CD8 Depleted, Non-Engrafting, HLA Mismatched Unrelated Donor Lymphocyte Infusion in Patients With MDA and Secondary AML
Phase: PHASE1
Conditions: Myelodysplastic Syndromes, Secondary Acute Myeloid Leukemia
Sponsor: H. Lee Moffitt Cancer Center and Research Institute

 
 
CD8-depleted non-engrafting HLA antigen-mismatched unrelated donor 
lymphocyte infusion in patients with myelodysplastic syndrome and secondary 
acute myeloid leukemia  
Protocol Number: 20042  
 
Principal Investigator: 
Hany Elmariah, MD, MS 
IND/IDE Sponsor/IND Number: 
17305 
Biostatistician: 
Dr. Jongphil Kim 
Radiation Oncologist: 
N/A 
Pathologist: 
N/A 
Radiologist: 
N/A 
Medical Monitor (If Applicable) 
Pending 
 
Additional sites: N/A 
Role 
Site 
 
 
 
 
 
Funded by: Pending. Draft or Version Number: v.10 
29 March 2023 

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Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................. v 
1 
PROTOCOL SUMMARY ............................................................................................................................... 1 
1.1 
Synopsis ................................................................................................................................................ 1 
1.2 
Schema ................................................................................................................................................. 3 
1.3 
Schedule of Activities (SoA) ............................................................................................................... 4 
2 
INTRODUCTION ............................................................................................................................................ 5 
2.1 
Study Rationale .................................................................................................................................... 5 
2.2 
Background........................................................................................................................................... 7 
2.2.1 
Cd4+ Lymphocytes and Antitumor immunity ............................................................... 7 
2.2.2 
CD8+ Depleted Donor Lymphocyte Infusion ................................................................ 9 
2.2.3 
Study Diseases............................................................................................................... 12 
2.3 
Risk/Benefit Assessment .................................................................................................................. 15 
2.3.1 
Known Potential Risks ................................................................................................... 15 
2.3.2 
Known Potential Benefits .............................................................................................. 15 
2.3.3 
Assessment of Potential Risks and Benefits .............................................................. 16 
3 
OBJECTIVES AND ENDPOINTS .............................................................................................................. 16 
4 
STUDY DESIGN ........................................................................................................................................... 16 
4.1 
Overall Design .................................................................................................................................... 16 
4.2 
Scientific Rationale for Study Design.............................................................................................. 17 
4.3 
Justification for Dose ......................................................................................................................... 17 
4.4 
End of Study Definition ..................................................................................................................... 18 
5 
STUDY POPULATION ................................................................................................................................ 19 
5.1 
Inclusion Criteria ................................................................................................................................ 19 
5.1.1 
Donor Selection .............................................................................................................. 21 
5.2 
Exclusion Criteria ............................................................................................................................... 20 
5.3 
Lifestyle Considerations .................................................................................................................... 20 
5.4 
Screen Failures .................................................................................................................................. 21 
5.5 
Strategies for Recruitment and Retention ...................................................................................... 21 
6 
STUDY INTERVENTION ............................................................................................................................ 21 
6.1 
Study Intervention(s) Administration ............................................................................................... 23 
6.1.1 
Study Intervention Description ..................................................................................... 23 
6.1.2 
Dosing and Administration ............................................................................................ 24 
6.1.3 
Duration of Therapy ....................................................................................................... 28 
6.1.4 
Definition of Dose-Limiting Toxicity ............................................................................. 30 
6.2 
Preparation/Handling/Storage/Accountability ................................................................................ 31 
6.2.1 
Acquisition and accountability ...................................................................................... 31 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ................................................ 31 
6.2.3 
Product Storage and Stability ....................................................................................... 32 
6.2.4 
Preparation ...................................................................................................................... 32 
6.3 
Measures to Minimize Bias: Randomization and Blinding ........................................................... 33 
6.4 
Study Intervention Compliance ........................................................................................................ 33 
6.5 
Concomitant Therapy ........................................................................................................................ 33 
6.5.1 
Rescue Medicine ............................................................................................................ 33 

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STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL .................................................................................................................... 34 
7.1 
Discontinuation of Study Intervention ............................................................................................. 34 
7.2 
Participant Discontinuation/Withdrawal from the Study ............................................................... 34 
7.3 
Lost to Follow-Up ............................................................................................................................... 34 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 35 
8.1 
SAFETY Assessments ...................................................................................................................... 35 
8.2 
Other Assessments ........................................................................................................................... 37 
8.3 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES OR OTHER ENDPOINTS ........... 38 
8.4 
Adverse Events and Serious Adverse Events ............................................................................... 41 
8.4.1 
Definition of AE ............................................................................................................... 41 
8.4.2 
Definition of SAE ............................................................................................................ 41 
8.4.3 
Classification of an AE .................................................................................................. 42 
8.4.3.2.2 Reactions mediated by human anti-mouse antibodies (HAMA) ...................................... 44 
8.4.4 
Time Period and Frequency for Event Assessment and Follow-Up ....................... 48 
8.4.5 
AE Reporting .................................................................................................................. 49 
8.4.6 
SAE Reporting ................................................................................................................ 50 
8.4.7 
Reporting Events to Participants ................................................................................. 51 
8.4.8 
Events of Special Interest ............................................................................................. 51 
8.4.9 
Reporting of Pregnancy ................................................................................................ 52 
8.5 
Unanticipated Problems .................................................................................................................... 52 
8.5.1 
Definition of UP............................................................................................................... 52 
8.5.2 
Unanticipated Problem Reporting ................................................................................ 52 
8.5.3 
Reporting Unanticipated Problems to Participants ................................................... 53 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 53 
9.1 
Statistical Hypotheses ....................................................................................................................... 53 
9.2 
Sample Size Determination .............................................................................................................. 54 
9.3 
Populations for Analyses .................................................................................................................. 54 
9.4 
Statistical Analyses............................................................................................................................ 54 
9.4.1 
General Approach .......................................................................................................... 54 
9.4.2 
Analysis of the Primary Endpoint(s) ............................................................................ 55 
9.4.3 
Analysis of the Secondary Endpoint(s) ....................................................................... 56 
9.4.4 
Safety Analyses .............................................................................................................. 56 
9.4.5 
Baseline Descriptive Statistics ..................................................................................... 57 
9.4.6 
Planned Interim Analyses ............................................................................................. 57 
9.4.7 
Subgroup Analyses ........................................................................................................ 57 
9.4.8 
Tabulation of Individual participant Data .................................................................... 57 
9.4.9 
Exploratory Analyses ..................................................................................................... 57 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 57 
10.1 
Regulatory, Ethical, and Study Oversight Considerations ........................................................... 57 
10.1.1 
Informed Consent Process ........................................................................................... 57 
10.1.2 
Study Discontinuation and Closure ............................................................................. 59 
10.1.3 
Future Use of Stored Specimens and Data ............................................................... 59 
10.1.4 
Key Roles and Study Governance .............................................................................. 60 
10.1.5 
Safety Oversight ............................................................................................................. 60 
10.1.6 
Clinical Monitoring .......................................................................................................... 61 

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10.1.7 
Quality Assurance and Quality Control ....................................................................... 61 
10.1.8 
Data Handling and Record Keeping ............................................................................ 61 
10.1.9 
Protocol Deviations ........................................................................................................ 62 
10.1.10 
Publication and Data Sharing Policy ........................................................................... 63 
10.1.11 
Conflict of Interest Policy .............................................................................................. 64 
10.2 
Abbreviations ...................................................................................................................................... 65 
10.3 
Protocol Amendment History ........................................................................................................... 66 
11 
REFERENCES ............................................................................................................................................. 68 
12 
Appendix ........................................................................................................................................................ 74 
12.1 
Appendix A: International Prognostic Scoring System- Revised of Myelodysplastic 
Syndrome(58) ........................................................................................................................................................ 75 
12.2 
Appendix B: Response Criteria According to the International Working Group(1) .................. 76 
12.3 
Appendix C: Evaluation and management of cytokine release syndrome(75) ......................... 77 
12.4 
Appendix D: Consensus conference clinical grading of acute GVHD(80) ................................ 80 
12.5 
Appendix E: Grading of chronic GVHD(81) ..................................................................................... 0 
12.6 
Appendix F: Criteria for Dose Escalation ......................................................................................... 3 
 

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STATEMENT OF COMPLIANCE 
The trial will be conducted in accordance with the ICH E6, the Code of Federal Regulations on the Protection of 
Human Subjects (45 CFR Part 46), and the Supporting Agency Terms. The Principal Investigator will assure that no 
deviation from, or changes to the protocol will take place without prior agreement from the sponsor and 
documented approval from the Institutional Review Board, except where necessary to eliminate an immediate 
hazard(s) to the trial participants. All personnel involved in the conduct of this study have completed Human 
Subjects Protection Training. 
 
I agree to ensure that all staff members involved in the conduct of this study are informed of their obligation 
to meet the above commitments. 
 
 
 
Principal Investigator: 
   
 Print/Type Name 
 
 
Signed: 
   
 Date: 
 

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1 
 
PROTOCOL SUMMARY 
1.1 SYNOPSIS  
Title: 
CD8-depleted non-engrafting HLA-mismatched unrelated donor lymphocyte 
infusion in patients with myelodysplastic syndrome and secondary acute 
myeloid leukemia 
Study Description: 
Phase 1b/2 study of CD8-depleted non-engrafting HLA-mismatched unrelated 
donor lymphocyte infusion in patients with myelodysplastic syndrome (MDS) 
after failure or intolerance of hypomethylating agents (HMAs) or untreated 
secondary acute myeloid leukemia (sAML)  
Objectives: 
1. Determine the maximum tolerated dose (MTD) of CD8-depleted non-
engrafting HLA-mismatched unrelated donor lymphocyte infusion (NE-
DLI) in patients with MDS or sAML 
 
2. Estimate the response rate to therapy with CD8-depleted NE-DLI in 
patients with MDS who have failed therapy with HMAs or untreated 
patients with acute myeloid leukemia (AML) secondary to an 
antecedent hematologic disease (sAML) 
 
Endpoints: 
Primary Endpoint:  
Determine the maximum tolerated dose (MTD) of CD8-depleted cells from HLA-
mismatched unrelated donors infused after induction chemotherapy for 
patients with advanced MDS after HMA failure or untreated sAML 
Secondary Endpoints:  
• 
Overall response rate according to the International Working Group 
(IWG) (see Appendix B).  
• 
Overall survival. 
• 
Progression-free survival.  
• 
Hematologic response/improvement by IWG 2006 criteria for MDS 
patients and the IWG 2003 criteria for AML (1, 2). 
• 
Occurrence of adverse events. 
• 
Unexpected transplant complications (eg, graft-versus-host disease 
[GvHD]). 
Study Population: 
Eligible patients will have a MDS with progression or lack of response after at 
least 4 cycles of a HMA or sAML. HMA failure will be defined by the IWG criteria 
or intolerance to the drug (1)  
Number of Patients: Expected 15 in the phase Ib cohort and 10 in the phase II 
cohort. 28 patients maximum. (The number of patients in the phase I cohort can 
be as high as 18 (6 patients for each dose level).) 
Phase: 
Phase 1b/2 

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Description of 
Sites/Facilities 
Enrolling Participants: 
H. Lee Moffitt Cancer Center – Single Site 
Description of Study 
Intervention: 
Eligible subjects will receive intensive cytotoxic induction chemotherapy with 
any standard of care cytarabine-based regimen. Twenty-four to 36 hours after 
chemotherapy cessation, subjects will receive NE-DLI infusion at a dose 
determined by their dose cohort in phase 1 or the recommended phase 2 dose 
in phase 2. Subjects will remain hospitalized to receive supportive care during 
the period of aplasia, and after being discharged from the hospital they will 
undergo weekly study visits until day 56 from DLI infusion for toxicity 
assessments 
Study Duration: 
3-4 years 
Participant Duration: 
12 months 
 
 

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1.2 SCHEMA 
 
Figure: Example schema in conjunction with 7+3 Chemo. Note that the regimen will vary based on the specific chemotherapy regimen 
chosen by the provider. 
 
 
 

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1.3 
SCHEDULE OF ACTIVITIES (SOA) 
For explanation of procedures, please see Section 8 (Study Assessments and Procedures). 
 
Initial 
Allowable time 
from start of 
induction 
Starting 
Day 0 
Day 28 
Day 
42 
Day 
56 
6 months 
+/- 2 
weeks 
1 yr +/- 2 
weeks 
History and physical 
X 
Within 30 days 
 
 
X 
X 
X 
X 
Performance status 
X 
Within 30 days 
 
 
 
 
 
 
Staging Bone Marrow Biopsy and 
aspirate for research 
X 
Within 30 days 
 
 
Xa 
 
 
 
Peripheral blood for research 
X 
 
Daily 
days 0-7 
 
Xa 
 
 
 
CBC & Diff 
X 
Within 7 days 
ANC<50
0- 
3x/week 
ANC>50
0- 
weekly to 
day 56 
 
X 
X 
X 
X 
CMP 
X 
Within 7 days 
Weekly 
to day 56 
 
X 
X 
X 
X 
CXR or Chest CT 
X 
Within 30 days 
 
 
 
 
 
 
Pregnancy test (women, childbearing 
age) 
X 
Within 30 days 
 
 
 
 
 
 
Human Immunodeficiency Virus 
X 
Within 30 days 
 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
 
MUGA or ECHO 
X 
Within 30 days 
 
 
 
 
 
 
HLA typing 
X 
 
 
 
 
 
 
 
Anti-donor HLA antibody testing 
X 
 
 
 
X 
 
 
 
Adverse Event Monitoring 
 
 
Weekly 
to day 56 
 
X 
X 
X 
X 
GvHD evaluation 
 
 
At onset, 
weekly 
until 
resolves 
 
X 
X 
 
 
Peripheral blood or bone marrow for 
donor chimerism 
 
 
 
 
Xa 
Xb 
 
 
CMV IgG 
X 
Within 30 days 
 
 
 
 
 
 
CMV PCR 
 
 
Weekly 
through 
day 56 
 
 
 
 
 
C Reactive Protein 
X 
Within 7 days 
Weekly 
through 
day 56 
 
 
 
 
 
Ferritin 
X 
Within 7 days 
Weekly 
through 
day 56 
 
X 
X 
 
 
Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count; CMP, comprehensive metabolic panel; CMV, cytomegalovirus; CXR, 
chest radiograph; CT, computerized tomography scan; diff, differential blood count; ECHO, echocardiogram; EKG, electrocardiogram; GvHD, graft-
versus-host disease; MUGA, multigated acquisition; PCR,, polymerase chain reaction  
Tracking of days begins from day of cell transfusion (eg, Day 56 is 56 days after initial cell infusion). Day of cell 
transfusion is marked as Day 0. Procedures can be performed ± 3 days from marked date, unless otherwise 
specified. aSpecified day 42 samples may be performed prior to day 42 if the patient has recovered from aplasia, 
defined as ANC >500/l. Otherwise, the specimen collections will be planned for day 42 +/-3 days.b Day 56 
chimerism measurement may be omitted if donor T-cell chimerism is < 5% on first measurement or if whole 
blood/marrow chimerism is < 5% when T-cell chimerism is not measurable. If donor chimerism >5% on day 56, 
then the investigator will continue performing monthly GVHD evaluations and chimerism until donor T-cell 
chimerism decreases to <5% or until 1 year, whichever comes first. 

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Pre-treatment research samples may be added on to previously collected samples (e.g., Moffitt Total Cancer Care) 
in lieu of collecting new samples. Screening tests obtained as standard of care prior to patient consent to this trial 
may be used for screening purposes if the tests fall within the allowable time window to the start of induction 
chemotherapy as defined in the table above. 
NOTE: In certain clinical circumstances (e.g. relapsed or terminally ill patients, risk of undue harm to patients, etc.) 
study tests may be omitted at the physician’s discretion. Additionally, tests may be rescheduled as clinically indicated. 
2 
 
INTRODUCTION 
2.1 STUDY RATIONALE  
Patients with MDS after hypomethylating agent failure and patients with sAML are characterized by extremely 
poor prognoses.(3-5) These 2 patient groups are treated with cytotoxic induction chemotherapy, and have poor 
rates of overall survival. Survival is improved among patients who achieve a complete remission and are then 
treated with an allogeneic-bone marrow transplant (alloBMT), although this currently represents a minority of 
patients. This patient population is in critical need for new therapeutic options. The goal of this study is to use a 
novel non-engrafting CD8+-depleted DLI product to improve remission rates in patients with MDS with minimal 
toxicity, ultimately to increase the number of patients able to receive curative bone marrow transplant (BMT). 
 
Several observations support the use of immunotherapy in treating MDS and sAML. AlloBMT, which elicits a graft-
versus-tumor immune response, cures MDS and AML in a proportion of patients.(5, 6) In addition, 
immunosuppressive therapy can induce clinical responses in a minority of patients with MDS who have 
hypocellular bone marrow.(7-9) Analyses of responders to immunosuppressive therapies suggest that correction of 
T-cell dysregulation is correlated with clinical benefit.(10) There are anecdotal reports of MDS patients undergoing 
non-myeloablative alloBMTs who, despite losing their allografts, experience sustained responses.(11-13) MDS and 
AML are characterized by upregulation of markers of antitumor exhaustion such as programmed cell death protein 
1 (PD1), PD1 ligand (PDL1), and cytotoxic T lymphocyte associated protein 4 (CTLA4) which is both predictive of 
hypomethylating agent (HMA) failure and exacerbated by HMA treatment.(14, 15) This suggests that strategies to 
reverse antitumor exhaustion should be effective in these diseases. Indeed, the therapeutic benefit of adoptive 
cellular therapies, such as DLI and microtransplantation, may result from this mechanism.(16-18)  
 

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We propose that a source of exogenous, immunocompetent, HLA-mismatched CD8+-depleted CD4+ DLI would be 
capable of reversing immune tolerance and eliciting a significant antitumor response in patients with MDS and 
sAML. CD8+ depletion is intended to allow CD4+ help to be provided to exhausted host CD8+ T cells, many of which 
express T-cell receptors specific to tumor epitopes.(19, 20) If cytotoxic-competent donor CD8+ T cells are included, 
they may blunt the activity of host CD8+ T cells via an allogeneic mixed lymphocyte reaction.(21) Additionally, 
higher doses of CD8+ T cells would increase the likelihood of engraftment and, subsequently, lead to GvHD, 
morbidity, and mortality. Unlike other types of cellular therapy, this strategy would not require a specific antigenic 
target.  
 
On the basis of prior literature, starting doses of 106 CD4+ T cells/kg (with <104 CD8+ T cells/kg) are likely to be 
well tolerated. Grafts containing <104 CD8+ T cells/kg do not cause severe GvHD, even among patients receiving 
lethal conditioning and no pharmacologic immunosuppression after transplantation.(22-24) The negligible risk of 
GvHD following CD8+ depletion permits the use of HLA-mismatched unrelated donors. By choosing HLA-
mismatched, unrelated donors, we hope to induce more significant antitumor alloreactivity, and avoid recipient 
alloimmunization against potential HLA-matched BMT donors. This allows the treatment to be used in the pre-
transplant setting. Additionally, eliminating HLA matching increases the donor pool, allows for a more readily 
available product, and lowers the cost of preparation.  
 
NE-DLI has the potential to be an easily prepared and accessible product. Without the need for HLA matching, it 
has the potential to be used as a standard infusional product, similar to packed red cell or platelet transfusions. 
Ultimately, it is feasible that this could become a widespread therapy that is accessible to smaller medical centers, 
as well as tertiary and quaternary referral centers.  
 
Initially, this therapy will be combined with induction chemotherapy to improve remission rates. If successful, the 
low expected toxicity could be a boon for affected patients, who are often frail and elderly. As NE-DLI will 

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ultimately be rejected by the recipient immune system, the therapeutic benefit may be temporary; therefore, the 
treatment is intended to serve as a bridge to consolidative BMT.  
 
2.2 BACKGROUND  
In 1966, Schwarzenberg, et al showed that transfusion of leukocytes could elicit remissions in patients with 
refractory acute leukemia.(25) Since that time, DLI has become a standard therapy, generally given after alloBMT 
to boost antitumor immunity and donor engraftment, but the therapeutic benefit is nearly offset by high rates of 
GvHD and mortality.(25-30) The effectiveness of DLI has provided clinical evidence that the immune system can be 
manipulated to successfully treat myeloid malignancies. Current investigations are, thus, aimed at harnessing 
antitumor immunity while decreasing toxicity. 
 
We propose that a novel therapy consisting of exogenous, immunocompetent, HLA-mismatched CD8+ depleted 
CD4+ DLI for hematologic malignancies would be capable of eliciting a significant antitumor response without the 
concomitant toxicities of standard DLI.  
2.2.1 
CD4+ LYMPHOCYTES AND ANTITUMOR IMMUNITY 
Antitumor immunity is a critical component in regulating the proliferation of cancer cells. CD4+ T cells are the 
conductors of the immunologic response, coordinating effector components that include CD8+ cytotoxic T cells, 
macrophages, B cells and natural killer cells. CD4+ helper T cells are also the coordinators of immune memory,(31) 
and are essential for an effective and sustained immune response against cancer cells. Tumors evade the immune 
system and induce T-cell tolerance through multiple mechanisms, including decreased expression of immunogenic 
antigens,(32, 33) manipulation of cytokine balance in the tumor microenvironment,(34, 35) and upregulation of 
immune checkpoint molecules, such as programmed cell death protein 1, PD1 ligand, and cytotoxic T lymphocyte 
associated protein 4.(36)  
In tumor‐induced tolerance, the CD4+ helper T cells that recognize the tumor and initiate the host defense 
mechanisms become paralyzed.(37) Without CD4+ help, CD8+ T cells, though capable of recognizing antigens, fail 

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to remember the encounter and ultimately lose the capacity to produce cytokines or kill target cells. Thus, the 
CD8+ T cells themselves become exhausted.(38) A variety of tumor and viral infection experimental models have 
shown that the provision of allogeneic CD4+ T-cell help can prevent CD8+ T cells from becoming exhausted(39) and 
can reverse established exhaustion, enabling CD8+ T cells to respond appropriately.(40, 41) This restoration of help 
addresses the fundamental driver of tumor evasion and offers a potential foundation for therapy of all cancers and 
premalignant syndromes. 
 
Among the strategies to restore antitumor immunity is allogeneic DLI. Although the mechanisms by which DLI 
stimulates an antitumor effect are not completely understood, there is evidence to support the effect is largely 
dependent on CD4+ T cells (Figure 1). Immunocompetent donor CD4+ helper T cells are capable of recognizing 
recipient HLA Class II antigen molecules as foreign, which initiates proliferation of alloreactive CD4+ T cells.(42-44) 
Once activated, donor T cells develop memory against the inciting antigen and initiate a signaling pathway 
mediated by antigen-presenting cells; this pathway is necessary to activate cytotoxic effector cells.(45) This process 
involves upregulation of CD40L ligand on the CD4+ T cells, which binds to CD40 on the antigen-presenting cell, (46, 
47) and subsequent upregulation of CD70 on the antigen-presenting cell that interacts with CD27 on CD8+ 
cytotoxic T cells.(31, 48) The CD4+ T cells additionally release cytokines that further stimulate the immune 
response.(49) Meanwhile, CD8+ cytotoxic T cells recognize foreign antigens, such as tumor antigens, presented on 
HLA class I proteins and, with the stimulatory signals provided from the CD4+ T cells, become active against the 
tumor.(40, 41) Without effective CD4+ T cells, the potency of antitumor immunity is lost, but through these 
mechanisms, introduction of foreign CD4+ T cells are expected to reactivate the antitumor immune response.  

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Figure 1: Schematic showing the role of CD4+ T helper cells in stimulating CD8+ T cells against leukemia antigens 
The role of CD4+ T lymphocytes in antitumor immunity suggests that an infusion of donor CD4+ T lymphocytes may 
be able to promote a response in any CD8+ T cells able to recognize foreign antigens on the HLA class I antigen 
molecule. Theoretically, a donor CD4+ T cell could stimulate either a host- or donor-derived tumor-specific CD8+ T 
cell to create an antitumor effect. However, this mechanism also creates the problem of stimulating donor-derived 
host-specific CD8+ T cells against the host, thus precipitating GvHD. Indeed, in clinical practice, DLI leads to a 
potent boost in antitumor immunity known as graft versus leukemia (GvL), but the therapeutic benefit is nearly 
offset by high rates of GvHD and associated mortality.(25-30) Separating GvL and GvHD is a key problem in 
maximizing the benefit of DLI and other allogeneic immune therapies.  
 
2.2.2 
CD8+ DEPLETED DONOR LYMPHOCYTE INFUSION 
The mechanism of DLI suggests that depletion of donor CD8+ T cells from the DLI product may preserve antitumor 
immunity while decreasing GvHD. Prior animal studies have shown that CD8+ depleted DLI creates an antitumor 

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effect and improved survival without concomitant GvHD.(24) That same study showed that CD4+ CD8+ DLI did 
cause GvHD, depletion of both CD4+ and CD8+ T cells led to a loss of antitumor immunity, and CD4- CD8+ 
lymphocytes led to GvHD without an antitumor effect. Further, infusion of CD4+ CD8- DLI in mice that lacked host 
CD8+ T cells led to a decrease of the therapeutic effect of the DLI. The findings suggest that the antitumor effect of 
DLI requires donor CD4+ and host CD8+ lymphocytes, while donor CD8+ cells are unnecessary and, indeed, 
detrimental, as they precipitate GvHD.  
 
In humans, CD8+-depleted DLI has been attempted in the post-transplant setting.(16, 23, 50) In a prospective study 
by Soiffer and colleagues, subjects with mixed chimerism were randomized to CD8+ depletion, receiving a median 
of 0.7 x 105 CD8+ cells/kg versus 32.0 x 105 CD8+ cells/kg in the unmanipulated cohort.(23) There was less GvHD in 
the CD8+ depleted cohort (0/9 versus 6/9), and incidences of relapse and mortality were similar in this small 
patient set. No other significant toxicities were reported. Hence, although sustained engraftment and resultant 
GvHD would be expected after DLI in the post-allogeneic transplantation setting, CD8+ depletion negated the 
incidence of GvHD while retaining antitumor activity. In a review of CD8+ depleted DLI cases in patients with 
relapsed chronic myeloid leukemia after BMT, response to therapy was most strongly correlated with the presence 
of host CD8+ T cells infiltrating the marrow.(16) Further, clinical responses to CD8+ depleted DLI were associated 
with down-regulation of genetic markers of exhaustion in the resident T-cell repertoire, consistent with the 
hypothesis that donor CD4+ T cells provided help to reverse CD8+ T-cell exhaustion.(16)  
 
Although DLI is commonly used after transplant, there is limited experience using this therapy before 
transplantation.(51) Recently, investigators in China developed a technique called “microtransplantation” in which 
patients were infused with HLA -mismatched, granulocyte colony-stimulating factor stimulated allogeneic 
lymphocytes from related donors in conjunction with standard chemotherapy, resulting in only temporary levels of 
donor microchimerism (<1% donor).(17, 18, 52) In their initial trial, elderly patients with AML treated with 
induction chemotherapy and microtransplantation achieved significantly higher remission rates than those with 
standard induction (80% versus 43%), improved disease free survival, and no incidence of GvHD or increase in 

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infectious complications.(52) They subsequently showed that combining microtransplantation with standard 
consolidation improved overall survival and disease free survival in comparison with historical controls in low- and 
intermediate-risk AML patients.(17) Most recently, Hu et al applied the technique in patients with MDS or AML 
from antecedent MDS.(18) The MDS group achieved complete remission rates of 52% and 2-year overall survival of 
85%, compared with complete remission of 36% and overall survival of 34% in the AML group. Again, no GvHD was 
observed. Some patients did experience infectious complications, though not significantly more than would be 
expected from chemotherapy alone. Although this microtransplantation technique did not involve CD8+ depletion, 
the authors did report an increase in host CD8+ T-cell response after treatment, suggesting the therapeutic 
mechanism may be reactivation of recipient-versus-tumor immunity through donor-cell help. Similar studies in the 
United States are ongoing and registered at clinicaltrials.gov.  
 
At Johns Hopkins, 10 heavily pre-treated patients with hematologic malignancies and no validated therapeutic 
options were treated on protocol J0551. All patients received conditioning of cyclophosphamide 50 mg/kg/day IV 
for 2 days. The next day, they received an infusion of related HLA-haploidentical leukocytes, from which CD8+ T 
cells had been removed. A dose of 1 x 105 CD4+ cells/kg was infused into 5 patients and was well tolerated without 
a dose-limiting toxicity (DLT). There was 1 partial response in a patient with MDS. Three patients received 1 x 106 
CD4+ cells per kilogram without DLT. One of these patients achieved complete remission of refractory diffuse large 
B cell lymphoma. One patient received 1 x 107 CD4+ cells per kilogram and had fatal cytokine release syndrome. 
Notably, that event occurred before widespread recognition of cytokine release syndrome and data to support 
new treatment options such as tocilizumab.(53) A final patient received 3x106 CD4+ cells; the patient did not suffer 
any treatment-related toxicities but there was no disease response to the treatment. This unpublished work 
helped determine the MTD with this conditioning regimen and cell source, and demonstrated responses to the 
procedure. 
 
In summary, CD4+ T-cell help is necessary for a potent antitumor immune response. Exhaustion of CD4+ T cells 
allows tumors to evade the immune system. Replacement of the exhausted CD4+ T cells with immune competent 

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CD4+ T cells from an allogeneic donor is expected to reignite the recipient’s antitumor immune response without 
significant risk of developing GvHD. 
2.2.3 STUDY DISEASES 
2.2.3.1 MYELODYSPLASTIC SYNDROME 
MDS is a clonal disorder of hematopoietic stem cells that results in defects in blood cell production and attendant 
morbidity and mortality.(54, 55) It is prevalent in older adults, (56) so low toxicity treatments are prioritized, 
although alloBMT is the only curative treatment modality. As MDS progresses, it has the potential to transform 
into AML, which is often managed aggressively with induction chemotherapy but portends a poor prognosis.(57) 
 
Management decisions in MDS are based on the Revised International Prognostic Scoring System (IPSS-R)a,(58) 
which includes cell counts, bone marrow blast percentage, and cytogenetics of the malignant clone to determine 
risk of death or transformation to AML. Very low and low-risk patients are managed with observation, 
erythropoiesis stimulating agents, or transfusion support, and median survival is measured in years. Higher risk 
patients have a median survival of less than 2 years, and therapy directed against the malignant cells is typically 
indicated.(58)  
 
The HMAS azacitidine and decitabine are the standard of care treatments in higher risk MDS. Both offer an 
improvement in rates of freedom from transfusion, and azacitidine has been shown to improve overall survival 
from 15 months to 24.5 months, compared to the investigators’ preselection of best supportive care, low-dose 
cytarabine, or intensive chemotherapy.(59) Although DNA methyltransferase inhibitors mark an improvement over 
chemotherapy, only 50% of patients will experience a response,(1, 3-5, 59) and the median duration of response is 
13.6 months. Clinical remissions in these studies last less than 4 months. The prognosis of patients who experience 
                                                                    
 
 
a See appendix A 

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progressive disease on azacitidine or decitabine is especially poor, with median survival of less than 6 months 
without treatment.(5, 60) 
 
Conventional AML-like induction chemotherapy is the standard of care for MDS patients who fail HMA therapy. 
This approach for patients with MDS after HMA failure can produce remissions, although infrequently, and survival 
is dismal. For example, Prebet et al showed intensive induction chemotherapy achieved complete remission in only 
3 out of 22 patients failing azacitidine and in 2 out of 10 patients failing decitabine, and median survival was less 
than 9 months (Figure 2).(5) Other studies have similarly reported median overall survival (OS) typically less than 1 
year once treatment is initiated.(57, 61, 62)  
 
 
Figure 2: Overall survival outcomes for MDS patients after HMA failure by treatment modality as reported by 
Prebet, et al.(63)  
 
As patients who do respond to induction chemotherapy achieve only a transient benefit, alloBMT is necessary to 
prevent relapse. This therapy is associated with the best outcomes in these patients, with reported median survival 
estimates as high as 19.5 months and long-term cure in approximately 40% of patients.(5) However, remission is 
typically required before BMT, which leaves the 80% of patients unable to achieve remission after HMA failure 

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ineligible for the procedure. Hence, novel therapies are needed to improve remission rates and make more 
patients eligible for alloBMT. 
 
2.2.3.2 SECONDARY ACUTE MYELOID LEUKEMIA 
AML is a clonal malignancy of myeloid cells characterized by an increase in abnormal, immature myeloid blasts and 
hematologic complications, including leukostasis, cytopenias, infections, bleeding, and death.(64) AML can arise de 
novo or through progression of an antecedent hematologic disorder (AHD), such as MDS or a myeloproliferative 
neoplasm. In patients diagnosed with MDS, the risk of progression to AML varies depending on severity of disease 
and cytogenetics, with high-risk patients progressing within months and low-risk patients progressing over many 
years.(58) As MDS is more prevalent in the elderly population, it follows that nearly 40% of AML cases in patients 
above 60 years of age are sAML arising from an antecedent disorder.(65)  
 
Although MDS patients must be monitored closely for risk of progression to AML, many elderly patients with sAML 
first present after transformation, unaware of having had an AHD. The 2008 World Health Organization 
(WHO)classification system describes criteria to identify these cases.(66) By these criteria, antecedent MDS can be 
presumed on the basis of dysplasia of at least 50% of cells in at least 2 lineages, or presence of characteristic 
cytogenetic abnormalities, such as monosomy 5 and monosomy 7.(66) Although not included in the WHO criteria, 
subsequent studies have shown that somatic mutations in multiple genes, including SRSF2, SF3B1, U2AF1, ZRSR2, 
ASXL1, EZH2, BCOR, or STAG2, are >95% specific to WHO-defined sAML.(67) The ability to identify sAML is 
important to guide treatment options and predict prognoses.  
 
The treatment paradigm for sAML is similar to MDS after HMA failure, often using induction chemotherapy 
followed by consolidative BMT when possible. However, the prognosis is quite poor, particularly in elderly 
patients, due both to disease biology and an inability to tolerate aggressive treatment. In one retrospective study, 
61 patients with sAML treated with induction chemotherapy had a 59% remission rate but only a 6.5 month 
median OS.(68) Another similar study evaluated outcomes of AML patients above 60 years of age who were 

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treated with induction chemotherapy.(65) The complete remission rate was 43%, although the median OS was 
only 107 days. This study included both patients with and without an AHD, but presence of an AHD was correlated 
with a worse prognosis. The discordance between remission rates and long-term survival in these studies 
highlights the fact that most of these patients will die from rapid relapse or treatment-related toxicities. For those 
patients with sAML who respond well to induction chemotherapy, alloBMT can yield 3-year survival rate as high as 
40%.(69) Even in the elderly population who account for most sAML cases, OS rates at 3 years approach 40%.(70)  
These studies demonstrate both the dismal prognosis of patients with sAML and the potential for a fortunate few 
to achieve long-term survival with BMT. It is estimated that only 6% of elderly patients with AML are able to 
proceed to BMT because of early relapse, inability to achieve remission, or decline in functional status during 
induction.(71) Hence, novel treatments to increase the number of patients able to undergo BMT could 
dramatically improve the overall prognosis of this disease.  
   
In summary, patients with MDS who have failed HMAs or progressed to sAML represent a population with an 
unmet clinical need. New treatment options are necessary to increase the number of patients who achieve 
complete remissions without significant treatment-related morbidity, thus making more patients eligible for 
curative BMT.  
2.3 RISK/BENEFIT ASSESSMENT  
 
2.3.1  KNOWN POTENTIAL RISKS  
The most serious potential risks of the proposed treatment are death, cytokine release syndrome, and prolonged 
aplasia.  
2.3.2 KNOWN POTENTIAL BENEFITS  
Patients eligible for this study will be those with MDS after HMA failure and/or those with sAML. These groups lack 
effective treatment options. NE-DLI is expected to improve remission rates in these patients to increase their 
likelihood of reaching potentially curative allogeneic BMT.  
 

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2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
 
Patients included in this study are those with aggressive myeloid malignancies who lack adequately effective 
treatment options. This study offers a novel therapeutic option that may improve their overall prognosis. The 
study is designed to minimize potential adverse effects of the therapeutic DLI. In addition to low starting doses of 
lymphocytes, safety is bolstered by the anticipated rejection of the infusion without engraftment. Although 
chemotherapy can allow persistence and activity of allogeneic cells, the intensity of AML induction chemotherapy 
is less than that which allows sustained donor chimerism.(72) Additionally, HLA mismatch increases the likelihood 
of graft rejection before engraftment(73), and CD8+ depletion decreases the likelihood of engraftment.(74) Finally, 
cytokine release syndrome can be managed successfully with supportive care and tocilizumab.(53) 
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION 
FOR ENDPOINTS 
Primary 
 
 
 
Determine the MTD of CD8+ 
depleted, non-engrafting, HLA-
mismatched, unrelated donor 
lymphocyte infusion in patients 
with MDS or sAML. 
 
The MTD of CD8+ depleted cells from HLA-
mismatched, unrelated donors infused after 
induction chemotherapy for patients with 
advanced MDS after HMA failure or untreated 
sAML. 
This is a new therapy and 
the optimal dose is 
unknown. Thus, dosing is 
the primary endpoint as 
this improves safety for 
patients on the trial.  
Secondary 
 
 
 
Estimate the response rate to 
therapy with CD8+ depleted non-
engrafting HLA-mismatched, 
unrelated donor lymphocyte 
infusion in patients with MDS 
who have failed therapy with 
HMAs or untreated patients 
with AML having transformed 
from an antecedent hematologic 
disease (sAML). 
 
1. Overall response rate according to the IWG.  
2 Overall survival 
3. Progression-free survival  
4. Hematologic response/improvement by IWG 
2006 criteria for MDS patients and the IWG 
2003 criteria for AML  
5. Occurrence of adverse events.  
6. Unexpected transplant complications (eg, 
GvHD). 
 
These are clinically 
significant endpoints for 
evaluating the efficacy of 
treatments for MDS and 
AML. 
 
 
4 
STUDY DESIGN  
4.1 OVERALL DESIGN 
This is a standard phase Ib/II trial design. All patients will be treated on a single arm. The objective of the phase I 
portion of the trial will be to determine the MTD of CD8+-depleted peripheral blood cell leukocytes (CD8- PBCs) 

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when infused after cytotoxic chemotherapy. In the phase II portion of the trial, the efficacy of the treatment 
combined with cytotoxic chemotherapy will be determined. This is a phase 1b/2 study with dose escalation for 
determination of the MTD/recommended phase 2 dose (RP2D) of NE-DLI via a traditional 3+3 dose-escalation 
schema (see Appendix F) followed by an expansion cohort for assessing safety profile and preliminary estimation 
of the overall response rate (complete response and partial response based on IWG definition). Eligible patients 
will be treated with NE-DLI in the pre-transplantation setting. 
 
4.2 SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
Patients with MDS who have failed HMAs or progressed to sAML represent a population with an unmet clinical 
need. New treatment options are necessary to increase the number of patients who achieve complete remissions 
without significant treatment-related morbidity, thus making more patients eligible for curative BMT. We propose 
that a source of exogenous, immunocompetent, HLA-mismatched CD8+-depleted CD4+ DLI would be capable of 
reversing immune tolerance and eliciting a significant antitumor response in patients with MDS and sAML. CD8+ 
depletion is intended to allow CD4+ help to be provided to exhausted host CD8+ T cells, many of which express T-
cell receptors specific to tumor epitopes.(19, 20) If cytotoxic-competent donor CD8+ T cells are included, they may 
blunt the activity of host CD8+ T cells via an allogeneic mixed lymphocyte reaction.(21) Additionally, higher doses 
of CD8+ T cells would increase the likelihood of engraftment and, subsequently, lead to GvHD, morbidity, and 
mortality. Unlike other types of cellular therapy, this strategy would not require a specific antigenic target. 
4.3 JUSTIFICATION FOR DOSE 
 
For the dose-escalation portion, a minimum of 2 subjects and a maximum of 18 subjects will be enrolled. There will 
be 3 dose levels in the dose-escalation portion. MTD is defined as the highest dose level with the probability of DLT 
less than 33% (DLT events are defined in Section 6). The DLT observation window will be within 56 days following 
NE-DLI. If none of 3 patients experience a DLT, we will escalate to the next dose level. If one of the 3 patients 
experiences a DLT, we will enroll 3 additional patients and treat at the same dose level. In this case, if none of 
these additional 3 patients experiences a DLT, we will escalate to the dosage to next level. If 2 or more patients 

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among 6 enrolled patients experience a DLT, or 2 or more patients in the 3 patient cohort experience DLT, the 
dosage level exceeds the MTD and the previous dose level will be declared as the MTD. If the first dose level 
exceeds the MTD, the study will be suspended. If no subjects experience a DLT at any dose level, the MTD will be 
level 3 (5 x 107 CD4+ T cells/kg).  
The RP2D is defined as the dose level selected to be evaluated in the expansion cohort or future phase II studies 
and will be equivalent to the MTD determined in the phase 1b portion of the study. Once we determine the RP2D 
level, we will enroll 10 additional eligible patients and treat with the RP2D level. The goal for this expansion cohort 
is to evaluate the safety and tolerability for patients who will be treated with NE-DLI in the RP2D dose level, and to 
evaluate the preliminary efficacy estimate for the ORR. Including the dose-escalation portion in which 3 or 6 
enrolled patients will have been treated at the RP2D level, a total of at least 13 patients will be treated at the RP2D 
level. Based on previous data,(18) the complete response rate using microtransplantation was in the range of 36%-
52% for MDS or AML from antecedent MDS patients. Hence, for the phase 2 portion, an ORR of 45% is considered 
clinically meaningful. With a sample size of 13 patients, if our ORR is 45%, the 95% CI (exact binomial) will be (18-
74%); if the ORR is 50%, the 95% CI will be (22-78%).  
 
4.4 END OF STUDY DEFINITION 
The phase I portion of the study will continue until reaching the MTD to determine the RP2D. The phase II portion 
of the study will continue until an additional 10 patients are treated at the RP2D. The maximum total number of 
patients enrolled in this study will be 28 (6 per dose level plus 10 in the expansion cohort). We expect that the 
accrual rate will be one patient per every 5-6 weeks. With the DLI observation window (56 days after DLI), the 
dose-escalation portion may take up to a maximum of 2.5 - 3 years to complete and an additional 1.5 years for the 
expansion cohort.  
A participant is considered to have completed the study if he or she has completed all phases of the study 
including the last visit or the last scheduled procedure shown in the SOA, Section 1.3. 
 

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The end of the study is defined as completion of the last visit or procedure shown in the SOA in the trial globally. 
 
5 
STUDY POPULATION 
5.1 
INCLUSION CRITERIA 
Eligible subjects will have MDS and have failed treatment previously with HMAs or AML arising from an AHD 
(sAML), defined as meeting either item 1 or item 2 in the inclusion criteria below. All patients must fulfill item 3. 
Eligible patients will be registered with the Clinical Research Office after signing the consent form. A registration 
may be cancelled in writing provided the subject has not begun protocol therapy. 
1. MDS having failed HMA: 
a. Age 18-79 years, inclusive 
b. Pathologically confirmed MDS or myelodysplastic/myeloproliferative overlap (MDS/MPN) 
c. 
IPSS-R score intermediate, high, or very higha 
d. Must have failed therapy with an HMA (defined as lack of response by IWG response criteria(1) 
or intolerance of the drug). 
2. sAML 
a. Pathologically confirmed AML according to WHO criteria. 
b. Evidence of an antecedent hematologic disorder (AHD) prior to acute leukemia including a 
known prior diagnosis of MDS, MPN, or MDS/MPN or data suggestive of an AHD such as 
cytopenias, fibrosis, macrocytic anemia, cellular or dysplasia at or prior to the time of diagnosis. 
If available, MDS-defining karyotypes (-7/del(7q), -5/del(5q), del(13q), del(11q), del(12p), t(12p), 
del(9q), idic(X)(q13), t(17p) (unbalanced translocations) or i(17q) (ie, loss of 17p), 
t(11;16)(q23;p13.3), t(3;21)(q26.2;q22.1), t(1;3)(p36.3;q21), t(2;11)(p21;q23), inv(3)(q21q26.2), 
t(6;9)(p23;q34)) or somatic mutations in multiple genes including p53, TET2, JAK2, CALR, MPL, 
ASXL1, RUNX1, SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 would also confirm 
eligibility. 
c. 
Age 18-79 years, inclusive 
d. May be previously untreated 
3. For both cohorts: 
a. ECOG performance status of 0-2 
b. Deemed eligible to receive cytotoxic chemotherapy 
c. 
Creatinine clearance (CrCl)>50ml/min 
d. Total bilirubin <2 mg/dL (except for patients with Gilbert’s disease), AST and ALT < 3x ULN 
e. Left Ventricular Ejection Fraction ≥ 50% 
                                                                    
 
 
a See Appendix A 
 

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f. 
Willing and able to participate in study assessments 
Enrollment will be open to all without prejudice.  
5.2 EXCLUSION CRITERIA 
Exclusion criteria: 
1. Patients who have had systemic chemotherapy or radiotherapy within 4 weeks (6 weeks for nitrosoureas 
or mitomycin C) prior to entering the study or those who have not recovered from adverse events due to 
agents administered more than 4 weeks earlier. Hydroxyurea and/or targeted therapies (including but not 
limited to BCL2 inhibitors, JAK2 inhibitors, FLT3 inhibitors) during this period may be given as a bridging 
therapy to maintain disease stability while awaiting trial treatment. Intrathecal chemotherapy within this 
time frame is permitted. Intrathecal chemotherapy may be continued during protocol therapy in order to 
consolidate or maintain a central nervous system (CNS) remission, but not to treat active CNS disease 
2. Acute promyelocytic leukemia, or the presence of t(15;17) 
3. Patients receiving any other investigational agents 
4. Uncontrolled concurrent illness including, but not limited to, ongoing and uncontrolled infection, 
symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric 
illness/social situations that would limit compliance with study requirements 
5. Pregnant women are excluded from this study because there is an unknown but potential risk for adverse 
events in the fetus. Breastfeeding should be discontinued if the mother is treated. These potential risks 
may also apply to other agents used in this study 
6. Patients who have any debilitating medical or psychiatric illness that would preclude their giving informed 
consent or their receiving optimal treatment and follow-up 
7. Patients with a poor functional status of ECOG 3-4, or otherwise deemed unfit to tolerate induction 
chemotherapy. 
8. Patients with blastic transformation of chronic myelogenous leukemia are ineligible 
9. Exposure to a humanized mouse chimeric antibody within 1 year of starting trial therapy,  as this could 
sensitize patients to components of the CD8 depletion column that may be present in small amounts in 
the cell product 
10. Prior allogeneic hematopoietic cell transplant. 
5.3 LIFESTYLE CONSIDERATIONS 
 
During this study, participants are asked to: 
• 
Refrain from consumption of red wine, Seville oranges, grapefruit or grapefruit juice, (pomelos, exotic 
citrus fruits, grapefruit hybrids, or fruit juices) from 1 day before the start of chemotherapy until after the 
final dose. 
• 
Abstain from alcohol for 24 hours before the start of each dosing session until after collection of the final 
PK and/or pharmacodynamic sample.  
• 
Abstain from use of tobacco products, as the use of nicotine-containing products (including nicotine 
patches) will not be permitted while they are in the clinical unit.  
• 
Minimize interactions with household contacts who may be immunocompromised. 
 

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5.4 SCREEN FAILURES 
 
Screen failures are defined as participants who consent to participate in the clinical trial but are not subsequently 
randomly assigned to the study intervention or entered in the study. A minimal set of screen failure information is 
required to ensure transparent reporting of screen failure participants, to meet the Consolidated Standards of 
Reporting Trials (CONSORT) publishing requirements and to respond to queries from regulatory authorities. 
Minimal information includes demography, screen failure details, eligibility criteria, and any serious adverse event 
(SAE). 
 
Screen failure individuals who do not meet the criteria for participation in this trial because of a reversible risk 
factor may be rescreened after resolution/treatment of the risk factor. Rescreened participants should be assigned 
the same participant number as for the initial screening. 
 
5.5 STRATEGIES FOR RECRUITMENT AND RETENTION 
 
Eligible patients will be identified and offered the study during clinic visits and/or hospital admissions. Referrals to 
the trial are expected from outpatient Leukemia clinic and BMT clinic.  
5.6 
DONOR CRITERIA 
5.6.1 DONOR ELIGIBILITY 
I. 
Inclusion Criteria: 
a. Age  18 
b. Weight  110 pounds 
c. 
HCT  30% 
d. PLT > 100k 
e. WBC  3.50k 
 
II. 
Exclusion Criteria: 
1. AB blood type 
2. Positive testing for or known history of a communicable infectious disease  
3. Unwilling or unable to provide informed consent 
4. Has a medical contraindication to blood donation  
5. Unwilling to donate during business hours, Monday-Friday 
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5.6.2 DONOR RECRUITMENT 
This trial will require unrelated subjects who are willing to donate blood to be used for NE-DLI, via either simple 
phlebotomy or mononuclear cell (MNC) collection by apheresis.  Moffitt will contract with the New York Blood 
Center (NYBC) to use their existing donor pool and to collect the cells and ship to Moffitt for processing and 
infusion.  In all cases, donor eligibility must be in compliance with 21 CFR 1271 and Foundation for the 
Accreditation of Cellular Therapy (FACT) standards. The donation procedure will be managed per institutional 
standard of care for apheresis/DLI donation and governed by the donor’s procedural consent.  
Recruitment: The New York Blood Center has an existing pool of volunteer donors who have agreed to be donors 
for blood product donations, either for standard of care or other clinical trials. The NYBC will invite donors from 
their existing pool to volunteer as donors for this trial. No donors with AB blood types will be recruited. Donors 
that respond to recruitment and agree to participate will undergo HLA testing and CMV testing, if not already 
performed as part of routine blood donation, and will be consented for possible cell collection. Because the 
desired donor requires only a class II HLA mismatch,  HLA testing should include at least HLA DRB1, DQB1, and 
DPB1 as these are the markers required for donor selection. 
  
Following consent, they will complete a health history questionnaire for eligibility as per FACT standards.  
 
Potentially eligible donors chosen to donate will undergo routine FACT work up including physical exam within 6 
months of cell donation and infectious disease testing and final donor clearance within 1 month of cell donation: 
1. Infectious disease markers to rule out communicable infectious diseases.  
a. Positive results on infectious disease testing:  
i. Subjects will be informed of all positive results per standard procedures.    
ii. Mandatory reporting: Reportable diseases or conditioning will be reported to the 
State Department of Health based on the state of residence of the donor.  
iii. A proportion of potential donors likely will be determined to be ineligible based 
upon serologic infectious disease testing. 
2. Donor clearance visit with a provider in the Moffitt Department of Blood and Marrow 
Transplantation/Cellular Immunotherapy or a provider at the contracted collecting institution 
including physical exam and review of systems and complete blood counts and comprehensive 
metabolic panel  
 
Size of the potential donor pool:  Based on the likelihood of finding HLA mismatched donors and the need for 
backup donors in case of unavailability of the preferred donor, the viability of this study will require a goal of at 
least 30 volunteer donors in the PDP. As experience with the conduct of this trial progresses, the target size may 
be adjusted upwards or downwards to reflect HLA patterns, donor suitability and sustainability, and on-call donor 
availability.  
 
5.6.3 DONOR SELECTION 
Donors will be chosen from a pool of previously HLA-typed healthy volunteers. Selection of donors will be 
performed by the study PI.  
1. The donor must satisfy criteria, including results of serologic testing for infectious diseases, established by 
the National Marrow Donor Program and the Foundation for the Accreditation of Cellular Therapies 
(FACT) for MNC, Apheresis, donation, and leukapheresis. 

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2. The donor may not be a first or second degree relative of the patient.  
3. There must be at least one HLA Class II (HLA-DRB1, HLA-DQB1, or HLA-DPB1) antigen mismatch between 
the donor and recipient in the donor anti-recipient (graft-versus-host) direction. 
4. The donor must not have a major ABO incompatible red blood cell type: 
a. If the recipient is blood type O, then the donor must be blood type O 
b. If the recipient is blood type A, then the donor must be blood type O or A 
c. 
If the recipient is blood type B, then the donor must be blood type O or B 
d. If the recipient is blood type AB, then the donor can be any blood type 
5. If recipient is cytomegalovirus (CMV) seronegative, then a CMV seronegative donor will be selected. 
6 
STUDY INTERVENTION 
6.1 STUDY INTERVENTION(S) ADMINISTRATION 
6.1.1 STUDY INTERVENTION DESCRIPTION 
Donors will have their blood collected via peripheral whole blood collection (450 ml into CPDA-1) or a 
leukapheresis procedure to collect mononuclear cells under steady state conditions (without mobilization). Each 
leukapheresis collection will be performed on a continuous flow cell separator (COBE Spectra or Spectra Optia, 
Terumo or equivalent device) using institutional standard operating procedures for MNC collection. The method of 
blood donation (phlebotomy versus leukapheresis) will be determined by estimating the volume of blood required 
to obtain the targeted CD4+ T-cell dose and based on standard institutional practices/preferences. Since the 
normal range of peripheral blood CD4+ T-cell counts is 0.5-1.5 x 106/ml, it is likely that simple phlebotomy will be 
sufficient for dose level 1 but leukapheresis will be required for dose levels 2 and 3. Based upon extensive prior 
experience, a 4-hour leukapheresis procedure should be sufficient to obtain 5 x 107 CD4+ T cells/kg of recipient 
ideal body weight (IBW). Target collections will be 30% more than the desired dose to accommodate for cell loss 
during the depletion process.  Cells collected remotely will be shipped overnight to the administering site for 
processing and infusion.    
The product will undergo CD8+ depletion in the Moffitt Cell Therapy Laboratory. All standard operating procedures 
will be followed. The product will be analyzed for nucleated cell count, viability, and surface expression of CD3, 
CD4, and CD8. CD8+ depletion will take place on the CliniMACS® Selection System (Miltenyi Biotec, Auburn, CA). 
The CD4 concentration will be used to calculate the patient dose. The calculated volume will be removed and 
prepared for infusion according to institutional standard operating procedures. The removed cells will either be 

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infused that evening or suspended in preservation medium (e.g., HypoThermosol, Biolife Solutions, Bothell, WA or 
similar) and infused the following day.  
6.1.2 DOSING AND ADMINISTRATION 
6.1.2.1 PRE-TREATMENT EVALUATION 
All patients will require documentation of a detailed history and physical examination and standard evaluation of 
cardiac, liver and renal function. All patients will undergo a bone marrow aspirate and biopsy for morphological, 
cytogenetic and flow cytometric evaluation upon registration on protocol, along with other standard disease 
evaluations (e.g., CT of chest, abdomen, pelvis) where applicable. An echocardiogram or MUGA scan must be 
performed no more than one month prior to enrollment. 
6.1.2.2 CHEMOTHERAPY 
All subjects will receive any standard AML induction chemotherapy regimen with cytarabine backbone as chosen 
by the treating provider. Patients may receive the induction chemotherapy regimen in the hospital or in the 
outpatient setting as per standard practice for the specific regimen. All subjects must be admitted to the hospital 
prior to infusion of NE-DLI. Due the chemotherapy, there is an expected period of aplasia with associated severe 
neutropenia (ANC < 500/microliter) that typically last for at least 3 weeks from the start of chemotherapy. Subjects 
should remain in the hospital until aplasia resolves (defined as ANC > 500/microliter) or until at least day 28, 
whichever comes first. After neutrophil recovery and post-neutrophil recovery bone marrow biopsy, patients in 
remission may be treated with standard consolidation chemotherapy to maintain disease response.  
Standard regimens include, but are not limited to: 
Table: Examples of standard regimens for AML induction chemotherapy with cytarabine backbone.  
Days, 
range 
Induction regimen, 
Chemotherapy  
Dose and Administration 
 
CLAG 
 
-5 to -1 
Cladribine 
5 mg/m2/day IV 
-5 to -1 
Cytarabine 
2 gm/m2/day IV 

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-6 to -1 
G-CSF 
300mcg daily SQ 
 
7+3 
 
-7 to -1 
Cytarabine 
100 mg/m2 IVCI x 7 days 
-7 to -5 
Idarubicin 
12 mg/m2 IVP x 3 days 
 
HiDAc, patient age < 60 y 
 
-5 to -1 
Cytarabine 
2 g/m2 IV over 3 hours Q12H (10 
doses) 
 
HiDAc, patient age > 60 y 
 
-5 to -1 
Cytarabine 
1 g/m2 IV over 3 hours Q12H (10 
doses) 
 
Note: A more exhaustive list is available in the National Comprehensive Cancer Network Clinical Practice 
Guidelines for Acute Myeloid Leukemia (nccn.org). Standard regimens that incorporate FDA approved 
targeted therapies with cytarabine-based chemotherapy are permitted. However, concurrent use of 
investigational agents is not permitted.   
  
Because of cell procurement and processing requirements, the first day of chemotherapy must be timed so that 
the donor leukapheresis occurs on a Monday, Tuesday, Wednesday, or Thursday; the cell processing occurs on a 
Tuesday, Wednesday, Thursday, or Friday; and the cell infusion occurs on a Tuesday, Wednesday, Thursday, Friday, 
or Saturday. Because the donor cells are infused into the patient 24 to 36 hours after the completion of 
chemotherapy, the acceptable start days of chemotherapy vary by regimen.  
In the case of a need to discontinue cytotoxic chemotherapy before infusion of cells, the patient will be taken off 
protocol because of toxicity. This will not be classified as a DLT in the phase 1 portion of the trial.  
6.1.2.3 DONOR LYMPHOCYTE INFUSION 
All patients will receive an infusion of mononuclear cells, apheresis products depleted of CD8+ T cells using the 
CliniMACS® system with CliniMACS® CD8 reagent. Products will be labeled as “T Cells, Apheresis: CD8 reduced.” 
The numbering of the dose levels is from the lowest to the highest cell dose. The first cohort of patients (dose level 
1) will receive induction chemotherapy followed by an infusion of CD8+ T cell–depleted donor lymphocyte infusion 
from an intentionally HLA-mismatched unrelated donor at a dose of 1 x 106 CD4+ T cells/kg of recipient IBW. The 

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cells will be infused from 24-36 hours after the cessation of induction chemotherapy. If criteria for dose escalation 
are met (section 10.4), patients on dose level 2 and 3 will receive cells containing an intended dose of 1 x 107 or 5 x 
107 cells/kg, respectively.  
Dose level 
CD4 T cells / kg 
1 
1 x 106 
2 
1 x 107  
3 
5 x 107 
 
The CliniMACS® system with CliniMACS® CD8 reagent is a medical device that is used to enrich or reduce CD8+ T 
cells from human blood products. The CliniMACS® System intended for selection of CD8+ cells comprises 4 primary 
components: 1) CliniMACS® CD8 Reagent – colloidal superparamagnetic iron dextran beads linked to a murine 
antibody against human CD8; 2) CliniMACS®plus Instrument – a software controlled instrument that processes the 
blood sample (cell product); 3) CliniMACS® Tubing Set, (Standard or LS) – a single-use, sterile, disposable tubing set 
with 2 proprietary cell selection columns; and 4) CliniMACS® PBS/EDTA Buffer – a sterile, isotonic phosphate-
buffered, 1 mM EDTA, saline solution, used as external wash and transport fluid for the in vitro preparation of 
blood cells. The system utilizes magnetic cell sorting (MACS®), a powerful tool for the isolation of many cell types6, 
to selectively enrich or deplete the cell population of interest. In this case, CD8+ T cells are labeled with a 
monoclonal antibody linked to superparamagnetic particles and then are depleted from the blood product by 
passage through the CliniMACS® system, which incorporates a strong permanent magnet and a separation column 
with a ferromagnetic matrix to remove the labeled cells. It is worth noting that the therapeutic agent in this trial, 
CD8+ T cell–depleted blood cells, comes out of the device and is not intended to contain any component of the 
device. 
Further information on the CliniMACS® system with CliniMACS® CD8 reagent can be found in the Investigator’s 
Brochure: http://medbioscience.com/catalog/Miltenyi%20Biotec/CliniMACS/CliniMACS.pdf 
Leukapheresis of an unmobilized donor is performed using the mononuclear cell setting within 48-72 hours prior 
to the scheduled time of infusion.  

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CD8 depletion is performed using the CliniMACS® magnetic cell separation system and the CliniMACS® CD8+ 
depletion reagent. Following CD8+ depletion, the cells are washed and pelleted. If not planned for immediate 
infusion (within 4 hours of CD8+ depletion), the cells will be resuspended to a concentration of 5 x 107 cells/ml in a 
preservation medium (e.g., HypoThermosol). 
As cells will be collected remotely, cells will be shipped at 2-8 degrees Celsius overnight to the administering site. 
CD8+ depletion will be performed at the receiving site using the CliniMACS® magnetic cell separation system and 
the CliniMACS® CD8+ depletion reagent as described above.  
Release testing of the CD8+ depleted product includes: 1) viability by tryptan blue dye exclusion and flow 
cytometry; 2) flow cytometry for CD3, CD4, and CD8 demonstrating that the number of CD3+CD8+ cells in the 
product is <3.2% the number of CD3+CD4+ cells; 3) sterility by gram stain.  
6.1.2.4 INFECTION PROPHYLAXIS AND THERAPY 
Antifungal prophylaxis will be administered according to institutional guidelines. Antifungal prophylaxis should 
begin no later than day 5 and continue until the absolute neutrophil count (ANC) is >500/microliter for 3 
consecutive days (or for 2 consecutive measurements over a 3 day period) and the patient is free of evidence of 
infection. Antifungal prophylaxis can be substituted for patient intolerance or at the discretion of the treating 
physician. 
Viral prophylaxis will be administered according to institutional guidelines. Standard options include, but are not 
limited to, valacyclovir (500mg po tid) or acyclovir (62.5 mg/m2 IV every 4 hours, if not tolerating oral medication) 
starting at day 1 of chemotherapy. 
Antibiotic prophylaxis will follow institutional guidelines for induction therapy for acute myeloid leukemia/MDS. 
Antibiotics will be administered at least until the ANC is >500/microliter following induction.  
Empiric or directed treatment of suspected or active infections will supersede prophylactic therapy. 
6.1.2.5 SUPPORTIVE CARE 

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Supportive care including fluids, anti-emetics, and tumor lysis management will be indicated according to 
institutional guidelines and the judgment of the treating physician. 
6.1.2.6 TRANSFUSION SUPPORT 
Platelet and packed red cell transfusions will be given per current institutional recommendations. 
6.1.2.7 TREATMENT SCHEME 
 
The figure shows the protocol schema as planned for patients undergoing standard 7+3 induction chemotherapy. 
The induction regimen may vary per judgment of the patient’s provider.  
6.1.3 DURATION OF THERAPY 
Subjects are eligible for only one lymphocyte infusion. For the dose-escalation portion, a minimum of 2 subjects 
and a maximum of 18 subjects will be enrolled. There will be 3 dose levels in the dose-escalation portion. MTD is 
defined as the highest dose level with the probability of DLT less than 33% (DLT events are defined in Section 6). 
The DLT observation window is defined in section 9.4.2. If none of 3 patients experiences a DLT, we will escalate to 
the next dose level. If one of the 3 patients experiences a DLT, we will enroll 3 additional patients and treat at the 
same dose level. In this case, if none of these additional 3 patients experiences a DLT, we will escalate to the 
dosage to next level. If 2 or more patients among 6 enrolled patients experience a DLT, or 2 or more patients in the 

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3 patient cohort experience DLT, the dosage level exceeds the MTD and the previous dose level will be declared as 
the MTD. If the first dose level exceeds the MTD, the study will be suspended. If no subjects experience a DLT at 
any dose level, the MTD will be level 3 (5 x 107 CD4+ T cells/kg).  
The RP2D is defined as the dose level selected to be evaluated in the expansion cohort or future phase II studies 
and will be equivalent to the MTD determined in the phase 1b portion of the study. Once we determine the RP2D 
level, we will enroll 10 additional eligible patients and treat with the RP2D level. The goal for this expansion cohort 
is to evaluate the safety and tolerability for patients who will be treated with NE-DLI in the RP2D dose level, and to 
evaluate the preliminary efficacy estimate for the ORR. Including the dose-escalation portion in which 3 or 6 
enrolled patients will have been treated at the RP2D level, a total of at least 13 patients will be treated at the RP2D 
level. Based on previous data,(18) the complete response rate using microtransplantation was in the range of 36%-
52% for MDS or AML from antecedent MDS patients. Hence, for the phase 2 portion, an ORR of 45% is considered 
clinically meaningful. With a sample size of 13 patients, if our ORR is 45%, the 95% CI (exact binomial) will be (18%-
74%); if the ORR is 50%, the 95% CI will be (22%-78%).  
The maximum total number of patients enrolled in this study will be 28 (6 per dose level plus 10 in the expansion 
cohort). We expect that the accrual rate will be one patient per every 5-6 weeks. With the DLI observation 
window, the dose-escalation portion may take up to a maximum of 2.5 - 3 years to complete and an additional 1.5 
years for the expansion cohort.  
6.1.3.1 DURATION OF FOLLOW-UP 
Patients will be followed for a minimum of 56 days after DLI (day 0 is defined as the date of DLI infusion) and then 
until initiation of conditioning therapy for allogeneic stem cell transplantation, death, disease progression/relapse, 
or 1 year following the date of infusion, whichever occurs first. Patients removed from study for unacceptable 
adverse events or who develop treatment-related adverse events will be followed until resolution or stabilization 
of the adverse event. Patients who proceed to allogeneic stem cell transplantation will be removed from the study.  

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Post-infusion monitoring: Whole marrow and/or peripheral blood T-cell (CD3) donor cell chimerism will be 
measured after neutrophil recovery (ANC > 500/l) and no later than the day 42 sample collections to rule out 
engraftment. If donor chimerism is detected, this test will be repeated on day 56 and then monthly until graft 
rejection is confirmed. Non-hematologic toxicity will be assessed weekly while subjects are hospitalized, and 
weekly until day 42. 
Disease assessment: Disease restaging requirements are designated in section 1.3. 
In addition to disease assessments specified above, results of additional disease assessments performed as 
standard of care will be collected for study purposes until death or disease progression, whichever occurs first.  
Off study criterion is the first event of the following: 
• 
Death 
• 
Disease progression 
• 
One year after infusion (final study visit) 
 
6.1.4 DEFINITION OF DOSE-LIMITING TOXICITY 
 
 
Number of Patients with DLT at a Given 
Dose Level 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level. 
≥ 2 
Dose escalation will be stopped. This dose level will be declared the 
maximally administered dose <highest dose administered>. Three (3) 
additional patients will be entered at the next lowest dose level if only 
3 patients were treated previously at that dose. 
1 out of 3 
Enter at least 3 more patients at this dose level. 
• 
If 0 of these 3 patients experience DLT, proceed to the next dose 
level. 
• 
If 1 or more of this group experience DLT, then dose escalation is 
stopped, and this dose is declared the maximally administered 
dose. Three (3) additional patients will be entered at the next 
lowest dose level if only 3 patients were treated previously at that 
dose. 
≤ 1 out of 6 at highest dose level below 
the maximally administered dose 
This is generally the recommended phase II dose. At least 6 patients 
must be entered at the recommended phase II dose. 
 
Dose-Expansion Cohorts 
 

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Once the RP2D is reached, a total of10 patients will be treated at this dose. For the expansion cohort, patients will 
continue to be monitored for occurrence of DLT. If 2 of the first 5 patients or if ≥ 2 of 6 patients experience DLT, 
the PI will discuss with all study investigators and with CTEP whether further addition of patients is needed to re-
assess the RP2D. 
 
Monitoring of safety and toxicity data will be captured in OnCore, Moffitt’s clinical trials database. 
 
Dose-limiting toxicities are defined as any of the following that is at least possibly attributable to the 
investigational treatment: 
1. Grade 2-4 infusion toxicity 
2. Grade 4 organ toxicity (as defined by NCI CTCAE v 5.0), not preexisting, not due to the primary 
malignancy, and not due to chemotherapy or infection 
3. Grade 3 organ toxicity that does not resolve within 14 days and that is not preexisting, not due to the 
primary malignancy, and not due to chemotherapy or infection 
4. Grade 2-4 acute GvHD 
5. Delayed neutrophil recovery: ANC >500/l does not occur within 56 days AND measurable donor 
chimerism is >5% 
6. Grade 3-4 cytokine release syndrome (CRS) (see Appendix C) (75) 
Any grade 5 reaction other than progressive disease 
 
6.2 PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
 
Donors will have their blood collected via peripheral whole blood collection (450 ml into CPDA-1) or a 
leukapheresis procedure will be performed to collect mononuclear cells under steady state conditions (without 
mobilization). Each leukapheresis collection will be performed using institutional standard operating procedures 
for MNC collection. The method of blood donation, phlebotomy versus leukapheresis, will be determined by prior 
to donation and based on the volume of blood required to obtain the targeted CD4+ T cell dose. 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
 
T Cells, Apheresis: CD8 Reduced and T Cells, Whole Blood: CD8 Reduced products are cell suspensions collected 
from allogeneic donors; the product is characterized by flow analysis (CD4+ cells and CD8+ cells). The product is 

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free of bacterial contamination as determined by gram stain analysis and is formulated in an infusion compatible 
solution. The T Cells, Apheresis: CD8 Reduced and T Cells, Whole Blood: CD8 Reduced products are provided as a 
fresh cell suspension. The product is a sterile suspension of mononuclear cells. If the cells will not be infused 
immediately, they may be suspended in preservation medium (e.g., HypoThermosol®). The volume in the bag will 
vary depending on the number of cells. Cell concentrations will be between 5x107 cells/ml to 1x108 cells/ml. The 
cells should be stored in a monitored 2-8°C refrigerator until ready for use. 
 
6.2.3 PRODUCT STORAGE AND STABILITY 
 
 
T Cells, Apheresis: CD8 Reduced and T Cells, Whole Blood: CD8 Reduced products are stored at 2-8°C until the time 
of use. The refrigerator units at clinical sites must be located in a controlled-access facility with an alarm 
monitoring system.  
 
Products are shipped according to established SOPs, FACT Standards and DOT regulations. Fresh product 
shipments are monitored with single trip data logger to provide continuous temperature monitoring.  
 
T Cells, Apheresis: CD8 Reduced and T Cells, Whole Blood: CD8 Reduced products should not be allowed to warm 
above 15°C during transport. A study was performed at Johns Hopkins University to evaluate the post-processing 
stability of T Cells, Apheresis: CD8 Reduced products (unpublished). Cell viability, as measured by trypan blue dye 
exclusion, was ≥88% for up to 72 hours after storage. 
6.2.4 PREPARATION 
 
T Cells, Apheresis: CD8 Reduced and T Cells, Whole Blood: CD8 Reduced products are cells that remain in an 
apheresis or whole blood product after depletion of CD8+ cells using the CliniMACS® system with the CliniMACS® 
CD8 reagent. The product is a sterile suspension of mononuclear cells suspended in HypoThermosol® or similar 
preservative. The volume in the bag will vary depending on the number of cells. Cell concentrations will be 
between 5x107 cells/ml to 1x108 cells/ml.  
 

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After manufacturing, the product is stored or shipped at 2-8°C prior to infusion. T Cells, Apheresis: CD8 Reduced 
and T Cells, Whole Blood: CD8 Reduced products should not be allowed to warm above 15°C during transport. 
Product infusion should follow established SOPs.  
 
6.3 MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
This is a single arm study. Patients will be treated in order of enrollment at the current dose level as delineated in 
the dose escalation schema. Neither the patients nor the trial physicians/providers will be blinded as all patients 
on trial will receive the same intervention. However, the final analysis will use de-identified raw data.   
6.4 STUDY INTERVENTION COMPLIANCE 
Data and safety monitoring will follow institutional protocol. Additionally, scheduled meetings will take place 
monthly and will include the protocol PI, clinical trial coordinator, and, when appropriate, the collaborators, 
subinvestigators, and biostatistician involved with the conduct of the protocol.  
During these meetings the investigators will discuss matters related to: safety of protocol participants, validity and 
integrity of the data, enrollment rate relative to expectation, characteristics of participants, retention of 
participants, adherence to protocol (potential or real protocol violations), data completeness, and progress of data 
for secondary objectives.  
Regulatory documents and case report forms will be monitored internally according to Moffitt Cancer 
Center Monitoring Policies. Monitoring will be performed regularly by the MCC Clinical Monitoring 
Core for accuracy, completeness, and source verification of data entry, validation of appropriate 
informed consent process, reporting of SAEs, and adherence to the protocol, Good Clinical Practice 
(GCP) guidelines, and applicable regulatory requirements. The Moffitt Protocol Monitoring 
Committee (PMC) meets monthly and reviews accrual, patterns and frequencies of all adverse 
events, protocol violations and when applicable, internal audit results.  
6.5 CONCOMITANT THERAPY 
 
Patients enrolled on this study may not be concurrently enrolled on another interventional therapeutic clinical 
trial. 
6.5.1 RESCUE MEDICINE 

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The study site will supply the rescue medication tocilizumab for the management of cytokine release syndrome 
(CRS). This rescue medicine will be obtained locally. Although the use of rescue medications is allowable at any 
time during the study, the use of rescue medications should follow the algorithm for treatment of CRS as described 
in Appendix C. The date and time of rescue medication administration as well as the name and dosage regimen of 
the rescue medication must be recorded. 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
7.1 DISCONTINUATION OF STUDY INTERVENTION 
Discontinuation from NE-DLI does not mean discontinuation from the study, and remaining study procedures 
should be completed as indicated by the study protocol. If a clinically significant finding is identified (including, but 
not limited to changes from baseline) after enrollment, the investigator or qualified designee will determine if any 
change in participant management is needed. Any new clinically relevant finding will be reported as an AE. 
The data to be collected at the time of study intervention discontinuation will include the following: 
• 
Cause of discontinuation 
• 
Most recent laboratory values 
• 
Evidence of adverse events 
• 
Disease status at the time of discontinuation 
 
7.2 PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Patients may elect to withdraw from the study at any point without any further justification.  
Additionally, patients may be removed from the study at the discretion of their medical providers on the basis of 
the following criteria: 
• 
Adverse events or any concern that continuing in the study would be harmful 
• 
Need for treatment not allowed in the study, including disease relapse while on study 
• 
Failure by a patient to comply with study protocol 
• 
Onset of pregnancy after enrolling in the study 
• 
Cancellation of the study  
7.3 LOST TO FOLLOW-UP 

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A participant will be considered lost to follow-up if he or she fails to return for 3 scheduled visits and if study site 
staff is unable to contact the participant.  
The following actions must be taken if a participant fails to return to the clinic for a required study visit: 
• 
The site will attempt to contact the participant and reschedule the missed visit for 1 month and counsel 
the participant on the importance of maintaining the assigned visit schedule and ascertain whether the 
participant wishes to and/or should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the investigator or designee will make every effort to 
regain contact with the participant (where possible, by making 3 telephone calls and, if necessary, by 
sending a certified letter to the participant’s last known mailing address or local equivalent methods). 
These contact attempts should be documented in the participant’s medical record or study file.  
• 
A minimum of 2 years should be spent attempting to locate the patient. If after that period of time the 
coordinator has sufficiently documented all failed attempts to locate the patient, including sending a 
certified letter with no response, then he or she will be considered to have withdrawn from the study for 
the primary reason of being lost to follow-up. 
8 
STUDY ASSESSMENTS AND PROCEDURES 
8.1 SAFETY ASSESSMENTS  
Safety assessments will be as follows: 
• 
History and physical: Initial and repeated at days 42 and 56, 6 months, and 1 year to evaluate for subjective 
or exam findings consistent with AEs.  
• 
Bone marrow biopsy: Day 42 to evaluate the efficacy of the intervention. Day 42 bone marrow biopsy may 
be performed earlier than the day 42 if the subject has recovered from aplasia, defined as recovery of the 
ANC >500/l.  
• 
Peripheral blood and bone marrow chimerism: To rule out donor engraftment and confirm rejection of the 
DLI product, donor cell chimerism will be measured after neutrophil recovery (ANC > 500/l) or on day 42 
+/- 3 days after infusion. Engraftment is necessary for GVHD, so product rejection increases the long term 
safety of the product.  
• 
Complete blood count (CBC): 3 times per week until ANC recovers to 500. Weekly through day 56. This 
assessment is necessary to monitor for hematologic toxicity. 
• 
Comprehensive metabolic panel (CMP): Weekly through day 56 to evaluate for toxicity to the liver, kidneys, 
or electrolyte derangements.  

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• 
C Reactive Protein (CRP) and Ferritin: Weekly through day 56 to screen for evidence of cytokine release 
syndrome (CRS) 
• 
Cytomegalovirus polymerase chain reaction (PCR): Weekly through day 56 to monitor for CMV reactivation 
while awaiting immune reconstitution.  
 
 
Initial 
Allowable time 
from start of 
induction 
Starting 
Day 0 
Day 
28 
Day 
42 
Day 
56 
6 months 
+/- 2 
weeks 
1 yr +/- 2 
weeks 
History and physical 
X 
Within 30 days 
 
 
X 
X 
X 
X 
Performance status 
X 
Within 30 days 
 
 
 
 
 
 
Staging Bone Marrow Biopsy and 
aspirate for research 
X 
Within 30 days 
 
 
Xa 
 
 
 
Peripheral blood for research 
X 
 
Daily 
days 0-7 
 
Xa 
 
 
 
CBC & Diff 
X 
Within 7 days 
ANC<500- 
3x/week 
ANC>500- 
weekly to 
day 56 
 
X 
X 
X 
X 
CMP 
X 
Within 7 days 
Weekly to 
day 56 
 
X 
X 
X 
X 
CXR or Chest CT 
X 
Within 30 days 
 
 
 
 
 
 
Pregnancy test (women, childbearing 
age) 
X 
Within 30 days 
 
 
 
 
 
 
Human Immunodeficiency Virus 
X 
Within 30 days 
 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
 
MUGA or ECHO 
X 
Within 30 days 
 
 
 
 
 
 
HLA typing 
X 
 
 
 
 
 
 
 
Anti-donor HLA antibody testing 
X 
 
 
 
X 
 
 
 
Adverse Event Monitoring 
 
 
Weekly to 
day 56 
 
X 
X 
X 
X 
GvHD evaluation 
 
 
At onset, 
weekly 
until 
resolves 
 
X 
X 
 
 
Peripheral blood or bone marrow for 
donor chimerism 
 
 
If 
suspected 
GvHD 
 
Xa 
Xb 
 
 
CMV IgG 
X 
Within 30 days 
 
 
 
 
 
 
CMV PCR 
 
 
Weekly 
through 
day 56 
 
 
 
 
 
C Reactive Protein 
X 
Within 7 days 
Weekly 
through 
day 56 
 
 
 
 
 
Ferritin 
X 
Within 7 days 
Weekly 
through 
day 56 
 
X 
X 
 
 
Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count; CMP, comprehensive metabolic panel; CMV, cytomegalovirus; CXR, 
chest radiograph; CT, computerized tomography scan; diff, differential blood count; ECHO, echocardiogram; EKG, electrocardiogram; GvHD, graft-
versus-host disease; MUGA, multigated acquisition; PCR,, polymerase chain reaction 
Tracking of days begins from day of cell transfusion (eg, Day 56 is 56 days after initial cell infusion). Day 
of cell transfusion is marked as Day 0. Procedures can be performed ± 3 days from marked date, unless 
otherwise specified. a Specified day 42 samples may be performed prior to day 42 if the patient has 

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recovered from aplasia, defined as ANC >500/l. Otherwise, the specimen collections will be planned for 
day 42 +/-3 days. bDay 56 chimerism measurement may be omitted if donor T-cell chimerism is < 5% on 
first measurement or if whole blood/marrow chimerism is < 5%. If donor chimerism >5% on day 56, then 
the investigator will continue measuring monthly until it decreases to <5% or until 1 year, whichever 
comes first. 
Pre-treatment research samples may be added on to previously collected samples (e.g., Moffitt Total Cancer Care) 
in lieu of collecting new samples. Screening tests obtained as standard of care prior to patient consent to this trial 
may be used for screening purposes if the tests fall within the allowable time window to the start of induction 
chemotherapy as defined in the table above.  
NOTE: In certain clinical circumstances (e.g. relapsed or terminally ill patients, risk of undue harm to patients, etc.) 
study tests may be omitted at the physician’s discretion. Additionally, tests may be rescheduled as clinically indicated. 
 
8.2 OTHER ASSESSMENTS 
Baseline evaluations to be performed within 30 days of initiating treatment: 
• 
Performance status: to confirm ECOG <2 
• 
Chest X-ray or Chest CT: To rule out active pulmonary infection 
• 
EKG and either MUGA or echocardiogram: To confirm adequate cardiac function 
• 
HIV testing: To rule out active HIV infection which would require treatment prior to initiating 
chemotherapy 
• 
Pregnancy testing: To rule out pregnancy as participation in this study may be dangerous to the 
fetus 
• 
Bone marrow biopsy: To confirm diagnosis and evaluate baseline disease status 
• 
CMV IgG: To allow for optimal, CMV matched donor selection  
Baseline evaluations to be performed anytime prior to enrollment:  
• 
HLA typing with anti-donor HLA antibody testing: To determine if this treatment increases 
production of anti-HLA antibodies.  
Baseline evaluations to be performed within 7 days of initiating therapy: 
• 
CBC: To confirm adequate hematologic parameters to proceed on study 
• 
CMP: To confirm adequate kidney/liver function to proceed on study 
• 
CRP and Ferritin: To risk stratify risk of cytokine release syndrome prior to treatment 

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• 
CMV PCR: To rule out active CMV infection and assist with donor selection  
NOTE: In certain clinical circumstances (e.g. relapsed or terminally ill patients, risk of 
undue harm to patients, etc.) study tests may be omitted at the physician’s discretion. 
Standard of care tests may be rescheduled as clinically indicated. 
8.3 BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES OR OTHER 
ENDPOINTS 
 
 
Specimen Type 
Baseline (Pre-
treatment) 
Days 0-7* 
Day 42** 
Peripheral blood sample 
X 
Daily 
X 
Bone marrow biopsy/aspirate 
X 
 
X 
 
* Research sample collection may be omitted if the day falls on a weekend or holiday. 
**May be obtained earlier than day 42 if ANC recovery to >500/uL. (See study calendar)  
 
8.3.1 PLANNED CORRELATIVE STUDIES 
Below are outlined correlative studies to be completed from the tissue samples. These will be performed 
as funding permits. Specific reagents and devices may be substituted for equivalent reagents/devices as 
available to the performing laboratory.  
Hypothesis: In patients with MDS/AML, allogeneic HLA-mismatched CD4+ DLI will result in reversal of 
immune exhaustion of recipient tumor specific CD8+ T cells.  
Aim 1: Determine if treatment of MDS/AML with HLA-mismatched CD4+ DLI results in reversal of 
expression of protein markers of immune exhaustion in recipient cytotoxic T cells. 
Markers of Immune Exhaustion 
Immune exhaustion is characterized by CD8+ T cell surface expression of inhibitory proteins such as 
PD-1, which contribute to treatment failure in MDS.(14, 36, 76-78)  Thus, we wish to assess whether HLA-
mismatched CD4+ DLI can reverse expression of negative immune checkpoints on exhausted CD8+ T cells 
in patients with MDS who failed standard therapy. All enrolled patients will have peripheral blood and bone 
marrow samples obtained at baseline prior to protocol chemotherapy, and at the time of hematologic 
recovery, 30-50 days after HLA-mismatched CD4+ DLI. Peripheral blood mononuclear cells (PBMCs) and 
bone marrow mononuclear cells (BMMCs) will be enriched using standard Ficoll-Hypaque methods. The 
cells will be stained with propidium Iodide/acridine orange and live cells will be counted using a Cellometer 
2000 automated cell counter (Nexcelom Biosciences, Lawrence, MA). The cells will be suspended in 
Recovery Freeze Medium (Invitrogen, Carlsbad, CA) and controlled rate frozen to -80°C before transfer to 
liquid nitrogen storage. In batch, baseline and post-treatment samples will be thawed, followed by isolation 
of CD4+ and CD8+ populations using MultiSort magnetic MicroBeads (Miltenyi Biotec, Gaithersburg, MD). 
For this CD4+ cells will be bead captured on one column and the flow through will be mixed with CD8 beads 
and then captured on a second column. Total RNA will be extracted from one million each of the isolated 

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CD4+ and CD8+ cells and stored for subsequent RNAseq analysis. The collected samples will be analyzed 
via multicolor flow cytometric assay to characterize expression of markers of immune exhaustion (TIM3, 
LAG-3, PD1, PDL1, CTLA4).(79-81) Pre-treatment and post-treatment values will be compared to 
determine if expression of exhaustion antigens is down regulated after HLA-mismatched CD4+ DLI therapy, 
thus predicting that immune exhaustion is reversed. The one-sample t-test will be used to assess if the 
markers are significantly changed after treatment. 
Host versus Donor Origin of CD8+ T Cells  
Because we hypothesize that reversal of immune exhaustion will specifically occur in the recipient’s 
cytotoxic T cells, we wish to identify if reinvigorated CD8+ T cells are derived from the donor or the host as 
donor derived CD8+ T cells would be expected to lead to engraftment and potentially GVHD. Since the 
CD4+ DLI is intentionally HLA mismatched, RNA sequencing can be used to distinguish and quantify donor 
versus recipient HLA types in the samples.(82)  We will isolate the tumor infiltrating cells in the marrow after 
HLA-mismatched CD4+ DLI and use RNA sequencing of HLA to determine if the cells are donor- or host-
derived. We will supplement this testing with standard clinical bone marrow chimerism studies. In 
combination, these tests will assess whether donor CD4+ T cells are rejected and will confirm that the 
immune competent CD8+ T cells are of host origin.      
Aim 2: In patients with MDS/AML who received HLA-mismatched CD4+ DLI therapy, characterize 
anti-leukemic activity and specificity of recipient marrow CD8+ T cells in response to HLA-
mismatched CD4+ DLI. 
Leukemia-Specific Cytolytic Activity of CD8+ T cells 
After treatment with HLA-mismatched CD4+ DLI, we anticipate a reversal of immune 
exhaustion and a subsequent host-versus-leukemia response. We will evaluate changes to 
cytolytic activity of the recipient cytotoxic CD8+ T cells before and after therapy, first, in cytotoxicity 
experiments at different effector:target (CTL:tumor cell) ratios using leukemia blasts collected 
from the pre-treatment samples. If the frequencies of tumor-reactive T cells required more 
sensitive methods, we would use ELISPOT testing. Specifically, this sensitive assay can quantify 
a successful response to HLA-mismatched CD4+ DLI characterized by an increase in the 
frequencies of CD8+ T cells secreting interferon-γ (IFN-γ) in response to cognate tumor antigen, 
using peripheral or BM malignant cells as targets.(83) Cytotoxicity and/or ELISPOT results will be 
compared between patients who achieve a remission and those who do not to show that clinical 
response is dependent on marrow infiltration by activated cytotoxic T cells. The Satterthwaite t-
test will be used to detect a significant difference in changes to cytolytic activity and increase in 
frequency of CD8+ T-cells before and after treatment, and between patients who achieve a 
remission and those who do not. The odds ratio will be estimated along with 95% confidence 
intervals by using the simple logistic regression or the generalized estimating equation (GEE) 
model. Building multivariable model will not be considered due to the small sample size issue. 
The data transformation (e.g., log2-transformation) may be considered to meet the normality 
assumption for the application of parametric analytic tools. The Wilcoxon singed-rank test or 
Wilcoxon rank-sum test may be considered as the secondary approaches if necessary. As this is 
an exploratory study, the multiplicity adjustment is not considered. 
Clonotype of Expanded CD8+ T cells 
We will assess whether these activated, tumor specific, cytotoxic T cells are clonal. In the post-
allogeneic transplant setting, prior studies have shown that response to CD8+ depleted DLI is most 
successful when there is a high number of exhausted cytotoxic T cells infiltrating the marrow (tumor)  prior 
to therapy.(80) It has also been shown that tumor infiltrating lymphocytes that express the highest frequency 
T cell receptor beta chain clonotypes are tumor specific.(84) Thus, we will use  T cell receptor beta chain 
complementarity-determining region 3 (TCRB CDR3) region sequencing (ClonoSeq, Adaptive 
Biotechnologies) to identify clonally-expanded, tumor specific lymphocytes in the marrow prior to HLA-
mismatched CD4+ DLI and will confirm that the T lymphocytes that become activated after HLA-

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mismatched CD4+ DLI share the same TCRB signatures and, thus, are also tumor specific. TCRB 
signatures will also be compared between responders and non-responders to support that clinical 
remissions are due to clonal T cell expansion rather than just a chemotherapy effect.       
Aim 3: In patients with MDS/AML who received HLA-mismatched CD4+ DLI therapy, characterize 
cytokine profiles from the blood associated with severe cytokine release syndrome.  
Plasma Cytokine Profile 
HLA-mismatched CD4+ DLI is expected to reverse immune exhaustion and stimulate an 
inflammatory response consistent with cytokine release syndrome (CRS), characterized by fevers 
and sometimes associated hypotension or hypoxia.(85) Thus, we aim to identify risk factors for 
severe CRS in patients treated with this novel therapy. Clinically, we will compare baseline 
variables of patients who experience severe CRS and those who do not, including HLA-
mismatched CD4+ DLI dose, degree of HLA mismatch, choice of induction chemotherapy, and 
disease burden, all factors that are predictive of CRS with other cell therapies.(86-88) In the 
setting of chimeric antigen receptor T cells (CAR-T) therapy, confirmation that CRS is correlated 
with high levels of interleukin-6 (IL-6) led to the effective use of IL-6 blockade as CRS 
treatment.(89-91) We will similarly evaluate cytokine profiles associated with CRS to identify 
potential targets for anti-CRS therapy. As CRS typically occurs in the first 7 days after other cell 
therapies, we will collect blood samples prior to therapy and then daily from the day of HLA-
mismatched CD4+ DLI infusion through day 7 after infusion. We will test plasma using the Ella 
multiplex cytokine assay to identify the T cell-derived cytokines (e.g., IFN-gamma, IL-6, IL-17, 
TNF-alpha) increased at baseline in patients who develop severe CRS and monitor which 
cytokines significantly increase with onset of CRS. For patients who experience CRS, we will also 
monitor changes to the cytokine profile after administration of IL-6 blockade (tocilizumab) and 
correlate temporally with clinical resolution. The association between severe CRS and clinical 
variables (e.g., DLI dose, degree of HLA mismatch, choice of induction chemotherapy, disease 
burden) and the change of cytokine profiles after treatment (e.g. IFN-gamma, IL-6, IL-17, TNF-
alpha) will be explored by the Satterthwaite t-test. Specifically, cytokine profiles will be measured 
at baseline, and days 0 through 7. The GEE model may be employed to assess change over time 
in the difference between two groups. Odds ratios will be estimated along with 95% confidence 
intervals by using the simple logistic regression or the GEE model. Other statistical considerations 
apply as discussed in Aim 2. These studies will confirm whether IL-6 is primarily implicated 
in causing toxicity with HLA-mismatched CD4+ DLI or if other cytokines may be more 
effective targets for managing this toxicity.  
 
Power calculations 
 
We expect up to 18 subjects will be enrolled in this phase I with cohort expansion study. 
For Aim 1, up to 18 subjects will have 80% and 98% power to detect an effect size of 0.7 and 
1.0 at a two-sided significance level of 5%. The one-sample t-test is used. For Aim 2 and 3, the 
maximum power is obtained when the sample size of two groups is identical (i.e., 9 subjects per 
group). For each endpoint of Aim 2 and 3, the study will have 85% power to detect an effect size 
of 1.5 at a two-sided significance level of 5% when sample size of each group is 9. If the sample 

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size of one group deviates from 9, the statistical power will decrease, accordingly. Two sample 
t-test is used for Aim 2 and 3, and no multiplicity adjustment is considered for power analyses. 
 
8.4 ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
8.4.1 DEFINITION OF AE 
AE means any untoward medical occurrence associated with the use of an intervention in humans, whether or not 
considered intervention related—21 CFR 312.32 (a). An adverse event may include any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom or disease temporally associated with the 
use of a medical treatment or procedure regardless of whether it is considered related to the medical treatment or 
procedure (attribution of unrelated, unlikely, possible, probable, or definite).  
8.4.2 DEFINITION OF SAE  
An SAE is any sign, symptom or medical condition that emerges during treatment or during a post-treatment 
follow-up period that (1) was not present at the start of treatment and is not a chronic condition that was part of 
the patient’s medical history, OR (2) was present at the start of treatment or as part of the patient’s medical 
history but worsened in severity and/or frequency during therapy, AND that meets any of the following regulatory 
criteria:  
• 
is fatal (i.e., results in death from any cause at any time) or life-threatening (i.e., the patient was in the 
view of the investigator, at immediate risk of death from the reaction as it occurred)  
• 
required or prolonged hospitalization (see exclusions below)  
• 
results in persistent or significant disability/incapacity  
• 
constitutes a congenital anomaly or a birth defect  
• 
is medically significant, may jeopardize the subject and may require medical or surgical intervention to 
prevent one of the outcomes listed above.  
Events not considered to be SAEs are hospitalizations for the:  

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• 
Routine treatment or monitoring of the studied indication, not associated with any deterioration in 
condition  
• 
Treatment, which was elective or pre-planned, for a preexisting condition that did not worsen  
Serious adverse events occurring in a patient from the first day of treatment and until 4 weeks after the last dose 
of treatment must be reported. The period after discontinuing study drug may be extended if there is a strong 
suspicion that the drug has not yet been eliminated.  
All serious adverse events must be followed to resolution (≤ 1 or baseline) or until considered stable or irreversible. 
8.4.3 CLASSIFICATION OF AN AE 
8.4.3.1 
SEVERITY OF EVENT 
All AEs will be graded using the CTCAE 5.0 criteria.  
For AEs not included in the protocol-defined grading system (grade 1 to 4), the following guidelines will be used to 
describe severity. 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic measures. 
Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug therapy or 
other treatment. Severe events are usually potentially life-threatening or incapacitating. Of note, the term 
“severe” does not necessarily equate to “serious”. 
The grade and severity of the event will be determined using the DCT/NCI Common Terminology Criteria, CTCAE 
v.5.0. All appropriate treatment areas should have access to a copy of the CTCAE version 5.0. Study staff must use 
one of the CTCAE criteria to define the event. Adverse events not included in the CTCAE v.5.0 should be reported 
and graded under the “Other” adverse event within the appropriate category and grade 1 to 5 according to the 
general grade definitions, mild, moderate, severe, life-threatening, fatal or disabling, as provided in the CTCAE.  

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The event will be determined to be expected or unexpected. The determination of whether an AE is expected is 
based on agent-specific adverse event information provided in Pharmaceutical Information. Unexpected AEs are 
those not listed in the agent-specific adverse event information provided in Section 9 Pharmaceutical Information.  
Based on this information, a decision will be made whether an adverse event should be reported as an expedited 
report (Serious Adverse Event 8.4.2) in addition to the routinely reported clinical data. All expedited adverse event 
reports will be submitted to the Institutional Review Board (IRB) and to the FDA. 
Safety will be evaluated based on the incidence, severity, duration, causality, seriousness, and type of adverse 
events (AEs), and changes in the patient’s physical examination, vital signs, and clinical laboratory results. 
Investigators will use the NCI CTCAE version 5.0 published November 27, 2017. 
(https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcae_v5_quick_reference_5x7.pdf) 
to assess the severity of adverse events. Observed or volunteered Grade 3-5 adverse events regardless of 
treatment group or causal relationship to study drug will be recorded on the adverse event pages of the case 
report form. 
There is a known rate of treatment-related mortality associated with induction chemotherapy in this population; in 
addition, severe infections are common. Therefore, adjudication that an adverse event is possibly related to the 
exposure, and not the disease or standard treatment (chemotherapy) will be an important role of the Protocol 
Monitoring Committee.  
 
8.4.3.1.1 LIST AND REPORTING REQUIREMENTS 
There are 2 broad categories of reportable adverse events: 1) adverse events that are attributable to use of the 
device, i.e. the CliniMACS® system with CliniMACS® CD8 Reagent; and 2) adverse events that are not attributable 
to the use of the device. All reporting of adverse events will be carried out according to current IRB and FDA 
guidelines.  

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8.4.3.2 ADVERSE DEVICE EFFECTS 
8.4.3.2.1 HYPERSENSITIVITY REACTIONS 
Use of the CliniMACS® System enables the infusion of CD8 depleted or selected cells that may contain residual 
amounts of unbound CliniMACS® CD8 Reagent (murine monoclonal antibody conjugated to an iron dextran 
moiety). Hypersensitivity reactions resulting from iron dextran therapy have been reported. The most dangerous 
complication is an anaphylactoid reaction similar to contrast media reactions. The anaphylactoid reaction is usually 
manifested during the first few minutes of the infusion (often during test dose administration). The most common 
hypersensitivity reaction occurring in more than 30% of patients given therapeutic levels of iron dextran is the 
development of arthralgia and fever within 24 to 48 hours after initiation of the intravenous therapy. 
8.4.3.2.2 REACTIONS MEDIATED BY HUMAN ANTI-MOUSE ANTIBODIES (HAMA) 
The CliniMACS® CD8 Reagent also contains a murine monoclonal antibody. One of the problems limiting the 
therapeutic use of murine monoclonal antibodies (mAbs) has been the generation of HAMA and subsequent 
anaphylaxis.(92)The most commonly reported adverse effects have been myalgia, arthralgia, increased peripheral 
edema and flu-like symptoms, the latter involving fever (70% to 73%), chills (46% to 53%), chest pain and tightness 
(14%), wheezing (11% to 14%) and vomiting (13% to 32%).(93-95) 
8.4.3.3 ADVERSE EVENTS NOT ATTRIBUTABLE TO THE DEVICE 
Four categories of device non-attributable adverse events will be monitored and, if necessary, reported: 1) non-
hematologic toxicities; 2) GvHD; 3) DLI-induced aplasia; and 4) CRS. Reportable adverse events are defined as non-
hematologic toxicities requiring hospitalization, grade 3 or 4 toxicities defined below, and grade II acute GvHD. 
Adverse events that are not considered serious will be included in the annual report. Serious adverse events 
include any death within the first 56 days after DLI, grade 2-4 acute GvHD, DLI-induced aplasia, ≥grade 3 CRS, and 
any adverse event that is deemed to be significant by the PI. 
Non-hematologic toxicities will be monitored daily while patient remains admitted (typically until ANC exceeds 
500/microliter, which usually occurs between days 21 and 42), then at clinic visits on day 42, day 56, 6 months, and 

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1 year. Toxicities will be graded using the National Cancer Institute’s Common Terminology Criteria for Adverse 
Events (CTCAE) Version 5.0.  
The following is a list of categories that will not be required to be recorded unless the event becomes a grade 4: 
• 
Allergy/Immunology 
• 
Auditory/Hearing 
• 
Cardiovascular (Arrhythmia) 
• 
Cardiovascular (General) 
• 
Coagulation 
• 
Constitutional symptoms 
• 
Dermatology/Skin 
• 
Endocrine 
• 
Hemorrhage 
• 
Hepatic 
• 
Infection/Febrile neutropenia 
• 
Lymphatics 
• 
Metabolic/Laboratory 
• 
Secondary Malignancy 
• 
Sexual/Reproductive Function 
A toxicity in the following categories will be captured as an adverse event if it reaches grade 3 or grade 4: 
• 
Gastrointestinal 
• 
Musculoskeletal 
• 
Neurology 
• 
Ocular/Visual 
• 
Pain 
• 
Pulmonary 
• 
Renal/Genitourinary 
8.4.3.3.1 GRAFT-VERSUS-HOST DISEASE  
GVHD is an unexpected but potential complication of this therapy. Patients will be monitored for GVHD until donor 
T-cell chimerism decreases to <5% or 1 year from infusion, whichever comes first.  
 

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Acute GvHD shall be graded clinically according to the criteria developed by the consensus conference on acute 
GvHD (see Appendix D).(96) All suspected cases of acute GvHD must be confirmed histologically by biopsy of an 
affected organ (skin, liver, or gastrointestinal tract). For purposes of reporting, a pathologist will be ultimately 
responsible for determining whether a patient does or does not have histologic evidence of GvHD. Diarrhea and/or 
hyperbilirubinemia in a patient with histologically documented skin GvHD may be assumed to be a manifestation 
of visceral GvHD and will be graded as such. All patients with histologically documented, clinical grade >2 acute 
GvHD should receive initial treatment with corticosteroids according to institutional preference. If skin GvHD 
resolves with treatment but suspected visceral GvHD does not, biopsy of the affected organ (liver or 
gastrointestinal tract) should be obtained to rule out other causes of hyperbilirubinemia and/or diarrhea. Steroid 
refractory acute GvHD will be treated according to institutional preferences. 
The following information shall be collected on all patients with acute GvHD: 
• 
Date of onset (defined as the date of first biopsy confirming GvHD) 
• 
GvHD evaluation form at the time of onset, weekly until GvHD resolves, and/or day 56 
• 
Initial overall clinical grade 
• 
Maximum overall clinical grade 
• 
Date of onset of grade 3-4 acute GvHD, if any 
• 
The occurrence and severity of acute and chronic GvHD after day 56 will be captured at the patient’s six-
month evaluation. 
All instances of grade 2-4 acute GvHD will be captured as serious adverse events.  
Chronic GvHD is not an expected complication of the investigational therapy as the DLI should not result in long-
term engraftment. Nonetheless, monitoring for chronic GvHD will follow the same procedures as acute GvHD with 
the following exceptions: 
• 
The severity of GvHD will be determined based on NIH 2014 Consensus Guidelines (see Appendix F).(97) 
• 
Systemic therapy with corticosteroids will be indicated for any patient with moderate or severe chronic 
GvHD. 
• 
Adjunctive immunosuppressive therapies for severe or steroid refractory chronic GvHD will be 
determined by the primary provider and institutional preference. 
8.4.3.3.2 DONOR LYMPHOCYTE INFUSION-INDUCED APLASIA  

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DLI-induced aplasia is defined as neutropenia (ANC <500/mL) on day 42 with evidence of donor chimerism (>5%) 
on the day 42 sample collection. In the case of DLI-induced aplasia diagnosed on day 42, a second chimerism 
analysis will be sent on day 56 to confirm. All cases of DLI-induced aplasia persisting to day 56 will be reported as 
serious adverse events. 
8.4.3.3.3 CYTOKINE RELEASE SYNDROME 
CRS is a recognized complication of the infusion of HLA-mismatched lymphocytes.(51, 98) CRS comprises a 
constellation of symptoms and signs including fever, hypotension, fluid extravasation leading to edema, ascites 
and hypoxia, diffuse erythema, diarrhea, and kidney dysfunction. The syndrome is accompanied by elevation in 
serum markers of inflammation including elevations in acute phase reactants such as interleukin-6, ferritin, and 
soluble IL-2 receptor. Recent reports demonstrate that CRS can be managed effectively, and symptoms mitigated, 
by treatment with the anti-IL-6 receptor antibody tocilizumab. (98) Grading of CRS and guidelines for treatment to 
be used in this study are included in Appendix C.(75) Any instances of CRS grade ≥3 will be reported as serious 
adverse events. 
8.4.3.4 
RELATIONSHIP TO STUDY INTERVENTION 
For all collected AEs, the clinician who examines and evaluates the participant will determine the AE’s causality 
based on temporal relationship and his/her clinical judgment. The degree of certainty about causality will be 
graded using the categories shown below. 
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test result, 
occurs in a plausible time relationship to drug administration and cannot be explained by concurrent 
disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be 
clinically plausible. The event must be pharmacologically or phenomenologically definitive, with use of 
a satisfactory rechallenge procedure if necessary. 
• 
Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs within a 
reasonable time after administration of the drug, is unlikely to be attributed to concurrent disease or 
other drugs or chemicals, and follows a clinically reasonable response on withdrawal (dechallenge). 
Rechallenge information is not required to fulfill this definition. 
• 
Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event occurred 
within a reasonable time after administration of the trial medication). However, other factors may have 

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contributed to the event (e.g., the participant’s clinical condition, other concomitant events). Although 
an AE may rate only as “possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related,” as appropriate. 
• 
Unlikely to be related – A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to drug administration makes a causal relationship improbable (e.g., the event did not 
occur within a reasonable time after administration of the trial medication) and in which other drugs or 
chemicals or underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments). 
• 
Not Related – The AE is completely independent of study drug administration, and/or evidence exists 
that the event is definitely related to another etiology. There must be an alternative, definitive etiology 
documented by the clinician. 
8.4.3.5 
EXPECTEDNESS  
The PI will be responsible for determining whether an AE is expected or unexpected. An AE will be considered 
unexpected if the nature, severity, or frequency of the event is not consistent with the risk information previously 
described for the study agent. 
8.4.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND 
FOLLOW-UP 
The occurrence of an AE or SAE may come to the attention of study personnel during study visits and interviews of 
a study participant presenting for medical care, or upon review by a study monitor. 
AEs can be spontaneously reported or elicited during open-ended questioning, examination, or evaluation of a 
patient. 
Grade 3-5 AEs, including local and systemic reactions not meeting the criteria for SAEs, will be captured on the 
appropriate CRF. Information to be collected will include event description, time of onset, clinician’s assessment of 
severity, relationship to study product (assessed only by those with the training and authority to make a diagnosis), 
and time of resolution/stabilization of the event. All AEs occurring while on study must be documented 
appropriately regardless of relationship. All AEs will be followed to adequate resolution. 
 

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Any medical condition that is present at the time that the participant is screened will be considered as baseline 
and not reported as an AE. However, if the study participant’s condition deteriorates at any time during the study, 
it will be recorded as an AE.  
 
Changes in the severity of an AE will be documented to allow an assessment of the duration of the event at each 
level of severity to be performed. AEs characterized as intermittent require documentation of onset and duration 
of each episode. 
 
All events beginning with start of study intervention until  the first event of either starting allogeneic transplant 
conditioning, confirmed disease relapse, death, or 1 year from the of the trial cell infusion will be reported. At each 
study visit, the investigator will inquire about the occurrence of AE/SAEs since the last visit. Events will be followed 
for outcome information until resolution or stabilization. 
8.4.5 AE REPORTING  
The PI and/or the research nurse will monitor each patient closely for the development of adverse events and 
toxicities and record all such events. AEs will be recorded and reported in our annual report. As some toxicities are 
expected from the chemotherapy, we will compare incidence of toxicities to historical experience when using 
chemotherapy without the investigational DLI product. The FDA require the timely reporting of adverse events 
(including toxic deaths). 
Each individual sign or symptom must be documented separately. Grade 3-5 adverse events (both expected and 
unexpected) will be captured on the appropriate study-specific CRFs.  
An investigator must verify the attribution of all adverse events. Evaluation of laboratory toxicities may be 
documented  on CRF provided the investigator signs it. However, if an action was conducted due to this 
abnormality (e.g., red blood cell transfusion due to low hemoglobin) this would be recorded on the AE form also. 

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AE reporting will begin at the time of cell infusion and continue through 1 year from day 0 or until loss to follow-
up, death, disease progression, or proceeding to allogeneic BMT, whichever comes first. Subjects who proceed to 
allogeneic BMT will come off of the trial and will no longer be followed for AEs.  
8.4.6 SAE REPORTING  
The study clinician will immediately report to the FDA any SAEs, whether or not considered study intervention 
related, including those listed in the protocol or investigator brochure and must include an assessment of whether 
there is a reasonable possibility that the study intervention caused the event. Study endpoints that are SAEs (e.g., 
all-cause mortality) must be reported in accordance with the protocol unless there is evidence suggesting a causal 
relationship between the study intervention and the event (e.g., death from anaphylaxis). In that case, the 
investigator must immediately report the event to the sponsor. 
 
All SAEs will be followed until satisfactory resolution or until the site investigator deems the event to be chronic or 
the participant is stable. Other supporting documentation of the event may be requested by the DCC/study 
sponsor and should be provided as soon as possible. 
 
It is the responsibility of the PI and the research team to ensure that SAEs are reported according to the Code of 
Federal Regulations, Good Clinical Practices, the protocol guidelines, the sponsor’s guidelines, and IRB policy. 
 
All adverse experience reports must include the patient number, age, sex, weight, severity of reaction (mild, 
moderate, severe), relationship to study drug (probably related, unknown relationship, definitely not related), date 
and time of administration of test medications, all concomitant medications, and medical treatment provided. The 
PI is responsible for evaluating all AEs to determine whether criteria for “serious” as defined above are present. 
Adverse drug reactions that are serious, unlisted/unexpected, and at least possibly associated to the drug, and that 
have not previously been reported in the IB, or reference safety information document should be reported 
promptly to the FDA in writing by each investigator/physician engaged in clinical research. A clear description of 

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the suspected reaction should be provided along with an assessment as to whether the event is drug or disease 
related.  
8.4.7 REPORTING EVENTS TO PARTICIPANTS  
 
As these medications and procedures are part of a Phase 1/2 protocol, their side effects have not been evaluated 
or approved for safety by the FDA. Although it is anticipated that this protocol is relatively safe, fatal complications 
are possible. The major hazards are infection and disease progression. It is therefore only appropriate to carry out 
this experimental procedure in the context of life threatening metastatic cancer. The major discomforts are 
nausea, mucositis, anorexia, diarrhea, fever and malaise. Physicians have an ethical responsibility to inform 
patients about AEs and SAEs. 
Patients will be addressed by a nurse or physician to disclose AEs and SAEs. These individuals will explain the 
AE/SAE in an honest, open, and direct manner in a private setting. The nurse or physician will be given a detailed 
description of the event, incident, experience or outcome, as well as an explanation as to why it is considered an 
AE/SAE. They will be informed of any changes to protocol or corrective actions that will result from the AE/SAE. 
The physician will continue to monitor the AE/SAE in the most appropriate manner for the situation and will, if 
necessary, withdraw the patient from the study. 
Other patients enrolled in the study will be informed of changes to protocol that are resultant of another 
participant’s AE/SAE. They will not be given any personal details of the patient of their AE/SAE in order to protect 
patient privacy. 
Patients will be encouraged to communicate any incidental side effects or information discovered during study 
procedures. Patients will be holistically monitored for signs of any unintentional or incidental effects of the study. 
Physicians will communicate any incidental findings that may affect the patient or the patients’ desire to 
participate in the study. 
8.4.8 EVENTS OF SPECIAL INTEREST  
Not applicable. 

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8.4.9 REPORTING OF PREGNANCY  
Pregnancy, although not itself an SAE, should also be reported on an SAE form or pregnancy form and be followed 
up to determine outcome, including spontaneous or voluntary termination, details of birth, and the presence or 
absence of any birth defects or congenital abnormalities. 
8.5 UNANTICIPATED PROBLEMS 
8.5.1 DEFINITION OF UP 
The OHRP considers UPs involving risks to participants or others to include, in general, any incident, experience, or 
outcome that meets all of the following criteria: 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document; and (b) the characteristics of the participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a reasonable 
possibility that the incident, experience, or outcome may have been caused by the procedures involved in 
the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
This definition could include an unanticipated adverse device effect, any serious adverse effect on health or safety 
or any life-threatening problem or death caused by, or associated with, a device, if that effect, problem, or death 
was not previously identified in nature, severity, or degree of incidence in the investigational plan or application 
(including a supplementary plan or application), or any other unanticipated serious problem associated with a 
device that relates to the rights, safety, or welfare of subjects (21 CFR 812.3(s)). 
8.5.2  UNANTICIPATED PROBLEM REPORTING  
The investigator will report UPs to the reviewing IRB and to the DCC/lead PI. The UP report will include the 
following information: 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome represents 
an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or are 
proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:  

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• 
UPs that are SAEs will be reported to the IRB, depending on their policy, and to the DCC/study sponsor 
within 7 working days of the investigator becoming aware of the event.  
• 
Any other UP will be reported to the IRB and to the DCC/study sponsor within 7 working days of the 
investigator becoming aware of the problem.  
• 
All UPs should be reported to appropriate institutional officials (as required by an institution’s written 
reporting procedures), the supporting agency head (or designee), and the OHRP within 7 working days of 
the IRB’s receipt of the report of the problem from the investigator. 
 
An investigator shall submit to the sponsor and to the reviewing IRB a report of any unanticipated adverse device 
effect occurring during an investigation as soon as possible, but in no event later than 7 working days after the 
investigator first learns of the effect (21 CFR 812.150(a)(1)), A sponsor who conducts an evaluation of an 
unanticipated adverse device effect under 812.46(b) shall report the results of such evaluation to the FDA and to 
all reviewing IRB's and participating investigators within 7 working days after the sponsor first receives notice of 
the effect. Thereafter the sponsor shall submit such additional reports concerning the effect as FDA requests (21 
CFR 812.150(b)(1)). 
8.5.3 REPORTING UNANTICIPATED PROBLEMS TO PARTICIPANTS  
As these medications and procedures are part of a Phase 1/2 protocol, they may cause wholly unknown and 
unexpected side effects. Unexpected problems will be reported to patients as soon as possible. 
A nurse or physician will be given a detailed description of the event, incident, experience or outcome, as well as 
an explanation as to why it is considered a UP. They will be informed of any changes to protocol or corrective 
actions that will result from the UP.  
Other patients enrolled in the study will be informed of changes to protocol that are resultant of another 
participant’s UP. They will not be given any personal details of the patient of their UP in order to protect patient 
privacy. 
9 
STATISTICAL CONSIDERATIONS  
9.1 STATISTICAL HYPOTHESES 
We hypothesize that a source of CD8+ depleted, non-engrafting, HLA-mismatched, unrelated donor 
lymphocyte infusion (NE-DLI) would be capable of reversing immune tolerance and eliciting a significant 
antitumor response in patients with MDS and secondary AML, without causing GVHD. 
Primary Endpoint:  

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• 
Determine the maximum tolerated dose (MTD) of CD8-depleted cells from HLA-mismatched 
unrelated donors infused after induction chemotherapy for patients with advanced MDS after 
HMA failure or untreated sAML 
 
Secondary Endpoints:  
To determine: 
 
• 
Overall response rate according to the International Working Group (IWG) (see Appendix B).  
• 
Overall survival. 
• 
Progression-free survival.  
• 
Hematologic response/improvement by IWG 2006 criteria for MDS patients and the IWG 2003 
criteria for AML (1, 2).  
• 
Occurrence of adverse events. 
• 
Unexpected transplant complications (eg, graft-versus-host disease [GvHD]). 
9.2 SAMPLE SIZE DETERMINATION 
See Sections 9.4.2 and 9.4.4 
9.3 POPULATIONS FOR ANALYSES 
 
Not Applicable 
9.4 
STATISTICAL ANALYSES 
9.4.1 GENERAL APPROACH 
This is a phase 1b/2 study with dose escalation for determination of the MTD/RP2D of NE-DLI via a traditional 3+3 
dose escalation schemaa followed by an expansion cohort for assessing safety profile and preliminary estimation of 
the overall response rate (ORR) (complete response and partial response based on International Working Group 
definition). Eligible patients will be treated with NE-DLI in the pre-transplantation setting.  
9.4.1.1 
ANALYSIS PLAN 
                                                                    
 
 
a See Appendix F 

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Toxicity events for NE-DLI will be descriptively summarized at each dose level. The response rates, including ORR 
and hematologic response by IWG 2006 criteria will be estimated via an exact binomial test with 95% confidence 
interval in combining patients treated at the RP2D dose level and the expansion cohort patients. Overall survival, 
defined from date of treatment to date of death or censored at last follow-up date, will be estimated via Kaplan-
Meier method (combining patients treated at the RP2D dose level and expansion cohort patients). Other outcomes 
such as independence from red blood cell transfusion or platelet transfusion for 56 consecutive days or more, 
occurrence of AE, unexpected transplant complications will be descriptively reported via summary statistics. 
9.4.2 ANALYSIS OF THE PRIMARY ENDPOINT(S) 
For the dose escalation portion, a minimum of two subjects and a maximum of 18 subjects will be enrolled. There 
will be three dose levels in the dose escalation portion. MTD is defined as the highest dose level with the 
probability of DLT less than 33% (DLT events are defined in Section 6). The DLT observation window will be until 
confirmation that the donor cells have been rejected (ANC > 500/microL AND confirmation of either bone marrow 
donor chimerism <5% or donor blood CD3+ chimerism <5%) OR within 56 days following NE-DLI, whichever occurs 
first. Per the traditional 3+3 schema, up to three patients may be enrolled concurrently and treated at the same 
dose level. The first three patients will be treated at dose level 1. For each dose level, if none of three patients 
experiences a DLT, we will escalate to the next dose level until reaching dose level 3. For each dose level, if one of 
the three patients experiences a DLT, we will enroll three additional patients and treat at the same dose level. In 
this case, if none of these additional three patients experiences a DLT, we will escalate the dosage to next level 
until reaching dose level 3. For each dose level, if two or more patients among six enrolled patients experience a 
DLT, or two or more patients in the three patient cohort experience DLT, the dosage level exceeds the MTD and 
the previous dose level will be declared as the MTD. If the first dose level exceeds the MTD, the study will be 
suspended. If no subjects experience a DLT at any dose level, the MTD will be level 3 (5 x 107 CD4+ T cells/kg). 
Similarly, if exactly one of six patients enrolled at dose level 3 experiences a DLT, then the MTD will be dose level 3. 
All patients in a dose level must be observed through the DLT window prior to enrolling patients at a higher dose 
level.  

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9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
The RP2D is defined as the dose level selected to be evaluated in the expansion cohort or future phase II studies 
and will be equivalent to the MTD determined in the Phase 1b portion of the study. Once we determine the RP2D 
level, we will enroll 10 additional eligible patients and treat with the RP2D level. The goal for this expansion cohort 
is to evaluate the safety and tolerability for patients who will be treated with NE-DLI in the RP2D dose level, and to 
evaluate the preliminary efficacy estimate for the ORR. Including the dose escalation portion in which 3 or 6 
enrolled patients will have been treated at the RP2D level, a total of at least 13 patients will be treated at the RP2D 
level. Based on previous data,(99) the complete response rate using microtransplantation was in the range of 36%-
52% for MDS or AML from antecedent MDS patients. Hence, for the phase 2 portion, an ORR of 45% is considered 
clinically meaningful. With a sample size of 13 patients, if our ORR is 45%, the 95% CI (exact binomial) will be (18-
74%); if the ORR is 50%, the 95% CI will be (22-78%).  The response rates, including ORR and hematologic response 
by IWG 2006 criteria will be estimated via an exact binomial test with 95% confidence interval in combining 
patients treated at the RP2D dose level and the expansion cohort patients.   
9.4.4 SAFETY ANALYSES 
We will monitor the toxicity events for the expansion cohort for every 2 patients after the first 3 patients. We will 
apply a Bayesian toxicity monitoring rule: if the posterior probability of experiencing DLT exceeding 0.25 is greater 
than 65%, we will hold the enrollment for reviewing the MTD. The prior distribution for this monitoring rule is a 
beta,(1,5) which translates to a 90% probability of DLT being between 1% and 45.1%. The stopping rules for toxicity 
monitoring and operating characteristics for expansion cohort are listed in the following:  
Table 1: Early stopping rule for monitoring toxicity 
 
Number of 
Patients 
3 
4-6 
7-10 
Number of 
Patients with DLT 
resulting in 
escalation 
suspension 
2 
3 
4 
 

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Table 2: Operating Characteristics of stopping rule for toxicity, based on 50,000 simulations: 
True Toxicity 
Rate 
Probability of declaring treatment 
too toxic 
Average sample size 
15% 
20% 
25% 
30% 
35% 
40% 
45% 
7.1% 
15.3% 
26.5% 
39.6% 
52.9% 
65.4% 
76% 
9.8 
9.5 
9.2 
8.7 
8.2 
7.7 
7.2 
 
 
9.4.5 BASELINE DESCRIPTIVE STATISTICS 
 
Not applicable 
 
9.4.6 PLANNED INTERIM ANALYSES  
 
See section 9.4.2. If the first dose level exceeds the MTD, the study will be suspended. 
 
9.4.7 SUBGROUP ANALYSES 
 
Not applicable 
 
9.4.8 TABULATION OF INDIVIDUAL PARTICIPANT DATA 
 
Not applicable 
 
9.4.9 EXPLORATORY ANALYSES 
Toxicity events for NE-DLI will be descriptively summarized at each dose level. Overall survival, defined from date 
of treatment to date of death or censored at last follow-up date, will be estimated via Kaplan-Meier method 
(combining patients treated at the RP2D dose level and expansion cohort patients). Other outcomes such as 
independence from red blood cell transfusion or platelet transfusion for 56 consecutive days or more, occurrence 
of AE, unexpected transplant complications will be descriptively reported via summary statistics. 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
10.1.1 INFORMED CONSENT PROCESS 
10.1.1.1 
CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS 
PROVIDED TO PARTICIPANTS 

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A study investigator, study research nurse, or clinical trial coordinator working on this study will obtain written 
informed consent. An explanation of the nature of study, its purpose, procedures involved, expected duration, 
potential risks and benefits will be provided to each participant by the investigator or the research nurse. Each 
participant will be informed that participation in the study is voluntary and that he may withdraw from the study 
at any time, and that withdrawal of consent will not affect his subsequent medical treatment. Participants will be 
allowed time needed to make an informed decision. Participants will be encouraged to ask questions about the 
study and the consent before signing the consent form. Consent forms will be filed with the Clinical Research 
Office and copies stored securely with the study coordinator. No patient will enter the study before his informed 
consent has been obtained. 
 
10.1.1.2 
CONSENT PROCEDURES AND DOCUMENTATION 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study and 
continues throughout the individual’s study participation. Consent forms will be IRB-approved and the participant 
will be asked to read and review the document.  
 
The investigator will explain the research study to the participant and answer any questions that may arise. A 
verbal explanation will be provided in terms suited to the participant’s comprehension of the purposes, 
procedures, and potential risks of the study and of their rights as research participants.  
 
Participants will have the opportunity to carefully review the written consent form and ask questions prior to 
signing. The participants should have the opportunity to discuss the study with their family or surrogates or think 
about it prior to agreeing to participate. The participant will sign the informed consent document prior to any 
procedures being done specifically for the study.  
 
Participants must be informed that participation is voluntary and that they may withdraw from the study at any 
time, without prejudice. A copy of the informed consent document will be given to the participants for their 

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records. The informed consent process will be conducted and documented in the source document (including the 
date), and the form signed, before the participant undergoes any study-specific procedures. The rights and welfare 
of the participants will be protected by emphasizing to them that the quality of their medical care will not be 
adversely affected if they decline to participate in this study. 
 
 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable cause. 
Written notification, documenting the reason for study suspension or termination, will be provided by the 
suspending or terminating party to study participants, investigator, funding agency, the IND sponsor and 
regulatory authorities. If the study is prematurely terminated or suspended, the PI will promptly inform study 
participants, the IRB, and sponsor and will provide the reason(s) for the termination or suspension. Study 
participants will be contacted, as applicable, and be informed of changes to study visit schedule. 
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping  
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
Study may resume once concerns about safety, protocol compliance, and data quality are addressed, and satisfy 
the sponsor, IRB and/or FDA. 
10.1.3 FUTURE USE OF STORED SPECIMENS AND DATA  
Data collected for this study will be analyzed and stored at the Moffitt Cancer Center. After the study is completed, 
the de-identified, archived data will be transmitted to and stored at the Moffitt Cancer Center, for use by other 
researchers including those outside of the study. Permission to use this data will be included in the informed 
consent.  
 

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These samples could be used to research the causes of myelodysplastic syndrome and/or acute myeloid leukemia, 
its complications and other conditions for which individuals with myelodysplastic syndromes/acute myeloid 
leukemia are at increased risk, and to improve treatment. The Moffitt Cancer Center will also be provided with a 
code-link that will allow linking the biological specimens with the phenotypic data from each participant, 
maintaining the blinding of the identity of the participant. 
 
During the conduct of the study, an individual participant can choose to withdraw consent to have biological 
specimens stored for future research. However, withdrawal of consent with regard to biosample storage may not 
be possible after the study is completed.  
 
When the study is completed, access to study data and/or samples will be provided through the Moffitt Cancer 
Center. 
10.1.4 KEY ROLES AND STUDY GOVERNANCE 
 
PI 
Hany Elmariah, MD, MS 
 
Moffitt Cancer Center 
12902 USF Magnolia Drive 
813-745-8248 
Hany.elmariah@moffitt.org 
10.1.5 SAFETY OVERSIGHT 
SAEs from this protocol will be reported concurrently to the IRB and the study sponsor. The Protocol Monitoring 
Committee (PMC) will review these SAEs in accordance with the protocol-specific DSMP. The data and safety plan 
will define dose limiting toxicities, rules for escalation of dose, and criteria for stopping the trial and defining the 
MTD according to rules set forth by this protocol. This trial will be continuously monitored by the PI and the research 
team and reviewed at bi-weekly Immunotherapy/Cell Therapy Research Group meetings. Safety and monitoring 
reports will be submitted to the PMC after completing each odd numbered dose level (ie, 1, 3, 5, etc.) or more 
frequently if requested by the PMC. A final safety and monitoring report will be submitted to the PMC within three 

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months of defining the MTD. This protocol will be subject to periodic internal audits based on risk or as 
recommended by the PMC. 
10.1.6 CLINICAL MONITORING 
MCC’s Internal Monitors will periodically monitor regulatory documents and CRFs according to the protocol 
specific CMP. Monitoring will include review of data for accuracy, completeness, and source verification, reporting 
of SAEs, and adherence to the protocol, GCP guidelines, and applicable regulatory requirements. 
10.1.7 QUALITY ASSURANCE AND QUALITY CONTROL 
Each clinical site will perform internal quality management of study conduct, data and biological specimen 
collection, documentation and completion. An individualized quality management plan will be developed to 
describe a site’s quality management. 
 
QC procedures will be implemented beginning with the data entry system and data QC checks that will be run on 
the database will be generated. Any missing data or data anomalies will be communicated to the site(s) for 
clarification/resolution. 
 
Following written SOPs, the monitors will verify that the clinical trial is conducted and data are generated and 
biological specimens are collected, documented (recorded), and reported in compliance with the protocol, ICH 
GCP, and applicable regulatory requirements (e.g., Good Laboratory Practices (GLP), Good Manufacturing Practices 
(GMP)).  
 
The investigational site will provide direct access to all trial related sites, source data/documents, and reports for 
the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory authorities. 
10.1.8 DATA HANDLING AND RECORD KEEPING  
10.1.8.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  

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Data will be captured in OnCore and/or Moffitt’s electronic CTMS. For each subject enrolled, the electronic CRF 
must be completed by the assigned data manager or other authorized study staff. Clinical data will be entered 
directly from the source documents. Any paper forms should be typed or filled out indelible ink and must be 
legible. Errors should be crossed out but not obliterated, the correction inserted, and the change initialed and 
dated by the investigator or his/her authorized delegate. This also applies to records for those subjects who fail to 
complete the study. If a subject stops dosing or terminates from the study, the dates and reasons must be noted 
on the CRF. If a subject terminates from the study because of a DLT, thorough efforts should be made to clearly 
document the outcome. 
 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site investigator. 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness of the data 
reported. 
10.1.8.2 
STUDY RECORDS RETENTION  
Study documents should be retained for a minimum of 10 years after the last approval of a marketing application 
in an ICH region and until there are no pending or contemplated marketing applications in an ICH region or until at 
least 10 years have elapsed since the formal discontinuation of clinical development of the study intervention. 
These documents should be retained for a longer period, however, if required by local regulations. No records will 
be destroyed without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to 
inform the investigator when there is no longer a need for these documents to be retained. Permission must be 
acquired from the State of Florida for document destruction after the 10-year minimum record-retention period 
described above has elapsed. 
10.1.9 PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, ICH GCP, or MOP requirements. The 
noncompliance may be either on the part of the participant, the investigator, or the study site staff. As a result of 
deviations, corrective actions are to be developed by the site and implemented promptly.  

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These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 QA and QC, Section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
 
It is the responsibility of the site investigator to use continuous vigilance to identify and report deviations within 7 
working days of identification of the protocol deviation, or within 7 working days of the scheduled protocol-
required activity. All deviations must be addressed in study source documents. Protocol deviations must be sent to 
the reviewing IRB per their policies. The site investigator is responsible for knowing and adhering to the reviewing 
IRB requirements. Further details about the handling of protocol deviations will be included in the MOP. Deviations 
must be entered into the CTMS. 
10.1.10 
PUBLICATION AND DATA SHARING POLICY 
This study will be conducted in accordance with the Food and Drug Administration (FDA) publication and data 
sharing policies and regulations for FDA funded research: 
FDA will ensure grantee and contractor compliance with planned publication access and data management 
requirements by requiring, as a term and condition of the grant or contract award, periodic reporting to 
contracting officer representatives and program officers as a part of regular grants and contract management. 
Extramural researchers will periodically report  
• all published articles resulting from research funding and the unique FDA manuscript repository identifier 
associated with the article; and  
• the status of data collection and preservation, including deviation from the approved data management plan.  
Failure to comply with the publication access and data management requirements—including the periodic 
reporting requirements―may serve as grounds to terminate the contract or cancel the grant. This decision rests 
with the contracting or grants management officer and will be made only after a careful review of all relevant facts 
and circumstances, and with appropriate consultation with the program office administering the grant or contract. 

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Prior to funding new grant and contract awards or continuing non-competing continuation awards, FDA will 
confirm awardees are in compliance with the FDA Public Access Policy. FDA will not award a grant or contract until 
an awardee has come into compliance with the policy. 
10.1.11 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by the pharmaceutical industry, is 
critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, analysis, 
publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who have a perceived 
conflict of interest will be required to have such conflicts managed in a way that is appropriate to their 
participation in the design and conduct of this trial. The study leadership in conjunction with the National Cancer 
Institute has established policies and procedures for all study group members to disclose all conflicts of interest 
and will establish a mechanism for the management of all reported dualities of interest. 
 

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10.2 ABBREVIATIONS 
 
MDS 
Myelodysplastic syndrome 
HMA 
Hypomethylating Agent 
sAML 
Secondary acute myeloid leukemia 
MTD 
Maximum Tolerated Dose 
IWG 
International Working Group 
GVHD 
Graft-versus-host disease 
ANC 
Absolute neutrophil count 
CBC 
Complete blood count 
CMP 
Comprehensive metabolic panel 
CMV 
Cytomegalovirus 
CXR 
Chest x-ray 
Diff 
Differential blood count 
Echo 
Echocardiogram 
EKG 
Electrocardiogram 
MUGA 
Multigated acquisition (cardiac) 
PCR 
Polymerase chain reaction 
Allo 
Allogeneic 
BMT 
Bone marrow transplant 
PD1 
programmed cell death protein 1 
PDL1 
PD1 ligand 
CTLA4 
cytotoxic T lymphocyte associated protein 4 
DLI 
Donor lymphocyte infusion  
NE-DLI 
Nonengrafting DLI (CD8 depleted DLI) 
AHD 
Antecedent hematologic disorder 
WHO 
World Health Organization 
RP2D 
Recommended phase 2 dose 
ATRA 
All trans-retinoic acid 
ECOG 
Eastern Cooperative Oncology Group 
CrCl 
Creatinine clearance 
IND 
Investigational New Drug Application 
IRB 
Investigational Review Board 
LVEF 
Left ventricular ejection fraction  
MCC 
Moffitt Cancer Center 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
MSDS 
Material Safety Data Sheet 
NIH 
National Institutes of Health 
NIH IC 
NIH Institute & Center 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SMC 
Safety Monitoring Committee 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
IBW 
Ideal Body Weight 

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CONSORT 
Consolidated Standards of Reporting Trials 
DLT 
Dose Limiting Toxicity 
ORR 
Overall Response Rate 
CRS 
Cytokine Release Syndrome 
CRF 
Case Report Forms 
FACT 
Foundation for the Accreditation of Cellular Therapy 
AE 
Adverse Event 
CTCAE 
Common Terminology Criteria for Adverse Events 
NCI 
National Cancer Institute 
HAMA 
Human anti-mouse antibodies 
mAbs 
Monoclonal antibodies 
UP 
Unanticipated Problems 
 
10.3 PROTOCOL AMENDMENT HISTORY 
 
Any changes to the protocol will be made in the form of an amendment and must be approved by the IRB before 
implementation. 
 
The table below is intended to capture changes of IRB-approved versions of the protocol, including a description of 
the change and rationale. A Summary of Changes table for the current amendment is located in the Protocol Title 
Page.  
 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
1/25/2019 
Daley Drucker formatted into MCC 
Template; minor editing and changes. 
All Moffitt Cancer Center Clinical 
Trial Protocols must be in the MCC 
template. 
2.1 
2/8/2019 
Dr. Elmariah added additional 
information and reviewed Version 2.0 
changes. 
Completed the template and 
reviewed for clarity. 
2.2 
2/11/2019 
Daley Drucker addressed Dr. Elmariah’s 
questions and provided secondary minor 
edits and reformatting. 
Answered Dr. Elmariah’s questions, 
provided consistent formatting. 
3.0 
2/12/2019 
Dr. Elmariah edits 
Approval of comments by Daley 
Drucker 
3.1 
2/14/2019 
Daley Drucker review and final edits. 
Finalizing format. 
3.2 
2/14/2019 
Paul Fletcher reviewed Drucker’s 
formatting before resubmission to PI. 
 
Finalize formatting 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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12 APPENDIX 

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12.1 APPENDIX A: INTERNATIONAL PROGNOSTIC SCORING SYSTEM- REVISED OF 
MYELODYSPLASTIC SYNDROME(58) 
Table 1: Prognostic score variables 
Prognostic 
Variable 
0 
0.5 
1 
1.5 
2 
3 
4 
Cytogenetics 
Very 
Good 
 
Good 
 
Intermediate 
Poor 
Very Poor 
BM Blast % 
<=2 
 
>2-<5% 
 
5-10% 
>10% 
 
Hemoglobin 
=>10 
 
8-<10 
<8 
 
 
 
Platelets 
=>100 
50-<100 
<50 
 
 
 
 
ANC 
=>0.8 
<0.8 
 
 
 
 
 
 
Table 2: Cytogenetic Risk Groups 
 
 
 
Table 3: Risk Categories 
 
 
RISK CATEGORY 
RISK SCORE 
Very Low 
≤1.5 
Low 
>1.5-3 
Intermediate 
>3-4.5 
High 
>4.5-6 
Very High 
>6 
 
 

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12.2 APPENDIX B: RESPONSE CRITERIA ACCORDING TO THE INTERNATIONAL WORKING 
GROUP(1) 
 
 
 
 

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12.3 APPENDIX C: EVALUATION AND MANAGEMENT OF CYTOKINE RELEASE 
SYNDROME(75) 
 

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12.4 APPENDIX D: CONSENSUS CONFERENCE CLINICAL GRADING OF ACUTE GVHD(96) 
Clinical Staging 
Stage 
Skin 
Liver: Total Bilirubin 
Intestinal Tract: Diarrhea 
0 
No rash 
<2.0 mg/dL 
<500 ml/day 
1 
<25% of skin surface 
2.0-3.0 
500-1000 ml/day 
2 
25-50% 
3.1-6.0 
1001-1500 ml/day 
3 
Erythroderma 
6.1-15.0 
>1500 ml/day 
4 
Erythroderma with bullae and 
desquamation 
>15.0 
Severe abdominal pain with or 
without ileus 
 
Clinical Grading 
Grade 
Skin* 
Liver 
GI 
I 
1-2 
0 
0 
II 
3 
1 
1 
III 
- 
2-3 
2-4 
IV 
4 
4 
- 
*Each column identifies minimum stage for organ grade.

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12.5 APPENDIX E: GRADING OF CHRONIC GVHD(97) 
  

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12.6 APPENDIX F: CRITERIA FOR DOSE ESCALATION 
Dose escalation will occur if 0 out of the first 3 patients experience DLTs. Rules for dose escalation are as follows: 
0/3 DLTs: Escalate the dose (dose levels 1-2), or halt dose escalation (dose level 3).  
1/3 DLTs: Study an additional 3 patients. If an additional patient experiences a DLT, halt dose escalation. If no 
other patient experiences DLT (that is if 1/6 patients on dose level experiences DLT), escalate the dose (dose levels 
1-2) or halt dose escalation (dose level 3).  
2/3 DLTs: Halt dose escalation. 
The decision to escalate the dose of donor peripheral blood cells and to enroll patients to the higher dose level 
may not be made until one of the following has occurred: 
1. The first 3 patients have been fully evaluated for DLTs and none has experienced a DLT.  
2. Six patients have been fully evaluated and only 1 has experienced a DLT. 
The DLT observation window continues until the first of the following:  
1. ANC > 500/microL AND confirmation the DLI has been rejected (either bone marrow donor chimerism 
<5% or donor blood CD3+ chimerism <5%) 
2. Within 56 days following NE-DLI  
If neither of these conditions has been met, patients will continue to be accrued to the current dose level. In the 
event that 0/3 patients experience DLT, accrual to the next higher dose level may proceed even if additional 
patients have been treated at the lower dose level and are not yet evaluable. However, if any of those patients 
experience DLT, accrual to the higher dose level will be suspended until a total of 6 patients have been treated and 
evaluated at the lower dose level. Further accrual to the higher dose level will not resume until the DLT incidence 
in the lower dose level is documented to be no greater than 1/6 patients. Accrual to the higher dose level will be 
stopped if the incidence of DLTs in the previous level exceeds 33% (for instance, if none of the first 3 patients 
experiences DLT, but the next 2, who were accrued during the observation period for the first 3 patients, do). 
DLTs are defined as any of the following that is at least possibly attributable to the investigational treatment: 
1. Grade 2-4 infusion toxicity 
2. Grade 4 organ toxicity (as defined by NCI CTCAE v 5.0), not preexisting, not due to the primary 
malignancy, and not due to chemotherapy or infection 
3. Grade 3 organ toxicity that does not resolve within 14 days and which is not preexisting, not due to the 
primary malignancy, and not due to chemotherapy or infection 
4. Grade 2-4 acute GvHD 
5. Delayed neutrophil recovery: ANC>500/l does not occur within 56 days AND measurable donor 
chimerism is >5% 
6. Grade 3-4 CRS; see Appendix for grading scheme) 
7. Any Grade 5 reaction other than progressive disease. 
 
If 2 or more of the first 6 patients on any dose level experience DLTs, accrual to the trial will be suspended and the 
sponsor of the IDE (the PI) will initiate a formal consultation with the FDA in order to determine whether the 

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observed toxicities are or are not attributable to the device. Subsequent accrual to the trial may resume only with 
permission from the FDA. Any questions or concerns as to the relationship of grade > 3 toxicities to use of the 
investigational device will be discussed with the appropriate FDA Clinical Review Staff.