Phase I/II Study of Donor Lymphocyte Infusion With Methotrexate GVHD Prophylaxis to Hasten Immune Reconstitution After CD34+ Cell-Selected Transplant
Phase: PHASE1
Conditions: Immunodeficiency
Sponsor: Wake Forest University Health Sciences

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Phase I/II Study of Donor Lymphocyte Infusion with Methotrexate GVHD Prophylaxis to 
Hasten Immune Reconstitution after CD34+ Cell-Selected Transplant 
 
[Companion study to A Phase II Study Using the CliniMACS® Device for CD34+ Cell 
Selection and T Cell Depletion for Graft-versus-Host Disease Prophylaxis in Alternative 
Donor Stem Cell Transplant Recipients] 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator 
Andrew Gilman, MD 
 
 
 
 
Levine Children’s Hospital 
 
 
 
 
Carolinas Medical Center 
 
 
 
 
1000 Blythe Boulevard  
 
 
 
 
Charlotte, NC 28203 
 
 
 
 
Phone: (704) 381 – 6847 
 
 
 
 
Fax:  
(704) 381 – 6841 
 
 
 
 
E-mail: Andrew.Gilman@carolinashealthcare.org 
 
 
Investigational New Drug# BB-IND-14045 
 
 
Version 1.9 (11/14/2014) 
 
Previous Versions: 
 
Version 1.8 (1/31/2014) 
 
Version 1.7 (7/11/2013) 
 
Version 1.6 (7/30/2012) 
Version 1.5 (3/14/2012) 
 
Version 1.4 (3/4/2011) 
 
Version 1.3 (12/14/2010) 
Version 1.2 (9/7/2010) 
 
Version 1.1 (11/30/2009) 
 
Version 1.0 (3/20/2009) 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
STUDY COMMITTEE .............................................................................................................................. 4 
SHORT ABSTRACT ................................................................................................................................. 5 
1.0 
GOALS AND OBJECTIVES ..................................................................................................... 8 
1.1 
PRIMARY OBJECTIVE .................................................................................... 8 
1.2 
SECONDARY OBJECTIVES .............................................................................. 8 
2.0 
BACKGROUND AND RATIONALE ....................................................................................... 8 
2.1 
INTRODUCTION ........................................................................................... 8 
2.2 
EXPERIENCE WITH DONOR T LYMPHOCYTE INFUSIONS (DLI) AFTER T CELL-DEPLETED 
TRANSPLANTS ........................................................................................................ 9 
2.3 
OTHER APPROACHES TO HASTEN IMMUNE RECONSTITUTION ............................... 10 
2.4 
RATIONALE FOR PLANNED APPROACH ............................................................ 10 
3.0 
STUDY DESIGN AND ELIGIBILITY CRITERIA .............................................................12 
3.1 
STUDY DESIGN .......................................................................................... 12 
3.2 
PATIENT INCLUSION CRITERIA ...................................................................... 12 
3.3 
PATIENT EXCLUSION CRITERIA ..................................................................... 13 
3.4 
PATIENT RECRUITMENT .............................................................................. 13 
3.5 
PATIENT REGISTRATION .............................................................................. 13 
3.6 
DONOR SELECTION CRITERIA ....................................................................... 13 
4.0 
INVESTIGATIONAL TREATMENT PLAN .........................................................................13 
4.1 
TREATMENT DOSAGE AND ADMINISTRATION .................................................... 13 
4.2 
DURATION OF THERAPY .............................................................................. 14 
5.0 
DOSE MODIFICATIONS AND TOXICITIES ....................................................................15 
5.1 
METHOTREXATE ....................................................................................... 15 
5.2 
RITUXIMAB .............................................................................................. 15 
5.3 
DOSE MODIFICATIONS ................................................................................ 16 
6.0 
SCHEDULE OF ASSESSMENTS ..........................................................................................17 
6.1 
TIME AND EVENTS TABLE ............................................................................ 17 
6.2 
PATIENT PRE-STUDY ASSESSMENTS ............................................................... 17 
6.3 
TREATMENT ASSESSMENTS .......................................................................... 17 
6.4 
FOLLOW UP ASSESSMENTS ........................................................................... 18 
7.0 
CRITERIA FOR TERMINATION ..........................................................................................18 
7.1 
CONDITIONS FOR TERMINATING THE STUDY .................................................... 18 
7.2 
CONDITIONS FOR INDIVIDUAL PATIENT TERMINATION ........................................ 18 
8.0 
STATISTICAL CONSIDERATIONS .....................................................................................18 
8.1 
STUDY DESIGN .......................................................................................... 18 
8.2 
SAMPLE SIZE ............................................................................................ 19 
8.3 
DATA MANAGEMENT PROCEDURES/PROCESS ................................................... 19 
8.4 
DATA ANALYSIS ......................................................................................... 19 
8.5 
ESTIMATED DURATION OF STUDY .................................................................. 21 
9.0 
CRITERIA FOR EVALUATION .............................................................................................21 
9.1 
MONITORED OUTCOMES .............................................................................. 21 

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9.2 
TOXICITY DEFINITIONS ............................................................................... 21 
10.0 
DATA SAFETY MONITORING PLAN .................................................................................21 
10.1 
OVERSIGHT AND MONITORING PLAN ............................................................... 22 
10.2 
MONITORING AND REPORTING GUIDELINES ..................................................... 22 
10.3 
REVIEW AND OVERSIGHT REQUIREMENTS ....................................................... 22 
11.0 
ETHICAL ASPECTS .................................................................................................................24 
11.1 
REGULATORY CONSIDERATIONS .................................................................... 24 
11.2 
INDEPENDENT ETHICS COMMITTEES/INSTITUTIONAL REVIEW BOARD .................. 24 
12.0 
DATA FORMS AND SUBMISSION SCHEDULE ..............................................................24 
12.1 
STUDY MONITORING ................................................................................... 24 
APPENDIX I - ACUTE GVHD STAGING AND GRADING FOR CHILDREN ........................26 
APPENDIX II - CHRONIC GVHD STAGING AND GRADING FOR CHILDREN .................27 
REFERENCES ..........................................................................................................................................31 

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STUDY COMMITTEE 
 
Principal Investigator 
Andrew Gilman, MD 
Director, Pediatric Blood and Marrow 
Transplantation 
Levine Children’s Hospital 
Carolinas Medical Center 
PO Box 32861 
Charlotte, NC 28232-2861 
Phone: (704) 381 – 6847 
Fax:  
 (704) 381 – 6841 
E-mail: 
Andrew.Gilman@carolinashealthcare.org 
 
Sub-Investigators 
Javier Oesterheld, MD 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard  
Charlotte, NC 28203 
Phone: (704) 381 – 9900  
Fax:  
 (704) 381 – 9901  
E-mail: 
Javier.Oesterheld@carolinashealthcare.org  
 
Michael J. Eckrich, MD, MPH 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard 
Charlotte, NC 28203 
Phone: (704) 381 – 9900  
Fax:  
 (704) 381 – 9901 
E-mail: 
Michael.Eckrich@carolinashealthcare.org 
 
Alexis F. Teplick, MD 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard 
Charlotte, NC 28203 
Phone: (704) 381 – 9900  
Fax:  
 (704) 381 – 9901 
E-mail: 
Alexis.Teplick@carolinashealthcare.org 
 
 
 
 
 
 
 
 
 
 
 
Study Coordinator  
Mark Cannon 
Levine Children’s Hospital 
Levine Cancer Institute 
Carolinas Medical Center 
1021 Morehead Medical Drive, Suite 
2200 
Charlotte, NC 28204 
Phone: (980) 442 – 2324 
Fax:     (980) 442 – 2301 
E-mail: 
Mark.Cannon@carolinashealthcare.org 
 
Statistician 
Anastasia Ivanova, PhD 
Associate Professor 
Dept. of Biostatistics 
School of Public Health 
CB# 7420 
3103-C McGavran-Greenberg Hall 
University of North Carolina at Chapel 
Hill 
Chapel Hill, NC 27599-7420 
Phone: (919)  –  843 8086 
Fax:  
 (919)  – 966 3804  
E-mail: aivanova@bios.unc.edu 
 
Data and Safety Monitoring Committee  
Chair: 
Paul Martin, MD 
Director, Long-Term Follow-Up program 
Fred Hutchinson Cancer Research 
Center 
Tel 206- 667-4415 
Fax 206- 667-5619 
Email: pmartin@fhcrc.org  
 
DSMC Coordinator 
Laura Hancock 
PBMTC Operations Center 
440 E. Huntington Drive, 4th Floor 
Arcadia, CA 91006-3776 
Ph:  (626) 241-1619 
Fax: (626) 445-4334 
email: lhancock@pbmtc.org 
 
 
 
 
 
 
 
 

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SHORT ABSTRACT 
 
A previous clinical trial has suggested that giving donor T cells after a mismatched T 
cell-depleted stem cell transplant resulted in recovery of T cells in the patient. However, 
this approach is associated with a risk of graft-versus-host disease (GVHD).  The 
purpose of this study is to determine whether giving a donor lymphoycyte infusion (DLI) 
with methotrexate GVHD prophylaxis can accelerate immune recovery in recipients of T 
cell-depleted stem cell transplants without causing significant GVHD.  At day 30 (up to 
Day +42 if necessary) after transplant, patients will receive DLI.  The patients will be 
monitored for immune recovery as measured by CD4 count. They will also be monitored 
for graft versus host disease (GVHD).  The dose of DLI will be escalated to determine a 
dose at which immune recovery is achieved without significant GVHD.  The starting 
dose will be 3 x 104 T cells/kg and the highest dose will be 10 x 104/kg.  In addition, all 
patients will receive methotrexate after the DLI to prevent GVHD.  All patients will be 
followed for 2 years after transplant to determine outcomes.    

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SCHEMA 
 
 
 
 
 
 
 
 
COHORT 1  
DLI  
(3 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
COHORT 2  
DLI  
(4 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
COHORT 3  
DLI  
(5 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
 
COHORT 4  
DLI  
(6 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
 
COHORT 5  
DLI  
(8 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
 
COHORT 6  
DLI  
(10 x 104/kg)   
+  
Methotrexate  
(10 mg/m2) 
Days: 1, 3, 10, 
17& 24 
[7.5 mg/m2] 
 31& 38 
[5 mg/m2] 
45, 52, 59, 66, 
73, 80 
RECIPIENT 
Day +30 after transplant (up to Day +42 if necessary) 
 
DONOR 
Apheresis of mobilized stem cells + Donor Lymphocytes 
(Cryopreserve aliquot for Donor Lymphocyte infusion) 

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LIST OF ABBREVIATIONS 
 
AE 
 
Adverse Event 
ALL 
 
Acute Lymphocytic Leukemia 
AML 
 
Acute Myelocytic Leukemia 
ANC 
 
Absolute Neutrophil Count 
ASCO  
American Society for Clinical Oncology  
BMT 
 
Bone Marrow Transplant 
BSA 
 
Body Surface Area 
CIBMTR 
Center for International Blood and Marrow Transplant Research 
CMV 
 
Cytomegalovirus 
CR 
 
Complete Response 
CRF 
 
Case Report Form 
CTC 
 
Common Toxicity Criteria 
DFS 
 
Disease-Free Survival 
DLI 
 
Donor Lymphocyte Infusion 
DLT 
 
Dose Limiting Toxicity 
EBV 
 
Epstein-Barr Virus 
EFS 
 
Event-Free Survival 
FDA 
 
Food and Drug Administration 
FHCRC 
Fred Hutchinson Cancer Research Center 
GCP 
 
Good Clinical Practice 
G-CSF  
human Granulocyte-Colony Stimulating Factor 
GMP   
Good Manufacturing Practice 
GVHD  
Graft Versus Host Disease 
HLA 
 
Human Leukocyte Antigen 
ICH 
 
International Conference on Harmonization 
IND 
 
Investigational New Drug 
IEC 
 
Independent Ethics Committee 
IRB 
 
Institutional Review Board 
JMML  
Juvenile Myelomonocytic Leukemia 
KIR 
 
Killer cell Immunoglobulin-like Receptor 
MDS 
 
Myelodysplastic Syndrome 
MRI 
 
Magnetic Resonance Imaging 
MTX 
 
Methotrexate 
PBSC  
Peripheral Blood Stem Cells 
PCR 
 
Polymerase Chain Reaction 
PI 
 
Principal Investigator 
PTLD   
Post-transplant Lymphoproliferative Disorder 
rATG  
polyclonal rabbit Anti-Thymocyte Globulin  
SAE 
 
Serious Adverse Event 
SCT 
 
Stem Cell Transplant 
TBI 
 
Total Body Irradiation 
TMP/SMX 
Trimethoprim-sulfamethoxazole 
TRM 
 
Transplant Related Mortality 
 

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1.0 
GOALS AND OBJECTIVES 
 
1.1 
Primary objective 
To assess the ability of a donor lymphocyte infusion given with methotrexate 
GVHD prophylaxis to accelerate immune recovery following transplantation of a 
CD34+selected, T cell-depleted stem cell product. 
 
1.2 
Secondary objectives  
1.2.1 To determine the optimum dose of DLI that will accelerate 
immune recovery without causing significant GVHD 
1.2.2 To determine the incidence and severity of GVHD after this 
treatment  
1.2.3 To determine the incidence of infection and EBV-related post-
transplant lymphoproliferative disease (PTLD) after this treatment 
 
 
2.0 
BACKGROUND AND RATIONALE 
 
2.1 
Introduction  
This study will be a companion study to a study which uses the CliniMACS® 
device for CD34+ (hematopoietic stem cell) cell selection and T cell depletion to 
prevent severe (grade III-IV), life-threatening acute graft-versus-host disease 
(GVHD) after alternative donor stem cell transplant. The purpose of this study is 
to investigate post-transplant immunotherapy to hasten immune recovery and 
decrease post-transplant infections. 
 
Blood stem cell and bone marrow transplants (SCT) can cure patients with 
leukemia, lymphoma, myelodysplastic syndrome, and bone marrow failure. Only 
15-25% of patients will have histocompatible (HLA-identical) family member 
donors. For patients without a matched family donor, alternative donors can 
include matched or mismatched volunteer unrelated adult donors or cord blood, 
or mismatched family members. A major complication of unmanipulated, 
unrelated adult donor or cord blood is severe (grade III-IV), life-threatening acute 
GVHD, which occurs in 25-30% of patients (1;2). Mismatched family member 
donor transplant is associated with a very high risk of severe acute GVHD (3). 
GVHD results in rash, diarrhea, and jaundice. GVHD is caused by T cells (or T 
lymphocytes). GVHD is amplified by inflammatory proteins (cytokines) that are 
released as a result of high-dose chemotherapy and radiation therapy given just 
prior to the transplant (4). Removal of the T cells from unrelated and 
mismatched related SCT can prevent severe GVHD. Older studies reported that 
T cell depletion is associated with a high incidence of graft rejection and 
leukemic relapse (5). More recent experience shows that newer techniques for T 
cell depletion and for transplantation have overcome these challenges (6-10). 
However, the low number of T cells given with the SCT results in minimal 
passive transfer of immunity and delayed immune recovery. This is associated 
with high risk of morbidity and mortality due to infections and remains as a 
challenge (6;11-13). 
 
We are participating in a study sponsored by University of California – San 
Francisco (UCSF # 01151) that is close to completing accrual. The study uses 
the CliniMACS® device for CD34+ cell selection and T cell depletion.to prevent 
graft-versus-host disease (GVHD) after mismatched family member donor 
transplant. 
The 
study 
uses 
the 
conditioning 
regimen 
(radiation 
and 
chemotherapy) established by Martelli and colleagues in Perugia, Italy (14).  The 
study attempts to improve immune recovery and decrease post-transplant 

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infections by the following: (1) giving a large dose of CD34+ cells, (2) reducing the 
dose of anti-thymocyte globulin (ATG) given to the patient before transplant to 
decrease the number of donor T cells that are killed after transplant, (3) giving a 
fixed small dose of T cells with the stem cell transplant. Immune recovery is 
judged by achievement of > 100 CD4+ T cells/uL of blood by 100 days after 
transplant. The study also allowed a donor T cell infusion of 3 x 104 T cells/kg to 
be given for patients who have serious viral infections or those who do not have 
a CD4+ T cells/uL by 100 days after transplant. The study has enrolled 17 of the 
planned 21 patient accrual. Sixty-five percent of patients remain alive with a 
median follow-up of 2 years (range 1 month – 4 years). Although overall survival 
is very good, immune recovery has not been accelerated in most patients and 
post-transplant infections have caused significant morbidity. Only 3/17 (18%) of 
patients achieved > 100 CD4+ T cells/uL by 100 days after transplant and 3/4 of 
these patients either had received donor T cell infusion for a serious viral 
infections prior to 100 days or had GVHD (in which case the T cells probably 
represented those causing the GVHD and not providing protection against 
infection). We have therefore developed this study to build on the success of the 
UCSF study, and add a new approach to hasten immune recovery and decrease 
post-transplant infections. 
 
Because the overall survival has been very good and because there has been no 
severe acute GVHD, we will extend this approach to patients with unrelated 
donors as well. 
 
2.2 
Experience with donor T lymphocyte infusions (DLI) after T cell-
depleted transplants 
As part of a retrospective review with UCSF, we recently reported giving low dose 
DLI (3-6 x 104/kg) to 16 recipients of mismatched related donor SCT (15). The 
DLI given were from the CD34-negative fraction of the G-CSF mobilized blood 
stem cells that were cryopreserved at the time of SCT. Ten of these patients were 
transplanted for malignancies or aplastic anemia, and 6 were transplanted for 
immunodeficiency. DLI were planned to be given to hasten immune recovery in 
patients that had a CD4+ T cell count < 100/mcl at 100 days after SCT for 
malignancy or at 4 weeks after SCT for immunodeficiency, or for the treatment 
of infection. DLI were given at a median 12 weeks (range 4-34 wks) after SCT. 
The median time to have a CD4+ T cell count > 100/mcl was 1 month (range 
0.5-5.5 months) after DLI. Four patients never achieved this response. Grade I-II 
GVHD was seen in 19% of patients and no severe GVHD occurred. Of the 15 
patients that survived more than 100 days, 20% developed chronic GVHD. Six 
patients died of pre-existing or new infections, including 2 in the context of 
treatment for chronic GVHD. These results suggest that DLI given at a median of 
12 weeks after a mismatched SCT are reasonably well tolerated and result in a 
recovery of T cells. However, this approach doesn’t address the marked risk of 
infection during the first few months after SCT.  
 
Lewalle et al. reported a dose-finding study of G-CSF mobilized mismatched 
related donor DLI in 11 adults (16). Nine patients given 1 x 104 CD3+ cells/kg 
starting one month after SCT and 4 developed Grade I to Grade III GVHD. Two 
patients were given a dose of 3 x 104 CD3+ cells/kg and both had Grade II 
GVHD. Both eventually died from complications of chronic GVHD. In 
comparison to our experience, this data suggests that either early (ie. 1 month) 
after SCT DLI and/or adult recipients is associated with an increased risk of 
GVHD. 
 

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Handgrettinger et al. reported the use of DLI in recipients of mismatched related 
donor SCT (17). The DLI were given from freshly collected blood (not from the G-
CSF mobilized stem cell collection). Eleven patients were given doses from 2.5-
10 x 104 CD3+/kg around 1 month after transplant. The DLI could be repeated 
at monthly intervals. Six patients developed acute GVHD – grade I in 2 patients 
(dose 2.5 x 104 for one and 5 x 104 x 2 for one), grade II in 3, patients and grade 
4 in 1 patient. Immune recovery was faster than usually reported for this type of 
transplant, with the median time to reach CD3+ cells > 100/mcl being 72 days 
after transplant. This study also suggests that DLI soon after SCT is associated 
with an increased risk of acute GVHD. 
 
2.3 
Other approaches to hasten immune reconstitution  
One approach to hasten immune recovery involves the administration of donor T 
lymphocytes that are not alloreactive (don’t recognize the transplant recipient’s 
tissues as different) and therefore not capable of causing GVHD. This approach 
involves the stimulation of donor blood with recipient blood and then the 
subsequent removal of activated (alloreactive) T cells. The basis for this 
approach is that after removal of alloreactive T cells, the T cells that remain are 
not alloreactive but can provide protection against infectious agents. The 
removal of the activated (alloreactive) T cells has been done using 
immunomagnetic selection (in a similar fashion to how CD34+ cells are obtained 
with the CliniMACS® device) with antibodies against CD25 or CD69, which are 
activation antigens on T cells (18). A similar approach uses an antibody against 
CD25 that has been linked to a toxin to kill the activated cells (19). The 
approaches for depletion of alloreactive T cells are technically cumbersome, 
require GMP facilities for cellular therapy, and have a potential risk of 
transmitting infection to the recipient as a result of the ex vivo manipulation. It 
is unlikely that such approaches will ever be widely available. 
 
An approach that would provide killing of alloreactive T cells in vivo would be 
feasible at a larger number of centers and would reduce costs. Such an 
approach has been used for mismatched related donor transplantation. Donor 
stem cells (not T cell depleted) were used and high-dose cyclophosphamide given 
3-4 days after transplantation to delete alloreactive T cells (20). Patients were 
also given cyclosporine and mycophenolate mofetil to prevent GVHD after 
transplant. The incidence of grade III-IV acute GVHD was 6%. However, the 
incidence of relapse was high and survival was only 36%. The study was 
conducted in adults with advanced hematological malignancies, preventing a 
comparison with our experience. 
 
2.4 
Rationale for planned approach 
This study will use a donor T lymphocyte (CD3+ cells) infusion (DLI) to help 
immune recovery. As discussed above, T cells cause GVHD. The likelihood of 
GVHD depends on the T cell dose given, when they are given, and if ATG is given 
around the time of T cell infusion. GVHD prophylaxis will be given with the 
donor T cell infusion to prevent severe GVHD. Our hypothesis is that the use of 
GVHD prophylaxis with methotrexate will prevent GVHD by killing T cells 
responding to alloantigens in the recipient while sparing a sufficient number of 
viral-specific T cells to provide some immunity and protection from infection. 
 
2.4.1 GVHD prophylaxis  
Murine models suggested that a delayed DLI separated from the 
transplant would prevent GVHD (21). However, the delay in humans 
probably needs to be 3 to 6 months and this leaves patients at high risk 
for infection. Early (ie. 1 month after transplant) DLI carries a significant 

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risk of severe acute GVHD when given without GVHD prophylaxis 
(14;22). 
This study will explore an approach that combines aspects of the 
approaches described above. Methotrexate, which is an anti-proliferative 
chemotherapy drug like cyclophosphamide, will be used. Methotrexate 
has been used for the prevention of GVHD for many years (23). This 
approach is also supported by laboratory data that showed that 
trimetrexate (a folate antagonist similar to methotrexate) can delete 
alloreactive T cells. Remaining lymphocytes were able to respond to cells 
from a third party and to candidal antigens (24).  
 
2.4.2 Rituximab  
Following a comprehensive review of study data in September 2014, it 
was noted that a significant number of subjects began to have B cell 
recovery as early as 8 weeks after transplant.  This recovery may be due 
to B cells in the donor lymphocyte infusion (DLI) and may have 
contributed to disease, such as post-transplant lymphoproliferative 
disease (PTLD) and autoimmune disease, in some patients.  We believe 
that we may be able to prevent these complications by giving rituximab 
with the DLI.  The risk of giving rituximab will be slower B cell 
reconstitution but we think that the benefit of preventing disease due to 
B cells will outweigh this risk.  A lower dose of rituximab than used with 
transplant was chosen because we are only trying to eliminate the B cells 
in the DLI and not those in the patient.  The dose that we will use has 
recently been reported in the setting of T alpha beta depletion where B 
cells are given at the time of transplant (31) 
  
2.4.3 Timing of T cell infusion 
T cells can be given with the stem cells at the time of transplant. As 
noted above, this has been done with T replete alternative donor 
transplants using post-transplant cyclophosphamide. Most approaches 
utilize very T cell-depleted transplants (12). The UCSF study used a very 
T cell-depleted transplant, but added back a fixed dose (3 x 104 CD3+ 
cells/kg) of T cells to the transplant. Immune recovery was not 
accelerated, perhaps due to the ATG that had been given prior to 
transplant. Residual ATG in the patient may have killed these T cells. 
 
We will delay the DLI until 30 days after the SCT. Reasons for the delay 
are: residual ATG in the patient should be less at that time point to 
decrease the chance of T cells being killed; delaying the DLI will allow 
engraftment and healing from the transplant conditioning regimen-
related toxicity.  Methotrexate given soon after the conditioning regimen 
adds to oral and GI mucositis and slows recovery of blood counts. 
Patients receiving CD34+ selected transplants with a large stem cell dose 
typically have very robust engraftment at one month after transplant and 
should be able to tolerate the methotrexate without a significant decrease 
in blood counts. 
 
2.4.4 Cell dose 
Our treatment plan will be to target the T cell dose at transplant to < 1 x 
104 CD3+ cells/kg recipient. A dose of < 1 x 104 CD3+ cells/kg should 
minimize the risk of GVHD from the transplant (12). We will then give 
DLI with methotrexate GVHD prophylaxis in an attempt to hasten 
immune reconstitution while preventing significant GVHD from these 
cells. 

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We have chosen 6 DLI (CD3+ or T cell) doses to evaluate. We are 
attempting to balance the chance of benefit from the DLI - faster immune 
recovery and fewer infections - with the risk of severe GVHD. Our 
experience in children with viral infections after this type of transplant is 
that 
they 
have 
benefited 
from 
anti-viral 
responses 
against 
cytomegalovirus, Epstein-Barr Virus, and adenovirus following DLI doses 
of 3 x 104/kg. Viral-specific T cells are present at a level of 1 per 1 x 104 
cells to 1 per 105 T cells CMV and EBV, respectively (25;26). A DLI dose 
of 3 x 104/kg to an average size patient weighing 30 kg will provide 9 x 
105 T cells. This should provide adequate viral-specific clones that can be 
expanded in vivo to prevent viral infections.  
 
We have seen GVHD in patients with a dose of 3 x 104 cells/kg. In the 
haploidentical donor setting, others have reported a T cell dose of > 1-5 x 
104/kg as the threshold for causing GVHD when no GVHD prophylaxis is 
used. The range of doses probably reflects different variables like time 
post-transplant, number of T cells in the PBSC graft, and conditioning 
regimen (16;17). Therefore, there will not be a cohort of patients receiving 
DLI for immune reconstitution without methotrexate. Our hypothesis is 
that the methotrexate prophylaxis will prevent GVHD and allow these 
doses of T cells to be given. If there is not acceleration of immune 
recovery and severe GVHD is not seen with a dose of 3 x 104 cells/kg, the 
dose of DLI will be escalated to a maximum of 1 x 105 cells/kg. We will 
not exceed this dose because we feel that the risk of severe GVHD 
outweighs the potential benefit at higher doses.  
 
 
3.0 
STUDY DESIGN AND ELIGIBILITY CRITERIA  
 
3.1 
Study design 
Patients will receive a donor lymphocyte infusion (DLI) on Day +30 (up to Day 
+42 if necessary) after transplant. The DLI dose will be escalated for each cohort 
as described below.  Rituximab 200 mg/m2 IV will be given on the day of DLI 
infusion prior to the infusion if possible.  It can be given +/- 1 day if necessary 
for logistical reasons.  After the DLI, patients will be given IV methotrexate as 
GVHD prophylaxis. The methotrexate dose will be as follows: 
Days 1, 3, 10, 17, 24: 10 mg/m2  
Days 31, 38: 7.5 mg/m2 
Days 45, 52, 59, 66, 73, 80: 5mg/m2   
Days 0 denotes day of donor lymphocyte infusion. 
 
 
3.2 
Patient inclusion criteria 
3.2.1 Patients treated on the study “A Phase II Study Using the 
CliniMACS® Device for CD34+ Cell Selection and T Cell Depletion 
for Graft-versus-Host Disease Prophylaxis in Alternative Donor 
Stem Cell Transplant Recipients”.  
3.2.2 All patients or legal guardians must have agreed to participate by 
signing an informed consent document. 
 
 
Day 
0 
Day 
1 
Day 
3 
Day 
10 
Day 
17 
Day 
24 
Day 
31 
Day 
38 
Day 
45 
Day 
52 
Day 
59 
Day 
66 
Day 
73 
Day 
80 
DLI 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
MTX 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

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3.3 
Patient exclusion criteria 
 
3.3.1 Active GVHD at the time when DLI are due  
3.3.2 History of acute GVHD > grade I prior to DLI 
3.3.3 Disease due to viral infection (eg. CMV) when DLI are due 
(asymptomatic viral replication or viral shedding is not a 
contraindication) 
3.3.4 Uncontrolled bacterial or fungal infection 
3.3.5 O2 saturation by pulse oximetry < 95% 
3.3.6 Bilirubin > 3mg/dL or ALT > 5 x upper limit of normal 
3.3.7 Creatinine > 3x baseline (baseline is creatinine at admission for 
transplant) 
3.3.8 ANC (WBC x % neutrophils + bands) < 500/ul 
3.3.9 Significant effusions (eg. pleural or pericardial) or ascites 
3.3.10 EBV-related PTLD 
3.3.11 Persistent or increasing mixed chimerism requiring therapeutic 
DLI as defined on the LCH 09-01 protocol 
 
 
3.4 
Patient recruitment 
All patients who have a transplant on the companion study using the 
CliniMACS® device (referenced above) will be evaluated for eligibility. Patients 
who meet the eligibility criteria will be offered the opportunity to participate in 
this study. 
 
3.5 
Patient registration 
The patients will be registered by completion of eligibility forms and each patient 
will be assigned a unique subject study ID.   
 
3.6 
Donor selection criteria 
The DLI donor will be the same as the donor for the stem cell transplant.  
 
 
4.0 
INVESTIGATIONAL TREATMENT PLAN 
 
4.1 
Treatment dosage and administration  
Patients will receive a donor lymphocyte infusion on Day +30 after transplant 
(may be given up to day +42 if necessary). The DLI dose for the first cohort will 
be 3 x 104cells/kg and will be obtained from cells cryopreserved from the G-
CSF-mobilized PBSC at the time of the transplant. The dose administered will be 
based on the cryopreserved dose. Dose escalation will continue as described in 
the Statistical section (8.0).  
 
4.1.1 Donor lymphocyte infusion (DLI) 
DLI will be cryopreserved at the time of the CD34+ selection from donors. 
The DLI will be prepared from the “negative” fraction of the stem cell 
harvest. DLI will be cryopreserved in bags containing 3-10 x 104 CD3+ 
cells/kg of recipient weight. 
 
DLI will be thawed and infused according to the LCH BMT procedures for 
thawing and infusion of cryopreserved hematopoietic progenitor cells. 
When a dose is needed that is less than a dose that was frozen, an 
aliquot of a volume proportional to the desired dose may be drawn into a 
syringe. 
 
4.1.2 Rituximab 

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Rituximab 200 mg/m2 IV will be given on the day of DLI infusion prior to 
the infusion if possible.  It can be given +/- 1 day if necessary for 
logistical reasons. 
 
4.1.3 Patients will receive methotrexate IV as follows: 
Day 1 (approximately 24 hrs after DLI infusion): methotrexate 10 mg/m2 
Day 3: methotrexate 10 mg/m2 
Day 10: methotrexate 10 mg/m2 
Day 17: methotrexate 10 mg/m2 
Day 24: methotrexate 10 mg/m2 
Day 31: methotrexate 7.5 mg/m2 
Day 38: methotrexate 7.5 mg/m2 
Day 45: methotrexate 5 mg/m2 
Day 52: methotrexate 5 mg/m2 
Day 59: methotrexate 5 mg/m2 
Day 66: methotrexate 5 mg/m2 
Day 73: methotrexate 5 mg/m2 
Day 80: methotrexate 5 mg/m2 
 
The dose should be given on these days whenever possible, especially for 
doses through Day +24.  Doses after day 3 dose may be given +/- 2 days 
if necessary. 
 
Concurrent use of other myelosuppressive drugs should be 
minimized to avoid neutropenia. 
 
4.1.4 There will be a dose escalation of DLI as follows: 
Cohort 1: DLI 3 x 104/kg  
Cohort 2: DLI 4 x 104/kg  
Cohort 3: DLI 5 x 104/kg  
Cohort 4: DLI 6 x 104/kg  
Cohort 5: DLI 8 x 104/kg  
Cohort 6: DLI 10 x 104/kg  
 
The dose escalation will occur separately for Cohorts A (mismatched 
 
related donors) and B (unrelated donors) as discussed in the statistical 
 
section.    
Details of the dose escalation are provided in the Statistical section (8.0). 
 
The DLI dose will be calculated using the weight used to calculate the 
CD34+ cell dose for PBSC transplant. At investigator discretion, the dose 
may be adjusted in the following situations: 
(1) the dose may be reduced to a dose based on current weight at time of 
DLI if the patient has lost more than 10% body weight from the weight 
used to calculate the CD34+ dose 
(2) the dose may be reduced to a dose based on adjusted body weight if 
the patient’s actual weight is greater than 120% of the ideal body weight. 
The ideal and adjusted body weights will be calculated according to the 
LCH Blood and Marrow Transplant Program High-dose Chemotherapy 
Orders policy. The investigator will consider factors such as disease for 
which the transplant was performed, patient’s body frame and build, etc. 
 
4.2 
Duration of therapy   
Patients will be followed for study outcomes for 2 years after transplant.  
 

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5.0 
DOSE MODIFICATIONS AND TOXICITIES 
 
5.1 
Methotrexate  
5.1.1 Availability  
Methotrexate is commercially available in a number of dosage forms: 
 
Solution for Injection: 2.5 mg/ml (2ml vial), or 25 mg/ml 
(2ml, 4ml, 8ml, and 10ml vials) 
 
Powder for Reconstitution/Injection: 20 mg, 100 mg, 1000 
mg 
 
Oral Tablet: 2.5 mg 
 
5.1.2 Storage and stability  
Store tablets and intact vials at room temperature and protect from light. 
Dilute powder with D5W or NS to a concentration of <25 mg/ml (for 20 
mg and 50 mg vials) or 50 mg/ml (for the 1g vial size). Further dilutions 
are stable for 24 hours at room temperature. Reconstituted solutions 
with a preservative may be stored under refrigeration for up to 3 months 
and for up to 4 weeks at room temperature.  
 
5.1.3 Preparation  
The 1 g vial may be diluted in 100 mL of saline or D5W. 
 
5.1.4 Administration  
Methotrexate, in this protocol, will be administered as a slow push IV. No 
leucovorin rescue is required at the doses used in this protocol. 
 
5.1.5 Toxicity  
Most 
common 
adverse 
effects 
are 
alopecia, 
rash, 
diarrhea, 
nausea/vomiting, and mucositis.   
 
Most serious adverse effects are skin ulceration, hyperuricemia, 
myelosuppression (usually with higher doses), LFT abnormalities, liver 
failure, hepatic cirrhosis/fibrosis/necrosis, acute renal failure, and 
interstitial pneumonitis. 
 
5.2 
Rituximab 
5.2.1 Availability 
Rituximab is commercially available as a solution for injection: 
 
10mg/ml (10ml and 50ml vials) 
 
5.2.2 Storage and stability 
Vials are stable a 2°C-8°C (36°F-46°F) protected from direct sunlight.  Do 
not freeze or shake.  Rituxmab solutions for infusion may be stored at 
2°C-8°C (36°F-46°F) for 24 hours and at room temperature for an 
additional 24 hours. 
 
5.2.3 Preparation 
Dilute to a final concentration of 1 mg/ml in an infusion bag containing 
either 0.9% Sodium Chloride, USP, or 5% Dextrose in Water, USP. 
 
5.2.4 Administration 
Infuse at an initial rate of (0.5 mg/kg/hour; Maximum of 50 mg/hour) 
for the first hour.  If no hypersensitivity or infusion related events 

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increase rate by (0.5 mg/kg/hour; Maximum 50 mg increase every 30 
minutes) every 30 minutes to a maximum rate of 3x maintenance IV 
fluids (Max 400mg/hr). 
5.2.5 Toxicity 
Most common side effects include fever, chills, nausea, weakness, 
headache, low blood pressure, itching, rash, bronchospasm, abdominal 
pain, vomiting, anemia, achy joints and muscles, dizziness, congestion, 
low blood counts, suppression of B lymphocytes resulting in a greater 
risk of infections. Rare side effects include angioedema, severe 
reactivation of hepatitis B infection, liver failure, angioedema involving 
the skin, mouth, and GI and GU tracts, and progressive multifocal 
leukoencephalopathy. 
 
5.3 
Dose Modifications 
Patients that develop neutropenia (ANC < 1000/ul) during the three months 
after DLI will be given G-CSF approximately 5 mcg/kg IV/SQ as needed. G-CSF 
will not be given within 24 hrs of a dose of methotrexate.  The investigator may 
decide to not give G-CSF depending on the clinical situation.  The reason should 
be documented.   
 
If a patient has an ANC < 500/ul when methotrexate is due, the methotrexate 
will be delayed until the ANC is > 500/ul.    
 
ANC is the absolute number of neutrophils + bands (% neutrophils + % bands X 
WBC).   
 
5.3.1 The dose of methotrexate will be adjusted as follows:  
 
 
 
 
 
 
If creatinine level is > 2x baseline, then leucovorin (10 mg/m2 IV or PO 
q6h for 4 doses) will be started 24 hours after the Methotrexate has been 
given.  The baseline creatinine is the creatinine at the time of transplant 
admission.   
 
5.3.2 Hold methotrexate dose in the presence of significant effusions or 
ascites.  
 
5.3.3 Hold methotrexate for serum ALT > 20x upper limit of normal 
(ULN). 
 
5.3.4 When a dose of methotrexate is held, it should be given as 
soon as the reason for holding has resolved (for example, ALT < 
20 X ULN). The investigator may choose to continue to hold it 
depending on the clinical scenario. The reason should be 
documented. 
 
5.3.5 A methotrexate dose may be held if required by the patient’s 
condition (eg. sepsis). Methotrexate may continue to be held for 
ongoing serious infection such as significant viral infection.  The 
reason should be documented and should also be discussed with 
the Principal Investigator. 
Bilirubin 
(mg/dl) 
%dose 
Creatinine mg/dl 
%dose 
< 3.0 
100 
=< 2x baseline  
100 
3.1 - 6.0 
50 
> 2x baseline 
50 
> 6.0 
HOLD 
> 3x baseline 
HOLD 

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6.0 
SCHEDULE OF ASSESSMENTS  
 
 
Timing of protocol therapy administration and response assessment studies are 
based on schedules derived from the experimental design or on established 
standards of care. Minor unavoidable deviations (up to 72 hours) from protocol 
directed therapy and/or disease evaluations for valid clinical, patient or family 
logistical, or facility or procedure and/or anesthesia scheduling issues are 
acceptable. Necessary minor deviations for common logistical or clinical reasons 
will not be considered violations at audit. Minor delays as described above will 
not be construed as prospectively planned or instituted on a routine basis so 
advance IRB approval will not be required. 
 
 
6.1 
Time and events table  
 
Assessment 
Pre-
study 
Prior 
to 
MTX* 
Follow up$© 
Off 
Study 
Day 
56 
Day 
100 
Day 
120 
6 
Mo 
9 
Mo 
12 
Mo 
24 
Mo 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
History and PE 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
O2 saturation 
X 
 
 
 
 
 
 
 
 
 
Bilirubin, ALT, Cr 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CBC, diff 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lymphocyte markers 
(CD3, CD4, CD8, 
CD19, CD16/56) 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
*MTX = methotrexate on days 1, 3, 10, 17 24, 31, 38, 45, 52, 59, 66, 73, and 80 
after DLI; The assessments are preferably performed within 2 days prior to 
methotrexate, but history, PE, and performance status may be up to 5 days 
prior to methotrexate.   
$Follow up is days/months after transplant.  Assessments can be +/- 7 days for 
evaluations at Day 56, 100 and 120.  The evaluation at Day 120 should be 
within 2 days of Day 120 if possible. Assessments can be within one month of 
target after Day 120. 
©If the patient has progression of primary disease or receives therapeutic DLI 
(eg. For the treatment of viral infection or relapse), chemotherapy, or a PBSC 
infusion then the above follow-up tests are optional at discretion of the 
physician.   
 
6.2 
Patient pre-study assessments 
The patients will have a full history and physical examination before 
participating in this study.  In addition, the assessments listed in section 6.1 
will be completed for all recipients.    
 
6.3 
Treatment assessments 

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The assessments listed in section 6.1 will be followed during the treatment 
portion of the protocol.  In addition, the recipients will be routinely evaluated for 
GVHD and other toxicities.     
 
6.4 
Follow up assessments 
Recipients will be followed for survival and development of GVHD up to 2 years 
after transplant for efficacy of the protocol.  After 2 years, the recipients will be 
followed as standard procedure for transplants.   
 
 
7.0 
CRITERIA FOR TERMINATION  
 
7.1 
Conditions for terminating the study 
The Principal Investigator may terminate the study for any of the following 
reasons: 
7.1.1 Significant toxicities 
7.1.2 If it becomes clear that the study treatment is less effective than 
other available treatments. 
 
7.2 
Conditions for individual patient termination 
The Principal Investigator may terminate the participation of an individual 
patient for any of the following reasons: 
 
7.2.1 Extraordinary medical circumstances 
If at any time the constraints of this protocol are detrimental to the 
patient's health, the patient will be removed from protocol therapy.  In 
this event: 
 
Principal Investigator will be notified. 
 
Document the reason(s) for withdrawal (Case Report Form). 
 
Follow the patient for survival. 
 
CHS IRB will be notified as part of the annual renewal unless the 
reason for removal is unexpected for the patient’s condition, 
transplant, and/or therapy, in which case the IRB will be notified 
promptly. 
 
7.2.2 Voluntary withdrawal 
The patient may at any time withdraw from protocol therapy.  The steps 
outlined above will be followed. 
 
7.2.3 Excessive toxicity 
Excessive toxicity will be determined by the Principal Investigator and 
steps outlined above will be followed.    
 
7.2.4 Investigator’s judgment 
The Principal Investigator or attending physician may deem that it is in 
the best interest of the patient to be removed from protocol. The steps 
outlined above will be followed.  The patients will be included in the 
intent-to-treat analysis.  
 
 
8.0 
STATISTICAL CONSIDERATIONS  
   
8.1 
Study design  

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The goal of the study is to determine a DLI dose that (1) results in a biological 
endpoint of CD4+ count > 100 at Day +120 after transplant and (2) avoids dose-
limiting toxicity defined as unacceptable GVHD. This will be accomplished by a 
dose escalation of DLI to a dose that results in a high percentage of patients 
with a CD4+ count > 100. Dose escalation will also be limited by the occurrence 
of dose-limiting toxicity, which will be unacceptable GVHD. Unacceptable GVHD 
will be defined as grade IV acute GVHD or an excessive rate of grade II/III acute 
GVHD as defined in Section 8.4.   Clinical success for a patient in this trial is to 
have successful immune recovery (CD4+ > 100 at Day +120) without 
experiencing any grade IV by Day +180. 
 
The study can be amended during its progress by adapting the methotrexate 
dosing if GVHD occurs and CD4+ recovery in the absence of GVHD doesn’t. This 
may include a change in the methotrexate dose or prolongation of methotrexate 
to more closely mimic long course methotrexate (15 mg/m2 on Day +1 followed 
by 10 mg/m2 on Days +3, 6, 11 and then weekly until Day +102). After a 
change in therapy, future patients will be considered as new cohorts even at the 
same DLI dose.  
 
8.2 
Sample size 
The study could enroll up to 46 patients (6 at each dose level plus up to 10 
additional patients at the estimated target dose) for each cohort (see below). It is 
unlikely that 6 patients will be enrolled at each dose level. It is more likely that 
enrollment will approximate the original estimate of 28 patients.  
 
8.3 
Data management procedures/process 
Proper use of the study design will be ensured by the Study Investigator 
informing the study statistician A. Ivanova (aivanova@bios.unc.edu or 919-843-
8086) whenever a grade IV acute GVHD (following DLI) occurs (except for 
unevaluable patients as defined below in section 8.4) or a new patient is ready 
for assignment.   
 
8.4 
Data analysis  
The target dose in this study is defined as the dose where:  
1) The probability of grade II and III GVHD is at most 33% and the 
probability of grade III GVHD is at most 17%; 
2) No grade IV GVHD events are observed; 
3) Probability of successful immune recovery (CD4+ > 100) by Day +120 
is at least 66%. 
The design for the dose-finding portion of the study is described below in 
subsection Dose finding design.  The following treatments with DLI will be 
considered: 
 
Cohort 1: DLI 3 x 10^4/kg  
 
Cohort 2: DLI 4 x 104/kg  
Cohort 3: DLI 5 x 104/kg  
Cohort 4: DLI 6 x 104/kg  
Cohort 5: DLI 8 x 104/kg  
Cohort 6: DLI 10 x 104/kg  
 
Patients will be evaluated for acute GVHD due to prophylactic DLI between the 
day of prophylactic DLI infusion and Day +180 after transplant. Patients that 
have not had grade II, III, or IV acute GVHD following prophylactic DLI but 
receive therapeutic DLI (eg. For the treatment of viral infection or relapse), 
chemotherapy, or a PBSC infusion prior to Day +180 will be considered 
unevaluable because these therapies may cause or prevent GVHD and affect the 

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determination of this endpoint. Patients that receive third party anti-viral cells 
within 14 days of Day +180 may be considered evaluable at the discretion of the 
Principal Investigator. The decision is based upon the fact that different cells 
may have a different risk of GVHD. In general, the risk of GVHD due to these 
cells will be very low. Patients will be replaced by enrollment of additional 
patients. 
 
There will be two cohorts of patients that will be analyzed separately: 
 
COHORT A: Recipients of transplant from mismatched related donors 
 
COHORT B: Recipients of transplant from unrelated donors 
 
 
 
8.4.1 Dose finding design 
All patients who meet the eligibility criteria will be offered the opportunity 
to participate in this study.   
 
The study design consists of two phases, a dose-escalation phase and 
dose confirmation phase.  If at any point one grade IV acute GVHD event 
is observed at a specific dose, no more patients will be assigned to this or 
higher dose levels unless the methotrexate dosing is modified.  In the 
dose-escalation phase, patients will be enrolled in groups of 3. The first 
group of 3 patients will be enrolled at the lowest of six dose levels. 
 
o 
If none of the initial three patients at a dose level 
experiences grade II/III GVHD and < 2/3 have a CD4+ 
count > 100 at Day +120, the next three patients will be 
assigned to the next higher dose level. 
o 
If 1 out of 3 patients has a grade III GVHD event or 1 – 2 
out of 3 patients have a grade II GVHD event and/or > 2/3 
have a CD4+ count > 100 at Day +120, the dose is 
repeated for the next group of patients.  
 
If < 2/6 grade II/III GVHD events and < 1/6 grade 
III GVHD events occur and < 4/6 patients have a 
CD4+ count > 100 at Day +120, the dose is 
increased for the next group of patients.  
 
If < 2/6 grade II/III GVHD events and < 1/6 grade 
III GVHD events are observed and > 4/6 patients 
have a CD4+ count > 100 at Day +120, this dose is 
selected for the dose confirmation phase.  
o 
If all 3 patients have grade II GVHD events or 2 grade III 
GVHD events are observed, 3 more patients are assigned 
to the next lower dose level, unless there are already 6 
patients at that dose level.  
 
Because of the relatively long time for a patient to be evaluable and the 
excellent early clinical outcomes on the study: 
o 
While the initial 3 patients at a dose level are being 
followed, additional patients may be enrolled at the same 
dose level if none of the initial 3 have grade II/III GVHD 
within 30 days of DLI. 
o 
While patients in the second group of 3 at a dose level are 
being followed, additional patients may be enrolled at the 
same dose level if: (a) 0-1 of the initial 3 patients had 
grade II GVHD and none had grade III GVHD, and (b) none 

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of the second group of 3 have grade II/III GVHD within 30 
days of DLI. 
o 
When patient 6 is being followed, additional patients may 
be treated at the next higher dose level if (a) none of the 
first 3 patients had grade II/III GVHD and < 2 of the first 
five evaluable patients have grade II/III GVHD, (b) patient 
6 did not have grade II/III GVHD within 30 days of DLI,  
(c) and < 3/5 patients have CD4+ count >100 at Day 
+120. Patient 6 will not be replaced if the patient becomes 
not evaluable. 
o 
If the above criteria are not met, then the additional 
patients will be enrolled at a previous, lower dose level. 
o 
When the patients being followed are at the initial dose (3 
x 10^4/kg), the additional patients will be treated at this 
dose. 
 
 
The dose-escalation phase is continued until the maximum dose of DLI is 
reached or until escalation is stopped for toxicity or efficacy.  
 
After the dose-escalation phase is completed and preliminary estimate of 
the target dose is obtained, up to 10 more patients will be assigned to the 
estimated target dose in each cohort (cohorts A and B).  
 
The confirmatory stage will be stopped and the dose re-evaluated if one 
of the following occurs: 
 
-  The lower bound of the 50% confidence interval for the rate of  grade II 
and III GVHD is above 33%  
-  The lower bound of the 50% confidence interval for the rate of  grade III 
GVHD is above 17%; 
-  Grade IV GVHD event is observed; 
-   The upper bound of the 50% confidence interval for the rate of  
successful immune recovery (CD4+ > 100) by Day +120 is lower than 
66%. If patients are having clinical benefit despite not achieving the 
target efficacy (CD4+ > 100 at Day +120) but are not having 
unacceptable rates of GVHD, then up to 10 more patients can still be 
entered.  
 
8.5 
Estimated duration of study    
The study will require 5 years for accrual and up to 2 years for follow-up 
studies. 
 
 
9.0 
CRITERIA FOR EVALUATION  
 
9.1 
Monitored outcomes   
9.1.1 The outcomes will be CD4 count at day +120 post-transplant, 
GVHD, infections, and EBV-related PTLD.   
 
9.2 
Toxicity definitions 
9.2.1 Toxicity will be graded according to the CTCAE v3.0.  
 
 
 
10.0 
DATA SAFETY MONITORING PLAN 

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10.1 
Oversight and monitoring plan 
The study will utilize the Data and Safety and Monitoring Committee (DSMC) of 
the Pediatric Blood and Marrow Transplant Consortium (PBMTC). The DSMC 
will be responsible for safeguarding the interests of participants in this trial. 
This responsibility will be exercised by providing recommendations for 
continuation or early termination of the trial, based on assessment of safety.  
The DSMC may also formulate recommendations related to the selection, 
recruitment or retention of participants, their management and adherence to 
protocol-specified regimens, and the procedures for data management and 
quality control. 
 
The DSMC will be advisory to Andrew Gilman, MD who will serve as sponsor and 
principal investigator and to his co-investigators.  Dr. Gilman and his co-
investigators will be responsible for promptly reviewing any recommendations 
and deciding how to respond. 
 
The DSMC will:  
 
Review the current protocol   
 
Review progress of the trial on a semi-annual basis 
 
Review all serious adverse events including expected and unexpected 
events according to PBMTC DSMC guidelines. 
 
10.2 
Monitoring and reporting guidelines 
The PI will conduct continuous review of data and patient safety at the weekly 
Blood and Marrow Transplant meeting where the results of each patient’s 
treatment are discussed.  The discussion will be documented in the minutes. 
The discussion may include for each dose level, the number of patients, 
significant toxicities as described in the protocol, dose adjustments, and 
observed responses.  Semi-annual reports will be submitted to the PBMTC 
DSMC for review according to PBMTC guidelines.  Unexpected grade 3 adverse 
events (AE), all grade 4-5 adverse events and all serious adverse events (SAE) 
related to study participation will be submitted to the FDA, IRB and DSMC.    
 
10.3 
Review and oversight requirements 
10.3.1 Adverse events definition 
An adverse experience is any untoward medical occurrence in a patient 
or clinical investigation subject administered a pharmaceutical product 
and which does not necessarily have a causal relationship with this 
treatment.  An adverse experience or event (AE) can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a 
medicinal (investigational) product, whether or not related to the 
medicinal (investigational) product. 
 
10.3.2 Adverse event recording 
Adverse events will be graded using the NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 3.0 found on the following 
website: 
http://ctep.cancer.gov/reporting/ctc.html 
for 
grading 
the 
severity of adverse events. All grade 3-5 adverse events will be recorded 
until 1 year post-transplant and all other endpoints in Section 9.1 will 
continue to be recorded through the duration of follow-up.  Grading of 
acute and chronic GVHD will be according to Appendix I and II.  
Causality of AE will be rated as definitely, probably, possibly, unlikely 
related, or unrelated to the DLI.  The investigator is responsible for 

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making an assessment of whether or not it is reasonable to suspect 
a causal relationship between the adverse event and the study 
treatment. 
 
10.3.3 Serious adverse events definition 
An unexpected adverse event is any adverse drug experience where the 
specificity or severity of which is not consistent with the current 
investigator brochure; or, if an investigator brochure is not required or 
available, the specificity or severity of which is not consistent with the 
risk information described in the general investigational plan or 
elsewhere in the current application. 
 
A serious adverse experience (SAE) or serious adverse drug reaction 
(ADR) is any adverse drug experience occurring at any dose that results 
in any of the following outcomes: 
10.3.3.1 
Death 
10.3.3.2 
A life-threatening adverse drug experience 
10.3.3.3 
Inpatient hospitalization or prolongation of existing 
hospitalization. 
10.3.3.4 
Persistent 
or 
significant 
disability/incapacity 
(a 
substantial disruption of a person’s ability to conduct 
normal life functions).  
10.3.3.5 
Birth defect/ congenital anomaly 
10.3.3.6 
Any important medical event that may not result in 
prior listed outcomes but, based upon appropriate 
medical judgment, may jeopardize the subject, and 
may require medical or surgical intervention to prevent 
one of the prior listed outcomes. 
 
10.3.4 Reporting procedures 
Reporting of serious adverse events will be in accordance with 21 CFR 
312.32 and 21 CFR 314.80.  If required, an NCI Adverse Drug Reaction 
(ADR) form will be completed and copies kept in the regulatory binder 
and submitted to the IRB.  
 
For all adverse events reported or observed, the information will be 
recorded in the Case Report Forms for that patient.  This will include a 
full description of the event, its severity or toxicity grade, the relationship 
to the study drug, and the treatment, outcome and sequelae of the event. 
 
FDA 
website 
for 
guidance 
in 
reporting 
serious 
adverse 
eventshttp://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSe
arch.cfm?fr+312.32 
 
 
MedWatch forms and information: 
Change this to FDA Adverse Event Reporting System  (AERS) 
http://www.fda/gov/medwatch/getforms.html 
 
If the SAE is death and determined to be possibly, probably, or definitely 
related to the investigational drug or any research related procedure, the 
event must be reported to the DSMC, or his designee within 24 business 
hours. The reporting procedure is by personal communication via phone 
or in person with written documentation of the 1:1 communication via e-
mail with a copy of the e-mail placed in the regulatory binder.  

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10.3.5 Review of adverse event rates 
Each semi-annual report will indicate if the AE incidence is considered to 
be higher than expected by the PI.  If at any time the Investigator stops 
enrollment or stops the study due to safety issues, the DSMC and IRB 
will be notified within 24 business hours via e-mail. A formal letter will 
be submitted within 10 business days and the FDA will be notified. 
 
10.3.6 Semi-annual review of study progress 
Principal Investigator is required to submit semi-annual study progress 
reports to determine whether accrual projections are being met, to 
summarize unexpected grade 3 adverse events, all grade 4 and 5 adverse 
events, SAE reports, and the rate of unexpected grade 3 and all grade 4-5 
AE’s.  In addition, progress on recruitment and subjects known response 
to the investigational therapy should be reported.   
 
These semi-annual reports are reviewed by the PBMTC DSMC.  These 
reports are scheduled for:  May and November as per DSMC procedures.  
The cut-off date for data will be 2 weeks prior to this date.  The reports 
will be submitted within at least 1 month from the scheduled date.  
 
 
11.0 
ETHICAL ASPECTS  
11.1 
Regulatory considerations 
This study will be reviewed by the Carolinas Healthcare System (CHS) 
Institutional Review Board. In addition, an Investigational New Drug 
application will be filed with the FDA for the companion protocol using 
the CliniMACS® device.  As per FDA regulations (21 CFR 312.33), annual 
reports will be submitted to the FDA within 60 days of the anniversary 
date that the IND went into effect. 
 
11.2 
Independent Ethics Committees/Institutional Review Board 
This protocol and the informed consent will be approved by the CHS IRB.  
The Principal Investigator is responsible for keeping the IRB advised of 
the progress of the study and of any changes made in the protocol prior 
to implementation.  The Principal Investigator will also keep the IRB 
informed of any significant adverse reactions, and any protocol 
exceptions or deviations.  Records of all study review and approval 
documents must be kept on file by the Principal Investigator and are 
subject to FDA inspection during or after completion of the study.  The 
IRB will receive notification of the termination of the study. 
 
 
12.0 
DATA FORMS AND SUBMISSION SCHEDULE  
12.1 
Study monitoring 
Forms will be completed and documented for each patient. The Completion 
Schedule below is intended as a reasonable guideline only. The exception is the 
Recipient Eligibility form which should be completed before the recipient 
receives study treatment. A research chart with completed paper case report 
forms and supporting documentation of reportable AEs and SAEs for all enrolled 
patients will be maintained in a locked cabinet in the BMT Research office.  
Patient enrollment information and toxicity/reporting information will also be 
entered by the coordinator into the BMT Research database. 
 
Forms 
Completion Schedule 

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Enrollment Forms: 
 
Recipient Eligibility 
 
Recipient Baseline Information 
Complete after recipient, parent, or 
guardian has signed consent and prior to 
treatment of the recipient 
Treatment Form 
Complete within one month after last dose 
of methotrexate 
Treatment Forms: 
 
Immune Recovery Form 
 
GVHD  
 
AE form 
Complete within one month of the time 
points listed in table 6.1.  
Toxicity Reporting: 
 
SAE Form 
 
CHS IRB Form 
 
FDA Medwatch Form 
Complete as required based on reporting 
criteria for each institution.   
 

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APPENDIX I - Acute GVHD staging and grading for children 
 
ORGAN 
STAGE 
DESCRIPTION 
SKIN 
1 
Maculopapular rash < 25% of 
BSA 
 
2 
25 – 50% of BSA 
 
3 
Generalized erythroderma 
 
4 
Desquamation and bullae 
 
 
 
LIVER 
1 
Bilirubin 2 - 3 mg/dL 
 
2 
Bilirubin 3.1 - 6 mg/dL 
 
3 
Bilirubin 6.1 - 15 mg/dL 
 
4 
Bilirubin > 15 mg/dL 
 
 
 
GUT 
1 
Diarrhea > 500 – 1000 ml/day 
(> 10 mL/kg - 20 mL/kg/day) 
OR persistent UGI symptoms 
 
2 
Diarrhea > 1000 – 1500 ml/day 
(> 20mL/kg – 30 mL/kg/day) 
 
3 
Diarrhea >1500 ml/day 
(>30 mL/kg/day) 
 
4 
Severe abdominal pain or ileus 
 
 
 
GRADE 
Skin 
    Liver 
 
Gut 
I 
1-2 
       0  
 
0 
II 
3 and/or 
       1      and/or 
1 
III 
-- 
     2-3      and/or 
2-3 
IV 
4 and/or 
       4      and/or 
4 
 
 
 
 
** Adapted from Glucksberg and Jacobsohn articles (27;28). 
 

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APPENDIX II - Chronic GVHD staging and grading for children 
 
Chronic GVHD grading will be performed with both a limited/extensive grading 
system and with the NIH Consensus scoring system. 
The limited/extensive grading will use the revised Seattle classification (29) 
shown below. 
 
 
 
 
 
 
 

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Grading of Chronic GVHD severity by NIH Consensus Guidelines using Organ Scoring Table (30) 
 
Severity 
 
 
Definition 
 
 
Mild 
 
Involves on 1 or 2 organs or sites (except the lung; see below), with no clinically significant 
functional impairment (maximum of score 1 in all affected organs or sites) 
 
 
Moderate 
 
(1) At least one organ or site with clinically significant but no major disability (maximum 
score of 2 in any affected organ or site OR (2) 3 or more organs or sites with no clinically 
significant functional impairment (maximum score of 1 in all affected organs or sites) OR a 
lung score of 1. 
 
 
Severe 
 
 
(1) Major disability caused by chronic GVHD (score of 3 in any affected organ) or site OR 
a lung score of > 2. 
 
 
 
 

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NIH Consensus Chronic GVHD Organ Scoring 
 

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Organ scoring of chronic GVHD. *AP may be elevated in growing children, and not reflective of liver dysfunction. 
†Pulmonary scoring should be performed using both the symptom and pulmonary function testing (PFT) scale whenever 
possible. When discrepancy exists between pulmonary symptom or PFT scores the higher value should be used for final 
scoring. Scoring using the Lung Function Score (LFS) is preferred, but if DLCO is not available, grading using FEV1 
should be used. The LFS is a global assessment of lung 
function after the diagnosis of bronchiolitis obliterans has already been established [29]. The percent predicted FEV1 and 
DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score as follows: > 80% = 1; 
70-79% = 2; 60-69% = 3; 50-59% = 4; 40-49% = 5; < 40% = 6. The LFS = FEV1 score + DLCO score, with a possible 
range of 2-12. GVHD indicates graft versus host disease; ECOG, Eastern Cooperative Oncology Group; KPS, Karnofsky 
Performance Status; LPS, Lansky Performance Status; BSA, body surface area; ADL, activities of daily living; LFTs, liver 
function tests; AP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper 
limit of normal. 
 
 

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