Clinical trial NCT04151706
Conditions: Acute Myeloid Leukemia Acute Lymphoblastic Leukemia Myelodysplastic Syndromes Acute Leukemia Chronic Myeloid Leukemia
Sponsor: Robert Lowsky
Start date: 2020-02-27; Completion date: 2023-09-08
Enrollment: 7 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This study will evaluate combining stem cells from the patient's matched sibling donor (a standard CD34-selected transplant) with a second infusion of white blood cells called "CD8 memory T-cells" from their sibling donor.

Detailed description:
Primary Objective: To determine the rate of graft versus host disease (GvHD) free, relapse free survival (GRFS) at one year following CD34 selected allogeneic hematopoietic cell transplantation using myeloablative conditioning combined with an infusion of phenotypic CD8+ memory T cells from human leukocyte antigen (HLA) matched donors for patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), or acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML).

Secondary Objective: To determine the rate of graft rejection, acute and chronic GvHD, non relapse mortality, relapse, overall survival, and disease free survival.

Arms: arm 1: Participants will be infused on Day 0 with donor derived CD34+ selected cells combined with CD8+CD45RA- T cells {CD Memory T Cells} following a standard myeloablative conditioning regimen that might consist of fTBI, Thiotepa, and Fludarabine or Busulfan and Cyclophosamide.
Interventions: intervention 1: CD8+ Memory T Cell Infusion intervention 2: Thiotepa intervention 3: Fludarabine intervention 4: Hyperfractionated TBI intervention 5: Busulfan intervention 6: Cyclophosphamide
Intervention descriptions: intervention 1: Allogeneic phenotypic CD8+ memory T cells from HLA matched donors infused at the time of hematopoietic cell transplantation intervention 2: 5 mg/kg/day: IV for 2 consecutive days (days -6 to -5) intervention 3: 25 mg/m2/day: IV for 5 consecutive days (days -6 to -2) intervention 4: Administered in 11 fractions of 125 cGy over 4 days (Total dose of 1375 cGy) intervention 5: 6 mg/kg/dose Q24h IV. Infused over 3 hours. 1 dose per day x 4 consecutive days x 3.6 mg/kg/dose = 14.4 mg/kg intervention 6: 60 mg/kg/dose Q24h IV. Infused over 2 hours. 1 dose per day x 2 consecutive days x 60 mg/kg/dose = 120 mg/kg

Protocol documents:
Prot_SAP_001.pdf: 
IRB-49023 
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12 February 2021 v4 
CD34-Selected Allogeneic Hematopoietic Cell Transplantation with Myeloablative 
Conditioning and CD8+ Memory T-cell Infusion For Patients with Myelodysplastic 
Syndrome, Acute Leukemia, and Chronic Myelogenous Leukemia 
Coordinating Center 
Stanford Cancer Center 
875 Blake Wilbur Dr 
Stanford, CA 94305 
Protocol Director 
Robert Lowsky, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr, Room H0101B, Stanford, CA 94305  
650-723-0822;  650-725-8950 
rlowsky@stanford.edu  
IRB-49023  
OnCore-BMT339 
NCT04151706 
IND 014844 
Version date:  12 February 2021 
SRC approval:  28 May 2019 
Co-Investigators 


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12 February 2021 v4 
Co-Investigators 
Wen-Kai-Weng, MD, PhD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
 wkweng@stanford.edu 
Robert Negrin, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
negrs@stanford.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sally Arai, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
sarai1@stanford.edu 
Laura Johnston, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
korb@stanford.edu 
 
 
 
 
 
 
 
 


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Co-Investigators 
David Miklos, MD, PhD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
dmiklos@stanford.edu 
Judith Shizuru, MD, PhD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA 94305 
650-723-0822;  650-725-8950 
Jshizuru@stanford.edu 
 
 
 
Everett Meyer, MD, PhD 
Division of Blood and Marrow Transplantation 
and Cellular Therapeutics Facility Laboratory 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
evmeyer@stanford.edu 
Andrew Rezvani, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
arezvani@stanford.edu 
Lori Muffly, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0822;  650-725-8950 
lmuffly@stanford.edu 
Samuel Strober, MD 
Divisions of Immunology & Rheumatology 
CCSR Building, Room 2215-C 
Stanford, California 94305 
650-723-6500;  650-725-6104 
sstrober@stanford.edu 
Michael Spinner, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Drive MC5623, Stanford, CA 94305 
650-723-0822;  650-725-8950 
mspinner@stanford.edu 
Parveen Shiraz, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-0820;  650-725-8950 
pshiraz@stanford.edu  
Surbhi Sidana, MD 
Division of Blood and Marrow Transplantation 
Stanford University Medical Center 
300 Pasteur Dr MC5623, Stanford, CA  94305 
650-723-1389;  650-725-8950 
 surbhi.sidana@stanford.edu 
Philip Lavori, PhD (Biostatistician) 
Department of Health Research and Policy 
Biostatistics Shared Resource, 
Redwood Building, T101E, Stanford, CA 94305 
650-725-6109;  650-725-6951 
lavori@stanford.edu 
 
 
 


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TABLE OF CONTENTS 
 
Protocol Synopsis 
4 
Protocol Schema 
5 
1. 
Objectives 
6 
2. 
Background 
8 
3. 
Participant Selection and Eligibility Criteria 
20 
4. 
Treatment Plan 
27 
5. 
Investigational Agent Information 
29 
6. 
Dose Modification 
30 
7. 
Adverse Events and Reporting Procedures 
30 
8. 
Correlative Studies 
31 
9. 
Study Calendar 
32 
10. 
Measurements 
32 
11. 
Regulatory Considerations 
33 
12. 
Statistical Considerations 
34 
13. 
References 
36 
Appendices 
40 
 


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Protocol Synopsis 
TITLE  
CD34-selected allogeneic hematopoietic cell transplantation with 
myeloablative conditioning and CD8+ memory T-cell infusion for 
patients with myelodysplastic syndrome, acute leukemia, and chronic 
myelogenous leukemia (CML)   
STUDY PHASE 
Phase 2 
STUDY DESIGN 
Single-arm, non-randomized safety and efficacy study using Simon 
stage 2 design  
STUDY SITE 
Stanford University Medical Center, CA 94305 
INDICATION 
Subjects with myelodysplastic syndrome (MDS), acute myeloid 
leukemia (AML), or acute lymphoblastic leukemia (ALL) eligible for 
full-dose myeloablative conditioning, or chronic myelogenous 
leukemia (CML) 
INVESTIGATIONAL 
PRODUCT  
Allogeneic phenotypic CD8+ memory T-cells from HLA-matched 
donors infused at the time of hematopoietic cell transplantation 
PRIMARY 
ENDPOINT and 
OBJECTIVE(S) 
To determine the rate of graft-versus-host-disease (GvHD)-free, 
relapse-free survival (GRFS) at one year post-transplant 
SECONDARY 
ENDPOINTS AND 
OBJECTIVE(S) 
To determine the rate of graft rejection, acute and chronic GvHD, 
non-relapse mortality, relapse, overall survival, disease-free survival, 
EBV and CMV reactivation, and the expansion and migration of EBV 
and CMV-reactive CD8+ memory T-cells 
STUDY 
OBSERVATION 
PERIOD 
The total duration of subject study participation is 1 year.  Subjects will 
be followed indefinitely for all outcomes including overall survival, 
disease-free survival, and late complications including chronic GvHD 
and infections. 
TREATMENT 
SUMMARY 
CD34 selection with immunomagnetic beads is an effective method of 
depleting alloreactive donor T-cells from a peripheral blood graft and 
results in a significant reduction in acute and chronic GvHD.  
However, depletion of donor T-cells also leads to an increased rate of 
disease relapse and infection.  We will test the hypothesis that 
combining phenotypic CD8+ memory T-cells to a purified CD34+ graft 
will retain the low incidence of GvHD, yet also provide enhanced 
antitumor activity, and transfer of EBV and CMV immunity. 
SAMPLE SIZE  
32 subjects and HLA-matched donors. 


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SUMMARY OF 
SUBJECT 
ELIGIBILTY 
Subjects with histologically-confirmed MDS, acute leukemia, or CML. 
Subjects age 18 to 65 years who are eligible for full-dose 
myeloablative conditioning. 
Subjects with HLA-matched donors. 
CONTROL GROUP 
Historically-matched control subjects with MDS, AML, ALL, or CML 
receiving CD34-selected transplants with myeloablative conditioning 
without CD8+ memory T-cell infusion 
Study Schema 
 
 
 
 
Donors will undergo apheresis for processing of CD34+ selected cells and CD8+CD45RA- TM cells.  These cells 
will be infused on Day 0 following a standard non-ATG containing regimen used for CD34-selected transplants 
consisting of fTBI (1375 cGy);  thiotepa (10 mg/kg);  and fludarabine (125 mg/m2) or non-TBI conditioning 
consisting of Busulfan (14.4 mg/kg) and Cyclophosphamide (120 mg/kg). 
TBI/Thiotepa/Flu or Bu/Cy 
D = 0 
Patient with MDS, acute leukemia, or 
CML.  Age 18-65 
HLA-matched donor 
3 mo. 
1 mo. 
Immune monitoring studies 
1
O endpoint: GRFS at 1 year 
2
O endpoints: 
• 
Graft rejection 
• 
Acute GVHD 
• 
Chronic GVHD 
• 
Non-relapse mortality 
• 
Disease relapse 
• 
MRD by flow cytometry 
• 
Overall survival 
• 
Progression free survival 
• 
CMV and EBV viremia 
• 
Ability to transfer CMV and EBV reactive T cell 
clones from donor to recipient 
6 mo. 
CD34 selection 
+ 
CD8+CD45RA- 
memory T cells 
(1-10 x 10
6/kg 
1 year 
G-SCF 


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1. 
OBJECTIVES 
1.1 
Objectives 
1.1.1  Primary Objective 
To determine the rate of graft-versus-host-disease (GvHD)-free, relapse-free survival (GRFS) at 
one year following CD34-selected allogeneic hematopoietic cell transplantation using 
myeloablative conditioning combined with an infusion of phenotypic CD8+ memory T-cells 
from HLA-matched donors for subjects with myelodysplastic syndrome (MDS), acute myeloid 
leukemia (AML), acute lymphoblastic leukemia (ALL), or chronic myelogenous leukemia 
(CML). 
1.1.2  Secondary Objectives 
To determine the rate of graft rejection, acute and chronic GvHD, non-relapse mortality, relapse, 
overall survival, and disease-free survival. 
1.1.3  Exploratory Objective 
To determine in some donor recipient pairs the expansion and migration of EBV and 
CMV-reactive CD8+ memory T-cells and correlation with EBV and CMV reactivation. 
1.2 ClinicalTrials.gov Outcomes 
1.2.1  Primary Outcome 
Title:  Graft-versus-Host Disease (GvHD)-free and relapse-free survival (GRFS)  
Description:  The rate of participants who do not experience GvHD and also do not 
experience relapse are collectively considered to be GRFS.  Relapse will be assessed 
according to the myelodysplastic syndrome or leukemia response criteria.  The participants 
will be assessed for GRFS though 1 year post-transplant.  The outcome will be reported as 
the number of participants, a number without dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  
1.2.2  Secondary Outcomes 
Title:  Graft Rejection  
Description:  Graft rejection will be determined on the basis of reaction against the donor 
hematopoietic cells.  The outcome will be reported as the number of participants who 
experience graft rejection though 1 year post-transplant, a number without dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  


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Title:  Acute Graft-versus-Host Disease (GvHD)  
Description:  The participants will be assessed for acute graft-versus-host disease (GvHD) 
though 1 year post-transplant.  The outcome will be reported as the number of participants 
who experience acute GvHD, a number without dispersion.  
Timeframe:  1 year  
Safety Outcome?:  Yes  
Title:  Chronic Graft-versus-Host Disease (GvHD)  
Description:  The participants will be assessed for chronic, steroid-requiring 
graft-versus-host disease (GvHD) though 1 year post-transplant.  The outcome will be 
reported as the number of participants who experience chronic GvHD, a number without 
dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  
Title:  Non-relapse Mortality  
Description:  Non-relapse mortality will be assessed as the number of participants who have 
died though 1 year post-transplant, without a relapse or recurrence of their myelodysplastic 
syndrome or leukemia.  Relapse will be assessed according to the myelodysplastic syndrome 
or leukemia response criteria.  The outcome will be reported as the number of affected 
participants, a number without dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  
Title:  Relapse  
Description:  Relapse will be assessed according to the myelodysplastic syndrome or 
leukemia response criteria.  The outcome will be reported as the number of participants who 
experience relapse though 1 year post-transplant, a number without dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  
Title:  Overall Survival (OS) 
Description:  Overall Survival (OS) will be assessed as the number of participants who 
remain alive at 1 year post-transplant.  The outcome will be reported as a number without 
dispersion.  
Timeframe:  1 year  
Safety Outcome?:  No  


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2. 
BACKGROUND 
2.1  
Allogeneic Hematopoietic Cell Transplantation for MDS, Acute Leukemia, and 
CML 
Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for subjects 
with high-risk MDS and acute leukemia.  Following allogeneic HCT, a significant percentage of 
subjects with otherwise incurable blood cancers using best of care non-transplantation therapies 
may achieve long-term disease control and are cured.  These patients are characterized by 
developing donor type hematopoiesis and have complete resolution of all disease-related signs, 
symptoms, and markers.  Generally, patients who undergo allogeneic HCT are doing so as a 
“last ditch” effort for long-term disease control and possibly cure as best of care non-transplant 
therapies are unlikely to provide this. 
The antitumor activity of allogeneic HCT is from the direct cytotoxic effect of the conditioning 
regimen combined with the immunologic graft-versus-tumor (GVT) effect mediated by 
alloreactive donor T-cells.  A myeloablative conditioning regimen is the current standard of care 
for patients with MDS and acute leukemia, as shown in a recent randomized controlled trial 
demonstrating significantly lower relapse rates and superior overall survival with myeloablative 
conditioning compared to reduced intensity conditioning (RIC) for fit patients age 18 to 65 with 
MDS or acute leukemia. 1 The main causes for treatment failure following allogeneic HCT are 
disease relapse, acute and chronic GvHD, and infections.  A strategy to augment donor-mediated 
GVT activity and improve infection control without increasing the risk of acute and chronic 
GvHD may improve transplant outcomes including overall survival. 
2.2  
CD34-Selected Allogeneic HCT for MDS, Acute Leukemia, and CML 
CD34 selection with immunomagnetic beads is an effective method of depleting alloreactive 
donor T-cells from a peripheral blood graft and results in a significant reduction in acute and 
chronic GvHD. 2-9  Studies of CD34-selected HCT for various hematologic malignancies 
performed at Memorial Sloan Kettering Cancer Center (MSKCC) reported incidences of acute 
GvHD (limited to Grade II) of 8%, and chronic GvHD of 9% in recipients of matched related 
grafts and incidences of acute and chronic GvHD of 9% and 29%, respectively, in recipients of 
matched unrelated donors. 3,4  None of the subjects received GvHD prophylaxis beyond T-cell 
depletion of the graft.  Results from single-center studies have been validated in the National 
Blood and Marrow Clinical Trials Network (BMT CTN) study 0303 in which 44 subjects with 
AML in CR1 or CR2 were conditioned with total body irradiation, thiotepa, and 
cyclophosphamide with rabbit antithymocyte globulin (ATG) followed by CD34-selected 
allogeneic HCT from an HLA-matched sibling donor.  The incidence of acute GvHD 
Grade II-IV was 22.7% and the incidence of extensive chronic GvHD was 6.8% at 24 months. 5  
Similar results have been reported in subjects with high-risk MDS.  In a recent study of 102 
subjects at MSKCC with advanced MDS who received CD34-selected HCT, the cumulative 
incidence of acute GHVD (Grade II to IV) at 180 days was 16% and the cumulative incidence of 
chronic GvHD at 1 year was 4%. 10  A recent analysis of the BMT CTN 0303 subjects compared 
to a subset of subjects on BMT CTN 0101 who received a conventional transplant for AML in 
CR1/CR2 showed a significantly lower rate of chronic GvHD at 2 years with a CD34-selected 
graft than a conventional graft (19% vs 50%, respectively, p < 0.001, Figure 1) with no 
difference in overall or disease-free survival. 11 


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Figure 1.  Cumulative incidence of chronic GvHD in recipients of CD34-selected T-cell 
depleted (TCD) grafts compared to recipients of unmanipulated grafts with 
post-transplant immunosuppressive therapy (IST), Pasquini, et al, J Clin Oncol 2012. 
Another advantage of CD34-selected HCT is that no post-transplant GvHD prophylaxis is 
required, thus eliminating the risks of side effects like nephrotoxicity associated with calcineurin 
inhibitors (CNIs) and the combined toxicity of an ablative regimen and methotrexate, which may 
exacerbate mucositis and cytopenias.  The ability to avoid post-transplant immunosuppression 
also makes CD34-selected HCT an ideal platform for post-transplant adoptive cellular therapies 
targeting minimal residual disease or infections. 12  
Subject accrual to a multicenter phase 3 trial (BMT CTN 1301, NCT02345850) was recently 
completed; this trial randomized subjects with MDS or acute leukemia to one of three arms 
following myeloablative conditioning and transplantation from an HLA-matched donor.  
Subjects were randomized to either: a CD34-selected graft, an unmanipulated graft followed by 
post-transplant cyclophosphamide (PTCy), or an unmanipulated graft with conventional 
post-transplant immunosuppressive therapy consisting of a CNI combined with methotrexate; all 
three arms are currently accepted standard-of-care approaches for GHVD prophylaxis.  The 
results from the trial may still be more than 18 months from being publically disclosed, albeit 
interim DSMB analyses throughout has found no evidence of superiority or inferiority in any of 
the three arm that was sufficient to warrant early closure for safety or efficacy concerns. 
2.3  
CD34+ Selection Impact on Disease Relapse, Infection, and Graft Rejection 
A major concern limiting the widespread adoption of CD34-selected, T-cell depleted (TCD) 
donor grafts is loss or attenuation of the GVT effect mediated by alloreactive donor T-cells, 
which appears to lead to a higher risk of disease relapse.  This was first demonstrated in patients 
with chronic myelogenous leukemia (CML), where a retrospective study of 46 patients who 
underwent TCD transplants were compared to 40 patients who received conventional grafts. 13  


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The 3-year probability of disease relapse was significantly higher in the TCD group than in the 
non-TCD group (62% vs 24%, p = .0003).  The infusion of supplemental donor T-cells was 
beneficial to the TCD group as after a donor lymphocyte infusion (DLI), 17 of 20 patients in the 
TCD group and 2 of 3 patients in the non-TCD group achieved a complete remission.  
While the CML experience clearly showed a higher risk for disease relapse with a TCD graft, 
this relationship has not been clearly demonstrated in patients with acute leukemia or MDS.  For 
example, in the BMT CTN 0303 study, the relapse rate for patients with acute leukemia in CR1 
receiving a CD34-selected graft was 17.4% at 36 months, similar to historical controls receiving 
conventional grafts. 5  Another retrospective study compared 115 patients who received TCD 
grafts after myeloablative conditioning at MSKCC to a cohort of 181 patients who received 
unmodified grafts after conditioning with busulfan/fludarabine and GvHD prophylaxis with 
tacrolimus and mini-methotrexate at MD Anderson Cancer Center (MDACC).  There were no 
significant differences in the rate of disease relapse at 3 years between the TCD and unmodified 
graft recipients (18% vs 25%, p = 0.3). 8  Similarly, in a study of 102 patients with advanced 
MDS who received CD34-selected grafts at MSKCC, the cumulative incidence of relapse was 
11.8% at 1 year and 15.7% at 2 years, 11 similar to historical controls receiving unmodified 
grafts.  The prospective randomized phase 3 BMT CTN 1301 study will be pivotal in comparing 
CD34-selected HCT to an unmodified graft with either PTCy or standard CNI-based 
immunosuppression in patients with advanced MDS and acute leukemia. 
Another important limitation of CD34-selected TCD grafts is the increased risk for infection, 
particularly viral reactivation with CMV and EBV, with a higher incidence of EBV-associated 
lymphoproliferative disorders. 14-18  Prior studies of CD34-selected HCT have demonstrated 
delayed reconstitution of CD4+ T-cells with a large proportion of patients having absolute CD4+ 
counts < 200 cells/uL for more than seven months, which may contribute to the increased risk for 
severe, recurrent, or opportunistic infections. 3  
Early studies of TCD grafts were limited by higher rates of graft rejection than with conventional 
grafts, with reported graft failure rates as high as 27%. 19-21  These clinical results confirmed 
pre-clinical data that donor-derived T-cells facilitate engraftment.  However, following 
modification of the conditioning regimen, and in particular the use of ATG to promote 
engraftment, several centers have reported consistent engraftment with CD34-selected TCD 
grafts. 2,5,22,23 
2.4.  
CD8+ Memory T-cells Have GVT Activity Without Causing Severe GvHD in 
Murine Models of Transplantation 
Murine studies showed that in many strain combinations memory T-cells, including memory 
CD4+ and memory CD8+ T-cells induce significantly less GvHD than naïve T-cells 
(CD62LhiCD44lo) or combinations of naïve and memory T-cells. 24-30 
Accordingly, our group at Stanford compared isolated naïve CD4+, CD8+, or total T-cells, 
and/or memory CD4+CD44hi;  CD8+CD44hi;  and total memory CD44hi T-cells from unprimed 
donors for their capacity to induce GvHD, promote chimerism and mediate antitumor activity 
against a naturally occurring B-cell lymphoma (BCL1) in an MHC-mismatched model. 31  Only 
the CD8+CD44hi memory T-cell subset containing both central and effector memory cells was 


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capable of eradicating the lymphoma cells without inducing GvHD.  In contrast, CD4+ and 
CD8+ naïve T-cells, memory CD44hi CD4+ T-cells, naive total T-cells, and memory CD44hi 
total T-cells either induced lethal GvHD or lacked potent antitumor activity.  The tumor-bearing 
recipients of CD8+CD44hi T-cells had a clear survival advantage over those given CD8+ naive 
T-cells because of the lethal GvHD induced by the latter.  The CD8+CD44hi T-cells were also 
used in a model of treatment of progressive lymphoma growth after BMT, and were able to 
promote complete chimerism and eradicate the tumor without causing GvHD. 
In other preclinical murine models of bone marrow transplantation using various MCH-matched 
strain combinations conducted by our group, the only cell subset that we could identify that 
provided GVT reactions against FBL3 leukemia tumor cells without inducing lethal GvHD was 
CD44hiCD8+ memory T-cells. 32 
Taken together, our studies and those by other groups suggest that CD8+ memory T-cells 
promote donor cell engraftment and retain GVT reactions without precipitating severe GvHD.   
2.5  
Methods for the Enrichment of Human CD8+ Memory T-cells 
Since we successfully treated progressive tumor growth after BMT without inducing GvHD in 
mice with purified donor CD8+ memory T-cells, we developed methods to translate this work to 
human clinical trials.  In studies using healthy donor PBMC apheresis collections and 
immunomagnetic bead separation on Miltenyi columns, we enriched for 
CD8+CD45RO+CD45RA- phenotypic memory T-cells to > 95% purity. 33 
Briefly, phenotypic CD8+ memory T-cells (CD8+CD45RA-) were isolated from healthy donors 
following one or two 12-liter volume apheresis collections.  The CD45RA-
 
cells were obtained 
by labeling peripheral blood collections with the research grade CliniMACS CD45RA 
Microbeads (Catalogue number 130-020-003) and Reagent (Miltenyi Biotec) followed by 
selection on the CliniMACS Plus instrument using program Depletion 3.2 with CliniMACS 
Depletion tubing sets.  The flow through CD45RA-
 
cells were labeled with the research grade 
CliniMACS CD8 Microbeads (Catalogue number 130-030-810) and Reagent and selected using 
program Enrichment 3.1 with standard CliniMACS tubing sets.  Post-selection products were 
washed and re-suspended in 100 mL of Normosol-R with 1% human serum albumin (HSA) and 
evaluated immediately and after 24 to 48 hours of storage at 4°C.  Products were infused fresh 
(ie, not cryopreserved) and there was no change in cell viability or phenotype during the 48-hour 
storage time compared to immediate analysis.  Cell viability (consistently > 90%), recovery and 
purity were determined by flow cytometry, and a representative example is shown in Figure 2 
with > 95% of cells consistent with the CD8+ memory T-cell subset.  


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Figure 2.  Representative flow analysis of peripheral blood apheresis collections from the pre-selection, 
post-CD45RA depletion, and CD8+ enrichment steps and stained for expression of CD4, CD8, CD45RA, and 
CD45RO.  Plots show CD45-gated events. 
Cell yields were determined using apheresis products from normal HCT donors and the mean 
CD8+ memory T-cell post-enrichment cell count obtained from a single 12-liter apheresis 
product was 8 x 108 with a range of 5 to 8 x 108 CD45RA-CD8+ cells.  Therefore, following 
2 standard volume aphereses or one large volume (18 to 22 liters) apheresis per donor, we 
estimated that the highest feasible dose of memory CD8+ T-cells available for infusion would be 
10 x 108 cells.  Assuming that most subjects weigh less than 100 kg, attainable numbers of CD8+ 
memory T-cells for a phase 1 dose escalation trial would be 1 x 106/kg;  5 x 106/kg;  and 
10 x 106/kg. 
In further studies, the purified phenotypic CD8+ memory T-cells were tested for immune 
reactivity in vitro by stimulation with irradiated allogeneic PBMCs from normal donors. 33 The 
responder CD8+ memory T-cells showed a modest increase in the mean 3H-thymidine 
incorporation in cultures with allogeneic stimulator cells compared to control cultures without 
allogeneic stimulators (Figure 3A).  The CD8+ memory T-cell supernatants showed a marked 
increase in the concentration of IFN-γ after culture with allogeneic stimulator cells and a 
minimal increase in the concentration of IL-2 (Figure 3B).  The results of the human mixed 
leukocyte response (MLR) experiments were consistent with the responses observed with CD8+ 
memory T-cells from mice where the murine responder cells also showed a modest increase in 
3H-thymidine incorporation after stimulation with allogeneic cells, and the production of IFN-γ 
was considerably greater than that of IL-2. 
CD
45
RA 
CDRO 
Unfractionated 
CD45RA-Depleted 
CD8 Enriched 
CD
45
RA 
CD8 


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Figure 3A.  Proliferation of CD8+CD45R- and CD4+ cells activated by co-culture with or without irradiated allogeneic 
stimulators (Stim) and assessed by 3H-thymidine uptake during the last 24 hours of culture, mean and standard deviations are 
shown (n = 4).  B.  Cytokine secretion assessment in CD8+CD45R- and CD4+ cells activated by co-culture with or without 
irradiated allogeneic stimulators (Stim).  Supernatants from 7-day cultures were analyzed by flow cytometry using Cytokine 
Bead Arrays for INFγ, IL-2, and TNFα (n = 4). 
2.6  
Phase 1 Trial of CD8+ Memory T-cell Infusion for Relapsed Hematologic 
Malignancies after Allogeneic HCT 
To test the DLI treatment concept of human donor-derived CD8+CD45RO+CD45RA- memory 
T-cells in patients, a 15-subject phase 1 safety and feasibility study was conducted under an 
approved IND (BB-IND-14844) entitled “A Phase I Study of CD8+CD45RO+CD45RA- Memory 
T-cell Donor Lymphocyte Infusion for Relapse of Hematolymphoid Malignancies Following 
Matched Related Donor Allogeneic Hematopoietic Cell Transplantation” (NCT01523223).  The 
study followed a standard 3+3 dose escalation format using a low CD8+ memory T-cell dose of 
1 x 106 cells/kg, an intermediate dose of 5 x 106 cells/kg and a final dose of 10 x 106 cells/kg.  
The objectives of the protocol were:   
1. To determine the feasibility of purifying allogeneic CD8+ memory T-cells suitable for 
clinical application and to determine the safety and maximum tolerated dose (MTD) of 
these cells in subjects with recurrent or refractory hematolymphoid malignancies 
following allogeneic HCT.   
A. 
B.


IRB-49023 
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12 February 2021 v4 
2. To determine disease response, time to disease progression, event-free survival, and 
overall survival following treatment with allogeneic CD8+ memory T-cells.   
3. To assess donor specific chimerism before and at designated time points after treatment 
with allogeneic CD8+ memory T-cells. 
Eligible subjects were 18 to 75 years of age.  Phenotypic CD8+ memory T-cells were isolated 
following 1 or 2 days of unmobilized donor apheresis using a tandem immunomagnetic selection 
strategy including CD45RA depletion followed by CD8+ enrichment on the CliniMACS Plus 
instrument.  Fifteen subjects received CD8+ memory T-cells at 3 dose levels.  The subject 
characteristics and outcomes are summarized in Table 1 and have been previously reported. 34 
Table 1. Subject Outcomes Following CD8+ Memory T-cell DLI for Disease Relapse after Allogeneic HCT 
Subject Diagnosis 
Disease Status at 
CD8+ Memory 
Infusion 
CD8+ 
Memory Dose 
(x 106/kg) 
Response 
following 
DLI 
Time from DLI to 
Relapse/Progression 
(Days) 
Time from DLI to 
Death or Last 
Follow-up (Days) 
Status at Last 
Follow Up 
1 
AML 
CR2 
1.0 
CR2 
973 
1057 
Expired 
2 
AML 
Active Disease 
1.0 
CR2 
480 
591 
Expired 
3 
AML 
CR3 
1.0 
CR3 
579 
1328 
Alive in CR 
4 
AML 
CR2 
5.0 
CR2 
120 
430 
Expired 
5 
CML 
CR3 
5.0 
CR3 
- 
1069 
Alive in CR 
6 
CLL 
Active Disease 
5.0 
PD 
NR 
168 
Expired 
7 
AML 
Active Disease 
7.8 
PD 
NR 
128 
Expired 
8 
AML 
Active Disease 
8.0 
PD 
NR 
151 
Expired 
9 
NHL 
Active Disease 
10.0 
PD 
NR 
495 
Alive with 
disease 
10 
NHL 
Active Disease 
5.2 
SD 
90 
412 
Alive with 
disease 
11 
AML 
CR2 
10.0 
CR2 
- 
328 
Alive in CR 
12 
AML 
CR3 
7.7 
CR3 
147 
286 
Alive with 
disease 
13 
NHL 
Active Disease 
10.0 
SD 
158 
194 
Expired 
14 
MM 
Active Disease 
10.0 
PD 
NR 
221 
Alive with 
disease 
15 
ALL 
Active Disease 
10.0 
PR 
- 
118 
Alive with 
disease 
AML, acute myeloid leukemia;  CML, chronic myeloid leukemia;  CLL, chronic lymphocytic leukemia;  
NHL, non-Hodgkin lymphoma;  MM, multiple myeloma;  ALL, acute lymphoblastic leukemia;  
CR, complete remission;  PD, progressive disease;  SD, stable disease;  PR, partial response;  NR, no response 
All subjects in dose levels 1 and 2 received the intended CD8+ memory T-cell dose of 
1 to 106/kg and 5 to 106/kg, respectively.  Five of the subjects treated at dose level 3 received the 
10 to 106/kg CD8+ memory T-cell dose; however, the intended dose could not be obtained in 


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12 February 2021 v4 
4 subjects, who received doses of 5.2 to 10x106/kg.  The maximum tolerated dose (MTD) was 
not reached. 
No adverse events occurred during CD8+ memory T-cell infusion, and no dose-limiting toxicities 
were observed.  GvHD developed in only 1 subject, was limited to Grade II involvement of the 
liver, and resolved following a course of corticosteroids. 
Thirteen subjects (87%) received cytoreductive therapy prior to CD8+ memory T-cell infusion, 
and 6 subjects (40%) had returned to complete remission (CR) at the time of infusion.  The 
median follow-up from the time of CD8+ memory T-cell infusion was 328 days (range, 
118 to 1328 days).  Ten subjects (67%) maintained or achieved an objective response (7 CR;  
1 partial response;  2 stable disease) for at least 3 months following CD8+ memory T-cell 
infusion, and four of these responders had active disease at time of infusion.  Among the 
10 responders;  7 have relapsed at the time of this report after a median 165 days (range, 
90 to 973 days) to relapse.  Eight subjects are currently alive;  3 are alive in CR and 5 are alive 
with active disease.  For the entire study cohort, the median event-free survival and overall 
survival following CD8+ memory T-cell infusion were 4.9 months (95% CI, 
1 month to 19.3 months) and 19.6 months (95% CI;  5.6 months to not reached), respectively.   
In summary, this phase 1 trial showed that CD8+ memory T-cell DLI is well tolerated and 
associated with a low incidence of GvHD.  The MTD was not reached even at the higher dose 
level of 10 x 106/kg; however, this yield was unable to be achieved using the current-cell 
selection strategy.  Some subjects achieved a prolonged CR in this small heterogeneous study 
population with heavily pretreated hematologic malignancies.  There did not appear to be a dose 
response to CD8 memory T-cell infusions as responses were observed at all doses used, and 
likewise there was not a dose that was associated with increase in toxicity or side effects.  
Importantly, this clinical trial defined that the dose of phenotypic CD8+CD45RO+CD45RA- TM 
cells reliably obtained for use in a clinical trial using our enrichment strategy is 1 to10 x 106/kg. 
2.7  
Phase 2 Trial of Consolidative CD8+ Memory T-cell Infusion for Subjects with 
Mixed Chimerism after Allogeneic HCT 
Based on the results of the Phase 1 study that confirmed safety and suggested some level of 
efficacy, we initiated a second clinical trial entitled “Post-Transplant Infusion of Allogeneic CD8 
Memory T-cells as Consolidative Therapy After Non-Myeloablative Allogeneic Hematopoietic 
Cell Transplantation in Patients with Leukemia and Lymphoma”, (NCT02424968) under 
IND BB-14844.  The study rationale is based on multiple publications, including our own, that 
showed that persistence of mixed hematopoietic cell chimerism after allogeneic HCT was 
associated with a significantly higher risk of disease relapse. 35-44  Once a patient’s disease has 
relapsed after allogeneic HCT, the ability to return the patient to a durable remission is poor.  
Therefore, a strategy that can help promote conversion from mixed chimerism (MC) to full 
donor chimerism (FC) and that does not promote significant GvHD has the potential to improve 
the outcomes of transplant recipients by reducing the risk of disease relapse. 
The primary objective of this study will be to determine the rate of conversion from MC to FC 
following a post-transplant infusion (day 30 to 70) of enriched donor phenotypic CD8+ memory 
T-cells in subjects with hematologic malignancies who received non-myeloablative HCT with 


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TLI-ATG conditioning.  It is hypothesized that subjects who convert from MC to FC will have a 
reduced risk of relapse similar to subjects who are achieve FC without DLI.  The secondary 
objectives are to determine the risk of disease relapse, overall and event-free survival, 
non-relapse mortality, and the incidence of acute and chronic GvHD following the infusion of 
allogeneic CD8+ memory T-cells. 
Eligible subjects are 18 to 75 years of age with a high-risk or refractory hematologic malignancy 
and who receive nonmyeloablative allogeneic HCT using TLI-ATG conditioning and cells from 
an HLA-matched donor and who have MC (< 95% donor CD3+ cells) at day +30.  Phenotypic 
CD8+ memory T-cells are isolated from HLA-matched donors following 1 or 2 days of apheresis 
using the tandem immunomagnetic selection strategy including CD45RA depletion followed by 
CD8+ enrichment on the CliniMACS Plus instrument.  Cell purity is determined by flow 
cytometry analysis.  CD8+ memory T-cells are planned for infusion between day +30 and 
day +70 at a dose of 1 to 10 x 106 cells/kg, the dose determined from the phase 1 trial 
(Section 2.6, above) to be the maximum dose that can be consistently collected from 
1 or 2 apheresis products. 
This study will follow a single-arm, Simon 2-stage trial principle.  In Simon stage 1, 9 subjects 
are planned for enrollment, and if three or fewer subjects responded, defined as the conversion 
from mixed to complete chimerism within 90 days of the CD8+ TM cell infusion, the study will 
be stopped for futility.  If 4 or more subjects respond in Simon stage 1, then in stage 2 enrollment 
will be planned to continue to a full sample of 18 subjects.  Success will be declared if a total of 
13 or more subjects respond. 
To date, 9 subjects have been enrolled on the trial and received prophylactic donor 
CD8+CD45RO+CD45RA- memory T-cells between day 30 to 70 post-transplant. 45  Following 
the CD8+ memory DLI, subjects were assessed for the development of acute and chronic GvHD, 
conversion from MC to FC (≥ 95% donor CD3+ chimerism) within 3 months, and for disease 
relapse.  Secondary outcomes included the incidence of adverse events, invasive infections, 
non-relapse mortality, overall survival (OS), and disease-free survival (DFS).  The subjects and 
their outcomes are shown in Table 2. 45 
Table 2.  Subject characteristics and outcomes following consolidative CD8+ memory T-cell DLI 
Age/sex 
Diagnosis and 
disease status 
Day of 
DLI 
CD3+ chimerism 
pre- and 3 months 
post-DLI 
Acute 
GvHD 
Chronic 
GvHD 
Remission 
status 
Surviving 
Follow up 
days 
post-HCT 
71F 
AML 
CR1 
+40 
88% 
97% 
None 
None 
CR, MRD 
negative 
Alive 
747 
67F 
AML 
CR2 
+33 
23% 
44% 
None 
None 
Relapsed 
(day +698) 
Alive 
730 
67M 
MDS 
PR 
+40 
40% 
19% 
None 
None 
Relapsed 
(day +88) 
Deceased 
128 
38F 
CHL 
PR 
+42 
84% 
99% 
Grade II 
(skin) 
Mild 
(joints) 
Relapsed 
(day +231) 
Alive 
474 
47M 
DLBCL 
PR 
+41 
88% 
95% 
None 
None 
CR 
Alive 
390 


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Table 2.  Subject characteristics and outcomes following consolidative CD8+ memory T-cell DLI 
Age/sex 
Diagnosis and 
disease status 
Day of 
DLI 
CD3+ chimerism 
pre- and 3 months 
post-DLI 
Acute 
GvHD 
Chronic 
GvHD 
Remission 
status 
Surviving 
Follow up 
days 
post-HCT 
60F 
DLBCL 
CR 
+47 
93% 
100% 
Grade II 
(skin) 
None 
Relapsed 
(day +141) Deceased 
187 
30F 
PMBCL 
PD 
+42 
88% 
95% 
None 
None 
CR 
Alive 
306 
47F 
ENKTL 
PR 
+62 
75% 
72% 
None 
None 
CR 
Alive 
152 
65M 
DLBCL 
CR 
+49 
92% 
96% 
None 
None 
CR 
Alive 
131 
AML = acute myeloid leukemia;  CHL = classical Hodgkin lymphoma;  CR = complete remission;  DLBCL = diffuse 
large B-cell lymphoma;  DLI = donor lymphocyte infusion;  ENKTL = extranodal NK/T-cell lymphoma;  
GvHD = graft-versus-host disease;  HCT = hematopoietic cell transplantation;  MDS = myelodysplastic syndrome;  
MRD = minimal residual disease;  PMBCL = primary mediastinal B-cell lymphoma;  PD = progressive disease;  
PR = partial remission 
Median age of the subjects was 60 years (range 30 to 71 years) with a median follow up of 
306 days post-HCT (range 131 to 747 days).  Diagnoses included non-Hodgkin lymphoma 
(n = 5), AML (n = 2), MDS (n = 1), and Hodgkin lymphoma (n = 1).  Median donor CD3+ 
chimerism at Day +30 was 88% (range 23 to 93%).   
The cell product was highly enriched for CD8+/CD45RA-/CD45RO+ cells, and flow analysis 
confirmed the selected cells were phenotypically consistent with the CD8+ T-effector memory 
subset with a post-enrichment purity of ≥ 96% (range 96.4 to 98.8%) in all subjects.   
CD8+ memory T-cell DLI was well-tolerated with no adverse events.  6 of the 9 subjects (67%) 
converted from MC to FC within 3 months post-DLI with a median donor CD3+ chimerism of 
97% (range 95 to 100%).  Two subjects developed Grade II acute GvHD (skin only) that was 
treated to resolution.  One subject developed mild chronic GvHD.  No subjects developed severe 
acute GvHD (Grade III to IV) or extensive chronic GvHD.  No subjects developed invasive 
infections, and non-relapse mortality was 0%.  Of the 3 subjects who failed to convert from MC 
to FC, 2 had residual disease at the time of transplant, 1 of whom had early disease relapse at 
Day +88.  4 of the 6 subjects who converted to FC were in complete remission (CR) at the last 
follow up.  1 of the 3 subjects with persistent MC was in CR at the last follow up.  For the entire 
study cohort, the median disease-free survival is 1.1 years and the median overall survival is not 
yet reached. 
As with the phase  1 study, the early results from our phase 2 study demonstrate (1) the 
feasibility of consistently obtaining a cellular product highly enriched for phenotypic CD8+ 
memory T-cells at a yield of 1 to 10 x 106 cells/kg from donor apheresis products;  (2) the safety 
of this cellular therapy with a very low incidence of mild GvHD and no cases of severe acute 
GvHD (Grade III-IV) or extensive chronic GvHD observed to date in any of the 24 subjects 
treated thus far on the phase 1 and phase 2 trials;  and (3) the efficacy of CD8+ memory T-cells 
in converting 6 of 9 subjects (67%) with MC to FC, recapitulating the results seen in our murine 
model.  Subjects continue to accrue to trial, and further accrual and longer follow up are needed 
to determine if this strategy will reduce relapse rates for high-risk subjects with MC. 


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2.8  
Proposed Study Rationale and Hypothesis 
CD34 selection with immunomagnetic beads is an effective method of depleting alloreactive 
donor T-cells from a peripheral blood graft, and results in a significant reduction in acute and 
chronic GvHD. 3,46.47  Yet in these trials, overall subject survival did not appear improved 
perhaps due to an increase in tumor relapse;  graft rejection;  and infection.  Our hypothesis is 
that combining CD8+CD45RA- TM phenotype cells to a purified CD34+ graft will retain the low 
incidence of GvHD, yet also provide protection from graft rejection;  protection from infection;  
and enhance anti-tumor activity compared with CD34+ selection alone.  The proposed trial has 
particular significance especially in context of the current BMT CTN Phase 3 randomized, 
multicenter trial (BMT CTN 1301) in which a T-cell depleted (TCD) CD34-selected graft was a 
SOC arm in subjects with hematologic malignancies undergoing myeloablative conditioning for 
HCT.   
2.9  
Study Overview  
This single institution, single arm, Simon 2-Stage trial will test the hypothesis that combining 
donor CD8+CD45RA- TM cells with donor CD34+ selected grafts will be safe, and that the 
adoptively transferred CD8+ TM cells into the recipient will enhance anti-tumor, and 
anti-infection reactions without aggravating GvHD compared with CD34+ selection alone.  The 
primary study objective is the one-year rate of GvHD-free, Relapse-Free Survival (GRFS) as 
previously defined. 48,49  As with other CD34-selected graft trials, post-transplant 
immunosuppression will not be used in this trial. 
3. 
PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES 
3.1  
Eligibility Criteria and Participant Eligibility Checklist 
Inclusion and Exclusion Criteria are provided on the Eligibility Checklist, following, and which 
may be extracted from this document for use in screening potential subjects. otherwise indicated: 
Pre-transplant screening: all studies to be completed within 70 days of beginning conditioning 
therapy, unless otherwise indicated: 
The Inclusion and Exclusion Criteria are provided on the Participant Eligibility Checklist 
following, which must be completed in its entirety for each subject prior to registration.  The 
checklist may be extracted from this document for use in screening potential subjects.  The 
completed, signed, and dated checklist must be retained in the subject’s study file, and the 
study’s Regulatory Binder.   
Pursuant to Stanford Medicine SOP “Confirmation of Participant Eligibility in Clinical Trials,” 
the treating Physician (investigator);  the Study Coordinator;  and an Independent Reviewer will 
verify that the subject’s eligibility is accurate;  complete;  and legible in source records.  A 
description of the eligibility verification process should be included in the EPIC or other 
Electronic Medical Record progress note. 


IRB-49023 
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12 February 2021 v4 
Participant Eligibility Checklist 
I. 
Protocol Information  
Protocol Title: CD34-Selected Allogeneic Hematopoietic Cell Transplantation with 
Myeloablative Conditioning and CD8+ Memory T-cell Infusion For 
Patients with Myelodysplastic Syndrome, Acute Leukemia, and 
Chronic Myelogenous Leukemia (CML)   
eProtocol number: 
OnCore number: 
IRB-49023 
BMT339 
Principal Investigator: Robert Lowsky, MD 
II. 
Subject Information  
Subject name / Unique ID: 
/ 
Gender  
  Male   
 Female   
III. 
Eligibility Criteria  
RECIPIENT Inclusion Criteria  
Pre-transplant screening: all studies to be completed within 70 days of beginning conditioning 
therapy, unless otherwise indicated.  


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12 February 2021 v4 
Prospective RECIPIENTS Must MATCH  
ALL these Inclusion Criteria to be Eligible 
Yes 
No 
Supporting 
Documentation * 
Acute leukemia, in morphologic complete remission;  
OR myelodysplasia with < 10% blasts in the marrow, 
and no circulating blasts that contain auer rods;  OR 
chronic myelogenous leukemia (CML) with any one of 
the following high-risk features: 
1. Chronic Phase CML yet with additional karyotypic 
features at diagnosis that are beyond the reciprocal 
translocation (9;22)(q34;q11) known as Philadelphia 
(Ph) chromosome. 
2. Resistant CML, defined as: 
• A failure of therapy to achieve a complete 
cytogenetic response within 12 months of 
diagnosis, 
• A failure of therapy to maintain a > 3 log-fold 
reduction in molecular response once beyond 
12 months of treatment, 
• Any new leukemia-associated 
chromosomal/sequence abnormality detected by 
conventional cytogenetics;  FISH;  molecular or 
next generation sequencing. 
• Enlarging spleen size despite ongoing treatment. 
3. Accelerated Phase CML, defined as: 
• 10% to 19% blasts in the blood and/or bone 
marrow.  
• > 20% basophils in the peripheral blood.  
• Any new leukemia-associated 
chromosomal/sequence abnormality detected by 
conventional cytogenetics;  FISH;  molecular or 
next generation sequencing. 
4. Blast Crises CML, defined as: 
• > 20% marrow or blood blasts. 
Subjects with chronic myelomonocytic leukemia 
(CMML) must have a WBC count ≤ 10,000 cells/μL and 
< 10% blasts in the marrow.  
 
 
 
1. Planned myeloablative conditioning regimen at 
Stanford University Medical Center. 
 
 
 


IRB-49023 
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12 February 2021 v4 
Prospective RECIPIENTS Must MATCH  
ALL these Inclusion Criteria to be Eligible 
Yes 
No 
Supporting 
Documentation * 
2. ≥ 18 years to < 66.0 years 
 
 
 
3. Males or females  
 
 
 
4. Karnofsky or Lansky Performance Score ≥ 70%. 
 
 
 
5. Must have an HLA-matched donor as follows:  donor 
must be an 8/8 match for HLA-A, -B and -C at 
intermediate (or higher) resolution, and -DRB1 at 
high resolution using DNA-based typing.  The donors 
must be willing to receive G-CSF followed by 
collection of cells by apheresis, and must meet the 
Program’s criteria for donation.  
 
 
 
6. Cardiac function:  Ejection fraction at rest ≥ 40%  
 
 
 
7. Serum creatinine value of < 1.5 mg/dL, or an 
estimated creatinine clearance greater than 
50 mL/minute (using the Stanford calculator for 
eGFR available in EPIC)  
 
 
 
8. Diffusing capacity of the lungs for carbon monoxide 
(DLCO) ≥ 50% (adjusted for Hgb)  
 
 
 
9. Forced vital capacity (FVC) ≥ 50%  
 
 
 
10. 
Forced expiratory volume (FEV1) ≥ 50%  
 
 
 
11. 
Total bilirubin < 2 times the upper limit of 
normal (ULN) (unless the elevated bilirubin is 
attributed to Gilbert’s Syndrome)  
 
 
 
12. 
Alanine aminotransferase (ALT) < 2.5 x ULN  
 
 
 
13. 
Aspartate aminotransferase (AST) < 2.5 x ULN  
 
 
 
14. 
Signed informed consent  
 
 
 


IRB-49023 
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12 February 2021 v4 
RECIPIENT Exclusion Criteria  
Prospective RECIPIENTS Must NOT Match  
ANY of These Exclusion Criteria  
Yes 
No 
Supporting 
Documentation * 
1. Prior autologous or allogeneic hematopoietic stem 
cell transplant  
 
 
 
2. Prior malignancies, except resected non-melanoma 
or treated cervical carcinoma in situ.  Cancer treated 
with curative intent ≥ 5 years previously is allowed.  
Cancer treated with curative intent < 5 years 
previously will not be allowed unless approved by 
the Protocol Officer or one of the Protocol Chairs  
 
 
 
3. Active CNS involvement by malignant cells  
 
 
 
4. Presence of fluid collection (ascites, pleural or 
pericardial effusion) that interferes with 
methotrexate clearance or makes methotrexate use 
contraindicated  
 
 
 
5.  Requirement for supplemental oxygen  
 
 
 
6. Uncontrolled bacterial, viral or fungal infections 
(currently taking medication and with progression 
or no clinical improvement) at time of enrollment  
 
 
 
7. History of uncontrolled autoimmune disease or on 
active treatment (defined as > 5 mg prednisone 
daily)  
 
 
 
8. Seropositive for HIV-1 or -2  
 
 
  
9. Seropositive for HTLV-I or –II  
 
 
 
10. Active Hepatitis B or C viral replication by PCR  
 
 
 
11. Documented allergy to iron dextran or murine 
proteins  
 
 
 


IRB-49023 
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12 February 2021 v4 
Prospective RECIPIENTS Must NOT Match  
ANY of These Exclusion Criteria  
Yes 
No 
Supporting 
Documentation * 
12. Pregnant (positive serum or urine βHCG) or 
breastfeeding)  
 
 
 
13. Females of childbearing potential (FCBP) or men 
who have sexual contact with FCBP unwilling to 
use an effective form of birth control or abstinence 
for one year after transplantation  
 
 
 
14. Unable to comply with the treatment protocol, 
including appropriate supportive care, follow-up 
and research tests  
 
 
 
15. Planned to receive post-transplant maintenance 
therapy except for FLT3 inhibitors or BCR-ABL 
tyrosine kinase inhibitors (TKIs)  
 
 
 
* All subject files must include supporting documentation to confirm subject eligibility.  The 
method of confirmation can include, but is not limited to, laboratory test results, radiology test 
results, subject self-report, and medical record review.  
V. 
Statement of Eligibility (RECIPIENT) 
By signing this form of this trial I verify that this subject is:  
 eligible / 
 ineligible  for 
participation in the study.  This study is approved by the Stanford Cancer Institute Scientific 
Review Committee, the IRB of record, and has finalized financial and contractual agreements as 
required by Stanford School of Medicine’s Research Management Group.  
Study Coordinator printed name: 
Date:  
Signature: 
 
Investigator printed name: 
Date:  
Signature: 
 
Triple-check reviewer printed name: 
Date:  
Signature: 


IRB-49023 
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12 February 2021 v4 
Protocol Title: CD34-Selected Allogeneic Hematopoietic Cell Transplantation with 
Myeloablative Conditioning and CD8+ Memory T-cell Infusion For 
Patients with Myelodysplastic Syndrome, Acute Leukemia, and 
Chronic myelogenous leukemia (CML)    
eProtocol number: 
OnCore number: 
IRB-49023 
BMT339 
Principal Investigator: Robert Lowsky, MD 
DONOR name / Unique ID: 
/ 
Gender  
  Male   
 Female   
DONOR Eligibility Criteria - Donors for this trial will follow the general standard of care that is 
used to select G-CSF-mobilized hematopoietic cell donors at Stanford University  
Prospective DONOR Must MATCH  
ALL these Inclusion Criteria to be Eligible 
Yes 
No 
Supporting 
Documentation * 
1. HLA-matched donor (matching at 8/8 antigens or 
alleles including HLA-A, -B, -C, and –DRB1). 
 
 
 
2. ≥ 18 years to < 66.0 years 
 
 
 
3. State of general good health  
 
 
 
4. Completed a donor evaluation with history, medical 
examination and standard blood tests within 60 days 
of starting the hematopoietic cell collection procedure.  
In order to fairly represent the interests of the donor, 
the donor evaluation and consent will be performed by 
a study team member other than the recipient’s 
attending physician. 
 
 
 
5. Hepatitis A, B and C, HIV-1 and -2, HTLV, VZV, 
EBV, HSV, West Nile virus, Syphilis Treponema, 
T cruzi (Chagas), CMV, and the MPX NAT IDT 
(HIV/HCV/HBV) will be tested as per national 
standard of care guidelines for transplant donors.  
Donors who are HIV-positive will be excluded.  
Donors who are positive by serology for 
Hepatitis B or C are eligible as long as PCR for 
RNA/DNA is negative 
 
 
 
6. White blood cell count > 3.5 x 109/L 
 
 
 


IRB-49023 
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12 February 2021 v4 
Prospective DONOR Must MATCH  
ALL these Inclusion Criteria to be Eligible 
Yes 
No 
Supporting 
Documentation * 
7. Platelets > 150 x 109/L 
 
 
 
8. Hematocrit > 35% 
 
 
 
9. Capable of undergoing leukapheresis 
 
 
 
10. Able to understand and sign informed consent 
 
 
 
DONOR Exclusion Criteria  
Prospective DONOR Must NOT Match  
ANY of These Exclusion Criteria  
Ye
s 
No 
Supporting 
Documentation * 
1. Psychological traits or psychological or medical 
conditions which make them unlikely to tolerate the 
procedure 
 
 
 
2. Pregnant or lactating female 
 
 
 
* All subject files must include supporting documentation to confirm subject eligibility.  The 
method of confirmation can include, but is not limited to, laboratory test results, radiology test 
results, subject self-report, and medical record review.  
V. 
Statement of Eligibility (DONOR) 
By signing this form of this trial I verify that this subject is:  
 eligible / 
 ineligible for 
participation in the study.  This study is approved by the Stanford Cancer Institute Scientific 
Review Committee, the IRB of record, and has finalized financial and contractual agreements as 
required by Stanford School of Medicine’s Research Management Group.  
Study Coordinator printed name: 
Date:  
Signature: 
 
Investigator printed name: 
Date:  
Signature: 
 
Triple-check reviewer printed name: 
Date:  
Signature: 
 


IRB-49023 
Page 27 of 43 
12 February 2021 v4 
3.2  
Recruitment and Informed Consent 
Subjects will be selected based upon the eligibility requirements highlighted above.  Informed 
consent forms will be approved by the Stanford University IRB, SRC and FDA.  No payment 
will be provided to participate in this protocol.  Costs for routine care will be borne by the 
subject’s insurance carrier.  Research costs including cell enrichment procedures of the CD8+ TM 
cells as well as all research studies will be borne by an NCI-sponsored Program Project Grant 
(PPG 4P01CA049605-27). 
3.3  
Potential Risk to the Recipient 
Subjects who undergo allogeneic HCT have risks for a variety of complications that include 
transplant-related deaths.  An Attending Physician will explain the risks associated with HCT in 
detail to the subject and their caregivers once it is deemed that treatment with this protocol is a 
feasible consideration and appropriate.  The physician involved in the consenting process will 
not be the primary physician who is involved in managing the care of the subject or donor.  A 
detailed discussion regarding the current alternative treatment which is a CD34-selected graft 
without a CD8+ memory T-cell infusion, an unmanipulated donor graft with SOC post-transplant 
immune suppression or post-transplant Cyclophosphamide will be discussed with the subject and 
their caregiver(s).  All inclusion criteria eligibility must be fulfilled.  The plan will also be 
discussed with the subject’s referring physician. 
Potential Sensitization to Murine Proteins:  Mouse protein antibodies are used in the CliniMACS 
processing procedures.  If the recipient has a pre-existing allergy, he or she may be at risk for 
allergic reactions during infusion of the processed cells; although the residual amount of murine 
protein in the final product is very low (estimated maximum dose for a 50 kg subject would be 
30 μg).  To date, no allergic reactions are reported in subjects receiving cells processed with the 
CliniMACS System.  Epinephrine and antihistamines will be available at the recipient's bedside 
during the PBSC infusion. 
3.4  
Protection Against Risks 
The patient-subject will receive a consent form with appropriate information concerning the cell 
infusion.  Risks include the toxicities of the acute and chronic GvHD, infection, marrow aplasia 
and cytopenia, disease progression, and the possible other unforeseen risks given the novel 
nature of the clinical trial.  All questions will be answered by the Attending Physician as well as 
Nurse Coordinator.  The physician involved in this process will not be the primary physician 
who is involved in managing the care of the patient-subject or donor.  Once a patient-subject has 
agreed to proceed with the trial a formal 1 to 1.5 hour consenting process will be performed by 
the Research Nurse and all questions will be answered.  Signed informed consent must be 
obtained prior to initiation of the protocol.  Risks to the donors are expected to be very low and 
are the same as for other donors who undergo collection for traditional HCT. 
To maintain confidentiality, study subject identification will not be revealed in any reports or 
publications resulting from the study.  Information will be provided to the study subjects’ 
physicians as necessary, to the IRB, DSMB, and FDA as necessary.  Case report forms and other 
study records will not identify the subjects by name.  To monitor data and safety of the research, 
the procedures as outlined in the “Data and Safety Monitoring Plan” (see below) will be 
followed. 


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3.5  
Potential Benefits of the Proposed Research to the Subjects 
This study represents a logical extension of our work on the use of CD8+ TM cells that have the 
potential to improve transplant outcomes by providing anti-tumor activity and enhancing 
post-transplant immune reconstitution with prevention from infection yet without increasing the 
risk of GvHD.  The potential benefits could be realized by patient-subjects who participate in this 
trial, yet it is understood that the trial concept and design is investigational. 
3.6  
Importance of the Knowledge to be Gained 
The goal of the trial is to test the treatment concept that adding HLA matched donor 
CD8+CD45RA- TM phenotype cells to purified CD34+ grafts given to recipients undergoing full 
dose conditioning and HCT for acute leukemia and MDS is safe, and will reduce the risk of 
disease relapse and infections without increasing the risk of GvHD.  If we are successful, it is 
likely that this strategy will improve overall subject survival.  As well, the strategy developed in 
the proposed trial can be effectively adapted to other clinical transplant settings, and therefore 
has applicability to treat diseases beyond acute leukemia and MDS.  The overall significance is a 
potential improvement in transplant outcomes. 
3.8  
Inclusion of Women and Minorities 
Women and minorities and are eligible for this protocol.  
3.9  
Subject Withdrawal  
Participation in the trial is voluntary.  Subjects will be advised that they may voluntarily 
withdraw from the study at any time and will be instructed to notify the investigator.  Subjects 
may choose to withdraw for any reason(s).  Subjects are not obligated to reveal their reason(s) 
for withdrawal to the sponsor.   
3.10  Subject Withdrawal from Data Collection  
If a subject explicitly states they do not wish to contribute further data to the trial their decision 
must be respected and notified to the sponsor in writing.  In this event details should be recorded 
in the subject’s hospital records, no further CRFs must be completed and no further data sent to 
the sponsor. 
4 TREATMENT PLAN  
Study Transplant Conditioning Regimen 
OPTION 1:  
Fractionated TBI will be delivered in the standardized format consisting of a total dose of 1375 
cGy given in fractions on days -10 -9,-8 and -7.  Sequential doses are administered in an 
anterior/posterior or lateral orientation.  The orientation of TBI chosen will be left to the 
discretion of the radiation oncologist but will remain consistent at our institution throughout the 
duration of the trial.  Full value lung blocks are not used yet compensators and lung blocks 
yielding a minimum of 800 cGy lung dose are allowed based on our institutional practice.  
Depending on the method of lung shielding employed, the blocked areas should be boosted with 
high-energy electrons or be otherwise radiated so that the cumulative chest wall dose is 
approximately 1300 cGy, so as to ensure that marrow sites in the ribs are adequately treated.  In 


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addition, male subjects receiving transplants for ALL or AML, use of boost to testes is allowed 
according to institutional practices. 
Thiotepa will be administered at 5 mg/kg/day intravenously over 4 hours for 2 consecutive days 
(Day -6 and -5).   
Fludarabine will be administered intravenously over 30 minutes at a total dose of 125 mg/m2 
divided into 5 daily doses of 25 mg/m2/day (Day -6 to -2). 
Treatment Schedule 
Days -10 to -7  
Fractionated TBI (1375 cGy), administered over 4 days 
Days -6 to -5  
Thiotepa 5 mg/kg/day:  IV for 2 consecutive days  
Days -6 to -2  
Fludarabine 25 mg/m2/day:  IV for 5 consecutive days  
OPTION 2: : Non TBI Conditioning – Busulfan/Cyclophosphamide 
Days -7 to -4:  
Busulfan:  3.6 mg/kg/dose Q24h IV.  Infused over 3 hours.  1 dose per day x 
4 consecutive days x 3.6 mg/kg/dose = 14.4 mg/kg  
Days -3 to -2  
Cyclophosphamide:  60 mg/kg/dose Q24h IV.  Infused over 2 hours.  1 dose 
per day x 2 consecutive days x 60 mg/kg/dose = 120 mg/kg 
Immune Suppression Medication 
Tacrolimus .01 mg/kg IV BID or .03 mg/kg PO BID  
1. Begin day -3  
2. Target serum level is 5-10 ng/ml  
3. If > 15 kg over IBW, dose based on adjusted IBW  
Dose adjusted based on creatinine, t. bilirubin and serum drug levels. 
Tacrolimus taper will begin around day +90 in the absence of GVHD and based on clinical 
decisions. The aim will be to discontinue Tacrolimus altogether if clinically applicable (no 
history of GVHD) by around day +180. In some patients who are considered higher risk for 
disease relapse, as determined by the medical team, the tacrolimus taper may begin in the 
absence of active GVHD by day +60. 
  
 
 
 
4.2  
Hematopoietic Stem Cell Graft Collection 
Transplant donors are considered research subjects by the FDA.  For HLA-matched donors, the 
consent process will be done at the Stanford University Medical Center along with routine donor 
consent procedures according to institutional guidelines.  The donor informed consent document 
for donors will be reviewed and approved by the Stanford University IRB. 


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4.3  
Mobilization of Donor 
Following screening and enrollment, donors will undergo 1 to 2 days of apheresis collections for 
the processing and enrichment for CD34+ cells and for CD8+CD45RA- TM cells as detailed in 
Section 5.  As per standard of care, donor mobilization will be using G-CSF (filgrastim or its 
biosimilar equivalent) and cell collection will be done using standard procedures for apheresis.  
A sample donor schedule is described below, and as per standard of care the schedule can shift 
one day up or down depending on apheresis and CTF (cell therapy facility) availability. In 
general, the donor will begin GCSF on Day -6 or Day -7 of the conditioning regimen at a dose of 
10 μg/kg/day actual body weight subcutaneously (rounded to a multiple of the nearest vial size of 
either 300 or 480 μg).  Based on the volume of GCSF, the dose may be split into 
multiple injection sites.  The mobilization phase will start on the 1st day of administration of 
G-CSF (Day -6 or Day -7) and continue until the final day of leukapheresis (Day -1 or Day -2).  
The donor will undergo their 1st apheresis on Day -2 or Day -3, and if it is determined that an 
additional apheresis is required to achieve adequate cell doses, then a 2nd apheresis will be on 
Day -1 or Day -2, respectively.  Therefore, the cell collection days for the donor will be Days -3 
and -2, or it can be Days -2 and -1.   
 
4.3  
Donor Leukapheresis  
Leukapheresis will be performed on a continuous flow cell separator according to institutional 
standards and will commence on the morning of the fifth day of G-CSF treatment.  The 
anti-coagulant used for the procedure will be acid citrate dextrose (ACD).  The volume of blood 
processed per leukapheresis session should be approximately three to four times total blood 
volume as tolerated by the donor.  It is expected that many donors will undergo 2 consecutive 
days of apheresis.  Concurrent plasma (about 200 mLs) will be collected for products to be 
stored overnight.  A unique identification and labeling system will be used to track the 
leukapheresis product from collection to infusion according to FACT/JACIE guidelines. 
5 
INVESTIGATIONAL AGENT INFORMATION 
5.1  
Investigational Agent:  CD8+ Memory T-Cell Selection 
The apheresis product will be enriched for CD8+CD45RA- memory T-cells.  In a procedure we 
successfully used in 2 prior clinical trials, we will enrich for donor CD8+ memory T-cells using a 
tandem immunomagnetic selection procedure that involves the CliniMACS Cell Selection 
System (Miltenyi Biotec).  Depletion of CD45RA expressing cells uses CliniMACS CD45RA 
Reagent (BB-MF-11872).  The CD45RA depleted fraction is then enriched for CD8-expressing 
cells by positive cell selection with CliniMACS CD8 Reagent (BB-MF-11704).  The cells will 
be infused in the usual manner on Day 0 and within 6 hours of the CD34+ selected stem cell 
infusion. 
In our two prior clinical trials we determined that 2 consecutive days of aphereses per donor will 
yield a target cell dose of 5 to 10 x 106 CD8+ memory T-cells/kg for all subjects, assuming most 
subjects weigh less than 120 kg.  We will use a dose range of 5 to 10 x 106 CD8+ memory 
T-cells/kg for the current study, as both dose levels (5 x 106/kg and 10 x 106/kg) were well 
tolerated in our prior phase 1 study.  We will not exceed a dose of 10 x 106 CD8+ memory 
T-cells/kg.  If the number of CD8+ memory T-cells falls below the desired threshold of 


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5 x 106/kg, the infusion will proceed as planned because we believe it is in the subject’s best 
interest to receive the cell infusion.  The CD8+ memory T-cell infusion has the potential to help 
achieve donor cell engraftment, provide anti-tumor reactions and protect from infections yet 
without increasing the GvHD risk.  It is expected that a cell yield of < 5 x 106/kg will be 
obtained less than 20% of the time. 
5.2  
Stability 
As part of our original IND, we submitted stability data to the FDA from validation runs that 
extended to 72 hours from the time of completion of first apheresis, and showed negligible loss 
in cell viability as all validation products continued with > 90% viability.  In addition, the results 
from our prior CD8+ memory T-cells trials confirmed stability data extending to 72 hours after 
completion of first apheresis from 8 additional donors, and all cases maintained  > 94% viability. 
5.3  
Reagents for cells for immediate release and conditions for storage 
The CD8 and CD45 reagent systems are pharmaceutical grade and both have Master Files that 
are BB-MF-11704 and BB-MF-11872, respectively.  Donors will undergo two consecutive 
apheresis days with the first day of apheresis being in the afternoon expected to begin around 
2 PM and be completed by 6 PM (the collection will be stored overnight in a 4-degree Celsius 
bio-fridge), followed by a second apheresis the next morning starting at around 7:30 AM.  The 
two days of collections will be batched for routine SOC CD34+ cell selection that will be 
performed on the same day as the second collection.  The CD34+ flow through will be diluted for 
overnight storage at 4 degrees Celsius with 1 volume of Normosol-R with 2% HSA.  Thereafter, 
the following morning the tandem immunomagnetic selection procedure for enrichment of 
CD45RA-CD8+ phenotype T-cells will be performed.  For subject comfort, the final volume of 
the infusate will be reduced to < or equal to 200 mL and the CD8+ memory T-cell concentration 
will range between 1 x 106 to 1 x 108 cells/mL.  The final infusate will be diluted in the Miltenyi 
Biotech Selection Buffers that contain PBS with 1mmolar EDTA and 0.5% HSA.  It is estimated 
that all CD45RA-CD8+ phenotype T-cell products will be infused within 72 hours from 
completion of the first apheresis. 
 
5.4  
Release Requirements of the Product 
Release of the selected cell products for infusion includes: 
• Negative Gram staining (no organisms detected). 
• Endotoxin levels ≤ 0.5 EU/milliliter of infused product volume 
• 80% cell viability as assessed by trypan blue or 7-AAD dye exclusion. 
• ≥ 80% of the CD3+ cells express the CD8 memory T-cell phenotype, here defined as: 
o CD8+/CD45RA-/CD45RO+ 
o ≤ 5% of cells with the CD3+/CD45RA+/CD45RO- phenotype 
o Sufficient cells with the CD8 memory T-cell phenotype to meet the desired dose, or 
at least the minimum dose of > 2 x 106 CD8+ memory T-cells/kg 


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In addition, post-selection sterility cultures in accordance with sterility testing guidelines 
(21CFR§610.12) will be conducted to confirm the sterility of the selected cells.  Any positive 
growth results obtained will be documented in the processing record of the selection and 
immediately communicated to the subject’s attending physician in accordance with the plan of 
action for the infusion of cellular products with microbial contamination.  Ancillary studies may 
include phenotypic analysis of the selected cells and cytokine secretion profiles from MLR 
assays may be used to further our understanding of the CD8+ memory T-cell population 
characteristics but do not constitute release criteria at this time.  If the cell product does not 
meet release criteria after manipulation, the product will not be infused. 
In the case(s) where the manipulated cell product does not meet release criteria, the subject will 
be removed from the study due to a failure to receive the investigational product.  If 2 of the first 
5 enrolled subjects fail to receive the CD8+ memory T-cell product due to a failure to satisfy the 
release criteria, a formal DSMC review will be initiated.  An enrolled subject who does not 
receive the CD8+ memory T-cell product will be removed from the study and replaced with 
another subject as the accrual target is 30 subjects who will be infused with CD8+ memory 
T-cells. 
6 
DOSE MODIFICATION  
As a single treatment transplant procedure, dose modification for any particular subject is not 
anticipated. 
7 
ADVERSE EVENTS AND REPORTING PROCEDURES 
7.1  
Adverse Events 
Hematopoietic cell transplantation (HCT) is an aggressive therapy for the treatment of a number 
of life-threatening disorders, including cancer.  In this setting, a very large number of Grade 1, 2 
and 3 adverse events (AEs) are expected to occur, regardless of whether or not a subject is 
participating in a research study.  In order to minimize the “background noise” and to focus on 
clinically significant, meaningful adverse events useful in the assessment of proposed trial the 
following AE reporting schema is proposed.  This schema is intended to capture all AEs that are 
clinically significant and/or impactful, but minimize “not informative” AE collection. 
Laboratory values without a clinical consequence or outcome will not be recorded as adverse 
events, unless deemed serious (see below). 
Grade 1 to 3 AEs not meeting these criteria will not be collected, except as follows.  Grade 1 to 3 
AEs resulting in subject termination or withdrawal from the study will be collected. 
An adverse event is considered a serious adverse event (SAE) if it fulfills one of the following 
SAE criteria per 21CFR§312.32(a):  
 Results in death. 
 Life-threatening (subject at risk of death at the time of the event). 
 Requiring new inpatient hospitalization or prolongation of existing hospitalization. 
 Resulting in persistent or significant disability. 
 Other medical events that may not be immediately life-threatening or result in death 
or hospitalization by may jeopardize the subject or require intervention to prevent one 
of the outcomes listed above 


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Grade 4 and higher AEs will be collected and documented with causality attribution on an 
adverse event log (AE log), and all AEs meeting the criteria of “serious” as defined at 
21CFR§312.32(a), including any that are otherwise Grade 1, 2 or 3 will be collected; identified 
as serious; and documented on an AE log, including causality attribution. 
All SAEs will be collected as outlined in Section 7.1 from date of CD8+ memory T-cell infusion 
through 365 days.  All SAEs will be tracked until resolution or at least 60 days after the study 
treatment. 
Vital signs for all subjects enrolled on the trial will be monitored for infusion-related toxicities 
(AEs) during the CD8 memory T-cell infusion, and every 30 minutes for 2 hours afterwards.  
Observed events will be recorded on the AE log.  In particular, subjects will be observed for 
hemodynamic instability, allergic reactions (eg, hives, rashes, angioedema), and fevers (> 38.3º). 
The development of acute GvHD, its date of onset and overall grade, is a key secondary outcome 
measure for subjects on this trial.  Acute GvHD will be recorded on an AE log. 
7.2  
Adverse Event Reporting 
All SAEs will be reported to the IRB;  to the IND;  and to the DSMC, either in an annual review 
or as an expedited report, in accordance with the established practices of the Stanford Division of 
Blood and Marrow Transplantation, as well as applicable guidelines and regulations.  AEs 
meeting the criteria specified for an IND Safety Report as defined at 21CFR§312.32(c)(1) will 
be collected; documented in the adverse event log; and reported to the IND (on a 
MedWatch 3500A form) in the timeframe specified.  The IND annual report will include 
summaries of the collected AEs, as specified by 21CFR§312.33. 
8 
CORRELATIVE/SPECIAL STUDIES 
None planned. 
9 
STUDY CALENDAR 
Pre-Enrollment 
requirements A 
On-study enrollment A = CD8+ 
Memory T-cell Infusion (Day 0) 
Time (days) from on-study 
enrollment ± 20 days 
 
 
Day +90 Day 180 Day 365 
CD34+ cell Infusion 
 
X 
 
 
 
Infusion of CD8+ memory 
T-cells 
 
X 
 
 
 
Informed consent 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Medical history 
X 
 
X 
X 
X 
Donor evaluation 
X 
 
 
 
 
Physical exam 
X 
 
X 
X 
X 
Fulfills inclusion criteria 
X 
 
 
 
X 
Has no exclusion criteria 
X 
 
 
 
 


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12 February 2021 v4 
Performance status 
X 
 
X 
X 
X 
Disease Status 
Assessment 
X 
 
X 
X 
X 
CBC with differential 
 
 
X 
X 
X 
Serum chemistry 
 
 
X 
X 
X 
STR analysis B 
 
 
X 
X 
X 
Assess for ≥ Grade 2 
acute GvHD C 
 
 
X 
X 
X 
Assess for moderate or 
severe chronic GvHD C 
 
 
X 
X 
X 
A  
All pre-transplant screening and procedures must be completed within 70 days of beginning conditioning therapy 
(unless otherwise indicated). 
B  
STR analysis will only be performed beyond Day +90 if there is concern about mixed chimerism or relapse. 
C  
Refers to if the event was recorded during the reporting periods of Day 0 to +90;  Day 91 to +180;  and 
Day 181 to +365. 


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10. 
MEASUREMENTS 
10.1  Primary and Secondary Objectives and Outcome measures 
Objectives and outcome measures are described at Section 1.1 and 1.2. 
11. 
REGULATORY CONSIDERATIONS 
11.1 
Institutional Review of Protocol 
The protocol, the proposed informed consent and all forms of participant information related to 
the study (eg, advertisements used to recruit participants) will be reviewed and approved by the 
Stanford IRB and Stanford Cancer Institute Scientific Review Committee (SRC).  Any changes 
made to the protocol will be submitted as a modification and will be approved by the IRB prior 
to implementation.  The Protocol Director will disseminate the protocol amendment information 
to all participating investigators. 
11.2 
Data and Safety Monitoring Plan 
The Stanford Cancer Institute Data and Safety Monitoring Committee (DSMC) will be the 
monitoring entity for this study.  The DSMC will audit study-related activities to determine 
whether the study has been conducted in accordance with the protocol, local standard operating 
procedures, FDA regulations, and Good Clinical Practice (GCP).  This may include review of the 
following types of documents participating in the study: regulatory binders, case report forms, 
eligibility checklists, and source documents.  In addition, the DSMC will regularly review 
serious adverse events and protocol deviations associated with the research to ensure the 
protection of human subjects.  Results of the DSMC audit will be communicated to the IRB and 
the appropriate regulatory authorities at the time of continuing review, or in an expedited 
fashion, as needed. 
11.3 
Data Management Plan 
The Protocol Director, or his/her designee, will prepare and maintain adequate and accurate 
participant case histories with observations and data pertinent to the study, in accordance with 
the established practices of the Stanford Division of Blood and Marrow Transplantation. Accrual 
rates will be reported to the Data Safety Monitoring Committee (DSMC) and data quality will be 
reviewed by the Study Statistician.  The DSMC of the Stanford Cancer Center will oversee this 
study and will receive regular reporting of SAEs and monitoring.  This Committee involves 
knowledgeable physicians, scientists and statisticians who oversee all phase I/II trials conducted 
under the auspices of the Stanford Cancer Center.  Individuals who serve on this Committee who 
are associated with this trial will recuse themselves from discussions of study results, and 
decisions about revision or closure.  Audits will be conducted as requested.  Data will also be 
submitted to the FDA as required by our IND and follow the appropriate reporting rules and 
regulations including an annual report. 
11.4  Good Clinical Practice Guidelines 
The clinical investigators assure that the clinical study is performed in accordance with the 
protocol, the ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996, the 
Declaration of Helsinki (Recommendations guiding physicians in Biomedical Research 
involving Human Subjects;  Helsinki 1964;  and as amended Tokyo 1975;  Venice 1983;  


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Hong Kong 1989;  Somerset West 1996;  Edinburgh 2000;  Seoul 2008;  and Fortaleza 2013) 
and applicable regulatory requirements. 
12. 
STATISTICAL CONSIDERATIONS 
12.1 
Statistical Design 
The study is a single arm, Simon 2-stage trial with a modification to avoid protracted temporary 
closure of accrual at the end of the first stage.  In single and multi-center studies consisting of 
over 1500 adult patients treated with SOC allogeneic HCT, the 1-year probability of GRFS was 
30%. 48,49  For Type 1 and 2 error rates of 0.10 and 0.2 respectively (power of 0.80), a control 
success rate of 0.30 and experimental rate of 0.50, the Simon optimal design calls for enrolling 
15 subjects in stage 1 and if 6 or more subjects achieve success, then in stage 2 continuing to 
enroll to a full sample of 32 subjects and declare success if a total of 13 subjects experience 
success.  In order to avoid a protracted temporary closure between the first and second stage, 
Day 100 GRFS will be evaluated at the 1st stage.  If ≥ 6 subjects in Simon stage 1 are alive 
without relapse or Grade III-IV GvHD at Day +100, then the full cohort of 32 subjects will be 
enrolled for stage 2.  Success will be declared if ≥ 13 subjects of 32 meet the primary endpoint of 
GRFS at 1 year, and if at least 6 of the stage 1 subjects ultimately meet that endpoint as well.  
With that proviso, this modified design has the same type 1 and 2 errors of a hypothetical 
standard Simon 2-stage design which would pause at the end of stage 1 until at least 6 subjects 
reach the 1-year GRFS outcome.  However, the modification modestly increases the expected 
number of subjects treated (under the null, as discussed below). 
Note that the end of stage 1 is defined as 100 days, while at the final analysis is for 1 year. 
Subjects who fail at 100 days will be regarded as failing in the time frame of the final analysis. 
No increase in type I or type II error is anticipated. 
12.1.1 Randomization 
This is a single arm, non-randomized study. 
12.2 
Interim analyses 
There is no formal plan for an interim analysis because the intrinsic nature of the Simon 
two-stage design is such that the study will not proceed to stage 2, unless the null hypothesis is 
rejected, because activation of the second stage depends on an adequate number of success 
observed in the first stage.  Therefore, the trial design incorporates stopping early for futility and 
safety.  It is possible that CD8+CD45RA- TM cells will induce acute GvHD.  If 2 of the first 
4 subjects develop Grade 2 or higher acute GvHD, or, if after 8 subjects, > 30% develop Grade 2 
or higher acute GvHD, then we will include a post-transplant GvHD prophylactic regimen.  
Toxicity, adverse events, and other safety endpoints will be monitored regularly and reported to 
the DSMB at each meeting or earlier.  The expected 100-day mortality rate for a myeloablative 
transplant is 15 to 20% based on our data as well as CIBMTR data.  The rate of mortality will be 
monitored up to 100 days after transplant, and if 2 of the first 4 enrolled subjects, or if 3 subjects 
in Simon stage one die within 100 days of transplant, this will trigger a DSMB safety review.  If 
2 subjects experience graft rejection in Simon stage one, this too will trigger a DSMB review. 


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12.3 
Primary endpoint  
The primary endpoint is GvHD-free, relapse-free survival (GRFS) at 1 year.  Development of 
acute GvHD Grade 3 or higher, extensive chronic GvHD requiring immunosuppression, relapse, 
or death will be considered failures for this endpoint. 
12.4  Sample Size 
12.4.1 Accrual estimates  
32 subjects is the full target accrual for the study.  
12.4.2 Sample size justification 
It is estimated that 1 subject per month will be accrued.  The total subject accrual is expected to 
take approximately 32 months.  Given that accrual rate, the 15th (last) subject recruited for 
stage 1 will reach the +100 day milestone at about 18 months of study time, and the first 
6 subjects recruited will have reached the 1-year point.  The entire first stage group of 
15 subjects will reach the 1-year milestone at month 27.  Thus, sometime between the end of 
months 18 and 27, it will be known whether the unmodified Simon trial would be stopped.  
During that interim period, up to 9 new subjects would be recruited.  This is an upper bound on 
the excess exposure under the null.  It is approached only if the +100 day GRFS rate is 
considerably larger than the 1-year GRFS rate null, so that few of the first-stage +100 day 
successes continue to 1 year.  We have no direct estimate of the failure rate over 1 year given 
GRFS at +100 days (under the experimental treatment), so we do not attempt to refine the 
calculation further. 


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13. 
REFERENCES 
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Transplantation for Acute Myeloid Leukemia and Myelodysplastic Syndromes.  J Clin Oncol.  
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2.  Papadopoulos EB, Carabasi MH, Castro-Malaspina H, et al.  T-cell-depleted allogeneic bone marrow 
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of graft-versus-host disease.  Blood.  1998;91(3):1083-1090. 
3.  Jakubowski AA, Small TN, Young JW, et al.  T-cell depleted stem-cell transplantation for adults with 
hematologic malignancies: sustained engraftment of HLA-matched related donor grafts without the use of 
antithymocyte globulin.  Blood.  2007;110(13):4552-4559. 
4.  Jakubowski AA, Small TN, Kernan NA, et al.  T-cell-Depleted Unrelated Donor Stem Cell Transplantation 
Provides Favorable Disease-Free Survival for Adults with Hematologic Malignancies.  
Biol Blood Marrow Transplant.  2011.  
5.  Devine SM, Carter S, Soiffer RJ, et al.  Low risk of chronic graft-versus-host disease and relapse associated 
with T-cell-depleted peripheral blood stem cell transplantation for acute myelogenous leukemia in first 
remission: results of the blood and marrow transplant clinical trials network protocol 0303.  
Biol Blood Marrow Transplant.  2011;17(9):1343-1351. 
6.  Castro-Malaspina H, Jabubowski AA, Papadopoulos EB, et al.  Transplantation in remission improves the 
disease-free survival of patients with advanced myelodysplastic syndromes treated with myeloablative 
T-cell-depleted stem cell transplants from HLA-identical siblings.  Biol Blood Marrow Transplant.  
2008;14(4):458-4568. 
7.  Perales MA, Jenq R, Goldberg JD, et al.  Second-line age-adjusted International Prognostic Index in patients 
with advanced non-Hodgkin lymphoma after T-cell depleted allogeneic hematopoietic SCT.  
Bone Marrow Transplant.  2010;45(9):1408-1416. 
8.  Goldberg JD, Linker A, Kuk D, et al.  T-cell-Depleted Stem Cell Transplantation for Adults with High-Risk 
Acute Lymphoblastic Leukemia: Long-Term Survival for Patients in First Complete Remission with a 
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APPENDICES 
APPENDIX A  GvHD SCORING CRITERIA 
Glucksberg clinical stage and grade of acute GvHD 
 
NIH consensus criteria for chronic GvHD severity with examples 
(comprehensive organ scoring criteria is reviewed in Jagasia, et al, BBMT 2014) 
 


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APPENDIX B.  RESPONSE CRITERIA by DISEASE HISTOLOGY 
AML Response Criteria (Dohner, et al, Blood 2017) 
 
 


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ALL Response Criteria (Brown, et al, JNCCN 2017) 
 
MDS Response Criteria (Cheson, et al, Blood 2006)