Phase 1-2 Trial for Patients With Advanced Hematologic Malignancies Undergoing Myeloablative Allogeneic HCT With a T-cell Depleted Graft With Infusion of Conventional T-cells and Regulatory T-cells
Phase: PHASE1
Conditions: Myeloid Leukemia, Chronic, Acute Myelogenous Leukemia, Myelodysplastic Syndromes (MDS), Lymphoma, Non-Hodgkin, Acute Lymphoblastic Leukemia (ALL), Myeloproliferative Syndrome, Acute Myeloid Leukemia, Acute Leukemia, Chronic Myelogenous Leukemia
Sponsor: Stanford University

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Phase 1-2 Trial for Patients with Advanced Hematologic Malignancies 
undergoing Myeloablative Allogeneic HCT with a T-cell Depleted Graft with 
Infusion of Conventional T-cells and Regulatory T-cells 
Stanford Cancer Institute 
875 Blake Wilbur Drive 
Stanford, CA 94305 
Single Center 
Protocol Director:  
Everett Meyer, MD, PhD 
 
 
 
650-725-5816 
 
Email: evmeyer@stanford.edu 
Stanford Cancer Institute Sub-Investigators:  
Robert Negrin, MD  
Sally Arai, MD  
Laura Johnston, MD  
Robert Lowsky, MD  
David Miklos, MD, PhD 
Lori Muffly, MD, MS  
Andrew Rezvani, MD  
Parveen Shiraz, MD 
Judith Shizuru, MD, PhD  
Wen Kai Weng, MD, PhD  
Rajni Agarwal, MD 
Biostatistician: 
Study Coordinators: 
  
  
 
 
 
 
   
  
            
  
or other designate 
IRB-21257 
BMT236 
IND 014686 
NCT01660607 
Date: 
04 February 2022 

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PROTOCOL SYNOPSIS 
 
TITLE 
Phase 1-2 Trial for Patients with Advanced Hematologic 
Malignancies undergoing Myeloablative Allogeneic HCT with a 
T-cell Depleted Graft with Infusion of Conventional T-cells and 
Regulatory T-cells 
STUDY PHASE 
Phase 1-2  
INDICATION 
Subjects with hematologic malignancies eligible for myeloablative 
allogeneic HCT using human leukocyte antigen (HLA)-matched 
donor or 9/10 HLA-matched donor (related or unrelated)  
INVESTIGATIONAL 
PRODUCT  
Purified regulatory T-cells (Treg) plus CD34+ hematopoietic 
progenitor cells (“CD34+ HSPC”)  
PRIMARY OBJECTIVE 
1. To determine the efficacy, safety and feasibility of administration 
of several dose combinations of conventional T-cells (Tcon) and 
regulatory T-cells (Treg) in subjects undergoing allogeneic 
hematopoietic cell transplantation (HCT) with HLA-matched 
donor or 9/10 HLA-matched donors (related or unrelated) using a 
T-cell depleted graft [CD34+ hematopoietic progenitor cells 
(“CD34+ HSPC”)], without immune-suppression.  
2. To determine the maximum tolerated dose (MTD) of infused 
regulatory and conventional T-cells in the matched donor setting 
3. Determine if concomitant single-agent immunosuppression is 
needed with fresh Treg cells (phase 2 stage 1) 
4. To determine 1-year GvHD-free relapse-free survival (GRFS) 
post-HCT (phase 2 stage 2) 
SECONDARY 
OBJECTIVES 
1. To determine the 1-year OS in subjects undergoing allogeneic 
HCT with matched donors. 
2. To measure the incidence and severity of acute and chronic graft 
vs host disease (GvHD) 
3. To measure incidence of serious infections 
HYPOTHESIS 
For subjects with hematologic malignancies undergoing allogeneic 
myeloablative (MA) HCT with a T-cell depleted graft, the infusion of 
naturally occurring regulatory T-cells  with conventional T-cells 
(T-cell addback) in pre-defined doses and ratios will reduce the 
incidence of acute GvHD while augmenting the graft vs leukemia 
effect and improving immune reconstitution. 

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TREATMENT 
SUMMARY 
Conditioning regimen will be determined by diagnosis: 
Acute leukemia:  high-risk CML;  myelodysplastic syndrome;  
myeloproliferative disorders:  
fTBI / VP16 / Cyclophosphamide  
   OR 
Busulfan / Cyclophosphamide 
OR 
Thiotepa/ Busulfan/ Fludarabine 
Lymphoma: non-Hodgkin; Hodgkin:  
BCNU / VP16 / Cyclophosphamide 
SUMMARY OF 
SUBJECT 
ELIGIBILITY 
CRITERIA 
See Section 3 for complete eligibility criteria.   
Subjects with the following diseases are eligible:  
• Acute leukemia, primary refractory or beyond CR1, or minimal 
residual disease (MRD) positivity. 
• Chronic myelogenous leukemia (accelerated, blast or second 
chronic phase, or first chronic phase that and has not achieved a 
molecular remission after ≥ 3 prior therapies 
• High-risk leukemia with any of the following features: 
o Acute myeloid leukemia (AML) in CR1 as follows: 
 Complex karyotype (≥ 3 clonal chromosomal abnormalities) 
 Any of the following high-risk chromosomal abnormalities:  
• Monosomal karyotype (-5, 5q-, -7, 7q-) 
• t(11q23); t(9;11);  inv(3);  t(3;3) t(6;9);  t(9;22) 
• Normal karyotype with FLT3-ITD mutation 
 Other high-risk features as determined by molecular studies, 
or clinical presentation as assessed by a study investigator 
o Other high-risk leukemia in CR1, as determined by a 
study investigator 
• Myelodysplastic syndromes 
• Myeloproliferative disorders 
• Non-Hodgkin lymphoma with poor risk features not suitable for 
autologous HCT 
• Hodgkin lymphoma with poor risk features not suitable for 
autologous HCT 
• Age ≤ 73 years old 
• Availability of an HLA-matched donor or 9/10 HLA-matched 
donor (related or unrelated) defined by Class I (HLA–A and -B) 
serologic typing (or higher resolution) and Class II (HLA-DRB1) 
molecular typing.  
• No prior myeloablative therapy or hematopoietic cell 
transplantation; prior allo transplants are excluded  

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SAMPLE SIZE  
Up to 88 subjects, consisting of: 
Phase 1 trial:  3 subjects per dose pair of regulatory T-cells and 
conventional T-cells (3 dose pairs to be studied).  Total of 
13 subjects. 
Phase 2 trial:  adaptive Simon 2-stage design. 
Initial stage cohort:  24 subjects 
Second stage cohort:  up to 51 subjects 
STATISTICAL 
CONSIDERATIONS 
This is a phase 1-2 dose finding trial, with 2 degrees of freedom in 
the definition of dose (numbers of Treg and Tcon, or, equivalently, the 
number of Tcon and the ratio of Treg to Tcon).  The study will 
nevertheless follow a traditional 3+3 model, as described in the 
diagram, to find the maximum tolerated dose (MTD) of Tcon at the 
minimum necessary ratio of Treg to Tcon. 
The Phase 2 portion of the trial will consist of an admissible Simon 
2-stage design (Simon, 1989). 
The 1st phase is designed to evaluate the need for 
immunosuppression (IS) by comparing IS + graft-engineered product 
to graft-engineered product alone.  In the first stage, the trial 
investigates if the rate of GVHD is non-inferior for the 
graft-engineered product with and without IS.  Define θ to be the 
difference in GVHD rates between the two arms:  (1) – (2), so that 
large θ is promising for the graft-engineered product (2).  To test for 
non-inferiority, we want to test H0:θ = -ϐ vs Hα:θ = 0, where ϐ is a 
specified margin of non-inferiority.  With 24 evaluable subjects this 
method has 80% power to test for a ϐ = 10% margin of 
non-inferiority at α = 0.5 (assuming a variance of 0.35).  
The 2nd phase will either use IS + graft-engineered product or 
graft-engineered product alone based on the first stage and evaluate 
1-year GRFS as the primary endpoint. The null hypothesis that the 
true 1-year GRFS is 40% will be tested against a one-sided 
alternative. The probability of early termination under the null 
hypothesis is 64%. In the first stage, 17 patients will be 
accrued. If there are 7 or fewer successes in these 17 patients, 
the study will be stopped. Otherwise, 24 additional patients will 
be accrued for a total of 41. The null hypothesis will be rejected 
if 22 or more responses are observed in 41 patients. This 
design yields a type I error rate of 5% and power of 80% when 
the true 1-year GRFS is 60%.  
 
 

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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
ADL 
Activities of daily living 
AE 
Adverse event 
BID 
Twice daily 
BSA 
Body surface area 
CBC 
Complete blood count 
CI 
Confidence interval 
CNS 
Central nervous system 
CRF 
Case report/record form 
CR 
Complete response 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTF 
Cell Therapy Facility  
DLT 
Dose-limiting toxicity 
DSMB 
Data safety monitoring board 
ECG 
Electrocardiogram 
EFS 
Event free survival 
GI 
Gastrointestinal 
GvHD 
Graft vs host disease 
GRFS 
GvHD-free, relapse-free survival 
HCT 
Hematopoietic cell transplantation 
Hgb 
Hemoglobin 
HIV 
Human immunodeficiency virus 
HPF 
High-power field  
HTN 
Hypertension 
IRB 
Institutional review board 
IV 
Intravenous 
LLN 
Lower limit of normal  
MA 
Myeloablative 
MLR 
Mixed lymphocyte reaction 
OS 
Overall survival  
PLT 
Platelet 
PD 
Progressive disease 
PFS 
Progression-free survival  
PR 
Partial response 
QD 
Once daily 
RR 
Response rate 
SAE 
Serious adverse event 
SD 
Stable disease 
Tcon 
Conventional T-cells 
Treg 
Regulatory T-cells 
TTP 
Time to progression 
ULN 
Upper limit of normal 
UNK 
Unknown 
URD 
Unrelated donor  
WBC 
White blood cell 
WHO 
World Health Organization 
 
 

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Cohort 1A 
Treg: 1x106/kg 
Tcon: 1x106/kg 
Min. 3 patients 
Add 3 patients 
at cohort dose 
No DLT 
Cohort 3  
Treg: 3x106/kg 
Tcon: 3x107/kg 
Min. 3 patients 
• Complete Phase I accrual with additional 12 recipients at MTD 
• Initiate Phase II accrual of 27 additional recipients at MTD  
Halt trial as per 
Stopping Rules 
DLT 
in any patient 
No DLT 
DLT 
in any patient 
Add 3 patients 
at cohort dose 
DLT 
in any patient 
No DLT 
Cohort 2 
Treg: 3x106/kg  
Tcon: 1x107/kg 
Min. 3 patients 
Cohort 2A 
Treg: 3x106/kg 
Tcon: 3x106/kg 
Min. 3 patients 
DLT 
in any patient 
No DLT 
No DLT 
Add 3 patients 
at cohort dose 
DLT 
in any patient 
No DLT 
No DLT 
DLT 
in any patient 
Cohort 1   
Treg: 1x106/kg 
Tcon: 3x106/kg 
3 patients 
Add 3 patients 
at cohort dose 
DLT 
in any patient 
DLT 
in any patient 
DLT 
in any patient 
No DLT 
Cohort 3A 
Treg: 1x106/kg 
Tcon: 1x107/kg 
Min. 3 patients 
No DLT 
DLT 
in any patient 
Add 3 patients 
at cohort dose 
Add 3 patients 
at cohort dose 
No DLT 
DLT 
in any patient 
No DLT 
No DLT 
DLT 
in any patient 
Dose Limiting Toxicity (DLT): 
- 
Acute GvHD > grade 2 
- 
Grade 4 neutropenia lasting to 28 days after HCT 
- 
Grade 3 to 5 cytokine/release syndrome/acute infusion reaction  
Figure 1.  Phase 1 Dose Escalation Schema of Regulatory 
T-cells (Treg) Conventional T-cells (Tcon) 

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1 
OBJECTIVES 
Primary Objectives 
 
1. To determine the efficacy, safety, and feasibility of administration of several dose 
combinations of conventional T-cells (Tcon) and regulatory T-cells (Treg) in subjects 
undergoing allogeneic hematopoietic cell transplantation (HCT) with HLA-matched 
donor or 9/10 HLA-matched donor (related or unrelated), using a T-cell depleted 
graft [CD34+ hematopoietic progenitor cells (“CD34+ HSPC”)], without 
immunosuppression (see Section 3.1 Recipient Inclusion Criteria for the 
protocol-specified definition of matched donor). 
2. To determine the maximum tolerated dose of infused Treg and Tcon cells with 
myeloablative allogeneic HCT using matching donors 
3. Determine if concomitant single-agent immunosuppression is needed with fresh 
Treg cells (phase 2 stage 1) 
4. To determine 1-year GvHD-free relapse-free survival (GRFS) post-HCT (phase 2 
stage 2). 
Secondary Objectives 
 
1. To determine the 1-year overall survival in subjects undergoing allogeneic 
hematopoietic cell transplantation with matched donors. 
2. To measure the incidence and severity chronic GvHD. 
3. To measure incidence of serious infections 
Exploratory Objective 
1. To measure immune reconstitution parameters. 
ClinicalTrials.gov outcomes are specified in Section 9.1. 
2. 
BACKGROUND 
The procedures for this study include the infusion of 3 phenotypically-distinct cell types 
obtained from the apheresis harvests.  The cellular infusions may be referred to herein as 
treatment components.  The treatment components used in Study IRB-21257 / BMT236 
are described as:  
• 
Conventional, unmanipulated, CD3+ T-cells (“Tcon cells” or “Tcon”).  These cells an 
unmanipulated aliquot of the apheresis harvests; are used in regular medical care; and 

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are not considered the investigational agent.  The Tcon cells will be administered to 
the patient-subject on Study Day 2. 
• 
Purified CD34+ hematopoietic progenitor cells (“CD34+ HSPC”).  The CD34+ HSPC 
will be administered to the patient-subject on Study Day 0. 
• 
Highly-purified regulatory T-cells (“Treg cells” or Treg) are defined as 
CD4+ CD25+ CD127- Foxp3+ regulatory T-cells.  The Treg cells will be administered 
to the patient-subject on Study Day 0. 
2.1 Background/Rationale/Pilot Data 
Background/Rationale/Pilot Data 
Patients with high risk hematologic malignancies such as leukemia and lymphoma 
beyond first remission or with refractory relapse are rarely cured with standard 
chemotherapy.  Myeloablative (MA) allogeneic HCT is their only chance for long term 
survival with disease free survival ranging from 10 to 50% and non-relapse mortality 
ranging from 30 to 50%.1,2  The early morbidity and mortality associated with aGvHD is 
a major factor limiting the success of HCT as is the long-term morbidity associated with 
chronic GvHD.  The incidence of aGvHD following allogeneic HCT from an 
HLA-matched sibling donor (MSD) is 20 to 60% despite the use of various 
immunosuppressive agents such as tacrolimus, cyclosporine, methotrexate, 
mycophenolate, anti-thymocyte globulin and corticosteroids.2,3,4,5  Accordingly, 
approximately one-third of patients who undergo allogeneic HCT using a MSD and a 
T-cell replete graft will develop chronic GvHD.6,7,8  
T-cell Depletion of Allografts: 
T-cell depletion (TCD) of donor grafts is an accepted method to prevent acute and 
chronic GvHD.  There is extensive experience with TCD of marrow grafts and such 
techniques have involved physical adsorption of T-cells to protein ligands such as lectins, 
elutriation and immuonadsorption or immunodeletion with T-cell or lymphocyte-specific 
monoclonal antibodies.  These methods have yielded results in marrow grafts ranging 
from 1.5 to 3.2log10 T-cell depletion.  Much less is published regarding TCD of PBSC 
allografts.  However, in light of the evidence that the risk of acute and/or chronic GvHD 
may be greater with PBSC grafts, strategies for TCD of PBSC allografts is of 
considerable interest.  Using an immunoadsorption column or immunoaffinity 
techniques, investigators have performed TCD of GCSF-mobilized PBSC grafts and 
obtained T-cell log depletions ranging from 1 to 2.9 log depletion and with the incidence 
of grade 2 to 4 aGvHD ranging from 0 to 4%.  When G-CSF mobilized grafts from 
haploidentical donors were utilized, an immunoaffinity system (CliniMACS) achieved a 
4 to 5 log CD3+ depletion and a median CD3+ dose of 0.2 x 105/kg.  The incidence of 
aGvHD was < 10% despite the use of haploidentical grafts and without use of any 
post-transplant GvHD prophylaxis.9-14  
Consequences of TCD include impaired immune recovery and increased incidence of 
relapse.  Persistent immune dysregulation is a known complication of allogeneic HCT 

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and can manifest as opportunistic infections especially in the setting of chronic GvHD.  
Peripheral blood T-lymphocyte counts do not return to normal levels until ~3 to 12 
months after HCT and functional T-cell recovery may be even more protracted especially 
after TCD HCT.9,13,15  Additionally, when marrow grafts are TCD in the MSD setting, 
the risk of leukemia relapse is significantly higher especially for advanced phase acute 
leukemia patients.15  
2.2 Purified Regulatory T-cells  
 To ameliorate the impaired immune recovery and address the increased relapse incidence 
after TCD allografting, we will infuse conventional donor T-cells (Tcon) or “T-cell 
addback” along with naturally occurring donor regulatory T-cells (Treg).  This allows 
assessment of the ability of Treg to reduce the extent and severity of GvHD and to 
ascertain the maximum tolerable dose of donor Tcon in relation to specific Treg doses.  To 
circumvent the potential acute GvHD associated with T-cell addback, the planned 
addition of Treg at appropriate numbers and ratios compared to Tcon will occur at a 
defined timepoint after hematopoietic cell infusion.  The scientific rationale for pursuing 
Treg in this setting arises from reports from several different laboratories demonstrating 
that highly purified Treg are capable of controlling GvHD and improving immune 
reconstitution in the preclinical setting.16,17,18,19  In a murine GvHD model induced by 
Tcon, we have previously demonstrated that adoptive transfer of Treg leads to abrogation 
of GvHD and accelerated donor lymphoid reconstitution of a diverse TCR-Vβ 
repertoire.20  Treg will be obtained by selection of donor T-cells expressing the surface 
phenotype CD4+CD25+CD127-.  This population has been shown to express high levels 
of intracellular FoxP3+ which is currently the most reliable marker of Treg activity based 
on in vitro assays and non-human animal models.  These cells also actively suppress the 
mixed lymphocyte reaction.  Preliminary studies in the setting of cord blood 
transplantation and haploidentical transplantation are highly supportive of this 
concept.21-22  
Dose Escalation  
A baseline cell dose of conventional T-cells of 1 x 106/kg will be used with escalation to 
the maximum tolerated dose up to 1 x 107/kg.  As demonstrated in mouse models of 
allogeneic HCT and in vitro assays, Treg can effectively suppress proliferation of 
alloreactive T-cells even when Tcon are added at 10-fold greater doses if the Treg are 
infused prior to the Tcon which allows for these cells to home to the proper 
microenvironment and proliferate.  The suppressive effect is most pronounced when Treg 
are added prior to Tcon.  Mice infused with donor Treg 48 hours before Tcon infusion show 
little, if any, evidence of GvHD despite significant MHC disparities.  This finding is 
replicated in the proposed transplant regimen.  In addition, the ratio of Tcon:Treg will be 
staggered to establish and confirm the ability of Treg to suppress alloreactivity at doses 
lower than the infused Tcon. 
In order to measure the impact of our intervention, at specified timepoints, before and 
after infusion of the planned dose of Treg and Tcon, we will measure immune 
reconstitution in the peripheral blood via immunophenotyping of B-cell;  T-cell;  and 
NK subsets.  To assess the contribution of thymopoiesis to T-cell immune recovery after 
HCT, we will measure peripheral recent thymic emigrants by determining the number of 

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T-cell receptor excision circles generated during intrathymic TCR-α rearrangement using 
real-time PCR assays from the peripheral blood.  
Phase 2 Simon 2-Stage  
Based on the prior protocol modifications and the use of fresh T-regulatory cells with 
single-agent immunosuppression prophylaxis, a safe and feasible dose has been established 
for further research.  Since 2 modifications were made during dose-finding and the initial 
phase 2 extension of the use of fresh donor Treg and also the use of single-agent GvHD 
prophylaxis with either tacrolimus or sirolimus, this aspect of the phase 2 extension study 
has a Simon Two-Stage design, with the 1st stage designed to test if the incidence of GVHD 
is different between subjects who receive Treg alone versus those who received single-agent 
prophylaxis and Treg.  A total of 24 subjects will be required (2 arms of 12 each).  For the 
2nd stage, the better strategy will be used in the phase 2 stage 2 extension:  Treg alone vs Treg 
plus single-agent prophylaxis.  In the event of non-inferiority of the Treg alone arm, stage 2 
will use this treatment strategy.  A further 51 subjects would be enrolled in the Phase 2 
Stage 2 extension, enough to determine if there is a significant increase in GFRS (60%) 
from historic rates (40%). 
Overview 
After obtaining the MTD is obtained from the phase 1 trial, we propose conducting a 
Simon 2-Stage phase 2 trial in which the 1st stage will determine if immunosuppression 
with graft product is required and the 2nd stage will determine the 1-year GRFS compared 
against historical control patients who underwent the same conditioning regimens but 
received a T-cell replete graft.  This protocol will be eligible to patients with high-risk 
hematologic malignancies, that is, patients beyond CR1, CR1 with high-risk features or 
with refractory disease who would be otherwise eligible for MA allogeneic HCT. 
3. 
PARTICIPANT SELECTION AND ENROLLMENT PROCEDURES 
All eligibility criteria of transplant recipients are to be satisfied within 30 days of 
initiation of the recipient’s conditioning regimen.  
All eligibility criteria of transplant donors are to be satisfied within 40 days of the donor 
leukapheresis procedures.  
For the purposes of this study, “registration” will be considered to be signing of the 
informed consent.  Enrollment will be considered to be confirmation of eligibility per the 
following criteria.  Initiation of study treatment will be the initiation of the conditioning 
regimen (infusion recipients) or mobilizing agents (cellular donors).  

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3.1 
Recipient Inclusion Criteria 
3.1.1  Patients with the following diseases that are histopathologically-confirmed are 
eligible 
 
Acute leukemia, primary refractory or beyond CR1, or minimal residual 
disease (MRD) positivity 
 
High-risk leukemia with any of the following features: 
• 
Acute myeloid leukemia (AML) in CR1 as follows: 
o Complex karyotype (≥ 3 clonal chromosomal abnormalities) 
o Any of the following high-risk chromosomal abnormalities:  
 
Monosomal karyotype (-5; 5q-; -7; 7q-) 
 
t(11q23); t(9;11);  inv(3);  t(3;3);  t(6;9);  t(9;22) 
 
Normal karyotype with FLT3-ITD mutation 
o Other high-risk features as determined by molecular studies, or 
clinical presentation as assessed by a study investigator 
• 
Other high-risk leukemia in CR1, as determined by a study investigator 
 
Chronic myelogenous leukemia (accelerated, blast or second chronic phase 
or first chronic phase that and has not achieved a molecular remission after 
three or greater therapies)  
 
Myelodysplastic syndromes  
 
Myeloproliferative syndromes  
 
Non-Hodgkin lymphoma with poor risk features not suitable for autologous 
HCT  
 
Hodgkin lymphoma with poor risk features not suitable for autologous HCT 
3.1.2  Age ≥ 18-years old, and for subjects in Cohort 1 only, ≤ 73 years of age  
At the start of Cohort 2A and beyond, eligibility will be expanded  
to allow pediatric subjects age ≥ 13 years old. 
3.1.3  Left ventricular ejection fraction (LVEF) ≥ 45% 
3.1.4  Diffusing capacity of the lungs for carbon monoxide (DLCO) ≥ 50% 
3.1.5  Calculated creatinine clearance ≥ 50 mL/min or creatinine < 2.0 mg/dL 
3.1.6  SGPT and SGOT ≤ 7.5 x ULN 
3.1.7  Total bilirubin ≤ 3 x ULN (patients with Gilbert’s syndrome may be included at 
the discretion of the PI or where hemolysis has been excluded 
3.1.8  Negative serum or urine beta-HCG test in females of childbearing potential  
3.1.9  Availability of a 10/10 or 9/10 HLA-matched donor (related or unrelated) 
defined by Class I (HLA–A and -B) serologic typing (or higher resolution) and 
Class II (HLA-DRB1) molecular typing.  If the donor is a 9/10 HLA-match, the 
mismatch may be a permissive or non-permissive allelic mismatch as assessed by 
an independent HLA and transplantation expert. 
3.1.10 Karnofsky performance status ≥ 70% 

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3.2 
Recipient Exclusion Criteria 
3.2.1  Seropositive for any of the following:  
HIV antibodies; hepatitis B surface antigen (sAg); hepatitis C antibodies 
3.2.2  Prior myeloablative therapy or hematopoietic cell transplant 
3.2.3  Candidate for autologous transplant 
3.2.4  HIV-positive 
3.2.5 
Active uncontrolled bacterial, viral or fungal infection, defined as currently 
taking antimicrobial therapy and progression of clinical symptoms. 
3.2.6 
Uncontrolled CNS disease involvement 
3.2.7 
Pregnant or a lactating female 
3.2.8 
Positive serum or urine beta-HCG test in females of childbearing potential  
3.2.9 
Psychosocial circumstances that preclude the patient being able to go through 
transplant or participate responsibly in follow-up care 
3.2.10   Recipients of prior allogeneic transplants 
3.3 
Donor Inclusion Criteria 
 
3.3.1 
Age ≥ 13 and ≤ 75 years of age  
3.3.2 
Karnofsky performance status of ≥ 70% defined by institutional standards or 
cleared by the NMDP for NMDP donors 
3.3.3 
Seronegative for HIV-1 RNA PCR;  HIV 1 and HIV 2 ab (antibody);  HTLV-1 
and HTLV-2 ab;  PCR+ or sAg (surface antigen) hepatitis B; or PCR or sAg 
negative for hepatitis C;  negative for the Treponema palladum antibody 
Syphillis screen;  and negative for HIV-1 and hepatitis C by nucleic acid 
testing (NAT) within 40 days of donor apheresis procedures.  
In the case that T palladum antibody tests are positive, donors must: 
• 
Be evaluated and show no evidence of syphilis infection of any stage by 
physical exam and history 
• 
Have completed effective antibiotic therapy to treat syphilis 
• 
Have a documented negative non-treponemal test (such as RPR) or in the 
case of a positive non-treponemal test must be evaluated by an infectious 
disease expert to evaluate for alternative causes of test positivity and confirm 
no evidence of active syphilitic disease 
3.3.4 
Must be a related or unrelated, HLA match or 9/10 HLA match to recipient. 
3.3.5 
Female donors of child-bearing potential must have a negative serum or urine 
beta HCG test  
3.3.6 
Capable of undergoing leukapheresis, have adequate venous access, and be 
willing to undergo insertion of a central catheter should leukapheresis via 
peripheral vein be inadequate 
3.3.7 
Agreeable to 2nd donation of PBPC (or bone marrow harvest) in the event of graft 
failure 

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3.3.8 
The donor or legal guardian greater than 18 years of age, capable of signing an 
IRB approved consent form. 
3.3.9 
Meet criteria for donation as specified by standard NMDP guidelines for NMDP 
unrelated donors. 
3.4 
Donor Exclusion Criteria 
3.4.1 
Evidence of active infection  
3.4.2 
HIV-positive 
3.4.3 
Medical, physical, or psychological reason that would place the donor at 
increased risk for complications from growth factor or leukapheresis 
3.4.4 
Lactating female  
3.5 
Informed Consent Process 
The BMT attending physician discusses the rationale, logistics, risks, and benefits of the 
study with the subject.  Alternative therapies are discussed.  The subject's bill of rights is 
reviewed.  The subject is given a copy of the consent form to take home and read.  Then a 
meeting is scheduled with a clinical nurse specialist or research nurse to review the rationale, 
logistics, treatment plan, risks, benefits and consent form.  The subject's bill of rights is 
reviewed with an emphasis placed on voluntary participation.  Subjects are given time to ask 
questions and do not need to sign the consent form until they are ready to proceed.  In 
general, there is one hour devoted to the consent discussion with a clinical nurse specialist or 
research nurse educator.  The BMT attending physician is available to discuss the study with 
the subject at any time.  For pediatric subjects, all efforts will be made to have both parents 
sign.  If one parent is unable to sign, the reason will be documented. 
During the consent teaching, the physician, clinical nurse specialist or research nurse 
frequently asks questions of the subject to assess their understanding and a consent note is 
written documenting the subjects understanding.  For subjects that are not fluent in English, 
the consent procedure is performed with an interpreter present (or via telephone), or a signer 
for those that are hearing impaired.  Consents are translated into Spanish as needed. 
Subjects are evaluated by a physician, nurse coordinator, or clinical nurse specialist and 
social worker.  Any member of the team can request a psychiatric evaluation if a concern of 
competency to provide consent is raised. 
4. 
TREATMENT PLAN 
4.1 Phase 2 Stage 1 treatment allocation  
Between Day -10 and Day -1, incoming subjects in Phase 2 Stage 1 will be assigned to 
GvHD prophylaxis or not.  An unblinded study coordinator will ascertain the consecutive 
number of the subject and the arm assignment and convey this to the investigator or study 
representative.  
4.2 Mobilization Therapy 
Following screening and enrollment, the donor will receive mobilization therapy per 
institutional guideline treatment with daily G-CSF (filgrastim or equivalent).  Recommended 

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dose is 16 µg/kg/day SQ (rounded off to the nearest vial size of either 300 or 480 µg).  The 
Mobilization Phase starts on the first day of administration of G-CSF and continues until the 
final day of leukapheresis. 
4.3 Cell Collection 
All cellular components will be obtained from matched donors mobilized with G-CSF 
[filgrastim (Neupogen) or equivalent] and collected by apheresis at Stanford Hospital and 
Clinics (SHC) or certified National Marrow Donor Program (NMDP) collection centers as 
per current institutional allogeneic transplant protocols. 
Leukapheresis will be performed on a continuous flow cell separator according to 
institutional standards and will start on Day +4 of G-CSF administration.  Leukapheresis of 
the donor will start on Day -3 of infusion with a target of ≥ 3 x 106 CD34+ cells/kg recipient 
body weight post-selection and a maximum dose of 10 x 106 CD34+cells/kg.  In order to 
have sufficient cells available for CD34 enrichment and Treg sorting procedures, donors will 
undergo apheresis collections on 2 or more consecutive days;  additional apheresis may be 
needed to ensure a sufficient T-cell count for above stated reasons;  an additional mobilizing 
agent such as plerixafor (Mozobil) may be used under these circumstances at the discretion of 
the attending physician.  Collections will occur on Day -3; Day -2; and potentially Day -1 of 
the CD34-enriched and Treg product infusions (Day 0).  The 1st day’s apheresis collection will 
be scheduled for afternoon hours and the 2nd day’s for early morning, thereby limiting the 
time from the end of the first collection to the infusion of the cellular products to less than 
72 hours.  
At the conclusion of each collection at Stanford, cellular products will be transferred to the 
cell processing facility.  For collection occurring outside of Stanford, products can be 
transferred on the same day, with the previous days product stored at 4ºC or according to 
institutional practice but not frozen.   
The first day’s collection (Day -3) will be kept under refrigeration as a quarantined product in 
a continuously monitored, secure refrigerator in accordance with apheresis collection storage 
standards.  Prior to being placed in storage, samples for cell count, flow cytometric analysis, 
and collection sterility testing will be aseptically removed by the cell processing facility staff 
and submitted to the designated group within the SHC Clinical Laboratory for assessment.  
The 2nd and 3rd days’ collections (Day -2, Day -1) will be transferred to the cell processing 
facility upon completion of apheresis.  The product will be sampled in the same manner for 
assessment but retained at room temperature for cell processing.  
After collection, the cellular harvests will be transferred via validated transport methods to 
Orca BioSystems, Inc, facility at 3400 Business Dr (Suite 140) in Sacramento, CA (95820).  
All cellular processing subsequent to December 2019 will occur at this location pursuant to 
Orca BioSystems IND 018542, to which the IND for this protocol is cross-referenced.  
4.4 Cell Processing 
The donor leukapheresis collections will be the source of the 3 infusions products: 
conventional T-cells (Tcon), regulatory T-cells (Treg), and CD34+ hematopoietic 
progenitor/stem cells.  CD34+ cell enrichment will be performed according to procedures 
given in the CliniMACS Users Operating Manual and institution SOPs.  Collections from 
Day -3 and Day -2 will be pooled for CD34 enrichment as described in the SOP “Selection of 
CD34+ Cells Using the CliniMACS System.”  Upon completion of the cell selection process, 
samples of the CD34-enriched product will be aseptically removed for release testing to 

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assess the cellular content, the cell doses and viability, sterility by Gram staining, and 
endotoxin content (table below), along with additional quality assessments including standard 
14-day sterility cultures.  
CD34+ HSPC release criteria 
Final Release Tests and Specifications for CD34+ HSPC Final Formulated Drug Product  
CD34 Post-selection 
Treatment 
Component Release 
Testing 
Assay Type 
Method 
Specification 
Purity 
Viability 
Dye exclusion  
(7AAD or Trypan Blue)  
≥ 70% * 
Purity / Identity 
CD34+ cells 
Flow Cytometry 
≥ 50% CD34+ cells* 
Potency 
CD3+ cell number 
Flow Cytometry 
≤ 5 x 104 cells/kg FIO 
CD34+ cell 
number  
Flow Cytometry 
3 ± 1 x 106 cells/kg 
FIO 
Safety 
Sterility 
Gram-stain 
Negative * 
Safety 
Sterility 
Bactec Method 
Negative / 
no growth observed 
Safety 
Endotoxin 
Endosafe-PTS Assay 
or Limulus amoebocyte 
assay (LAL) 
5 EU/kg/hour * 
*  Release specification for CD34+ HSPC Drug Product  
FIO:  For information only. 
After cell selection and sampling, the CD34-enriched products will be securely stored at 
2 to 8°C in a continuously monitored refrigerator until released for infusion.  Product labeling 
will be compliant with ISBT 128 specifications.  

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Treg release criteria 
Release Tests and Specifications for Treg Cells Final Formulated Drug Product 
Treg Cells Drug 
Product Release 
Testing 
Assay Type 
Method 
Specification 
Identity / Potency 
FoxP3+ Cells 
Flow Cytometry 
≥ 80% * 
Potency 
Cell count 
Automated Cell Count 
To meet cohort 
dose* 
(3 ± 1 x 106 cells/kg  
for Cohort 2A)  
Purity 
Viability 
Dye exclusion  
(7AAD or Trypan Blue)  
≥ 70% * 
Safety 
Sterility 
Gram Stain 
Negative * 
Safety 
Sterility 
14-day sterility 
Negative  
Safety 
Endotoxin 
Endosafe-PTS Assay 
or LAL 
5 EU/kg/hour * 
*  Release specification for Treg Drug Product  
After processing by Orca BioSystems, Inc, the cellular components (drug products), will be 
transferred via validated transport methods to BMT-CTF, which is the “cellular pharmacy” 
for the Stanford University Medical Center.  The BMT-CTF will dispense the cellular 
components to the respective subjects.  
4.5 Infusion of CD34+ Cells and Treg cells 
The selected CD34+ cells will be infused fresh into the subject according to institutional 
guidelines.  The day that the subject receives the donor CD34+ peripheral blood stem cells 
(PBSC) will be defined as Subject Day 0. Infusion of cell products to occur prior to cell 
expiration which will be determined by the cell lab. 
Treg cells meeting the release criteria will be infused on Day 0 after CD34+ cell infusion.  The 
cells will be infused through a central venous catheter or a peripheral IV of at least 18 gauge.  
Treg cell infusion will take place over 5 minutes or less.  It is anticipated that the volume of 
the cellular products will range from 5 mL to 45 mL. 
After Treg infusion, subjects will be monitored for vital signs every 30 minutes including 
pulse oximetry following infusion for at least 4 hours with evaluation every 30 minutes.  
Recipients will be monitored for infusional toxicity and treated as needed.  Benadryl and 
hydrocortisone will be utilized should a reaction occur and epinephrine will be available at 
the bedside. 

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4.6 Conditioning Regimen 
The conditioning regimen that will be used will be determined by subject diagnoses.  
Regimens will be assigned as follows: 
ACUTE LEUKEMIA, CHRONIC MYELOGENOUS LEUKEMIA (HIGH RISK), 
MYELODYSPLASTIC SYNDROME AND MYELOPROLIFERATIVE DISORDERS will be 
offered 1 of the 3 following regimens (TBI-based or chemotherapy-based):  
Day -8 Day -7 Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 
Day 0 
Day +1 Day +2 
fTBI 
fTBI 
fTBI 
fTBI 
VP16 
rest 
Cy 
Rest* 
CD34+ HSPC and 
Treg infusion 
rest 
Tcon 
infusion 
Doses:   Fractionated total body irradiation (fTBI):  1320 cGy  
 
 
VP16:  60 mg/kg x 1 dose 
 
 
Cyclophosphamide(Cy):  60 mg/kg x 1 dose 
OR 
Day -7 Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 
Day 0 
Day +1 
Day +2 
Bu 
Bu 
Bu 
Bu 
Cy 
Cy 
Rest* 
CD34+ HSPC and 
Treg infusion 
rest 
Tcon 
infusion 
 
Doses:   Busulfan:  3.6 mg/kg Q24h  
 
 
Cyclophosphamide:  60 mg/kg x 2 doses  
 
OR 
 
Day -6 Day -5 Day -4 Day -3 Day -2 Day -1 
Day 0 
Day +1 
Day +2 
Bu 
Flu 
Bu 
Flu 
Bu 
Flu 
TTP 
TTP 
Rest* 
CD34+ HSPC 
and Treg 
infusion 
Rest 
T con 
infusion 
 
Doses:  Thiotepa:        5 mg/kg x 2 doses 
             Busulfan:        3.2 mg/kg x 3 doses 
 
Fludarabine:  50 mg/m2 x 3 doses 
 
* Two days of rest are allowed at the discretion of the treating physician. 
 
 
 
NON-HODGKIN LYMPHOMA, HODGKIN LYMPHOMA 
Day -6 
Day -5 
Day -4 
Day -3 
Day -2 
Day -1 
Day 0 
Day +1 
Day +2 
Carmustine 
rest 
VP16 
rest 
Cy 
Rest* 
CD34+ HSPC and 
Treg infusion 
rest 
Tcon 
infusion 
 
Doses:   
Carmustine:  
300 mg/m2  
 
VP16:   
60 mg/kg x 1 dose 
 
Cyclophosphamide:   
100 mg/kg x 1 dose 

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4.7 Graft-vs-Host Disease Prophylaxis 
For subjects who are determined to receive GvHD prophylaxis in the phase 2 stage 1 or 
phase 2 stage 2 extension, this will be administered corresponding to the specific 
conditioning regimen utilized and as follows: 
For subjects receiving fTBI, VP-16 and Cy: 
Subjects will start tacrolimus at Day +3 at an initial dose of 0.015 mg/kg/day 
intravenous infusion with a target of 4 to 8 ng/mL. 
For subjects receiving Bu and Cy: 
Subjects will start tacrolimus at Day +3 at an initial dose of 0.015 mg/kg/day 
intravenous infusion with a target of 4 to 8 ng/mL.  
For subjects receiving carmustine, VP-16 and Cy: 
Subjects will start tacrolimus at Day +3 at an initial dose of 0.015 mg/kg/day 
intravenous infusion with a target of 4 to 8 ng/mL.  
For subjects receiving Thiotepa, Fludarabine and Busulfan: 
Subjects will start tacrolimus at Day +3 at an initial dose of 0.015 mg/kg/day 
intravenous infusion with a target of 4 to 8 ng/mL.  
For subjects with a 9/10 HLA matched non-permissive donor: 
Subjects will receive two options for GVHD prophylaxis, depending on primary 
treating physician discretion: 
Cyclophosphamide at Day +5 as a single infusion dose of 50mg/kg and Tacrolimus 
at Day +6 at an initial dose of 0.015 mg/kg/day intravenous infusion with a target 
of 4 to 8 ng/mL. 
Or 
Tacrolimus at day +3 at an initial dose of 0.015 mg/kg/day intravenous infusion 
with a target of 4 to 8 ng/mL. and at day +4 CellCept (Mycophenolate mofetil) 
1,000 mg BID.  
Should a subject become intolerant of their specific GvHD prophylaxis or other reasons to 
change are determined by the treating physician then prophylaxis can be altered at the 
discretion of the treating physician, with the recommendation that tacrolimus, sirolimus, or 
mycophenolate mofetil be utilized. 
Should GvHD occur, the appropriate treatment schedule and dose will be initiated.  
Recipients who develop acute GvHD will be treated at the discretion of the treating 
physician.  
4.8 
Infusion of Treg and Tcon cells will proceed as follows:  
On Day 0:  
*  Infusion of regulatory T-cells with dose based on assigned cohort  

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On Day 2  
*  Infusion of conventional T-cells with dose based on assigned cohort. 
As shown in the Schema in Figure 1, box “Cohort 1a,” dose escalation will start with: 
• 
Treg dose = 1 x 106/kg Treg enriched by immunomagnetic selection of CD25+ cells and 
further purified by flow cytometric cell sorting of the CD4+CD127- population). 
• 
Tcon dose = 1 x 106/kg Tcon administered as unfractionated mobilized peripheral blood 
cells containing specified CD3+ cell dose 
4.9 
Cell Source 
All cellular components will be obtained from matched donors mobilized with G-CSF 
[filgrastim (Neupogen) or equivalent] and collected by apheresis at Stanford Hospital and 
Clinics (SHC) or certified National Marrow Donor Program (NMDP) collection centers as 
per current institutional allogeneic transplant protocols.  The clinical protocol requires 
3 cellular products for infusion as indicated in Figure 2, below: 
1) CD34+ HSPC:  The cell processing facility currently performs CD34 cell 
immunomagnetic selection using the CliniMACS (Miltenyi Biotec) system.   
The CD34+ HSPC will be infused on Day 0.  
2) Treg:  The selection of Treg will include a tandem selection procedure utilizing 
immunomagnetic selection on the CliniMACS Cell Selection System (Miltenyi Biotec) 
followed by flow cytometric cell sorting on an Influx high-speed cell sorter.  Treg will be 
enriched by immunomagnetic selection of the CD25+ population and highly-purified to 
≅ 90% FoxP3+ cells by cell sorting of the CD25+ cell fraction gated for selection of the 
CD4+CD127- population.  In our experience, cells recovered through this process are 
typically > 90% FoxP3+ (range 86% to 99%) with a mean of 95%, FoxP3+ cells in sorted 
Treg performed to date.  Treg isolation will use the flow-through fraction from the CD34 
selection as the cell source as depicted in Figure 2.   
The Treg product will be infused on Day 0 after the CD34+ are infused.  There will be 
a minimum of a 1-hour interval between the end of the CD34+ product infusion and the 
start of the Treg infusion.  
3) Tcon.  This designation is used to indicate the infusion product containing the 
conventional T-cell dose.  The Tcon product will be obtained from the Day 2 or Day 3 
leukapheresis product prior to pooling with the Day 1 product.  Cell dose of the Tcon 
infusion product will be based on the CD3+ cell content in order to assess GvHD risk.  
The Tcon product will be cryopreserved until the planned infusion on Day +2 post-HCT.  
Flow cytometry to measure initial CD3 and CD34 percentages of leukapheresis product 
will be performed in the BMT-CTF located in Stanford Hospital.  Flow cytometry of 
selected CD34+ products is performed by the SHC Clinical Laboratory Flow Cytometry 
service.  

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4.10 
Supportive Care Guidelines 
4.10.1 Venous Access 
Recipients will have appropriate long-term central venous access placed, per institutional 
standard practice, prior to beginning the conditioning regimen. 
4.10.2 Irradiation of blood products 
All blood products, except the infused CD34+ cells, Treg and Tcon will be irradiated in 
accordance with institutional standards. 
 

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Figure 2
CD4+/CD127dim cells 
• Cell count/viability 
• Sterility 
• Flow assessment 
• CFU assay  
CliniMACS CD25 selection 
CD34 selected cells 
CD34 flow through cells 
CELL SELECTION
CliniMACS CD34 selection 
Fresh HSPC  
for infusion  
Day 0 
CD25-selected cells 
Flow cytometric cell sorting 
Cryopreservation 
PRODUCT QC
Pooled apheresis product 
• Cell count/viability 
• Sterility 
• Flow assessment 
• Cell count/viability 
• Sterility 
• Flow assessment
Fresh Treg  
for infusion  
Day 0 
• Cell count/viability 
• Sterility 
• Flow assessment 
• CFU assay 
• Endotoxin assay 
• FoxP3 assay 
Tcon  
for infusion  
Day +2 
Apheresis collection 
Day -1 
Apheresis collection 
Day -2 

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4.10.3 Anti-bacterial Prophylaxis 
A quinolone or appropriate alternative if quinolone-allergic will be given according to 
institutional guidelines. 
Pneumocystis pneumonia prophylaxis will be provided for adults and children according 
to institutional guidelines.  
4.10.4 Anti-viral Prophylaxis 
EBV surveillance via quantitative PCR on recipient serum will be performed every 
2 weeks starting at Day +14 until Day +100, then accordance with institutional guidelines 
with active treatment initiated upon documented evidence of infection.  Treatment with 
rituximab shall be in accordance with institutional guidelines and will be initiated upon 
documented evidence of infection. 
4.10.5 Anti-fungal Prophylaxis 
Fungal prophylaxis will be provided for adults and children according to institutional 
guidelines.  
4.10.6 Antibiotic treatment for culture positive results from graft products. 
If bacterial culture results from graft products become positive, recipients will be initiated 
on appropriate treatment-dose antibiotic unless the culture positivity is determined to be a 
false positive due to contamination.  Subjects who have bacterial culture positivity from 
their donor products will not be removed from the trial or censored from analysis.  
4.11 
Management of Graft Failure   
Primary graft failure is defined as the failure to achieve an absolute neutrophil count of 
> 500 cells/µL after Day +30.  Secondary graft failure is defined as a sustained loss of 
hematopoiesis after engraftment has occurred.  Subjects experiencing primary and/or 
secondary graft failure will be removed from the study and treated according to 
institutional guidelines at the investigator's discretion. 
4.12 
Management of Post-Transplant Relapse 
Subjects who relapse with their disease after transplant will be removed from the study 
and will be treated according to institutional guidelines. 
4.13 
Criteria For Removal From Study 
Subjects may be removed from study if any one of the following events occur: 
• 
Significant protocol violation or noncompliance, either on the part of the subject or 
investigator. 
• 
Graft failure 
• 
Refusal of the subject to continue treatment and/or observations. 
• 
Decision by the Investigator, or subject's physician that removal from the study is in 
the subject's best medical interest. 
• 
Unrelated medical illness or complication. 

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• 
Lost to follow-up. 
• 
Disease progression or a requirement for alternative therapy.  
4.14 
Duration of Therapy 
The active duration of therapy is during the conditioning regimen and up to Day +100 
after transplantation and follow up for up two years after transplantation. 
4.15 
Duration of Follow Up 
Afterwards, the subject will be followed per treating physician until death even in the 
event of graft failure or disease progression. 
 
4.16 
Alternatives 
After discharge from the hospital after the transplant procedure, the subject will be 
required to live in the local area up to at least day +100 after transplant so that the subject 
can be monitored closely for signs of infection, graft vs host disease or other 
complications.  If the subject chooses not to enroll in this protocol, the subject will be 
offered conventional chemotherapy and/or palliative care.  
4.17 
Compensation 
Subjects will not receive any compensation for participation in this study.   
4.18 
Evaluations During the Study 
DONOR Evaluation/Monitoring  
NOTE:  The following laboratory tests/evaluations will be performed for all donors 
registered in the study within 40 days prior to donor leukapheresis.  
SEE TABLE 1 FOR DONOR EVALUATION SCHEDULE  
4.18.1 Baseline 
4.18.1.1 
Complete medical history and physical examination, and review of systems 
4.18.1.2  
Prothrombin time and partial thromboplastin time 
4.18.1.3 
Serum chemistries including electrolytes, glucose, ALT, AST, creatinine, 
bilirubin, alkaline phosphatase, albumin 
4.18.1.4  
Serum or urine Beta HCG for female donors 
4.18.1.5  
Serologies for:  HIV including HIV 1 and 2 antibodies, HTLV I/II Ab; 
Hepatitis B including HBsAg, HBcAb; Hepatitis C ab; RPR for syphilis; 
Cytomegalovirus (CMV); Epstein-Barr Virus (EBV); Herpes Simplex Virus 
(HSV); Varicella zoster Virus  
4.18.1.6  
ABO/Rh typing and HLA typing: HLA-A,B,C loci (typed by serology) and 
HLA-DRB1, DQ, DP loci 
4.18.1.7  
Baseline samples for chimerism studies 

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4.18.1.8 
For NMDP donors, the study team will utilize standard NMDP guidelines 
and clearance as the baseline evaluation 
4.18.1.9 
Donor Research Samples: 
a. Phenotypic analysis of T cells subsets, particularly absolute counts and 
percentage of CD4+ CD25+ CD127- Foxp3+ regulatory T-cells in donor 
peripheral blood by immunoflow cytometry. 
b. Mixed lymphocyte reaction (MLR) to show investigational drug product 
suppression potential in-vitro by mixing effector cells (donor Tregs) with 
activated responder cells (donor PBMCs). Responder cell numbers assessed 
after 72 hours by immunoflow cytometry. 
c. Immune reconstitution assessment using T-cell and B-cell receptor gene 
rearrangement test. 
Donor research samples are needed to characterize the Treg in the donor in order to understand 
how this might influence the efficacy of the clinical trial results. In graft engineering, the 
measurement of cell subsets at the time of collection is important for understanding if a sufficient 
collection can be achieved.  
 
For example, CD34+ cells are measured in order to see if there are sufficient cells for 
hematopoietic engraftment. It is critical to determine whether the count of Tregs in the donor prior 
to collection correlates to the total Treg collected and outcome. This is accounted for through flow 
cytometry (a); to determine if these Tregs are functional, accounted for by MLR (b); and need to 
know if factors in the donor graft like T-cell and B-cell diversity affect immune reconstitution 
after transplantation (c) as Tregs are known to influence this. 
4.19.2 
Graft Evaluation 
4.19.1. 
All donor apheresis collections will be assessed as follows. 
a. Cell count by validated cell counting system 
b 
Viability by popidium iodide (PI) or 7-aminoactinomycin D (7AAD) exclusion 
(flow cytometric assay) 
c. Sterility in accordance with 21CFR§610.12 using Fluid Thioglycollate medium 
and Tryptic Soy Broth 14-day cultures. 
d. Phenotypic analysis by flow cytometry for cell surface expression of CD34; 
CD3; CD19; CD25; CD127 and intracellular FoxP3 expression. 
4.19.2. 
Final Products for infusion, including CD34-selected and Treg fractions will be 
assessed prior to infusion as follows. 
a. Cell count by hemocytometer. 
b. Viability by PI, 7AAD (flow cytometric assay); or trypan blue exclusion as 
according to institutional SOPs. 
c. Sterility in accordance with 21CFR§610.12 using Bactec 14-day cultures will be 
initiated but will not be completed prior to infusion.  Gram stain will be completed 
on the CD34-enriched and sorted Treg products prior to release for infusion.  

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d. Phenotypic analysis by flow cytometry for cell surface expression of CD34; CD3; 
CD19; CD25; CD127; and intracellular FoxP3 expression. 
e. Endotoxin testing post-selection on CD34+ enriched and sorted Treg products.  
4.19.3. 
Tcon for infusion may be thawed bedside for immediate infusion or in lab for 
post-thaw assessment and/or dosing per institutional SOPs. 
a. Cell count by hemacytometer. 
b. Viability by PI, 7AAD (flow cytometric assay); or trypan blue exclusion as 
according to institutional SOPs. 
4.20 
Evaluation/Monitoring – Recipient 
NOTE:  The following laboratory tests/evaluations will be performed for all recipients 
registered in the study within 30 days prior to initiation of conditioning regimen, except as 
indicated. Additionally, as per our institutional practice, all subjects will be closely monitored 
in our out-patient Infusion Treatment Area (ITA) from discharge up to Day +100.  These 
visits in the ITA typically occur 3x week during Day +30 to Day +60 and then are reduced to 
either once to twice/week depending on subject acuity.  Each visit entails lab tests including a 
CBC; comprehensive metabolic panel; and history and physical exam by a transplant MD and 
physician assistant or nurse practitioner.  
4.20.1 Eligibility and other pre-infusion evaluations 
4.20.1.1  
Medical history including bone marrow biopsy/aspirate for confirmation of 
histologic and/or morphologic diagnosis and disease status and staging 
4.20.1.2  
Physical examination including performance status (ECOG) 
4.20.1.3  
Informed consent per institutional standard timing 
4.20.1.4  
Vital signs including weight, blood pressure, pulse rate and respiratory rate  
4.20.1.5  
Pulmonary testing (DLCO), cardiac performance (LVEF) 
4.20.1.6  
Automated CBC (WBC, RBC, hematocrit, hemoglobin, platelets) with 
differential 
4.20.1.7  
Prothrombin time (PT), partial thromboplastin time (PTT) 
4.20.1.8 
Serum chemistries including electrolytes; glucose; ALT; creatinine; bilirubin; 
alkaline phosphatase; albumin 
4.20.1.9  
Serum or urine Beta HCG for female recipients 
4.20.1.10 
Urinalysis 
4.20.1.11 
Quantitative Immunoglobulins for IgG; IgA; IgM 
4.20.1.12 
Serologies for:  HIV (including HIV-1 RNA PCR); HIV 1+2 Ab; 
HTLV I/II Ab; cytomegalovirus (CMV); hepatitis B (including HBsAg and 
HBcAb); hepatitis C ab; Syphilis; and if indicated, Epstein-Barr virus (EBV); 
herpes simplex virus (HSV); varicella zoster virus (VZV).  Also HLA-typing 
for HLA-A,B,C loci (typed by serology) and HLA-DRB1, DQ, DP loci 
(typed by PCR/SSOP if available) * 
* HLA typing per standard institutional timing. 

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4.20.1.13 
Within 40 days of the cellular infusions, sample of peripheral blood to site 
laboratory for percentage and absolute cell count by immunoflow cytometry 
of CD3; CD4; CD5; CD8; CD 19/20; CD16/56; CD45RA; CD45RO; 
CD62L; and CD44.  
4.20.2 
Follow Up Evaluations (Days +30, +60, +90, +180 and +365, except as indicated)  
4.20.2.1 
Physical examination with vital signs 
4.20.2.2 
Assessment of relapse of disease using bone marrow aspirate/biopsy or other 
tests such as PET/CT scan as clinically indicated on Days +100; +180; 365; 
and then yearly for 2 years post-HCT. 
4.20.2.3 
Automated CBC (WBC; RBC; hematocrit; hemoglobin; platelets) with 
differential 
4.20.2.4  
Serum chemistries: glucose; electrolytes; creatinine; ALT; bilirubin; 
alkaline phosphatase; albumin 
4.20.2.5 
Sample of peripheral blood to site laboratory for absolute count and 
percentage immunoflow cytometry of CD3;  CD4;  CD5;  CD8;  CD19/20;  
CD44;  CD45RA;  CD45RO;  CD62L;  CD16/56, and lineage-specific 
chimerism studies.  TREC analysis will also be performed.  These samples 
will also be collected on Days 0 (prior to the infusion), +7, and +14.  
4.20.2.6   
Quantitative Immunoglobulin levels to include: IgG;  IgA;  IgM 
4.20.2.7  
Summary of acute GvHD (Appendix C) 
4.20.2.8  
Assessment for the presence of chronic GvHD (Appendix C) 
4.21 
Potential Benefits  
Subjects who participate in this study have the potential of achieving a durable remission 
and long term survival from their hematologic malignancy.  Additionally, because 
post-transplant immune suppression will not be given as part of protocol, there may be a 
smaller risk of serious infections.  The risk of graft vs host disease also may be reduced 
since the subject is receiving a T-cell depleted graft.  Additionally, the infusion of as 
regulatory T-cells may also reduce the risk of GvHD. 
5. 
INVESTIGATIONAL AGENT/DEVICE/PROCEDURE INFORMATION 
5.1 Investigational Agent 
This protocol is submitted to FDA IND 14686.  The specific investigational agent for 
which this IND was obtained was for “CD4+CD25+CD127-foxp3+ regulatory T-cells” 
referred to as Treg cells.  The IND application was submitted 4 April 2011, and was 
approved to proceed on 3 June 2011.  
6. 
ADVERSE EVENTS AND REPORTING PROCEDURES 
6.1 Potential Adverse Events 
Recipients of allogeneic hematopoietic cell transplantation face significant risks.  The 
risk of treatment related morbidity and mortality following allogeneic transplant is related 

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to the underlying disease, disease status at the time of transplant, prior therapies and other 
coexisting health problems.  
Significant risks include: 
1)  Infection.  Risks include bone marrow suppression, hospitalization, invasive 
procedures, prior exposure to micro-organisms and immunosuppressive medications 
for the prevention and treatment of graft vs host disease.  Infections may be caused 
by bacteria; viruses; protozoa; or fungus.  
2)  Organ toxicity.  There is a risk of life-threatening toxicity to the heart; lungs; liver; 
kidney; and brain.  Risks include prior therapy; concurrent health problems; and the 
high-dose chemotherapy and radiation used in the preparatory regimen. 
3)  Graft versus host disease.  Acute GvHD can range from a mild and treatable 
problem to a life-threatening complication.  Chronic GvHD can vary from a mild to 
an extensive disorder affecting almost any body tissue.  Chronic GvHD can 
negatively affect functional status and quality of life. 
4)  Secondary malignancies.  Radiation, chemotherapy and immunosuppressive 
medications increase the risk of secondary malignancies. 
5)  Graft failure.  Although rare, graft failure can be a fatal complication. 
6)  Infertility in both genders and premature menopause in women at risk. 
7)  Relapse.  All subjects face a risk of relapse.  The risk is highest for those subjects 
with active or resistant disease at the time of transplantation. 
6.2 Adverse Event Reporting 
Definitions: 
Unanticipated Problem:  any incident, experience, or outcome that meets all of the 
following criteria:  (1) Is unexpected in terms of nature, severity, or frequency in relation 
to (a) the research  risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and (b) 
the characteristics of the subject  population being studied; and  (2) Is related or possibly 
related to participation in the research; and  (3) places subjects or others at a greater risk 
of harm (including physical, psychological, economic, or social harm) than was 
previously know or recognized. 
Adverse Event:  Any untoward medical occurrence in a clinical investigation subject 
administered a pharmaceutical product, medical treatment or procedure and which does 
not necessarily have to have a causal relationship with this treatment.  An adverse event 
can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), a symptom, or disease temporally associated with the use of a medicinal 
product, medical treatment or procedure whether or not considered related to medicinal 
product or treatment. 
Life-threatening Adverse Event:  Any adverse event that places the subject, in the view 
of the investigator, at immediate risk of death from the reaction.  

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Unexpected Adverse Event:  An adverse event, the nature or severity of which is not 
consistent with the applicable product information (Investigator’s Brochure, product 
insert).  For studies that do not involve investigational products or devices, an unexpected 
adverse event is an adverse event that is not described in the transplant medical literature 
or consent form. 
Serious Adverse Event (SAE):  Any adverse event occurring that results in any of the 
following outcomes: death, a life-threatening adverse event, a persistent or significant 
disability/incapacity, a congenital anomaly, requires intervention to prevent permanent 
impairment or damage (21CFR§312.32).  The PI is also bound by 45 CFR§46, Subpart A 
which is the “Common Rule” for the Protection of Human Subjects, therefore, the 
Stanford IRB definition applies (report “unanticipated problems” involving risks to study 
participants or others).  All grades 3 to 5 organ-related adverse events not due to the 
primary malignancy or pre-existing condition and concomitant medications will be 
collected from Day 0 through 90 days after the Tcon dose which is given on Day +2, ie, 
through Day +32 post-HCT. 
Grade 4 infusion reactions will be reported expeditiously and as a separate listing in 
the FDA Annual Report.  
Distinction between Serious and Severe:  The term severe is used to describe the 
intensity (severity) of a specific event, for example mild, moderate or severe.  The event 
itself however, may be of relatively minor medical significance, for example a severe 
headache.  This is not the same as serious, which is based on the subject/event outcome 
and is usually associated with events that pose a threat to the subject’s life or functioning.  
Seriousness, not severity, serves as a guide for defining regulatory obligations.  
Hematopoietic cell transplantation (HCT) is an aggressive therapy for the treatment of a 
number of life threatening malignant and non-malignant disorders.  Individuals 
presenting for HCT generally have exhausted other avenues of therapy that will result in 
any lasting benefit.  The treatment related mortality (TRM) of autologous transplantation 
is approximately 5%.  The TRM of a sibling myeloablative allogeneic transplant is 
approximately 20% and the TRM for an unrelated myeloablative allogeneic transplant 
ranges from 20 to 50%.  
As an aggressive therapy HCT is associated with a large number of significant adverse 
and serious adverse events.  The toxicities associated with HCT are related to the 
following:  
1) Underlying disease 
2) Therapy antecedent to HCT 
3) Health status of the transplant recipient including co-existing conditions 
4) High-dose preparative regimen employed in preparation for transplant 
5) Therapies directed at reducing transplant related complications 
(eg, immunosuppressants for the prevention of GvHD), and  
6) Treatment of complications of HCT.  
The use of toxicity grading scales such as the NCI CTC is a standard in the medical 
community for the reporting of SAEs in the investigation of new drugs or devices.  The 

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use of this type of scale is less helpful in the evaluation of SAEs associated with a 
treatment, such as HCT.  In an effort to report to regulatory agencies the toxicities that 
are relevant and meaningful for the evaluation of risks and benefits to potential HCT 
recipients the following guidelines will serve to determine what is reported as SAEs in 
recipients of HCT. 
The following SAEs require expedited reporting (as soon as possible but within at least 7 
calendar days of the investigator learning of the event): 
1) Deaths  
Death while the subject is receiving treatment on this protocol up to 90 days 
post-transplant if it is felt to be treatment related 
This includes deaths from the common and expected grade 3-4 toxicities noted below.  
Deaths that occur outside of Stanford will be reported whenever possible.  It must be 
noted that obtaining detailed information on the cause and circumstances of a death 
occurring at another institution can be difficult.  Excludes deaths related to relapse of 
underlying disease, which will be reported at the time of protocol renewal.  
2) All serious and unexpected toxicities.  
Defined as those toxicities not identified in the transplant literature or in the consent 
form. 
The following will generally not be reported as SAEs: 
1) Hospitalizations.  
Approximately 50% of allogeneic transplant recipients will be readmitted to the hospital.  
The most common indications for readmission of an allogeneic HCT recipient are fever, 
failure to maintain nutritional status and graft versus host disease. 
2) Relapse of disease. 
Relapse unfortunately remains a significant problem following both autologous and 
allogeneic transplantation.  The risk of relapse is influenced by both subject and disease 
variables.  The risk of relapse following allogeneic transplant is extremely dependent on 
the disease being treated but ranges from 10% (for subjects with severe aplastic anemia) 
to 80% (for subjects with refractory acute leukemia). 
3) Common and expected Grade 3 to 4 toxicities of HCT that are well-described in 
the transplant literature, the product inserts or stated in the consent form and do not 
result in death.  
This includes but is not limited to neutropenia, thrombocytopenia, anemia, thrombotic 
microangiopathy, bleeding requiring transfusions, edema, hypertension, hypotension, 
gastritis, mucositis, nausea, vomiting, diarrhea, hematuria, central venous catheter 
infections, febrile episodes, sepsis, mental status changes, insomnia, mood alterations, 
seizures, tremor, pain, hypoxia, pleural effusion, pneumonitis, incontinence, infertility, 
laboratory abnormalities, veno-occlusive disease, graft failure, cardiac arrhythmias and 
graft versus host disease.  

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Secondary Malignancies. 
The occurrence of secondary malignancies and associated mortality is a known risk of 
cancer therapies.  The occurrence of secondary malignancies will be reported at the time 
of the protocol annual review. 
 
6.3 
Adverse Event Reporting 
Protocol directors/principal investigators (PDs/PIs) are responsible for reporting any 
serious adverse events (SAEs) to both the IRB and regulatory authorities.  Stanford’s 
Human Subject Manual defines reportable events as “all serious adverse events, related 
or unrelated to the study treatment, occurring at Stanford or elsewhere, and unanticipated 
problems.”  The panel has established the reporting timeline as within five to fifteen days 
of first learning of the event.  PDs and PIs must also report any SAEs to the 
biopharmaceutical sponsor or FDA (when the PD acts as sponsor-investigator) according 
to 21CFR§312.32.  By definition, serious adverse event is any untoward or unexpected 
event or medical occurrence, associated with the use of a drug or device or procedure that 
at any dose: results in death; is life-threatening; requires in-patient hospitalization or 
prolongation of existing hospitalization; results in persistent or significant 
disability/incapacity; or is a congenital anomaly/birth defect.  The term “unexpected” is 
used to further quantify an event/experience when it is not consistent with the current 
investigator brochure or with the risk information in the investigational plan.  
“Associated”  with the investigational drug/biologic/device means that there is a 
reasonable possibility that the experience/event may have been caused by the drug or was 
contributed to, at least in part, by the drug or device or procedure.  Finally, a “life 
-threatening” event means that in the view of the investigator, the event places the subject 
or subject at immediate risk of death from the reaction as it occurred.  Timelines for 
reporting SAEs to sponsors or the FDA are more specific and depend on the outcome of 
the event (hospitalization, disability, death).  
PDs and PIs are responsible for adhering to the timelines for reporting SAEs.  When the 
protocol director is the principal investigator for an NIH/NCI/NHLBI sponsored study, 
the investigator must notify the Investigational Drug Branch (IDB) of the NCI or NHLBI 
by telephone of all Grade 4 and Grade 5 expected and unexpected events (see the 
Common Toxicity Criteria Index at www.ctep.nih.gov) within 24 hours of learning of the 
event.  Protocol-specific adverse event case report forms must be completed and 
continued close follow-up is required until the event resolves.  
When a protocol director is both the principal investigator and sponsor of a clinical trial 
(investigator-initiated), the requirements for reporting all SAEs to the FDA and other 
investigators rest with the PD.  Specifically, the PD must notify the FDA of any 
study-related death or life-threatening event via telephone within 24 hours of learning of 
the event.  A written safety report (MedWatch 3500A) must be sent to the FDA within 
seven days.  Any other investigators who are participating in the study must be notified 
via an IND Safety Report within 15 days of learning of the event.  
6.4 Action Plan for Positive Results on Treatment Component Safety Testing 
In the unlikely event that a positive sterility test result is obtained in either cell product 
manufacturing facility after administration of any treatment component , (ie, Tcon;  

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CD34+ HSPC;  or Treg) to a study subject (whether fresh or cryopreserved), or after 
administration of the product to the subject, the following steps will be initiated 
IMMEDIATELY: 
a. Clinical Laboratory personnel will notify the BMT-CTF, via the BMT-CTF IND Manager 
or Medical Director, of positive test results.  
b. BMT-CTF personnel will notify the IND Sponsor-Investigator (who is also the Stanford 
Protocol Director / Principal Investigator) at 650-906-8582.  Information provided to the 
Sponsor-Investigator / Protocol Director will include identification of which treatment 
component(s) are affected/were administered to the subject.  The Sponsor-Investigator will 
be updated with any substantive changes, including the final report on the identification and 
sensitivity from the positive sterility test.  
c. If the BMT-CTF personnel are unable to reach Sponsor-Investigator within 15 minutes, the 
in-patient attending physician caring for the subject on the hospital service will be 
contacted via hospital page with direct communication.  NOTE:  If not within 15 minutes, 
the Sponsor-Investigator will be notified as soon as possible.  The Sponsor-Investigator 
and/or designee will contact the attending physician, who will determine the extent of the 
work-up of a positive culture in consultation with appropriate infectious disease 
consultants, as well as determine an appropriate action treatment plan.  
d. The Sponsor-Investigator/attending physician will discuss the positive results with the 
subject, and specify the clinical therapy, antibiotic regimen and/or monitoring plan.  
e. A contaminated sample of a treatment component that has been administered to a subject 
will be handled in the same fashion as a Grade 4/5 toxicity.  The IND Sponsor-Investigator 
in the role of the Stanford Protocol Director will be responsible to notify the IRB of the 
event via an Unanticipated Problem (UP) report within 5 working days.  If the infused 
treatment component is the HSPC or Treg drug product, the , the Sponsor-Investigator will 
also notify the FDA via an expedited 7-day IND Safety Report.  
In addition to the above, appropriate Safety reporting will be done as per Volume 6 AS 3.1 
Sterility Testing.  A sample of each product is retained by Quality Systems and will be sent to 
the Microbiology Laboratory for repeat testing and speciation.  An 
Out-of-Specification (OOS) Investigation will be conducted by the Quality Systems staff of 
the manufacturing laboratory including root cause analysis, review of viable environmental 
monitoring results collected at the time of manufacturing on personnel, equipment and 
reagents.  Whether or not attribution is established, a formal Corrective and Preventive 
Action plan will be issued by BMT-CTF Quality staff and appropriate remediation will be 
performed including retraining of manufacturing personnel, elimination of any contaminated 
reagents and re-cleaning of the production facility followed by viable microbiological 
monitoring to establish effectiveness of cleaning. 
 

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STUDY CALENDAR 
Assessment Schedule - RECIPIENT 
Protocol IRB-21257 / BMT236 
Subject Name 
MRN 
 
 
 
 
 
Last, First 
nnnnnn-n 
 
 
 
 
 
mm/dd/yyyy 
 
D0+ 
 
 
BMT Date: 
mm/dd/yyy 
Pre-conditioning 
Day 0 
D+7 
±1 day 
D+14 
±1 day 
D+30  
±7 days 
D+60  
±7 days 
D+90 
±10 days 
D+180  
±14 days 
D+365  
±14 days 
2 years 
±4 weeks 
Study Assessments 
Baseline 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
Eligibility checklist 
X 
 
 
 
 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
 
 
 
 
Beta HcG for females 
X 
 
 
 
 
 
 
 
 
 
Pre transplant Virology Panel 
X 
 
 
 
 
 
 
 
 
 
HLA Typing 
X 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
 
 
 
Pulmonary Function Tests  
X 
 
 
 
 
 
 
 
 
 
Cardiac Function Evaluation 
X 
 
 
 
 
 
 
 
 
 
Eligibility checklist 
X 
 
 
 
 
 
 
 
 
 
Flow Cytometry 
X 
 
 
 
X 
X 
X 
X 
X 
 
(CD3,CD4,CD5,CD8,CD19/20, CD16/56, 
CD45RA, CD45RO, CD62L, CD44) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Serum Ig levels (IgA, IgG, IgM) 
X 
 
 
 
X 
X 
X 
X 
X 
 
TREC 
X 
 
 
 
X 
X 
X 
X 
X 
 
EBV  
X 
 
 
 
X 
X 
X 
X 
X 
 
GvHD ASSESSMENT  
 
 
 
 
X 
X 
X 
X 
X 
X 

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BMT Date: 
mm/dd/yyy 
Pre-conditioning 
Day 0 
D+7 
±1 day 
D+14 
±1 day 
D+30  
±7 days 
D+60  
±7 days 
D+90 
±10 days 
D+180  
±14 days 
D+365  
±14 days 
2 years 
±4 weeks 
CBC w/ Diff 
X 
 
 
 
X 
X 
X 
X 
X 
 
Serum Chemistry  
X 
 
 
 
X 
X 
X 
X 
X 
 
Karnofsky performance score 
X 
 
 
 
X 
X 
X 
X 
X 
 
BMBx, Aspirate, Cytogenetics 
X 
 
 
 
 
 
X 
X 
X 
 
Lineage specific chimerism studies 
 
 
 
 
X 
 
X 
X 
X 
 
CT/PET scan as clinically indicated 
 
 
 
 
 
 
X 
X 
X 
 
 
Table 3 
DONOR ASSESSMENT SCHEDULE 
ASSESSMENT SCHEDULE - DONOR 
 
 
 
 
 
 
 
 
 
 
 
 
Study Assessments 
BaseLine 
* 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
Eligibility checklist 
X 
 
 
 
 
 
CBC w/ Diff 
X 
 
 
 
 
 
Chem 7 
X 
 
 
 
 
 
Pre transplant Virology Panel 
X 
 
 
 
 
 
EBV 
X 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
PE 
X 
 
 
 
 
 
Serum or urine Beta HCG *** 
X 
 
 
 
 
 
ABO/RH typing 
X 
 
 
 
 
 
HLA typing **** 
X 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
Chest Xray 
X 
 
 
 
 
 
* 
Within 40 days prior to leukapheresis 
 
 
 
 
** 
Electrolytes;  glucose;  ALT;  AST;  creatinine;  bilirubin; alkaline phosphatase; 
albumin 

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*** Female donors 
 
 
 
 
 
 
**** HLA-A,B,C loci (typed by serology) and HLA-DRB1,DQ,DP loci 
 

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7. 
MEASUREMENT OF EFFECT 
7.1 Anti-tumor Effect 
Subjects will undergo disease evaluations on Day +100, +180, +365 and then annually until 5 
years from HCT date.  The testing should be done +/- 14 days from targeted evaluation date 
although the Day + 180 and yearly evaluations will be done as close to the date as possible. 
7.1.1 Definitions 
7.1.1.1  Acute Leukemia – relapsed defined as 
a.  Leukemic blasts > 20% are documented in the blood or bone marrow after HCT 
b.  Leukemic blasts > 5% and < 20% are documented in the blood or bone marrow 
and supported by reappearance of cytogenetic abnormality or 
c.  Leukemic blasts > 5% and < 20% are documented in the blood or bone marrow 
on at least 2 occasions  
d.  Leukemia detected at an extramedullary site or the appearance of new dysplastic 
changes within the bone marrow or % blasts  
7.1.1.2  Chronic Myelogenous Leukemia – relapsed defined as 
a.  Immature hematopoietic cells are persistently documented in the peripheral 
blood, or 
b. Myeloid hyperplasia in the bone marrow in the presence of a cytogenetic relapse 
as defined by: 
- 50% of the metaphases exhibit the characteristic 9;22 translocation with at least 
10 metaphases analyzed or 
- At least 1 metaphase exhibits the 9;22 translocation on each of 2 separate 
consecutive examinations at least 1 month apart, regardless of number of 
metaphases analyzed 
7.1.1.3  Myelodysplastic syndrome 
a.  Appearance of pre-transplant morphologic abnormalities detected in 
2 consecutive bone marrow specimens taken at least 1 month apart 
b. Satisfying above criteria for evaluation into acute leukemia 
c. Reappearance of pre-transplant cytogenetic abnormalities in at least 50% of 
metaphases with at least ten metaphases examined or 
d. Reappearance of pre-transplant cytogenetic abnormality in at least 2 metaphases 
on each of two separate consecutive examinations at least 1 month apart, 
regardless of the number of metaphases analyzed 
7.1.1.4  Hodgkin and Non-Hodgkin lymphoma (relapse defined below)  
Lymph nodes should be considered abnormal if the long axis is more than 1.5 cm 
regardless of the short axis.  If a lymph node has a long axis of 1.1 to 1.5 cm, it 

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should only be considered abnormal if its short axis is more than 1.0 cm.  Lymph 
nodes ≤ 1.0 x ≤ 1.0cm will not be considered as abnormal for relapse or progressive 
disease. 
i. 
Appearance of any new lesion more than 1.5 cm in any axis during or at the end of 
therapy, even if other lesions are decreasing in size.  Increased FDG uptake in a 
previously unaffected site should only be considered relapsed or progressive 
disease after confirmation with other modalities.  In patients with no prior history 
of pulmonary lymphoma, new lung nodules identified by CT are mostly benign.  
Thus, a therapeutic decision should not be made solely on the basis of the PET 
without histologic confirmation. 
ii. 
At least a 50% increase from nadir in the SPD of any previously involved nodes or 
in a single involved node, or the size of other lesions (eg, splenic or hepatic 
nodules).  To be considered progressive disease, a lymph node with a diameter of 
the short axis of less than 1.0 cm must increase by ≥ 50% and to a size of 
1.5 x 1.5 cm or more than 1.5 cm in the long axis. 
iii. 
At least a 50% increase in the longest diameter of any single previously identified 
node more than 1 cm in its short axis 
iv. 
Lesions should be PET positive if observed in a typical FDG-avid lymphoma or 
the lesion was PET positive before therapy unless the lesion is too small to be 
detected with current PET systems (< 1.5 cm in its long axis by CT).  Measurable 
extranodal disease should be assessed in a manner similar to that for nodal 
disease.  For these recommendations, the spleen is considered nodal disease.  
Disease that is only assessable (eg, pleural effusions, bone lesions) will be 
recorded as present or absent only, unless, while an abnormality is still noted by 
imaging studies or physical examination, it is found to be histologically negative.  
In clinical trials where PET is unavailable to the vast majority of participants, or 
where PET is not deemed necessary or appropriate for use, response should be 
assessed as above, but only using CT scans.  However, residual masses should not 
be assigned CRu status, but should be considered partial responses. 
 
8. 
DATA REPORTING / REGULATORY CONSIDERATIONS 
8.1 Monitoring plan 
The Monitoring Entity (ME) that will be responsible for monitoring the data collected, 
including data related to unanticipated problems and adverse events, and their respective 
roles (eg, Stanford Cancer Center, CRC, investigator, sponsor, coordinating or statistical 
center, independent medical monitor, DSMB/DSMC, or some other entity).  If there is no 
ME, then the Protocol Director (PD) is responsible for this function. 
Primary internal data monitoring will be performed by the Principal Investigator and the 
study coordinator.  The PI will review data to assure the validity of data, as well as the 
safety of the subjects.  The PI will also monitor the progress of the trial.  The PI is 
responsible for maintaining the clinical protocol, reporting adverse events, assuring that 
consent is obtained and documented, reporting of unexpected outcomes, and reporting the 

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status of the trial in the continuing renewal report submitted annually to the IRB and 
SRC.  
The DSMC of the Stanford University Cancer Center will provide oversight of the study.  
This multidisciplinary committee monitors the safety of participants in clinical trials, and 
the conduct, progress, validity, and integrity of the data for all investigator initiated 
clinical trials at the Stanford University Clinical Cancer Center.  The Stanford DSMC 
audits all investigator-initiated studies on an annual basis and submits a DSMC report to 
the Principal Investigator.  
The content of this DSMC report is comprised of an overall rating of the study audit, 
(Outstanding, Satisfactory, Minor Deficiencies, or Major Deficiencies) that is a 
composite of overall study conduct and the findings of the audit session.  In rating the 
conduct of the study, the DSMC categorizes deviations as Major, Moderate or Minor.  
The report also contains the specific recommendation from the committee, which can 
consist either of Full Approval, Conditional Approval, Suspension of defer to the SRC 
for closure. 
8.2 
Stopping rules (for the individual subject and for the study as a whole) 
As indicated in the dose escalation schema (Figure 1), any recipients within a dose cohort 
showing GvHD > Grade 2 will require trigger the inclusion of three additional recipients 
at that dose.  Dose escalation will proceed if none of the additional recipients show 
GvHD > Grade 2; Grade 4 neutropenia lasting to 28 days after HCT; or Grade 3 to 5 
cytokine release syndrome / acute infusion reaction. 
8.3 Data management  
i)  Research records are maintained in a secure office of the data management staff.  
Electronic data is maintained in a secure database.  
ii)  Research samples are sent to the SCTT lab for coding prior to the freezing or 
processing of the cell.  A link between the sample and the subject is maintained with 
access to the link limited to study personnel. 
iii)  Only study personnel within the Stanford BMT program have access to the electronic 
database, the research records or the link between research samples and identifying 
information.  Stanford researchers outside of the BMT program do not have access to 
PHI. 
iv)  Research records are kept in a locked file cabinet within an office of the data 
management staff.  Access to the data management office is limited to key access.  
v)  The BMT Database System Administrator limits access to the database to research 
personnel within the BMT program.  Data sets can be generated to share with 
investigators outside the program by de-identifying the data. 
8.4 Confidentiality  
All subjects undergoing hematopoietic cell transplantation at Stanford are assigned a SPN 
(Stanford Patient Number) and data is entered into a secure database.  Access to the 
database is limited to study personnel and password protected. 

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Subjects will not be directly identified in any publications, presentations or 
correspondence.  
All staff within the BMT Program are required to complete HIPAA training.  All research 
staff must complete the IRB human subjects research training. 
The subject’s data are identified by SPN number alone in the research documents with 
the information about the code kept in a secure location.  Access to the research database 
is limited to approved clinical and research staff of the BMT Division.  The code is 
maintained by the BMT Database System Administrator. 
PHI is collected and recorded at the time of the subject’s first consultation with the BMT 
program.  Additional information is collected when the subject is enrolled in a clinical 
trial.  PHI may include some or all of the following:  
1. Names  
2. Social Security numbers  
3. Telephone numbers  
4. All geographic subdivisions smaller than a State, including street address, city, county, 
precinct, zip code, and their equivalent geocodes, except for the initial three digits of 
a zip code, if, according to the current publicly available data from the Bureau of the 
Census: (1) The geographic unit formed by combing all zip codes with the same three 
initial digits contains more than 20,000 people; and (2) The initial 3 digits of a zip 
code for all such geographic units containing 20,000 or fewer people is changed to 
000.  
5. All elements of dates (except year) for dates directly related to an individual, including 
birth date, admission date, discharge date, date of death; and all ages over 89 and all 
elements of dates (including year) indicative of such age, except that such ages and 
elements may be aggregated into a single category of age 90 or older  
6. Fax numbers  
7. Electronic mail addresses  
8. Medical record numbers  
9. Health plan beneficiary numbers  
10. Account numbers  
11. Certificate/license numbers  
12. Vehicle identifiers and serial numbers, including license plate numbers  
13. Device identifiers and serial numbers  
14. Web Universal Resource Locations (URLs).  
15. Internet Protocol (IP) address numbers  
16. Biometric identifiers, including finger and voice prints  
17. Full face photographic images and any comparable images; 

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Referring physicians are routinely sent letters by the BMT physicians to update them on 
their subject's status.  Specific information regarding the study would not be routinely 
shared with the referring physician, however, we would share with referring physician’s 
information regarding the expected side effects of the treatment, necessary precautions 
and also the appropriate follow-up.  
Other outside agencies that may receive information include:  
•  The Office for Human Research Protections in the US Department of Health 
and Human Services  
•  The Food and Drug Administration  
•  Federal and regulatory agencies as required  
•  Center for International Blood and Marrow Transplant Research (CIBMTR)  
•  The National Marrow Donor Program or donor registry from which the cells 
were obtained  
•  Foundation for the Accreditation of Cellular Therapy  
•  The National Institutes of Health 
•  Pediatric Blood and Marrow Transplant Consortium 
•  Children's Oncology Group 
•  Data Safety Monitoring Board 
•  Other investigators with IRB-approved projects 
8.5 
Reporting Mechanisms 
The Principal Investigator of the trial (or designee) is responsible for the coordination and 
development of all protocol amendments.  Any changes to the protocol or consent will be 
made in the form of an amendment and will be approved prior to implementation and this 
process should be included in the monitoring plan.  
Changes or amendments to the protocol or consent will be reported to the IRB, FDA and 
NHLBI.  
All lRB actions will be reported to the DSMC, NHLBI, and FDA.  
 
9. 
STATISTICAL CONSIDERATIONS 
This is a phase 1 dose escalation trial followed by a phase 2 trial at the MTD.  The addition of 
planned numbers and ratios of Treg compared to Tcon will occur at defined time points after 
hematopoietic cell infusion.  Each cohort will have 3 subjects per group.  The initial doses and 
ratios utilized will be 1 x 106/kg of T reg cells to 1 x 106/kg of Tcon  cells at a 1:1 ratio.  In order to 
progress to the next dose level, there must be no evidence of Grade 3 or 4 acute GvHD.  If a 
single subject develops Grade 3 or 4 acute GvHD, an additional 3 subjects will be treated at the 
same dose level.  If a 2nd subject develops Grade 3 or 4 GvHD, then the dose escalation will be 
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9.1 Endpoints 
A. Endpoint for Safety 
This is a phase 1 dose finding trial with two degrees of freedom in the definition of dose 
(numbers of  Treg  and Tcon , or, equivalently, the number of Tcon and the ratio of Treg to 
Tcon).  These ‘dose pairs’ act in different directions to produce the resulting rates of 
GvHD, and presumably, anti-tumor activity.  The study will follow a traditional 3+3 
model, as described in the diagram to find the maximum tolerated dose of Tcon at the 
minimum necessary ratio of Treg to Tcon  There will be 3 subjects in each cohort with a 
minimum of 3 subjects in each cohort.  At all levels, progression to the next cohort will 
be dependent on no occurrence of > grade 2 acute GvHD or any other DLT which 
includes Grade 4 neutropenia lasting to 28 days after HCT and Grade 3 to 5 
cytokine release syndrome / acute infusion reaction. 
If at any point, a predefined DLT is observed, then the cohort is expanded to (no more 
than) 6 total at that dose pair.  If no further grade 3 or 4 GvHD or pre-defined DLT is 
observed (so the total is now 1/6), the MTD is declared and in that case the cohort at that 
dose pair is expanded by up to 12 subjects (in 2 cohorts of 6) for a total of 18 subjects. 
However, if at any point in the 12-subject expansion, the observed Grade 3 or 4 GvHD 
rate or pre-defined DLT rises to 50% or above, the next lower dose of Tcon at the ratio of 
1:1 Treg to Tcon is expanded as the MTD.  Thus, if in either cohort of 3 subjects at any 
dose level there are 2 or more with Grade 3 to 4 GvHD, or pre-defined DLT we will 
de-escalate to the lower dose. 
B. Endpoint for Feasibility 
The feasibility of Treg isolation is another primary endpoint of this trial.  If the Treg yield 
is below the dose for a specified cohort, the subject will be placed in the previous cohort.  
As this is a dose escalation trial that includes administering higher doses of Treg cells, 
consideration has been taken in the event of failure to isolate the specified target dose of 
Treg cells.  We have determined that 40% represents the “unacceptable’ failure rate to 
reach target Treg  dose.  In the event that the observed failure rate in a dose cohort implies 
that the true failure rate exceeds 40%, with 80% confidence, then we will de-escalate T 
reg dose and treat subjects on a previous dose level.  We will assess the failure rate after 
each cohort of 6 and in the expanded cohort of 12.  If we see 4 failures out of 6 tries in a 
cohort, or 7 failures out of 12, this feasibility stopping rule will be triggered and the dose 
will be de-escalated.  See table below. 
Observed Failures / Subject cohort 
80% Lower Confidence Limit for 
True Failure Rate 
4 / 6 
0.41 
7 / 12 
0.42 

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9.1.1 Primary endpoint 
Phase 1 Primary Objective  
The Primary Objective of the phase 1 part of this trial is to determine the safety and 
feasibility of administration of conventional T-cells and regulatory T-cells to subjects 
undergoing allogeneic hematopoietic cell transplantation with matched related donors, 
and to find the maximum tolerated dose of Tcon at the minimum necessary ratio of Treg to 
Tcon.  Thus, the primary endpoint in each subject is the occurrence of Grade 3 or 4 
GvHD, as defined above. 
Phase 2 Primary Objective  
Once the MTD is determined, it is proposed to expand the cohort at that dosage into a 
single-arm, phase 2 admissible Simon 2-stage proof-of-concept study, continuing with 
HLA-matched donors (related or unrelated).  The Primary Objective of the phase 2 
Stage 1 portion is to determine the need for concomitant immune suppression with fresh 
Tregs, and the phase 2 Stage 2 portion is GvHD-free, relapse-free survival post-HCT.  
Historical control data from Stanford will be used as the comparator, documented as 
~40% 1-year GRFS in a comparable patient population receiving T-cell replete grafts.  
GRFS is defined as the minimum time from HCT to relapse; GvHD; or death.  The 
anticipated 1-year GRFS probability is 60% and the lower boundary for proceeding to a 
possible phase 3 trial is an GRFS probability of > 0.40.  The primary hypothesis can be 
described as H0: p ≤ 0.40 versus H1: p > 0.40.  When the true GRFS percentage is 60% 
with a sample size of 41, there is 80% power at one-sided α = .05 to rule out a GRFS 
percentage of < 40%. 
An “Admissible Simon’s two-stage design” (Simon, 1989) will be used. The null 
hypothesis that the true 1-year GRFS is 40% will be tested against a one-sided 
alternative. The probability of early termination under the null hypothesis is 64%. In the 
first stage, 17 patients will be accrued. If there are 7 or fewer successes in these 17 
patients, the study will be stopped. Otherwise, 24 additional patients will be accrued for a 
total of 41. The null hypothesis will be rejected if 22 or more responses are observed in 
41 patients. This design yields a type I error rate of 5% and power of 80% when the true 
1-year GRFS is 60%.  
 
Design  
r1  n1  r  
n  
EN.p0  
PET.p0  
Error  
Power  
Admissible  
7  
17  
21  
41  
25.62782  
0.6405077  
0.04733667  
0.8009427  
Phase 2 Primary Outcome (for ClinicalTrials.gov)  
Title:  GvHD-free Relapse-free Survival (GRFS) 
Description:  Clinical effect was assessed as graft vs host disease (GvHD)-free relapse 
free survival (GRFS), as determined by per protocol analysis.  GvHD-free is defined as 

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no GvHD symptoms, and relapse free survival is defined as survival at 12 months 
without relapse. 
Time Frame:  12 months 
Safety Issue:  Yes 
9.1.2 Stopping Guidelines 
Stopping Rule for Grade 3 to 4 acute GvHD 
Interim analyses for safety will be conducted for every 5 subjects accrued.  The incidence 
of Grade 3 to 4 acute GvHD will be closely monitored and the following stopping rules 
will apply.  Based on historical control data observed in a similar patient population, we 
anticipate an observed rate of Grade 3 to 4 acute GvHD to be 15% and we would not 
accept a true rate > 30%.  The stopping rule is triggered when the 80% one-sided lower 
confidence limit for the rate exceeds 30%. 
Observed Cases of  
Grade 3 to 4 GvHD / Subjects in Cohort 
Action 
3 / 5 * 
Stop accrual 
5 / 10 * 
7 / 15 * 
9 / 20 * 
10 / 25 * 
12 / 30 * 
14 / 35 * 
16 / 40 * 
18 / 45 * 
20 / 50 * 
22 / 55 * 
24 / 60 * 
26 / 65 * 
28 / 70 * 
30 / 75 * 
* protocol Phase 2 component / adaptive Simon 2-stage design 

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Stopping Rule for Primary Graft Failure 
Interim analyses for safety will be conducted for every 5 subjects accrued.  The incidence of 
primary graft failure (Grade 4 neutropenia lasting to 28 days after HCT) will be closely 
monitored and the following stopping rules will apply.  Based on historical control data observed 
in a similar patient population, we anticipate an observed rate of primary graft failure to be 20%, 
and we would not accept a true rate > 30%.  The stopping rule is triggered when the 80% 
one-sided lower confidence limit for the rate exceeds 30%. 
Observed Cases of  
Primary Graft Failure / Subjects in Cohort 
Action 
3 / 5 * 
Stop accrual 
4 / 10 * 
5 / 15 * 
6 / 20 * 
8 / 25 * 
9 / 30 * 
10 / 35 * 
11 / 40 * 
12 / 45 * 
13 / 50 * 
14 / 55 * 
15 / 60 * 
16 / 65 * 
17 / 70 * 
18 / 75 * 
* protocol Phase 2 component / adaptive Simon 2-stage design 

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Stopping Rule for Acute Cytokine Release Syndrome / Infusion Reaction 
Interim analyses for safety will be conducted for every 5 subjects accrued.  The incidence of 
acute cytokine release syndrome / infusion reaction will be closely monitored and the following 
stopping rules will apply.  Based on historical control data observed in a similar patient 
population, we anticipate an observed rate of acute cytokine release syndrome to be 10% and we 
would not accept a true rate > 20%.  The stopping rule is triggered when the 80% one-sided lower 
confidence limit for the rate exceeds 20%. 
Observed Cases of  
Grade 3 to 4 GvHD / Subjects in Cohort 
Action 
2 / 5 * 
Stop accrual 
3 / 10 * 
3 / 15 * 
4 / 20 * 
5 / 25 * 
5 / 30 * 
6 / 35 * 
7 / 40 * 
8 / 45 * 
9 / 50 * 
10 / 55 * 
11 / 60 * 
12 / 65 * 
13 / 70 * 
14 / 75 * 
* protocol Phase 2 component / adaptive Simon 2-stage design 
Stopping Rules for evaluation of 9/10 HLA matched non-permissive subgroup 
The same stopping rules will be applied as for HLA-matched as described above. Subjects from 
9/10 HLA matched non-permissive subgroup will be monitored separately and in event that a 
stopping rule is triggered, accrual will be stopped for review. 
9.1.3 
Secondary endpoints 
9.1.3.1 One-year OS  
For overall survival (OS), the event analyzed is death from any cause.  Alive at 
the time of last observation will be censored.   

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9.1.3.2 Incidence and severity of chronic GvHD 
Chronic GvHD scored according to the first day of chronic GvHD onset will be 
used to calculate cumulative incidence curves.  Subjects will be followed for 
2 years from the Day 0 (day of CD34+ peripheral blood stem cell infusion) for 
estimation of cGvHD incidence.  See Appendix C. 
9.1.3.3  Incidence of serious infections 
Microbiologically and/or radiographically documented infections will be 
reported by source of infection (virus, bacteria, fungus), site of disease, date of 
onset and resolution, if any.  
9.1.3.4 Immune reconstitution parameters 
This will be measured in all subjects by: 
1.  The rate of peripheral blood repopulation by CD3;  CD4;  CD8;  CD45RA;  
CD45RO;  CD16/56;  CD19;  CD20;  CD44;  and CD62L 
2.  The rate of blood repopulation with recent thymus emigrants measured by T-cell 
receptor excision circle-positive T-cells (TRECs) via quantitative PCR. 
3.  The serum levels of IgA;  IgG;  and IgM. 

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Phase 2 Secondary Outcomes (for ClinicalTrials.gov)  
Title:  Dose-limiting Toxicity  
Description:  Dose-limiting Toxicity (DLT) was assessed as: 
• Absolute neutrophil count <500/µL, to 28 day 
• Cytokine release syndrome/acute infusion reactions as Common Terminology Criteria 
for Adverse Events (CTCAE) Grade 3 to 5 
• Grade 3 to 4 acute GvHD.  GvHD was staged as follows: 
+ 1: Skin: rash <25%. Liver: bilirubin (BIL) 2-3mg/dL. Gut: diarrhea (DIA) 500-
1000mL/day 
+ 2: Skin: rash 25-50%. Liver: BIL 3-6mg/dL. Gut: DIA 1001-1500mL/day 
+ 3: Skin: rash > 50%. Liver: BIL 6-15mg/dL. Gut: DIA >1501-2000mL/day 
+ 4: Skin: generalized erythroderma. Liver: BIL >15mg/dL. Gut: DIA >2001mL/day 
GvHD was graded as follows. 
+ 1: Skin Stage 1-2; No Liver stage; No Gut stage 
+ 2: Skin Stage 1-3 ; Liver Stage 1; +/- Gut Stage 1 
+ 3: Skin Stage 2-3, Liver Stage 2-4; +/- Gut Stage 2-3 
+ 4: Skin Stage 2-4; Liver Stage 2-4; +/- Gut Stage 2-4 
The outcome is reported as the number of participants who received both Treg and Tcon cell 
infusions and had DLT events, per treatment level, a number without dispersion. 
Time Frame:  28 days 
Safety Issue:  Yes 
Title:  Concomitant Single-agent Immunosuppression 
Description:  During study Phase 2, Stage 1, concomitant single-agent 
immunosuppression was assessed as in participants receiving fresh Treg cells.  The 
outcome is reported as number of such participants who received single-agent 
immunosuppression, by treatment level, a number without dispersion.  
Time Frame:   
Safety Issue:   

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Title:  1-year Overall Survival (OS) 
Description:  Overall Survival (OS) at 1 year was assessed as the number of participants 
per treatment level that received the hematopoietic cell transplant (HCT), and remained 
alive 12 months later, a number without dispersion.  
Time Frame:  12 months  
Safety Issue:  No 
Title:  Incidence and Severity Chronic Graft vs Host Disease (cGvHD) 
Description:  The incidence and severity of chronic graft vs host disease (cGvHD) was 
assessed in participants who received the hematopoietic cell transplant (HCT).   
Stage of chronic GvHD was assessed as follows. 
• Stage 1: Skin: rash <25% of skin. Liver: bilirubin 2-3mg/dL. Gut: diarrhea 500-
1000mL/day 
• Stage 2: Skin: rash 25-50% of skin. Liver: bilirubin 3-6mg/dL. Gut: diarrhea 1001-
1500mL/day 
• Stage 3: Skin: rash > 50% of skin. Liver: bilirubin 6-15mg/dL. Gut: diarrhea >1501-
2000mL/day 
• Stage 4: Skin: generalized erythroderma. Liver:  bilirubin >15mg/dL. Gut: diarrhea 
>2001mL/day 
Grade of chronic GvHD was determined as follows. 
• Grade 1: Skin Stage 1-2; No Liver stage; No Gut stage 
• Grade 2: Skin Stage 1-3 ; Liver Stage 1; +/- Gut Stage 1 
• Grade 3: Skin Stage 2-3, Liver Stage 2-4; +/- Gut Stage 2 to 3 
• Grade 4: Skin Stage 2-4; Liver Stage 2-4; +/- Gut Stage 2 to 4 
The outcome is reported as the number of participants by cGvHD grade and treatment 
level, a number without dispersion.  
Time Frame:  24 months 
Safety Issue:  Yes 
Title:  Serious Infections  
Description:  Participants were assessed for infections judged serious by 21CFR§312.32.  
The outcome is reported as the number of serious infections per treatment level, in 
participants who received the hematopoietic cell transplant (HCT), a number without 
dispersion.  
Time Frame:  24 months 
Safety Issue:  Yes 

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9.2. Plan of Analysis 
9.2.1 Background and Demographic Characteristics 
We will tabulate and summarize all background and demographic characteristics. 
9.2.2 Evaluation of Efficacy 
The OS and GRFS estimates at the MTD dose pair will be calculated according to the 
methods of Kaplan and Meier and will be calculated from Day 0 of infusion of CD34+ 
selected peripheral blood stem cells.  At the sample size (41) of the expanded cohort, the 
distance from the proportion surviving 1 year and the exact one-sided lower 90% 
confidence limit of that proportion is no more than 12%, illustrating the precision of the 
estimates of survival parameters. 
9.2.3 Methods for handling missing data and non-adherence to protocol 
No missing data expected; in the event of non-adherence, DLTs will be counted in any 
subject who receives any dose of cells. 
9.2.4 Evaluation of Conduct of trial (including accrual rates, data quality) 
Accrual rates will be reported to the DSMC and data quality will be reviewed by the 
Study Statistician. 
9.2.5 Methods for Correlative Studies 
Parameters of immune reconstitution and other secondary outcomes will be correlated 
with dose of each type of T-cell, using standard regression methods, assuming a range of 
doses are explored.  The results will be descriptive, given the early phase of the study and 
small sample size, but estimates of confidence intervals for all summaries will be 
provided. 
9.3 
Sample Size 
9.3.1 
Accrual estimates 
The number of subjects accrued during the Phase 1 component of the trial was 13.  The 
maximum tolerated dose (MTD) was determined to be 3 x 106 Tcon cells/kg in 
combination with 3 x 106 Treg cells/kg. 
This study’s admissible Simon two-stage design is intended to evaluate the 
immune-suppressant (IS) combination, as well as efficacy and toxicity of the 
graft-engineered product.  
In study phase 2, Stage 1, the trial will enroll 24 subjects to 1 of 2 arms, each received the 
established MTD of 3 x 106 Tcon cells in combination 3 x 106 Treg cells.  In this stage, the 
two treatments will be graft-engineered product + IS vs graft-engineered product alone.  
In the 1st stage, the trial investigates if the rate of GvHD is non-inferior for the 
graft-engineered product with and without IS. 
In study phase 2, Stage 2, if the GvHD rate is found to be non-inferior between the 
2 arms, the 2 arms will be pooled for the interim analysis evaluating the primary endpoint 
and the remaining subjects enrolled in the 2nd stage will be administered the 

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graft-engineered product alone.  Based on the admissible Simon two-stage design [2] the 
null hypothesis that the true 1-year GRFS is 40% will be tested against a one-sided 
alternative. The probability of early termination under the null hypothesis is 64%. In the 
first stage, 17 patients will be accrued. If there are 7 or fewer successes in these 17 
patients, the study will be stopped. Otherwise, 24 additional patients will be accrued for a 
total of 41. The null hypothesis will be rejected if 22 or more responses are observed in 
41 patients. This design yields a type I error rate of 5% and power of 80% when the true 
1-year GRFS is 60%. See a table below.  
Design  
r1  n1  r  
n  
EN.p0  
PET.p0  
Error  
Power  
Admissible  
7  
17  
21  
41  
25.62782  
0.6405077  
0.04733667  
0.8009427  
  
9.3.2 
Sample size justification 
The phase 1 dose escalation trial will accrue 3 subjects/dose level.  Such a cohort size 
provides a traditional level of confidence in the safety of each level before proceeding to 
the next.  The implied target of about 33% Grade 3 or 4 GvHD rate is considered 
appropriate given the prognosis of the disease under standard therapy and the risk:benefit 
tradeoff.  At the sample size of the expanded cohort, the distance from the proportion 
surviving 1 year and the exact one-sided lower 90% confidence limit of that proportion is 
no more than 18%, illustrating the precision of the phase 1 experiment.  In the event that 
2 of 3 subjects experience Grade 3 or 4 GvHD at any dose level, then subsequent subjects 
will be treated at the previous dose level following the Schema in Figure 2.  
The Phase 2 part of this has a planned sample size of 41 subjects based on the true EFS 
being 60%.  This sample size provides 80% power at α = .05 to rule out a EFS percentage 
of < 40%.  Given the complexity of the intervention, we consider the 60% EFS target as 
the smallest improvement on our historical control rate of 40% that would justify 
continuing to use this treatment concept as the basis for further development. 
9.3.3 
Definition of Dose Limiting Toxicity 
a.  Grade 4 neutropenia lasting to 28 days after HCT 
b.  Grade 3 to 4 acute GvHD 
c.  Grade 3 to 5 cytokine/release syndrome/acute infusion reaction  
9.4 Interim analyses 
The interim analyses consist of monitoring of the Grade 3 or 4 GvHD rates in successive 
cohorts defined by the dose pairs.  The stopping rules are as described above.  The 
confidence limits for secondary parameters will be presented at their nominal values as is 
traditional in phase 1.  The stopping rules for the Phase 2 portion are outlined in 
Section 9.1.2.  An “efficacy futility” interim analysis is not proposed due to the time lag 
between treatment and ascertainment of the primary outcome (1-year EFS rate).  

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10 
CONSENT FORMS 
10.1 Donor Consent Form 
Provided separately. 
10.2 Recipient Consent Form 
Provided separately. 
11 
INVESTIGATOR AND FACILITIES INFORMATION 
Provided separately. 
 

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12 
APPENDICES 
A. Participant Eligibility Checklist  
ELIGIBILITY CRITERIA FOR RECIPIENT 
In order for the subject to be eligible, all inclusion criteria must be yes and all exclusion criteria 
must be no. 
Inclusion Criteria 
Histopathologically-confirmed diagnosis of one of the following: 
_____ Acute leukemia with refractory disease or beyond CR1, or minimal residual disease (MRD) 
positivity 
_____ High-risk leukemia with any of the following features 
_____ Acute myeloid leukemia (AML) in CR1 as follows 
_____ Complex karyotype (≥ 3 clonal chromosomal abnormalities) 
_____ Any of the following high-risk chromosomal abnormalities:  
_____ Monosomal karyotype (-5;  5q-;  -7;  7q-) 
_____ t(11q23);  t(9;11);  inv(3);  t(3;3);  t(6;9);  t(9;22) 
_____ Normal karyotype with FLT3-ITD mutation 
_____ Other high-risk features as determined by molecular studies, or clinical 
presentation as assessed by a study investigator 
_____ Other high-risk leukemia in CR1, as determined by a study investigator 
_____ Chronic myelogenous leukemia (accelerated, blast or second chronic phase or first chronic phase 
that and has not achieved a molecular remission after three or greater therapies) 
_____ Myelodysplastic syndromes 
_____ Myeloproliferative syndromes 
_____ Non-Hodgkin lymphoma with poor risk features not suitable for autologous transplantation 
_____ Hodgkin lymphoma with poor risk features not suitable for autologous transplantation 
_____ Age ≥ 18-years old and for subjects in Cohort 1 only, ≤ 73 years of age.  At the start of Cohort 
2A and beyond, eligibility will be expanded to allow pediatric subjects age ≥ 13 years old. 
_____ Left ventricular ejection fraction (LVEF) ≥ 45% 
_____ Diffusing capacity of the lungs for carbon monoxide (DLCO) ≥ 50% corrected for hemoglobin 
_____ Creatinine clearance calculated ≥ 50 mL/min or creatinine < 2.0 mg/dL 
_____ SGPT and SGOT ≤ 7.5 x ULN; 
_____ Total bilirubin ≤ 3 x ULN (patients with Gilbert’s syndrome may be included at the discretion of 
the PI or where hemolysis has been excluded 
_____ Negative serum or urine beta-HCG test in females of childbearing potential  
_____ Availability of HLA-matched donor or 9/10 HLA-matched donor (related or unrelated) defined 
by Class I (HLA–A and -B) serologic typing (or higher resolution) and Class II (HLA-DRB1) 
molecular typing.   
_____ Karnofsky Performance Status ≥ 70% 
Exclusion Criteria (Recipient) 
_____ Seropositive for any of the following:  
HIV ab;  hepatitis B sAg;  hepatitis C ab 

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_____ Prior myeloablative therapy or hematopoietic cell transplant 
_____ Candidate for autologous transplant 
_____ HIV-positive 
_____ Active uncontrolled bacterial, viral or fungal infection, defined as currently taking antimicrobial 
therapy and progression of clinical symptoms. 
_____ Uncontrolled CNS disease involvement 
______Pregnant or lactating female 
_____ Positive serum or urine beta-HCG test in females of childbearing potential  
_____ Psychosocial circumstances that preclude the patient being able to go through transplant or 
participate responsibly in follow-up care 
_____   Recipient of prior allogeneic transplants 
 
______________________________________________ 
_____________________ 
Treating Physician Signature/Print Name 
 
Date 
______________________________________________ 
_____________________ 
Research Coordinator Signature/Print Name 
 
Date 
______________________________________________ 
_____________________ 
Other Third Signature/Print Name 
 
Date 

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ELIGIBILITY CRITERIA FOR DONOR 
In order for the donor to be eligible, all inclusion criteria must be yes and all exclusion criteria must 
be no. 
Inclusion Criteria 
_____  ≥ 13 and ≤ 75 years of age  
_____ Karnofsky performance status ≥ 70% defined by institutional standards cleared by the NMDP for 
NMDP donors 
_____ Medical history and PE confirm good health status as defined by institutional standards or the 
NMDP for NMDP donors  
_____ Seronegative for HIV-1 RNA PCR;  HIV 1 and HIV 2 ab (antibody);  HTLV-1 and HTLV-2 ab;  
PCR+ or sAg (surface antigen) hepatitis B ;  or PCR+ or sAg for hepatitis C;  negative for the 
Treponema palladum antibody Syphillis screen;  and negative for HIV-1 and hepatitis C by 
nucleic acid testing (NAT) within 40 days of donor apheresis procedures  
In the case that T palladum antibody tests are positive, donors must: 
• 
Be evaluated and show no evidence of syphilis infection of any stage by physical exam 
and history 
• 
Have completed effective antibiotic therapy to treat syphilis  
• 
Have a documented negative non-treponemal test (such as RPR) or in the case of a 
positive non-treponemal test must be evaluated by an infectious disease expert to evaluate 
for alternative causes of test positivity and confirm no evidence of active syphilitic 
disease 
_____ Must be a related or unrelated, HLA-matched donor or 9/10 HLA-matched donor. 
_____ Female donors of child-bearing potential negative serum or urine beta-HCG test  
_____ Capable of undergoing leukapheresis, have adequate venous access, and be willing to undergo 
insertion of a central catheter should leukapheresis via peripheral vein be inadequate 
_____ Agreeable to 2nd donation of PBPC (or bone marrow harvest) in the event of graft failure 
_____ Donor or donor’s legal guardian ≥ 18 years of age, capable of signing an IRB approved consent 
form. 
_____ Meet criteria for donation as specified by standard NMDP guidelines for NMDP unrelated 
donors. 
Exclusion Criteria 
 
_____ Evidence of active infection  
_____ HIV-positive 
_____ Medical, physical, or psychological reason that would place the donor at increased risk for 
complications from growth factor or leukapheresis 
_____ Lactating female 

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______________________________________________ 
_____________________ 
Treating Physician Signature/Print Name 
 
Date 
______________________________________________ 
_____________________ 
Research Coordinator Signature/Print Name 
 
Date 
______________________________________________ 
_____________________ 
Other Third Signature/Print Name 
 
Date 

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B. AE and SAE Reporting Guidelines 
ADVERSE EVENT MONITORING AND REPORTING 
Definitions: 
Unanticipated Problem 
Any incident, experience, or outcome that meets all of the following criteria: (1).  Is expected in 
terms of nature, severity, or frequency in relation to (a) the research risks that are described in the 
IRB-approved research protocol and informed consent document; Investigator’s Brochure or 
other study documents, and (b) the characteristics of the subject population being studied; and  
(2) Is related or possibly related to participation in the research; and  (3)  Places subjects or others 
at a greater risk of harm (including physical, psychological, economic, or social harm) than was 
previously know or recognized. 
Adverse Event (AE) 
Any untoward medical occurrence in a clinical investigation subject administered a 
pharmaceutical product, medical treatment or procedure and which does not necessarily have to 
have a causal relationship with this treatment.  An adverse event can therefore be any unfavorable 
and unintended sign (including an abnormal laboratory finding), a symptom, or disease 
temporally associated with the use of a medicinal product, medical treatment or procedure 
whether or not considered related to medicinal product or treatment. 
Life-threatening Adverse Event 
Any adverse event that places the subject, in the view of the investigator, at immediate risk of 
death from the reaction.  
Unexpected Adverse Event 
An adverse event, the nature or severity of which is not consistent with the applicable product 
information (Investigator’s Brochure, product insert).  For studies that do not involve 
investigational products or devices, an unexpected adverse event is an adverse event that is not 
described in the transplant medical literature or consent form. 
Serious Adverse Event (SAE) 
Any adverse event occurring that results in any of the following outcomes:  death, a life-
threatening adverse event, a persistent or significant disability/incapacity, a congenital anomaly, 
requires intervention to prevent permanent impairment or damage(21CFR§312.32).  The PI is 
also bound by 45 CFR§46, Subpart A which is the “Common Rule” for the Protection of Human 
Subjects, therefore, the Stanford IRB definition applies (report “unanticipated problems” 
involving risks to study participants or others). 
Distinction between Serious and Severe 
The term severe is used to describe the intensity (severity) of a specific event, for example mild, 
moderate or severe.  The event itself however, may be of relatively minor medical significance, 
for example a severe headache.  This is not the same as serious, which is based on the 
subject/event outcome and is usually associated with events that pose a threat to the subject’s life 
or functioning.  Seriousness, not severity, serves as a guide for defining regulatory obligations. 

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Hematopoietic cell transplantation (HCT) is an aggressive therapy for the treatment of a number 
of life threatening malignant and non-malignant disorders.  Individuals presenting for HCT 
generally have exhausted other avenues of therapy that will result in any lasting benefit.  The 
TRM of a sibling myeloablative allogeneic transplant is approximately 20% and the TRM for an 
unrelated myeloablative allogeneic transplant ranges from 20 to 50%.  In the setting of a 
non-myeloabative allogeneic transplant from a sibling donor the TRM is approximately 10% and 
ranges from 20 to 50% for unrelated donors.  
As an aggressive therapy HCT is associated with a large number of AEs and SAEs.  The 
toxicities associated with HCT are related to the following:  1) the underlying disease;  2) therapy 
antecedent to HCT;  3) the health status of the transplant recipient including co-existing 
conditions;  4) the preparative regimen employed in prior to transplant;  5) therapies directed at 
reducing transplant related complications (eg, immunosuppressants for the prevention of GvHD);  
and 6) the treatment of complications of HCT.  
The use of toxicity grading scales such as the NCI CTC is a standard in the medical community 
for the reporting of AEs and SAEs in the investigation of new drugs or devices.  The use of this 
type of scale is less helpful in the evaluation of AEs and SAEs associated with a treatment, such 
as HCT.  In an effort to report to regulatory agencies the toxicities that are relevant and 
meaningful for the evaluation of risks and benefits to potential HCT recipients the following 
guidelines will serve to determine what is reported as AEs and SAEs. 
The following SAEs require reporting to the CCTO.  If the event is unexpected, it will also 
require reporting to the IRB: 
1)  Deaths  
All deaths while the subject is receiving treatment on a protocol up to 90 days after last dose 
of protocol treatment or any death that occurs more 90 days (allogeneic) after protocol 
treatment has ended that is felt to be treatment related. 
This includes deaths from the common and expected Grade 4 toxicities noted below.  Deaths 
that occur outside of Stanford will be reported whenever possible.  It must be noted that 
obtaining detailed information on the cause and circumstances of a death occurring at another 
institution can be difficult.  Excludes deaths related to relapse of underlying disease, which 
will be reported at the time of protocol renewal.  
2) All serious and unexpected toxicities.  
Defined as those toxicities not identified in the transplant literature, product inserts or in the 
consent form. 
The following will generally not be reported as AEs or SAEs: 
1)  Hospitalizations.  
Approximately 50% of allogeneic transplant recipients will be readmitted to the hospital.  
The most common indications for readmission of an allogeneic HCT recipient are fever; 
failure to maintain nutritional status; and graft vs host disease. 
 
 

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2)  Relapse of disease. 
Relapse unfortunately remains a significant problem following both autologous and 
allogeneic transplantation.  The risk of relapse is influenced by both subject and disease 
variables.  
3)  Common and expected Grade 4 toxicities of HCT that are well described in the transplant 
literature, the product inserts or stated in the consent form and do not result in death.  This 
includes but is not limited to neutropenia;  thrombocytopenia;  anemia;  thrombotic 
microangiopathy;  bleeding requiring transfusions;  edema;  hypertension;  hypotension;  
gastritis;  mucositis;  nausea;  vomiting;  diarrhea;  hematuria;  central venous catheter 
infections;  febrile episodes;  sepsis;  mental status changes;  infections;  insomnia;  mood 
alterations;  seizures;  tremor;  pain;  hypoxia;  pleural effusion;  pneumonitis;  incontinence;  
infertility;  laboratory abnormalities;  veno-occlusive disease;  graft failure;  cardiac 
arrhythmias and graft versus host disease.  
4)  Secondary Malignancies. 
The occurrence of secondary malignancies and associated mortality is a known risk of cancer 
therapies.  The occurrence of secondary malignancies will be reported at the time of the 
protocol annual review.  

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C. Grading Graft vs Host Disease 
 
Grading of Individual Organ Systems 
 
Organ 
Stage  
Description 
Skin 
+1 
+2 
+3 
+4 
Maculo-papular eruption over < 25% of body area 
M-P eruption over > 25 to 50% of body area 
Generalized erythroderma 
Generalized erythroderma with bullous formation and often with 
desquamation 
 
Liver 
+1 
+2 
+3 
+4 
Bilirubin 2.0 to 3.0 mg/dL; SGOT 150 to 750IU 
Bilirubin 3.1 to 6.0 mg/dL 
Bilirubin 6.1 to 15.0 mg/dL 
Bilirubin >15.0 mg/dL 
 
Gut 
+1 
+2 
+3 
+4 
Diarrhea > 30 mL/kg or > 500 mL/day 
Diarrhea > 60 mL/kg or > 1000 mL/day 
Diarrhea > 90 mL/kg or > 1500 mL/day 
Diarrhea > 90 mL/kg or > 2000 mL/day; or severe abdominal pain with or 
without ileus 
 
 
Overall Grade 
 
Grade 
Skin 
Liver 
 
Gut 
ECOG Performance 
I 
+1 to +2 
0 
 
0 
0 
II 
+1 to +3 
+1 
and/or 
+1 
0 to 1 
III 
+2 to +3 
+2 to +4 
and/or 
+2 to +3 
2 to 3 
IV 
+2 to +4 
+2 to +4 
and/or 
+2 to +4 
3 to 4