Statistical analysis for the ‘Early DLI’ cohort ………………………………………...
53
12.0
OBTAINING INFORMED CONSENT…………………………………………………
55
12.1
Informed Consent Prior to Research Interventions…………………………………….
55
12.2
Consent at Age of Majority…………………………………………………………….
55
12.3
Consent When English is Not the Primary Language………………………………….
55
13.0
References………………………………………………………………………………
56
A
Appendix A……………………..……………………………………….………………
59
B
Appendix B……………………..……………………………………….………………
60
C
Appendix C……………………..……………………………………….………………
60
D
Appendix D……………………..……………………………………….………………
61
E
Appendix E………………………………………………………………………………
65

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
STUDY SUMMARY
Protocol Title
TCRαβ-depleted progenitor cell graft with additional memory T-cell DLI, plus selected use of
blinatumomab, in naïve T-cell depleted haploidentical donor hematopoietic cell transplantation
for hematologic malignancies
Principal Investigator:
Brandon M. Triplett, MD
Brief Overview
Study Population:
Patients less than or equal to 21 years old with high-risk hematologic malignancies who would
likely benefit from allogeneic hematopoietic cell transplantation (HCT). Patients with a suitable
HLA matched sibling or unrelated donor identified will be eligible for participation ONLY if the
donor is not available in the necessary time.
Additional eligibility criteria are specified to assure sufficient multi-organ system function.
Intervention, Brief Outline, and Objectives of Treatment Plan:
In this study, participants with high-risk hematologic malignancies undergoing hematopoietic
cell transplantation (HCT), who lack an available suitable human leukocyte antigen (HLA)
matched related/sibling donor (MSD) or matched unrelated donor (MUD), will receive a TCRαβ-
depleted haploidentical donor HCT with additional memory cell DLI. One course of
blinatumomab will be empirically added for patients with CD19+ malignancy.
The assessments and follow-up evaluations noted in the protocol follow the St. Jude standard
operating procedures (SOP) for all recipients of allogeneic HCT.
The primary objective of the study for the original cohort is to determine the minimum effective
dose for prophylactic CD45RA-depleted DLI when given in the early post-engraftment period. In
addition, we will assess efficacy of TCRαβ-depleted progenitor cellgraft with additional memory
T cell DLI, plus selected use of blinatumomab, in haploidentical donor hematopoietic cell
transplantation for hematologic malignancies as measured by 1 year EFS (events = relapse, death).
For the Amendment 5 expansion (early DLI) cohort, the primary objective is to assess the
feasibility and safety of early CD45RA-depleted DLI administration.
Secondary and Exploratory aims will assess overall survival, event-free survival, risk of relapse,
graft versus host disease (GVHD), transplant related mortality (TRM), transplant related
morbidity, and immune reconstitution.

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
IND/IDE:
IDE 16971
Criteria for Evaluation – Safety and Efficacy
Safety:
The primary measures of safety will be the rate of therapy related death and the rate of severe
graft versus host disease.
Ongoing assessment of toxicity will be done using the NCI CTCAE version 5
Acute and chronic graft-vs.-host disease will be evaluated using established staging/grading
criteria and expert consensus guidelines.
Efficacy:
The primary measure of efficacy for memory T-cell DLI will be achievement of a peripheral
blood donor memory T-cell count of at least 300/µL at 4 weeks following the prophylactic DLI.
The primary measure of efficacy for the trial will be event-free survival at one year (events =
relapse, death)
Neutrophil and platelet engraftment will be determined using the parameters put forth by the
Center for International Blood and Marrow Transplant Research. Assessments will be made upon
review of daily complete blood count and serial (typically weekly) chimerism studies.
Bone marrow studies for disease status evaluation will be performed at approximately 21 days,
100 days, and 1 year post-transplant. Testing will include evaluations for minimal residual disease.
Immune reconstitution will be determined by standard blood testing of lymphocyte subsets.
Research tests required for participation will examine lymphocyte phenotype and function.
Statistical Considerations and Data Analysis
Study Design: Phase II
Randomization: No
Sample Size: Initial study: Up to 70; Early DLI cohort: Up to 30
Data Analyses:
Anticipated primary completion date: 12/31/2026
Anticipated study completion date: 12/31/2027
Time frame for primary outcome measure: 1 year post-transplant

Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Data Management including statistical evaluations
Protocol compliance, data collection including safety data, and reporting will be carried out by
the Department of Bone Marrow Transplantation and Cellular Therapy Research Office.
Statistical considerations and ongoing analysis will be conducted by Dr. Subodh Selukar and
designated associates within the St. Jude Department of Biostatistics.
Human Subjects:
The risks to participants are primarily related to the cellular infusions and the conditioning
regimen. The proposed regimen and donor cells may induce serious and possibly fatal disorders
such as GVHD, veno-occlusive disorder, and post-transplant lymphoproliferative disease.
Because of the required conditioning, recipients are at high-risk for serious and possibly life-
threatening infection, bleeding, and anemia. Adverse events will be treated, monitored, and
reported appropriately.
Possible benefits of participation include obtaining and/or sustaining disease remission. In
addition, there is the possibility of psychological benefit from knowing participation has helped
researchers gain more understanding about the efficacy of haploidentical HCT.
Alternatives
to
participation
are
identified
as
conventional
(standard)
transplantation,
chemotherapy without transplant, including palliative therapy, other research treatment if
available, and/or supportive therapy alone.
The possible benefits, alternatives to participation, and side effects, including that there may be
unknown side effects of treatment, are detailed in lay language within the respective informed
consent document.

HAP2HCT 1
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
1.0 OBJECTIVES
1.1 Primary Objective
1.1.1 Determine the maximum effective dose for prophylactic CD45RA-depleted DLI
when given in the early post-engraftment period.
1.1.2 Assess the efficacy of TCRαβ-depleted progenitor cell graft with additional memory
T-cell DLI, plus selected use of blinatumomab, in haploidentical donor hematopoietic
cell transplantation for hematologic malignancies as measured by 1 year EFS (events
= relapse, death)
1.1.3 Assess the feasibility and safety of early CD45RA-depleted DLI administration in
the Amendment 5 expansion (early DLI) cohort
1.2 Secondary Objectives
1.2.1 Assess the safety and feasibility of the addition of blinatumomab in the early post-
engraftment period in patients with CD19+ malignancy.
1.2.2 Estimate the incidence of neutrophil and platelet engraftment, malignant relapse,
event-free survival per disease subgroups (e.g. ALL vs AML), and overall survival
at one-year post-transplantation.
1.2.3 Estimate incidence and severity of acute and chronic (GVHD).
1.2.4 Estimate the rate of transplant related mortality (TRM) in the first 100 days after
transplantation.
1.2.5 To measure and describe the pharmacokinetics of rabbit ATG in HCT recipients on
this study.
1.3 Exploratory Objectives
1.3.1
Record
immune
reconstitution
parameters,
including
chimerism
analysis,
quantitative lymphocyte subsets, T cell receptor excision circle (TREC) analysis,
V-beta spectratyping, and lymphocyte phenotype and function.
1.3.2
Describe the use of additional CD45RA-depleted DLI for recipients who have
severe viral infections, disease recurrence or progression, or poor immune
reconstitution. Assess and record efficacy of CD45RA-depleted DLI for these
conditions, and all adverse events that are related to CD45RA-depleted DLI.
1.3.2.1 Assess the degree of T-cell expansion in the early post HCT period and at 4
weeks post-DLI in the early DLI cohort compared to the original cohort.
1.3.2.2 Assess the cumulative incidence of composite viremia (including CMV, EBV,
and adenovirus) in the early DLI cohort compared to the original cohort; and
assess the duration of CMV viremia in the Amendment 5 early DLI cohort
compared to the original cohort.

HAP2HCT 2
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
2.0 BACKGROUND AND RATIONALE
2.1 Overview
Allogeneic HCT is a potential curative therapy for various pediatric hematologic
malignancies, however approximately 25-60% of eligible pediatric recipients will not have
a human leukocyte antigen (HLA)-matched related/sibling donor (MSD) or a HLA
matched unrelated donor (MUD).1-4 The lack of an adequate MUD for a substantial number
of patients, and the lengthy duration of the donor search process in those with high risk of
relapse, have prompted the search for alternative donors, including haploidentical family
donors and unrelated umbilical cord blood (UCB) grafts. 3,4
Nearly all patients have a readily available mismatched family member (haploidentical)
donor, and haploHCT is an effective therapy for patients with hematologic malignancies.5,6
This institution has a track record of success using haploidentical donors for patients with
hematologic malignancies.7 However, in our early studies, profound T-cell depletion of
these mismatched grafts was utilized for avoidance of GVHD – thereby prolonging donor
adaptive immune recovery and attenuating T-cell mediated GVL. Recently, we have found
that selective depletion of CD45RA+ cells provides efficient depletion of B cells and acute
GVHD inducing naïve T cells, while preserving hematopoietic progenitor cells and
memory T cells. Specifically, the recent study HAPNK1 utilized selective T-cell depletion
of the donor progenitor cell graft with CD45RA-depletion, which provided adoptive
transfer of diverse donor memory T cell populations. Although treatment related death and
overall outcomes have been better than previous studies, there have been significant
toxicity burden with cytokine release syndrome and severe acute GVHD in a subset of
patients. There is also evidence in the literature that TCRγδ T cells likewise may facilitate
engraftment and have anti-leukemia effects (see section 2.3). The combination of reduced
intensity conditioning regimen, administration of donor TCRγδ T cells, and precise dosing
of donor memory T cells has the potential to provide low regimen-related toxicity, a low
rate acute and chronic GVHD, enhanced donor immune recovery to prevent prolonged
immunodeficiency, and a low rate of disease recurrence, which would likely maintain the
excellent disease-free survival seen in HAPNK1 and lead to an enhanced quality of life.
Herein, we propose a single institution prospective phase II trial to determine the maximum
effective dose for CD45RA-depleted DLI when given in the early post-engraftment period;
and to estimate the efficacy of TCRαβ-depleted haploidentical donor progenitor cell graft
and CD45RA-depleted DLI following reduced intensity conditioning regimen that avoids
radiation in patients with high risk hematologic malignancies This study will evaluate the
safety and efficacy of this HCT procedure, through the estimation of donor engraftment,
survival, relapse, acute and chronic GVHD, chimerism, toxicity, and disease recurrence.
2.2 Haploidentical HCT
Haploidentical donors are viable alternative donors since family members are highly
motivated and readily available for most patients. Along with other institutions, St. Jude
has shown haploidentical HCT to be an effective therapy for patients with hematologic
malignancies. Due to the high potential for GVHD with the degree of HLA mismatch seen
in haploHCT, haploidentical grafts are often extensively T-cell depleted prior to infusion.

HAP2HCT 3
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Early results with T-cell depleted haploHCT were disappointing due very high rates of
graft failure and infections. Significant progress was initially achieved with the use of
mega-dose of T-cell depleted hematopoietic progenitor cells (HPC) after high-intensity
conditioning. Aversa and colleagues initially reported successful long-term engraftment
with minimal GVHD in 43 patients with leukemia (including 15 children) using high doses
of CD34+ enriched haploidentical cells, purified by positive (CD34+) and negative (CD3-
lectin agglutination) selection. Utilizing haploidentical grafts and megadose cell therapy
(>1 x 107 CD34+ cells/kg), Handgretinger and colleagues reported similar results in 23
pediatric patients. Prompt engraftment was associated with a low incidence of GVHD,
even without the use of pharmacologic GVHD prophylaxis.
Building on the initial studies above, St. Jude has gained considerable expertise with
haploidentical HCT in children. Over the past 16 years (2001-2016), 365 mismatched
related (haploidentical) donor HCT have been performed at St. Jude. Initially, most
haploHCT performed in this institution were for patients with relapsed and refractory
hematologic malignancies, however as it became a more effective therapy, it has become
a primary transplant option for patients in remission who lack a well matched donor.
The current haploHCT study HAPNK1 was activated in 2013. Preliminary results analyzed
as of August 2017 (n=49) showed that early event free survival (EFS) for standard high
risk (non-refractory) patients on HAPNK1 (black) was substantially improved compared
to previously completed haploHCT trials (blue) at St. Jude, however the EFS for refractory
patients on HAPNK1 (red) did not improve compared to previously completed haploHCT
trials (green) (events = relapse, death).
Years after transplant
0
0.5
1
1.5
2
2.5
3
Event Free Survival
0
0.2
0.4
0.6
0.8
1
HAPNK1-standard risk
Completed haploHCT trials-standard risk
Completed haploHCT trials-refractory
HAPNK1-refractory

HAP2HCT 4
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Based on this experience, it is clear that refractory patients need a specialized protocol
designed differnently and non-refractory (standard) HCT patients. Therefore, standard
non-refractory high risk malignancies will be treated on this study, and refractory patients
who are receiving their first allogeneic HCT will be transplanted on a specialized refractory
(REF) protocol.
2.3 Rationale for Immunomagnetic T-Cell Depletion (TCD) of Hematopoietic Progenitor Cell
Graft
As mentioned above, T-cell depleted haploidentical HCT were reasonably effective with
the infusion of a large number of CD34+ cells (>107 CD34+ cells/kg). However, with little
to no adoptive transfer of donor T-cell immunity, T-cell functional reconstitution was
substantially delayed. Donor T cells in the haploidentical graft play a major role in
mediating engraftment and GVL, but also GVHD - as few as 3 x 104 conventional T
cells/kg can cause GVHD. Methods of TCD have been developed to generate a graft with
very low T-cell content, such that the risk of GVHD is decreased. T cells can be removed
from the graft by direct removal of T cells (negative selection) or selection of CD34+
progenitors (positive selection). The transplantation program at St. Jude has had success
using either method of TCD in haploHCT.
Although positive selection allows for extensive TCD, the graft is devoid of other
important cell populations such as NK cells, myeloid cells, dendritic cells, and monocytes.
Several previous institutional haploHCT studies utilized CD3+ cell depletion by
CliniMACS, which preserves these innate immune facilitating populations. This method
substantially, but non-selectively, depletes all T-cell populations. Although GVHD
inducing naïve T cells are adequately depleted, potentially beneficial T-cell populations
that may not contribute substantially to GVHD such as gamma/delta (γ/δ) T cells and
memory T cells are likewise depleted in similar scale.
A newer form of immunomagnetic TCD available is α/β T-cell depletion by CliniMACS.
TCD of mobilized peripheral blood progenitor cell products has been shown to effectively
and consistently deplete α/β T cells approximately 4 log (10,000 fold).8,9 This level of α/β
T-cell depletion is greater than the level of TCD with CD3+ cell depletion by CliniMACS.
Importantly, this method has a CD34+ cell recovery comparable to the CD34+ enrichment
method of TCD, and preserves a γ/δ T-cell dose >100 fold greater than the residual α/β T-
cell dose. This is desirable because α/β T cells cause GVHD, and γ/δ T cells do not appear
to cause GVHD.10,11 In addition, there is evidence that γ/δ T cells have anti-leukemia
effects, and may facilitate engraftment. Additionally, higher γ/δ T-cell recovery in the
early post-HCT period has been associated with improved leukemia free survival.12 Our
experiences with TCRαβ-depletion performed by the St. Jude Human Applications
Laboratory in two institutional protocols (REFNK1, REF2HCT) show similar results, with
a greater than 90% recovery of CD34+ cells, CD56+ cells, and γ/δ T cells, and with a
greater than 4 log depletion of α/β T cells.

HAP2HCT 5
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
There is extensive publication record for use of TCRαβ-depleted haploidentical donor
progenitor cell grafts in HCT for patients with malignant and nonmalignant diseases.13-16
A most recent manuscript from Locatelli et al. described a cohort of 80 patients with
hematologic malignancy, all in CR, who received myeloablative conditioning that included
ATG, and TCRαβ-depleted haploidentical donor progenitor cell grafts.16 3 year leukemia
free survival was similar to HAPNK1 results at 70.7% (61-81%); TRM was similarly low
and relapse rate appeared to be slightly higher at 24.3% (16-36%). Importantly, a majority
received myeloablative TBI to achieve this good overall outcome. In this study then, we
will utilize a similar progenitor cell graft that is selectively depleted of naïve T cells by
removing alpha/beta (α/β) T cells and B cells by targeting TCRαβ and CD19 for depletion
by CliniMACS.
2.4 Rationale for CD45RA-depleted DLI
As mentioned above, extensive TCD has significant negative effects on the time to donor
immune competency. Donor T cells are critical for reconstituting the allogeneic host
immune system, and lymphocyte recovery is an important determinate of outcome post-
transplant. Transplants that employ T-cell depletion result in elimination of most memory
T cells, leading to protracted immune dysfunction - an effect that becomes more severe
with higher intensity conditioning regimens. This results in an increased rate of
opportunistic infections. Indeed, viral infections have been a major cause of death of
children receiving haploidentical transplants. The majority of these infections occur within
the first 6 months following transplantation, when T-cell immunity is the lowest.
Reconstitution of immunity can be partially restored by therapeutic infusions of donor
viral specific cytotoxic lymphocytes. However, in addition to requiring significant
resources and expertise, the cells must be engineered and selected for specific infections
and specific HLA - making this approach impractical for broad rapid application at this
time. The provision of a diverse population of donor memory T cells may provide
protection against several dangerous viral infections in the early post-transplant period.
The first 26 patients on HAPNK1 were analyzed for the incidence of viremia in the first 6
months after transplantation that included memory T-cell populations. Compared to the
two prior haploidentical donor protocols that utilized CD3 depletion (HIFLEX and
HAPREF), the incidence of CMV viremia (19.2% vs. 56.1%) and adenoviral viremia
(3.8% vs 19.2%) were significantly reduced.17 In addition, early T-cell reconstitution was
markedly higher in recipients of CD45RA-depleted grafts with a median T-cell count at
Day +30 of 550/uL vs 10/uL. Additional preliminary analysis as of August 2017 (HAPNK1
n=49) revealed that treatment related mortality was substantially lower in standard (non-
refractory) HAPNK1 patients (green) compared to observed rates in the same patients from
prior trials (black, red, blue), likely due to enhanced donor immune recovery and viral
control described above.

HAP2HCT 6
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Months after Transplant
0
2
4
6
8
10
12
CIN of TRM
0
0.2
0.4
0.6
0.8
1
standard (non-refractory) patients only
For this reason, the provision of donor CD45RA-depleted (memory) T cells will be an
important part of this therapeutic protocol.
However, giving large numbers of donor memory cells with the progenitor cell graft is
associated with some untoward effects. Most commonly that manifests as cytokine release
syndrome. A increasingly described syndrome with T-cell therapies that variably manifests
with high spiking fevers, diarrhea, liver dysfunction, acute kidney injury, fluid overload,
need for noninvasive or invasive respiratory support, tachycardia with hypotension, and
cardiomyopathy. Patient CRP level has been observed to correlate with CRS severity in
the literature,18 and has consistently been elevated in HAPNK1 patients compared to prior
haploHCT trials as noted in the figure below.
HAPNK1
Completed haploHCT trials-using ATG
Completed haploHCT trials-using Campath
Completed haploHCT trials-using OKT3

HAP2HCT 7
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Additionally, although memory T cells are known to be less alloreactive than naïve T cells,
and have demonstrated less GVHD in experimental systems, there may still be
manifestations of donor memory T-cell activity in the form of GVHD. As of August 2017,
the rate of severe (grade III-IV) acute GVHD has been similar on HAPNK1 (green)
compared to prior haploHCT trials that utilized CD3 depletion (blue, red) despite a median
T cell dose of 68 million/kg, and many recipients have received more than 100 million
(1x108)/kg, whereas CD3-depletion provides a median T-cell dose less than 1x105/kg (or
1000 fold less).
Months after Transplant
0
2
4
6
8
10
12
CIN of Acute GVHD III-IV
0
0.2
0.4
0.6
0.8
1
Although the rate of moderate to severe acute GVHD is similar, the pattern appears to be
different. Specifically, 6 of the first 13 patients on HAPNK1 with grade III-IV acute GVHD
manifested as isolated severe (stage 3 or 4) gut GVHD with no skin or liver involvement.
When all 88 patients with acute GVHD on all haploHCT protocols were reviewed, no other
patients presented with isolated severe gut GVHD. This suggests that a subset of the severe
GVHD seen with donor memory cells may be driven by organ specific antigen exposure
as opposed to the more a more universally expressed “self” antigen that is thought to drive
GVHD in conventional HCT. Because mucositis is a nearly universal regimen related
toxicity from preparative regimens of this nature, and because significant microbial antigen
exposure will occur the pre-engraftment period with the absence of neutrophils and the
presence of a disrupted mucosal barrier, it is plausible that delaying donor memory cell
infusion until the post-engraftment period may reduce this gut specific memory T-cell
GVHD phenomenon. This will also avoid the memory T cells from experiencing high
inflammatory cytokine exposure that is known to occur with monocyte and neutrophil
engraftment.
Completed haploHCT trials-using Campath
Completed haploHCT trials-using OKT3
Completed haploHCT trials-using ATG
HAPNK1

HAP2HCT 8
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The infusion of conventional T cells from haploidentical donors has been associated with
a significant increase in risk of acute GVHD at doses above 1x105/kg. This has been seen
with both DLI and with the progenitor cell graft during transplantation. With haploidentical
donor memory cell infusion however, we have not seen this same kind of threshold so far.
As of August 2017 (n=49) on HAPNK1, the infused T-cell dose given to patients who
developed grade III-IV acute GVHD was not significantly different from the doses given
to those patients who did not develop grade III-IV acute GHVD (p = 0.1 with Wilcoxon
rank sum test, see figure below).
Even if there is truly no dose above which the risk of severe acute GVHD is prohibitive,
perhaps the more important question is, “what is the minimally effective dose that will
provide the benefits of adoptive transfer of donor memory cells?”. To date, we have only
anecdotal cases to explore this question. Patient #41 on HAPNK1 experienced graft
rejection and was successfully salvaged with the same donor but with a CD3-depleted
graft. Therefore, he did not have successful adoptive transfer of donor memory cells. His
first several months were plagued with chronic infections and severe T-cell lymphopenia.
Nearly 4 months after his initial transplant, he had an absolute lymphocyte count (ALC) of
100/uL and no detectible T cells. After receiving CD45RA-depleted DLI at 1x105 T
cells/kg, his ALC rose to 600, and his T-cell count rose to 330/uL, and he had clearance of
his chronic adenovirus infection. This indicates that a dose of 1x105/kg has the potential to
effectively enhance donor reconstitution and protect against viral infection. Critically, this
dose is known to be safe even for conventional DLI. As of July 2018, a total of 4 patients
have received CD45RA-depleted DLI at doses ranging from 1x105 T cells/kg to 1 x 107/kg.
There has been no development of GVHD following these eight administrations of
CD45RA-depleted DLI in these four patients.

HAP2HCT 9
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Therefore, we propose to give CD45RA-depleted DLI in the early post-engraftment period,
with the goal of determining the maximum effective dose as measured by memory T-cell
count at 4 weeks post-DLI. (this measurement must occur any time from day 28 to day 42
after DLI, with DLI infusion as day 0). After the first 10 recipients at each dose level, if
the median memory T cell count is less than 300/uL, then subsequent patients will undergo
dose escalation to the next dose level (0.5 log higher). (See table below)
Although 300/uL is somewhat arbitrary,
this will allow recipeients to achieve a
similar early memory T-cell recovery to
patient on HAPNK1, where the median T-
cell count at Day +60 was 380/uL.17 (See
figure)
2.5 Rationale for the Reduced Intensity Conditioning Regimen
The immunologic GVL effect after allogeneic HCT has allowed reduction in the intensity
of conditioning, as HCT was no longer reliant purely on the cytotoxicity of high dose
chemo-/radio-therapy to eradicate disease. Reduced-intensity conditioning (RIC) regimens
have the potential to improve the outcomes of allogeneic HCT by decreasing the acute
TRM.19-21 Fludarabine-based RIC regimens are safe and effective alternatives to total body
irradiation (TBI) in haploidentical HCT.22-24 In addition, intensification of immunoablative
conditioning using ATG, OKT3, or alemtuzumab, results in the decrease incidence of
GVHD and graft rejection as well.24-27 In our first RIC haploidentical HCT protocol
REFSCT, we evaluated a RIC regimen for pediatric patients with refractory hematological
malignancies or considered at high risk for TRM with a full myeloablative regimen. The
Dose level
-1
Add MMF through Day +60
1
1x10^5/kg
2
1x10^6/kg
3
1x10^7/kg

HAP2HCT 10
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
RIC regimen consisted of fludarabine (200mg/m2), thiotepa (10mg/kg), melphalan (120
mg/m2) and Orthoclone-OKT3 and enrolled 25 patients from 2003-2005.
Out of the 25 participants, 22 engrafted with a median of 10 days (range, 7-12) and 3
experienced graft failure. The patients with graft failure, one patient had disease relapse
and 2 were salvaged with stem cells infusions from their first transplant donor. The median
time to platelet engraftment to 20,000/mm3 and 50,000/mm3 was 17 days (range, 12-36)
and 17 days (range, 12-76), respectively. The cumulative incidence of overall grade III-
IV acute GVHD and chronic GVHD was 8% and 28%, respectively. Of those participants
who died, 13 were due to relapsed disease (at a median of 105 days post-HCT). Only 4
(12%) died of TRM, despite most of the patients having received one prior allogeneic HCT
- 1 died of GVHD, 1 of infection, 1 of hemorrhage, and 1 of cardiomyopathy. In summary,
RIC allows for engraftment with an acceptable rate for graft failure, GVHD, and TRM.
Two subsequent haploidentical HCT studies at St. Jude (HAPREF and HIFLEX) also
utilized non-TBI preparative regimens with a fludarabine, thiotepa, melphalan backbone.
Twenty-seven patients with high risk hematologic malignancies were transplanted on one
of these two protocols with OKT3 as in vivo T cell depletion, followed by infusion of a
CD3+ depleted graft. The one-year EFS was 51.9% ± 9.6%. These studies have
demonstrated the EFS and TRM in TCD RIC HCT from mismatched haploidentical donor
compare favorably with reports from MUD.
Importantly however, OKT3 was an important component of the preparative regimen for
these transplants, and it is no longer commercially available. Therefore, the institutional
front-line haploidentical donor HCT trial (HIFLEX) substituted Campath for OKT3. Early
results indicate that survival is similar between patients on HIFLEX treated with OKT3 versus
Campath (data not shown). However, requirement for the use of therapeutic DLI rose
substantially. Only 1 of the 11 patients who received OKT3 on HIFLEX required more than
2 DLI in the first 100 days post-HCT. In contrast, 9 of the first 14 patients who received
Campath on HIFLEX required more than 2 DLI in the first 100 days post-HCT. This is likely
due to Campath’s anti-NK cell effects, leading to an increased clinical need for early T-cell
add back in the form of DLI. Therefore, in the subsequent protocol HAPNK1, 8 Gy total
lymphoid irradiation (TLI) was used in the preparative regimen along with cyclophosphamide
(60mg/kg), added to the backbone of fludarabine (150mg/m2), thiotepa (10mg/kg), and
melphalan (140mg/m2) to facilitate engraftment, and no serotherapy was utilized. As detailed
above, preliminary results show donor engraftment to be reliably obtained in the early post-
transplant period as graft failure occurred in 2 of 57 (3.5%) as of March 2018. Although TLI
has been well tolerated in patients receiving their first HCT on HAPNK1, the heart, gut, and
to a lesser extent the liver, receives significant radiation exposure with TLI. We have utilized
ATG in place of TLI in our protocol for repeat allogeneic transplantation protocol
(REF2HCT), because many patients will have already received significant radiation exposure,
and are more heavily pre-treated than their HAPNK1 counterparts. This protocol incorporates
TCRab-depleted progenitor cell graft and CD45RA-depleted progenitor cell graft, and as of
March 2018 there have been 0 patients experiencing graft failure in the first 14 recipients. In
addition, Locatelli et al, utilized ATG in their preparative regimens for patients with
hematology malignancy who received a TCRab-depleted haploidentical donor graft, and the
rate of graft failure was acceptable with 2 of 80 (2.5%) experiencing graft failure.16 Therefore,

HAP2HCT 11
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
we hypothesize that donor engraftment can still be reliably obtained in this population without
radiation if ATG is added to the chemotherapy backbone.
On this study, with the use of ATG in the preparative regimen, no additional pharmacologic
acute GVHD prophylaxis will be added. The optimal dose and timing of ATG administration
is unknown. Locatelli et al found that while 30% of patients experienced grade I-II acute
GVDH, no single dcase of visceral GVHD or Grade III-IV acute GVHD was recorded.16
Therefore, we will utilize the same schedule of ATG administration in this protocol. Optional
PK testing will be offered to trial participants so that we can better define the exposure and
duration of ATG when dosed in this manner in recipients of TCD haploidentical donor HCT.
This data will allow us to optimize ATG dose and timing in future therapeutic protocols.
2.6 Rationale for Blinatumomab
Blinatumomab is a bispecific CD19-directed CD3 T-cell engager that mediates formation
of a synapse between T cells and the CD19+ target cell, resulting in lysis of that CD19+
cell.28 It was granted accelerated approval by the FDA on 12/3/2014.28 Multiple clinical
trials have demonstrated the efficacy of blinatumomab for patients with refractory B-cell
malignancies.29-31
An early study included 21 patients with refractory disease at an minimal residual disease
level of burden. Blinatumomab dosing was 15μg/m2/day for 28 days, and 16 of 20
evaluable patients responded by becoming MRD negative.29 Fevers were common, but
there was no mention of cytokine release syndrome (CRS), and one patient experienced a
seizure that was reversible. Following an initial dip, T-cell numbers quickly returned to
baseline consistent with T-cell re-distribution, and then 19 of the 20 patients who
completed therapy had a significant increase in T-cell numbers at a median of 17 days into
treatment.32 The contribution of this T-cell expansion was predominately from the effector
memory T-cell (TEM) fraction.29,32
This same group also treated 36 adults with active disease (>5%), and saw 25 responders
amongst 36 adult patients.30 That includes 8 responders amonst the 15 patients who
relapsed after a prior allogeneic HCT. Fever was again common (>80%), and several cases
of more severe CRS were noted. Two grade 4 CRS were associated with a disease burden
in excess of 50% blasts in the marrow. A dose finding portion of this study determined that
starting at 15μg/m2/day was associated with a high rate of adverse events (and serious
advese events), and the preferred dose was ultimately chosen to be 5μg/m2/day x 1 week,
followed by 15μg/m2/day x 3 weeks, to comprise a full 28 day course.30 Also noted was
6 patients with temporary or permanent discontinuation of blinatumomab for neurologic
or psychiatric adverse events, all of which resolved within 72 hours. 9 responders
subsequently received allogeneic HCT as consolidative therapy, and toxicity was no
greater than expected following blinatumomab. The authors recommend HCT as standard
of care post-blinatumomab if response obtained. Long term follow-up of these patients
confirmed that long term survivors had a more pronounced T-cell expansion, and that the
expansion was predominantly TEM; whereas non responders had the lowest T-cell and TEM
counts.33 They also noted that patients who failed to respond with the first course, were
unlikely to respond after a second course.

HAP2HCT 12
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Recently, a large multicenter phase 2 trial in which 189 adults with relapsed/refractory B
cell malignancies received blinatumomab was published.31 Dosing was in graduated
fashion, with a lower dose the first week. The overall response rate was 43%, and was 45%
in the 64 patients with a prior allogeneic HCT.
The response rate in those with less than 50% blasts in their bone marrow was 73%. With
dexamethasone pre-treatment in all patients, only 3 (2%) experienced grade 3 CRS, though
neurologic toxicities were more common with 11% grade 3 and 2% grade 4 events.
Tremor, dizziness, and confusion were most common neurologic events.
There is also 9 additional patients in the literature who have been given blinatumomab for
post-transplant relapse.34 4 of the 9 achieved CR after a single course. Two who failed to
respond to a first course at 5μg/m2/day, did respond to a second course at 15μg/m2/day;
3 had no response. There was no difference in the starting T-cell numbers in responders
and non-responders, though both were generally under 200 T-cells/μL despite being a
median of over 8 months post-transplant.34 Fever, fatigue, and drop in blood pressure were
common, except for one non-responder and 1 patient with minimal disease burden. They
found that T-cell to blast ratio was important, and patients who did the best had a relatively
high number of T cells and relatively low disease burden.34
There is a strong rationale for the addition of blinatumomab for patients with CD19+
hematologic malignancy in this trial. Firstly, prognosis is uniformly poor in patients who
relapse after allogeneic HCT, and therefore any therapy that has the potential to reduce
postHCT relapse has the potential to increase long term survival. Additionally,
blinatumomab has demonstrable efficacy in patients with chemotherapy refractory B-cell
malignancies, and it has efficacy in the post-transplant setting. Blinatumomab appears to
work best when a low disease burden is present, and this is likely to be the case in the early
post-transplant period.
It also appears to need a minimum host T-cell presence, with
preference for the presence of TEM cells. In HAPNK1, which also utilized CD45RA
depletion, there was a robust early recovery of T cells, with 11 of the first 16 patients having
normal CD8+ T-cell numbers at Day +30.35 An abundance of both effector and central
memory T cells was also noted at Day +30 in HAPNK1 recipients confirming successful
adoptive transfer. Therefore, the good blinitumomab activity would be predicted to be
present early after CD45RA-depletion DLI due to the adoptive transfer of large numbers
of donor memory T cells. Finally, memory T cells have documented GVL effects (see
section 2.4), however their specificity will be MHC restricted, as demonstrated by 2 of the
first 4 relapses on HAPNK1 showing haplotype loss.36 Blinatumomab provides a direct
antigen recognition that is not MHC restricted and does not require T cells that recognize
leukemia or patient specific peptides, although antigen specific therapy to CD19 can
likewise be circumvented by antigen loss.37 The combination of donor memory cells and
blinatumomab however could provide graft-versus-malignancy effects in both MHC
restricted and whole antigen specific mechanisms, which may reduce mutational escape.
Combinatorial immune therapy is likely to be a major component of future cancer therapy.
Bispecific T-cell Engager (BiTE) are currently in development for AML and multiple solid
tumors.38-41 Therefore, successful incorporation of a BiTE such as blinatumomab into HCT
that provides donor memory cells may provide a robust platform for additional diseases.

HAP2HCT 13
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
For Amendment 2, the minimum peripheral blood CD3+ count to receive blinatumomab
will be set at 150/µL, and patients must be at least 28 days (4 weeks) following their
CD45RA-depleted (memory T-cell) DLI. These new parameters are being added due to
the experience with the first 16 patients to receive blinatumomab on this protocol.
None of the 4 patients who had a CD3+ count less than 150/µL at the initiation of the
blinatumomab infusion experienced response (B cell depletion). However, 11 of the 12
patients with a CD3+ count of 150/µL or greater showed effective depletion of circulating
B cells. In addition, none of the 3 patients who initiated blinatumomab at less than 28 days
from the protocol mandated memory T-cell DLI showed a response.
2.7 Minimal Residual Disease
Detection of minimal residual disease (MRD) prior to HCT can have a significant impact
on prognosis.42 Detection of leukemic cells that are below the limits of detection by
standard morphologic examination allow early interventions when the patients are MRD
positive but still in remission. Therefore, detection of MRD following transplant and early
intervention may improve post-transplant clinical outcome. For these reasons, the testing
of MRD prior to transplantation and as a part of disease evaluations post-transplantation is
now an established standard clinical procedure.
2.8 Rationale for Present Study
For many patients with high risk hematologic malignancies that have a poor prognosis
with conventional chemotherapy, allogeneic HCT offers an increased chance of cure, yet
is fraught with several hazards.
HCT utilizing a related haploidentical donor offers a unique advantage in that it can be
performed quickly, the donor is readily available and highly motivated, the degree of HLA
mismatching may provide stronger graft-vs.-leukemia effects (GVL), and accessibility of
the donor facilitates post-transplant cellular therapeutics such as donor lymphocyte
infusion (DLI). Memory T cells and TCRγδ T cells have demonstrated antileukemic
effects, and may provide useful anti-leukemic effects in the early post-transplant phase.
Furthermore, for this often heavily pretreated patient population, it is important to explore
new regimens using novel conditioning components and novel GVHD prevention
strategies to take advantage of the immunologic anti-cancer effects of allogeneic HCT and
to decrease transplant-related toxicity and mortality.
In this study, we will build upon the improved outcomes seen with the use of CD45RA-
depletion of donor progenitor cell grafts in HAPNK1, by attempting to identify the lowest
effective dose of haploidentical donor memory T cells in the early post-transplant setting.
We hypothesize that this will allow us to maintain the improvement in efficacy through
retention of the benefits of donor memory cells such as enhanced viral control, reduction
in therapy related mortality, and possibly reduction in relapse; while simultaneously
reducing the overall toxicity of the HAPNK1 regimen by giving the memory cells after
engraftment and avoiding radiation therapy in the preparative regimen. We will also assess
the safety and tolerability of the addition of blinatumomab in those patients with CD19+
malignancies. At the same time, we will describe the regimen-related toxicities,

HAP2HCT 14
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
lymphohematopoietic reconstitution, graft failure, and incidence of acute and chronic
GvHD.
2.9 Rationale for Early DLI Cohort in Amendment 5
Following the dose escalation period of this study in which 29 patients received escalating
doses of CD45RA-depleted DLI per protocol (Dose level 1 n=9; Dose level 2 n=10; Dose
level 3 n=10), the maximum effective dose was determined to be Dose level 3 = 1x10^7/kg.
The infusion of the memory DLI were well tolerated, and one month post infusion, there
was a significant increase in the median number of CD3 T-cells, including CD8 and
CD45RO+ T-cell subsets (**p<0.01 and ***p<0.001, Fig 1A). There was also significant
expansion of virus-specific T-cells (VSTs) directed towards Cytomegalovirus (CMV),
Adenovirus (AdV), BK, or HHV-6 as shown by Elispot assays (**p<0.01, Fig. 1B).43
While this robust donor memory cell recovery appeared to shorten the duration for those
patients who experienced viremia, 29 of 50 (58%) patients still experienced viremia
(CMV/EBV/adenovirus). Evaluation of all patients who received memory DLI per
protocol found that the median day of DLI infusion was Day +28.5 and the mean day of
infusion was Day +32 (min 22, max 57). This means that the robust expansion of donor
memory T-cells noted above typically did not occur until about two months following
transplant, the time period when patients are most at risk of serious transplant related
complications. The primary goal of this Amendment 5 expansion (early DLI) cohort
then is to move up (closer to the date of transplant) the donor memory DLI infusion by
two weeks.

HAP2HCT 15
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
To accomplish this goal, the preparative regimen will have one alteration – the doses of
ATG will be moved back (away from the date of transplant) by 7 days. Instead of ATG
infusions on Day -5, -4, and -3 the infusions will instead occur on Day -12, -11, and -10.
(See section 4.1.2) This will minimize any potential ATG effects on the early infusion
of donor memory cells.
The goal day of memory DLI infusion at the determined MED of 1x10^7/kg then will
be around Day +14, with the goal of most (if not all) infusions to occur on Day +10 to
Day +21. Infusions after Day +21 are allowed if the patient was otherwise not suitable
for infusion prior to Day +21.
3.0 RESEARCH PARTICIPANT ELIGIBILITY CRITERIA AND STUDY
ENROLLMENT
According to institutional and NIH policy, this study will accession patients regardless of
gender and ethnic background. Institutional experience confirms broad representation in this
regard. However, pregnant and lactating females are excluded from participation as the short
and long-term effects of the preparative agents, study infusion (recipients) as well as the long-
term effects of mobilization and apheresis procedure (donors) on a fetus and a nursing child
through breast milk are not entirely known at this time.
3.1 Inclusion Criteria for Transplant Recipient
3.1.1 Age less than or equal to 21 years.
3.1.2Does not have a suitable HLA-matched sibling donor (MSD) or volunteer 10/10 HLA-
matched unrelated donor (MUD) available in the necessary time for progenitor cell
donation.
3.1.3Has a suitable single haplotype matched (≥4 of 8) family member donor.
3.1.4 High risk hematologic malignancy.
3.1.4.1 High risk ALL in CR1.
Examples include, but not limited to: t(9;22) with persistent or recurrent
transcript, hypodiploid cytogenetics, MRD >1% at the end of induction,
M2 or greater marrow at the end of induction, recurrent or rising MRD
after induction, Infants with MLL fusion or t(4;11), relapse after prior
CART therapy.
3.1.4.2 ALL in High risk CR2.
Examples include, but not limited to t(9;22), BM relapse <36 mo CR1 or
<6mo after completion of therapy, any T-ALL, very early (< 6mo CR1)
isolated CNS relapse, late BM relapse with poor response to standard
reinduction therapy(e.g. MRD positive or recurrence after two blocks),
relapse after prior CART therapy.
3.1.4.3 ALL in CR3 or subsequent.

HAP2HCT 16
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
3.1.4.4 AML in high risk CR1 (diagnosis of AML includes myeloid sarcoma).
Examples include but not limited to: preceding MDS or MDS-related
AML, FAB M0, FAB M6, FAB M7 with high risk genetics such as ML
not t(1;22), MRD > 0.1% after two cycles of induction, MRD > 1% after
one cycle of induction, FLT3-ITD in combination with NUP98-NSD1
fusion or WT1 mutation, any high risk cytogenetics such as: DEK-
NUP214
[t(6;9)],
KAT6A-CREBBP
[t(8;16)],
RUNX1-CBFA2T3
[t(16;21)], -7, -5, 5q-, KMT2A-MLLT10 [t(6;11)], KMT2A-MLLT4
[t(10;11)], inv(3)(q21q26.2), CBFA2T3-GLIS2 [inv(16)(p13.3q24.3)],
NUP98-KDM5A [t(11;12)(p15;p13)], ETV6-HLXB [t(7;12)(q36;p13)],
NUP98-HOXA9 [t(7;11)(p15.4;p15)], NUP98-NSD1.
3.1.4.5 AML in CR2 or subsequent.
3.1.4.6 Therapy related AML, with prior malignancy in CR > 12mo
3.1.4.7 MDS, primary or secondary
3.1.4.8 NK cell, biphenotypic, or undifferentiated leukemia/lymphoma in CR1 or
subsequent.
3.1.4.9 CML in accelerated phase, or in chronic phase with persistent molecular
positivity or intolerance to tyrosine kinase inhibitor.
3.1.4.10 Hodgkin lymphoma in CR2 or subsequent after failure of prior autologous
HCT, or unable to mobilize progenitor cells for autologous HCT.
3.1.4.11Non-Hodgkin lymphoma in CR2 or subsequent after failure of prior
autologous HCT, or unable to mobilize progenitor cells for autologous
HCT.
3.1.4.12 JMML
3.1.5 If prior CNS leukemia, it must be treated and in CNS CR
3.1.6 Does not have any other active malignancy other than the one for which this HCT
is indicated.
3.1.7 No prior allogeneic HCT, and no autologous HCT within the previous 12 months.
3.1.8 Patient must fulfill pre-transplant organ function criteria:
3.1.8.1 Left ventricular ejection fraction > 40%, or shortening fraction ≥25%.
3.1.8.2 Creatinine clearance (CrCl) or glomerular filtration rate (GFR) ≥50
ml/min/1.73m2.

HAP2HCT 17
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
3.1.8.3 Forced vital capacity (FVC) ≥50% of predicted value; or pulse oximetry
≥92% on room air if patient is unable to perform pulmonary function
testing.
3.1.8.4 Karnofsky or Lansky (age-dependent) performance score ≥50 (See
APPENDIX A).
3.1.8.5 Bilirubin ≤3 times the upper limit of normal for age.
3.1.8.6 Alanine aminotransferase (ALT) or Aspartate aminotransferase (AST) ≤
5 times the upper limit of normal for age.
3.1.8.7 Not pregnant. If female with child bearing potential, must be confirmed
by negative serum or urine pregnancy test within 14 days prior to
enrollment.
3.1.8.8 Not breast feeding
3.1.8.9 Does not have current uncontrolled bacterial, fungal, or viral infection.
3.2 Inclusion Criteria for Haploidentical Donor
3.2.1 At least single haplotype matched (≥4 of 8) family member
3.2.2 At least 18 years of age.
3.2.3 HIV negative.
3.2.4 Not pregnant as confirmed by negative serum or urine pregnancy test within 14 days
prior to enrollment (if female).
3.2.5 Not breast feeding.
3.2.6 Regarding donation eligibility, is identified as either:
3.2.6.1
Completed the process of donor eligibility determination as outlined in 21
CFR 1271 and agency guidance; OR
3.2.6.2
Does not meet 21 CFR 1271 eligibility requirements, but has a declaration
of urgent medical need completed by the principal investigator or physician
sub-investigator per 21 CFR 1271.
3.3 Research Participant Recruitment and Screening
This study will be posted on http://clinicaltrials.gov. Potential transplant recipients are
typically referred from their primary clinical service at St. Jude, from the Physician
Referral Office for non-St. Jude patients, or are patients currently being treated by the
Department of BMTCT. Each patient considered for transplantation is registered on the
“Transplant List,” an ongoing list used to provide the clinical service with information
regarding new patients for consultation and under consideration for transplantation and the
status of those undergoing pre-evaluation. The patient remains on this list until a decision
has been confirmed for acceptance or rejection based on several factors, primarily
completion of prior therapy and/or clinical status. The proposed patient schedules on this
form are often subject to change due to various reasons such as family travel issues, donor
availability for procurement of progenitor cells, recipient clinical status, etc. This list is a
confidential document, updated and maintained by the Department of BMTCT
Coordinators.

HAP2HCT 18
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
3.4 Enrollment on Study at St. Jude
A member of the study team will confirm potential participant eligibility as defined in
Section 3.1 - 3.2, and register the participant in OnCore. A research participant-specific
consent form and assent document (where applicable) will be generated. The entire signed
consent/assent form(s) must be scanned into the Electronic Health Record (EHR) by the
study team designee.
4.0 TREATMENT PLAN
4.1.1
Preparative regimen for original cohort (through Amendment 4)
DAY
MEDICATION
DOSE
DOSE #
-9
Cyclophosphamide
60 mg/kg intravenous once daily
1 of 1
-8
Fludarabine*
30 mg/m2 intravenous once daily
1 of 5
-7
Fludarabine*
30 mg/m2 intravenous once daily
2 of 5
-6
Fludarabine*
30 mg/m2 intravenous once daily
3 of 5
-5
Fludarabine*
30 mg/m2 intravenous once daily
4 of 5
ATG (rabbit)
1mg/kg intravenous daily
1 of 1
-4
Fludarabine*
30 mg/m2 intravenous once daily
5 of 5
ATG (rabbit)
2mg/kg intravenous daily
1 of 2
-3
Thiotepa
5 mg/kg intravenous twice daily
1,2 of 2
ATG (rabbit)
2mg/kg intravenous daily
2 of 2
-2
Melphalan**
70 mg/m2 intravenous once daily
1 of 2
-1
Melphalan**
70 mg/m2 intravenous once daily
2 of 2
0
HPC,A Infusion(TCRα/β+ and CD19+ depleted)
+1
HPC,A infusion (if needed to achieve goal CD34+ cell dose, see section 4.5)
+2-5
--
+6
G-CSF
5mcg/kg subcutaneous or intravenous daily until ANC >2000
for 2 consecutive days, or as clinically indicated
TBD¶
CD45RA-
depleted DLI
Dose based on dose level (See section 2.4 and 4.7)
TBD‡
Blinatumomab
5mcg/m2/day x 7days, then 15mcg/m2/day x 21days
*
Fludarabine dosing will be 1 mg/kg/day for patients < 10kg
** Melphalan dosing will be 2.3 mg/kg/day for patients < 10kg
¶
CD45RA-depleted DLI will be given at least two weeks after engraftment (see section 4.7)
‡ Blinatumomab will be given at least four weeks post-DLI, and only to patients with CD19+
malignancies, and only once the peripheral blood CD3+ count is at least 150/µL. (see
section 4.2)

HAP2HCT 19
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
4.1.2 Preparative regimen for early DLI cohort (Amendment 5)
DAY
MEDICATION
DOSE
DOSE #
-12
ATG (rabbit)
1mg/kg intravenous daily
1 of 1
-11
ATG (rabbit)
2mg/kg intravenous daily
1 of 2
-10
ATG (rabbit)
2mg/kg intravenous daily
2 of 2
-9
Cyclophosphamide
60 mg/kg intravenous once daily
1 of 1
-8
Fludarabine*
30 mg/m2 intravenous once daily
1 of 5
-7
Fludarabine*
30 mg/m2 intravenous once daily
2 of 5
-6
Fludarabine*
30 mg/m2 intravenous once daily
3 of 5
-5
Fludarabine*
30 mg/m2 intravenous once daily
4 of 5
-4
Fludarabine*
30 mg/m2 intravenous once daily
5 of 5
-3
Thiotepa
5 mg/kg intravenous twice daily
1,2 of 2
-2
Melphalan**
70 mg/m2 intravenous once daily
1 of 2
-1
Melphalan**
70 mg/m2 intravenous once daily
2 of 2
0
HPC,A Infusion(TCRα/β+ and CD19+ depleted)
+1
HPC,A infusion (if needed to achieve goal CD34+ cell dose, see section 4.5)
+2-5
--
+6
G-CSF
5mcg/kg subcutaneous or intravenous daily until
ANC >2000 for 2 consecutive days, or as clinically
indicated
+10-21
CD45RA-depleted DLI
1x10^7 CD3+ cells/kg ¶
TBD‡
Blinatumomab
5mcg/m2/day x 7days, then 15mcg/m2/day x 21days
*
Fludarabine dosing will be 1 mg/kg/day for patients < 10kg
** Melphalan dosing will be 2.3 mg/kg/day for patients < 10kg
¶ CD45RA-depleted DLI will be given around Day +14 (goal range Day 10 to 21)
‡ Blinatumomab will be given at least four weeks post-DLI, and only to patients with CD19+
malignancies, and only once the peripheral blood CD3+ count is at least 150/µL. (see section
4.2)
Cellular infusion procedures and monitoring
For the proper infusion procedures and monitoring of the HPC product please refer to
BMTCT SOP 40.02 “Hematopoietic Progenitor Cell Infusion – FRESH (Allogeneic): IV
Push and IV Drip” or SOP 40.03 “Hematopoietic Progenitor Cell Infusion – FROZEN: IV
Push”. Please note that all relevant SOPs can be found on the BMTCT Clinical Transplant
Program intranet page.

HAP2HCT 20
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Importantly, during the cellular infusions, monitoring of vital signs, breath sounds, heart
rate, pulse oximetry, and I/O will be done per the established transplant nursing procedure,
as well as appropriate Department of BMTCT SOPs, then documented on the Cellular
Product Infusion Record. If a reaction is suspected at any time during the infusion, the
nurse will 1. Stop the infusion, 2. Notify the Attending Transplant Physician immediately,
3. NOT discard the product until physician orders are given. Proper documentation
(symptoms of patient, vital signs, actions taken, outcome, and follow-up) will be completed
in the Cellular Product Infusion Record.
General treatment related comments:

The Hematopoietic Progenitor Cell, Apheresis (HPC,A) infusion(s) may be delayed
by approximately 24 hours in order to accommodate progenitor cell collection with
the donor, the Blood Donor Center and/or HAL as well as the research participant
clinical condition.

The term “every” used in tables is an approximate term meaning that these
medications noted will be administered approximately “every” 12 hours. The drug
administration timing in the case of “every 12 hours” may be modified by
approximately +/- 4 hours or as clinically indicated such as in the case of surgical
procedures or to accommodate other necessary medication, blood product delivery,
or procedures (such as a needed CT scan). The term “day” does not refer to an
absolute calendar day. It refers to a general 24-hour period.

Dosing for the medications cyclophosphamide, fludarabine, thiotepa, and
melphalan may be modified for research recipients based upon actual body weight
or adjusted ideal body weight when clinically indicated. Mesna will be
administered for prevention of hemorrhagic cystitis from the medication
cyclophosphamide. In general, mesna is administered at 15 mg/kg/dose prior to
cyclophosphamide
and
at
approximately
3,
6,
and
9
hours
after
the
cyclophosphamide infusion, to give a 1:1 ratio of mesna:cyclophosphamide.
Mesna dose and administration schedule may vary based on physician
recommendation.

Criteria for medication calculations based on body weight/body surface area and
other medication related information can be found in the St. Jude Formulary or the
St. Jude Dept of Pharmaceutical Sciences intranet website. Medication doses may
be rounded to the nearest integer or to the nearest appropriate quantity when
clinically or pharmaceutically indicated as per the MD and PharmD.

This treatment plan includes ATG in the preparative regimen and selective T-cell
depletion for GVHD prophylaxis. No post-transplant pharmacologic GVHD
prophylaxis will be routinely given. In the event the participant develops GVHD,
treatment will be according to the SOPs of the St. Jude Department of BMT&CT.
4.2
Blinatumomab Administration

HAP2HCT 21
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Blinatumomab will be given to patients with a history of CD19+ malignancy as determined by
St. Jude hematopathologist review of current and historical specimens and reports. The PI
had a personal patient (unpublished) that received CD19+ directed therapy, and developed
CD19 negative leukemia (by flow cytometry) and was then transplanted, and relapsed with
CD19+ disease again. Therefore, patients who were previously CD19+ and became CD19
negative after antigen specific therapy, will still be offered blinatumomab at the discretion
of their primary transplant physician after a discussion of the risks and benefits.
Blinatumomab dosing will begin no sooner than 4 weeks after CD45RA-depleted DLI and no
later than Day +180. There must be no acute GVHD or it must be quiescent. ALT must be
less than 5x ULN, bili less than or equal to 1.5x ULN, and creatinine less than or equal to
1.5x ULN. Tylenol and/or Benadryl may be given as pre-medication. Dexamethasone will
not routinely be given as pre-medication, but its use is not prohibited.
Dosing will be 5mcg/m2/day x 7days, then 15mcg/m2/day x 21days. Dosing will be based
on actual BSA with no dose adjustment for obese patients. For patients greater than or
equal to 45kg, dosing will be 9mcg/day x 7 days, then 28mcg/day x 21 days.
Blinitumomab infusion will be held for Grade 3 neurologic toxicity or any other relevant
Grade 3 or 4 toxicity. Missed days will not be made up by adding extra days to the
originally planned 28 day course. If the interruption is greater than 14 days, blinatumomab
will be permanently discontinued. Blinatumomab may also be discontinued for disease
progression despite blinatumomab, or if continued administration is not deemed beneficial
in the investigator’s opinion. Specific recommendations for withholding, permanently
discontinuing, or dose alteration of blinatumomab due to toxicity are in the chart that
follows:

HAP2HCT 22
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The median onset for cytokine release syndrome (CRS) is 2 days33, therefore patients will
be admitted to the hospital for a minimum of 72 hours for the initial dosing of
Blinatumomab. Dexamethasone does not inhibit cytotoxic activity of BiTE activated T
cells, and therefore it may be given for patients who experience high grade CRS.44
However, it can be generally immune suppressive and lympholytic, therefore an IL-6
inhibitor such as tocilizumab is the preferred first agent for CRS.45
As described in Section 11.1, if two patients in the first six experience permanent
discontinuation of blinatumomab (as described above), then the study will be placed on
hold to further blinatumomab administration; and protocol modification will occur to
adjust dosing and premedication, or to remove blinatumomab administration. Because the
use of blinatumomab at this time point is novel and the safety is unknown, the provision
of blinatumomab to the first six qualified recipients will be staggered. If one patient is on
blinatumomab, a second patient cannot start blinatumomab therapy until the prior patient
has received at least 72 hours at the second dose level (i.e. after 10 days total blinatumomab
dosing) without interruption. If the prior patient experiences interruption, then the next
patient cannot begin blinatumomab until the prior patient successfully completes the four

HAP2HCT 23
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
week course. No more than two patients may receive blinatumomab at the same time. Once
one permanent discontinuation has occurred, only one patient is allowed to receive
blinatumomab at a time. (i.e. after one permanent discontinuation for safety reasons, each
prior patient must successfully complete blinatumomab dosing before a subsequent patient
can initiate blinatumomab.)
This sequential step-wise approach prevents more than two patients in the first six from
experiencing blinatumomab toxicity of a severity that requires permanent discontinuation.
It also allows a second patient to initiate blinatumomab without waiting for the prior patient
to complete their course if no prior permanent discontinuation has occurred. Because
protocol accrual is anticipated to be approximately 10 recipients per year, and because
about 33% of recipients will be CD19+ (and therefore qualify for blinatumomab),
significant overlap that would trigger this staggering rule is expected to be rare.
It is the treatment with blinatumomab that will be staggered and not enrollment onto the
protocol. Many patients enrolled on this protocol have limited other potentially curative
therapy available and will have a short time period during which they are suitable
transplantation (due to disease or clinical condition factors). To delay or reschedule a HCT
due to waiting for another patient to complete blinatumomab places significant risk on
prospective patients who have the potential to benefit from this transplantation therapy,
even if blinatumomab may not be available. Furthermore, if a patient were not to receive
blinatumomab, the patient would still be evaluable for other study endpoints including the
primary objective.
4.3Donor Selection
If more than one family member donor is suitable, then donor selection will be based on
the preference of the primary transplant attending. Factors in selection will include degree
of KIR mismatching, donor-recipient matching of CMV serology, donor-recipient red
blood cell compatibility, degree of HLA matching, size of the potential donor, previous
use as a donor, presence of donor-specific antibody, and overall health and availability of
the potential donor.
Donor eligibility for cell collection will be determined through the guidelines outlined in
21 CFR 1271 and the Guidance for Industry: Eligibility Determination for Donors of
Human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps). Potential donors
will undergo an initial screening process that will include at least a complete physical
exam, history and testing for relevant communicable diseases. Physical exams to evaluate
donor candidacy will be conducted by a non-Department of BMTCT physician
(St. Jude or non-St. Jude). For subsequent therapeutic cell collection procedures, if a
complete screening procedure has been performed within the previous 6 months, an
abbreviated donor screening procedure may be used for these repeat donations. The
abbreviated screening procedure must determine and document any changes in the donor’s
medical history since the previous donation that would make the donor ineligible, including
changes in relevant social behavior.
If a donor is determined to be ineligible, the donor is not automatically excluded. Part 21
CFR 1271.65 (b)(1)(i) allows use of ineligible donors who are first or second degree blood
relatives. In this situation, the physician will document the necessity of using the ineligible
donor by providing a statement of "Urgent Medical Need" as explained in the 21 CFR

HAP2HCT 24
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
1271.3 (u). The cell therapy products will be labeled as required in 21 CFR 1271.65 (b)(2).
Recipients or their legal guardians will be informed of the use of an ineligible donor.
Please see Departmental SOP 30.05 “Determination of Eligibility and Suitability for
Progenitor Cell and Lymphocyte Cell Donors” for additional information. Please note that
all relevant SOPs can be found on the BMTCT Clinical Transplant Program intranet page.
4.4Donor Mobilization and Graft Collections
A G-CSF (or biosimilar) mobilized peripheral blood progenitor cell product (identified as
HPC,A) is the preferred progenitor cell graft source. Our desired target goal will be 5 x
106 CD34+ cells/kg. This number of cells will be necessary to provide an adequate graft,
following the various ex vivo manipulations, for prompt reconstitution. More then one
collection may be needed to achieve this goal. Donors will undergo a standard
hematopoietic progenitor cell mobilization regimen consisting of 5 days of G-CSF given
subcutaneously at 10 micrograms/kilogram. The graft will be collected by leukapheresis
on day 5 (and 6 if needed) of G-CSF. The HPC product will typically be collected and
infused fresh, however there may be patients or logistical situations that require the HPC
product to be collected early, processed, and stored frozen.
The decision to use a fresh versus frozen HPC will be made by the PI and/or primary
transplant attending based on patient and donor factors, as well as potential scheduling
conflicts.
DONOR MOBILIZATION TIME LINE (if fresh HPC product)
DAYS
MEDICATION
APHERESIS
Day -5
G-CSF* 10 mcg/kg/day SC**
Day -4
G-CSF* 10 mcg/kg/day SC**
Day -3
G-CSF* 10 mcg/kg/day SC**
Day -2
G-CSF* 10 mcg/kg/day SC**
Day -1
G-CSF* 10 mcg/kg/day SC**
Apheresis for HPC graft
Day 0
G-CSF* 10 mcg/kg/day SC**
(if needed)
Apheresis for HPC graft
(if needed)
*Or biosimilar
** G-CSF may be reduced if the donor’s WBC is >75.0 x 106/mL
The dose of G-CSF may require modification based on the complete blood counts (CBC).
If the donor’s white blood count (WBC) is >75.0 x 106/ml the dose of cytokine
administered will be reduced. The guidelines for dose modification can be found in the St.

HAP2HCT 25
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Jude Children's Research Hospital Department of BMT and CT SOP 30.06.00 “The
practice for the evaluation, preparation and care of allogeneic and autologous donors
mobilized with growth factor.” Please note that all relevant SOPs can be found on the
BMTCT Clinical Transplant Program intranet page.
The daily leukapheresed volumes for HPC,A collection is generally 3–4 total blood
volumes.
Two additional days of leukapheresis may be performed at the physician’s
discretion (no more than 3 total) to reach the cell dose target, however, this is expected to
be rare.
Leukapheresis may be terminated early upon request of donor, or when deemed medically
necessary per the judgment of the treating sub-investigator physician or principal
investigator. All products will be collected as per Foundation for the Accreditation of
Cellular Therapy (FACT) and AABB guidelines. Donors will be monitored during the
period of the mobilization and leukapheresis procedure for adverse events (Appendix C).
If we are unable to collect the minimum dose of 2 x 106 CD34+ cells/kg of recipient weight
from the first donor, then an alternative family member may be used if he/she fulfills all
the donor criteria described in section 3.2.
4.5 Progenitor Cell Graft Preparation
Graft evaluation and preparation will take place in the Human Applications Laboratory
(HAL) in the Department of BMTCT using established SOP.
The HPC product(s) will be TCD using the investigational CliniMACS device and TCRαβ
Biotin reagent and CD19 reagent, and tubing set as directed by the manufacturer (Miltenyi
Biotech).
See section 5.2 for additional CliniMACS device information. Briefly,
hematopoietic progenitor cells collected by apheresis (HPC,A) from the mobilized donor
will be initially assessed in the HAL and stored overnight at 4oC, if necessary. The product
will be washed to remove platelets and adjusted to an appropriate cell concentration for
incubation with Biotinylated CliniMACS TCRαβ reagent (biotin conjugated anti TCRαβ
antibody) and CD19 reagent in the manufacturer provided media. The cells will be washed
to remove unbound reagent before labeling with paramagnetic beads conjugated with anti-
biotin antibody). These cells will be applied to the CliniMACS device and the depletion
will be performed as described by the manufacturer.
After depletion is complete, the cells will be washed and resuspended in an infusion grade
solution. The graft product will be enumerated and assessed for viable CD34+ cell , α/β T-
cell, and CD19+ cell content by flow cytometry. The processed HPC,A product will be
infused fresh or cryopreserved for future use after completion of release testing and
evaluation. Cryopreservation will be performed per SOPs of the Human Applications
Laboratory.
Target cell doses are listed in the following table:
Initial HPC Graft
Target Dose
Minimum Dose
Maximum Dose
CD34+ cells/kg
≥5 x 106
2 x 106
50 x 106
TCRαβ+ cells/kg
≤0.1 x 106
N/A
*0.1 x 106
* Maximum TCRαβ+ cell dose includes the sum of all TCRαβ-depleted products.

HAP2HCT 26
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
If the target CD34+ dose is not achieved in a single collection, up to two additional
collections may be performed (see Section 4.4). These additional collections may be TCD
on the CliniMACS device by TCRαβ-depletion, or by CD34+ cell enrichment (if the
maximum TCRαβ+ cell dose has been reached). There is no target, minimum, or maximum
cell dose for residual CD19+ cells.
4.6 Additional Progenitor Cell Graft Administration
Infusion of an additional HPC graft from the original or an alternative haploidentical donor
may be performed for participants when clinically indicated for graft failure, poor immune
reconstitution, or poor hematopoietic recovery. The use of and content of a conditioning
regimen is left to the discretion of the PI and/or primary transplant attending such that the
most appropriate therapy is chosen for the clinical situation. Participants who receive a
conditioning regimen may remain on therapy but will not have further immune
reconstitution and evaluation research labs collected.
The HPC graft will typically be obtained by apheresis (HPC,A) and be infused fresh. The
target CD34+ dose for this additional infusion is ≥5 x 106 cells/kg. If the participant has
quiescent or active BOOP, BOS, acute Grade III-IV GVHD, or any other reason that a
severely T-cell depleted graft may be indicated, then a graft from the donor processed on the
CliniMACSTM device using either CD34+ selection or CD3+ depletion methodology may be
utilized.
4.7 Donor Lymphocyte Infusions
4.7.1 Prophylactic CD45RA-depleted DLI
This donor product will be processed for CD45RA+ depletion using the
investigational CliniMACS device as directed by the manufacturer (Miltenyi
Biotech). See section 5.2 for additional CliniMACS device information. Briefly,
therapeutic cells collected by apheresis (MNC,A) from a non-mobilized donor will
be initially assessed in the HAL and stored overnight at 4oC, if necessary. The product
will be washed to remove platelets and adjusted to an appropriate cell concentration
for incubation with the CliniMACS CD45RA Microbead reagent in the manufacturer
provided media. The cells will be washed to remove unbound microbeads. These
cells will be applied to the CliniMACS device and the depletion will be performed
as described by the manufacturer. The CD3+ cell target dosing will be determined
by the protocol determined dose level (see section 2.4 or the table below). The goal
CD45RA-depletion ≥2.0 log10 depletion of CD45RA+ cells, however a depletion t
hat does not achieve this target level may still be released to the patient as long as th
e maximum dose of CD3+CD45RA+ cells is no greater than 0.05x106/kg.
Dose level
CD3+ dose
0
1x10^5/kg + MMF through
Day +60
1
1x10^5/kg
2
1x10^6/kg
3
1x10^7/kg

HAP2HCT 27
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The donor non-moblized product may be collected anytime after donor consent is
obtained, but care must be made to ensure patient is very likely to proceed with
transplantation if the collection is performed prior to the patient initiating therapy.
The collection may occur at any time prior to the initiation of G-CSF for mobilization,
or any time that is at least 10 days after completion of mobilization. The DLI product
may be collected as a whole blood unit donation or by leukapheresis (preferred). If
the DLI is collected by standard phlebotomy, the volume to be collected would be
approximately 300 ml whole blood. If the DLI is collected by leukapheresis, the
amount to be processed would be approximately 2 total blood volumes.
Protocol prescribed CD45RA-depleted DLI for the original cohort was given no
sooner than 2 weeks following donor neutrophil engraftment, and no later than Day
+60. Patient had to be sufficiently recovered from mucositis to the point that they
were tolerating PO feeds or approximately 50% of caloric needs by NG tube with
absence of, or improving, diarrhea; and resolution of any narcotic need for abdominal
pain. In addition, there must be no acute GVHD or it must be quiescent. ALT must
be less than 5x ULN, bili less than or equal to 1.5x ULN, and creatinine less than or
equal to 1.5x ULN.
For the early DLI cohort (Amendment 5), the goal is to give the primary
prophylactic CD45RA-depleted DLI at the MED of dose level 3 (1x10^7/kg) around
Day +14, with a goal range of Day +10 to +21. As above, any DLI given after Day
+60 will not be considered prophylactic DLI per protocol and will have to be given
based on clinical need (see Section 4.7.2.2). Unlike above, patients do not have to be
recovered from mucositis. However, like above, there must be no acute GVHD or it
must be quiescent. ALT must be less than 5x ULN, bili less than or equal to 1.5x
ULN, and creatinine less than or equal to 1.5x ULN.
4.7.2 Additional DLI (based on clinical need)
Additional DLI may be given as needed as detailed below. It may be given with or
without preceeding lymphodepleting chemotherapy. Recipients who are further out
from transplant and with substantial lymphocyte engraftment may quickly tolerize
the
newly
infused
donor
lymphocytes.
In
such
patients,
lymphodepleting
chemotherapy may be needed for effective DLI activity. Regimen will vary based on
patient condition, underlying disease, etc. Regimen will be determined by attending
physician and/or PI, but in general would be low dose, expected to have minimal
direct toxicity, and have limited or very transient effects on ANC.
Regimens for lymphodepleting chemotherapy will be defined in a separate document
(non-protocol treatment plan; NPTP) and will require specific consent to the NPTP.
4.7.2.1 Conventional Donor Lymphocyte Infusions
DLI may be administered from the original donor for <100% donor chimerism,
serious viral reactivation or infection, any evidence of disease/recurrence, or poor
immune recovery (such as lymphopenia).

HAP2HCT 28
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Decreased donor chimerism is defined as any single chimerism test
that is not 100% donor
Serious viral reactivation is defined as any virus detected in the
blood by PCR, with a 1 log increase in viral load despite antiviral
medication.
Serious viral infection is defined as any infectious disease from an
identified virus that has progressed in severity despite antiviral
therapy (if available) or shows no improvement despite 1 week of
antiviral therapy (if available)
Any evidence of disease/recurrence is defined as any flow
cytometry, PCR, NGS (such as RNAseq), cytogenetic, or any other
validated molecular testing that detects the presence of the original
hematologic malignancy within the limits of that particular test.
The DLI collected may be collected as a whole blood unit donation or by
leukapheresis. If the DLI is collected by standard phlebotomy, the volume to be
collected would be 300 - 500 ml whole blood. If the DLI is collected by leukapheresis,
the amount to be processed would be approximately 2 total blood volumes.
Prior to administration of DLI, any active immunosuppression should be withdrawn
and the recipient should have no active GVHD. The initial dose will typically be 2.5
or 5 x 104 CD3+/kg. Subsequent doses will be administered at approximately 2 to 4-
week intervals with escalating doses of T cells if no moderate or severe GVHD occurs
with the prior DLIs. The typical initial dose escalation for patients on this protocol is
presented in the following table:
CONVENTIONAL DLI DOSE AND SCHEDULE
DLI
Dose(104 CD3+/kg)
Comments
Initial Dose
2.5
Approximately 2-4 week interval
If no moderate or severe GVHD
Dose #2
5
Dose #3
10
Although this algorithm will be appropriate for a majority, the treating transplant
attending physician may alter the dose and/or interval of DLI based on response to
previous DLI, the severity of the clinical situation, and the condition of the patient.
There is a low threshold for providing an initial dose of 2.5 x 104 CD3+/kg. DLI at
this dose level have not been associated with GVHD. Therefore, DLI may be initiated
for any chimerism less than 100% in a patient who is off immune suppression. The
risk of GVHD increases with increasing doses.
4.7.2.2 CD45RA-depleted Donor Lymphocyte Infusions (additional)

HAP2HCT 29
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Low dose conventional DLI has been found to be very effective for treatment of low
level mixed chimerism, but it’s efficacy in serious viral infections and recurrent
disease (even at MRD levels) is poor. Although prophylactic memory T-cell DLI is
given as a part of this transplant, some patients may have recurrence or persistence
of lymphopenia due to infections or post-transplant immunosuppressive drugs (such
as corticosteroids). Therefore selected patients may benefit from additional adoptive
transfer of donor memory cell populations in the post-transplant setting. This would
include: 1. Patients with viral reactivation or infections that are not responding to
antiviral therapy or historically have a poor response to antiviral therapy and cannot
receive viral specific CTL due to ineligibility or unavailability; 2. Patients with
persistant <100% donor chimerism despite low dose conventional DLI; 3. Patients
who have evidence of disease/recurrence, particularly molecular or MRD levels of
relapse; 4. Patients who have poor immune recovery (such as lymphopenia), defined
as ALC <500/uL and/or TCRαβ T-cell count <300/uL. To receive additional
CD45RA-depleted DLI, patients must meet at least one of the above criteria and be
at least 4 weeks following preemtive DLI. They should preferrably be off immune
suppression and must have no evidence of active GVHD (any prior GVHD must be
quiescent). These patients will be allowed to receive CD45RA-depleted DLI in the
following doses:
CD45RA-DEPLETED DLI DOSE AND SCHEDULE
DLI
Dose(106 CD3+/kg)
Comments
Initial Dose
0.1
Approximately 2-4 week interval
If no moderate or severe GVHD
Dose #2
1
Dose #3
10
If the prophylactic DLI failed to achieve the goal memory cell reconstitution, the
initial dose for this additional CD45RA-depleted DLI may be started at the next
dose level up from the prophylactic dose provided.
The DLI may be collected as a whole blood unit donation or by leukapheresis. If the
DLI is collected by standard phlebotomy, the volume to be collected would be
approximately 300 ml whole blood. If the DLI is collected by leukapheresis, the
amount to be processed would be approximately 2 total blood volumes. The
collected donor lymphocytes would then undergo CD45RA depletion as decribed
above (section 4.7.1).
The release criteria for the CD45RA-depleted DLI will include a maximum dose of
CD3+CD45RA+ cells no greater than 0.05x106/kg.
Conventional DLI and CD45RA-depleted DLI cannot be given within 14 days of
each other.
4.8 Quality Assurance of Cellular Products
Quality assurance for cell products is overseen by the TPQ Quality Assurance division,
which authorizes release of all products. Only trained cell processors will process the cell

HAP2HCT 30
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
products. A labeling and product tracking system is in place to ensure that the correct cells
are infused into the research participant.
Assays of cell numbers and immunophenotyping will be performed both before cell
processing and at critical stages of the process. These values will be recorded according to
SOP of the HAL. All products will be tested for viability and sterility (culture and Gram
stain). Culture results are not available before infusion of cell products. If the gram stain is
positive, the research participant/parent and/or guardian will be informed of this event and
of the risks of proceeding prior to infusion. Positive results will be investigated as per the
variance procedures of the HAL. The IRB and FDA will be notified, if at any time after
infusion, cell product was determined to be contaminated.
5.0 DRUG/DEVICE/BIOLOGIC AGENT INFORMATION
5.1 Medications
Anti-thymocyte globulin (rabbit) (Thymoglobulin®, rabbit ATG)
Source &
Pharmacology
Anti-thymocyte globulin 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. The mechanism of
action by which polyclonal antilymphocyte preparations suppress immune
responses is not fully understood. Possible mechanisms by which anti-
thymocyte globulin may induce immunosuppression in vivo include: T-
cell clearance from the circulation and modulation of T-cell activation,
homing and cytotoxic activities. Anti-thymocyte globulin (rabbit)
includes antibodies against T-cell markers such as CD2, CD3, CD4, CD8,
CD11a, CD18, CD25, CD44, CD45, HLA-DR, HLA Class 1 heavy
chains, and 2 microglobulin. T-cell depletion is usually observed within
a day from initiating anti-thymocyte globulin therapy.
Formulation
and Stability
Anti-thymocyte globulin (refrigerated) is available as sterile, lyophilized
powder to be reconstituted with sterile diluent (both lyophilized powder
and diluent should be at room temperature before reconstitution).
Reconstituted solutions provide a final concentration of 5mg/ml x 5ml.
The product must be further diluted in normal saline prior to
administration. Infusion solutions may be administered over 2 to 6 hours
depending on dose and should be prepared immediately prior to
administration.
Supplier
Commercially available
Toxicities
Frequently reported evens include fever, chills, leukopenia, pain,
headache, abdominal pain, diarrhea, hypertension, nausea,
thrombocytopenia, peripheral edema, dyspnea, asthenia, hyperkalemia,
tachycardia. The most serious toxicity is that of anaphylaxis. Supportive
medical resources must be readily available for patient management
anaphylaxis precludes further administration of the drug. The dose must
be administered over at least 4 hours and the patient pretreated with
antihistamine, corticosteroid, and antipyretic.
Route
Intravenous

HAP2HCT 31
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Blinatumomab (Blincyto)
Source &
Pharmacology
Blinatumomab is a bispecific CD19-directed CD3 T-cell engager
indicated for the treatment of Philadelphia chromosome-negative relapsed
or refractory B-cell precursor acute lymphoblastic leukemia. It activated
endogenous T cells by connecting CD3 in the T-cell receptor complex
with CD19 on benign and malignant B cells. Blinatumomab mediates the
formation of a synapse between the T-cell and the tumor cell,
upregulation of cell adhesion molecules, production of cytolytic proteins,
release of inflammatory cytokines, and proliferation of T cells, which
result in redirected lysis of CD19+ cells.
During the continuous intravenous infusion over 4 weeks, the
phamacodynamic response was characterized by T-cell activation and
initial redistribution, reduction in peripheral B cells, and transient
cytokine elevation.
The pharmacokinetics of blinatumomab appear linear over a dose range
from 5 to 90 mcg/m2/day in adult patients. Following continuous
intravenous infusion, the steady-state serum concentration was achieved
within a day and remained stable over time.
Formulation
and Stability
Blinatumomab is available as 35mcg of lyophilized powder in a single-
use vial for reconstitution.
Supplier
Commercially available
Toxicities
The most common adverse reactions (≥20%) were pyrexia, headache,
perhipheral edema, febrile neutropenia, nausea, hypokalemia, tremor,
rash, and constipation. Cytokine Release Syndrome and Neurologic
toxicities that are severe, which can be life-threatening or fatal, have
occurred with blinatumomab and necessitate interruption or
discontinueation of blinatumomab.
Route
Continuous intravenous infusion
Cyclophosphamide (Cytoxan)
Source
&
Pharmacology
Cyclophosphamide is a nitrogen mustard derivative. It acts as an alkylating
agent that causes cross-linking of DNA strands by binding with nucleic
acids and other intracellular structures, thus interfering with the normal
function of DNA. It is cell cycle, phase non-specific. Cyclophosphamide is
well absorbed from the GI tract with a bioavailability of >75%. It is a
prodrug that requires activation. It is metabolized by mixed function
oxidases in the liver to 4-hydroxycyclo-phosphamide, which is in
equilibrium with aldophosfamide. Aldofosfamide spontaneously splits into
nitrogen mustard, which is considered to be the major active metabolite,
and
acrolein.
In
addition,
4-hydroxycy-clophosphamide
may
be
enzymatically
metabolized
to
4-ketocyclophosphamide
and
aldophosfamide
may
be
enzymatically
metabolized
to
carboxyphosphamide
that
is
generally
considered
inactive.
Cyclophosphamide and its metabolites are excreted mainly in the urine.

HAP2HCT 32
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Dose adjustments should be made in patients with a creatinine clearance of
<50 ml/min.
Formulation
and Stability
Cyclophosphamide is available in vials containing 100, 200, 500, 1000 and
2000mg of lyophilized drug and 75 mg mannitol per 100 mg of
cyclophosphamide. Both forms of the drug can be stored at room
temperature. The vials are reconstituted with 5, 10, 25, 50 or 100 ml of
sterile water for injection, respectively, to yield a final concentration of 20
mg/ml.
Reconstituted solutions may be further diluted in either 5%
dextrose or 0.9% NaCl containing solutions. Diluted solutions are
physically stable for 24 hours at room temperature and 6 days if
refrigerated, but contain no preservative, so it is recommended that they be
used within 24 hours of preparation.
Supplier
Commercially available
Toxicities
Dose limiting toxicities of cyclophosphamide includes BM suppression
and cardiac toxicity. Cardiac toxicity is typically manifested as congestive
heart failure, cardiac necrosis or hemorrhagic myocarditis and can be fatal.
Hemorrhagic cystitis may occur and necessitates withholding therapy. The
incidence of hemorrhagic cystitis is related to cyclophosphamide dose and
duration of therapy. Forced fluid intake and/or the administration of mesna
decreases the incidence and severity of hemorrhagic cystitis.
Other
toxicities reported commonly include nausea and vomiting (may be mild
to
severe
depending
on
dosage),
diarrhea,
anorexia,
alopecia,
immunosuppression and sterility. Pulmonary fibrosis, SIADH, anaphylaxis
and secondary neoplasms have been reported rarely.
Route
Intravenous infusion
Fludarabine (Fludara)
Source
&
Pharmacology
Fludarabine phosphate is a synthetic purine nucleoside analog. It acts by
inhibiting DNA polymerase, ribonucleotide reductase and DNA primase
by competing with the physiologic substrate, deoxyadenosine triphosphate,
resulting in inhibition of DNA synthesis. In addition, fludarabine can be
incorporated into growing DNA chains as a false base, thus interfering with
chain elongation and halting DNA synthesis.
Fludarabine is rapidly
dephosphorylated in the blood and transported intracellularly via a carrier-
mediated
process.
It
is
then
phosphorylated
intracellularly
by
deoxycytidine kinase to the active triphosphate form. Approximately 23%
of the dose is excreted as the active metabolite in the urine (with dosages
of 18-25 mg/m2/day for 5 days). Renal clearance appears to become more
important at higher doses, with approximately 41-60% of the dose being
excreted as the active metabolite in the urine with dosages of 80-260
mg/m2.

HAP2HCT 33
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Formulation
and Stability
Fludarabine is supplied in single-dose vials containing 50 mg fludarabine
as a white lyophilized powder and 50 mg of mannitol. The intact vials
should be stored under refrigeration. Each vial can be reconstituted by
adding 2 ml of sterile water for injection resulting in a final concentration
of 25 mg/ml. Because the reconstituted solution contains no antimicrobial
preservative, the manufacturer recommends that it should be used within
8 hours of preparation. The solution should be further diluted in 5%
dextrose or 0.9% NaCl prior to administration.
Supplier
Commercially available.
Toxicities
The major dose-limiting toxicity of fludarabine is myelosuppression.
Nausea and vomiting are usually mild. Side effects reported commonly
include anorexia, fever and chills, alopecia and rash. Neurotoxicity can be
manifested by somnolence, fatigue, peripheral neuropathy, mental status
changes, cortical blindness and coma and is more common at high doses.
Neurotoxicity is usually delayed, occurring 21-60 days after the completion
of a course of therapy and may be irreversible. Side effects reported less
commonly include diarrhea, stomatitis, increased liver function tests, liver
failure, chest pain, arrhythmias and seizures. Pulmonary toxicity includes
allergic pneumonitis characterized by cough, dyspnea, hypoxia and
pulmonary infiltrates.
Drug induced pneumonitis is a delayed effect,
occurring 3-28 days after the administration of the third or later course of
therapy. Administration of corticosteroids usually results in resolution of
these symptoms.
Route
Intravenous
G-CSF (Filgrastim, NeupogenÒ)
Source &
Pharmacology
G-CSF (granulocytic colony stimulating factor), is a biosynthetic
hematopoietic agent that is made using recombinant DNA technology in
cultures of Escherichia coli. G-CSF stimulates production, maturation and
activation of neutrophils. In addition, endogenous G-CSF enhances certain
functions of mature neutrophils, including phagocytosis, chemotaxis and
antibody--dependent cellular cytotoxicity.
Formulation
and Stability
G-CSF is supplied in vials containing 300 mcg and 480 mcg of G-CSF at
a concentration of 300mcg/ml. The intact vials should be stored under
refrigeration. The vials can be left out of refrigeration for 24 hours, but
should be discarded if left at room temperature for longer periods of time.
G-CSF can be drawn up into tuberculin syringes for administration and
stored under refrigeration for up to 7 days prior to usage. G-CSF can be
further diluted for intravenous infusion in 5% dextrose. Do not dilute in
saline---precipitate may form. If the final concentration of this product is
<15 mcg/ml, it is recommended that albumin be added to a final
concentration of 2mg/ml (0.2%) to minimize adsorption of the drug to
infusion containers and equipment.
Supplier
Commercially available.
Toxicities
G-CSF causes marked leukocytosis. Adverse reactions reported commonly

HAP2HCT 34
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
include bone pain, thrombocytopenia, diarrhea, nausea, rash, alopecia,
fever, anorexia and pain or bruising at the injection site. Allergic reactions,
MI, atrial fibrillation, and splenomegaly have been reported rarely. G-CSF
is contraindicated in research participants with allergy to E. coli derived
products.
Route
Intravenous or subcutaneous.
Melphalan (L-phenylalanine mustard, phenylalanine mustard, L-PAM, L-sarcolysin,
Alkeranâ)
Source
&
Pharmacology
Melphalan, a derivative of nitrogen mustard, is a bifunctional alkylating
agent. Its chemical name is 4-[bis(2-chloroethyl)amino]-L-phenylalanine,
and it has a molecular weight of 305.20. Melphalan is active against tumor
cells that are actively dividing or at rest. Its cytotoxicity is thought to be
due to inter-strand cross-linking with DNA, probably by binding at the N7
position of guanine.
Melphalan is highly protein bound and does not
penetrate well into the cerebral spinal fluid. Elimination half-life after
intravenous administration in adults is approximately 75 minutes.
Elimination appears to be primarily by chemical hydrolysis, but caution
should be used in patients with renal impairment. Plasma concentrations
of melphalan after oral administration are highly variable, possibly due to
incomplete absorption, variable “first pass” hepatic metabolism or rapid
hydrolysis. Area under the plasma concentration-time curves for orally
administered
melphalan
is
approximately
60%
of
intravenously
administered melphalan in adult studies.
Formulation
and Stability
Available as 2 mg tablets for oral administration. This medication is stable
at room temperature until expiration date on the packaging. Intravenous
formulation is supplied as 50 mg freeze dried glass vial. Each 50 mg vial is
supplied in a carton containing a 10 ml vial of sterile diluent. Lyophilized
melphalan should be stored at controlled room temperature and protected
from light. Each vial is marked with its expiration date. The melphalan for
injection must be reconstituted immediately prior to infusion by rapidly
adding the contents of the diluent vial (10 ml) to the freeze dried powder
with a 20 gauge or larger sterile needle and immediately shaking vigorously
until a clear solution is obtained. This results in a 5 mg/ml solution. The
dose should then be diluted in 0.9% NaCl for injection to a final
concentration of not greater than 0.45 mg/ml. The resulting admixture
should be infused over a minimum of 15 minutes. The infusion should be
completed within 60 minutes of reconstitution. Do not refrigerate the
reconstituted melphalan.
Supplier
Commercially available
Toxicities
Melphalan is cytotoxic and caution should be used in handling and
preparing the solution or administering the tablets. Use of gloves is
recommended, and if contact with skin or mucosa occurs, immediately
wash thoroughly. Second cancers such as acute non-lymphocytic leukemia,
myeloproliferative syndrome, and carcinoma have been reported in patients
taking melphalan alone or in combination with other chemotherapy or

HAP2HCT 35
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
radiation.
Melphalan
causes
suppression
of
ovarian
function
in
premenopausal women, with a significant number of patients having
amenorrhea. Testicular suppression (reversible and irreversible) has been
reported. The most common adverse reaction is myelosuppression.
Irreversible BM failure has been reported. Gastrointestinal side effects
reported include nausea/vomiting, diarrhea and oral mucosa ulceration.
Hepatic toxicity has occurred, including veno-occlusive disease.
Acute
hypersensitivity reactions occur in about 2.4% of patients, and can include
anaphylaxis. Hypersensitivity reactions were characterized by urticaria,
pruritus, and edema. Some patients exhibited tachycardia, bronchospasm,
dyspnea
and
hypotension
that
responded
to
antihistamines
and
corticosteroids. Other side effects that have been reported include skin
ulceration or necrosis at injection site, vasculitis, alopecia, hemolytic
anemia, pulmonary fibrosis, and interstitial pneumonitis.
Route
Intravenous
Mesna (Mesnex)
Source
&
Pharmacology
Mesna is a synthetic sulfhydryl (thiol) compound. Mesna contains free
sulfhydryl groups that interact chemically with urotoxic metabolites of
oxaza-phosphorine derivatives such as cyclophosphamide and ifosfamide.
Oral bioavailability is 50%. Upon injection into the blood, mesna is
oxidized to mesna disulfide, a totally inert compound. Following
glomerular filtration, mesna disulfide is rapidly reduced in the renal tubules
back to mesna, the active form of the drug. Mesna and mesna disulfide are
excreted primarily via the urine.
Formulation
and Stability
Mesna is available in 2 ml, 4 ml and 100 ml amps containing 100 mg/ml
of mesna solution. The intact vials can be stored at room temperature.
Mesna may be further diluted in 5% dextrose or 0.9% NaCl containing
solutions. Diluted solutions are physically and chemically stable for at
least 24 hours under refrigeration.
Supplier
Commercially available
Toxicities
Mesna is generally well tolerated. Nausea and vomiting, headache,
diarrhea, rash, transient hypotension and allergic reactions have been
reported. Patients may complain of a bitter taste in their mouth during
administration. Mesna may cause false positive urine dipstick readings for
ketones.
Dosage
and
Administration
Mesna is generally dosed at approximately 25% of the cyclophosphamide
dose. It is generally given intravenously prior to and again at 3, 6 and 9
hours following each dose of cyclophosphamide.
Route
Intravenous
Thiotepa (ThioplexÒ by Immunex) (TESPA, TSPA)
Source
&
Pharmacology
Thiotepa is a cell-cycle nonspecific polyfunctional alkylating agent.
It
reacts with DNA phosphate groups to produce cross-linking of DNA

HAP2HCT 36
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
strands leading to inhibition of DNA, RNA and protein synthesis. Thiotepa
is extensively metabolized in the liver to metabolites that retain activity,
primarily triethylene-phosphoramide (TEPA).
The main route of
elimination is via the urine, mainly as metabolites; the elimination half-life
of the thiotepa is 2.5 hours, and that of TEPA is 17.6 hours.
Formulation
and Stability
Thiotepa is supplied in single-use vials containing 15 mg of lyophilized
thiotepa, 80 mg NaCl and 50 mg NaHCO3. The intact vials should be
stored under refrigeration and protected from light. Each vial should be
reconstituted with 1.5 ml of sterile water for injection to yield a
concentration of 10 mg/ml. Further dilution with sterile water for injection
to a concentration of 1 mg/ml yields an isotonic solution; if larger volumes
are desired for intracavitary, intravenous infusion, or perfusion therapy,
this solution may then be diluted with 5% dextrose or 0.9% NaCl containing
solutions. The 10 mg/ml reconstituted solution is chemically stable when
stored in the refrigerator for up to 5 days, however, it is recommended that
solutions be prepared just prior to administration since they do not contain
a preservative. Reconstituted solutions should be clear to slightly opaque:
the solutions may be filtered through a 0.22 micron filter to eliminate haze.
Supplier
Commercially available; manufactured by Immunex
Toxicities
Dose limiting toxicity is myelosuppression. The leukocyte nadir may occur
at any time from 10 to 30 days. Other toxicities include pain at the injection
site, nausea and vomiting, anorexia, mucositis, dizziness, headache,
amenorrhea, interference with spermatogenesis, and depigmentation with
topical use. Allergic reactions, including skin rash and hives, have been
reported rarely. Rare cases of apnea, hemorrhagic cystitis, and renal failure
have occurred. Thiotepa is mutagenic, carcinogenic, and teratogenic in
animals. Pregnancy category D.
Route
Intravenous infusion.
5.2 CliniMACSTM System
The mechanism of action of the CliniMACS Cell Selection System is based on magnetic-
activated cell sorting (MACS). The CliniMACS device is a powerful tool for the isolation
of many cell types from heterogeneous cell mixtures, (e.g. apheresis products). These can
then be separated in a magnetic field using an immunomagnetic label specific for the cell
type of interest, such as CD3+ human T cells.
The cells to be isolated are specifically labeled with super-paramagnetic particles by an
anti-body directed toward a cell surface antigen. After magnetic labeling, the cells are
separated using a high-gradient magnetic separation column as described below. The
magnetically labeled cells are retained in the magnetized column while the unlabeled cells
flow through the column for collection. The retained cells are eluted by removing the
magnetic field from the column, washing the cells out and collecting them in a separate
container from the unlabeled cells.
The super-paramagnetic particles are small in size (about 50 nm in diameter) and are
composed
of
iron
oxide/hydroxide
and
dextran
conjugated
to
monoclonal
antibodies. These magnetic particles form a stable colloidal solution and do not precipitate

HAP2HCT 37
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
or aggregate in magnetic fields. The antibody conjugated beads used in this system are
highly specific (e.g. CD3+ conjugated beads). High-gradient MACS technology has been
shown to achieve rapid and highly specific depletion or enrichments of large numbers of
target cells from BM, cord blood, and normal peripheral blood mononuclear cells.
The CliniMACS device incorporates a strong permanent magnet and a separation column
with a ferromagnetic matrix to separate the cells labeled with the magnetic particles. The
high-gradient system allows the application of strong magnetic forces and a rapid de-
magnetization. Small ferromagnetic structures, such as the column matrix, placed in a
magnetic field concentrate this homogenous field and thereby produce high magnetic
gradients. In their immediate proximity, the ferromagnetic structures generate magnetic
forces 10,000-fold greater than in the absence of those structures enabling the retention of
magnetically labeled cells. After removing the column from the magnet, the rapid de-
magnetization of the column matrix allows the release of retained cells.
The CliniMACS device is comprised of a computer controlled instrument incorporating a
strong permanent magnet, a closed-system sterile tubing set containing columns with a
coated ferromagnetic matrix and a paramagnetic, cell specific, labeling reagent. The
instrument will separate the cells in a fully automated process yielding a cell population
highly depleted of a specific cell population. The CliniMACS device is not licensed by the
FDA for TCRαβ depletion or CD45RA depletion and therefore is investigational.
The CliniMACS device has separate programs that allow cell selection procedures
optimized for either depletion (e.g. CD3+, TCRαβ+, CD45RA+) or selection of a target cell
population (e.g. CD34+ or CD56+ cells). The basic mechanism is the same for either
application; target cells are "tagged" with super-paramagnetic particles and eventually
separated from the unlabeled cells using the CliniMACS device as described above. The
desired target cells can either be infused or discarded appropriately.
6.0
REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS
6.1 Schedule of Evaluations
All evaluations for these participants will be carried out as outlined in Appendix D, and
guided by the Standard Operating Procedures (SOPs) of the St. Jude Children's Research
Hospital, Department of BMTCT, for recipients of allogeneic stem cell transplantation.
Copies of these SOPs and ongoing updates can be found at the following site: Please note
that all relevant SOPs can be found on the BMT&CT Clinical Transplant Program intranet
page.
Furthermore, to accommodate the research studies, flexibility in the date is allowed without
a deviation from protocol.
The degree of flexibility in the timing is also provided in
Appendix D.
6.2 Long-term Follow-up Evaluations
In general, recipients of allogeneic HCT at St. Jude are seen at least annually until 10 years
post-transplant in the Department of BMTCT outpatient clinic or in the after completion

HAP2HCT 38
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
of therapy (ACT) clinic. For the purpose of this study, research participants will be
followed to year 1 post-transplantation.
6.3 Evaluation for Chimerism and Engraftment
Evaluation for chimerism and engraftment will be performed on bone marrow or peripheral
blood samples according to the timelines noted in Appendix D. Bone marrow chimerism
studies will be conducted on or about the following time points: day +21, day +100, and
year one post-transplant. However, for research participants who have less than 100%
donor chimerism at or about day +21 post-transplant, a repeat bone marrow study (to
include chimerism) may be performed as indicated.
The time to neutrophil and platelet engraftment will be recorded. Neutrophil engraftment
will be defined as the first of 3 consecutive days of an absolute neutrophil count (ANC)
greater than or equal to 500/mm3. Time to platelet engraftment will be designated as the
time to platelet count exceeding 20,000/mm3 and 50,000/mm3 without a platelet transfusion
in the preceding seven days.
Chimerism studies using peripheral blood will be obtained on an approximate weekly basis
according to the evaluation schedule noted in Appendix D. Additional bone marrow and/or
peripheral blood chimerism studies may be performed throughout the course of this study
when clinically indicated. Chimerism studies derived from bone marrow may be used in
lieu of a specified peripheral blood sample if a bone marrow sample is available. In the
event of graft failure/rejection, subsequent chimerism studies may be held as they would
not be clinically indicated at that time.
If there is an initial decrease in donor chimerism to less than 90% at any time on peripheral
blood studies, a bone marrow examination will subsequently be performed within
approximately two weeks to confirm this initial decline. In addition, chimerism analysis
may be performed in subsets of lymphocytes, granulocytes, and monocytes for research
participants with increasing host chimerism until the research participant attains 100%
donor chimerism. Chimerism studies will be performed in the St. Jude Department of
Pathology using standard techniques. Chimerism studies will be reported in the database
as donor percentages.
6.4 Evaluation for Immune Reconstitution
Performed according to the schedule outlined in Appendix D until the immune parameters
recover to normal level or donor pattern:
6.4.1
Lymphocyte subsets study: Flow cytometry enumeration.
6.4.2
Quantitative immunoglobulins: IgG, IgM, and IgA levels.
6.4.3
VBETA/TREC Research: Thymic output and T cell repertoire.
6.4.4
Lymphocyte Research: Lymphocyte subset number and function.
6.4.5
Phenotype Research: Phenotype number and function.
6.4.6
IR-Phenotype: Immune reconstitution of memory and naïve T cells will be
investigated in depth in the Youngblood Lab. This may include phenotypic subset

HAP2HCT 39
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
characterization, functional correlates, and analysis of the epigenetic signatures of
these populations.
6.4.7
T-Function: T-cell function will be investigated in depth in the Thomas Lab. This
may include antigen-specific T-lymphocyte response to viral infections, such as
herpes viruses (CMV, HSV and VZV) and respiratory viruses (influenza and RSV).
6.5 Evaluations for ATG pharmacokinetics testing (optional)
PK testing of rabbit ATG will be obtained in HCT recipients to evaluate drug exposure and
to determine the length of time that ATG remains active following HCT. 2mL of peripheral
blood will be drawn into a heparinized (dark green top) tube at the following timepoints
for the original cohort: pre- and post- 3rd dose of ATG on Day -3, then once each on Day
-1, 0, +2, +4, +7, +14, and +21 (total samples = 9). Sample collection date may be adjusted
±2 days as needed (such as to accommodate blood volume concerns, line access issues,
etc.). The ±2 days adjustment will not apply to the Day -3, -1, 0, +2 and +4 samples.
For the early DLI cohort, samples will be obtained pre- and post- 3rd dose of ATG on Day
-10, then once each on Day -8, -7, -5, -3, 0, +7, and +14 (total samples = 9). The ±2 days
adjustment will not apply to the Day -10, -7, -5, and -3 samples.
6.6 Minimal Residual Disease Evaluation
Minimal residual disease (MRD) assays in BM by immunologic and molecular methods54-
55 will be performed for those research participants who have had this test performed during
prior therapy for their disease at St. Jude only or those for who samples of diseased marrow
were available to identify a leukemic marker for MRD testing.
MRD assays may be
performed more frequently in participants with increasing host chimerism. Tests will be
performed in the appropriate St. Jude laboratories.
6.7 Research Tests on Haploidentical Donor (optional)
Donors will be offered the option for participation in research studies of immune
reconstitution. These tests will be obtained after consent and preferably prior to growth
factor administration. Lymphocyte subset analysis of the donor appears to allow for the
prediction of the reconstitution of the lymphocyte subsets in the research participant after
transplantation. Data in larger donor/research participant pairs will help to verify these
observations. A list of these optional research studies are noted in Appendix D and detailed
below:
6.7.1
Lymphocyte subsets study: Flow cytometry enumeration.
6.7.2
VBETA/TREC Research: Thymic output and T cell repertoire.
6.7.3
Lymphocyte Research: Lymphocyte subset number and function.
6.7.4
Phenotype Research: Phenotype number and function.
6.7.5
IR-PHENOTYPE, and T-FUNCTION: In depth characterization of donor
lymphocytes

HAP2HCT 40
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
7.0 EVALUATION CRITERIA
7.1Toxicity Evaluation Criteria
Adverse event (AE) monitoring for on-study research participants will be assessed using the NCI
Common terminology Criteria for Adverse Events Version 5.0. The standard procedures for
adverse event collection and monitoring are noted in Appendix C. An exception will be made
with the staging and grading of acute and chronic GVHD, which will be assessed by the
criteria defined in Appendix B.
7.2 Hematologic Recovery Criteria
Post-transplant hematologic recovery will be determined using the engraftment criteria as
follows:
7.2.1Neutrophil engraftment will be defined as the first of 3 consecutive tests performed on
different days of an ANC ≥500/mm3 with evidence of donor cell engraftment.
7.2.2Platelet engraftment will be defined as the first of 3 consecutive tests performed on
different days of a platelet count ≥20,000/mm3 with no platelet transfusions in the
preceding 7 days.
7.3 Graft Failure Criteria
7.3.1 Primary graft failure will be defined as an ANC never meeting or exceeding 500/mm3
for 3 consecutive tests performed on different days and no evidence of donor
chimerism (<5%) by day +30 post-HCT.
7.3.2 Secondary graft failure will be defined as a decline in ANC to<500/mm3 with a decline
in donor chimerism to <5% in research participants with prior engraftment.
8.0 CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF-STUDY
CRITERIA
8.1 Recipient Criteria
Recipient research participants will remain on study until one of the following occurs:
8.1.1
Withdrawal from protocol.
8.1.2
Death.
8.1.3
Donor unable to provide the HPC required for intended recipient to undergo the
primary HCT procedure (refer to donor off-study criteria).
8.1.4
Unable to be contacted and/or effectively monitored by the Principal Investigator
(PI) and/or designees for follow-up (lost to follow-up).
8.1.5
One year after HPC infusion (i.e. has completed the year +1 post-primary transplant
evaluation).

HAP2HCT 41
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
8.1.6
Development of a significant change in health status at any point of therapy which
would render receipt of the transplantation procedure or continuation in the study
medically unsafe or not in the participant’s best interest.
8.2 Off-therapy Criteria
Recipient research participants may remain on study, but considered off therapy, for
monitoring if one of the following occurs (off therapy participants monitored for disease
status and survival only):
8.2.1
Requires conventional chemotherapy for confirmed (generally >5%) disease
relapse.
Epigenetic or targeted therapy, immune therapy (such as repeat dosing of
blinatumomab, or use of checkpoint inhibitor), and low dose lymphodepleting
chemotherapy with DLI, even if molecularly detectible disease is present, would
not trigger this off-therapy criteria.
8.2.2
Experiences graft failure/rejection, requiring non-protocol therapy other than as
described in Section 4.6 of this protocol.
8.2.3
Noncompliance with protocol medications/administrations and/or required follow-
up evaluations.
8.2.4
Positive pregnancy test post-HPC infusion.
8.2.5
Recipient requires an additional HPC infusion, and unable to receive these cells
due to donor issues.
8.3 Donor Criteria
Donor research participants will remain on study until one of the following occurs:
8.3.1
Withdrawal from protocol. Donor research participants may withdraw their consent
to participate at any time.
Physician may withdraw donor at any time that
continuation in the study is deemed medically unsafe or no longer in the donor’s
best interest.
8.3.2
Day of transplant recipient or transplant donor death, whichever occurs first.
8.3.3
Development of a change in health status, including positive pregnancy test or a
clinically significant risk for or positive testing for communicable disease, which
in the opinion of the PI, would render the donor medically ineligible to serve (or
continue to serve) as a therapeutic cell donor.
8.3.4
Unable to be contacted and/or effectively monitored by PI and/or designees for
follow-up as judged by the PI (including non-compliance and lost to follow-up).
8.3.5
Once the PI has determined that the transplant recipient does/will not require an
additional infusion(s) of the donor’s cells for the purpose of this protocol, the donor
will be taken off study seven days post final cell collection procedure.
9.0 SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS
9.1 Reporting Adverse Experiences and Deaths to St. Jude IRB

HAP2HCT 42
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The principal investigator is responsible for promptly reporting to the IRB any adverse
events that are unexpected/unanticipated, serious, and that may represent potential harm
or increased risk to research participants. When an unexpected death occurs, the PI should
report it to the Director of Human Subject’s Protection immediately. A reportable event
submission should follow within 48 hours of notification of the event.
Serious, unexpected, and related or possibly related events must be reported within 10
business days of notification of the event. At the same time, the investigator will notify the
study sponsor and/or the FDA, as appropriate. All other SAEs, including expected death,
and all captured AEs will be reported to the IRB at the time of the continuing reviews, with
the following exception:

Any grade III-IV infusion reactions will be reported as soon as possible but every
effort should be made to assure reporting is no more than within 10 business days
of the event.
For this research study, recipient participants will be followed for all NCI Grade III-V
adverse events from the start of conditioning and throughout the first year post HCT,
regardless of their relationship to the treatment given. In addition, clinically significant
NCI Grade I-II adverse events that are judged to be related/possibly related may be
collected per the discretion and judgment of the PI. Examples of “clinically significant
Grade I-II adverse events could include, but are not limited to: events meeting criteria for
SAE, infections requiring oral systemic therapy, VOD or hemorrhagic cystitis. GVHD
events will be recorded on an ongoing basis regardless of stage or grade using the criteria
defined in Appendix Band will not be graded according to NCI criteria.
With regard to the haploidentical donor participants, they will be followed for all SAEs
and any clinically significant AEs, per the judgment of the PI, that are deemed related to
the mobilization and/or apheresis procedure from the time of the initiation of mobilization
growth factors, until 7-days post last day of the final apheresis procedure. If the transplant
recipient requires a second HPC infusion, meaning that the donor is required to undergo
the mobilization and apheresis procedure again, collection of this donor safety data will
restart upon the initiation of the subsequent mobilization procedure and continue until 7-
days post the last day of this apheresis procedure. Timelines for reporting of these donor
events to the institutional and federal governing agencies will be according to the same
timelines utilized for the recipients. A listing of the captured donor safety data will be
provided in a separate table from the transplant recipients within each respective continuing
review report.
The following definitions apply with respect to reporting adverse experiences:
Serious adverse event – any adverse event temporally associated with the participant’s
participation in research that meets any of the following criteria:

results in death;

is life-threatening (places the participant at immediate risk of death from the event
as it occurred);

requires inpatient hospitalization or prolongation of existing hospitalization

results in a persistent or significant disability/incapacity;

results in a congenital anomaly/birth defect; or

HAP2HCT 43
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024

any other adverse event that, based upon appropriate medical judgment, may
jeopardize the participant’s health and may require medical or surgical intervention
to prevent one of the above outcomes.
Unexpected adverse event – any adverse event meeting any of the following criteria:

an event for which the specificity or severity is not consistent with the protocol
related documents, including the applicable investigator brochure, IRB approved
consent form, IND/IDE application or any of the product labeling or package
inserts;

an event for which the observed rate of occurrence is significantly increased above
what is expected or credible baseline rate for comparison;

an event for which the occurrence is not consistent with the expected natural
progression of any underlying disease, disorder, or condition of the participant(s)
experiencing the adverse event and the participant’s predisposing risk factor profile
for the adverse event.
The principal investigator is responsible for reviewing the aggregate toxicity reports and
reporting to the IRB if the frequency or severity of serious toxicities exceed those expected
as defined in the protocol or based on clinical experience or the published literature. Any
proposed changes in the consent form or research procedures resulting from the report are
to be prepared by the study team and submitted with the report to the IRB for approval.
9.2 Reporting to St. Jude Institutional Biosafety Committee
Continuing review reports will be sent to the Institutional Biosafety Committee (IBC) on
at least an annual basis using the most current version of the continuing review form found
on the IBC website. The safety reports, sent to the IRB for both the donors and recipients,
will be simultaneously forwarded to the IBC. Therefore, reporting for safety events to this
committee will be according to the same timelines as reporting to the IRB. This includes
notification of achievement of MTD (if/when applicable). As per the direction of the IBC,
only those protocol revisions and amendments directly related to the CliniMACS
processing and related reagent(s) will require review and consideration by the IBC. Other
revisions/amendments will be noted in the IBC continuing review report.
9.3 Reporting to FDA (21CFR§312.32 Safety reports)
The FDA will be notified in writing (IDE safety report) of any serious and unexpected AE
associated with an investigational treatment or device; or any results from laboratory
animal tests that suggest a significant risk for human subjects including reports of
mutagenicity, teratogenicity, or carcinogenicity.
Each notification to the FDA should be made as soon as possible and no later than 15
calendar days after the sponsor’s initial receipt of the information. The FDA may
require additional data to be submitted. In each written IND safety report, the sponsor
shall identify all safety reports previously filed with the IND concerning a similar adverse
experience, and shall analyze the significance of the adverse experience in light of the
previous, similar reports where applicable.

HAP2HCT 44
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The sponsor shall also notify the FDA by telephone, email, or by facsimile transmission of
any unexpected fatal or life-threatening experience associated with the use of the
investigational device as soon as possible but no later than 7 calendar days after the
sponsor’s initial receipt of the information. Any grade III-IV infusion reactions will be
reported as soon as possible but every effort should be made to assure reporting is no more
than within 7 business days of the event. Follow-up information to a safety report must be
submitted as soon as the relevant information is available.
If the results of further investigation show an AE that was not initially determined to be
reportable should later be deemed reportable, the sponsor shall inform the FDA of the
event in a written safety report as soon as possible, but no later than 15 calendar days after
the determination is made. Results of the investigation of other safety information shall be
submitted, as appropriate, in an information amendment or annual report.
Continuing
review reports, which will include the up-to-date clinical and safety data, will be submitted
to the FDA at least annually.
9.4 Reporting to St. Jude Office of Regulatory Affairs
Copies of all correspondence to the St. Jude IRB, including SAE reports are provided to
the St. Jude Regulatory Affairs Office. All FDA related correspondence and reporting will
be conducted through the Regulatory Affairs Office. Adverse event reporting and annual
reporting will be in accord with the FDA Title 21 CFR312.32 and Title 21 CFR312.33,
respectively. The Regulatory Affairs Office can be reached at 901-595-2347 (secondary
contact: St. Jude Vice President of Clinical Trials Administration 901-595-2876).
9.5 Continuing Review Reports
Continuing review reports of protocol progress and summaries of adverse events will be
filed with the St. Jude IRB, and IBC at least annually. Continuing review reports to all
regulatory authorities will be structured in a manner so that any infusion toxicities or stem
cell product related variances will be reported in separate listings from all other required
elements.
9.6 Reporting to Miltenyi Biotec
Data related to device malfunction will be reported to Miltenyi Biotec, the manufacturer
of the CliniMACS system. These device related malfunctions as well as any device related
adverse events will be reported so the manufacturer can collect data and address those
issues appropriately.
9.7 Reporting to the Center for International Blood and Marrow Transplant Research
The Transplant Program at St. Jude is required by the federal government to report
transplant information to the Center for International Blood and Marrow Transplant
Research (CIBMTR). The CIBMTR is a research partnership of the International Bone
Marrow Transplant Registry, the National Marrow Donor Program (NMDP), and the
Foundation for the Accreditation of Cellular Therapy (FACT). This organization is
responsible for the collection and maintenance of a standardized data warehouse registry
of autologous and all allogeneic (related and unrelated donor) transplants performed in the
United States.

HAP2HCT 45
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The Office of General Counsel, U.S. Department of Health and Human Services, had
deemed the CIBMTR not a covered entity under the Privacy Rule (45 CFR 164.512), 45
CFR Parts 160 and 164, and the Health Insurance Portability and Accountability Act
(HIPPA) of 1996. For this reason, the submission and disclosure of certain protected health
information (PHI), including that required for CIBMTR, is allowable without the
individual’s authorization (i.e. consent is waived) when such disclosure is made to public
health authorities authorized by law for the purpose of preventing or controlling disease,
injury, or disability.
Data resulting from this transplant procedure will be sent for general registry purposes to
comply with the federal government requirements. This information for both donor and
recipient is submitted using a unique participant identification number. The information
submitted for haploidentical recipients is less extensive than recipients of other donor
products. For this reason, variables submitted may include but are not limited to the
transplant recipient’s date of birth, country/state of current residence, diagnosis, basic
lympho-hematopoietic reconstitution (e.g. date of ANC and platelet engraftment), post-
HCT disease status, and basic AEs (e.g. GVHD- yes or no), survival status, date/cause of
death.
9.8 Data and Safety Monitoring Board
This study has been referred to the St. Jude Data and Safety Monitoring Board (DSMB)
for regular monitoring, and will be sent to the DSMB upon approval by the St. Jude CT-
SRC and IRB. The DSMB is charged with advising the Director and other senior leaders
of St. Jude Children’s Research Hospital (SJCRH) on the safety of clinical protocols being
conducted by SJCRH investigators and on their continuing scientific validity.
10.0 DATA COLLECTION, STUDY MONITORING, AND CONFIDENTIALITY
10.1Data Collection and Submission
The Clinical Research Associates (CRAs) and CRA-RNs, within the Department of
BMTCT will assure protocol compliance, and conduct all clinical and safety data
collection. Data will be entered into the St. Jude institutional databaseat the time it is
obtained from the electronic health record.. The PI and/or designee will be responsible
for the review of data for accuracy and completeness. The PI and/or designee will review
the study data at least monthly.
10.2Study Monitoring
This study will be monitored according to a study specific clinical trial monitoring plan
(CTMP). The CTMP is based on the current St. Jude Children’s Research Hospital
Institutional Plan for Data and Safety Monitoring of Clinical Trials (DSMP) which
outlines the monitoring strategy based on the risk of the study with an emphasis on
participant safety, data quality, and human subjects protections. Additionally, the CTMP

HAP2HCT 46
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
is a supplement to the protocol, Clinical Trial Operations SOPs and study specific
materials which will aid in the monitoring of this study.
The Investigator will permit study-related monitoring by providing direct access to
source data and to the participants’ medical histories. The Investigator will also maintain
an Investigator Site File with essential documents according to ICH and all applicable
regulatory guidelines and make the file accessible for monitoring visits as needed.
The Monitor will visit the study site at regular intervals during the study. Visits may be
performed onsite, remotely, or in a hybrid fashion (both onsite and remote) as needed for
the sponsor (SJCRH) to fulfill their obligations. Site monitoring is conducted according
to applicable ICH and GCP guidelines to ensure the protection of participants’ rights and
well-being, protocol adherence, quality of data (accurate, complete, and verifiable),
study treatment accountability, compliance with regulatory requirements, and continued
adequacy of the investigational site and its facilities.
10.3Confidentiality
Unique patient numbers will be used in place of an identifier such as a medical record
number. This unique patient number will be used to identify any data that is released to
persons or agencies outside of the study team. No research participant names will be
recorded on the data collection forms. The list containing the unique patient number and
the medical record number will be maintained in a password protected file that is
accessible only to study team members.
10.4 Genomic Data Sharing
Genomic and epigenomic data may be shared through the St. Jude Cloud, the Database
for Genotypes and Phenotypes (dbGAP) and the Gene Expression Omnibus (GEO),
which are both run by the NIH, and the Sequence Read Archive (SRA). Prior to
submitting data, data will be stripped of identifiers such as name, date of birth, medical
record number, and any other information that could be used to identify participants and
will be fully de-identified by standards consistent with the Common Rule and HIPAA.
The genotype data will be made publicly available no later than six months after
completion of sequencing and analysis for all patients on the study, or the date of initial
publication, whichever comes first.
11.0 STATISTICAL CONSIDERATIONS
11.1 Statistical Design and Analysis for the Primary Objectives and Stopping Rules (original
cohort)
This is a phase I/II study with two different phases, to determine the maximum effective
dose (MED) for CD45RA-depleted DLI when given in the early post-engraftment period
in phase 1 and, then to assess efficacy of TCRαβ-depleted progenitor cell graft with
additional effective memory T cell DLI defined in phase 1, plus selected use of
blinatumomab, in Haploidentical Donor Hematopoietic Cell Transplantation for
Hematologic Malignancies in phase 2. The statistical design for phase 1 will be ad-hoc
design. The statistical design for phase 2 will evaluate, using a group sequential design,
evidence that the new treatment strategy would be expected to result in 1-year EFS in an
acceptable rate of patients treated with effective dose determined from phase 1. The

HAP2HCT 47
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
statistical design for the Phase 2 study is an expansion cohort at the effective dose
determined in the Phase 1 portion of the study. Those treated at the effective dose in the
Phase 1 study will be evaluated for efficacy as part of the Phase 2 study.
Phase 1: Maximum effective dose
For phase 1, a maximum effective dose of CD45RA-depleted DLI is defined as the
maximum value of doses that satisfy the rate of patients with their memory T cell count
measured at week 4 post-DLI more than 300/uL is more than 50% and the toxicity of
grade 3-4 aGVHD is less than 0.2. Only acute GVHD with an onset date within 28 days
of the CD45RA-depleted DLI will be considered to be caused by the DLI, all other acute
GVHD will be attributed to the transplant in general and not the DLI. Although it is not
possible to accurately attribute any GVHD following DLI to the DLI itself versus the
overall transplant regimen, we feel that 28 days is an appropriate demarcation for the
following reasons: 1) clinical experience shows that positive effects of DLI (such as
correction of chimerism) are typically seen at 7 to 14 days after DLI, therefore it is
reasonable to postulate that untoward T-cell alloreactivity (such as GVHD) would occur
in the same time frame; 2) it is a long standing standard practice across institutions (and
in this protocol) to repeat DLI within 4 weeks if the desired efficacy was not attained and
if there is no GVHD, indicating that the general consensus based on clinical experience
is that the duration of activity following DLI is approximately 4 weeks.
We note that for one specific dose level, the actual dose re-infused to the patients may be
slightly different from the target dose. However, per intent to treat, the patients will still
be evaluable for that specific dose level on this study. To determine the MED of
CD45RA-depleted DLI, we will start to treat patients from dose level 1 and will enroll
a cohort of 10 patients at each dose level (See table below for each dose level). We
conclude that a dose level X is the MED only if it satisfies 1) among a cohort of 10
patients are treated at dose level X, if 6 or more patients with their memory T cell counts
are ≥300/uL at week 4 post-DLI and 7 or more patients without grade 3-4 aGvHD, 2)
dose level X+1 is too toxic (≥four patients experience grade 3-4 aGVHD) or not
efficacious (<6 patients with their memory T cell counts are ≥300/uL). Then this MED
will be assessed for efficacy and toxicity in phase 2.
There is no preliminary or published literature estimating the rates of patients with grade
3-4 aGvHD and with memory T cell count ≥300/uL post DLI for the proposed four dose
levels, thus we considered two scenarios of projected multinomial probability
distribution for grade 3-4 aGvHD and memory T cell count ≥300/uL for four dose levels
as pair 1 of [0.05, 0.1, 0.15, 0.25] and [0.2, 0.4, 0.8, 0.9] and pair 2 of [0.05, 0.1, 0.15,
0.4] and [0.2, 0.4, 0.8, 0.9] in our simulations below. If there is no correlation between
event of grade 3-4 aGvHD and event of memory T cell counts are ≥300/uL, the empirical
probabilities that dose levels 2 and 3 would be MED are 0.21 and 0.74 for pair 1 and 0.58
and 0.35 for pair 2 using the diagram below, based on 1000 simulations, respectively.
Dose level
CD3+ dose
0
1x10^5/kg + MMF through
Day +60
1
1x10^5/kg
2
1x10^6/kg
3
1x10^7/kg

HAP2HCT 48
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Similarly, if there is some mild correlation of 0.2 between grade 3-4 aGvHD and memory
T cell count ≥300/uL, the corresponding simulated probabilities were similar with less
than 2% difference. This was obtained using rmvbin function in bindata package to
simulate the correlated multivariate binary random variables. This is a phase I part to
determine MED, thus, this calculation is only for illustrative purpose and the proposed
determination rule is “ad hoc” in nature. However, it was deemed reasonable in PI’s
evaluation.
Otherwise, diagram below shows that at the current dose level X, if four or more patients
experience grade 3-4 aGVHD but there are still ≥6 patients with their memory T cell
counts are more than 300/uL at week 4 post-DLI, then this level of CD45RA-depleted
DLI is too toxic and we will move one dose level down X-1 if level X-1 is not
investigated; otherwise, Dose X-1 is MED.
Otherwise, if less than four patients experience grade 3-4 aGVHD but there are 5 or less
patients with their memory T cell counts are less than 300/uL at week 4 post-DLI, then
this level of CD45RA-depleted DLI is not efficacious and we will move one dose level
up X+1;
Otherwise, if at any time point, four patients experience grade 3-4 aGVHD but there are
5 or more patients with their memory T cell counts are less than 300/uL at week 4 post-
DLI, then this level of CD45RA-depleted DLI is too toxic and not efficacious; we will
stop and conclude that no MED of CD45RA-depleted DLI is determined if the previous
dose does not meet condition 1) above.
If a patient dies from TRM or disease progression within 4 weeks post DLI, it will be
counted as a failure in terms of the memory T cell count ≥300/uL at week 4 post-DLI.

HAP2HCT 49
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Phase 2: Efficacy and stopping rules
For phase 2, based on HAPNK1 data frozen on 01/04/2018, 30 out of 36 non-refractory
patients without any events (relapse or death) in the first 1 year post transplantation in
the standard arm of HAPNK1 (EFS rate is 0.83 (95% CI: 0.67, 0.94) based on exact
binomial distribution) (Figure in section 2.2). It may further be noted that on HAPNK1
standard arm there was 8 patients censored within 1 year follow-up because they have
not finished their 1-year follow-up. Thus, with assumption of no censoring expected
during 1-year time period we can approximate the EFS at 1-year using Binomial
distribution. That is, each patient will be followed for one-year and will be counted as a
success if the patient survived without any events (death due to any cause or relapse). If
any patient is lost follow up within 1 year, we will count it as a failure for EFS. Denote
p be the proportion of patients surviving at 1-year without these events mentioned.
Although the final EFS number may be slightly smaller after all 8 censored patients have
been followed for 1 year, based on the data above, we have assumed an EFS rate of 0.83.
Using a one sample test with four looks based on a gamma family spending functions
with parameter γ = 1 for assessing a single proportion with H0: p≥0.83 vs H1: p<0.83,
East 6 gives 85.6% power to detect a decline from 83% to 70% with 0.15 of type I error
rate and a sample size of 50 patients. Based on this design, if we observe 6 failures in the
first 20 patients, 9 failures in the first 30 patients, 11 failures in the first 40 patients, or
13 failures in the 50 patients within the first one year post transplantation, this would
suggest that the proposed treatment approach is inferior.
#Patients
Cum α Spent
Boundary
#Events
20
0.078
0.711
≥6
30
0.107
0.728
≥9
40
0.131
0.744
≥11
50
0.150
0.754
≥13
In addition, we will closely monitor the trial for early excessive toxicities in terms of the
safety of Blinatumomab administration, severe acute GvHD (aGvHD), and therapy
related mortality (TRM). TRM is any death in remission and related to protocol therapy.
Safety of Blinatumomab administration, aGvHD, and TRM will be monitored for 100
days from the date of transplantation. These endpoints will be monitored independently.
A patient who experiences grade 3-4 aGVHD and the TRM will count towards both
rules.
Below is toxicity monitoring related to Blinatumomab administration. If the drug is held
for more than 2 weeks, it will be permanently discontinued. To assure the safety of
Blinatumomab administration on this study, if two or more out of the first 6 patients
experience Blinatumomab permanent discontinuation due to toxicity, then the study will
be placed on hold and the Blinatumomab regimen will be adjusted to a lower dose and/or

HAP2HCT 50
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
premedication will be added. If less than 2 out of the first 6 recipients experience
Blinatumomab permanent discontinuation due to toxicity, then the remaining CD19+
recipients may continue to receive Blinatumomab at the proposed dosing.
The
investigators expect that there will be 15 to 20 patients with a CD19+ leukemia in the 50
or 70 planned recipients (see below).
Out of 34 patients on the standard arm of HAPNK1 patients, 7 patients experienced grade
3-4 aGVHD within 100 days post transplantation (0.206, 95% CI: 0.087~0.379 based on
exact binomial distribution). Thus, consideration to stopping/amending the trial would
be given if there is evidence suggesting that the rate of grades III-IV aGVHD in 100 days
post transplantation is greater than 20%. The planned interim evaluation time points and
stopping rules are provided in Table below. This plan is based on one sample test with
four looks based on a gamma family spending functions with parameter γ = 2.5 to reject
a high rate of grades III-IV aGVHD of 30% with a type I error rate of 0.2. Based on the
Table, if we observe 6, 9, 12, and 15 patients with grade III-IV acute GVHD within the
first 100 days post transplantation in the first 20, 30, 40, and 50 evaluable research
participants treated, respectively, then we will temporarily halt the trial and consider
amending the protocol. Based on these stopping rules, if the true grade 3-4 aGVHD is
0.4, the respective probability of stopping the trial early for excess of grade 3-4 aGVHD
would be 0.87, 0.91, 93%, and 0.95. It may be noted that the above stopping rules are “ad
hoc” in nature. However, the proposed stopping rules were deemed reasonable in PI’s
evaluation.
Stopping Rules for Toxicity of Grade III-IV aGVHD within the first 100 days post
transplantation.
#No. of Research
Participants Enrolled
Cum α Spent
Boundary
# Grade 3-4 aGVHD
Observed
20
0.138
0.298
≥6
30
0.169
0.293
≥9
40
0.188
0.286
≥12
50
0.200
0.283
≥15
Out of 34 patients on the standard arm of HAPNK1 patients, 1 patients experienced TRM
within 100 days post transplantation (0.03, 95% CI: 0.001~0.15 based on exact binomial
distribution). There, besides the stopping rules for monitoring grade 3-4 aGVHD, we
will closely monitor the trial for too much TRM, that is, if there are 5 or more patients
experience TRM within 100 days post transplantation in the first 25 patients or 8 or more
patients experience TRMs in the 50 patients, the study will be paused and assessed for
potential changes to the treatment. Based on these stopping rules, if the true TRM rate is
0.2, the respective probability of stopping the trial early for excess of TRM would be

HAP2HCT 51
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
0.58 and 0.81, respectively. It may be noted that the above stopping rules are “ad hoc” in
nature. However, the proposed stopping rules were deemed reasonable in PI’s evaluation.
With this study plan, the minimum number of patients (whom received protocol defined
dosing of CD45RA-depleted DLI and are evaluable at the 4 week time point post DLI)
needed for the Phase I portion of the study would be 10 on dose 1. The maximum number
of patients (whom received protocol defined dosing of CD45RA-depleted DLI and are
evaluable at the 4 week time point post DLI) for Phase I would be 30 patients (10 patients
on each dose level 1-3). The Phase II expansion cohort will include 40 additional patients
(are evaluable at the 1-year time point post transplantation) treated at the effective dose
of CD45RA-depleted DLI determined in the Phase I trial. So the total number of patients
for the phase I of the study would be minimum of 10 and maximum of 30. Thus, for both
phase 1 and phase 2 of the study, we will need in total a minimum of 50 patients and a
maximum of 70 patients.
Based on our St. Jude protocol HAPNK1, the total 34 standard risk patients were enrolled
on the study in three years and 3 months with excluding 14 months temporary pause for
accrual. With an estimated accrual rate of about 10 participants per year, the study is
expected to last for a minimum of 5 years to enroll 50 patients and a maximum of 7 years
to enroll 70 patients.
After the study is finished, for the second primary objective, the number of patients
without events of relapse and death along with 95% Blyth-Still-Casella confidence
interval will be estimated based on the binomial distribution. In addition, to account for
potential lost follow-up, the Kaplan-Meier estimate of EFS along with its standard error
will also be calculated, where EFS = min (date of last follow-up, date of relapse, date of
death due to any cause) – date of transplant, and all participants surviving at the time of
analysis without events will be censored. As a secondary analysis, we will also perform
comparisons of EFS of the study cohort with the standard risk patients in HAPNK1
cohort. The EFS will be compared using the log-rank test method based on a two-sided
test.
We propose to analyze and report all outcomes from this original cohort after the primary
objective follow-up is complete: when the last evaluable participant enrolled on the
original cohort has been followed for one-year post transplant.
11.2 Statistical Analysis for Secondary Objectives (to be done for each cohort individually)
The final results of these secondary objectives are expected to be available when the last
evaluable participant has been followed for one-year post-transplant.
11.2.1 Assess the safety and feasibility of the addition of blinatumomab in the early
post-engraftment period in patients with CD19+ malignancy.
Giving blinatumomab in the early post-transplant period is novel, and the safety
and feasibility of this approach is unknown. Therefore, we will use an ad hoc

HAP2HCT 52
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
design very similar to to the traditional 3+3 phase I approach to describe toxicity
and assess safety in many pediatric oncology trials. As described in section 11.1
above,
if
two
patients
out
of
6
experience
blinatumomab
permanent
discontinuation due to toxicity (as instructed in section 4.2), then the study will
be placed on hold and the blinatumomab regimen will be adjusted to a lower dose
and/or premedication will be added. If at least 5 of 6 patients are able to
successfully receive at least 21 of planned 28 days of blinatumomab without
permanent discontinuation, then the addition of blinatumomab in the early post-
engraftment period will be considered safe and feasible, and dosing will continue
per protocol.
11.2.2 Estimate the incidence of malignant relapse, EFS and OS at one-year post-
transplantation
The estimate of cumulative incidence of relapse will be estimated using
Kalbfleisch-Prentice method. Death without relapse is the competing risk event.
The Kaplan-Meier estimates of OS and EFS along with their standard errors will
be calculated using the SAS macro (bmacro251-Excel2007\kme) available in the
Department of Biostatistics at St. Jude, where OS = min (date of last follow-up,
date of death) – date of transplant and all participants surviving at the time of
analysis without events will be censored, and EFS = min (date of last follow-up,
date of relapse, date of graft failure, date of death due to any cause) – date of
transplant, and all participants surviving at the time of analysis without events
will be censored. The analysis for this objective will be performed when the last
evaluable participant has been followed for one-year post transplant.
11.2.3 Estimate the incidence and severity of acute and chronic GVHD
The cumulative incidence of acute and chronic GVHD will be estimated using
Kalbfleisch-Prentice method. Death without acute and chronic GVHD is the
competing risk event. The severity of acute GVHD and chronic GVHD will be
described. The analysis for this objective will be performed when the last
evaluable participant has been followed for 1 year post-transplant.
11.2.4 Estimate the rate of transplant related mortality in the first 100 days after
transplantation
The cumulative incidence of transplant related mortality will be estimated using
the same method as used in evaluating Objective 11.2.2. Deaths before day 100
because of other reasons are the competing risk events. The analysis for this
objective will be performed when the last evaluable participant has been followed
for 100 days post-transplant.
11.2.5 To measure and describe the pharmacokinetics of rabbit ATG in HCT recipients
on this study.
Pharmacokinetic analysis of rabbit ATG will be performed in the Department of
Pharmaceutical Sciences using standard methods.
11.3 Analysis for Exploratory Objectives (to be done for each cohort individually)
The final results of these exploratory objectives are expected to be available when the
last evaluable participant has been followed for one-year post-transplant.

HAP2HCT 53
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
11.3.1
Record immune reconstitution parameters, including chimerism analysis,
quantitative lymphocyte subsets, T cell receptor excision circle (TREC) analysis,
V-beta spectratyping, and phenotype and functional analysis of reconstituted
lymphocyte subsets.
All immune reconstitution measures will be descriptively analyzed.
11.3.2 Describe the use of additional CD45RA-depleted DLI for recipients who have
<100%
donor
chimerism,
viral
reactivation
or
infection,
evidence
of
disease/recurrence or poor immune recovery (such as lymphopenia). Assess and
record efficacy of CD45RA-depleted DLI for these conditions, and all adverse
events that are related to CD45RA-depleted DLI.
The use of additional CD45RA-depleted DLI for recipients who have <100%
donor chimerism, viral reactivation or infection, evidence of disease/recurrence
or poor immune recovery (such as lymphopenia) will be recorded, including dose
given, date given, and indication for use and and will be assessed descriptively.
To assess efficacy, the resolution of viral infection, resolution of molecular or
frank disease, or the correction of severe lymphopenia will be noted. All adverse
events that are at least possibly related to CD45RA-depleted DLI and any episode
of GVHD will be recorded and will be assessed descriptively.
11.4 Statistical analysis specific to the Early DLI cohort
11.4.1 Early DLI cohort Primary objectives
The early DLI cohort will recruit and transplant 30 patients with the primary
objective to assess the feasibility and safety of early CD45RA-depleted DLI
administration. We will restart the enrollment count at the time of the first patient
enrolled onto the early DLI cohort, and these patients will be summarized
separately from the previous cohorts for monitoring and analysis.
Feasibility will be measured by the days from HCT to DLI infusion in the early
DLI cohort with the goal to reduce the mean days to DLI infusion by 14 days
from the original phase 2 HAP2HCT cohort (mean: 32.2 days, SD: 8.0). This
endpoint will be reported descriptively. There will be no stopping rule for this
objective.
Safety will be measured by the occurrence of grade III-IV aGVHD in the first
100 days after transplant, and the proposed regimen will be declared safe based
on the stopping rule proposed in the table below. This rule was constructed to
have a high probability of detecting a grade III-IV aGVHD rate of 28% (which is
consistent with the HAPNK1 100-day cumulative incidence estimate) and a low
probability of triggering the stopping rule at an 8% rate (consistent with the
observed 4 events within 100 days in the original HAP2HCT cohort) using the
exact binomial distribution. The 100-day cumulative incidence of grade III-IV
aGVHD will be estimated using the Kalbfleisch-Prentice method and reported

HAP2HCT 54
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
with a 95% confidence interval. Death without grade III-IV aGVHD is the
competing risk.
Stopping rule for Early DLI cohort based on occurrence of 100-day grade III-IV
aGVHD
Analysis
Cumulative
sample size
Cumulative
aGVHD III-
IV event
threshold
True 100-day aGVHD III-IV rates
0.08
0.1
0.15
0.2
0.25
0.28
0.3
Probability
of crossing
the
threshold
at a given
analysis
Interim
10
3
0.04
0.07
0.18
0.32
0.47
0.56
0.62
Final
30
7
0.00
0.01
0.07
0.17
0.25
0.26
0.26
Probability
of
declaring
the
treatment
acceptable
< 6 events,
declare
treatment
acceptable
0.96
0.92
0.75
0.50
0.28
0.17
0.12
We do not plan to pause enrollment for the interim analysis, anticipating that the
risks due to delaying transplantation for eligible subjects are higher than the risks
of grade III-IV aGVHD for patients enrolled while the first 10 transplanted
patients are still in their 100-day safety follow-up windows. With the expected
accrual rates, we also expect very few patients to be enrolled while the first 10
patients are in their safety follow-up.
After the 10th transplanted patient
completes their safety follow-up, the cumulative number of patients experiencing
grade III-IV aGVHD will be compared to the stopping rule. Any patients after
the 10th transplanted patient will not be included for this calculation, but their
available data will be summarized separately and reported for monitoring. If the
interim monitoring threshold is met, the trial will be suspended temporarily, and
study therapy will be reviewed by the study team and appropriate regulatory
committees. Based on this review, the study may resume as written, with
modifications, or be terminated. If the trial does not meet the monitoring
threshold, then enrollment will continue as planned.
The planned sample size of 30 patients has high power (>99%) to detect a 14 day
decrease in mean days to DLI infusion using a one-sample t-test at the one-sided
5% level with a null hypothesis that the early DLI cohort only reduces the mean
time to DLI by 7 days or less (assuming an identical SD as the original cohort).
The sample size is also informed by feasibility based on the original cohort
accrual and with the goal to generate adequate data for the safety and exploratory
objectives.

HAP2HCT 55
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Based on the experience with the original cohort, we anticipate very few patients
will be unable to receive DLI, and patients who fail to receive DLI will not be
replaced.
11.4.2 Additional Early DLI cohort Exploratory objectives
11.4.2.1 Assess the degree of T-cell expansion in the early postHCT period and at
4 weeks post-DLI in the Amendment 5 early DLI cohort compared to the original
cohort.
T-cell expansion at 4 weeks post-DLI and throughout the early postHCT period
will be descriptively compared between patients transplanted on the original
cohort and those transplanted on the early DLI cohort.
11.4.2.2 Assess the cumulative incidence of composite viremia (including CMV,
EBV, and adenovirus) in the Amendment 5 early DLI cohort compared to the
original cohort; and assess the duration of CMV viremia in the Amendment 5
early DLI cohort compared to the original cohort.
The cumulative incidence of composite viremia (including CMV, EBV, and
adenovirus) will be estimated using the Kalbfleisch-Prentice method with death
without composite viremia as the competing risk. The difference in 60-day
cumulative incidence of composite viremia between the Early DLI and original
cohorts will be computed and reported with a 95% confidence interval.
Among the patients experiencing CMV viremia, the duration of CMV viremia
will be measured as the days from CMV viremia onset until resolution. This will
be compared descriptively between the early DLI and original cohorts.
There will be no stopping rule for the exploratory assessments.
12.0 OBTAINING INFORMED CONSENT
12.1Informed Consent Prior to Research Interventions
The PI or physician sub-investigator will conduct the signature authorization portion of
the consent process. Authorization for the recipient procedure should be conducted in
the presence of an independent witness, such as a nurse from the St. Jude Department of
Nursing or the St. Jude Institutional Review Board Ombudsperson/Patient Advocate, as
applicable and available to serve as a witness.
12.2Consent at Age of Majority

HAP2HCT 56
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
The age of majority in the state of Tennessee is 18 years old. Research participants must
be consented at the next clinic visit after the 18th birthday. If an affiliate is located in a
country or state where a different age of majority applies, the location must consent the
participants according to their local laws.
12.3Consent When English is Not the Primary Language
When English is not the patient, parent, or legally authorized representative’s primary
language, the Social Work department will determine the need for an interpreter. This
information documented in the participant’s medical record. Either a certified interpreter
or the telephone interpreter’s service will be used to translate the consent information.
The process for obtaining an interpreter and for the appropriate use of an interpreter is
outlined on the Interpreter Services, OHSP, and CTO websites.

HAP2HCT 57
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
13.0 REFERENCES
1.
Westoff CF. Fertility in the United States. Science 1986;234:554-9.
2.
Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from
unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med
1993;328:593-602.
3.
Barker JN, Wagner JE. Umbilical-cord blood transplantation for the treatment of
cancer. Nat Rev Cancer 2003;3:526-32.
4.
Beatty PG, Clift RA, Mickelson EM, et al. Marrow transplantation from related
donors other than HLA-identical siblings. N Engl J Med 1985;313:765-71.
5.
Aversa F, Terenzi A, Tabilio A, et al. Full haplotype-mismatched hematopoietic
stem-cell transplantation: a phase II study in patients with acute leukemia at high risk of
relapse. J Clin Oncol 2005;23:3447-54.
6.
Ciceri F, Labopin M, Aversa F, et al. A survey of fully haploidentical hematopoietic
stem cell transplantation in adults with high-risk acute leukemia: a risk factor analysis of
outcomes for patients in remission at transplantation. Blood 2008;112:3574-81.
7.
Leung W, Campana D, Yang J, et al. High success rate of hematopoietic cell
transplantation regardless of donor source in children with very high-risk leukemia. Blood
2011;118:223-30.
8.
Schumm M, Lang P, Taylor G, et al. Isolation of highly purified autologous and
allogeneic
peripheral
CD34+
cells
using
the
CliniMACS
device.
J
Hematother
1999;8:209-18.
9.
Li Pira G, Malaspina D, Girolami E, et al. Selective Depletion of alphabeta T Cells
and B Cells for Human Leukocyte Antigen-Haploidentical Hematopoietic Stem Cell
Transplantation. A Three-Year Follow-Up of Procedure Efficiency. Biol Blood Marrow
Transplant 2016;22:2056-64.
10.
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;155:21-3.
11.
Daniele N, Scerpa MC, Caniglia M, et al. Transplantation in the onco-hematology
field: focus on the manipulation of alphabeta and gammadelta T cells. Pathology,
research and practice 2012;208:67-73.
12.
Perko R, Kang G, Sunkara A, Leung W, Thomas PG, Dallas MH. Gamma delta T
cell reconstitution is associated with fewer infections and improved event-free survival
after hematopoietic stem cell transplantation for pediatric leukemia. Biol Blood Marrow
Transplant 2015;21:130-6.
13.
Bertaina A, Merli P, Rutella S, et al. HLA-haploidentical stem cell transplantation
after removal of alphabeta+ T and B cells in children with nonmalignant disorders. Blood
2014;124:822-6.
14.
Airoldi I, Bertaina A, Prigione I, et al. gammadelta T-cell reconstitution after HLA-
haploidentical
hematopoietic
transplantation
depleted
of
TCR-alphabeta+/CD19+
lymphocytes. Blood 2015;125:2349-58.
15.
Balashov D, Shcherbina A, Maschan M, et al. Single-Center Experience of
Unrelated and Haploidentical Stem Cell Transplantation with TCRalphabeta and CD19
Depletion in Children with Primary Immunodeficiency Syndromes. Biol Blood Marrow
Transplant 2015;21:1955-62.

HAP2HCT 58
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
16.
Locatelli F, Merli P, Pagliara D, et al. Outcome of children with acute leukemia
given HLA-haploidentical HSCT after alphabeta T-cell and B-cell depletion. Blood
2017;130:677-85.
17.
Triplett BM, Muller B, Kang G, et al. Selective T-cell depletion targeting CD45RA
reduces viremia and enhances early T-cell recovery compared with CD3-targeted T-cell
depletion. Transpl Infect Dis 2018;20.
18.
Teachey DT, Lacey SF, Shaw PA, et al. Identification of Predictive Biomarkers for
Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute
Lymphoblastic Leukemia. Cancer Discov 2016;6:664-79.
19.
Chen YB, Spitzer TR. Current status of reduced-intensity allogeneic stem cell
transplantation using alternative donors. Leukemia 2008;22:31-41.
20.
Aversa F, Terenzi A, Felicini R, et al. Haploidentical stem cell transplantation for
acute leukemia. Int J Hematol 2002;76 Suppl 1:165-8.
21.
Aversa F, Velardi A, Tabilio A, Reisner Y, Martelli MF. Haploidentical stem cell
transplantation in leukemia. Blood Rev 2001;15:111-9.
22.
Chen X, Hale GA, Barfield R, et al. Rapid immune reconstitution after a reduced-
intensity conditioning regimen and a CD3-depleted haploidentical stem cell graft for
paediatric refractory haematological malignancies. Br J Haematol 2006;135:524-32.
23.
Benaim E, Hale G, Horwitz E, et al. Reduced-intensity conditioning (RIC) in
haploidentical transplantation. Blood 2004;104:590a.
24.
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 Transplant 2002;30:359-
66.
25.
O'Donnell PV, Luznik L, Jones RJ, et al. Nonmyeloablative bone marrow
transplantation from partially HLA-mismatched related donors using posttransplantation
cyclophosphamide. Biol Blood Marrow Transplant 2002;8:377-86.
26.
Rizzieri DA, Koh LP, Long GD, et al. Partially matched, nonmyeloablative
allogeneic transplantation: clinical outcomes and immune reconstitution. J Clin Oncol
2007;25:690-7.
27.
Ogawa H, Ikegame K, Yoshihara S, et al. Unmanipulated HLA 2-3 antigen-
mismatched
(haploidentical)
stem
cell
transplantation
using
nonmyeloablative
conditioning. Biol Blood Marrow Transplant 2006;12:1073-84.
28.
Przepiorka D, Ko CW, Deisseroth A, et al. FDA Approval: Blinatumomab. Clinical
cancer research : an official journal of the American Association for Cancer Research
2015;21:4035-9.
29.
Topp MS, Kufer P, Gokbuget N, et al. Targeted therapy with the T-cell-engaging
antibody blinatumomab of chemotherapy-refractory minimal residual disease in B-lineage
acute lymphoblastic leukemia patients results in high response rate and prolonged
leukemia-free survival. J Clin Oncol 2011;29:2493-8.
30.
Topp MS, Gokbuget N, Zugmaier G, et al. Phase II trial of the anti-CD19 bispecific
T cell-engager blinatumomab shows hematologic and molecular remissions in patients
with relapsed or refractory B-precursor acute lymphoblastic leukemia. J Clin Oncol
2014;32:4134-40.

HAP2HCT 59
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
31.
Topp MS, Gokbuget N, Stein AS, et al. Safety and activity of blinatumomab for
adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a
multicentre, single-arm, phase 2 study. Lancet Oncol 2015;16:57-66.
32.
Klinger M, Brandl C, Zugmaier G, et al. Immunopharmacologic response of
patients with B-lineage acute lymphoblastic leukemia to continuous infusion of T cell-
engaging CD19/CD3-bispecific BiTE antibody blinatumomab. Blood 2012;119:6226-33.
33.
Zugmaier G, Gokbuget N, Klinger M, et al. Long-term survival and T-Cell kinetics
in adult patients with relapsed/refractory B-precursor acute lymphoblastic leukemia who
achieved minimal residual disease response following treatment with Anti-CD19 BiTE(R)
antibody construct blinatumomab. Blood 2015.
34.
Schlegel P, Lang P, Zugmaier G, et al. Pediatric posttransplant relapsed/refractory
B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the
T-cell engaging bispecific antibody blinatumomab. Haematologica 2014;99:1212-9.
35.
Triplett BM, Shook DR, Eldridge P, et al. Rapid memory T-cell reconstitution
recapitulating CD45RA-depleted haploidentical transplant graft content in patients with
hematologic malignancies. Bone Marrow Transplant 2015;50:1012.
36.
Crucitti L, Crocchiolo R, Toffalori C, et al. Incidence, risk factors and clinical
outcome of leukemia relapses with loss of the mismatched HLA after partially
incompatible hematopoietic stem cell transplantation. Leukemia 2015;29:1143-52.
37.
Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained
remissions in leukemia. N Engl J Med 2014;371:1507-17.
38.
Krupka C, Kufer P, Kischel R, et al. CD33 target validation and sustained depletion
of AML blasts in long-term cultures by the bispecific T-cell-engaging antibody AMG 330.
Blood 2014;123:356-65.
39.
Walter RB. Biting back: BiTE antibodies as a promising therapy for acute myeloid
leukemia. Expert review of hematology 2014;7:317-9.
40.
Stieglmaier J, Benjamin J, Nagorsen D. Utilizing the BiTE (bispecific T-cell
engager) platform for immunotherapy of cancer. Expert opinion on biological therapy
2015;15:1093-9.
41.
Huehls AM, Coupet TA, Sentman CL. Bispecific T-cell engagers for cancer
immunotherapy. Immunology and cell biology 2015;93:290-6.
42.
Leung W, Pui CH, Coustan-Smith E, et al. Detectable minimal residual disease
before hematopoietic cell transplantation is prognostic but does not preclude cure for
children with very-high-risk leukemia. Blood 2012;120:468-72.
43.
Swati Naik, Renee M. Madden, Ewelina Mamcarz, et al. CD45RO+ T-Cell Add
Back
and
Prophylactic
Blinatumomab
Administration
Post
Tcrαβ/CD19-Depleted
Haploidentical
Transplantation
in
Pediatric
Patients
with
High
Risk
Acute
Leukemia. Blood 2021; 138 (Supplement 1): 2897. doi: https://doi.org/10.1182/blood-
2021-149671
44.
Brandl C, Haas C, d'Argouges S, et al. The effect of dexamethasone on polyclonal
T cell activation and redirected target cell lysis as induced by a CD19/CD3-bispecific
single-chain antibody construct. Cancer immunology, immunotherapy : CII 2007;56:1551-
63.

HAP2HCT 60
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
45.
Teachey DT, Rheingold SR, Maude SL, et al. Cytokine release syndrome after
blinatumomab treatment related to abnormal macrophage activation and ameliorated
with cytokine-directed therapy. Blood 2013;121:5154-7.

HAP2HCT 61
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
APPENDIX A:
KARNOFSKY PERFORMANCE STATUS SCALE
≥16 YEARS OLD
Score
General Description
100
Normal. No complaints. No evidence of disease.
90
Able to carry on normal activity. Minor signs or symptoms of disease.
80
Normal activity with effort. Some signs or symptoms of disease.
70
Care of self. Unable to carry out normal activity or to do active work.
60
Requires occasional assistance, but is able to care for most of his needs.
50
Requires considerable assistance and frequent medical care.
40
Disabled. Requires special care and assistance.
30
Severely disabled. Hospitalization is indicated although death is not imminent.
20
Hospitalization necessary, very sick, active support treatment necessary.
10
Moribund. Fatal processes progressing rapidly.
0
Dead.
LANSKY PERFORMANCE STATUS SCALE
< 16 YEARS OLD
Score
General Description
100
Fully active, normal
90
Minor restrictions in physically strenuous activity
80
Active, but tires more quickly
70
Both greater restriction of and less time spent in play activity
60
Up and around, but minimal active play; keeps busy with quieter activities
50
Gets dressed but lies around much of the day, no active play but able to participate in
all quiet play and activities
40
Mostly in bed; participates in quiet activities
30
In bed; needs assistance even for quiet play
20
Often sleeping; play entirely limited to very passive activities
10
No play; does not get out of bed
0
Unresponsive

HAP2HCT 62
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
APPENDIX B
CRITERIA FOR ACUTE AND CHRONIC GVHD EVALUATION
The St. Jude Department of BMTCT Clinical Research Office standard operating procedure for
the evaluation of acute GVHD (SOP 20.01 Acute Graft-versus-Host Disease) and the evaluation
of chronic GVHD (SOP 20.02 Chronic Graft-versus-Host Disease) will provide guidance on the
evaluation, collection and reporting of acute and chronic GVHD for this clinical trial. The
current version of this document, as well as ongoing updates, can be found on the BMTCT
Clinical Transplant Program intranet page.
APPENDIX C
CRITERIA FOR ADVERSE EVENT (AE) EVALUATION AND REPORTING
The St. Jude Department of BMTCT Clinical Research Office standard operating procedure for
the documenting and reporting of adverse (SOP 10a Capturing and Reporting of Adverse Events
for HPC Transplant Patients) will provide guidance on the evaluation, collection and reporting of
adverse events for this clinical trial. The current version of this document, as well as ongoing
updates, can be found on the BMTCT Clinical Transplant Program intranet page.

HAP2HCT 63
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
APPENDIX D
Recommended testing and evaluation schedule for Transplant Recipient
STANDARD OF
CARE STUDIES
SAMPLE
VOLUME
PRE
PRE
DLI
MONTH 1
4 weeks post
DLI
MONTH 2
MONTH 3
MONTH 6
MONTH 12
Pregnancy Test
PB
2 ml
X
As clinically indicated
Physical Exam
N/A
N/A
X
Weekly
X
X
GVHD
Assessment
N/A
N/A
Weekly
CBC with diff.
PB
0.5-2 ml
X
Daily until engrafted, then weekly
X
X
Chemistry
PB
0.25-2ml
X
Weekly
X
X
Viral
surveillance
(BMTPCR)
PB
4 ml
X
Weekly
As clinically indicated
Chimerism
PB
1-2 ml
Weekly upon engraftment
X
X
BM
2 ml
X
X
X
Disease Status
Evaluation
N/A
N/A
X
X
X
X
MRD Bone
Marrow
BM
3 ml
X
X
X
X
Lymphocyte
Subset Study
PB
2.5-4 ml
X
X
X
X
X
X
Weekly during Blinatumomab administration
Lymphocyte
Subset with
RARO
PB
2.5- 4 ml
X
X
Quantitative
Immunoglobulin
s
PB
2 ml
X
X
X

The information derived from or noted on the physical examinations, standard tests, and other assessments that comprise standard of care for recipients are not required to be transcribed
onto case report forms and/or entered into the database. In reference to section 6.1 Evaluations, the above-indicated follow-up regimen for these evaluations is guided by the SOPs of the
Department of BMTCT, for recipients of allogeneic stem cell transplantation. As these evaluations are considered standard clinical care (non-research), variations in frequency (more or
less frequent) of these evaluations can occur due to the participant’s current clinical condition and will not be noted as protocol deviations with exception of the pre and post DLI Lymph
Subset with RARO..

Disease status evaluations/BM testing results obtained prior to enrollment may be used for the baseline/pre-infusion assessments.

Additional chimerism testing may be required as clinically indicated and described in Section 6.3.

Lymphocyte subset studies may be omitted without variance when the absolute lymphocyte count (ALC) is zero.

In the event of graft failure/rejection, the post failure/rejection bone marrow, chimerism and several applicable immune studies would not be clinically indicated and these studies may
be held.

HAP2HCT 64
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
APPENDIX D (continued)
Immune reconstitution testing and evaluation schedule for Transplant Recipient
RESEARCH STUDIES
SAMPLE
VOLUME
PRE
MONTH 1
MONTH 2
MONTH 3
MONTH 6
MONTH 12
VBETA/TREC
RESEARCH
PB
8 mL
X
X
X
LYMPHOCYTE
RESEARCH
PB
12 mL
X
X
X
X
X
X
PHENOTYPE
RESEARCH
PB
5 mL
X
X
X
X
X
X
IR-PHENOTYPE
(Youngblood Lab*)
PB
5 mL
X
X
X
X
X
X
T-FUNCTION
(Thomas Lab*)
PB
5 mL
X
X
X
X
X
X
BM
1 mL
X
X
X

VBETA/TREC Research and Lymphocyte Research results will be maintained in the Immune Monitoring Core database.

Phenotype Research results will be maintained in the Flow Core database.

For RESEARCH studies, the posted volumes are the minimum volumes required to perform the respective protocol evaluations.

When blood volumes exceed established safety parameters (3% of patient blood volume in 24hr) priority for blood draws go to clinically necessary labs first, then research
labs in order from top to the bottom of this table. However, for the pre-transplant time point the priority for research labs is from the bottom to the top of this table. Any
omissions in testing due to this safety parameter will not constitute a protocol deviation or variance.

The PRE-transplant bone marrow for T-FUNCTION is an optional test that may be omitted at the discretion of the PI.
*
Research testing results will be maintained in a secured database in the respective co-investigator laboratory database.
APPENDIX D (continued)

HAP2HCT 65
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
ATG PK Testing for original cohort
OPTIONAL
Evaluation for
ATG PK
Testing
RESEARCH
STUDY
SAMPLE
VOLUME
Day -3
(Pre 3rd
dose)
Day -3 (post
3rd dose)
D -1
D 0
D +2
D +4
D +7
D +14
D +21
ATG PK
PB
2 mL
X
X
X
X
X
X
X
X
X

Sample collection date may be adjusted +2 days as needed (such as to accommodate blood volume concerns, line access issues, etc.) The +2 days adjustment will not
apply to the Day -3, -1, 0, +2 and +4 samples.
ATG PK Testing for early DLI cohort
OPTIONAL
Evaluation for
ATG PK
Testing
RESEARCH
STUDY
SAMPLE
VOLUME
Day -10
(Pre 3rd
dose)
Day -10
(post 3rd
dose)
D -8
D -7
D -5
D -3
D 0
D +7
D +14
ATG PK
PB
2 mL
X
X
X
X
X
X
X
X
X

Sample collection date may be adjusted +2 days as needed (such as to accommodate blood volume concerns, line access issues, etc.) The +2 days adjustment will not
apply to the Day -10, -8, -7, and Day-5 samples.
APPENDIX D (continued)

HAP2HCT 66
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
Research testing for DONOR
Prior to initial stem cell collection procedure:
OPTIONAL research immune studies testing schedule
Evaluation
Volume Requirement
Flow cytometry enumeration
Lymphocyte Subset Study = 4 mL
Thymic output and T cell repertoire
VBETA/TREC Research = 17 mL
Donor baseline lymphocyte number and function (Immune Monitoring Core)
Lymphocyte Research = 12 mL
Donor baseline phenotype number and function (Flow Core)
Phenotype Research = 5mL
Donor baseline immune function (Youngblood Lab*)
IR-PHENOTYPE = 5mL
Donor baseline immune function (Thomas Lab*)
T-FUNCTION = 5mL

All donor research testing to be collected prior to growth factor administration and progenitor cell collection. These optional research tests may be collected at
separate times.

Research testing results will be maintained in a secured database in the respective co-investigator laboratory database.

HAP2HCT 67
Amendment 5.0, dated: 03/01/2024
Protocol document date: 03/01/2024
APPENDIX D (continued)
Research Study Evaluation Target Windows
Several laboratory tests can only be processed on weekdays; therefore, if the scheduled evaluation
falls on a weekend, or during a holiday period, an adjustment in the follow-up visit is expected and
would not be noted as a protocol variation. Additionally, in order to accommodate such logistical
constraints, evaluation/collection dates of all protocol assessments (required and optional
research), may be performed within a reasonable window of the intended date following the
guidelines provided in the table below:
If the Planned Evaluation
Time Point is:
Window
Weekly
+ 3 Days
Month 1
Week 2 to Week 6
Month 2
Week 7 to Week 11
Month 3
Week 12 to Month 4
Month 6
Month 5 to Month 7
Month 9
Month 8 to Month 10
Month 12
Month 10 to Month 14
APPENDIX E
The St. Jude Department of BMTCT Clinical SOPs for standard of care for all allogeneic stem cell
infusion recipients and stem cell donors will provide guidance on the evaluation, ongoing clinical
care and follow up for this clinical trial. The current versions of these SOPs, as well as ongoing
updates, of these documents can be found on the BMTCT Clinical Transplant Program intranet
page.