A Phase 1 Trial of CMV RNA-Pulsed Dendritic Cells With Tetanus-Diphtheria Toxoid Vaccine in Pediatric Patients and Young Adults With WHO Grade IV Glioma, Recurrent Malignant Glioma, or Recurrent Medulloblastoma
Phase: PHASE1
Conditions: Glioblastoma, Malignant Glioma, Medulloblastoma Recurrent, Pediatric Glioblastoma Multiforme, Pediatric Brain Tumor, Recurrent, Pediatric Brain Tumor
Sponsor: Gary Archer Ph.D.

CONFIDENTIAL 
The information contained in this document is regarded as confidential, and may not be disclosed to another party unless such disclosure is 
required to initiate the study, to conduct study-related activities, or to comply with national, state, or local laws and regulations.  Written 
authorization from the coordinating site and sponsor is required for disclosure otherwise. 
 
 
A Phase I Study 
 
ATTAC-P: A Phase 1 Trial of CMV RNA-Pulsed Dendritic Cells 
with Tetanus-Diphtheria Toxoid Vaccine in Pediatric Patients 
and Young Adults with WHO Grade IV Glioma, Recurrent 
Malignant Glioma, or Recurrent Medulloblastoma 
 
 
 
 
 
 
 
Sponsor: PI – Duke Cancer Institute 
 
 
Funding 
Source: 
Departmental Discretionary Funds from SPORE CEP 
 
 
Protocol 
Source: 
PI - Duke Cancer Institute 
 
 
Duke 
IRB#: 
Pro00092868 
 
 
IND# 
17868 
 
Principal Investigator 
Daniel Landi, MD 
DUMC Box 102382 
Durham, NC 27710 
Pager: (919) 206-3405 
Phone: (919) 613-7504 
daniel.landi@duke.edu 
  
Sub-Investigator(s) 
Gary Archer, PhD (IND holder) 
gary.archer@duke.edu 
 
David Ashley, MBBS, FRACP, PhD 
david.ashley@duke.edu 
Henry Friedman, MD 
henry.friedman@duke.edu 
 
John Sampson, MD, PhD 
john.sampson@duke.edu 
 
Kristin Schroeder, MD, MPH 
kristin.schroeder@duke.edu 
 
Statistician 
James E. Herndon II, PhD 
james.herndon@duke.edu 
 
Lead Study Coordinator 
Charlene Flahiff, MS, CCRP 
cflahiff@duke.edu 
 
Regulatory Coordinator 
Elizabeth Miller 
elizabeth.s.miller@duke.edu 
 
Data Manager 
Eric Lipp 
eric.lipp@duke.edu 
Original version: 
5/10/18 
 
Amended version 1: 
7/27/2018 
Modifications requested by CPC in Section 13.1; clarifications to inclusion criteria in Section 10.1 
Amended version 1.1: 
8/6/2018 
Change in diagnosis for newly diagnosed population from GBM to WHO Gr.IV glioma in Sections 4.1, 4.3, 
5, 7, 8.1, 10.1, 11.7.1, and 14.4.2; revisions made to the observation period for unacceptable toxicity in 
Sections 4.6, 8.1.7, 14.1, and 14.4.2.1; added language regarding risk to benefit ratio and patient assent 
in Sections 6.3, 15.3, and 15.4.  
Amended version 2: 
11/16/2018 
Clarify schema in Section 5; include description of bridge chemotherapy for recurrent patients after 
leukapheresis while their CMV-DCs are being generated (see Sections 4.3 and 8.1); Remove restriction of 
only up to 2 patients with prior craniospinal radiation as the 1st patient met this description and was able 
to generate enough PBMCs for at least 3 vaccines with one leukapheresis procedure (Sections 4.6, 
14.4.1, 14.6); revisions to inclusion and exclusion criteria in Section 10; revisions to schedule of events in 
20182259
IRB Approved at the 
Protocol Level 
Jun 13, 2019

Table 1 in Sections 11 and 11.4; corrections made to Table 2; new Section 8.1.3; change study pause for 
safety evaluation from after the 3rd patient to after the 4th patient (Sections 4.6 and 14.4.2.2). 
Amended version 3: 
4/12/2019 
Increase eligible age for patients with WHO grade III or IV glioma (Sections 4.1, 7, 8.1, 10.1, 14.4.2); 
grade IV patients without measurable/progressive disease can be included on recurrent arm (Sections 
4.1, 4.3, 5, 7, 8.1, 10.1); allow 2nd leukapheresis, if applicable, to be obtained at additional times during 
the study (Sections 4.3, 8.1, 8.1.1, 11.5, 14.4); if patients PBMCs generate only 1-2 vaccines, they may 
remain on study and receive all vaccines generated (Sections 4.3, 4.6, 8.1, 8.1.1, 9, 11.5, 11.7.1, 14.6); 
add exclusion criterion for steroid dose (Section 10.2); patients who are receiving bevacizumab may 
continue to do so (10.1, 10.2); window of time for obtaining blood for immune monitoring altered 
(Sections 8.1, 11 [Table 1], 11.4); bridge therapy option for all patients, not just recurrent added 
(Sections 4.3, 8.1, 8.1.3). 

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1 TABLE OF CONTENTS 
2 
LIST OF TABLES .................................................................................................................. 4 
3 
LIST OF ABBREVIATIONS ................................................................................................. 5 
4 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY .................................................. 6 
4.1 
Purpose ............................................................................................................................. 6 
4.2 
Background and Significance........................................................................................... 6 
4.3 
Design and Procedure....................................................................................................... 6 
4.4 
Selection of Subjects ........................................................................................................ 7 
4.5 
Duration of Study ............................................................................................................. 7 
4.6 
Data Analysis and Statistical Considerations ................................................................... 7 
5 
STUDY SCHEMA.................................................................................................................. 8 
6 
BACKGROUND AND SIGNIFICANCE .............................................................................. 9 
6.1 
Study Disease ................................................................................................................... 9 
6.2 
Study Agent ...................................................................................................................... 9 
6.2.1 
Pre-clinical experience ............................................................................................ 10 
6.2.2 
Clinical experience .................................................................................................. 11 
6.3 
Study Purpose/Rationale ................................................................................................ 13 
7 
OBJECTIVES AND ENDPOINTS ...................................................................................... 14 
8 
INVESTIGATIONAL PLAN ............................................................................................... 14 
8.1 
Study Design .................................................................................................................. 14 
8.1.1 
Removal of PBMCs by Leukapheresis ..................................................................... 15 
8.1.2 
Concomitant Temozolomide and Radiation Therapy (Newly Diagnosed WHO 
Grade IV Glioma only) ........................................................................................................... 15 
8.1.3 
Optional “Bridge” Treatment .................................................................................. 16 
8.1.4 
Dose Intensified Temozolomide Therapy ............................................................... 16 
8.1.5 
Tetanus and Diphtheria Toxoid Booster and Site Preconditioning ........................ 16 
8.1.6 
DC Vaccination ........................................................................................................ 16 
8.1.7 
Definition of Unacceptable Toxicity ........................................................................ 16 
8.1.8 
Safety Considerations ............................................................................................. 17 
8.1.9 
Concomitant Medications ....................................................................................... 20 
8.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration............................. 20 
8.3 
Rationale for Correlative Studies ................................................................................... 20 

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8.4 
Definition of Evaluable Subjects, On Study, and End of Study .................................... 21 
8.5 
Early Study Termination ................................................................................................ 21 
9 
STUDY DRUG ..................................................................................................................... 21 
9.1 
Names, Classification, and Mechanism of Action ......................................................... 21 
9.2 
Packaging and Labeling ................................................................................................. 21 
9.3 
Supply, Receipt, and Storage ......................................................................................... 22 
9.4 
Dispensing and Preparation ............................................................................................ 22 
9.5 
Compliance and Accountability ..................................................................................... 22 
9.6 
Disposal and Destruction ............................................................................................... 22 
10 SUBJECT ELIGIBILITY ..................................................................................................... 22 
10.1 Inclusion Criteria ............................................................................................................ 22 
10.2 Exclusion Criteria ........................................................................................................... 23 
11 SCREENING AND ON-STUDY TESTS AND PROCEDURES ....................................... 25 
11.1 Screening Examination .................................................................................................. 26 
11.2 Run-In Period ................................................................................................................. 26 
11.3 Leukapheresis #1 ............................................................................................................ 26 
11.4 Treatment Period ............................................................................................................ 26 
11.5 Follow-up Period ............................................................................................................ 27 
11.6 End of Study ................................................................................................................... 28 
11.7 Early Withdrawal of Subject(s) ...................................................................................... 28 
11.7.1 Criteria for Early Withdrawal .................................................................................. 28 
11.7.2 Follow-up Requirements for Early Withdrawal ...................................................... 28 
11.7.3 Replacement of Early Withdrawal(s) ...................................................................... 28 
11.8 Study Assessments ......................................................................................................... 28 
11.8.1 Medical History ....................................................................................................... 28 
11.8.2 Physical Exam .......................................................................................................... 28 
11.8.3 Radiographic Review ............................................................................................... 28 
11.8.4 Correlative Assessments ......................................................................................... 29 
12 SAFETY MONITORING AND REPORTING.................................................................... 30 
12.1 Adverse Events ............................................................................................................... 30 
12.1.1 AEs of Special Interest ............................................................................................. 31 
12.1.2 Reporting of AEs...................................................................................................... 31 
12.2 Serious Adverse Events .................................................................................................. 31 
12.2.1 Reporting of SAEs .................................................................................................... 31 

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12.3 Safety Oversight Committee (SOC) ............................................................................... 32 
12.4 External Data and Safety Monitoring Board (DSMB) ................................................... 32 
13 QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 32 
13.1 Monitoring ...................................................................................................................... 32 
13.2 Audits ............................................................................................................................. 32 
13.3 Data Management and Processing ................................................................................. 33 
13.3.1 Case Report Forms (CRFs) ....................................................................................... 33 
13.3.2 Data Management Procedures and Data Verification ............................................ 33 
13.3.3 Study Closure .......................................................................................................... 33 
14 STATISTICAL METHODS AND DATA ANALYSIS ...................................................... 34 
14.1 Analysis Sets .................................................................................................................. 34 
14.2 Patient Demographics and Other Baseline Characteristics ............................................ 34 
14.3 Treatments ...................................................................................................................... 34 
14.4 Primary Objective .......................................................................................................... 34 
14.4.1 Primary Objective #1: Feasibility ............................................................................ 34 
14.4.2 Primary Objective #2: Safety .................................................................................. 34 
14.4.3 Exploratory Objectives ............................................................................................ 36 
14.5 Interim Analysis ............................................................................................................. 36 
14.6 Sample Size Calculation................................................................................................. 36 
15 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ............................................. 37 
15.1 Regulatory and Ethical Compliance ............................................................................... 37 
15.2 DUHS Institutional Review Board and DCI Cancer Protocol Committee .................... 37 
15.3 Informed Consent ........................................................................................................... 37 
15.4 Privacy, Confidentiality, and Data Storage .................................................................... 38 
15.5 Data and Safety Monitoring ........................................................................................... 38 
15.6 Protocol Amendments .................................................................................................... 38 
15.7 Records Retention .......................................................................................................... 38 
16 REFERENCES ..................................................................................................................... 39 
 
2 LIST OF TABLES 
Table 1: Schedule of Events .......................................................................................................... 25 
Table 2: Schedule of Blood Draw Volumes ................................................................................... 30 
 
 

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3 LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
ATRT 
Atypical Teratoid-Rhabdoid Tumor 
BTIP 
Brain Tumor Immunotherapy Program 
CBC 
Complete Blood Count 
CMP 
Comprehensive Metabolic Panel 
CMV 
Cytomegalovirus 
CMV-DCs 
Long-Lamp CMV RNA-pulsed Dendritic Cells (study drug) 
CNS 
Central Nervous System 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DC 
Dendritic Cell 
DCI 
Duke Cancer Institute 
DI 
Dose Intensified  
DSMB 
Data and Safety Monitoring Board 
DUHS 
Duke University Health System 
ELISPOT 
Enzyme-Linked Immunospot 
FDA 
Food and Drug Administration 
GBM 
Glioblastoma 
GCP 
Good Clinical Practice 
Gd 
Gadolinium  
GM-CSF 
Granulocyte Macrophage-Colony Stimulating Factor 
GMP 
Good Manufacturing Practice 
ICH 
International Conference on Harmonization   
IFC 
Imaging Flow Cytometry 
IRB 
Institutional Review Board 
IV 
Intravenously 
KPS 
Karnofsky Performance Status 
LPS 
Lansky Performance Status 
MG 
Malignant Glioma 
MRI 
Magnetic Resonance Imaging 
NCI 
National Cancer Institute 
OARC 
Office of Audit, Risk, and Compliance 
OS 
Overall Survival 
PBMCs 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PD 
Progressive Disease 
PFS 
Progression Free Survival 
PI 
Principal Investigator 
PJP 
Pneumocystis jiroveci pneumonia 
PNET 
Primitive Neuro-Ectodermal Tumor 
Pp65-flLAMP Full-Length LAMP Protein 
RDSP 
Research Data Security Plan 
RT 
Radiation Therapy 
SAE 
Serious Adverse Event 
SOC 
Safety Oversight Committee 
Td 
Tetanus Toxoid 
TILs 
Tumor Infiltrating Lymphocytes 
TMZ 
Temozolomide  
ULN 
Upper Limit Normal  
VDLNs 
Vaccine-Site Draining Lymph Nodes  

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4 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
4.1 Purpose 
The purpose of this study is to determine the feasibility and safety of administering CMV RNA-pulsed dendritic 
cells (CMV-DCs) to children and young adults up to 35 years old with WHO grade IV glioma, recurrent 
malignant glioma, or recurrent medulloblastoma.  Evidence for efficacy will also be sought. 
 
Primary Objectives: 
1. Determine the feasibility of producing CMV-DCs from the leukapheresis product collected from children 
and young adults up to 35 years old with WHO grade IV glioma, recurrent malignant glioma, or recurrent 
medulloblastoma.   
2. Determine the safety of CMV-DCs in eligible patients with WHO grade IV glioma, recurrent malignant 
glioma, or recurrent medulloblastoma 
 
Exploratory Objectives: 
1. Describe change from baseline in T cell response to CMV-DC vaccination as measured by ELISPOT 
2. Describe the overall survival (OS) and progression-free survival (PFS) in children and young adults up to 35 
years old with WHO grade IV glioma, recurrent malignant glioma, or recurrent medulloblastoma.   
 
Hypothesis: 
Treatment with CMV-DCs will be feasible and safe in children and young adults up to 35 years old with WHO 
grade IV glioma, recurrent malignant glioma, or recurrent medulloblastoma.   
4.2 Background and Significance 
The incidence rate of childhood (ages 0-14 years old) primary malignant and non-malignant brain and Central 
Nervous System (CNS) tumors in the United States (U.S.) is 5.54 cases per 100,000 with an estimated 3,560 
new cases expected to be diagnosed in the U.S. in 2018 [1].  While significant improvements have been made 
in the treatment of some of these children, recurrent brain tumors are generally incurable and are routinely 
treated with maximal surgical resection followed by radiation therapy (RT) and concomitant chemotherapy 
[2].   Even with improved understanding about molecular changes in pediatric tumors, only a small number of 
targeted therapies have demonstrated clinical benefit.  Immune therapies, with their potential for highly 
specific tumor killing or ability to recruit the endogenous immune system against a tumor, hold great potential 
and are being pursued.  Whereas immune checkpoint blockade has shown some promise in children with rare 
hyper-mutated brain tumors, responses have not been durable in most cases and these agents have limited 
clinical impact for most pediatric brain tumors, due in part to the lower tumor mutational burden in most 
pediatric tumors [2, 3].  Given that the mutational landscape and immune environment in children vary from 
that of adults, pediatric-focused studies are necessary to optimize immune-based therapies for treating 
children and young adults. 
4.3 Design and Procedure 
This will be a phase 1 study evaluating CMV-DC administration with Td preconditioning and GM-CSF adjuvant 
in children and young adults up to 35 years old with WHO grade IV glioma, recurrent malignant glioma, or 
recurrent medulloblastoma.  This safety study will enroll a maximum of 10 patients.  Eligible patients will 
undergo leukapheresis to obtain autologous cells for preparation of CMV-DCs.  If necessary, patients who 
received prior craniospinal radiation (which may cause long-term suppression of their PBMCs) may undergo a 
second leukapheresis procedure, in an attempt to obtain adequate PBMCs to generate at least three DC 
vaccines. 
 
If the number of PBMCs collected from the leukapheresis (or two leukapheresis procedures for those with 
previous craniospinal radiation) is less than the number needed to make at least one vaccine, the patient will 
be withdrawn from the study.   
 

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Newly Diagnosed Patients:  After the leukapheresis procedure is completed, patients with newly diagnosed 
WHO grade IV glioma will undergo standard of care radiation therapy (RT) with or without concurrent TMZ 
prior to dose intensified (DI) adjuvant TMZ.   
 
Recurrent Patients or WHO IV patients without recurrence:  After leukapheresis procedure(s) are completed, 
patients may be given “bridge” therapy while the CMV-DCs are being generated (≥ 3 weeks) if needed.  This 
decision regarding bridge therapy will be made by the treating neuro-oncologist on a case-by-case basis 
depending upon the patient’s clinical needs.  Leukapheresis procedure(s) will ideally be completed prior to 
initiation of bridge therapy; however, the leukapheresis procedure(s) may be done during bridge therapy, at 
the discretion of the PI.   
 
Review of Appropriateness of Continuing Protocol Participation:  For all patients, eligibility criteria must be 
confirmed prior to proceeding with DI TMZ.  Patients will not proceed with DI TMZ if they do not have 
sufficient PBMCs to generate CMV-DCs for at least one vaccine.  If bridge therapy is provided for patients 
during the time the vaccines are being generated (≥ 3 weeks or recurrent patients and ≥ 4 weeks for newly 
diagnosed patients), the patient must still meet the inclusion and exclusion criteria regarding prior treatments 
described in Section 10, in order to proceed with 21-day DI TMZ.  Newly diagnosed patients must complete 
the full course of radiation to remain eligible for the study (minimum ~54 Gy).  Eligibility for newly diagnosed 
patients will be confirmed after they have completed the standard of care radiation therapy; patients may be 
removed from study at that time due to not meeting eligibility or at the discretion of the PI.   
 
Initiation of Experimental Therapy for All Patients:  For patients continuing protocol participation, a single cycle 
of 21-day DI TMZ will be administered.  On day 22 (+3 days) of their DI TMZ cycle, patients will receive the 
study drugs (Td preconditioning and CMV-DC vaccine containing GM-CSF, Vaccine #1) intra-dermally; Vaccine 
#2 and #3 will occur at 2-week intervals (± 2 days) after Vaccine #1.  Study drug administration will be divided 
equally between left and right inguinal regions.  All further available vaccines will occur monthly, 
approximately every 4 weeks (± 5 days).  Preconditioning with Td will occur 6-24 hours prior to Vaccine #1 and 
Vaccine #4+, if further vaccines are available.  Patients who are ≥ 18 years of age will also receive a Td booster 
during screening.  Patients will receive as many vaccinations as can be made from their leukapheresis 
procedure(s).   
 
Feasibility will be defined based on the production of at least three CMV-DC vaccines of 2x107 cells from the 
initial leukapheresis or initial two leukapheresis procedures for patients who have had previous craniospinal 
radiation (only expected in recurrent medulloblastoma patients).      
4.4 Selection of Subjects 
Please see Section 10. 
4.5 Duration of Study 
Unless an unacceptable toxicity is observed, patients will receive at least all the CMV-DC vaccines generated 
for them.  While the patient remains on study, they will be followed with MRI and clinical evaluations every 
three months, at the discretion of the treating physician, as possible.  Upon progression or 
removal/withdrawal from the study, patients who have received at least one CMV-DC vaccine will be followed 
periodically, if possible, for progression, future medications to treat their brain tumor, and survival.   
4.6 Data Analysis and Statistical Considerations 
The primary objectives for this study include the assessment of feasibility for generating at least three vaccines 
from the pre-treatment leukapheresis of eligible patients and the safety of treating these patients with the 
CMV-DC vaccine.  
 
Feasibility will initially be assessed separately among patients with and without prior craniospinal radiation.  A 
maximum of 10 patients may be accrued to this study in total to achieve the number of evaluable patients. 

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Only patients who have one or more vaccinations generated from their leukapheresis will begin treatment 
with the CMV-DC vaccines.  Safety is defined in terms of the proportion of patients who experience an 
unacceptable adverse event as defined in Section 8.1.7 after Td preconditioning, prior to initial vaccination 
and before the ~30 days follow-up after the 3rd vaccination, but before the 4th vaccination, if given.  Safety 
monitoring rules that are provided in Section 14.4.2.2 combine the treatment experiences of patients with and 
without prior craniospinal radiation.  After 4 patients have been enrolled and treated (i.e. the last patient has 
received Vaccine #3 and been observed for 30 days after Vaccine #3), the study will be paused for evaluation 
of safety before proceeding with further enrollment.  A patient will be considered evaluable for the 
assessment of safety if the patient experiences an unacceptable adverse event before the time when Vaccine 
#4 would be administered, or if the patient receives three vaccines and completes the endpoint observation 
period that extends ~30 days after Vaccine #3 (approximately to the time that the 4th vaccine would be 
administered).   
 
Survival and PFS will be described, as well as the percent change in pp65-specific T cell responses. 
5 STUDY SCHEMA 
 
  
 
 
 
 
 
 

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6 BACKGROUND AND SIGNIFICANCE 
6.1 Study Disease 
The incidence rate of childhood (ages 0-14 years old) primary malignant and non-malignant brain and Central 
Nervous System (CNS) tumors in the United States (U.S.) is 5.54 cases per 100,000, with an estimated 3,560 
new cases expected to be diagnosed in the U.S. in 2018 [1].  While significant improvements have been made 
in the treatment of some of these children, recurrent brain tumors are generally incurable and are routinely 
treated with maximal surgical resection followed by radiation therapy (RT) and concomitant chemotherapy 
[2].   Even with improved understanding about molecular changes in pediatric tumors, only a small number of 
targeted therapies have demonstrated clinical benefit.  Immune therapies, with their potential for highly 
specific tumor killing or ability to recruit the endogenous immune system against a tumor, hold great potential 
and are being pursued.  Whereas immune checkpoint blockade has shown some promise in children with rare 
hyper-mutated brain tumors, responses have not been durable in most cases and these agents have limited 
clinical impact for most pediatric brain tumors, due in part to the lower tumor mutational burden in most 
pediatric tumors [2, 3].  Given that the mutational landscape and immune environment in children vary from 
that of adults, pediatric-focused studies are necessary to optimize immune-based therapies for treating 
children and young adults.   
 
CMV antigens have been identified in pediatric tumors including glioblastoma (GBM) and medulloblastoma, 
and may make excellent anti-tumor immunotherapeutic targets [4, 5]. Vaccination and adoptive T cell 
strategies targeting cytomegalovirus (CMV) in humans in other contexts have been found to be safe and 
effective. Previous studies examining adult patients with GBM have demonstrated the feasibility, safety, 
immunogenicity, and potential clinical efficacy of this approach [6] and Duke is currently participating in a 
multicenter, phase 2 study (Pro00084478) of CMV RNA-pulsed dendritic cells (DCs) in adult patients with GBM.   
 
This study combines temozolomide (TMZ) therapy, tetanus toxoid (Td), and granulocyte macrophage-colony 
stimulating factor (GM-CSF) with the CMV-DC study drug to treat children and young adults with GBM, 
recurrent malignant glioma, or recurrent medulloblastoma.  Temozolomide (TMZ) has been widely used to 
treat brain tumors and is routinely given to children with high grade gliomas, regardless of the fact that it is 
not curative and shows modest efficacy in children. Therapeutic TMZ induces lymphopenia, typically a 
negative effect of chemotherapy; however, repeated DC vaccination during recovery from lympho-depletion 
can induce potent humoral and cellular immunologic responses [7-9]. GM-CSF is a powerful adjuvant capable 
of stimulating macrophage function, inducing proliferation and maturation of DCs, and is able to enhance T-
lymphocyte stimulatory function. It has been safely used in children with solid tumors following chemotherapy 
to counteract neutropenia [10, 11]. Intradermal administration of GM-CSF enhances the immunization efficacy 
at the site of administration in a dose-dependent fashion. Significant anti-tumor immunity has been 
demonstrated in preclinical murine studies in which irradiated, stably transfected tumor cell lines secreting 
GM-CSF have protected against subsequent tumor challenge, especially against intracerebral tumors. Finally, 
Td is a routinely used vaccine in the normal human population that has been shown in pilot studies to function 
as a potent adjuvant that may enhance DC trafficking to vaccine-site draining lymph nodes (VDLNs). Our 
previous studies have shown that successful DC migration to VDLNs may be a requisite for clinical activity of 
RNA-pulsed DCs, and administration of Td prior to vaccination may improve the effectiveness of DC vaccines in 
patients with GBM. 
6.2 Study Agent 
DCs are potent immuno-stimulatory cells that continuously sample the antigenic environment of the host and 
specifically activate CD4+ and CD8+ T cells and B cells [12, 13]. They are part of many networks of the immune 
system, and DCs represent a promising biologic entity for use as an immunotherapy. Potent immune 
responses and encouraging clinical results have been seen in Phase 1 and 2 human clinical trials in systemic 
cancers treated with DCs [14-30]. 
 

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Numerous animal studies [31, 32], including many of our own [33-35], and a few clinical studies [31, 36] have 
demonstrated potent antitumor responses using DC-based immunotherapy against adult malignant gliomas 
(MGs).  Specifically, CMV-targeted vaccines have been tested in adult patients at Duke in numerous IRB-
approved studies, including Pro00003877 (ATTAC) and ATTAC-GM, Pro00000580 (ERADICATE), Pro00000581 
(REGULATE), Pro00054740 (ELEVATE), Pro00065241 (AVERT), and Pro00084478 (ATTAC-II). 
 
Adjuvants frequently used with vaccination include Freund’s incomplete adjuvant, bacilli Calmette-Guerin, QS-
21, and diphtheria toxoid.  Supplemental cytokines have been used as well for the adjuvant immunological 
effects [37]. Granulocyte macrophage-colony stimulating factor (GM-CSF) has been commonly used, as it is 
commercially available and well tolerated.  GM-CSF is capable of stimulating macrophage function, inducing 
proliferation and maturation of DCs, and is able to enhance T-lymphocyte stimulatory function.  Intradermal 
administration of GM-CSF enhances the immunization efficacy at the site of administration in a dose 
dependent fashion at an optimal dose of 125 µg [38].  Significant anti-tumor immunity has been demonstrated 
in preclinical murine studies in which irradiated, stably transfected tumor cell lines secreting GM-CSF have 
protected against subsequent tumor challenge, especially against intracerebral tumors [39, 40].  Furthermore, 
the potency of GM-CSF has been demonstrated in a phase 1 clinical trial in melanoma patients vaccinated with 
irradiated autologous melanoma cells engineered to secrete GM-CSF [41].  The immunization sites were 
intensely infiltrated with T lymphocytes, DCs, macrophages, and eosinophils in 100% of evaluable patients.  
Extensive tumor destruction was seen in 11 of 16 patients.  Both cytotoxic T cells and antibody responses were 
associated with this tumor destruction. Hence, GM-CSF has an extensive track record as both a growth factor 
and an adjuvant, is commercially available, and has an acceptable toxicity profile. Our experience in multiple 
studies at Duke, including the ACTIVATE study (Pro00000580), supports the use of GM-CSF and sets the 
precedent for this phase 1 study. 
 
Td is a routinely used vaccine in the normal human population that has been shown in pilot studies to act as a 
potent adjuvant to enhance DC trafficking to VDLNs [6].  Our previous studies have shown that successful DC 
migration to VDLNs may be a requisite for clinical activity of RNA-pulsed DCs and, as such, administration of Td 
prior to vaccination may improve the effectiveness of DC vaccines in patients. 
6.2.1 
Pre-clinical experience 
Systemic immunization using DCs co-cultured with uncharacterized tumor homogenate [34], whole tumor 
RNA [35], unidentified peptides eluted from tumor cells by gentle acid washing [32], or a distinct peptide 
encompassing the tumor-specific EGFRvIII mutation [33] have been shown to induce humoral and cell 
mediated systemic immune responses and to prolong the survival of rodents with brain tumors. 
In our laboratory [34], inbred VM/Dk mice received three or four weekly intraperitoneal injections of 
autologous bone marrow-derived DCs transiently co-cultured with tumor homogenate. The homogenate was 
derived from a syngeneic murine astrocytoma cell line derived from a spontaneously occurring astrocytoma in 
the inbred VM/Dk mouse strain. Splenocytes from mice immunized in this way were able, in vitro, to lyse the 
astrocytoma cell line that was used to generate the tumor homogenate. They were also able to lyse other 
astrocytoma cell lines derived from the same inbred mouse strain, but they had no effect against syngeneic 
fibroblasts. Similarly, these immunized mice also demonstrated a significantly increased antibody titer against 
the astrocytoma cell line used to generate the homogenate. In addition, mice immunized with DCs transiently 
co-cultured with tumor homogenate that were subsequently challenged with a lethal dose of this astrocytoma 
cell line intracerebrally were found to have a median survival >160% longer than those immunized with DCs 
cultured without tumor homogenate (P=0.016). In addition, 50% of the mice treated with the tumor 
homogenate-supplemented DCs survived long-term without any evidence of tumor growth and also survived a 
re-challenge of tumor cells indicating that a sustained anti-tumor immune response had been established. 
These findings are especially significant in light of the fact that the astrocytoma cell line used is known to 

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secrete the immunosuppressive agent transforming growth factor-β (TGF-β1), which is secreted by most 
human gliomas [42-46]. 
In another report from our laboratory [35], C57BL/6 mice received three weekly intraperitoneal injections of 
autologous bone-marrow derived DCs co-cultured with tumor homogenate or whole tumor RNA derived from 
the poorly immunogenic B16F10 melanoma cell line.  Standard in vitro cytotoxicity assays again revealed that 
splenocytes harvested from mice immunized with DCs transiently co-cultured with either tumor-derived 
homogenate or whole tumor RNA were able to lyse B16F10 melanoma cells, but not unrelated tumor cells 
from the same major histocompatibility complex (MHC2) background. In this experiment, mice immunized 
with autologous bone-marrow derived DCs co-cultured with tumor homogenate or whole tumor RNA 
increased median survival by >233% (P=0.0006) and 48% (P=0.0001), respectively, relative to mice immunized 
with DCs co-cultured with tumor homogenate or whole tumor RNA derived from an unrelated tumor with the 
same MHC background. In addition, 8/13 (61.5%) in the specific homogenate group and 4/10 (40%) in the 
specific RNA group survived beyond the endpoint of the study without evidence of tumor. Immunization of 
mice with pre-existing tumors with specific tumor homogenate also demonstrated the potency of this 
immunization approach by increasing survival by 62.5% relative to controls. An inflammatory infiltrate 
composed of mononuclear cells and polymorphonuclear leukocytes was only identified in mice treated with 
DCs co-cultured with tumor homogenate that matched the intracerebral tumor challenge. 
In a recent report from another laboratory [32], the survival of tumor-bearing rats injected subcutaneously 
with autologous bone marrow-derive DCs co-cultured with peptides eluted from tumor cells with a gentle acid 
wash was significantly prolonged compared to tumor-bearing rats receiving equivalent numbers of DCs co-
cultured with peptides acid-eluted from normal astrocytes (P< 0.05).  Median survivals in these groups were 
35 and 22 days, respectively. In addition, three of the twelve rats (25%) treated with DCs co-cultured with 
acid-eluted tumor peptides remained alive at the end of the experiment. In addition, immuno-histochemical 
analysis of five animals from each group in this experiment documented an increased peri-tumoral and 
intratumoral infiltration of CD8+T cells, and to a lesser extent CD4+ T cells and macrophages, in the group 
treated with DCs co-cultured with peptides acid-eluted from tumor cells when compared to controls. 
6.2.2 Clinical experience 
We have compared the ability to generate DCs from adult patients with malignant brain tumors and patients 
undergoing craniotomy for non-tumor related procedures using a simple DC generation method described 
previously [47] to generate human DCs by culturing peripheral blood mononuclear cells (PBMCs3) in media 
supplemented with granulocyte-macrophage colony stimulating factor (GM-CSF4) and interleukin-4 (IL-45).  
The phenotype of DCs from both tumor and normal populations were identical and were characterized as 
being highly positive for HLA-ABC and HLA-DR, the co-stimulatory molecules CD80 and CD86, and the 
DC/monocyte marker CD11c, but negative for the monocyte marker CD14.  The cells were negative for the B 
and natural killer (NK6) cell lineage markers, CD19 and CD56, respectively, which is consistent with published 
DC phenotypes. 
 
The number of clinical trials using DC immunotherapy in adult patients with MGs has been on the rise.  A 
handful of these adult studies are summarized below.  In the published study by Yu et al.[31], patients 
received biweekly intradermal injections of peripheral blood derived DCs pulsed with uncharacterized 
peptides eluted from the surface of autologous glioma cells by gentle acid washings.  All patients were 
required to complete a course of RT and were off steroids at the time of immunization.  Toxicity was minimal 
and included only mild fever and lymphadenopathy.  There was no clinical or radiographic evidence of 
                                                 
1 TGF-β, transforming growth factor-β 
2 MHC, major histocompatibility complex 
3 PBMCs, peripheral blood mononuclear cells 
4 GM-CSF, granulocyte-macrophage colony stimulating factor 
5 IL-4, interleukin-4 
6 NK, natural killer 

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autoimmune encephalomyelitis in any patient and no serious adverse events occurred.  The immunization 
resulted in enhanced cytotoxic T lymphocyte (CTL7) activity in 4/7 patients and both cytotoxic and memory T-
cells were found to have infiltrated the patient’s tumors whom underwent reoperation after immunization.  
Although this study was performed in a selected population of patients, the median survival of 455 days in the 
treated group compared very favorably with an institutional control group where median survival was only 
257 days.  Unfortunately, no clinical responses were seen and any antigen-specific immune response could not 
be characterized because the immunizing antigens were not characterized. 
 
In another Phase 1/2 trial, tumor lysate pulsed DCs were given safely to ten patients who received 
immunizations every 3 weeks for a minimum of one and a maximum of 10 immunizations.  There were only 
two minor clinical responses seen.  Of 5 patients evaluated by enzyme-linked immunospot (ELISPOT8) before 
and after vaccination, T-cells reactive against tumor lysate-pulsed DCs were increased in two patients [48].  
 
In a more recent study, De Vleeschouwer et al. [49] investigated the safety and clinical response in both adults 
and children > 3 years of age with recurrent MG to DCs pulsed with tumor lysate.  Fifty-six patients underwent 
a surgical resection followed by intradermal injection of DCs on one of three different treatment schedules: 
DCs at week 1 and 3 and then every 4 weeks, 5 DC vaccinations every 2 weeks and then every 4 weeks, or 4 
weekly DC vaccinations with intradermal boosts of autologous tumor lysate.  No serious adverse events were 
seen and the authors concluded that the DC regimen was safe in both children and adult patients.  Univariable 
analysis indicated that younger age and total resection were better prognostic factors for this DC treatment 
regimen. 
 
In addition to the aforementioned studies, a handful of other studies have been performed with DCs in the 
pediatric population. One study by Ardon et. al. [50] demonstrated the clinical feasibility of using DCs loaded 
with autologous tumor cell lysate in children with recurrent malignant glioma. In this study, 45 children were 
divided up into groups that received identical vaccination schedules to those listed in the De Vleeschouwer 
study described above with one additional group that had the DCs grown with different enhancing molecules.  
Each DC vaccine was given intra-dermally at a median dose of 2.8x106 cells.  Overall, the study determined 
that DC vaccination was safe and feasible in children with high grade glioma, medulloblastoma, primitive 
neuro-ectodermal tumor (PNET), ependymoma, and atypical teratoid-rhabdoid tumor (ATRT).  Additionally, 
this DC vaccination method resulted in a subgroup of long-term survivors with an OS over 24 months.  Another 
study using tumor lysate-loaded DCs in children with WHO grade III and IV glioma demonstrated that DC 
treatment was tolerable and feasible in pediatric patients, albeit with some limitations [51].  Three children 
(one with recurrent GBM, one with newly diagnosed GBM, and one with anaplastic astrocytoma) received 2-4 
DC vaccinations at 1x106 cells per injection.  In summary, there is experience in administering DC vaccines to 
children with brain tumors and studies confirm that there are no major toxicities associated with the 
treatment.   
 
Our research team has extensive experience with DC vaccines in adults including, most recently, a clinical trial 
of 11 patients with newly diagnosed glioblastoma who were given upfront DI TMZ, followed by administration 
of CMV-targeted DC vaccines (2x107 cells) administered bilaterally in the groin [52].  The primary objective of 
this study was to evaluate the safety and feasibility of vaccinating newly diagnosed patients with CMV-
targeted DCs mixed with GM-CSF after DI TMZ.  No patients experienced DC-related AEs during the study; 
however, there was one reaction to GM-CSF after vaccine 8, which was alleviated by removing the GM-CSF 
from the DC vaccine mixture.  Another study performed at our center involved CMV-targeted DC vaccine 
administration (2x107 cells) following Td preconditioning [6].  Patients given Td had increased migration of DCs 
to VDLNs and showed a significant increase in both progression-free survival (PFS) and overall survival (OS) as 
compared to patients who were preconditioned with mature DCs.  Additional studies we have performed 
include EGFRvIII-targeted DC vaccine studies in GBM [53, 54].  And, importantly, we have also administered DC 
                                                 
7 CTL, cytotoxic T lymphocyte 
8 ELISPOT, enzyme-linked immunospot 

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vaccines to children with recurrent medulloblastoma in the RE-Match study (Pro00018020), giving our 
research team extensive experience with DC vaccines.        
6.3 Study Purpose/Rationale 
While significant improvements have been made in the treatment of children with these diagnoses, the 
outcome for those with recurrent brain tumors remains grim.  Immune therapies, with their potential for 
highly specific tumor killing and/or ability to recruit the endogenous immune system against a tumor, hold 
great potential and are being pursued.  Whereas immune checkpoint blockade has shown some promise in 
children with rare hyper-mutated brain tumors, responses have not been durable in most cases and these 
agents have limited clinical impact for most pediatric brain tumors, due in part to the lower tumor mutational 
burden (TMB) in most pediatric tumors [55].  Given that the mutational landscape and immune environment 
in children vary from that of adults, pediatric-focused studies are necessary to optimize immune-based 
therapies for treating children and young adults.  
 
One such immune-based therapy being studied in children is the use of primed, autologous dendritic cell 
vaccines.  As mentioned above in Section 6.2.2, a handful of studies have been performed with DCs in the 
pediatric population [50, 51].  These previous studies have used autologous tumor lysate to prime the 
autologous DCs; however, this requires that the patient undergo surgery to obtain a tumor sample for 
priming.  To avoid that potentially unnecessary surgery, specifically for those with recurrent tumor, our study 
proposes the use of an antigen that is highly expressed on brain tumors, Cytomegalovirus (CMV).  CMV 
antigens have been identified in pediatric tumors including glioblastoma (GBM) and medulloblastoma, and 
may make excellent anti-tumor immunotherapeutic targets [4, 5].  Vaccination and adoptive T cell strategies 
targeting CMV in humans in other contexts have been found to be safe and effective.  Previous studies 
examining adult patients with GBM have demonstrated the feasibility, safety, immunogenicity, and potential 
clinical efficacy of this approach [6] and Duke is currently participating in a multicenter, phase 2 study 
(Pro00084478) of CMV RNA-pulsed dendritic cells (DCs) in adult patients with GBM.  
 
We have demonstrated in murine models that DCs loaded with tumor-specific antigens in the form of peptides 
or RNA can induce potent and specific humoral and cell-mediated immune responses that are effective against 
murine intracerebral (i.c.) tumors, including a syngeneic murine astrocytoma, without inducing autoimmunity 
[33, 34, 56].  Our previous clinical experience has also shown that DC vaccines, in combination with standard 
of care radiation therapy and chemotherapy, are capable of generating potent, tumor-specific immune 
responses and clinical radiographic responses in patients with malignant gliomas.  We and others have also 
shown that antigens derived from CMV are contained within malignant gliomas and may serve as potent and 
specific immunotherapy targets.  Vaccination and adoptive T cell strategies targeting CMV in humans in other 
contexts, including the targeting of lesions within the CNS, have been found to be safe and effective.  We have 
also shown that DCs generated from patients with GBM and loaded with pp65-LAMP mRNA are capable of 
generating CD4+ and CD8+ T cells that produce IFN-γ and kill malignant astrocytes infected with CMV in an 
antigen-specific fashion.  We have found that tumor infiltrating lymphocytes (TILs) isolated from these 
patients are significantly enriched for T cells that specifically recognize CMV antigens, suggesting that this 
response may be important in the biology of these tumors.  We have recently demonstrated that fusion of the 
immuno-dominant antigen CMV pp65 to full-length LAMP targeting protein enhances activation and 
expansion of CMV-specific T cells in vitro.  Based on previous clinical trials and our experience in DC 
administration in adults with brain tumors, we are proposing a CMV-DC vaccine study in children and young 
adults that incorporates DI TMZ, Td preconditioning, and a GM-CSF adjuvant. 
 
TMZ is used as a standard of care chemotherapy for patients with high grade glioma.  Therapeutic TMZ 
induces a profound lymphopenia that may inhibit anti-tumor vaccination; however, we have shown in an 
animal model, that TMZ-induced lympho-depletion actually enhances the induction of antigen-specific 
immune responses after active and adoptive immunotherapy.  This suggests that DI TMZ may enhance the DC 
vaccination immune response, and therefore this tactic will be applied to this study. 
 

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Td is a routinely used vaccine in the normal human population that we have shown in pilot studies may 
function as a potent adjuvant to enhance DC trafficking to VDLNs [6]. Our previous clinical studies at Duke 
have shown that successful DC migration to VDLNs may be a requisite for clinical activity of RNA-pulsed DCs 
and administration of Td prior to vaccination may improve the effectiveness of DC vaccines in patients with 
GBM.  The CMV-DC vaccines with the combination of Td preconditioning and GM-CSF adjuvants should result 
in a favorable risk to benefit ratio that provides a safe and feasible treatment regimen for children and young 
adults with WHO grade IV glioma, recurrent malignant glioma, and with recurrent medulloblastoma. 
7 OBJECTIVES AND ENDPOINTS 
 
 
Objective 
Endpoint 
Analysis 
Primary 
Determine the feasibility of 
producing CMV-DCs from the 
leukapheresis product collected from 
children and young adults up to 35 
years old with WHO grade IV glioma, 
recurrent malignant glioma, or 
recurrent medulloblastoma.   
Percentage of patients for whom three or 
more vaccines can be generated from the pre-
treatment leukapheresis 
See Section 14.4.1 
Primary 
Determine the safety of intradermal 
administration of CMV-DCs in 
children and young adults up to age 
35 with WHO grade IV glioma, 
recurrent malignant glioma, or 
recurrent medulloblastoma. 
Percentage of patients that experience 
unacceptable toxicity from CMV-DC 
administration 
See Section 14.4.2 
Exploratory 
Describe changes in T cell response 
to CMV-DC vaccination 
Percent change in pp65-specific T cell 
responses assessed by Imaging Flow 
Cytometry (IFC)  
See Section 14.4.3.1 
Exploratory 
Describe OS and PFS 
Percentage of patients alive without disease 
progression 6 months after initiation of DI 
adjuvant TMZ with CMV-DC vaccination (6-
month PFS) 
 
Percentage of patients alive 1 year after 
initiation of DI adjuvant TMZ with CMV-DC 
vaccination (1-year OS) 
See Section 14.4.3.2 
 
8 INVESTIGATIONAL PLAN 
8.1 Study Design 
This is a phase 1 study to confirm the safety of CMV-DCs in children and young adults up to 35 years old with 
WHO grade IV glioma, recurrent malignant glioma, or recurrent medulloblastoma.  .  Eligible children or adult 
patients will undergo a leukapheresis (3 blood volumes) for peripheral blood mononuclear cells (PBMCs).  A 
maximum of 10 patients with WHO grade IV glioma, recurrent malignant glioma, or recurrent 
medulloblastoma will be treated with CMV-DCs on this phase 1 safety study.  Patients who received prior 
craniospinal radiation will be recruited into a separate cohort as described in Section 14.4.2.  Patients who 
have previously undergone craniospinal radiation may have a second leukapheresis procedure. 
 
All patients will undergo leukapheresis, as soon as possible after enrollment onto the study.  After the 
leukapheresis procedure is completed, patients with newly diagnosed WHO grade IV glioma will undergo 
standard of care radiation therapy (RT) with or without concurrent TMZ prior to dose intensified (DI) adjuvant 
TMZ.  Also, after the leukapheresis procedure is completed, patients may be given “bridge” therapy while the 
CMV-DCs are being generated, if needed.  This decision regarding bridge therapy will be made by the treating 
neuro-oncologist on a case-by-case basis depending upon the patient’s clinical needs.  Leukapheresis 

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procedure(s) will ideally be completed prior to initiation of bridge therapy; however, the leukapheresis 
procedure(s) may be done during bridge therapy, at the discretion of the PI. 
 
Review of Appropriateness of Continuing Protocol Participation:  For all patients, the eligibility criteria must be 
confirmed prior to proceeding with DI TMZ.  Patients will not proceed with DI TMZ if they do not have 
sufficient PBMCs to generate CMV-DCs for at least 1 vaccine.  If bridge therapy is provided during the time the 
vaccines are being generated, the patient must still meet the inclusion and exclusion criteria regarding prior 
treatments described in Section 10, in order to proceed with 21-day DI TMZ.  Newly diagnosed patients must 
complete the full course of radiation to remain eligible for the study (minimum ~54 Gy).  Eligibility for newly 
diagnosed patients will be confirmed after they have completed the standard of care radiation therapy; 
patients may be removed from study at that time due to not meeting eligibility or at the discretion of the PI.   
 
Initiation of Experimental Therapy for All Patients:  For patients continuing protocol participation, a single cycle 
of 21-day DI TMZ will be administered.   Patients who received prior craniospinal radiation may undergo a 
repeat leukapheresis to obtain adequate numbers of PBMCs to generate at least one DC vaccine.  DI TMZ 
treatment will be at 50-100 mg/m2/day for 21 days.  On Day 22 (+3 days) of the single cycle of DI TMZ, patients 
will receive preconditioning with Td (1 Lf) in the right inguinal region, 6-24 hours later they will receive their 
first CMV-DC vaccination (Vaccine #1) given intra-dermally, divided equally between both inguinal regions.  
Vaccine #2 and Vaccine #3 will occur at 2-week (±2 days) intervals following Vaccine #1.   
 
After the first three vaccines, patients will be vaccinated approximately every 4 weeks (±5 days). Patients will 
continue with monthly vaccinations until they have used up all of the prepared CMV-DCs or until tumor 
progression (whichever comes first).  All patients will undergo vaccine site preconditioning with Td (1 Lf) 6-24 
hours prior to Vaccine #1, Vaccine #4, and all subsequent monthly vaccines.  The Td preconditioning site will 
alternate between the right and left inguinal region with Td prior to Vaccine #1 given in the right inguinal 
region, Td prior to Vaccine #4 given in the left inguinal region, Td prior to Vaccine #5 given in the right inguinal 
region, and so on.  
 
Blood for immune monitoring will be collected during the initial leukapheresis, prior to Vaccine #1, ~ 1 week 
(±5 days) after Vaccine #3, and ~1 week (±5 days) after the child’s last vaccine.  Patients will have an MRI 
within 2 weeks of starting DI TMZ as a baseline MRI, ~3-4 weeks after Vaccine #3 (at the Vaccine #4 visit, if 
possible), and then every 3 months at the discretion of the treating physician.  Patients will be followed for 
progression and future medications to treat their brain tumor until death.     
8.1.1 Removal of PBMCs by Leukapheresis 
Prior to the initiation of radiation therapy in newly diagnosed WHO grade IV glioma patients or prior to 
DI TMZ initiation in all recurrent patients, subjects will undergo a leukapheresis procedure.  The 
leukapheresis procedure will process three blood volumes of the patient’s blood.  Patients who have 
received prior craniospinal radiation may undergo a second leukapheresis, at the discretion of the 
treating physician, to generate up to three vaccines to meet the feasibility endpoint.  As long as at least 
one vaccine is generated, the patient will remain on the study.    
8.1.2 Concomitant Temozolomide and Radiation Therapy (Newly Diagnosed WHO 
Grade IV Glioma only) 
Children or young adults with newly diagnosed WHO grade IV glioma who are enrolled onto the study 
will undergo standard of care radiation therapy with or without concomitant TMZ, at the discretion of 
their treating physician, prior to initiating the first cycle of adjuvant DI TMZ.  Patients must complete 
the full course of radiation therapy (minimum ~54 Gy), in order to continue onto the DI TMZ portion of 
the study. 
 
If TMZ is administered concomitantly with radiation therapy, its management will be done by the 
patient’s treating physician in accordance with institutional practices.   

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8.1.3 Optional “Bridge” Treatment  
Patients may be given “bridge” therapy after leukapheresis during the time needed to generate their 
CMV-DCs.  This decision will be made by the treating neuro-oncologist on a case-by-case basis 
depending upon the patient’s clinical needs.  This includes the type of bridge therapy that is most 
appropriate for the given patient, e.g., metronomic dosed chemotherapy such as daily etoposide or 
cyclophosphamide.  If bridge therapy is provided during the time the vaccines are being generated, the 
patient must still meet the inclusion and exclusion criteria regarding prior treatments described in 
Section 10, in order to proceed with 21-day DI TMZ.  Patients receiving the Tumor Treating Fields 
(TTFields) device called Optune™ can initiate DI TMZ immediately after ceasing device use and do not 
have to wait ≥ 1 week. 
8.1.4 Dose Intensified Temozolomide Therapy 
All patients will receive one cycle of DI TMZ.  For patients with newly diagnosed WHO grade IV glioma, 
this adjuvant DI TMZ cycle will begin ≥ 4 weeks after completing radiation therapy.  For recurrent 
patients, DI TMZ will begin ≥ 3 weeks after leukapheresis.  DI TMZ is administered once per day at 50-
100 mg/m2/day for 21 consecutive days of a 28-day cycle.  Dose reduction of DI TMZ may occur, if 
necessary, at the discretion of the treating physician.  The Principal Investigator may also choose to 
discontinue DI TMZ early due to declining blood counts.    
8.1.5 Tetanus and Diphtheria Toxoid Booster and Site Preconditioning 
Potentially eligible patients who are ≥ 18 years of age will receive a Td booster (5 Lf) during screening. 
 
Subjects will undergo vaccine site preconditioning with a one-fifth dose of Td (1 Lf) intradermally 6-24 
hours prior to Vaccine #1, Vaccine #4, and at all subsequent vaccines.  Td preconditioning will be done 
in the right inguinal region for Vaccine #1, in the left inguinal region for Vaccine #4, in the right inguinal 
region for Vaccine #5, and continuing in alternating regions until all vaccines have been given. 
8.1.6 DC Vaccination 
On day 22 (+3 days) of the DI TMZ cycle, patients will receive Td preconditioning and 6-24 hours later 
will receive their first intradermal CMV-DC immunization (Vaccine #1).  The two subsequent 
immunizations (Vaccine #2 and #3) will be given every 2 weeks (± 2 days) for a total of three doses.  
Each immunization will be divided equally between both inguinal regions with a total volume of ~200 μL 
per side.  Vaccine target dose is 2x107 cells. 
 
After vaccination, patients will be monitored for thirty minutes to one hour post-immunization for the 
development of any adverse effects.  The immunization procedures will be supervised by a nurse or 
physician that has completed an Advanced Cardiac Life Support (ACLS) course.  A cardiac resuscitation 
cart will be available in the immediate vicinity when performing these immunizations in case of severe 
allergic reactions.  After Vaccine #3, subsequent vaccines will be done every 4 weeks (± 5 days) until the 
patient has completed all of their available vaccines or until tumor progression, whichever comes first.  
If patients cannot receive the vaccine within the aforementioned time windows due to an 
uncontrollable circumstance such as an adverse event, manufacturing delay, natural disaster, etc., a 
maximum 2-week delay in the vaccine may occur.  If the patient’s vaccine cannot be given within 2 
weeks outside of the original window, the patient will be withdrawn from the study. 
8.1.7 Definition of Unacceptable Toxicity 
Toxicities will be graded according to the National Cancer Institute (NCI) Common Terminology Criteria 
for Adverse Events (CTCAE) version 5 criteria.   The timeframe for assessment of unacceptable toxicity 
begins with Td preconditioning prior to the initial vaccination and ends ~30 days after Vaccine #3, at the 
time that Vaccine #4 may be given.   

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Any Grade 3 or any Grade 4 non-hematologic toxicity at least possibly related to Td preconditioning or 
DC vaccine, other than the exceptions listed below, any life-threatening event deemed probably or 
definitely related to the study drug (with the exception of the Grade 4 non-hematologic toxicities that 
are reversible within 2 weeks), or any treatment-related death will be considered unacceptable.  In 
addition, any Grade 2 or higher autoimmune toxicities, particularly those affecting vital organs (e.g. 
cardiac, renal, CNS), will be considered an unacceptable toxicity if it occurs after Td preconditioning and 
before vaccine #4 is administered.  
 
Exceptions to these unacceptable toxicities are as follows: 
• 
Seizures: Due to the nature of the disease under investigation in this protocol, patients may have 
pre-existing seizures or be susceptible to new seizures as a result of the underlying disease 
process.  Although seizures may be defined as Grade 3 or 4 toxicities under NCI CTC, and will be 
reported as such in this protocol, seizures will not be considered an unacceptable toxicity if, in 
the opinion of the Principal Investigator (PI) they have not increased in frequency or can be 
attributed to another recognized cause of increasing seizure frequency such as sub-therapeutic 
anti-convulsant levels or biopsy proven tumor progression. 
• 
New or worsening neurologic deficits: Due to the nature of the disease under investigation in this 
protocol, patients may have an increase in pre-existing neurologic deficits or may develop new 
neurologic deficits as a result of tumor invasion. A new neurologic deficit, which resolves within 2 
weeks after initiation of medical therapy, will not be considered an unacceptable toxicity. New 
neurological symptoms will not be an unacceptable toxicity if they can be ascribed to tumor 
progression (e.g. documented with histopathologic analyses of biopsy tissue), or they respond to 
treatment (e.g. oral steroids) within 2 weeks.  
• 
Thromboembolism:  While deep vein thrombosis (DVT) is not as common in children as in adults 
with malignant glioma, patients may have undiagnosed pre-existing DVTs or be susceptible to the 
development of DVTs due to the underlying disease process.  Although DVT may be defined as 
Grade 3 or 4 toxicities under NCI CTC, and will be reported as such in this protocol, DVT will not 
be considered an unacceptable toxicity in this protocol. 
• 
Syndrome of Inappropriate Antidiuretic Hormone (SIADH): Due to the high incidence of SIADH in 
this patient population, patients may be susceptible to the development of SIADH due to the 
underlying disease process.  Although SIADH may be defined as Grade 3 toxicity under NCI CTC, 
and will be reported as such in this protocol, SIADH will not be considered an unacceptable 
toxicity in this protocol unless it is refractory to medical management. 
• 
Muscle Weakness and Weight Gain:  Due to the high incidence of muscle weakness and weight 
gain in patients taking steroids in this patient population, patients may be susceptible to the 
development of muscle weakness or weight gain which is due to steroids alone.  Although muscle 
weakness may be defined as Grade 3 or 4 toxicity and weight gain ≥ 20% may be defined as 
Grade 3 toxicity under NCI CTC, and will be reported as such in this protocol, muscle weakness or 
weight gain will not be considered an unacceptable toxicity in this protocol if the patient has 
required steroids greater than physiologic doses in the interval between the immunization and 
the development of the toxicity. 
8.1.8 Safety Considerations 
8.1.8.1 
Allergic Reactions to DC Immunization 
Injection of DCs may result in an allergic reaction, which could include redness and swelling at 
the injection site, itching, hives, low blood pressure, difficulty breathing, or in the most 
extreme circumstances, death.  In addition, if the immune system becomes overly activated, 
potential discomforts may include pain, redness and swelling at the injection site.   

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8.1.8.2 
Reactions to GM-CSF 
GM-CSF is routinely used to promote white blood cell recovery in patients receiving 
chemotherapy where it is given at much higher doses and for a longer duration than what will 
be given in this study.  Side effects at the standard (higher) dose of GM-CSF may include mild, 
temporary fever, skin reactions at the injection site, fluid retention, shortness of breath, rapid 
or irregular heartbeat, laboratory abnormalities (possibly suggesting kidney or liver damage), 
and GM-CSF tolerance , which would mean it may not be effective if given in the future.  These 
events occurred rarely, were usually mild to moderate in severity, and were usually reversible.  
It is unlikely that such reactions will be observed with the lower doses of GM-CSF being used in 
this study. 
8.1.8.3 
Cerebral Edema 
Cerebral edema may be secondary to the disease process itself, a previous surgical procedure, 
necrosis from previous radiation, or inflammation due to immune infiltration of the brain or 
destruction of tumor cells.  Symptoms may include, but are not limited to, severe headache, 
confusion, lethargy, unresponsiveness, coma, or focal neurological deficits.  Patients will be 
monitored throughout the course of the study and those patients with any signs or symptoms 
of cerebral edema may need their steroid doses increased, treatment with an osmotic diuretic, 
or surgical decompression.  Edema that fails to respond to aggressive therapy may lead to 
permanent neurological impairment.  The probability of this risk can be predicted to some 
degree based upon tumor size, location, pre-operative neurological impairment, and post-
operative course prior to DC injections.  Patients will be monitored throughout the course of 
the study.  
8.1.8.4 
Risk of Infection 
The DC injections may include the risk of infection due to potential contamination of the DC 
product in the laboratory.  This may result in localized redness, swelling, or induration at the 
injection site.  In the most extreme situation, this may lead to systemic bacterial/fungal sepsis 
and possibly death.  The probability of this risk is relatively low, given the small injection 
volume (1 mL divided between > 2 intradermal locations) and the fact that the DCs will be 
strictly tested for sterility prior to each injection.  The risk of infection due to potential 
contamination of the DCs in the laboratory will be minimized by biosafety quality assurance 
and testing.  All cell cultures will be handled under sterile conditions in a core tissue culture 
facility dedicated to the processing of human cells, the MPACT facility.  Prior to injection into 
patients, DCs must pass sterility tests for aerobic, anaerobic, and fungal organisms.  Following 
injections, patients will be monitored throughout the course of the study for any signs and 
symptoms of infection. If an active infection is suspected, patients will be cultured and treated 
with appropriate antibiotics.  
8.1.8.5 
Delayed Autoimmune Diseases 
It is possible that delayed autoimmune disease(s) may develop as a result of injection with DCs.  
This means that the immune system may be stimulated to attack natural tissue in the body.  
Animal studies have reported the development of autoimmunity in the context of vaccination 
and recovery from lymphopenia.  However, our current experience with DC vaccination in 
glioma patients has not demonstrated evidence of autoimmunity in treated patients.  
Furthermore, the doses of TMZ used in this study for induction of lymphopenia are standard 
doses administered to patients with brain tumors.  It therefore, is unknown what the risk of 
delayed autoimmune disease is for this study.  
8.1.8.6 
Risks of Phlebotomy 
Drawing blood or inserting an intravenous catheter into an arm vein may result in bruising or 
swelling in the area of the insertion, bleeding at the site of the needle puncture, light 

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headedness, fainting and very rarely, local infection, which may be severe.  These risks are 
reduced by the fact that the blood will be drawn by a qualified physician, nurse or 
phlebotomist (a professional trained to draw blood). 
8.1.8.7 
Risks of Leukapheresis 
As with any donation of blood, a variety of minor reactions may occur with leukapheresis, 
which include fainting, dizziness, or nausea.  Uncommon but serious complications may also 
result, which include bleeding, infection, an adverse reaction to the anticoagulant or 
replacement fluids, hypocalcemia, hypotension, shock, convulsions, air emboli, heart failure, or 
the inability to transfuse blood back into the patient.  These risks are reduced by the fact that 
the procedure will be performed by qualified staff at a specialized clinical hemapheresis unit.  
Patients will be carefully monitored throughout the procedure by trained nursing and medical 
staff.  Calcium gluconate (2 mg PO) will be given to minimize the risks of hypocalcemia, fluid 
supplementation will be given to minimize hypotension, and blood will be routinely screened 
for HIV, hepatitis, and syphilis to minimize the risk of transmitting infection. 
8.1.8.8 
Risks of Magnetic Resonance Imaging (MRI) 
The risks and/or discomforts associated with the performance of MRI include the anxiety 
produced from being in a tight, enclosed space (claustrophobia).  In addition, the machine 
operates using a large and powerful magnet.  The magnetism of the machine attracts certain 
metals, therefore, people with these metals in their bodies (specifically pacemakers, infusion 
pumps, metal aneurysm clips, metal prostheses, joints, rods or plates) will be excluded from 
the study. Patients will also be checked to make sure that they do not bring any metal objects 
into the MRI facility.  Dental fillings are less affected by the magnetic fields generated and are 
therefore permitted.  It will be asked that patients let the physicians conducting this study 
know of any metal in their bodies other than dental fillings.   
8.1.8.9 
Allergic Reactions to Contrast Agents 
During the MRI, patients will be given a contrast agent.  The agent is given routinely to obtain 
enhanced MRI scans of the brain.  The agent is administered through the vein and requires the 
placement of an intravenous (IV) catheter.  The catheter placement is similar to drawing blood 
except that the catheter remains in the vein during the time the agent is actively delivered.  
The risks of a blood draw and insertion of a catheter are similar.  There have been a few, rare 
cases of allergies to the agent used in MRI contrast enhanced scans. Patients with any known 
severe allergies to contrast agents will be excluded from the study.  Patients with mild allergies 
(i.e., rash only) will be pretreated with Tylenol and Benadryl prior to injection of the contrast 
agent. 
8.1.8.10 
Risks of Temozolomide 
TMZ has been well tolerated by both adults and children with the most common toxicity being 
mild myelosuppression.  Other, less likely, potential toxicities include nausea and vomiting, 
constipation, headache, alopecia, rash, burning sensation of skin, esophagitis, pain, diarrhea, 
lethargy, hepatotoxicity, anorexia, fatigue and hyperglycemia.  Hypersensitivity reactions have 
not yet been noted with TMZ.  As in the case with many anti-cancer drugs, TMZ may be 
carcinogenic.  Rats given TMZ have developed breast cancer.  The significance of this finding 
for human is not presently known.  
 
Based upon degree of myelosuppression experienced by the patient, dose reduction of DI TMZ 
may occur, if necessary, at the discretion of the treating physician.  The Principal Investigator 
may also choose to discontinue DI TMZ early for a patient if the patient’s blood counts drop 
too low.  Vaccines will still be administered per protocol, unless otherwise contra-indicated. 

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8.1.8.11 
Unknown Risks 
The overall risk classification of this research is unknown.   
8.1.9 Concomitant Medications 
8.1.9.1 Steroids 
Corticosteroids should be used at the lowest dose to control symptoms of edema and mass effect, 
and discontinued, if possible. Use of corticosteroids should be recorded in the electronic database. 
8.1.9.2 Anticonvulsants 
Anticonvulsants drugs should be used or continued, if indicated. Use of such anticonvulsants should 
be recorded in the electronic database. 
8.1.9.3 Growth Factors 
Routine use of growth factors outside of the protocol study drug (i.e. G-CSF, GM-CSF, and 
erythropoietin) is not permitted.  However, therapeutic use of G-CSF in patients with serious 
neutropenic conditions, such as sepsis, may be used at the investigator’s discretion. Use of such 
growth factors should be recorded in the electronic database. 
8.1.9.4 Anti-emetics  
The use of anti-emetics will be at the investigator’s discretion. Use of anti-emetics should be 
recorded in the electronic database. 
8.1.9.5 Proton Pump Inhibitors 
The use of proton pump inhibitors (e.g. rabeprazole, omeprazole, pantoprazole, lansoprazole or 
esomeprazole) is allowed on this study. 
8.1.9.6 Febrile Neutropenia 
Febrile neutropenia should be managed according to the local institutional guidelines.  Measures 
include laboratory testing, blood and urine cultures, and institution of broad spectrum antibiotics. 
8.1.9.7 Pneumocystis jiroveci pneumonia (PJP) prophylaxis 
The use of medication (i.e., Bactrim) for PJP prophylaxis in patients on chronic steroids is 
recommended, but is at the investigator’s discretion. 
8.1.9.8 Neurosurgical Procedures 
If a neurosurgical procedure is required for a reason other than tumor progression (i.e. the onset of 
hydrocephalus), these procedures should be documented, but will not constitute criteria for 
declaring the patient “off study.” 
8.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
The dose for this trial was selected based on the DC cell dose used in two completed clinical trials that were 
done in adults with newly diagnosed WHO grade IV malignant glioma (IRB# 8108 and Pro00003877).  More 
than 24 adult patients have been treated at this dose at Duke alone.  Additionally, this same dose is being used 
on a larger, randomized phase 2 multicenter clinical trial in adult newly diagnosed WHO grade IV malignant 
glioma (Pro00084478).  The dose for children in this study will be the same dose that has been given safely to 
adults, 2x107 cells/injection; details on the dosing regimen can be found in Section 11.4.     
8.3 Rationale for Correlative Studies 
Autologous DCs that are generated ex vivo and re-injected into advanced cancers, such as GBM, have shown 
promise but still have limited efficacy due in part to the lack of understanding of their mechanism after they 
are injected [6].  Given that the CMV-DC vaccine is meant to stimulate the immune system, we will test the 

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subject’s blood for immune response.  Specifically, the immune monitoring blood will be used to detect pp65 T 
cell responses following vaccination using IFC and possibly ELISPOT.  Briefly, patient’s immune cells from the 
blood will be stimulated with pp65 to measure IFNγ, CCL3, IL-2, and TNF-α response via IFC by intracellular 
cytokine staining.  The pp65 protein is expressed on numerous types of brain tumor cells and therefore serves 
as a great target for DC vaccination.  Studying the T cell response to pp65 will inform us if the DCs are 
stimulating an immune reaction to the pp65 protein, a significant factor for the efficacy of the CMV-DC 
vaccine.     
8.4 Definition of Evaluable Subjects, On Study, and End of Study 
All patients who undergo leukapheresis will be included in analyses relating to feasibility. 
 
Patients without an unacceptable toxicity who do not receive the first three DC vaccinations at 2x107 cells per 
dose will not be evaluable for the initial assessment of unacceptable toxicity.  Any patient who receives any 
dose of CMV-DCs will be evaluable for efficacy of the agent. 
8.5 Early Study Termination 
This study can be terminated at any time for any reason by the PI-sponsor.  If this occurs, all subjects on study 
should be notified as soon as possible.  Additional procedures and/or follow up should occur in accordance 
with Section 11.7, which describes procedures and process for prematurely withdrawn patients. 
 
9 STUDY DRUG 
Each patient will undergo at least one leukapheresis, while patients who have had previous craniospinal radiation 
may undergo up to two leukapheresis procedures, if necessary, to generate the three DC vaccines needed to be 
considered feasible.  As long as at least one vaccine is generated, the patient will continue on study.   Part of the 
contents from the leukapheresis will be used for DC generation.  Each leukapheresis procedure will consist of 
processing three blood volumes per patient.   
 
DCs will be generated from the first leukapheresis (or first and second, if required, for patients with previous 
craniospinal radiation) in vitro by 7-day culture with GM-CSF and IL-4.  PBMCs for in vitro generation of DCs will be 
obtained by leukapheresis at the Duke Apheresis Unit and transported to the Cell Processing facility.  For patients 
without sufficient venous access for leukapheresis, a temporary intravenous catheter may be inserted. 
 
At the end of the 7-day incubation for generation of DCs, a sample of the media is taken for mycoplasma testing, 
the cells are then harvested and electroporated with pp65-flLAMP mRNA.  The DCs are placed in a flask with AIM V 
media containing GM-CSF + IL-4 + TNF-α + IL-6 + IL-1β at 37OC, 5% CO2 for 18-20 hours for maturation.  The cells 
are washed twice with PBS and frozen at 5x106 cells/mL in 90% autologous human AB serum (Valley Biomedical, 
Winchester, VA 22602), 10% DMSO and 5% glucose in a controlled-rate freezer at a rate of 1oC/minute.  The DCs 
are then stored until needed at –135oC.  After freezing, an aliquot of cells is thawed for QA/QC.  This testing will 
look at viability (>70%), endotoxin content (<5 E.U. /Kg B.W.), mycoplasma contamination (negative), and sterility 
testing for aerobic and anaerobic bacterial cultures (1x106 DCs) and fungal cultures (1x106 DCs). 
9.1 Names, Classification, and Mechanism of Action 
CMV-DCs are autologous dendritic cells derived from PBMCs loaded with RNA encoding the human CMV 
matrix protein pp65 as a fusion protein with the full-length LAMP protein (pp65-flLAMP) plus GM-CSF and Td 
vaccine as adjuvants.   DCs work by activating the host immune system, specifically CD4+ and CD8+ T cells and 
B cells.  Activation of these immune cells by a specific antigen, such as pp65, stimulates these immune cells to 
attack cells that display that antigen, such as brain tumor cells, resulting in cell killing. 
9.2 Packaging and Labeling 
• 
For CMV-DCs: 
Name 

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MRN 
DOB  
Drug: pp65DCs 
Lot #: Lot 001 
Caution New Drug Limited By Federal Law To Investigational Use 
• 
For Td used in preconditioning, per Investigational Chemotherapy Services at Duke 
9.3 Supply, Receipt, and Storage 
The DCs will be stored in a locked liquid nitrogen freezer in the Molecular Products and Cellular Therapies 
(MPACT) cGMP facility. The Nautilus LIMS (Laboratory Information Management System) database will track 
receipt and storage location.   
 
MPACT Facility 
Duke ICS Pharmacy 
CMV pp65-flLAMP mRNA-
pulsed DCs 
Td used for 
preconditioning 
9.4 Dispensing and Preparation 
For each vaccination, cells that have passed QA/QC will be rapidly thawed at 37oC, washed three times with 
phosphate buffered saline (PBS) and counted. The cell concentration will be adjusted to 4x107 cells/mL and 
DCs will be re-suspended in preservative free saline and placed into a sterile tuberculin syringe with a 27 
gauge needle. 
 
For all DC preparations, from the final preparation, a sample of cells will be sent for Gram stain and endotoxin 
testing prior to administration.  DC vaccination will not be given until endotoxin testing has been passed (< 5.0 
E.U./Kg) and the Gram stain has been found to be negative.  An aliquot of cells will also be sent for aerobic and 
anaerobic bacterial cultures (1x106 DCs) and fungal cultures (1x106 DCs). 
 
The CMV-DCs will be delivered from the MPACT facility directly to the clinic under the supervision of the 
trained research staff.  Each patient vaccine will be transported to the clinic in a separate cooler.   
9.5 Compliance and Accountability 
All DC vaccines will be stored in the MPACT facility in a temperature-controlled, locked access controlled 
storage unit.  A drug log sheet will be used to track and document the drug. The products will be signed out 
and distributed by appropriate personnel.  The Duke Brain Tumor Immunotherapy Program (BTIP) personnel 
use safe medication practices to reduce the risk of medication errors and adverse events when setting up 
study drug procedures.  Investigational drugs are ordered, received, stored, and dispensed for BTIP protocols 
that are approved by the Duke University Health System (DUHS) Institutional Review Board (IRB).  
Investigational drugs are stored separately from other drugs in an area of limited access and in accordance 
with special storage requirements.  They are clearly labeled with the identity of the study drug and other 
control numbers.  All drug transfers, receipts, and disposal are recorded in the Duke Nautilus system. 
9.6 Disposal and Destruction 
All excess study drug and any item that contacts the study drug will be autoclaved and properly disposed 
according to institutional procedures. 
10 SUBJECT ELIGIBILITY 
10.1 Inclusion Criteria 
1. Age requirements: 
a. ≤ 35 years for patients with grade IV glioma or recurrent WHO grade III glioma 
b. 3-35 years old for patients with recurrent medulloblastoma 

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2. Newly diagnosed or recurrent WHO grade IV glioma, recurrent WHO grade III glioma, or recurrent 
medulloblastoma (multifocal/disseminated disease is eligible, at the discretion of the PI)  
3. Patients with WHO grade IV glioma who received surgery and radiation are eligible even without 
recurrence or progression 
4. Patients must have recovered from all previous treatments, including chemotherapy, radiation therapy, 
surgery, and other immunotherapies, etc. 
a. If the patient was receiving bevacizumab at the time of enrollment, the treating oncologist has 
the discretion of administering and adjusting bevacizumab 10 mg/kg every 14 days. The rationale 
for continuing patients on bevacizumab is to prevent rebound cerebral edema commonly seen 
after stopping this agent. 
5. Laboratory Studies: 
a. Platelets ≥ 100,000 cells/mm3  
b. Creatinine ≤ 1.2 x ULN  
c. 
Total bilirubin, AST, ALT, alkaline phosphatase ≤ 2.5 x ULN  
d. Neutrophil count ≥ 1000 cells/mm3 
e. Hemoglobin ≥ 9 g/dl (can be transfused) 
6. Able to undergo brain MRI with and without contrast 
7. Karnofsky Performance Status (KPS) ≥ 70 or Lansky Performance Status (LPS) ≥ 70 
8. A signed informed consent form approved by the IRB will be required for patient enrollment into the 
study.  Patients (if 18 years old or older) or their parent(s) or guardian(s) (if younger than 18 years old) 
must be able to read and understand the informed consent document and must sign the informed 
consent indicating that they are aware of the investigational nature of this study.   
9. For females of childbearing potential, negative serum pregnancy test within 48 hours of leukapheresis  
10. Females of childbearing potential must be willing to use acceptable contraceptive method to avoid 
pregnancy throughout the study and for at least 24 weeks after the last dose of study drug 
11. Males with female partners of childbearing potential must agree to practice adequate contraceptive 
methods throughout the study and should avoid conceiving children for 24 weeks following the last dose 
of study drug 
12. Newly diagnosed WHO grade IV glioma patients only: must be expected to complete standard of care 
radiation (minimum ~54 Gy) 
10.2 Exclusion Criteria 
1. Prior invasive malignancy (except for non-melanomatous skin cancer) unless disease free for ≥ 3 years. 
(For example, carcinoma in situ of the breast, oral cavity, and cervix are all permissible)  
2. Disease outside of the CNS  
3. HIV, Hepatitis B, or Hepatitis C seropositive 
4. Known active infection requiring IV antibiotics or active immunosuppressive disease 
5. Severe, active co-morbidity, defined as follows: 
a. Unstable angina and/or congestive heart failure requiring hospitalization 
b. Transmural myocardial infarction within the last 6 months 
c. 
Acute bacterial or fungal infection requiring intravenous antibiotics at initiation of XRT/TMZ 
d. Chronic Obstructive Pulmonary Disease exacerbation or other respiratory illness requiring 
hospitalization or precluding study therapy at initiation of XRT/TMZ for newly diagnosed patients 
or at initiation of DI TMZ for recurrent patients 
e. Hepatic insufficiency resulting in clinical jaundice and/or coagulation defects 
f. 
Acquired Immune Deficiency Syndrome (AIDS) based upon current CDC definition. The need to 
exclude patients with AIDS from this protocol is necessary because the treatments involved in 
this protocol may be significantly immunosuppressive 
g. 
Patients with autoimmune disease requiring medical management with immunosuppressant(s) 
h. Major medical illnesses or psychiatric impairments that, in the investigator's opinion, will prevent 
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i. 
Active connective tissue disorders such as lupus or scleroderma that, in the investigator’s 
opinion, place the patient at high risk for radiation toxicity 
6. Pregnant or lactating women 
7. Prior allergy to TMZ, GM-CSF, gadolinium (Gd), or Td 
8. Prior history of brachial neuritis or Guillain-Barré syndrome 
9. Patients treated on any other therapeutic clinical protocols within 30 days prior to study entry 
10. Patients receiving > 0.1mg/kg or 4mg/day dexamethasone or equivalent 
11. For recurrent patients only: Patients who have not recovered from the toxic effects of prior chemo- 
and/or radiation therapy9.  Guidelines for this recovery period are dependent upon the specific 
therapeutic agent being used: 
a. Patients who have received chemotherapy or bevacizumab ≤ 4 weeks [except for nitrosourea (6 
weeks) or metronomic dosed chemotherapy such as daily etoposide or cyclophosphamide (1 
week)] prior to starting the study drug unless patients have recovered from the side effects of 
such therapy. If the patient was receiving bevacizumab at the time of enrollment, the treating 
oncologist has the discretion of administering and adjusting bevacizumab 10 mg/kg every 14 
days.  
b. Patients who have received immunotherapy ≤ 4 weeks prior to starting the study drug unless 
patients have recovered from the side effects of such therapy 
 
 
                                                 
9 Please note that patients who are receiving treatment with a Tumor Treating Fields (TTFields) device such as Optune™ may 
continue to use this device during leukapheresis and during the ≥ 3 weeks before DI TMZ if recurrent.  Patients should discontinue 
use of Optune™ just prior to beginning DI TMZ and remain off Optune™ for the duration of time they are in the Treatment Period of 
the study (i.e., while they are receiving study vaccines).   

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11 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
Table 1: Schedule of Events 
Description 
Screening 
Leukapheresis 
#1 
At start 
of 
Adjuvant 
DI TMZ 
Cycle 
Vaccine 
#1 
Vaccine 
#2 
Vaccine 
#3 
Vaccine 
#4+10 
Post-vaccine 
(follow-up 
period) 
Week 
 
 
 
 
2 weeks 
(± 2 
days) 
after 
Vaccine 
#1 
2 weeks 
(± 2 
days) 
after 
Vaccine 
#2 
4 weeks 
(± 5 
days) 
after 
Vaccine 
#3 
Until 
progression 
or death 
Day 
 
 
 
Day 22 
(+3 days) 
of DI 
TMZ  
 
 
 
 
General 
Evaluations 
 
Physical Exam 
X 
 
X 
X11 
 
X 
X 
X 
Neurologic Exam 
X 
 
X 
X11 
 
X 
X 
X 
Performance Status 
X 
 
X 
X11 
 
X 
X 
X 
Adverse Events 
 
X12 
Continuous 
Laboratory 
Evaluations 
 
CBC w/diff 
X 
X 
X 
X 
X 
X 
X 
 
CMP 
X 
X 
X 
X 
X 
X 
X 
 
Serum Pregnancy 
Test, if applicable 
X 
X13 
 
 
 
 
 
 
Whole blood for 
immunologic 
analysis  
 
X14 
 
X14 
 
X14 
 
X14 
Leukapheresis 
 
X15 
 
 
 
 
 
 
Disease 
Evaluations 
 
MRI 
X16 
 
 
 
 
 
X16 
X16 
Treatment 
 
TMZ 
 
 
X17 
 
 
 
 
 
CMV-DCs 
 
 
 
X18 
X18 
X18 
X18 
 
Td booster 
X19 
 
 
 
 
 
 
 
Td preconditioning 
 
 
 
X20 
 
 
X20 
 
 
                                                 
10 Subsequent vaccines occur approximately every 4 weeks (± 5 days) until all CMV-DC vaccines generated from the patient have 
been used. 
11 Prior to Td preconditioning 
12 AESIs related to leukapheresis only (see Section 12.1.1).  
13 Serum pregnancy test, if appropriate, within 48 hours of leukapheresis.  If screening pregnancy test is within 48 hours of 
leukapheresis, it does not have to be repeated. 
14 Up to 76.5 mL in yellow top ACD tubes (not to exceed 3 mL/kg in a 24 hr period or 7 mL/kg in an 8 week period) and 1 red top (3 
mL) prior to Vaccine #1, 1 week (± 5  days) after Vaccine #3, and 1 week (± 5 days) after the last vaccine.  At leukapheresis #1, 3 
yellow top tubes (25.5 mL) and 1 red top tube (3 mL), 
15 An additional leukapheresis may be done on patients who have received prior craniospinal radiation if more cells are needed. 
16 MRI must be done within 2 weeks prior to initiating the first cycle of DI TMZ as a baseline (screening MRI). For newly diagnosed 
patients, this baseline MRI must be done after completing XRT/TMZ and before initiating DI TMZ. For patients with recurrent 
medulloblastoma, a baseline MRI of the spine must also be done. If nothing is detected on the baseline MRI, then no further spinal 
MRIs need to occur; spinal MRIs should continue if there is spinal detection.  MRI will also be done 3-5 weeks after Vaccine #3 (at 
Vaccine #4 visit) and then every 3 months, at the discretion of the treating physician, and at progression. 
17 21 days of daily TMZ at a dose of 50-100 mg/m2/day. 
18 Administer Vaccine #1 intra-dermally at day 22 (+ 3 days) of the first adjuvant TMZ cycle. Vaccines #2 and #3 will occur at 2 
week intervals (± 2 days). Vaccines #4+, if available, will be given every 28 days (± 5 days). Each vaccination will be equally 
divided between both inguinal regions. Monitor for 30-60 minutes post-immunization for any adverse effects. 
19 For patients ≥ 18 years of age. 
20 Td preconditioning (1 Lf) 6-24 hours prior to Vaccine #1, Vaccine #4, and all subsequent vaccines. 

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11.1 Screening Examination 
An informed consent must be signed by the patient and/or the parent(s) or legal guardian(s) before any study-
specific screening procedure takes place.   
 
Pre-treatment evaluations within 2 weeks before leukapheresis #1 to determine eligibility and as a baseline 
will include the following, unless otherwise indicated: 
• 
History and physical exam, including a full neurologic assessment and KPS or Lansky Performance 
Status  
• 
Laboratory Evaluations: 
o 
CBC with differential 
o 
CMP 
o 
Beta-HCG, if appropriate (within 48 hours of leukapheresis) 
• 
Baseline MRI of the brain (within 2 weeks before starting adjuvant DI TMZ). *For newly diagnosed 
patients, this baseline MRI must be done after completing XRT/TMZ. 
• 
Td booster (patients ≥ 18 years old only) 
 
If a subject does not undergo leukapheresis, minimal records regarding the subject and the reason for screen 
failure will be retained in the study database. 
11.2 Run-In Period 
Not applicable. 
11.3 Leukapheresis #1 
• 
CBC with differential 
• 
CMP 
• 
Serum pregnancy test (within 48 hours of leukapheresis) 
• 
Leukapheresis  
• 
Blood for immune monitoring (1 red top, 3 yellow top tubes) 
• 
AESI collection 
11.4 Treatment Period 
Adjuvant DI TMZ Cycle 
• 
Physical Exam 
• 
Neurologic Exam 
• 
Performance Status 
• 
CBC with differential 
• 
CMP 
• 
21-day DI TMZ 
 
Vaccine #1 (day 22 + 3 days): 
• 
Physical Exam 
• 
Neurologic Exam 
• 
Performance Status 
• 
Td preconditioning (1 Lf) 6-24 hours prior to Vaccine #1 
• 
CBC with differential 
• 
CMP 
• 
Whole blood for immunologic analysis (up to 76.5 mL in 9 yellow top ACD tubes, not to exceed 3 
mL/kg in a 24 hr period or 7 mL/kg in an 8 week period, and 1 red top tube) 
• 
CMV-DCs 
• 
AE collection 
 

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Vaccine #2 (2 weeks ± 2 days after Vaccine #1): 
• 
CBC with differential 
• 
CMP 
• 
CMV-DCs 
• 
AE collection 
 
Vaccine #3 (2 weeks ± 2 days after Vaccine #2): 
• 
CBC with differential 
• 
CMP 
• 
CMV-DCs 
• 
Whole blood for immunologic analysis (up to 76.5 mL in 9 yellow top ACD tubes, not to exceed 3 
mL/kg in a 24 hr period or 7 mL/kg in an 8 week period, and 1 red top tube) (1 week ± 5 days 
after Vaccine #3) 
• 
AE collection 
 
Vaccine #4+ (every 4 weeks ± 5 days after Vaccine #3): 
• 
Physical Exam 
• 
Neurologic Exam 
• 
Performance Status 
• 
CBC with differential 
• 
CMP 
• 
Td preconditioning (1 Lf) 6-24 hours prior to each vaccine 
• 
CMV-DCs 
• 
MRI at Vaccine #4 visit (~3-5 weeks after Vaccine #3) and then every 3 months, at the discretion of 
the treating physician 
• 
Whole blood for immunologic analysis (up to 76.5 mL in 9 yellow top ACD tubes, not to exceed 3 
mL/kg in a 24 hr period or 7 mL/kg in an 8 week period, and 1 red top tube) (1 week ± 5 days after 
the last Vaccine given to the patient) 
• 
AE collection 
11.5 Follow-up Period 
Until Progression or Death: 
• 
Physical Exam 
• 
Neurologic Exam 
• 
Performance Status 
• 
MRI 
 
Patients may not be treated with any other modality unless progressive tumor is noted or they are otherwise 
removed from the study.  Patients will be considered off study if they do not have enough PBMCs to generate 
at least one DC vaccine, upon tumor progression, or upon treatment of the tumor with another modality.  
When subjects are considered off study, this indicates that subjects will no longer be obligated to undergo 
study-related tests and procedures, but the data described below will still be collected from these subjects as 
feasible.  Patients who generate one or two vaccines after leukapheresis will have the option to receive these 
vaccines per the study schedule.  Patients who received prior craniospinal radiation can undergo second 
leukapheresis during or after administration of vaccine(s).  Subjects will be followed for serious adverse events 
(SAEs) for 30 days after coming off study.  Collection of the additional data listed in the following sentence 
from off study subjects will be performed, if possible, but is not mandatory and will not be considered a 
deviation if the data cannot be obtained.  Subjects’ medical records will be reviewed for the remainder of their 
life, in order to collect data on subsequent treatments, disease progression, tumor size/volume, and survival. 

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11.6 End of Study 
The study will be considered complete once enrollment has been met, follow-up procedures outlined in 
Section 11.4 have been conducted on all subjects, and data analysis is concluded.  The study may also be 
terminated early for any reason by the PI-sponsor. 
 
Subjects that are lost to follow-up will be documented in the patient record and in the 21 CFR Part 11 
database.  In the compliant database, the subject will be marked as “Patient Status Unknown,” along with a 
corresponding explanation, if any. 
11.7 Early Withdrawal of Subject(s) 
11.7.1 Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time.  The PI may also withdraw a subject from 
the study at any time based on his/her discretion.  Reasons for PI-initiated withdrawal may include, but 
is not limited to the following: 
- 
Adverse events 
- 
Abnormal laboratory values 
- 
Abnormal test procedure results 
- 
Protocol deviation 
- 
Administrative issues 
- 
Disease progression 
- 
Pregnancy 
- 
Unable to generate one CMV-DC vaccine 
- 
A >2 week delay in vaccine administration, outside of the allowable window 
- 
Unable to complete at least ~54 Gy of radiation (newly diagnosed WHO grade IV glioma patients 
only) 
 
Subjects will be withdrawn from the study if they do not receive any CMV-DC vaccinations due to 
contamination or any other inadequacy in the first batch of CMV-DC vaccines from the initial 
leukapheresis.  
11.7.2 Follow-up Requirements for Early Withdrawal 
Subjects should be seen in clinic or contacted at a minimum of every 3 months for 1 year. 
11.7.3 Replacement of Early Withdrawal(s) 
Subjects that withdraw from the study prior to leukapheresis will not be evaluable for any objective; 
these subjects will be replaced.  See Section 14.6 for related details. 
11.8 Study Assessments 
11.8.1 Medical History 
Standard medical history will be obtained and documented per institutional guidelines. 
11.8.2 Physical Exam 
Standard physical exam and neurological assessment will be conducted and documented per 
institutional and Preston Robert Tisch Brain Tumor Center (PRTBTC) guidelines. 
11.8.3 Radiographic Review 
The modified RANO provides clinical guidelines for continuing therapy beyond suspected radiographic 
progression if the treating physician believes there may be a therapeutic benefit and provides criteria 

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for defining progression and early drug failure while also allowing for the possibility of 
pseudoprogression.  
 
An update to the RANO criteria proposes strategies to establish a general framework for response 
assessment in neuro-oncology that is agnostic to the mechanism of action of the particular therapy 
(anti-angiogenic, immunotherapy, etc.) to address the challenges associated with interpretation of 
radiographic changes in patients with brain tumors in clinical trials.  
 
The modified RANO proposes to use the post-radiation time point as the baseline for response 
evaluation and to consider only objectively defined, measurable enhancing disease in the definition of 
response and progression (i.e. exclusion of qualitative assessed T2/FLAIR changes). 
 
Patients will be defined as progressive if they meet any of the following criteria: 
1. At least two sequential scans separated by ≥4 weeks both exhibiting ≥25% increase in sum of 
products of perpendicular diameters of enhancing lesions. The first scan exhibiting ≥25% increase 
in sum of products of perpendicular diameters of enhancing lesions should be compared to the 
smallest tumor measurement obtained either at baseline (if no decrease) or best response (on 
stable or increasing steroid dose) and is noted as “preliminary progressive disease (PD).” If the 
second scan at least 4 weeks later exhibits a subsequent ≥25% increase in sum of products of 
perpendicular diameters of enhancing lesions relative to the “preliminary PD” scan, it is considered 
“confirmed PD” and the patient should discontinue therapy, if not, this scan showing “preliminary 
PD” is noted as “pseudoprogression” and the patient should continue on therapy until a second 
increase in tumor size relative to the pseudoprogression scan is observed.  
2. In the case where the baseline or best response demonstrates no measurable enhancing disease 
(visible or not visible), then any new measurable (>10mm x 10mm) enhancing lesions are 
considered PD after confirmed by a subsequent scan ≥4 weeks exhibiting ≥25% increase in sum of 
products of perpendicular diameters of enhancing lesions relative to the scan first illustrating new 
measurable disease. The first scan exhibiting new measurable disease is noted as “preliminary PD.” 
If the second scan at least 4 weeks later exhibits a subsequent ≥25% increase in sum of products of 
perpendicular diameters of enhancing lesions relative to the “preliminary PD” scan it is considered 
“confirmed PD” and the patient should discontinue therapy. If the second scan at least 4 weeks 
later exhibits SD, CR, PR, or becomes non-measurable, this scan showing “preliminary PD” is noted 
as “pseudoprogression”, PsP, and the patient should continue on therapy until a second increase in 
tumor size relative to the “preliminary PD”, or PsP, scan is observed. Note that any new measurable 
(>10mm x 10mm) enhancing lesions on the subsequent scan following the preliminary PD scan 
should not be immediately considered confirmed PD, but instead should be added to the sum of bi-
dimensional products or total volume representing the entire enhancing tumor burden. 
3. Clear clinical deterioration not attributable to other causes apart from tumor (e.g. seizures, 
medication adverse effects, therapy complications, stroke, infection) or attributable to changes in 
steroid dose. 
4. Failure to return for evaluation as a result of death or deteriorating condition. 
11.8.4 Correlative Assessments 
Whole blood will be obtained for tests of anti-tumor immune responses at leukapheresis #1, just prior 
to Vaccine #1, ~1 week after Vaccine #3, and ~1 week after the patient has received their final CMV-DC 
vaccination.  Because of the large volume of the whole blood draws taken for immune monitoring, it 
will be essential to carefully consider the weight of each child (under the age of 18) when determining 
whether a blood draw may occur or how much blood may be drawn at a given time point.  In the 
pediatric population, the maximum blood volumes are 3 mL/kg in a 24 hour period or 7 mL/kg in an 8 
week period.  Table 2 contains predicted blood draw volumes that are required for the planned study 
assessments during the indicated periods. 
 

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Table 2: Schedule of Blood Draw Volumes 
 
Maximum blood draw volumes 21 
Screening 
Leukapheresis #1 
Adjuvant TMZ Cycle 
Final Vaccine 
Day 
 
Within 2 weeks of 
screening 
~22 
~38 
~52 
~1 week after 
final Vaccine 
CBC & CMP 
6 mL 
6 mL 
6 mL 
6 mL 
6 mL 
 
Pregnancy 
2 mL 
2 mL 
 
 
 
 
Whole blood 
immunologic 
analysis 
 
28.5 mL (1 red top 
tube and 3 yellow 
top ACD tubes) 
Up to 76.5 
mL22 (9 yellow 
top ACD tubes) 
+ 3 mL (1 red 
top tube) 
 
Up to 76.5 mL 22 
(9 yellow top 
ACD tubes) + 3 
mL (1 red top 
tube) 
Up to 76.5 mL 22 
(9 yellow top 
ACD tubes) + 3 
mL (1 red top 
tube) 
Total over 
period 
8 mL 
36.5 mL 
85.5 mL 
6 mL 
85.5 mL 
79.5 mL 
 
Max volume in 8 week period (Leukapheresis #1 and Adjuvant TMZ Cycle):  213.5 mL 
12 SAFETY MONITORING AND REPORTING 
The PI is responsible for the identification and documentation of adverse events (AEs) and serious adverse events 
(SAEs), as defined below.  At each study visit, the PI or designee must assess, through non-suggestive inquiries of 
the subject or evaluation of study assessments, whether an AE or SAE has occurred. 
12.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a subject receiving study drug and which does 
not necessarily have a causal relationship with this treatment.  For this protocol, the definition of AE also 
includes worsening of any pre-existing medical condition.  An AE can therefore be any unfavorable and 
unintended or worsening sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of CMV-DCs or Td preconditioning, whether or not related to the use of study drug(s).  
But laboratory value changes that require therapy or adjustment in prior therapy are considered adverse 
events. 
 
From the time of the Td preconditioning prior to Vaccine #1 through the End of Study visit (as defined in 
Section 11.6), as well as any Special Interest AEs related to leukapheresis (as defined in Section 12.1.1), all AEs 
must be recorded in the subject medical record and adverse events case report form. 
 
AEs will be assessed according to the CTCAE version 5.  If CTCAE grading does not exist for an AE, the severity 
of the AE will be graded as mild (1), moderate (2), severe (3), life-threatening (4), or fatal (5). 
 
Attribution of AEs will be indicated as follows: 
                                                 
21 Volumes for standard lab tests are based on DUHS Clinical Labs volumes for patients age 8-15.  For patients in the lower age 
range of this study and/or with a low body weight, lower pediatric volumes (for patients 0-8) may be used.  Some volumes may be 
higher for patients > 15 years old. 
22 Volumes of whole blood for immunologic analysis will be reduced as necessary to not exceed 3 mL/kg in a 24 hour period or 7 
mL/kg in an 8 week period. 

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- 
Definite:  The AE is clearly related to the study drug(s) 
- 
Probably:  The AE is likely related to the study drug(s) 
- 
Possible:  The AE may be related to the study drug(s) 
- 
Unlikely:  The AE is doubtfully related to the study drug(s) 
- 
Unrelated:  The AE is clearly NOT related to the study drug(s) 
12.1.1 AEs of Special Interest 
Adverse events will not be collected until the patient has received their first Td preconditioning prior to 
CMV-DC Vaccine #1, unless they are considered an Adverse Event of Special Interest related to the 
leukapheresis procedure.  Only these special interest adverse events will be collected prior to the first 
Td preconditioning, all other events occurring prior to the first Td preconditioning administration will 
not be recorded or monitored.   
 
Special Interest Adverse Events that may occur during the leukapheresis procedure include: 
• 
Allergic Reaction 
• 
Anaphylaxis 
• 
Pre-syncope 
• 
Syncope 
• 
Vasovagal reaction 
 
Special Interest Adverse Events that may occur after the procedure, but may still be related to 
leukapheresis include: 
• 
Vascular Access Complications 
• 
Venous Injury 
12.1.2 Reporting of AEs 
A database of all adverse events (not just those considered related to the study drug) will be 
maintained in 21 CFR Part 11 Compliant database. The event will be categorized by organ system, 
relationship to treatment, its grade of severity, and resolution. The Principal Investigator and study 
statistician will periodically review the collective adverse events with the intention of identifying any 
trends or patterns in toxicity. If any such trends are identified, depending on their severity and 
frequency, a protocol amendment will be considered. 
12.2 Serious Adverse Events 
An AE is considered “serious” if in the opinion of the investigator it is one of the following outcomes: 
- 
Fatal 
- 
Life-threatening 
- 
Constitutes a congenital anomaly or birth defect 
- 
A medically significant condition (defined as an event that compromises subject safety or may 
require medical or surgical intervention to prevent one of the three outcomes above) 
- 
Requires inpatient hospitalization or prolongation of existing hospitalization 
- 
Results in persistent or significant incapacity or substantial disruption to conduct normal life 
functions 
12.2.1 Reporting of SAEs 
All serious and unexpected AEs should be reported immediately to the PI or designee.  Only AEs that 
the PI determines to be serious, unanticipated, and related or possibly/probably (i.e. more likely than 
not) related to the research must be reported to the IRB and the Food and Drug Administration (FDA).  
Fatal or life-threatening, unexpected adverse events will be reported to the FDA by telephone, 
facsimile, or in writing as soon as possible, but no later than 7 calendar days after first knowledge by the 
sponsor followed by as complete a report as possible within 8 additional calendar days.  Serious, 
unexpected adverse events that are not fatal or life-threatening will be reported to the FDA by 

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telephone, facsimile, or in writing as soon as possible, but no later than 15 calendar days after first 
knowledge by the sponsor. 
 
All adverse events that are considered serious, unanticipated, and related or possibly related to study 
drug(s) (as defined by 21CRF312.32[a]) will be reported to the IRB and the FDA using the appropriate 
SAE report form.  At the time of the annual progress report to the IRB and the FDA, a summary of the 
overall toxicity experience will be provided. 
12.3 Safety Oversight Committee (SOC) 
The Duke Cancer Institute SOC is responsible for annual data and safety monitoring of DUHS sponsor-
investigator phase 1 and 2, therapeutic interventional studies that do not have an independent Data Safety 
Monitoring Board (DSMB). The primary focus of the SOC is review of safety data, toxicities and new 
information that may affect subject safety or efficacy. Annual safety reviews include but may not be limited to 
review of safety data, enrollment status, stopping rules if applicable, accrual, toxicities, reference literature, 
and interim analyses as provided by the sponsor-investigator.  The SOC in concert with the DCI Monitoring 
Team (see Section 13.1 for Monitoring Team description) oversees the conduct of DUHS cancer-related, 
sponsor-investigator greater-than-minimal-risk intervention studies that do not have an external monitoring 
plan, ensuring subject safety and that the protocol is conducted, recorded and reported in accordance with 
the protocol, standing operating procedures (SOPs), Good Clinical Practice (GCP), and applicable regulatory 
requirements. 
12.4 External Data and Safety Monitoring Board (DSMB) 
Due to a potential conflict of interest in other PI-initiated studies at Duke University Medical Center, an 
external DSMB-Plus was created.  The current study will be monitored by this DSMB-Plus.  The external DSMB-
Plus will be responsible for safeguarding the interests of trial subjects and assessing the safety of the 
interventions during the trial. The DSMB-Plus will provide recommendations about stopping or continuing 
enrollment in the trial.  To contribute to enhancing the integrity of the trial, the DSMB-Plus may also formulate 
recommendations relating to the selection, recruitment, and retention of subjects and their management.  
Additional details regarding the responsibility of the DSMB-Plus and its chair may be found in the charter 
document. 
13 QUALITY CONTROL AND QUALITY ASSURANCE 
13.1 Monitoring 
The Duke Cancer Institute (DCI) Monitoring Team will conduct monitoring visits to ensure subject safety and to 
ensure that the protocol is conducted, recorded, and reported in accordance with the protocol, standard 
operating procedures, good clinical practice, and applicable regulatory requirements.  As specified in the DCI 
Data and Safety Monitoring Plan, the DCI Monitoring Team will conduct routine monitoring after the third 
subject is enrolled, or annually if less than 3 subjects enroll in the first year, followed by annual monitoring of 
1 – 3 subjects until the study is closed to enrollment and subjects are no longer receiving study interventions 
that are more than minimal risk.   
 
Additional monitoring may be prompted by findings from monitoring visits, unexpected frequency of serious 
and/or unexpected toxicities, or other concerns and may be initiated upon request of DUHS and DCI 
leadership, the DCI Cancer Protocol Committee, the SOC, the PI, or the IRB.  All study documents must be 
made available upon request to the DCI Monitoring Team and other authorized regulatory authorities, 
including but not limited to the National Institute of Health, NCI, and the FDA.  Every reasonable effort will be 
made to maintain confidentiality during study monitoring. 
13.2 Audits 
The Duke Office of Audit, Risk, & Compliance (OARC) office may conduct confidential audits to evaluate 
compliance with the protocol and the principles of Good Clinical Practice (GCP).  The PI agrees to allow the 

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OARC auditor(s) direct access to all relevant documents and to allocate his/her time and the time of the study 
team to the OARC auditor(s) in order to discuss findings and any relevant issues. 
 
OARC audits are designed to protect the rights and well-being of human research subjects. OARC audits may 
be routine or directed (for cause). Routine audits are selected based upon risk metrics generally geared 
towards high subject enrollment, studies with limited oversight or monitoring, Investigator initiated 
Investigational Drugs or Devices, federally-funded studies, high degree of risk (based upon adverse events, 
type of study, or vulnerable populations), phase 1 studies, or studies that involve Medicare populations. 
Directed audits occur at the directive of the IRB or an authorized Institutional Official. 
 
OARC audits examine research studies/clinical trials methodology, processes and systems to assess whether 
the research is conducted according to the protocol approved by the DUHS IRB. The primary purpose of the 
audit/review is to verify that the standards for safety of human subjects in clinical trials and the quality of data 
produced by the clinical trial research are met. The audit/review will serve as a quality assurance measure, 
internal to the institution. Additional goals of such audits are to detect both random and systemic errors 
occurring during the conduct of clinical research and to emphasize “best practices” in the research/clinical 
trials environment. 
13.3 Data Management and Processing 
13.3.1 Case Report Forms (CRFs) 
The electronic CRF (eCRF) will be the primary data collection document for the study.  The CRFs will be 
updated in a timely manner following acquisition of new source data.  Only the PI, the study 
coordinator, the data management team, and the clinical trials manager are permitted to make entries, 
changes, or corrections in the eCRF. 
 
An audit trail will be maintained automatically by the electronic CRF management system.  All users of 
this system will complete user training, as required or appropriate per DCI requirements and other 
regulations. 
13.3.2 Data Management Procedures and Data Verification 
Access to electronic databases will be managed by the PRTBTC Data Manager. 
 
Completeness of entered data will be checked automatically by the eCRF system, and users will be 
alerted to the presence of data inconsistencies.  Additionally, the data management team and the 
statistical team will cross-reference the data to verify accuracy.  Missing or implausible data will be 
brought to the attention of the PI requiring appropriate responses (i.e. confirmation of data, correction 
of data, completion or confirmation that data is not available, etc.). 
 
The database will be reviewed and discussed prior to database closure, and will be closed only after 
resolution of all remaining queries.  An audit trail will be kept of all subsequent changes to the data. 
13.3.3 Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following activities: 
• 
Data clarification and/or resolution 
• 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
• 
Review of site study records for completeness 
• 
Shipment of all remaining laboratory samples to the designated laboratories 
 

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14 STATISTICAL METHODS AND DATA ANALYSIS 
All statistical analysis will be performed under the direction of the statistician designated in key personnel.  Any 
data analysis carried out independently by the investigator must be approved by the statistician before publication 
or presentation. 
14.1 Analysis Sets 
Any patient who undergoes leukapheresis will be included in analyses of feasibility. 
 
Any patient who experiences an unacceptable toxicity from the time of Td preconditioning until 30 days after 
the 3rd vaccination or any patient who remains on study until 30 days after the 3rd vaccination and has no 
unacceptable toxicity will be included in the primary safety analyses.  For all other safety analyses, all patients 
who receive at least 1 vaccination will be included.   
 
Efficacy analyses will include all patients who receive at least 1 vaccination. 
 
Analyses of T cell response data will include all patients who received at least 3 vaccines and have assessments 
completed before Vaccine #1 and after Vaccine #3. 
14.2 Patient Demographics and Other Baseline Characteristics 
A summary of clinical and socio-demographic characteristics will be generated.  Categorical descriptors will be 
summarized using frequency distributions; whereas, interval variables will be summarized using percentiles, as 
well as means and standard deviations. 
14.3 Treatments 
Among patients who initiate DI TMZ, a frequency distribution will be generated for the number of vaccines 
received by each patient.   
14.4 Primary Objective 
This study has two primary objectives:  the assessment of feasibility of manufacturing at least three CMV-DCs 
from a single leukapheresis (or two leukapheresis procedures for those who have received previous 
craniospinal radiation) of eligible patients, and the safety of intradermal administration of CMV-DC. 
14.4.1 Primary Objective #1: Feasibility  
Feasibility will be defined in terms of the percentage of patients for whom three or more vaccines can 
be generated from the pre-treatment leukapheresis. 
 
This study was originally designed to assess feasibility within two separate cohorts:  patients with prior 
craniospinal radiation and patients without prior craniospinal radiation.   Prior to an amendment 
(protocol version 2, dated 11/16/2018), up to 2 patients were to be accrued to the cohort with prior 
craniospinal radiation with the stipulation that the limit of 2 patients would be removed if 3 or more 
vaccines could be generated from the pre-treatment leukapheresis procedures in both patients.  Given 
that the first two patients with prior craniospinal radiation had 3 or more vaccines generated, the 
limitation was removed in protocol version 2. 
14.4.2 Primary Objective #2: Safety 
The second primary objective of this study is to assess the safety of intradermal administration of CMV-
DCs in in patients up to age 35  with WHO grade IV glioma, recurrent malignant glioma (WHO grade III-
IV glioma), and with recurrent medulloblastoma.   

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14.4.2.1 Variable 
The percentage of patients who experience an unacceptable adverse event as defined in 
Section 8.1.7 after Td preconditioning and within the 30-day window after Vaccine #3, prior to 
the 4th vaccination. 
 
A patient will be considered evaluable for the primary assessment of safety if the patient 
experiences an unacceptable adverse event during this noted period, or if the patient receives 
3 vaccines and completes the endpoint observation period of 30 days after Vaccine #3. 
14.4.2.2 Adverse Event Monitoring 
The experience of patients treated with CMV-DC will be continuously monitored to determine 
whether an unacceptable adverse event as defined by Section 8.1.7 occurs.  If any patient 
experiences a grade 5 treatment-related adverse event, the initiation of CMV-DC treatment 
will be suspended until all data are carefully reviewed.  The review will result in a decision as to 
whether treatment and accrual will continue with or without modification, or whether the 
study will be permanently terminated.  Otherwise, the following guidelines will be used to 
determine whether accrual should be suspended and adverse event data reviewed: 
 
Number of 
Patients Treated 
Who are 
Evaluable for 
Assessment of 
Toxicity 
Criteria for Accrual Suspension 
3 
≥2 with unacceptable adverse event 
4 
≥2 with unacceptable adverse event 
5 
≥2 with unacceptable adverse event 
6 
≥3 with unacceptable adverse event 
7 
≥3 with unacceptable adverse event 
8 
≥3 with unacceptable adverse event 
9 
≥4 with unacceptable adverse event 
10 
≥4 with unacceptable adverse event 
 
Though the study targets the treatment of 6 patients with CMV-DC who are evaluable for the 
assessment of adverse event per section 14.4.2.1, it is possible that up to 10 patients will be 
treated, depending on the number of accrued subjects that are deemed feasible and as such 
should be treated with CMV-DCs. 
 
After 4 patients have been enrolled and treated (i.e. the last patient has received Vaccine #3 
and been observed for 30 days after Vaccine #3), the study will be paused for evaluation of 
safety before proceeding with further enrollment.   
14.4.2.3 Other Safety Analyses 
Additional summaries of adverse events will be tabulated.  In these additional summaries, all 
patients who receive at least one vaccination will be included.  Different summaries will be 
generated for different purposes: 
 
• 
For the summary of adverse events and serious adverse events within the 
ClinicalTrials.gov report, the percentage of patients who experience each type of adverse 
event regardless of attribution will be summarized. 

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• 
For the manuscript, adverse events that are possibly, probably, and definitely treatment-
related will be summarized.  For each type of toxicity, the maximum grade experienced by 
each patient will be summarized with frequency distributions. 
• 
Two tabulations will be generated for review by the Safety Oversight Committee including 
one that includes all toxicities regardless of attribution, and another that includes only 
toxicities that are possibly, probably, and definitely related to study regimen.  For each of 
these tabulations, the maximum grade of each type of toxicity experienced by each 
patient will be summarized with frequency distributions. 
14.4.3 Exploratory Objectives 
This study includes two exploratory objectives that examine T cell response and efficacy.  Given the 
overall small sample size of this study, all exploratory analyses will be descriptive. 
14.4.3.1 Exploratory Objective #1: T cell response 
For each patient, the percent change in pp65-specific T cell responses assessed by 
polyfunctional T cell responses, including but not limited to IFNγ, CCL3, IL-2, and TNFα, 
between pre-chemotherapy baseline (initial leukapheresis), post-chemotherapy baseline (just 
prior to Vaccine #1), and the follow-up assessment 1 week (± 5 days) after Vaccine #3 and ~1 
week after the administration of the final vaccine will be calculated.  A one-sample Wilcoxon 
test or t-test will assess whether significant changes in these outcomes occurred between 
baseline and post-Vaccine #3. 
14.4.3.2 Exploratory Objective #2: Survival and Progression-Free Survival 
Survival, defined as the time between initiation of DI TMZ and death, will be censored at last 
follow-up for patients alive at the time of analyses.  Progression-free survival (PFS), defined as 
the time between initiation of DI TMZ and progression, or death if the patient dies without 
documentation of progression.  A patient that is alive without disease progression at the time 
of analyses will have PFS censored at the time of last follow-up.  The Kaplan-Meier estimator 
will be used to describe the survival and PFS of the patients treated on this protocol.  Estimates 
of 6-month, 1-year, 2-year, and 3-year OS and PFS will be generated. 
14.5 Interim Analysis 
No interim analyses of efficacy will be conducted.  Interim analysis of safety is described in Section 14.4.2. 
14.6 Sample Size Calculation 
All patients accrued to the study will be treated regardless of ability to generate at least three CMV-DC 
vaccines from the pre-treatment leukapheresis (or 2 leukaphereses for those who have had previous 
craniospinal radiation). 
 
The goal is to treat at least 6 patients with CMV-DC who are evaluable for the assessment of treatment 
safety.  As noted in Section 14.4.2.2, the actual number that will be treated may be greater than 6 and as 
many as 10 patients.  
 
Assuming we are targeting 10 patients to be treated with CMV-DC, then the probability of terminating early 
as a function of the true, though unknown, probability of an unacceptable adverse event is tabulated below.  
Included is also the probability of early study termination based upon the monitoring rules described in 
Section 14.4.2.2. 
 
True Unknown Probability of 
Unacceptable Adverse Event 
Probability of Terminating Early 
Average Number of Patients 
Treated 
0.1 
0.092 
9.6 
0.2 
0.322 
8.3 

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0.3 
0.596 
6.8 
0.4 
0.791 
5.5 
0.5 
0.920 
4.6 
 
15 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
15.1 Regulatory and Ethical Compliance 
This protocol was designed and will be conducted and reported in accordance with the International 
Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice, the 
Declaration of Helsinki, and applicable federal, state, and local regulations. 
15.2 DUHS Institutional Review Board and DCI Cancer Protocol Committee 
The protocol, informed consent form, advertising material, and additional protocol-related documents must 
be submitted to the DUHS IRB and DCI Cancer Protocol Committee (CPC) for review.  The study may be 
initiated only after the PI has received written and dated approval from the CPC and IRB. 
 
The PI must submit and obtain approval from the IRB for all subsequent protocol amendments and changes to 
the informed consent form.  The CPC should be informed about any protocol amendments that potentially 
affect research design or data analysis (i.e. amendments affecting subject population, inclusion/exclusion 
criteria, agent administration, statistical analysis, etc.). 
 
The PI must obtain protocol re-approval from the IRB within 1 year of the most recent IRB approval.  The PI 
must also obtain protocol re-approval from the CPC within 1 year of the most recent IRB approval, for as long 
as the protocol remains open to subject enrollment. 
15.3 Informed Consent 
The informed consent form must be written in a manner that is understandable to the subject population.  
Prior to its use, the informed consent form must be approved by the IRB. 
 
The PI or authorized key personnel will discuss with the potential subject and/or the potential subject’s 
parents or guardians the purpose of the research, methods, potential risks and benefits, subject concerns, and 
other study-related matters.  This discussion will occur in a location that ensures subject privacy and in a 
manner that minimizes the possibility of coercion.  Appropriate accommodations will be made available for 
potential subjects or parents/guardians who cannot read or understand English or are visually impaired.  
Potential subjects and/or their parents or guardians will have the opportunity to contact the Principal 
investigator or authorized key personnel with questions, and will be given as much time as needed to make an 
informed decision about participation in the study.  Written assent is required for subjects who are aged 12 to 
17 years old.  Verbal assent is required for subjects who are aged 6 years old to 11 years old.  In the instances 
where a child’s assent is required, the child should be given an explanation of the proposed research 
procedures in a language that is appropriate to the child's age, experience, maturity, and condition. 
 
Before conducting any study-specific procedures, the PI or designee must obtain written informed consent 
from the subject or a legally acceptable representative.  The original informed consent form will be stored 
with the subject’s study records, and a copy of the informed consent form will be provided to the subject 
and/or the subject’s parent or guardian.  The PI is responsible for asking the subject or their parent/guardian 
whether the subject’s primary care physician should be notified about participation in the study.  If the subject 
or their parent/guardian agrees to such notification, the PI will inform the subject’s primary care physician 
about the subject’s participation in the clinical study. 

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15.4 Privacy, Confidentiality, and Data Storage 
The PI will ensure that subject privacy and confidentiality of the subject’s data will be maintained.  Research 
Data Security Plans (RDSPs) will be approved by the appropriate institutional Site Based Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects during the course 
of the study.  Prospective participants will be consented in an exam room where it is just the research staff, 
the patient and his family, if desired. For all future visits, interactions with research staff (study doctor and 
study coordinators) regarding research activities will take place in a private exam room. All research related 
interactions with the participant and their parent/guardian, if applicable, will be conducted by qualified 
research staff who are directly involved in the conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock and key 
accessible only by the research staff. Subjects will be identified only by a unique study number and subject 
initials.  Electronic records of subject data will be maintained using a dedicated 21 CFR Part 11 compliant 
database, which is housed in an encrypted and password-protected file on a secure network drive.  Access to 
electronic databases will be managed by the PRTBTC Data Manager.  The security and viability of the IT 
infrastructure will be managed by the DCI and/or Duke Medicine.   
 
Upon completion of the study, research records will be archived and handled per DUHS HRPP policy.  Subject 
names or identifiers will not be used in reports, presentations at scientific meetings, or publications in 
scientific journals. 
15.5 Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and Safety Monitoring Plan.   
15.6 Protocol Amendments 
All protocol amendments must be initiated by the PI and approved by the IRB prior to implementation.  IRB 
approval is not required for protocol changes that occur to protect the safety of a subject from an immediate 
hazard.  However, the PI must inform the IRB and all other applicable regulatory agencies of such action 
immediately. 
15.7 Records Retention 
The PI will maintain study-related records until the youngest child on study is 21 years old, or for at least six 
years after study completion, whichever is longer (Duke policy). 
 
 
 
 

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