Conditions: Glioblastoma
Keywords: Glioblastoma Dendritic cells Temozolomide Tetanus Khasraw Immunotherapy Vaccine
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 6
Healthy volunteers: no

Brief summary:
This single-arm phase II study will assess the impact of tetanus pre-conditioning and adjuvant Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) on overall survival of patients newly diagnosed with World Health Organization (WHO) Grade IV glioblastoma who have undergone definitive tumor resection, are cytomegalovirus (CMV) positive and unmethylated, and completed standard temozolomide (TMZ) and radiation treatment. After completion of the standard of care radiotherapy with concurrent TMZ, patients will receive 1 cycle of dose-intensified TMZ followed by pp65-loaded dendritic cell (DC) vaccination beginning on day 23.

Detailed description:
Approximately 64 patients with resected, newly-diagnosed WHO Grade IV glioma who are CMV positive and in which the Methylguanine Methyltransferase (MGMT) is not methylated will be accrued to this study before standard of care radiation therapy (RT) and concurrent TMZ, with the goal of treating 48 patients with dose-intensified temozolomide and pp65 loaded dendritic cell vaccine after completion of standard RT and TMZ.

All enrolled patients will undergo a leukapheresis for the generation of DCs. Patients will then receive approximately 6 weeks of the standard of care radiation therapy (RT) and concurrent TMZ at a standard targeted dose of 75 mg/m2/day. For patients whose initial leukapheresis yields less than 3 vaccines, repeat leukapheresis may be obtained. At the post-RT clinic visit, a single post-RT cycle of dose-intensified TMZ (100 mg/m2/day for 21 days) will be given. On day 23 (± 2 days) of the cycle, patients will receive the first of 3 pp65 DC vaccines. Vaccines #1-3 will be given every two weeks (± 2 days). All patients will receive up to a total of 10 DC vaccines, with vaccines administered every 35 days (± 7 days) after the third vaccine, given bilaterally at the groin site unless progression occurs with no further cycles of TMZ. DC vaccines will be given intradermally (i.d.) and divided equally to both inguinal regions. Before the first DC vaccination, patients will receive 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly into the deltoid muscle to ensure adequate immunity to the tetanus antigen. Patients will undergo leukapheresis again for immunologic monitoring with a specific assessment of baseline antigen-specific cellular and humoral immune responses if needed for further DC generations 14 (± 2) days after vaccine #3. Prior to pp65 DC vaccination #4,(3±1) weeks after leukapheresis 2, the vaccine site will receive a pre-conditioning intradermal injection of Td. Up to 16 patients will receive 111-Indium labeled DCs at the 4th vaccine followed by SPECT/CT imaging immediately, and at 1 and 2 days after injections.

Arms:
arm 1: This single-arm phase II study will assess the impact of tetanus pre-conditioning and adjuvant GM-CSF on overall survival of newly diagnosed glioblastoma (GBM) patients who have undergone definitive resection, are unmethylated, and completed standard temozolomide and radiation treatment. All enrolled patients will undergo a leukapheresis for the generation of DCs. Patients will then receive approximately 6 weeks of standard of care radiation therapy (RT) and concurrent TMZ. A single post-RT cycle of dose intensified TMZ (100 mg/m2/day for 21 days) will then be given. On day 23 (± 2 days) of the cycle, patients will receive the first of 3 pp65 DC vaccines every 2 weeks. All patients will receive up to a total of 10 DC vaccines

Interventions: intervention 1: Human CMV pp65-LAMP mRNA-pulsed autologous DCs containing GM CSF intervention 2: Temozolomide intervention 3: Tetanus-Diphtheria Toxoid (Td) intervention 4: GM-CSF intervention 5: 111-Indium-labeling of Cells for in vivo Trafficking Studies
Intervention descriptions: intervention 1: 2x10\^7 human CMV pp65-LAMP mRNA-pulsed autologous DCs are given intradermally and bilaterally at the groin site (divided equally to both inguinal regions). Patients will receive up to a total of 10 DC vaccines. intervention 2: Temozolomide is a chemotherapy drug given to all enrolled patients at the post-RT clinic visit as dose-intensified TMZ (100 mg/m2/day for 21 days). intervention 3: Before the first DC vaccination, patients will receive 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly into the deltoid muscle to ensure adequate immunity to the tetanus antigen. Prior to pp65 DC vaccination #4,(3±1) weeks after leukapheresis 2 the vaccine site will receive a pre-conditioning intradermal injection of Td (1 flocculation unit (Lf), in 0.3 mL of saline for a total of 0.4 mL). intervention 4: Granulocyte macrophage-colony stimulating factor (GM-CSF) is a sterile, white, preservative-free lyophilized powder in a vial containing 250 mcg that will be reconstituted in 0.5 mL of sterile water for injection and used as an adjuvant with the DC vaccine. intervention 5: 111-In-labeled DCs are 2 x 10\^7 pp65-LAMP mRNA loaded mature DCs labeled with 111-In (50 μCi / 5 x 10\^7 DCs) and given i.d. as the fourth vaccine. In up to 16 patients, the fourth vaccine will be labeled with 111-In (50 μCi / 5 x 10\^7 DCs) prior to injection.

Protocol document:
Prot_SAP_001.pdf: 
I-ATTAC 
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I- ATTAC: 
Improved Anti-Tumor Immunotherapy Targeted Against 
Cytomegalovirus in Patients with Newly-Diagnosed WHO Grade IV 
Unmethylated Glioma (NCT03927222) 
 
Sponsor: 
PI - Duke Cancer Institute 
 
Funding Source: 
NIH Grant 
 
Protocol Source: 
PI - Duke Cancer Institute 
 
Duke IRB#: 
Pro00090683 
 
IND#: 
16301 
 
Principal Investigator 
Mustafa Khasraw, M.D. 
mustafa.khasraw@duke.edu 
Sub-Investigator(s) 
Annick Desjardins, M.D. 
annick.desjardins@duke.edu 
 
Henry Friedman, M.D. 
 
henry.friedman@duke.edu 
 
Margaret Johnson, M.D. 
margaret.johnson@duke.edu 
 
Katherine Peters, M.D. 
katherine.peters@duke.edu 
 
Kristen Batich 
kristen.batich@duke.edu 
 
John Sampson, M.D., Ph.D. 
john.sampson@duke.edu 
 
Daniel Landi, MD 
daniel.landi@duke.edu 
 
David Ashley, MBBS, Ph.D.  
david.ashley@duke.edu 
Statistician 
James E. Herndon II, Ph.D. 
james.herndon@duke.edu 
 
Lead Study Coordinator 
Nicole Cort 
nicole.cort@duke.edu 
 
Regulatory Coordinator 
Chasiety Turner 
chasiety.turner@duke.edu 
 
Therapeutic Material Manufacture 
Manager 
Gary Archer, Ph.D. 
gary.archer@duke.edu 
 
Data Manager 
Eric Lipp 
eric.lipp@duke.edu 
 
 
 
 
 


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Version Date 
Description of Changes 
Original version: 
20180108 
 
Amended version: 
20180205 
Title page for Dr. Ashley’s degrees; amended objectives/endpoints 
for grant submission (Sections 8 & 15); amended total number of 
subjects enrolled and updated number of subjects for endpoint 
evaluations (Sections 9 & 12); included a CBC with all immune 
monitoring time points (Section 12); clarified Td pre-conditioning 
dose and toxicity definition (Section 9); definition for progression & 
DC migration better described, as well as subclinical automimmunity 
included (Section 12). 
Amended version 
20180316 
In response to FDA information request: For Grade 3 toxicity 
management, immunization will be withheld until NCI CTC toxicity 
improves to a Grade 1 or less (Section 9.1.3);  
Amended version 
20190318 
CTCAE version number updated to 5, Updated the missing footnote 
8 (Table 4. Schedule of Events), Title page changed the Lead clinical 
and regulatory coordinator, updated Sub-Investigator list, specified 
the size of the blood draws in the footnotes of the table (Table 4. 
Schedule of Events), all mentions of CBC specified auto differential, 
We added a specification that if leukapheresis 2 must occur earlier 
than scheduled, that there will be a blood draw performed at that 
scheduled time point, added an interim analysis in Section 15.8, 
added a section on Adverse events of Special Interest (Section 13.2) 
Amended version 
20190529 
Updated the study schema (Section 6), Updated the acceptable 
steroid dosage, Modified the inclusion criteria to reflect radiation 
requirements for eligibility (Section 11), Updated the MGMT testing 
details to include use of CARIS services (Section 12.1).Clarified Td 
booster IM can be given on the day or before vaccine #1 as long as it 
is given prior to vaccine#1 (Table 4. Schedule of Events).Updated 
criteria for early withdrawal to >4 weeks of RT (Section 12.6.1). 
Included details of companion leukapheresis study that can likely be 
used for DC vaccine manufacture (Section 10.1 12.2); Vaccines 4-10 
will be given every 35 (±7) days after third vaccine. 
Amended version 
20191009 
Dr. Mustafa Khasraw, M.D will now be the PI for the study. The PI 
change is reflected throughout the protocol. Clarified statements in 
Table 4. Schedule of Events footnote. 
Amended version 
20200212 
Subject eligibility criteria updated to make it consistent across 
studies in the center. KPS ≥ 70%, Serum creatinine level was 
changed to ≤ 3 times institutional upper limit of normal for age, 
serum aspartate aminotransferase (AST) ≤ 3 times institutional upper 
limit of normal for age (Section 11). 
Amended version 
20200409 
Updates to title page.  Added description of the use of bevacizumab 
(reduced dose) to treat inflammatory reactions secondary to the 
immune response expected with DC vaccination (Sections 9.1, 
9.1.3).  Change in immune monitoring blood draws (amounts, tube 
types, timing) (Sections 12.2, 12.7.5).  Table 4 in Section 12 has been 
completely revised.  Change in how many weeks RT should be 
started after surgery and PI discretion language added (Section 9.1). 
Change in hemoglobin and bilirubin inclusion criteria (Section 11).  
Change in timing of MMSE at baseline (Section 12.1).  Revised 
language regarding initial tissue testing (Section 12.1).  Change in 
timing for early withdrawals being replaced (Section 12.6.3).  Curran 
Status removed (Section 12.7.2).  Formatting changed and 
clarification made in Section 15.6.5. 
Amended version 
20220204 
Updates made to staff on the face page.  Removed 111In-labeled DCs 
and associated SPECT/CT scan at Vaccine #4 as objective already 
met in an earlier completed study (see Schema in Section 6, Table 4, 
and Sections 12.7.4 and 15.6.1).  Slight revision to exclusion criteria 
related to current/recent participation in another study in Section 11.  
Revise exclusion criteria in Section 11 to remove mention of SPECT 


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scan and to allow for patients with MRI-compatible devices.  Schema 
in Section 6 re-done so now an editable picture.   
 
 


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1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ........................................................................................................ 4 
2 
LIST OF FIGURES ................................................................................................................ 7 
3 
LIST OF TABLES .................................................................................................................. 7 
4 
LIST OF ABBREVIATIONS ................................................................................................. 8 
5 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY ................................................ 11 
6 
STUDY SCHEMA................................................................................................................ 12 
7 
BACKGROUND AND SIGNIFICANCE ............................................................................ 13 
7.1 
Study Disease ............................................................................................................. 13 
7.1 
Study Agent ................................................................................................................ 13 
7.1.1 
Pre-Clinical Experience .......................................................................................... 19 
7.1.2 
Clinical Experience ................................................................................................. 20 
7.2 
Study Purpose/Rationale............................................................................................. 23 
8 
OBJECTIVES AND ENDPOINTS ...................................................................................... 24 
9 
INVESTIGATIONAL PLAN ............................................................................................... 25 
9.1 
Study Design............................................................................................................... 25 
9.1.1 
Definition of Unacceptable Toxicities .................................................................... 25 
9.1.2 
Dose Modification .................................................................................................. 26 
9.1.3 
Safety Considerations ............................................................................................. 26 
9.1.4 
Missed Doses .......................................................................................................... 27 
9.1.5 
Concomitant Medications ....................................................................................... 27 
9.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration ......................... 28 
9.3 
Rationale for Correlative Studies ............................................................................... 28 
9.4 
Definition of Evaluable Subjects, On Study, and End of Study ................................. 28 
9.5 
Early Study Termination ............................................................................................ 28 
10 STUDY DRUG ..................................................................................................................... 28 
10.1 
Names, Classification, and Mechanism of Action ..................................................... 28 
10.2 
Packaging and Labeling of Study Agents ................................................................... 30 
10.3 
Supply, Receipt, and Storage ...................................................................................... 30 
10.4 
Dispensing and Preparation ........................................................................................ 31 
10.5 
Compliance and Accountability ................................................................................. 31 
10.6 
Disposal and Destruction ............................................................................................ 31 


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11 SUBJECT ELIGIBILITY ..................................................................................................... 31 
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES ....................................... 33 
12.1 
Screening Examination ............................................................................................... 33 
12.2 
Treatment Period ........................................................................................................ 34 
12.3 
End of Treatment ........................................................................................................ 38 
12.4 
Follow-up Period ........................................................................................................ 38 
12.5 
End of Study ............................................................................................................... 38 
12.6 
Early Withdrawal of Subject(s) .................................................................................. 38 
12.6.1 Criteria for Early Withdrawal ................................................................................. 38 
12.6.2 Follow-up Requirements for Early Withdrawal ..................................................... 38 
12.6.3 Replacement of Early Withdrawal(s) ...................................................................... 39 
12.7 
Study Assessments ..................................................................................................... 39 
12.7.1 Medical History ...................................................................................................... 39 
12.7.2 Physical Exam ......................................................................................................... 39 
12.7.3 Use of Antihistamines ............................................................................................. 39 
12.7.4 Radiologic Evaluations ........................................................................................... 39 
12.7.5 Immunologic Assessments...................................................................................... 40 
13 SAFETY MONITORING AND REPORTING.................................................................... 41 
13.1 
Adverse Events ........................................................................................................... 41 
13.1.1 Reporting of AEs .................................................................................................... 42 
13.2 
Adverse Events of Special Interest ............................................................................. 42 
13.3 
Serious Adverse Events .............................................................................................. 42 
13.3.1 Reporting of SAEs .................................................................................................. 43 
13.4 
External Data and Safety Monitoring Board .............................................................. 43 
14 QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 44 
14.1 
Monitoring .................................................................................................................. 44 
14.2 
Audits.......................................................................................................................... 44 
14.3 
Data Management and Processing .............................................................................. 45 
14.3.1 Study Documentation.............................................................................................. 45 
14.3.2 Data Management ................................................................................................... 45 
14.3.3 Data Management Procedures and Data Verification ............................................. 46 
14.3.4 Coding ..................................................................................................................... 46 
14.3.5 Study Closure .......................................................................................................... 46 
15 STATISTICAL METHODS AND DATA ANALYSIS ...................................................... 46 


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15.1 
Study Design Overview .............................................................................................. 46 
15.2 
Analysis Sets............................................................................................................... 46 
15.3 
Patient Demographics and Other Baseline Characteristics ........................................ 47 
15.4 
Treatments .................................................................................................................. 47 
15.5 
Primary Objective ....................................................................................................... 47 
15.5.1 Variable ................................................................................................................... 47 
15.5.2 Statistical Hypothesis, Model, and Method of Analysis ......................................... 47 
15.5.3 Handling of Missing Values, Censoring, and Discontinuation............................... 47 
15.6 
Secondary Objectives ................................................................................................. 48 
15.6.1 Secondary Objective #1:  Migration and Survival .................................................. 48 
15.6.2 Secondary Objective #2:  CCL3 and Survival ........................................................ 49 
15.6.3 Secondary Objective #3:  Polyfunctionality and Survival ...................................... 49 
15.6.4 Secondary Objective #4:  TRegs ............................................................................... 50 
15.6.5 Secondary Objective #5:  Toxicity ......................................................................... 51 
15.7 
Exploratory Objectives ............................................................................................... 52 
15.7.1 Exploratory Objective #1:  Migration and CCL3 ................................................... 52 
15.7.2 Exploratory Objective #2:  Polyfunctionality and Migration ................................. 53 
15.7.3 Exploratory Objective #3:  Polyfunctionality and CCL3 ....................................... 53 
15.8 
Interim Analysis ......................................................................................................... 53 
15.9 
Sample Size Calculation ............................................................................................. 53 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ............................................. 54 
16.1 
Regulatory and Ethical Compliance ........................................................................... 54 
16.2 
DUHS Institutional Review Board and DCI Cancer Protocol Committee ................. 54 
16.3 
Informed Consent ....................................................................................................... 54 
16.4 
Privacy, Confidentiality, and Data Storage ................................................................ 55 
16.5 
Data and Safety Monitoring ....................................................................................... 55 
16.6 
Protocol Amendments ................................................................................................ 55 
16.7 
Records Retention....................................................................................................... 56 
16.8 
Conflict of Interest ...................................................................................................... 56 
16.9 
Registration Procedure ............................................................................................... 56 
17 REFERENCES ..................................................................................................................... 57 
18 APPENDICES ...................................................................................................................... 63 
18.1 
Standard Radiation Therapy ....................................................................................... 63 
18.2 
Temozolomide Therapy .............................................................................................. 63 


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2 LIST OF FIGURES 
Figure 1. Study Schema ................................................................................................................ 12 
Figure 2: Homeostatic Lymphodepletion Induced by Temozolomide Enhances Antigen-Specific 
Immune Responses. ...................................................................................................................... 17 
Figure 3: Polyclonal Antibody Median Fluorescence Index (MFI) Against GM-CSF is Plotted 
Over Time Coinciding With Vaccine and Apheresis Administrations in One subject.  Anti-GM-
CSF Antibodies (IgG+IgM) Increases Over Time With Repeated Vaccinations Using 
Recombinant GM-CSF and Begin to Decrease After the Adjuvant Was Removed. .................... 21 
Figure 4: IgE Antibody Median Fluorescence Index (MFI) Against GM-CSF is Plotted Over 
Time Coinciding With Vaccine and Apheresis Administrations in One Subject.  IgE Antibody 
Fluorescence Against GM-CSF Peaks at Vaccine#8, Consistent With the Subject’s Anaphylactic 
Episode at This Time Point Before Slowly Decreasing Back to Baseline After Adjuvant 
Removal. ....................................................................................................................................... 22 
 
3 LIST OF TABLES 
Table 1. Objectives and Endpoints ............................................................................................... 24 
Table 2. Storage of Study Agents ................................................................................................. 31 
Table 3. Dispensing of Study Agents ........................................................................................... 31 
Table 4. Schedule of Events.......................................................................................................... 36 
 
 


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4 LIST OF ABBREVIATIONS 
Ab 
Antibody 
ABC 
Automated Blood Count 
ACD 
Acid Citrate Dextrose 
ACLS 
Advanced Cardiac Life Support 
ACTH 
Adrenocorticotropic Hormone 
AE 
Adverse Event 
AIDS 
Acquired Immune Deficiency Syndrome 
ALT 
Adoptive Lymphocyte Transfer  
APAAP 
Alkaline Phosphatase Antialkaline Phosphatase Complex 
ANC 
Absolute Neutrophil Count  
AST 
Aspartate Aminotransferase 
AT 
Ambient Temperature 
AUC 
Area Under the Curve 
BCR 
B Cell Receptor 
β-HCG 
Beta-Human Chorionic Gonadotropin 
BMT 
Bone Marrow Transplant 
BTIP 
Brain Tumor Immunotherapy Program  
BTSC 
Brain Tumor Stem Cells 
Ca++ 
Calcium 
caBIG 
Cancer Biomedical Informatics Grid  
CAP 
College of American Pathologists 
CBC 
Complete Blood Count  
CCL3 
Chemokine (C-C motif) ligand 3 
cDNA 
Complimentary Deoxyribonucleic Acid 
CFA 
Complete Freund’s Adjuvant 
CFC 
Cytokine Flow Cytometry 
cGMP 
Current Good Manufacturing Practice 
CLIA 
Clinical Laboratory Improvement Act 
CLN 
Cervical lymph Nodes 
Cmax 
Maximum Concentration of Drug in Plasma or Serum 
CMP 
Comprehensive Metabolic Panel 
CMV 
Cytomegalovirus 
CNC 
Clinical Neurologic Change 
CNS 
Central Nervous System 
COI 
Conflict of Interest 
Con-A 
Concanavalin A 
CPC 
Cancer Protocol Committee 
CT 
Computed Tomography 
CTL 
Cytotoxic T-Lymphocyte 
DAR 
Drug Accountability Record 
DC 
Dendritic Cell 
DCI 
Duke Cancer Institute 
DI-TMZ 
Dose-Intensified Temozolomide 
DLT 
Dose Limiting Toxicity 
DNA 
Deoxyribonucleic Acid 
DOB 
Date of Birth 
DSMB 
Data Safety Monitoring Board  
DSMP 
Data Safety and Monitoring Plan 
DTH 
Delayed-type Hypersensitivity 


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DTPA 
DiethyleneTriamine Penta-acetic Acid  
DUHS 
Duke University Health System 
eCRF 
Electronic Case Report Form 
IEC 
Institutional Ethics Committee 
EAE 
Experimental Autoimmune Encephalomyelitis 
EBRT 
External Beam Radiation Therapy 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-linked Immunospot 
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII 
Epidermal Growth Factor Receptor variant type III 
EGFRvIII-KLH 
EGFRvIII conjugated to Keyhole Limpet Hemocyanin 
FACS 
Fluorescence Activated Cell Sorting 
FDA 
Federal Drug Administration 
FEV 
Forced Expiratory Volume 
FFPE 
Formalin-Fixed, Paraffin-Embedded  
GMP 
Good Clinical Practice 
GBM 
Glioblastoma  
GM-CSF 
Granulocyte Macrophage Colony Stimulating Factor 
HAMA 
Human Anti-Murine Antibody  
H&E 
Hematoxylin and Eosin  
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HMO 
Health Maintenance Organization 
HRPP 
Human Research Protection Program 
I.C. 
Intracerebral 
ICF 
Informed Consent Form  
ICH 
International Conference on Harmonisation 
ICS 
Investigational Chemotherapy Services 
ID 
Identification 
I.D. 
Intradermal 
IDH 
Isocitrate Dehydrogenase 
IE1 
Immediate Early Gene 1 Protein 
IFN-γ 
Interferon-gamma 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IHC 
Immunohistochemistry 
IL-2 
Interleukin-2 
IL-4 
Interleukin-4 
IL-12 
Interleukin-12 
IL-13 
Interleukin-13 
I.M. 
Intramuscular 
In 
Indium  
IND 
Investigational New Drug  
iRANO 
Immunotherapy Response Assessment in Neuro-Oncology 
IRB 
Institutional Review Board 
ISH 
In Situ Hybridization  
IT 
Information Technology 
IUD 
Intra-Uterine Device 
I.V. 
Intravenous 
KLH 
Keyhole Limpet Hemocyanin 
KPS 
Karnofsky Performance Status 
LAMP 
Lysosomal-associated Membrane Protein 
Lf 
Flocculation unit 


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LIMS 
Laboratory Information Management System 
MAb 
Monoclonal Antibody 
MFI 
Median Fluorescence Index    
MG 
Malignant Glioma 
MGMT 
Methylguanine Methyltransferase 
MHC 
Major Histocompatibility Complex 
mL 
MilliLiter 
MMSE 
Mini-Mental Status Examination 
MPACT 
Molecular Products and Cellular Therapies 
MRI 
Magnetic Resonance Imaging 
mRNA 
Messenger Ribonucleic Acid 
MTD 
Maximally Tolerated Dose 
NA 
Non-adherent 
NCI CTC 
National Cancer Institute Common Toxicity Criteria 
NCI CTCAE 
National Cancer Institute Common Toxicity Criteria of Adverse Events 
ng 
NanoGram 
NIH 
National Institutes of Health 
NK 
Natural Killer 
OARC 
Office of Audit, Risk and Compliance 
OS 
Overall Survival 
OT-I 
Ovalbumin-specific T cells 
OVA 
Ovalbumin 
PBLs 
Peripheral Blood Lymphocytes 
PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PCR 
Polymerase Chain Reactions 
PD 
Progressive Disease 
PHA 
Phytohemagglutinin 
PFS 
Progression Free Survival 
PI 
Principle Investigator 
PO 
By Mouth 
PRTBTC 
Preston Robert Tisch Brain Tumor Center 
PT 
Prothrombin Time 
PTT 
Partial Thromboblastin Time 
QA/QC 
Quality Assurance/Quality Control 
RDSP 
Research Data Security Plan 
RIO 
Research Integrity Office 
RNA 
Ribonucleic Acid 
RT 
Radiation Therapy 
RT-PCR 
Reverse Transcriptase Polymerase Chain Reaction 
SAE 
Severe Adverse Event 
S.C. 
Subcutaneous 
SFC 
Spot-Forming Cells  
SOC 
Standard of Care  
SOCOMM 
Safety Oversight Committee 
SOP 
Standard Operating Procedure 
SPECT 
Single-Photo Emission Computerized Tomography 
TCR 
T cell Receptor 
TD 
Tetanus-Diphtheria  
TGF-β 
Transforming Growth Factor-β 
TH2 
T helper type 2 
TILs 
Tumor-Infiltrating Lymphocytes  
TMZ 
Temozolomide 


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TNF-α 
Tumor Necrosis Factor-α 
TRegs 
Regulatory T cells 
TTP 
Time to Progression 
TTRNA 
Total Tumor mRNA 
VDLNs 
Vaccine-site Draining Lymph Nodes 
WBI 
Whole Body Irradiation 
WBRT 
Whole Brain Radiation Therapy  
WHO 
World Health Organization  
5 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
 Research Summary is available upon request.  


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6 STUDY SCHEMA 
 
Figure 1. Study Schema 
 
 
 
Newly diagnosed GBM 
CMV+, unmethylated 
Resection 
Enrollment† 
Leukapheresis*† 
DC generation 
Failed QC – Patient replaced 
Standard of care external beam radiation 
(XRT) with concurrent TMZ (as per SOC) 
Standard of care single cycle DI TMZ 
DC vaccine #1*– 3  
CMV pp65 RNA-pulsed DCs on Days 23, 
37, and 51 of this cycle 
Leukapheresis* 
Skin preparation* with Td (1 Lf) on Day before Vaccine #4 
DC vaccine #4* 
Continue with vaccines #5 – #10 
CMV DCs every month 
Radiographic or clinical tumor progression* 
Stereotactic biopsy or resection 
Immunohistochemical studies 
*Immunologic monitoring from each 
leukapheresis, prior to vaccines #1 and #4-10; 
and prior to Td pre-conditioning and at 
progression 
Safety monitoring throughout DC vaccination 
†Patient may enroll and undergo leukapheresis 
under an umbrella study (Pro00102797), 
instead of i-ATTAC.  If so, enrollment on i-
ATTAC will occur after DC generation, 
RT/TMZ, and single cycle TMZ before 1st DC 
vaccine. 


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7 BACKGROUND AND SIGNIFICANCE 
7.1 Study Disease 
Malignant primary brain tumors are more common than Hodgkin’s disease and account for more 
human deaths than melanoma or than cancer of the bladder or kidney. Despite aggressive, 
computer-guided tumor resection [1], high-dose external beam radiation therapy (XRT), and multi-
mechanistic chemotherapy delivered at toxic doses, most patients with malignant primary brain 
tumors live < 15 months from the time of diagnosis, and patients with recurrent tumors usually 
survive < 12 weeks [2-7]. The estimated cost of treatment for each patient with a malignant brain 
tumor is between $30,000 and several hundred thousand dollars annually. Thus, the annual 
treatment cost alone for these patients, not mention the lost earning potential of afflicted 
individuals, is greater than the entire annual budget of the National Institute of Neurological 
Diseases and Stroke. In fact, conventional therapy for patients with malignant brain tumors is the 
most expensive medical therapy per quality-adjusted life-years (QALYs) saved currently provided 
in the United States [8, 9]. Moreover, the non-specific nature of standard of care therapy for brain 
tumors often results in incapacitating damage to surrounding normal brain and systemic tissues 
[10, 11]. Thus, in order to be more effective, therapeutic strategies need to precisely target tumor 
cells while minimizing collateral damage to neighboring eloquent cerebral cortex. The rationale 
for employing the immune system to target brain tumors is based on the premise that the inherent 
biologic specificity of immunologic reactivity could meet the urgent need for more specific and 
less toxic therapy. 
7.1 Study Agent 
Human Cytomegalovirus pp65- Lysosomal-Associated Membrane Protein mRNA-Pulsed 
Autologous Dendritic Cell Vaccines Containing Granulocyte Macrophage-Colony 
Stimulating Factor 
 
Dendritic cells (DCs) are potent immunostimulatory 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 at the crossroads of many of the elegant networks of the immune system, and DCs 
represent the most promising contemporary biologic entity for realizing the promise of 
immunotherapy. Potent immune responses and encouraging clinical results have been seen in 
Phase I and II human clinical trials in systemic cancers [14-30]. 
 
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 [31]. 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 cell 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 [32].  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 [33, 34].  Furthermore, 
the potency of GM-CSF has been demonstrated in a Phase I clinical trial in melanoma patients 
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immunization sites were intensely infiltrated with T cells, DCs, macrophages, and eosinophils in 
100% of evaluable patients.  Extensive tumor destruction was seen in 11 of 16 patients.  Both 
cytotoxic T-cell and antibody responses were associated with this tumor destruction. Hence, GM-
CSF has an extensive track record both as a growth factor and an adjuvant, is commercially 
available and has an acceptable toxicity profile. In a recently published study from our laboratory, 
we reported that immediately following a course of dose-intensified temozolomide (DI-TMZ), 
given over 21 consecutive days, the administration of three sequential Cytomegalovirus (CMV)-
specific DC vaccines resulted in significantly increased CMV-specific cellular responses and 
superior overall survival (OS). Median progression free survival (PFS) and OS for this cohort were 
25.3 months [95% confidence interval (CI), 11.0–∞] and 41.1 months (95% CI, 21.6–∞), 
exceeding projected survival using recursive partitioning analysis (RPA) and matched historical 
controls. Moreover, four patients remained progression-free at 59 to 64 months from diagnosis. 
No known prognostic factors (i.e. age, KPS, IDH-1/2 mutation, and MGMT promoter methylation) 
were biased to predict more favorable outcomes for the patients in this cohort [36]. 
 
Human CMV is an endemic β-Herpesvirus that does not usually cause significant clinical disease 
[37]. During primary maternal infection, however, human CMV can cause severe encephalitis in 
fetuses and lead to congenital brain defects. Human CMV disease is also a significant problem in 
immunocompromised adults such as organ transplant recipients or patients with Acquired Immune 
Deficiency Syndrome (AIDS) [37]. Herpesviruses have also been implicated in a number of human 
malignancies including lymphoma, nasopharyngeal cancer, cervical cancer, and Kaposi’s sarcoma 
[38, 39]. Recently, expression of proteins unique to human CMV has been reported within a large 
proportion of malignant tumors including colorectal carcinoma, prostate cancer, and malignant 
astrocytomas [40-42]. Universal detection of the human CMV immunodominant protein pp65, 
immediate early gene 1 protein (IE1), and several other early antigens was demonstrated using 
immunohistochemistry (IHC) in Grade II-IV astrocytomas [42]. Presence of the virus in these 
samples was confirmed with in situ hybridization (ISH), polymerase chain reactions (PCR) for 
human CMV-specific glycoprotein B (UL55), electron microscopic detection of intact virions [42], 
and direct detection of the virus from fresh operative samples in the shell vial assay  (unpublished 
data). Notably human CMV antigens were not detected in surrounding normal brain samples, 
meningiomas, or brains affected by ischemia, Alzheimer’s disease, paraneoplastic encephalitis, or 
Cryptococcal cerebritis. 
 
The presence of highly-immunogenic human CMV antigens within malignant gliomas (MGs) 
affords a unique opportunity to target these tumors immunologically. There is a vast amount of 
experience with both the safety and efficacy of immunotherapy targeting human CMV [37], and 
the presence of this virus within brain tumors may allow this experience to be leveraged toward 
the effective eradication of MG expressing human CMV antigens. Adoptive T cell therapy has 
been used to safely and successfully protect against CMV reactivation in myelodepleted bone 
marrow transplant (BMT) patients [43-46]. In addition, T cell mediated immunotherapy has 
proven highly effective in the treatment of CMV-associated disease within the central nervous 
system (CNS) [47] and in the treatment of acute CMV infections [44, 48]. Tumors associated with 
other human Herpesviruses, such as Epstein-Barr virus-associated lymphoma, including tumors 
within the CNS, have also been effectively treated and even large tumors have been cured by 
immunotherapy [49-54]. More recently, a vaccine directed against the potent viral antigens of 


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human papilloma virus has also been shown to reduce the incidence of human papilloma virus-
related cervical intraepithelial neoplasia in a prospective, randomized, double-blind trial [55]. 
 
The potential for non-specific targeting of normal tissues is thought to be minimal in seropositive 
patients. After initial infection, CMV establishes lifelong latency in the infected individual, with 
cells of the myeloid lineage constituting a major reservoir for persistence of the virus. Virus can 
be detected within myeloid progenitors in the bone marrow, with a small portion of these cells 
demonstrating viral deoxyribonucleic acid (DNA) replication without any detectable gene 
expression [56, 57]. Also a small proportion  (typically 1 in 1,000 to 1 in 10,000) of peripheral 
blood monocytes can be found to contain CMV DNA, while detection of viral ribonucleic acid 
(RNA)  (gene expression) is not detected [58-60].   
 
Vaccination specifically against CMV[61-65] has effectively reduced the risk of viral infection 
and transmission to fetuses in animal models[66-68] and in clinical trials [61, 65, 69-72]. Human 
clinical trials have also demonstrated some benefit of administering neutralizing antibodies in the 
treatment of human CMV infection [73-76], highlighting the importance of the development of 
vaccination strategies that elicit both cellular and humoral immune responses. DCs strongly 
activate both T cell and B cell responses in vivo [13], and DCs pulsed in vitro with CMV antigens 
have been shown to be potent inducers of CMV-specific cytotoxic T-lymphocyte (CTL) responses 
in several studies [77-81], in addition to our own work which is outlined below. 
 
The use of RNA to encode tumor antigens for DCs was pioneered at Duke University in Dr. 
Gilboa’s laboratory, but the ability of RNA-loaded DCs to stimulate potent antitumor immunity 
has been independently confirmed in murine and human systems [82-87]. In fact, there is 
accumulating evidence that RNA transfection represents a superior method for loading antigens 
onto DCs [84, 88]. This novel and innovative approach to DC antigen loading has multiple 
conceptual advantages over other forms of antigen delivery as well. RNA-based antigen loading 
does not require knowledge of major histocompatibility complex (MHC) restriction, and responses 
are not restricted to single MHC haplotypes or to a narrow B or T cell repertoire. This diversity 
increases the likelihood of inducing effective and sustained antitumor immune responses by 
simultaneous activation of both CTLs and helper T-cells [89-91]. Furthermore, in direct 
comparisons, RNA-loaded DCs have been found to be better stimulators of antigen-specific T-
cells than other approaches[88]. Finally, RNA also carries a significant safety advantage, not 
possessed by other nucleic acid or viral vectors, in that it cannot be integrated permanently into 
the host genome. In addition to the preliminary data we present below, Kobayashi et al.[92] have 
demonstrated that tumor mRNA-loaded DCs can elicit a specific CD8+ CTL response against 
autologous tumor cells in patients with MG. 
 
Temozolomide (TMZ) 
 
TMZ, a methylating agent with superior blood-brain barrier penetration, has recently been shown 
to increase survival by a small, but statistically significant, 2.5 months in a subset of patients with 
newly-diagnosed GBM if given in conjunction with XRT following initial tumor resection of the 
tumor [93-95]. Leukopenia, diminished white blood cell counts in the host, is essentially the only 
known human toxicity of TMZ treatment. Although initially counter-intuitive, this TMZ-induced 
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subsequent homeostatic proliferation of newly generated lymphocytes that it induces. In this 
protocol, patients will receive standard of care six week course of TMZ concurrent with XRT 
following initial resection of the tumor, followed by a dose-intensified 21-day cycle of TMZ prior 
to DC vaccination. We believe that the lymphopenia induced by therapeutic TMZ treatment, if 
carefully timed before vaccination, will actually enhance the proliferation and maintenance of 
these tumor-specific T-cells through the natural forces that drive T cell homeostatic proliferative 
recovery. Thus, this combination strategy will uniquely exploit the toxicity of one effective therapy 
for MG to enhance another already promising therapy, immunotherapy. In preparation for this 
protocol, we have evaluated, in animal models, TMZ and sublethal whole body irradiation (WBI), 
as a positive control, as methods for induction of treatment-induced lymphopenia in order to 
determine the ability of TMZ-induced lymphodepletion to enhance active vaccination and adoptive 
immunotherapies. These studies were initially performed in a murine T cell receptor (TCR) 
transgenic model in which the antigen-specific T-cells can be followed in vivo in mice receiving 
adoptive transfer of lymphocytes. In this model system, we found that adoptive lymphocyte 
transfer (ALT) coupled with DC vaccination (DC + ALT) is a potent mechanism for inducing 
antigen-specific T-cell expansion after TMZ treatment. TMZ was found to be an effective agent 
for inducing homeostatic proliferation of transferred CD4+ and CD8+ T cells and for enhancing 
DC or peptide vaccinations with or without ALT (Figure 2). 
 
Markedly elevated levels of antigen-specific T-cells were achieved and maintained in mice 
receiving ALT after therapeutic TMZ-induced lymphodepletion compared to normal hosts 
receiving ALT. The precursor frequency of ovalbumin (OVA)-specific CD8+ T cells in the 
peripheral blood of untreated mice receiving OVA peptide vaccine after transfer of OVA antigen-
specific (OT-I) transgenic T cells was 1.5%. Mice pretreated with TMZ and subsequent OVA 
peptide vaccination had achieved a mean precursor frequency of 54%, exhibiting markedly 
increased levels of OVA-specific T-cells in the blood (P < 0.0001). Similar results were found in 
mice receiving DC vaccines, with untreated animals having an average precursor frequency of 
3.6% and TMZ pretreated animals achieving an average of 11.9% OVA-specific CD8+ T-cells 
after a single vaccination. These results demonstrate that active and adoptive immunotherapy 
administered during hematopoeitic recovery from TMZ treatment may be a very effective way to 
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Figure 2: Homeostatic Lymphodepletion Induced by Temozolomide Enhances Antigen-Specific Immune 
Responses. Untreated mice or mice receiving non-myeloablative high-dose temozolomide treatment (5 days x 60 
mg/kg) were administered a mixture of OVA-specific and normal lymphocytes intravenously and received either no 
vaccine, vaccination with DCs transfected with OVA RNA, or OVA peptide vaccination in complete Freund’s 
adjuvant.  The peripheral blood of treated animals was collected and analyzed 6 days after vaccination for the 
expansion of OVA-specific T cells in the circulation using αCD8 monoclonal antibodies and a specific tetramer.  As 
shown above, mice treated with temozolomide prior to ALT demonstrated markedly increased T cell expansion after 
vaccination compared to untreated hosts receiving ALT. 
 
111Indium-labeling of Cells for in vivo Trafficking Studies 
 
The in vivo distribution of DCs will be evaluated using 111Indium (111In)-labeled pp65-lysosomal-
associated membrane protein (pp65-LAMP) messenger RNA (mRNA)-loaded mature DCs. 111In-
labeling has been used extensively for evaluation of adoptively transferred tumor infiltrating 
lymphocytes, natural killer cells, granulocytes, DCs, and whole blood leukocytes, for in vivo 
localization studies in humans [96-99].  DCs will be labeled at the Duke Radiopharmacy Lab 
according to standardized protocols. Briefly, 2 x 107 pp65-LAMP mRNA-loaded mature DCs will 
be labeled with 111In (50 μCi / 5 x 107 DCs) (Nycomed-Amersham, Chicago, IL) prior to injection. 
Intradermal DC injections sites will be carefully marked at 10 cm from the groin crease.  Gamma 
camera (dual-headed) images will then be taken immediately after injections (0 hour), and at 24 
and 48 hours after the DC vaccination to compare DC migration from the inguinal intradermal 
injection sites to the inguinal vaccine site-draining lymph nodes (VDLNs). Regions of interest will 
represent the quantity of 111In signal and be gated on VDLNs with calculations corrected for 
isotope decay. The quantity of DC uptake in VDLNs will serve as the migration efficiency of the 
cellular vaccine, and these migration rates will be analyzed for correlation with OS in these 
patients. This will serve to address the secondary objective if migration rates of DC vaccines given 
after Td pre-conditioning correlate with OS of patients receiving this therapy. Beginning with 
Untreated
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protocol version 20220204, 111Indium-labeling of cells has been discontinued as the objective has 
already been met in a previously completed study entitled ELEVATE (Pro00054740). 
 
Tetanus-Diphtheria Toxoid (Td) 
 
The current use of Td toxoid is for active immunization in children and adults against infection 
with the bacteria Clostridium tetani and Corynebacterium diphtheria. Tetanus infection is 
manifested primarily by neuromuscular dysfunction caused by a potent exotoxin released by C. 
tetani. Diphtheria is an acute toxin-mediated infectious disease caused by toxigenic strains of C. 
diphtheriae. Protection against disease is due to the development of neutralizing antibodies to the 
diphtheria toxin. Td toxoids adsorbed are readily available as several approved administrations 
[i.e. Daptacel (DTaP), Infanrix (DTap), Tenivac (Td adult), Boostrix (Tdap)][100, 101]. Protection 
against disease is due to the development of neutralizing antibodies to the tetanus toxin. A serum 
tetanus antitoxin level of at least 0.01 IU/mL, measured by neutralization assays, is considered the 
minimum protective level. A level ≥0.1 IU/mL by ELISA has been considered as protective[102]. 
A serum diphtheria antitoxin level of 0.01 IU/mL, measured by neutralization assays, is the lowest 
level giving some degree of protection; a level of 0.1 IU/mL by enzyme-linked immunosorbent 
assay (ELISA) is regarded as protective. Diphtheria antitoxin levels ≥1.0 IU/mL by ELISA have 
been associated with long-term protection[103]. 
 
Following deep subcutaneous/intramuscular (s.c./i.m.) administration of the tetanus toxoid 
vaccine, toxoid molecules are taken up at the vaccination site by immature DCs, which are 
professional antigen-presenting cells. Within these cells, they are processed through the endosomal 
pathway (involving the phagolysosome) where they are bound to MHC type II molecules on the 
surface of DCs. The MHC II:toxoid complex then migrates to the cell surface. This cellular 
processing is similarly thought to occur for native immature DCs at the skin site and for vaccinated 
DCs. Upon encounter with tetanus toxoid, the now activated mature DC at the vaccine site migrates 
along lymph channels to the VDLN where they encounter naive TH2 cells, each with their own 
unique T cell receptor (TCR). Identifying and then binding of the MHC II:toxoid to the specific 
TH2 receptor then activates the naive T cell, causing it to proliferate. Simultaneously, toxoid 
molecules not taken up by DCs pass along lymph channels to the same draining lymph nodes 
where they come into contact with B cells, each with their own unique B cell receptor (BCR). 
Binding to the B cell through the specific immunoglobulin receptor that recognizes tetanus toxoid 
results in the internalization of toxoid, processing through the endosomal pathway and presentation 
on the cell surface as an MHC II:toxoid complex, similarly to DCs undergoing the same process 
[104].  
 
These two processes occur in the same part of the lymph node with the result that the B cell with 
the MHC II:toxoid complex on its surface now comes into contact with the activated TH2 whose 
receptors are specific for this complex. The process, termed linked recognition, results in the TH2 
activating the B cell to become a plasma cell with the production initially of IgM, with a later 
switch to IgG antibodies produced. Additionally, a subset of these B cells becomes memory 
cells[104]. 
 
The novelty of using Td toxoid vaccination lies in the ability of this potent recall antigen to enhance 
antitumor responses as part of a cancer vaccination protocol. Td toxoid induces an inflammatory 


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milieu within the intradermal vaccine site, thereby promoting the migration of injected tumor-
specific DCs. Additionally, in the context of vaccinating the host with tumor-derived peptides, 
conditioning the vaccine site with Td toxoid has demonstrated enhanced immunogenicity with 
these peptides.  
 
Our data from the ATTAC clinical trial (NCT00639639) demonstrating the capacity to enhance 
DC migration to VDLNs via Td pre-conditioning of the vaccine site offer potential therapeutic 
interventions whereby we can enhance the immunologic responses to ultimately overcome the 
inherent challenges in faithfully eradicating established tumors. In a completed randomized 
clinical trial, we found that migration of injected DCs to VDLNs following vaccine site pre-
conditioning with Td toxoid was significantly increased compared to controls and that the 
efficiency of DC migration was strongly associated with clinical outcomes of patients with newly-
diagnosed GBM, the most fatal type of malignant brain tumors. To address this observation, we 
took our Td pre-conditioning platform back into the preclinical setting using transgenic mouse 
models and were able to corroborate the effects of Td pre-conditioning on increasing the lymph 
node homing of intradermally administered DCs. Moreover, Td pre-conditioning at a single 
vaccine site increased the migration of a bilateral DC vaccine to both inguinal lymph nodes. 
Regardless of the side of the Td intradermal skin prep, DC migration to bilateral inguinal VDLNs 
was increased to similar magnitudes, supporting a systemic response to recruit peripherally 
administered DCs.  
 
Our Td pre-conditioning platform in the context of DC vaccination also elicited superior anti-
tumor responses compared to controls receiving DC vaccines without Td pre-conditioning. In our 
clinical trial, patients with newly diagnosed GBM who were administered the Td skin prep before 
DC vaccination revealed significantly longer progression-free and overall survival rates compared 
to the control cohort. In evaluating the relationship between DC migration and clinical responses, 
we observed a modest positive association between levels of DC migration and survival. In our 
preclinical model, Td pre-conditioning prior to vaccination with tumor antigen-specific DCs 
dramatically suppressed the growth of established and highly aggressive B16-F10/OVA tumors. 
The use of Td with a DC vaccine increased antitumor responses in an antigen-specific manner, as 
non-specific DC vaccines were not potentiated with Td pre-conditioning. Furthermore, in a 
challenge setting, where mice are administered the treatment platform prior to challenge with 
tumor inoculation, Td pre-conditioning at the vaccine site induced a significant survival benefit 
compared to controls. 
     
7.1.1 Pre-Clinical Experience 
In our laboratories and those of others, systemic immunization using DCs co-cultured with 
uncharacterized tumor homogenate [105], whole tumor RNA [106], unidentified peptides eluted 
from tumor cells by gentle acid washing [107], or a distinct peptide encompassing the tumor-
specific epidermal growth factor receptor variant type III (EGFRvIII) mutation [108], 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 [105], 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 
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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 rechallenge of tumor cells 
indicating that a sustained antitumor immune response had been established. These findings are 
especially significant in light of the fact that the astrocytoma cell line used is known to secrete the 
immunosuppressive agent transforming growth factor-β (TGF-β) which is secreted by most human 
gliomas [109-113]. 
 
In another report from our laboratory [106], 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 MHCbackground. 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. In these mice, an 
inflammatory infiltrate composed of mononuclear cells and polymorphonuclear leukocytes was 
identified, only in mice treated with DCs co-cultured with tumor homogenate that matched the 
intracerebral tumor challenge. 
 
In a recent report from another laboratory [107], the survival of tumor-bearing rats injected 
subcutaneously with autologous bone marrow-derived 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, immunohistochemical analysis of five animals from 
each group in this experiment documented an increased peritumoral 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.  
 
7.1.2 Clinical Experience 
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In two previous studies conducted under Investigational New Drug (IND)-12839 and one under 
IND-13240, we have vaccinated patients with newly-diagnosed GBM with DCs loaded with 
mRNA encoding the human CMV matrix protein pp65. To date, a total of 59 patients have received 
545 vaccinations with pp65-loaded DCs.   
 
One patient in the ATTAC-GM trial (NCT00639639, IND-12839), which utilized pp65-specific 
DC vaccines admixed with GM-CSF, had a severe Grade 3 immunologic reaction after receiving 
vaccine #8 [114]. The patient developed flushing of the face, reddening of the eyes, nausea, tunnel 
vision, headache, raised red bumps on the chest, back, antecubital spaces of both arms, and large 
red swollen areas in both injection sites. Allergy and Immunology was consulted and 
recommended testing the individual components of the vaccine for hypersensitivity. The 
components were separated into two parts: GM-CSF and DCs pulsed with pp65 RNA. 
Immunologic workup for this patient revealed sensitization to the GM-CSF component of the 
vaccine and the production of high levels of anti-GM-CSF autoantibodies during vaccination 
(Figure 3, Figure 4). Removal of GM-CSF from the DC vaccine allowed continued vaccination 
(total of 10 vaccines) without incident for this patient. No other study drug AEs were detected in 
this study. The allergic reaction is detailed in the MEDWATCH submitted May 16, 2011, IND 
12839 serial number 34.   
 
 
Figure 3: Polyclonal Antibody Median Fluorescence Index (MFI) Against GM-CSF is Plotted Over Time 
Coinciding With Vaccine and Apheresis Administrations in One subject.  Anti-GM-CSF Antibodies 
(IgG+IgM) Increases Over Time With Repeated Vaccinations Using Recombinant GM-CSF and Begin to 
Decrease After the Adjuvant Was Removed.   
 
 
 
Phereesis 1
Vaccine 1
Vaccine 2
Vaccine 3
Pheresis 2
Vaccine 4
Vaccine 5
Vaccine 6
Vaccine 7
Vaccine 8
Vaccine 9
Vaccine 10
Post-vaccines
0
10
20
30
40
IgG+gM (poly)
MFI
Anti-GMCSF (IgG + IgM) Antibodies


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Figure 4: IgE Antibody Median Fluorescence Index (MFI) Against GM-CSF is Plotted Over Time Coinciding 
With Vaccine and Apheresis Administrations in One Subject.  IgE Antibody Fluorescence Against GM-CSF 
Peaks at Vaccine#8, Consistent With the Subject’s Anaphylactic Episode at This Time Point Before Slowly 
Decreasing Back to Baseline After Adjuvant Removal. 
Intradermal, intravenous and intralymphatic migration studies using 111In labeled DCs have been 
successfully done in both animal models and patients with no adverse effects [99, 115-117].   
 
Under IND-12839, we have used labeled DCs with 111In and used single-photo emission 
computerized tomography (SPECT) imaging to track the migration to the VDLNs.  This work was 
published in Nature [118].   
 
There is a vast amount of experience with both the safety and efficacy of immunotherapy targeting 
human CMV [37].  Vaccination specifically against CMV has effectively and without toxicity, 
reduced the risk of viral infection and transmission to fetuses in clinical trials [61, 65, 69-72]. 
Adoptive T-cell therapy has been used to safely and successfully protect against CMV reactivation 
in myelodepleted bone marrow transplant patients [43, 44, 46, 119].  In addition, T cell-mediated 
immunotherapy has proven highly effective in the treatment of CMV-associated disease within the 
central nervous system [47] and in the treatment of acute CMV infections [44, 48].  Tumors 
associated with other human Herpesviruses, such as Epstein-Barr virus-associated lymphoma, 
including tumors within the CNS, have also been effectively treated and even large tumors have 
been cured by immunotherapy [49-52, 54, 120]. The pp65 minimal epitope peptide (495-503) was 
shown to be safe in doses up to 10mg in hematopoietic stem cell transplant patients, with grade 1 
adverse events being most commonly reported in approximately 10-15% of the participants and 
was successful in expanding CMV reactive T-cells [121]. 
Phereesis 1
Vaccine 1
Vaccine 2
Vaccine 3
Pheresis 2
Vaccine 4
Vaccine 5
Vaccine 6
Vaccine 7
Vaccine 8
Vaccine 9
Vaccine 10
Post-vaccines
3
4
5
6
7
8
IgE
MFI
Anti-GMCSF (IgE Isotype)


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7.2 Study Purpose/Rationale 
Our Overall Goal in this project is to extend our substantial pre-clinical and clinical findings and 
verify these in a larger cohort. We hypothesize that CMV pp65-loaded DCs given with Td and 
GM-CSF to a larger group of patients with TMZ-induced lymphodepletion will confirm our 
previous findings by inducing significant DC migration to draining lymph nodes, producing high 
levels of Chemokine (C-C motif) ligand 3 (CCL3) and polyfunctional CMV pp65-specific T cells 
systemically, and confirming extra-ordinary PFS and OS.  
 
We will conduct a larger Phase 2 trial of CMV pp65-loaded DC vaccination in patients with GBM. 
We will confirm our observations from smaller studies, in order to determine if a Phase 3 trial is 
warranted and to estimate patient numbers that might be required. Patients with CMV positive, 
newly-diagnosed GBM will receive serial vaccines with CMV pp65-loaded DCs with Td and GM-
CSF vaccine site pre-conditioning after standard of care treatment with concurrent external beam 
radiation and TMZ chemotherapy. Only patients that are methylguanine-DNA methyltransferase 
(MGMT) unmethylated will be enrolled because adjuvant TMZ shows very little benefit in that 
population so it can be avoided after the initial treatment that induces transient lymphodepletion. 
 
Next, we will also confirm predictive biomarkers of survival. In our prior studies, DC migration 
to draining lymph nodes and systemic CCL3 and CMV pp65-specific polyfunctional T cells 
predicted PFS and OS. Here we will collect samples to confirm these predictive biomarkers 
prospectively.   
 
Strikingly, Td-treated patients had systemically enhanced DC migration to VDLNs and 
significantly improved PFS and OS compared to controls (median PFS 18.5 vs 10.8 months, OS 
25.7 vs 18.5 months) with half the Td cohort living > 44.1 months from diagnosis. Our murine 
studies corroborated that Td pre-conditioning systemically enhanced DC migration and suppressed 
tumor growth. In the proposed clinical study, we aim to enhance the migration of CMV pp65 RNA-
pulsed DCs with Td and adjuvant GM-CSF and will assess if increased migration using these 
strategies can be predictive of patient survival outcomes.  
 
 


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8 OBJECTIVES AND ENDPOINTS 
 
 
Table 1. Objectives and Endpoints 
 
Objective 
Endpoint 
Analysis 
Primary 
Determine if Td preconditioning of 
pp65 loaded DC vaccination with 
GM-CSF extends overall survival. 
Median survival from initiation of dose 
intensive adjuvant temozolomide. 
See Section 15.5 
Secondary 
Assess the association between 
migration of CMV pp65 RNA-
pulsed DCs with GM-CSF to site-
draining inguinal lymph nodes after 
Td pre-conditioning and subsequent 
survival. 
(PLEASE NOTE: With Protocol 
v.20220204, this objective has been 
removed as it was already addressed 
in a previous study.) 
For survival after vaccine 4, the hazard ratio 
associated with a 1 unit increase in migration, 
defined as the maximum percentage of 111In-
labeled DCs reaching inguinal nodes during 
the 48 hours after the 4th vaccination. 
See Section 15.6.1 
Secondary 
Assess the association between 
CCL3 measured post-Td pre-
conditioning and subsequent 
survival. 
For survival after vaccine 4, the hazard ratio 
associated with a 1-unit increase in CCL3 
measured post-Td pre-conditioning at vaccine 
4. 
See Section 15.6.2 
Secondary 
Assess the association between fold 
changes in T cell polyfunctionality 
and subsequent survival. 
 
Mean fold change from baseline in the 
frequency of pp65 antigen-specific CD8+ T 
cells producing three or more cytokines (IFNγ, 
CCL3, IL-2, TNFα, CD107a) at leukapheresis 
2 14± (4) days after vaccine 3. 
 
Mean fold change from baseline in the 
frequency of pp65 antigen-specific CD8+ T 
cells producing three or more cytokines (IFNγ, 
CCL3, IL-2, TNFα, CD107a) at vaccine 6. 
 
For survival after vaccine 4, the hazard ratio 
associated with a 1-fold increase in the 
frequency of pp65 antigen-specific CD8+ T 
cells producing three or more cytokines (IFNγ, 
CCL3, IL-2, TNFα, CD107a). 
See Section 15.6.3 
Secondary 
Assess whether TReg levels remain 
low without adjuvant temozolomide. 
Maximum mean peak increase from vaccine 1 
in percent TReg of CD4+ T cells. 
See Section 15.6.4 
Secondary 
Assess the safety of DI-TMZ 
treatment followed by DC 
vaccination. 
Proportion of patients who experience 
unacceptable toxicity. 
See Section 15.6.5 
Exploratory  
Assess the association CCL3 levels 
and migration. 
Mean fold change in CCL3 between pre- and 
post-Td pre-conditioning. 
 
Correlation between CCL3 change from fold 
change in CCL3, and migration. 
See Section 15.7.1 
Exploratory  
Assess the association between 
pp65-specific polyfunctional CD8+ 
T cells and increased DC migration. 
Correlation between fold change from baseline 
(pre-vaccine 1) in polyfunctional CD8+ T 
cells and migration. 
See Section 15.7.2 
Exploratory  
Assess the association between 
pp65-specific polyfunctional CD8+ 
T cells and increased serum CCL3. 
Correlation between fold change in 
polyfunctionality and fold change in CCL3  
See Section 15.7.3 


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9 INVESTIGATIONAL PLAN 
9.1 Study Design 
Approximately 64 patients with resected, newly-diagnosed WHO Grade IV glioma who are CMV+ 
and in which the MGMT is not methylated will be accrued to this study before standard of care 
radiation therapy (RT) and concurrent TMZ, with the goal of treating 48 patients with dose-
intensified temozolomide and pp65 loaded dendritic cell vaccine after completion of standard RT 
and TMZ. 
 
All enrolled patients will undergo a leukapheresis after resection for harvest of peripheral blood 
lymphocytes (PBLs) for generation of DCs. Patients will then receive approximately 6 weeks of 
standard of care radiation therapy (RT) and concurrent TMZ at a standard targeted dose of 75 
mg/m2/d (please see Package Insert uploaded in electronic IRB for standard therapy). Patients 
should start RT within approximately 7 weeks of surgery.  [PI discretion can be used in this 
situation to allow patients outside of this window to enroll.] Patients who either experience 
progressive disease during radiation, are dependent on steroid supplements above 2 mg/day at time 
of first vaccination, are unable to tolerate TMZ, or whose DCs fail to meet release criteria will be 
withdrawn from the study and replaced. For patients whose initial leukapheresis yields less than 3 
vaccines, repeat leukapheresis may be obtained a minimum of 2 weeks from the previous 
leukapheresis (and may be repeated as needed) if pre-apheresis blood work is within the Apheresis 
Center’s parameters and as long as this does not cause a significant delay in treatment for the 
patient. At the post-RT clinic visit the patient’s MRI and blood work is evaluated before starting a 
single post-RT cycle of dose intensified TMZ (100 mg/m2/day for 21 days). On day 23 (± 2 days) 
of the cycle, patients will receive the first of 3 pp65 DC vaccines. DC vaccines #1-3 will be given 
every two weeks (± 2 days). All patients will receive up to a total of 10 DC vaccines, with vaccines 
administered every 35 days (± 7 days) after the third vaccine, given bilaterally at the groin site 
unless progression occurs with no further cycles of TMZ. DC vaccines will be given intradermally 
(i.d.) and divided equally to both inguinal regions. Before the first DC vaccination, patients will 
receive 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly into the deltoid 
muscle to ensure adequate immunity to the tetanus antigen. The Td vaccine will be obtained 
through Duke Investigational Chemotherapy Services (ICS) Pharmacy. Patients will undergo 
leukapheresis again for immunologic monitoring with specific assessment of baseline antigen-
specific cellular and humoral immune responses if needed for further DC generations 14 (± 4) days 
after vaccine #3. Prior to pp65 DC vaccination #4, (3±1) weeks after leukapheresis 2 the vaccine 
site will receive a pre-conditioning intradermal injection of Td (1 flocculation unit (Lf), in 0.3 mL 
of saline for a total of 0.4 mL).   
 
As part of standard care for these patients, upon tumor progression, participants may undergo 
stereotactic biopsy or resection. As this is not a research procedure consent will be obtained 
separately. However, if tissue is obtained, it will be used to confirm tumor progression 
histologically and to assess immunologic cell infiltration and pp65 antigen escape at the tumor 
site.  No other treatment intervention is allowable while on study, unless the patient needs 
bevacizumab at 7.5 mg/kg IV approximately every 3 weeks per the “Special Considerations” in 
Section 9.1.3. 
 
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Toxicities will be graded according to the National Cancer Institute Common Toxicity Criteria of 
Adverse Events (NCI CTCAE) version 5 criteria. An unacceptable toxicity is defined as any Grade 
3 or greater toxicity that is possibly, probably, or definitely attributed to the pre-conditioning agent 
Td or pp65 DC vaccine that does not resolve to baseline in 2-3 weeks; any Grade 3 hypersensitivity 
reactions or autoimmune toxicity requiring steroids or hormone replacement; and is not due to 
progressive disease, or any life–threatening event not attributable to concomitant medication, co-
morbid event, or disease progression. Rules for monitoring unacceptable toxicity are provided in 
Section 15.6.5. 
 
9.1.2 Dose Modification 
TMZ will be administered concomitantly with standard external beam RT under the direction of 
the oncologist.  Please see TMZ package insert uploaded in electronic IRB for standard of care 
treatment guidelines.  The dose of the concomitant TMZ with RT may be adjusted at the discretion 
of the treating oncologist. 
 
The study drug (CMV pp65 RNA-pulsed DCs with GM-CSF) dose will not be modified in this 
trial.  Those subjects whose cells fail to meet release criteria will be removed and replaced. 
 
9.1.3 Safety Considerations 
Management of Toxicities 
If a Grade 3 NCI CTC or greater toxicity is seen that is not attributable to a concomitant 
medication, co-morbid event, or disease progression that has been documented radiographically 
or clinically, the next immunization for that patient will be withheld for up to 2 months or until the 
NCI CTC toxicity improves to a Grade 1 or less. However, planned procedures requiring 
hospitalization, or long-term clinical decline that is now seen in patients years from whole brain 
radiation therapy (WBRT), which are clearly not related to study drug, but are the natural 
development common in this patient population, will not be considered an unacceptable toxicity 
nor will have immunizations withheld. 
 
Special Consideration 
Due to the impact of high dose steroids on the development of an optimal immune response, if a 
patient demonstrates neurologic or cerebral radiographic signs suggestive of a localized 
inflammatory reaction, secondary to the immune response triggered by DC vaccination, that 
requires an increase in dexamethasone dose, every effort should be made to not increase the dose 
above 4 mg per day at any time. Instead, patients should be treated with bevacizumab at the 
reduced dose of 7.5 mg/kg IV approximately every 3 weeks.  If a patient requires planned treatment 
for their tumor with bevacizumab > 7.5 mg/kg approximately every 3 weeks, they will be 
considered off study and enter the follow-up phase. Neuroimaging (MRI) will be performed 
according to protocol schedule and, at that time, it will be assessed whether further treatment with 
bevacizumab is needed to control the cerebral inflammation. Bevacizumab will not be provided 
by the study. Every attempt should be made to reduce or discontinue dexamethasone, when 
clinically possible, so as not to mitigate immune response. 
 
If there are AEs or other circumstances prohibiting the use of bevacizumab, corticosteroids, 
surgery, or other interventions deemed more appropriate for the patient by the treating physician 
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Adverse Event Reporting and Documentation 
An “Adverse Event” will be defined as any adverse change from the subject’s pre-treatment 
baseline condition (which is assessed at the post-RT/TMZ clinic visit), including any clinical or 
laboratory test abnormality that occurs during the course of research. Adverse events will be 
categorized and graded in accordance with the NCI CTCAE (Version 5). 
 
A “Serious Adverse Event” will be defined as an undesirable sign, symptom or medical condition 
which: 1) is fatal or life threatening; 2) requires inpatient hospitalization for > 24 hours or a 
prolongation of existing hospitalization; 3) results in persistent or significant disability/incapacity; 
4) constitutes a congenital anomaly or a birth defect and/or; 5) medically significant such that it 
may jeopardize the subject, and may require medical or surgical intervention to prevent one of the 
outcomes listed above. 
 
A summary of all adverse events (not just those considered related to study drug) that occur 
following RT/TMZ will be kept which will categorize the event by organ system, relationship to 
treatment, its grade of severity, and resolution. Periodic review by the primary investigator (PI) 
and weekly review at the Duke Preston Robert Tisch Brain Tumor Center (PRTBTC) Adverse 
Event (AE) meeting of the collective adverse events will occur 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. 
 
All adverse events which are serious and unexpected should be reported immediately to Dr. 
Mustafa Khasraw M.D. at 919-684-5301 during regular business hours or at 919-2060493 after 
hours and to the Federal 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 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 the 
research (as defined by 21CRF312.32[a]) will be reported to the Duke University Medical Center 
Institutional Review Board (IRB) using the appropriate serious adverse event (SAE) report form. 
At the time of the annual progress report to the Duke University Medical Center IRB, a summary 
of the overall toxicity experience will be provided. 
 
9.1.4 Missed Doses 
To ensure that repetitive DC vaccines will be given to patients, the initial 3 will be given every 2 
weeks (± 2 days), and the monthly DCs will be given every 35 days (± 7 days).  Therefore, with a 
margin of 2 days all patients will receive up to 10 DC vaccines or until progression, whichever 
comes first. At the discretion of the study PI, patients who miss vaccine administration for 
whatever reason will have their appointment re-scheduled to administer the vaccine as soon as 
possible and the study PI will assess whether this deviation needs to be filed with the IRB. For 
continued non-compliance with the scheduled vaccine appointments, the subject may be removed 
from the trial at the discretion of the study PI. 
 
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Concomitant medications will be managed by the treating oncologist and recorded at each study 
visit by the study coordinator. 
9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
 
In our previous randomized clinical trial of 12 patients with newly-diagnosed GBM, we evaluated 
the impact of vaccine site pre-conditioning with Td toxoid [118]. Patients randomized to Td 
showed increased dendritic cell migration bilaterally and significantly improved progression-free 
and overall survival. Furthermore, we observed a modest association between effective DC 
migration to VDLNs and clinical outcomes. Based on the small sample size of the initial study, we 
would like to conduct a validation study with Td pre-conditioning that is powered sufficiently to 
evaluate the impact of DC migration on clinical outcomes.  
9.3 Rationale for Correlative Studies 
Please see Section 12.7.4 111Indium-labeling of Cells for in vivo Trafficking Studies.  PLEASE 
NOTE:  With approval of Protocol v.20220204, this correlative study will no longer be conducted. 
9.4 Definition of Evaluable Subjects, On Study, and End of Study 
Subjects evaluable for the analysis of the primary endpoint of overall survival will include all 
patients who initiate DI-TMZ.  
 
Once the patient signs an informed consent form (ICF), that subject will be considered “on study.” 
Rationale for taking patient off protocol treatment will be documented. 
9.5 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 12.6, which describes procedures and process for 
prematurely withdrawn patients. 
10 STUDY DRUG 
10.1 Names, Classification, and Mechanism of Action 
 
Tetanus-Diphtheria Toxoid (Td Toxoid adsorbed) 
 
Td is indicated for active booster immunization against tetanus, diphtheria, and pertussis as a single 
dose; substitute 1-time dose of Tdap for Td booster, then standardly boost with Td every 10 years. 
Please refer to Section 9.1 on the use of Td in this protocol. 
 
DC Vaccine 
 
Human CMV pp65-LAMP mRNA-pulsed autologous DCs with GM-CSF is the name of the study 
drug given with every vaccine. This vaccination will then consist of administration of 2 x 107 CMV 
pp65-LAMP mRNA loaded mature DCs as described above.  In up to 16 patients, the fourth 
vaccine will be labeled with 111In (50 μCi / 5 x 107 DCs) (Nycomed-Amersham, Chicago, IL) prior 
to injection.  The class of action for all study drugs is a biological with the exception of the tetanus 
toxoid, which is an antitoxin. 


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GM-CSF (LEUKINE®; Sargramostim) 
 
GM-CSF will be obtained from commercial supply and stored in the ICS, as a sterile, white, 
preservative-free powder lyophilized powder in a vial containing 250 mcg to be reconstituted in 
0.5 mL of sterile water for injection.  
 
Leukapheresis and Dendritic Cell Vaccine Generation  
 
At least two leukaphereses may be performed on each patient enrolled on this protocol. The 
leukapheresis will be used for DC generation and immunologic monitoring. If the subjects were 
part of the companion study (Pro00102797) and have already undergone the leukapheresis 
procedure the DCs from that study will be used for the current study and any unused vaccine 
products will be handled as indicated by the patient in the companion study. All leukaphereses 
will be approximately a 4-hour leukapheresis. It is estimated that 10-12 L of blood will be 
processed during this leukapheresis. An additional 4-hour leukapheresis will be requested from 
patients with positive immunological responses to the therapy for additional immunologic 
monitoring studies or for patients who need additional DCs generated to ensure a total of 10 
vaccines. 
 
DCs will be generated from the first leukapheresis in vitro by 7-day culture with GM-CSF and IL-
4. Peripheral blood mononuclear cells (PBMC) 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 DC, a sample of the media is taken for 
mycoplasma testing, the cells are then harvested and electroporated with pp65-LAMP mRNA. The 
DCs are placed in a flask with AIM V media 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 2-4 x 
107cells/mL in 90% autologous human AB serum (Valley Biomedical, Winchester, VA 22602), 
10% DMSO and 5% dextrose.  
 
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 (1 x 106 DCs) and fungal cultures (1 x 106 DCs). 
 
For each vaccination, cells that have passed quality assurance/quality control (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 5 x 107 cells/mL and DCs will be resuspended in 
preservative free saline and GM-CSF and placed into a sterile tuberculin syringe with a 25-gauge 
needle. 
 
For all DC preparations, and 111In-labeled DCs, 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 
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to be negative. An aliquot of cells will also be sent for aerobic and anaerobic bacterial cultures (1 
x 106 DCs) and fungal cultures (1 x 106 DCs). 
 
The SOP MPACT-QA-0017 Responding to Sterility Test Results describes the roles, 
responsibilities, and actions that should be taken in the event of a positive, delayed, incomplete, or 
invalid sterility test result for a product manufactured in the MPACT facility after it has been 
administered to a study subject.  Briefly, personnel informed of the result will notify MPACT 
Management who is responsible for notifying all relevant study personnel.  The Principal 
Investigator or treating Neuro-Oncologist (or his or her designee) will notify  the patient. The 
patient will be asked to be evaluated by a physician within 48 business hours. Based on the route 
of administration of the investigational product, appropriate cultures (aerobic, anaerobic, and 
fungal) will be ordered for the subject (blood cultures or wound cultures) along with CBC and 
CMP tests.  If the patient has or develops a temperature ≥38.5°C or clinical evidence of infection, 
the patient will be treated expectantly with antibiotics based on the sensitivities of the organisms 
identified from the immunization product, as medically necessary. An independent infectious 
disease consultation will be obtained to guide further therapy if medically necessary. MPACT 
management and MPACT QA will perform an investigation into the sterility tst result.  If there are 
remaining vials from the affected batch, a vial will be sent for repeat sterility testing, and the 
remainder of the batch will be quarantined until re-testing is complete with negative results.  
Administration of subsequent products from the affected batch will be held, and upcoming study 
visits should be rescheduled as necessary. Immunizations will proceed only if the patient fully 
recovers and subsequent samples are found to be sterile. 
10.2 Packaging and Labeling of Study Agents 
 
• For CMV pp65-LAMP mRNA-pulsed DCs and CMV pp65-LAMP mRNA loaded mature 
DCs labeled with 111In (50 μCi / 5 x 107 DCs): 
Vaccine Label 
Patient Name / MRN 
DOB / ALPS # 
Trial Name and Number 
Date 
FOR AUTOLOGOUS USE ONLY 
NOT EVALULATED FOR INFECTIOUS SUBSTANCES 
Caution: New Drug Limited by Federal Law to Investigational Use 
• For tetanus diphtheria toxoid used in pre-conditioning 
Name 
MRN 
DOB  
Drug: Td  
• Td™ (Tetanus diphtheria toxoid adsorbed); Stored at 4oC used as Td booster vaccine 
Drug: Td 
Lot #  
Expiration Date 
10.3 Supply, Receipt, and Storage 
The DCs will be stored in a locked liquid nitrogen freezer in the Molecular Products and Cellular 
Therapies (MPACT) current Good Manufacturing Practice (cGMP) facility. The Nautilus 


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Laboratory Information Management System (LIMS) database will track receipt and storage 
location. 
 
Table 2. Storage of Study Agents 
MPACT Facility 
Duke ICS Pharmacy 
Duke Radiopharmacy 
CMV 
pp65-LAMP 
mRNA-pulsed DCs 
Td used for booster and for 
pre-conditioning 
CMV 
pp65-LAMP 
mRNA loaded mature 
DCs labeled with 111In 
 
10.4 Dispensing and Preparation 
The pp65 DC vaccines will be delivered from the MPACT facility directly to the clinic under the 
supervision of the trained research staff. Each labeled patient vaccine will be transported to the 
clinic in a separate cooler.  DCs will be administered according to protocol. The patient’s name, 
Study identification (ID), date of birth (DOB), and Duke history number will be double verified 
prior to DC administration as is standard Duke transfusion procedure.  
 
The radiolabeled DC products used with the migration studies will be prepared from the pp65 DC 
vaccines sent from the MPACT facility to the Radiopharmacy and dispensed radiolabeled from 
the Duke Radiopharmacy.  
 
Table 3. Dispensing of Study Agents 
MPACT Facility 
Duke ICS Pharmacy 
Duke Radiopharmacy 
CMV 
pp65-LAMP 
mRNA-pulsed DCs 
Td used for booster and for 
pre-conditioning  
CMV 
pp65-LAMP 
mRNA loaded mature 
DCs labeled with 111In 
 
10.5 Compliance and Accountability 
All DC vaccines will be stored in the Molecular Products and Cellular Therapies (MPACT) cGMP 
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 the 
MPACT personnel. The MPACT 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 MPACT protocols that are approved by the Duke 
University Health System (DUHS) 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. 
10.6 Disposal and Destruction 
Radioactively labeled drug will be disposed of according to standard Duke radiation safety 
practices. Un-labeled drug will be autoclaved. 
11 SUBJECT ELIGIBILITY 
 
Inclusion Criteria  


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• Age ≥18 years of age. 
• Newly diagnosed World Health Organization (WHO) Grade IV Glioma with definitive 
resection prior to consent, with residual radiographic contrast enhancing disease on the 
post-operative computed tomography (CT) or Magnetic Resonance Imaging (MRI) of <1 
cm in maximal diameter in any plane.  
• Able to receive standard of care radiation and chemotherapy for approximately 6 weeks 
duration and of more than 54GY 
• MRI post RT does not show progressive disease outside the radiation field  
• Enough tumor tissue available for determination of MGMT gene promoter status (must be 
unmethylated) or prior pathology report available confirming MGMT gene promoter 
status. 
• CMV Seropositive. 
• KPS of ≥ 70%. 
• Hemoglobin ≥ 9.0 g/dl, absolute neutrophil count (ANC) ≥ 1,000 cells/µl, platelets ≥ 
100,000 cells/µl prior to starting TMZ cycle 1 (patient must meet these criteria within 4 
weeks after the end of XRT/TMZ to be eligible). 
• Serum creatinine ≤ 3 times institutional upper limit of normal for age, serum aspartate 
aminotransferase (AST) ≤ 3 times institutional upper limit of normal for age and  
• Bilirubin ≤ 1.5 times upper limit of normal prior to starting TMZ cycle 1 (Exception: 
Patient has known Gilbert’s Syndrome or patient has suspected Gilbert’s Syndrome, for 
which additional lab testing of direct and/or indirect bilirubin supports this diagnosis.  In 
these instances, a total bilirubin of ≤ 3.0 x ULN is acceptable.). 
• Signed informed consent approved by the Institutional Review Board.  
• Female patients must not be pregnant or breast-feeding. Female patients of childbearing 
potential (defined as < 2 years after last menstruation or not surgically sterile) must use a 
highly effective contraceptive method (allowed methods of birth control, [i.e. with a failure 
rate of < 1% per year] are implants, injectables, combined oral contraceptives, intra-uterine 
device [IUD; only hormonal], sexual abstinence or vasectomized partner) during the trial 
and for a period of > 6 months following the last administration of trial drug(s). Female 
patients with an intact uterus (unless amenorrhea for the last 24 months) must have a 
negative serum pregnancy test within 48 hours prior to first study procedure 
(leukapheresis). 
• Fertile male patients must agree to use a highly effective contraceptive method (allowed 
methods of birth control [i.e. with a failure rate of < 1% per year] include a female partner 
using implants, injectables, combined oral contraceptives, IUDs [only hormonal], sexual 
abstinence or prior vasectomy) during the trial and for a period of > 6 months following 
the last administration of trial drugs. 
 
Exclusion Criteria: 
• Pregnant or breast-feeding. 
• Women of childbearing potential and men who are sexually active and not willing/able to 
use medically acceptable forms of contraception. 
• Patients with known potentially anaphylactic allergic reactions to gadolinium- 
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• Patients who cannot undergo MRI due to obesity or to having certain metal in their bodies 
(specifically non-MRI compatible pacemakers, infusion pumps, metal aneurysm clips, 
metal prostheses, joints, rods, or plates). 
• Patients with evidence of tumor in the brainstem, cerebellum, or spinal cord, radiological 
evidence of multifocal disease, or leptomeningeal disease. 
• Severe, active comorbidity, including any of the following: 
- Unstable angina and/or congestive heart failure requiring hospitalization; 
- Transmural myocardial infarction within the last 6 months; 
- Acute bacterial or fungal infection requiring intravenous antibiotics at the time of 
study initiation; 
- Chronic obstructive pulmonary disease exacerbation or other respiratory illness 
requiring hospitalization or precluding study therapy; 
- Known hepatic insufficiency resulting in clinical jaundice and/or coagulation 
defects; 
- Known Human Immunodeficiency Virus (HIV) and Hepatitis C positive status; 
- Major medical illnesses or psychiatric impairments that, in the investigator's 
opinion, will prevent administration or completion of protocol therapy; 
- Active connective tissue disorders, such as lupus or scleroderma that, in the opinion 
of the treating physician, may put the patient at high risk for radiation toxicity. 
• Co-medication that may interfere with study results; e.g. immuno-suppressive agents other 
than corticosteroids. 
• Prior, unrelated malignancy requiring current active treatment with the exception of 
cervical carcinoma in situ and adequately treated basal cell or squamous cell carcinoma of 
the skin. (Treatment with tamoxifen or aromatase inhibitors or other hormonal therapy that 
may be indicated in prevention of prior cancer disease recurrence, are not considered 
current active treatment.) 
• Patients are not permitted to have had any other conventional therapeutic intervention other 
than steroids prior to enrollment outside of standard of care chemotherapy and radiation 
therapy.  Patients who receive previous inguinal lymph node dissection, radiosurgery, 
brachytherapy, or radiolabeled monoclonal antibodies will be excluded. 
• Current, recent (within 4 weeks of the administration of this study agent), or planned 
participation in an experimental anti-cancer drug study. 
• Known history of autoimmune disease (with the exceptions of medically-controlled 
hypothyroidism and Type I Diabetes Mellitus). 
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
12.1 Screening Examination 
The screening examination will take place at the Duke PRTBTC clinic visit. An informed consent 
must be signed by the patient before any screening procedure takes place. Tumor MGMT promotor 
methylation status will be reviewed and only patients with non-methylated tumors will be enrolled.  
MGMT gene promoter methylation status is obtained at Duke using validated testing from 
LabCorp by PCR and/or Caris by pyrosequencing. If MGMT gene promoter methylation status 
has already been conducted outside of Duke, the results will be used as long as the testing was 
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Patients whose methylation status is inconclusive will not be eligible to participate in the study. 
The baseline physical and neurologic examination with KPS score along with standard of care 
blood work and CMV immune screen will be performed and documented by the neuro-oncology 
team and verified by the study team during this PRTBTC clinic visit. All subject data is standard 
of care evaluation that occurs for all patients being seen in the PRTBTC. If the subject is considered 
a screen failure prior to vaccine treatment, the source documents for electronic data entry will be 
obtained from the Duke electronic medical record. 
 
Initial clinical evaluations will also include a baseline and study eligibility MRI (with and without 
contrast) of the brain per standard of care for comparison to subsequent MRI images.  Mini-mental 
status examination (MMSE) will be performed at the post-RT/TMZ visit. 
 
The patient must have had a definitive resection.  Residual radiographic contrast enhancement on 
post-resection CT or MRI must not exceed 1 cm in diameter in any planes at time of consent.  
Patients with progressive disease after radiation will not be a candidate for the vaccine despite 
being previously consented and will be removed from the study and replaced (please see Section 
12.7.4 for radiologic evaluations).  
 
During the initial intake process within the Preston Robert Tisch Brain Tumor Center, all patients’ 
tissue is tested for diagnosis and IDH-1/2 mutation analysis through either the Duke Pathology 
Department or CARIS.  The study will collect this information in order to ensure baseline 
molecular diagnostics of the tumor for each patient is known.   
 
At the discretion of the study investigator, for surgical blocks containing initial resection and 
biopsy at recurrence (if applicable), formalin-fixed paraffin-embedded slides may be requested for 
immunohistochemistry assays for the detection of CMV. These slides for CMV detection will not 
exceed 10% of the original specimen block, so as to preserve adequate tissue should patients prefer 
future diagnostic studies. Patient consent will be obtained for obtaining these slides in either 
scenario if a surgery/biopsy was performed at Duke University or at an external institution.  
 
If resection is done outside of Duke University Hospital, formalin-fixed paraffin-embedded slides 
sectioned from outside blocks, not to exceed 10% of surgical specimen block tissue, prepared on 
Fischer Plus glass or Histostix coated slides when available will be mailed to: 
 
Mustafa Khasraw M.D.,  
Duke Brain Tumor Immunotherapy Program 
Department of Neurosurgery  
The Preston Robert Tisch Brain Tumor Center at Duke 
DUMC Box 3624 
047 Baker House, Trent Drive Duke University Medical Center 
Durham, North Carolina 27710, USA 
 
After patients have been consented, they will be entered into the electronic Research system.  
12.2 Treatment Period 
All enrolled patients will then undergo a leukapheresis for generation of DCs and for baseline 
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have already undergone the leukapheresis procedure the relevant DCs from that study will be used 
for the current study and any unused vaccine products will be handled as indicated by the patient 
in the companion study. Within 48 hours of leukapheresis, patients will have blood samples taken 
for the following tests as required by the Duke Apheresis Center: CBC with auto differential, CMP, 
ionized Calcium, and Beta-Human Chorionic Gonadotropin (β-HCG) (for females of child-bearing 
potential). Total estimated blood volume required for these evaluations is 12-15 mL. For patients 
without sufficient venous access for leukapheresis, a temporary central intravenous catheter may 
be inserted. To prevent the development of hypocalcemia from the citrate used for leukapheresis, 
all patients will be instructed to take oral Tums, 2 tablets three times a day and at bedtime the day 
before and the day of the leukapheresis procedure. Patients who have lower levels of calcium will 
be treated per Apheresis lab standard protocols under the direction of apheresis attending 
physician. This first leukapheresis will be approximately a 4-hour leukapheresis, and it is estimated 
that 10-12 L of blood will be processed during this leukapheresis. One red top tube 10 mL draw 
tube will be obtained prior to leukapheresis #1 for immune monitoring.  Nine yellow ACD 8.5 mL 
tubes and 2 red top 10 mL draw tubes will be obtained prior to leukapheresis #2. 
 


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Table 4. Schedule of Events 
 
CMV Screening 
Main Consent11 
 
Leukapheresis #17 
RT/TMZ  
Post-RT DI TMZ 
Cycle 1 
Td Booster 
& 
Vaccine #1 
Vaccine #2 
Vaccine #3 
Leukapheresis #27 
Td Pre-
conditioning8 
Vaccine #4 
 
  
Vaccines #5-10 
Progression9 
Time and 
Range 
 
 
 
Within 7 
weeks of 
resection 
Within 4 (-2) 
weeks of RT 
Day 23 
 (+/-2 days) Td 
booster given 
before vaccine #1 
2 weeks 
post 
Vaccine #1 
 (+/-2 days) 
2 weeks post 
Vaccine #2 
(+/-2 days) 
14 (+/-4) 
days after 
vaccine #3 
3 (+/-1) week 
after 
leukapheresis 
#2, 12-24 
hours prior to 
vaccine #4 
3 (+/-1) week 
after 
leukapheresis 
#2 
 
Every 35 
(+/-7) days 
after 
previous 
vaccine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CMV 
Screening 
Consent  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Main Consent10 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
Medical History/ 
Baseline 
Symptoms  
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor 
Pathology2 
X1 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Physical Exam, 
Neurological 
Exam, KPS, 
Vitals3 
X1 
 
 
 
X 
X 
X 
X 
X 
X 
 
 
X 
 
MMSE3 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
MRI4 
X1 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
CMV IgG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC (with auto 
differential)5 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
CMP5 
 
 
X 
 
X 
X 
X 
X 
X 
X 
 
 
X 
 
Beta HCG 
quantitative 
(WOCBP only) 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
Ionized 
Calcium 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
Immune 
Monitoring 
Blood6 
 
 
X 
(1R) 
 
X 
(9Y, 2R) 
X 
(9Y, 2R) 
 
X 
(9Y, 2R) 
X 
(9Y, 2R) 
X 
(2R) 
X 
(9Y, 2R) 
 
X 
(9Y, 2R) 
X 
(9Y, 
2R) 
SOC 
XRT/TMZ 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Dendritic Cell 
Vaccine 
 
 
 
 
 
X 
X 
X 
 
 
X 
 
X 
 


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Con meds and 
AEs 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
 
1Can be performed prior to any ICF being signed as part of standard of care evaluation. 
2Patients must have a WHO Grade IV Glioma with definitive resection prior to enrollment, with residual radiographic contrast enhancement on most recent CT or MRI of <1 cm in 
maximal diameter in any plane.  Methylation status must be checked per SOC practices (via LabCorp, CARIS, etc.) and patient must be unmethylated in order to be eligible to 
proceed with signing consent to undergo leukapheresis. 
3Clinical evaluations with each vaccine will include a general physical examination, complete neurologic examination, KPS rating, and vitals (prior to vaccine and 30 mins post 
vaccine).  MMSE will be performed at the Post-RT/TMZ visit only.  
4A baseline and study eligibility MRI (with and without gadolinium enhancement) of the brain per standard of care for comparison to subsequent MRI images will also be obtained. 
MRI or contrasted CT, every 8 ± 4 weeks per PRTBTC standard of care (may be adjusted by the treating neuro-oncologist). 
5CBC (with auto differential) and CMP to confirm eligibility, as per standard of care, and as per this schedule of events. Whenever blood for immune monitoring is obtained CBC 
with auto differential and CMP will be done to assess absolute number of TRegs. 
6Blood work for immunologic monitoring will be drawn before leukapheresis #1 (1 red top 10mL tube prior to leukapheresis), on the day of Post-RT/TMZ visit (9 yellow ACD 
8.5mL tubes and 2 red top 10mL tubes), on the day of but prior to vaccine #1 and vaccine #3 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tubes), on the day of Td preconditioning 
but before the administration of Td (2 red top 10mL tubes), at leukapheresis #2 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tubes) when leukapheresis is performed 14 (+/-4) 
days post vaccine #3, at the time of vaccine 4 (9 yellow ACD 8.5mL tubes and 2 red top 10mL tubes), and then at vaccines 5 through 10 (9 yellow ACD 8.5mL tubes and 2 red top 
10mL tubes) and/or at progression (whichever comes first). If an additional leukapheresis must occur earlier than 14 (± 4) days after vaccine 3, then no immune monitoring will be 
drawn.  All patients will undergo at least 2 leukapheresis procedures – prior to radiation (1 red top 10mL tube) and approximately 2 weeks post vaccine #3 (9 yellow ACD 8.5mL 
tubes, 2 red top 10mL tubes)  See footnote 7.  For subjects who complete all 10 study vaccines without progressing, an attempt will be made to obtain blood for immunologic 
monitoring 2-3 times a year at standard Duke Preston Robert Tisch Brain Tumor Center visits and at progression (whichever comes first).  
7Leukapheresis #1 will be scheduled following consent.  Per standard Duke Apheresis Protocol, all subjects will have CBC with auto differential, CMP, ionized Calcium, and βHCG 
(WOCBP only) within 48 hours prior to the procedure(s). Leukapheresis #2 will occur approximately 14 (± 4) days after third immunization to obtain PBMCs for Immunologic 
Monitoring and generation of additional DCs for continued vaccinations.  Additional leukapheresis can be obtained up to every 2 weeks if needed according to the Duke Apheresis 
Center protocol, but these will likely be needed less than every 2 months throughout the study to generate enough DCs to continue vaccinations. No immune monitoring blood will 
be drawn at the additional leukapheresis procedures; only at leukapheresis #1 prior to radiation and at leukapheresis #2 approximately 2 weeks post vaccine #3.  Additional 
leukapheresis following leukapheresis #1 will be represented as: Leukapheresis #1a, Leukapheresis #1b, etc.  Additional leukapheresis following Leukapheresis #2 will be represented 
as: Leukapheresis #2a, Leukapheresis #2b, etc.  
8On the day before the fourth DC vaccine, patients will receive vaccine site pre-conditioning strategy. A single dose of Td toxoid (1 flocculation unit, Lf) will be administered to a 
single side of the groin 12-24 hours prior to the fourth DC vaccine, which is always given bilaterally at the groin site. Standard of care blood for CBC (with auto differential) and 
CMP will be drawn during the pre-conditioning visit. 2 red top 10 mL draw tubes for immune monitoring will be obtained on the same day of the pre-conditioning visit but prior to 
patients receiving Td pre-conditioning. 
9As part of standard care for these patients, upon tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research procedure consent will be 
obtained separately. However, if tissue is obtained, it will be used to confirm tumor progression histologically and to assess immunologic cell infiltration and pp65 antigen escape at 
the tumor site. 
10When CMV and/or methylation status is unknown, patients may sign CMV and/or leukapheresis consent for the Umbrella study (Pro00102797).  This allows patients to undergo 
CMV screening and/or leukapheresis prior to signing main consent for this study.  If patients sign the leukapheresis consent for the Umbrella study prior to leukapheresis, they will 
sign main consent for this study at the Post-RT/TMZ visit. 


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12.3 End of Treatment 
Vaccines are given as described above for a total of 10 or until progression (whichever comes 
first).  Once the vaccinations are complete, the treatment phase of the study will be over and the 
follow-up period will begin. 
12.4 Follow-up Period 
Patients will be followed for survival, progression, and subsequent therapies only, and this data 
will be recorded by the research team.  For recording of subsequent therapies, the type of 
therapy(ies) that the subject receives will be recorded with approximate start and stop date, if 
possible. For subjects who complete all 10 study vaccines without progressing, an attempt will be 
made to obtain blood for immunologic monitoring 2-3 times a year at standard Duke Preston 
Robert Tisch Brain Tumor Center visits.  If the subject progresses in the follow-up period, blood 
for immunological monitoring will be drawn at the time of progression, if possible.     
12.5 End of Study 
Rationale for taking a patient off study will be documented (see Section 12.6.1). In the Follow-up 
Period, patients will be followed for survival, progression, and subsequent therapies only, and this 
data will be recorded by the research team. 
12.6 Early Withdrawal of Subject(s) 
 
12.6.1 Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time. Subjects may be withdrawn by the 
PI and considered screen failures prior to vaccine therapy and 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 are not limited to the following: 
• 
Inability to complete approximately >4 weeks of RT with TMZ as per standard of care. 
• 
Inability to tolerate TMZ  
• 
Patients with an active infection requiring treatment or having an unexplained febrile 
illness (Tmax > 99.5° F). 
• 
Patients requiring an increase in corticosteroids, with the exception of nasal or inhaled 
steroid, such that at the time of first vaccination they require a dose above 2 mg of 
dexamethasone / day. Once vaccinations have been initiated, if patients subsequently 
require increased steroids, they will still be permitted to remain on the study, but every 
effort will be made to minimize steroid requirements. 
• 
Adverse events 
• 
Abnormal laboratory values 
• 
Abnormal test procedure results (DCs fail to meet release criteria; inability to tolerate 
leukapheresis) 
• 
Protocol deviation 
• 
Administrative issues 
• 
Disease progression 
• 
Pregnancy 
 
12.6.2 Follow-up Requirements for Early Withdrawal 


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Consented subjects that are withdrawn prior to cycle 1 of post-RT DI TMZ for any of the 
aforementioned reasons will be considered eligibility failures and thus will not be followed for 
survival.  All other subjects will be followed by the study coordinator until death or are lost to 
follow-up. 
 
12.6.3 Replacement of Early Withdrawal(s) 
As noted in Section 12.6.2, subjects who voluntarily withdraw prior to cycle 1 of post-RT DI TMZ 
will not be followed for survival, and will be replaced. 
12.7 Study Assessments 
 
12.7.1 Medical History 
Medical history will be obtained from the Duke electronic system and from the subject and/or 
family at the screening visit and reviewed at each study visit. This data may include the following: 
- 
All past medical and surgical history; 
- 
Current medications; 
- 
Changes in physical or neurologic symptoms; 
- 
Any adverse events. 
 
12.7.2 Physical Exam 
Vital signs and physical and neurologic examinations will be assessed and recorded along with a 
KPS score prior to enrollment and at each visit.   
 
12.7.3 Use of Antihistamines 
Subjects will be advised to avoid antihistamine use 48 hours prior to each vaccine administration, 
the day of vaccine administration, and for 48 hours following each vaccine administration.  If the 
subject has a pre-existing condition that requires antihistamine usage, the PI and the treating 
oncologist will decide if it is safe and appropriate for the subject’s antihistamines to be held before 
and following vaccine administrations. 
 
12.7.4 Radiologic Evaluations 
Patients with newly diagnosed GBM will be imaged by MRI as per standard of care for eligibility 
and baseline measurements, and to assess progression prior to vaccine therapy; although the 
purpose of this study is not to detect tumor responses, any evidence of tumor response will be 
determined according to the Duke PRTBTC standard operating procedure (SOP). Immunotherapy 
Response Assessment in Neuro-Oncology (iRANO) criteria [122, 123] established by consensus 
conference, will be used for assessment of response and pseudoprogression following 
immunotherapy.  If pseudoprogression is suspected, the subject will continue with vaccine therapy 
for a minimum of 2-3 subsequent months so long as subject remains clinically and radiographically 
stable compared to the MRI showing suspected pseudoprogression.  If the subject continues to do 
well beyond the 3 months, they will continue on study as planned. Subjects demonstrating 
definitive progression will be removed from study.  Tumor progression will need to be documented 
histologically, unless there are clinical contraindications, to exclude inflammatory responses 
presenting as radiographic or clinical changes, which could indicate potentially toxic or therapeutic 
responses and not tumor progression. If tissue is obtained through the Duke Brain Tumor 
Biorepository, it will be used to confirm tumor progression histologically and to assess 
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site. Upon progression, patients may be treated on other therapies as directed by the treating 
Oncologist.  
 
12.7.5 Immunologic Assessments 
Immunological response evaluations will be conducted as described in Table 4. All patients will 
undergo at least 2 leukapheresis procedures – prior to radiation (1 red top 10mL tube) and 
approximately 2 weeks post vaccine #3 (9 yellow ACD 8.5mL tubes, 2 red top 10mL tubes).   If 
an additional leukapheresis must occur earlier than 14 (± 4) days after vaccine 3, then no immune 
monitoring will be drawn at this additional leukapheresis. For subjects who complete all 10 study 
vaccines without progressing, an attempt will be made to obtain blood for immunologic monitoring 
2-3 times a year at standard Duke neuro-oncology visits and at progression (whichever comes 
first). The immune monitoring blood that is collected prior to the initiation of post RT-TMZ cycle 
1 (on the same day) will be the baseline for Treg analysis. The immune monitoring blood drawn at 
pre-vaccine #1 will be the baseline for polyfunctional T cell analysis. The blood drawn on the same 
day as (but prior to) Td preconditioning will be analyzed for the baseline CCL3.  A comparison of 
pre-therapy lymphocyte functions to those at intervals after each immunization will be made. 
These tests may provide evidence for the development of immune responses following DCs 
immunization and will play an important role in the design of future DC-based clinical trials.  
Cellular immune responses to pp65 will be measured by polyfunctional T cell assays using 10-12 
color panels previously optimized in our laboratory, which includes detection of CCL3 on T cells. 
The effect of treatment on cytokines and other soluble factors will be assayed in the serum/plasma. 
Serum/plasma will be measured to determine levels of CCL3 from the blood drawn at the Td 
preconditioning visit, and at the vaccine #4 visit. TReg kinetics will be measured by using a 
constellation of markers specific for TRegs such as foxp3, CD25 and CD4. Whenever blood for 
immune monitoring is obtained, we will submit a CBC with auto differential to assess absolute 
number of TRegs.  These will be drawn with SOC blood draws whenever possible. 
 
In addition, blood and tumor samples will be stored for possible future research in the Duke BTIP 
Laboratory where all other samples from this study are stored.  The samples being stored are 
ONLY for this study.  Our mouse studies being done in the BTIP Lab are identifying markers that 
may influence migration, therefore, as other markers are identified, we would like to investigate 
whether these same markers are identified in human subjects enrolled on this trial. 
 
Polyfunctional Flow Cytometry 
Peripheral blood will be drawn into 9 yellow (ACD) tubes containing acid citrate dextrose and 2 
red top tubes for serum.  PBMCs will be separated by density gradient centrifugation using 
Leukosep tubes.   
 
Polyfunctional T cell analysis will be done in Dr. Sanchez-Perez’s immunology laboratory Dr. 
Sanchez-Perez’s immunology laboratory conducts all the clinical immune monitoring for the Duke 
Brain Tumor Center. Polyfunctional T cell panels have been developed in our laboratory and 
optimized for detection of CCL3 in T cells. This process involves the rapid early detection and 
analysis of the production of Tumor Necrosis Factor-α (TNF-a), CCL3, IFN-γ, interleukin-2 (IL-
2), and CD107a (a marker of cell cytotoxicity) prior to cellular secretion following antigen-specific 
stimulation in vitro. The functional CD4 and CD8 immune response of the patients will be 
monitored using a 10-12-color assay. To detect an increase in polyfunctional T cells we will 
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samples from pre-vaccine 1, and second leukapheresis (or blood draw) occurring 14 ± (4) days 
after vaccine #3. 
 
Isolated PBMCs will be stimulated for 6 hours with the pool of 138 peptides spanning the entire 
pp65 gene. The cells will be stained for the surface markers CD3 (to identify it as a T cell), CD4 
(to define as specific helper cell), CD8 (to define antigen specific cytotoxic T cells) and then 
submitted for intracellular cytokine staining. This procedure allows for the highly sensitive 
detection of TNF-a, CCL3, IFN-γ, IL-2 and CD107a secreting cells, which are assumed to be 
specifically responding to the stimulating antigen.  
 
The maturation state and the activation status of samples will be detected by polychromatic flow 
cytometry using optimized panels which will include CD3, CD4, CD8, CCR7, CD45RA, HLA-
DR, CD69 and HLA-DR. Levels of naïve  (TN – CD45RA+CCR7+), central memory (TCM – 
CD45RA-CCR7+), effector memory (TEM – CD45RA-CCR7-), or terminally differentiated 
effector memory (TEMRA – CD45RA+CCR7+) T cells . 
  
Serology/Multiplex Platform 
 
CCL3 and levels of other inflammatory soluble factors may be assayed by multiplex platform. The 
effect of treatment on CCL3 Serum/plasma will be assayed (2 red top 10 mL tubes) to determine 
if levels of CCL3 are impacted by Td pre-conditioning and will be drawn prior to the Td pre-
conditioning as described above, and at the vaccine #4 visit (9 yellow ACD 8.5 mL tubes,  , 2 red 
top 10 mL tubes) 24 hours after Td pre-conditioning.  Antibody levels to the immunizing antigen 
may be measured. 
 
TReg Kinetics 
 
TRegs will be analyzed by flow cytometry using a constellation of markers specific for TRegs 
included CD4, CD25 and foxp3. For TReg analysis, the time points will be leukapheresis 1, before 
DI TMZ in the Post-RT TMZ, pre-vaccine 1, leukapheresis 2, pre-vaccine 4, pre-vaccine 5, & pre-
vaccine 6. We will analyze the percent of Foxp3, CD25, CD4 T cells over these time points. 
13 SAFETY MONITORING AND REPORTING 
13.1 Adverse Events 
An AE is any untoward medical occurrence in a subject receiving pre-conditioning or the study  
vaccine 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 the DCs, whether 
or not related to use of the DCs. Abnormal laboratory findings without clinical significance (based 
on the PI’s judgment) should not be recorded as AEs. But, laboratory value changes that require 
therapy or adjustment in prior therapy are considered adverse events. 
 


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Adverse events will be collected from the time of administration of the first DC vaccine through 
1 month after the subject’s last vaccine, all AEs must be recorded in the subject’s 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: 
- 
Definite:  The AE is clearly related to the study drug 
- 
Probably:  The AE is likely related to the study drug 
- 
Possible:  The AE may be related to the study drug 
- 
Unlikely:  The AE is doubtfully related to the study drug 
- 
Unrelated:  The AE is clearly NOT related to the study drug 
 
Attribution of AEs will be determined for each study intervention individually (pp65 DC vaccine, 
pre-conditioning agent [Td], TMZ, bevacizumab). 
 
13.1.1 Reporting of AEs 
A summary of all adverse events (not just those considered related to the study drug) will be kept 
which will categorize the event by organ system, relationship to which treatment, its grade of 
severity, and resolution. Periodic review by the PI and monthly review at the PRTBTC Adverse 
Event meeting of the collective adverse events will occur 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. 
 
13.2 Adverse Events of Special Interest 
Adverse events will not be collected until the patient has received the DC vaccine, 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 DC vaccine, all other events occurring 
prior to DC vaccine 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 
13.3 Serious Adverse Events 
 


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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. 
 
13.3.1 Reporting of SAEs 
All SAEs should be reported immediately to Dr. Mustafa Khasraw M.D. at 919-684-5301 during 
regular business hours or at (Pager: 919-206-0493) after hours and to the 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 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 
the research (as defined by 21CRF312.32[a]) will be reported to the Duke University Medical 
Center IRB and the FDA using the appropriate SAE reporting process. At the time of the annual 
progress report to the Duke University Medical Center IRB and the FDA, a summary of the 
overall toxicity experience will be provided.  Safety Oversight Committee (SOCOMM) 
 
The Duke Cancer Institute (DCI) SOCOMM is responsible for annual data and safety monitoring 
of DUHS sponsor-investigator phase I and II, 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 includes 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 14.1 for 
Monitoring Team description) oversees the conduct of DUHS cancer-related, sponsor-investigator 
therapeutic intervention and prevention 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, SOPs, Good Clinical Practice (GCP), and applicable regulatory 
requirements.  
13.4 External Data and Safety Monitoring Board  
The Principal Investigator and Sub-Investigators must comply with applicable federal, state, and 
local regulations regarding reporting and disclosure of conflict of interest. Conflicts of interest 
(COI) may arise from situations in which financial or other personal considerations have the 
potential to compromise or bias professional judgment and objectivity. Conflicts of interest include 


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but are not limited to royalty or consulting fees, speaking honoraria, advisory board appointments, 
publicly-traded or privately-held equities, stock options, intellectual property, and gifts. 
 
The Duke University School of Medicine’s Research Integrity Office (RIO) reviews and manages 
research-related conflicts of interest. The Principal Investigator and Sub-Investigators must report 
conflicts of interest annually and within 10 days of a change in status, and when applicable, must 
have a documented management plan that is developed in conjunction with the Duke RIO and 
approved by the IRB/Institutional Ethics Committee (IEC). 
 
Due to potential for COI in relation to proprietary interest in the pp65CMV DC vaccine, a Data 
Safety and Monitoring Board (DSMBplus) has been established.  The Duke PRTBTC DSMBplus 
Charter is available upon request. 
14 QUALITY CONTROL AND QUALITY ASSURANCE 
14.1 Monitoring 
The 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, 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 sponsor, the 
Principal Investigator, 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, National Cancer Institute, and the FDA. Every reasonable effort 
will be made to maintain confidentiality during study monitoring. 
 
14.2 Audits 
The Duke University Office of Audit, Risk and Compliance (OARC) may conduct audits to 
evaluate compliance with the protocol and the principles of GCP.  The PI agrees to allow the 
auditor(s) direct access to all relevant documents and to allocate his/her time and the time of the 
study team to the 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 I studies, or 
studies that involve Medicare populations. Directed audits occur at the directive of the IRB or an 
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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. 
14.3 Data Management and Processing 
 
14.3.1 Study Documentation 
Study documentation includes but is not limited to source documents, case report forms, 
monitoring logs, appointment schedules, study team correspondence with sponsors or regulatory 
bodies/committees, and regulatory documents that can be found in the DCI-mandated “Regulatory 
Binder”, which includes but is not limited to signed protocol and amendments, approved and 
signed informed consent forms, FDA Form 1572, College of American Pathologists (CAP) and 
Clinical Laboratory Improvement Act (CLIA) laboratory certifications, and clinical supplies 
receipts and distribution records. 
 
Source documents are original records that contain source data, which is all information in original 
records of clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial. Source documents include but are not limited to hospital 
records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation 
checklists, pharmacy dispensing records, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate copies, microfiches, photographic 
negatives, microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at 
the laboratories and at medico-technical departments involved in the clinical trial. When possible, 
the original record should be retained as the source document. However, a photocopy is acceptable 
provided that it is a clear, legible, and an exact duplication of the original document. 
 
14.3.2 Data Management 
The subject’s medical records will be the primary source document for the study. Source 
documents include all information in original records and certified copies of original records of 
clinical findings, observations, or other activities in a clinical investigation used for reconstructing 
and evaluating the investigation.1 Source documentations may also include paper eligibility 
checklists, data flowsheets, patient reported outcomes and other paper documents. The PI, study 
coordinator, study research nurse, data management team and all associated study key personnel, 
are permitted to make entries, changes, or corrections in the source documents or database per the 
study delegation of authority log. 
 
Errors on the source documents will be crossed out with a single line, and this line will not obscure 
the original entry. Changes or corrections will be dated, signed, initialed, and explained (if 
necessary). Database changes will be tracked via electronic trail automatically. 
                                                 
1 In 21 CFR 312.62(b), reference is made to records that are part of case histories as “supporting data;” the ICH guidance for 
industry 
E6 
Good 
Clinical 
Practice: 
Consolidated 
Guidance 
(the 
ICH 
E6 
guidance) 
(available 
at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm) 
uses 
the 
term 
“source 
data/documents.” For the purpose of this guidance, these terms describe the same information and have been used interchangeably. 


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14.3.3 Data Management Procedures and Data Verification 
The DCI Information Technology (IT) Shared Resource has developed Title 21 CFR Part 11 
compliant databases for cancer clinical trials. DCI IT has extensive expertise in database quality 
assurance, data standards, and use of the Cancer Biomedical Informatics Grid (caBIG) tools to 
support cancer researchers.  
 
Data queries will be generated automatically by the electronic case report form (eCRF) system. 
These data queries signify the presence of data inconsistencies. The study and data management 
team will cross-reference the data to verify accuracy. Missing or implausible data will be 
highlighted for 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. 
 
14.3.4 Coding 
All medical terms will be coded using CTCAE (version 5). 
 
14.3.5 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. 
 
 
15 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. 
15.1 Study Design Overview 
This single-arm phase II study will assess the impact of tetanus pre-conditioning and adjuvant 
GM-CSF on overall survival of newly diagnosed GBM patients who have undergone definitive 
resection, are CMV+ and unmethylated, and completed standard temozolomide and radiation 
treatment.  After completing standard of care radiotherapy with concurrent temozolomide, patients 
will receive 1 cycle of dose intensified TMZ followed by pp65 DC vaccination beginning on day 
23.   
15.2 Analysis Sets 
All patients who initiate dose-intensified TMZ after completion of standard of care RT and TMZ 
will be included in the primary efficacy analyses, and toxicity summaries. 


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Subject to the availability of data, analyses of correlative objectives will include all or some of the 
patients who undergo vaccine 4.  For analyses of migration, the first 16 patients who reach vaccine 
4 will undergo migration analyses.  For CCL3, all patients who reach vaccine 4 will be included 
in statistical analyses.  For analyses of polyfunctionality, the first 24 patients who reach vaccine 4 
will be considered.   
15.3 Patient Demographics and Other Baseline Characteristics 
Summaries of clinical and socio-demographic characteristics will be generated within four groups 
of patients:  (1) All patients who sign informed consent prior to the initiation of standard of care 
temozolomide and radiation, (2) All patients who start dose intensified TMZ, (3) All patients who 
receive at least one pp65 DC vaccination, and (4) All patients who receive vaccine 4.  Categorical 
descriptors will be summarized using frequency distributions; whereas, interval variables will be 
summarized using percentiles, as well as means and standard deviations. 
15.4 Treatments 
Among patients who initiate dose intensified TMZ, a frequency distribution will be generated for 
the number of vaccines received by each patient.   
15.5 Primary Objective 
The primary objective of this study is to determine if Td preconditioning of pp65 loaded DC 
vaccination with GM-CSF extends survival relative to a historical benchmark. 
 
15.5.1 Variable 
Overall Survival (OS) is defined as the time between initiation of dose-intensified TMZ and death, 
or last follow-up if the patient remains alive at the time of analysis. 
 
15.5.2 Statistical Hypothesis, Model, and Method of Analysis 
The Kaplan-Meier estimator will graphically describe OS. Median OS will be estimated, with 95% 
confidence intervals, as well as 12, 18, 24, 36, and 48-month survival rates.  A one-sample logrank 
test [124, 125] will compare this curve to that reported by Gilbert [126] in which the median OS 
from the start of adjuvant temozolomide treatment was 14.0 months (95% CI: 12.9 to 14.7 months) 
for patients with newly diagnosed GBM that are unmethylated.  An intent-to-treat approach will 
be used in analyses. 
 
The statistical hypothesis and power calculations are provided in Section 15.9. 
 
Given the possibility that the characteristics of patients who have enrolled on this protocol differs 
from that described by Gilbert [127] for RTOG 0825, additional analyses will be conducted using 
the nomogram published by Gittlemen [128] for RTOG 0525 and 0825.  For each patient, the 
probability of 24-month survival will be estimated from the nomogram based upon the patients’ 
baseline characteristics.  From these individualized estimates of 24-month survival, an overall 
estimate of 24-month survival will be generated with 95% confidence interval.  That confidence 
interval will be compare to the actual 24-month survival confidence interval observed. 
 
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Patients who withdraw from the study without an allowance for survival follow-up, or are lost to 
follow-up will have OS censored at the date that the patient was last know alive. 
15.6 Secondary Objectives 
This study has five secondary objectives.  The first 3 secondary objectives examine the impact of 
functional measures of CMV pp65-loaded DC vaccine efficacy on survival post-vaccine.  These 
measures include migration (Section 15.6.1), fold change in CCL3 (Section 15.6.2), and fold 
change in pp65 antigen-specific polyfunctionality (Section 15.6.3).  Power calculations will be 
provided for each of the secondary objectives.  Power calculations for the three functional 
predictors of survival post-vaccine 4 will be adjusted to account for multiple comparisons using a 
Bonferroni correction, and will used a type I error rate of 0.0167. 
 
The other secondary objectives include an assessment of whether the percent of TReg of CD4+ T 
cells remains stable or decreases without additional temozolomide after vaccine 1 (Section 15.6.4), 
and an assessment of the safety of the protocol’s treatment regimen (Section 15.6.5). 
 
15.6.1 Secondary Objective #1:  Migration and Survival 
 
PLEASE NOTE:  The study originally planned to address secondary objective #1 as described 
below.  However, with the activation of Protocol v.20220204, the plans for examining the 
relationship between migration and survival have been dropped. 
 
Objective 
To assess the association between migration of CMV pp65 RNA-pulsed DCs 
with GM-CSF to site-draining inguinal lymph nodes after Td pre-conditioning 
and survival after vaccine # 4. 
Analysis Set All patients who undergo migration studies at vaccine 4.  Due to budgetary 
considerations, only the first 16 patients who reach vaccine 4 will undergo 
migration studies. 
Outcome 
Survival from vaccine 4. 
Hypothesis 
Patients with greater migration will have longer survival. 
Analysis 
Plan 
The Cox proportional hazards model will assess the impact of migration on 
survival after vaccine #4.  Migration is defined as the maximum percentage of 
111In-labeled DCs reaching inguinal nodes during the 48 hours after the 4th 
vaccination.  The hazard ratio associated with a 1-unit change in migration will 
be estimated with 95% confidence intervals. 
Power 
Calculations 
Cox proportional hazards model will assess the impact of peak migration at 24 
or 48 hours after vaccine 4 on subsequent survival, defined as the time between 
vaccine 4 and death, or last follow-up if the patient remains alive at the time of 
analysis.  Data from ATTAC [129] show that the standard deviation (SD) for 
peak migration is 7.86 and that the hazard ratio associated with 1 unit increase in 
migration is approximately 0.86.  Assuming SD=7.86, there would be 80% power 
to detect a hazard ratio of 0.86 assuming a two-tailed test (α=0.0167) within a 
Cox model after 8 deaths [130, 131].  Assuming the median OS post-vaccine 4 
is 24 months, approximately 24 months after the 16th patient undergoes migration 
studies will 8 deaths be observed. 
 


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15.6.2 Secondary Objective #2:  CCL3 and Survival 
 
Objective 
To assess the association between CCL3 measured post-Td administration and 
survival after vaccine # 4. 
Analysis Set All patients who undergo assessment of CCL3 after Td administration.  All 
patients who reach vaccine 4 when Td is administered will be included in 
analyses.  Forty (40) patients are anticipated. 
Outcome 
Survival from vaccine 4. 
Hypothesis 
Greater levels of CCL3 are associated with longer survival. 
Analysis 
Plan 
The Cox proportional hazards model will assess the impact of CCL3 on survival 
post-vaccine 4. The hazard ratio associate with a 1-unit increase in CCL3 will be 
estimated with 95% confidence intervals. 
Power 
Analyses 
Cox proportional hazards model will assess the impact of CCL3 on survival post-
vaccine 4.  Within ATTAC [129], the standard deviation for CCL3 was 12.9 and 
the hazard ratio for survival post-vaccine 4 was approximately 0.95 for a 1 unit 
increase in CCL3.  Assuming SD=12.9, there would be 80% power to detect a 
hazard ratio of 0.95 with a two-tailed test (α=0.0167) within a Cox model under 
these assumptions after 24 deaths [130, 131]. If we assume that the median OS 
post-vaccine 4 is 24 months, then 24 deaths should be observed approximately 
20 months after the 40th patient receives vaccine 4.  
 
15.6.3 Secondary Objective #3:  Polyfunctionality and Survival 
 
Objective 
To assess the association between fold changes in T cell polyfunctionality from 
baseline to pheresis 2, and survival after vaccine #4. 
Analysis Set All patients who have a baseline (pre vaccine 1) and polyfunctionality assessed 
at leukapheresis 2 who receive vaccine # 4.  Polyfunctionality is assessed at 
leukapheresis so that results do not reflect the impact of Td-preconditioning post-
vaccine 4 assessment of polyfunctionality. Due to budgetary considerations, only 
the first 24 patients who reach vaccine 4 will be analyzed. 
Outcome 
Survival from vaccine 4. 
Hypothesis 
Greater fold changes in polyfunctionality are associated with longer survival. 
Analysis 
Plan 
The mean fold change from baseline to leukapheresis 2 will be summarized for 
the frequency of pp65 antigen-specific CD8+ T cells producing three or more 
cytokines (IFNγ, CCL3, IL-2, TNFα, CD107a).  Cox proportional hazards model 
will assess the association between fold change increase between baseline and 
the leukapheresis 2 in the frequency of pp65 antigen-specific CD8+ T cells 
producing three or more cytokines (IFNγ, CCL3, IL-2, TNFα, CD107a), and 
survival post-vaccine 4. The hazard ratio associate with a 1-unit fold change in 
polyfunctionality will be estimated with 95% confidence intervals.  
Power 
Analyses 
Cox proportional hazards model will assess the impact of fold change of pp65-
specific polyfunctional CD8+ T cells between baseline and leukapheresis 2 on 
survival after vaccine #4.  Data from Pro00000580 ERADICATE [132] shows 
that the SD for fold change in polyfunctionality among vaccinated patients is 
1.589, and that the hazard ratio associated with 1 unit increase in fold change is 


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approximately 0.609.  Assuming SD=1.589, there would be 80% power to detect 
a hazard ratio of 0.6 assuming a two-tailed test (α=0.0167) within a Cox model 
after 16 deaths [130, 131]. If we assuming that the median OS post-vaccine 4 is 
24 months, then 16 deaths should be observed approximately 38 months after the 
24th patient receives vaccine 4. 
Additional 
Analyses 
Many of the 24 patients who have assessments at pre-vaccine 1 and leukapheresis 
2 will also have an assessment of polyfunctionality at pre-vaccine 6.  Among 
those patients who have all 3 assessments, a paired comparison of the fold change 
in polyfunctionality at pheresis 2 and vaccine 6 will be conducted using either a 
paired t-test or a Wilcoxon signed rank test.  These analyses will assess whether 
levels of polyfunctionality are maintained in the absence of DI-TMZ.  In addition, 
the Wilcoxon rank sum test will assess whether significant fold changes occur at 
pheresis 2. 
 
15.6.4 Secondary Objective #4:  TRegs 
  
Objective 
To assess whether percent TReg levels increase further after vaccine 1  
Analysis Set All treated patients 
Outcome 
Maximum peak increase from vaccine 1 in percent TReg of CD4+ T cells 
Hypothesis 
With no additional temozolomide treatment after vaccine 1, the level of percent 
TReg will not significantly increase above vaccine 1 levels.  Rather there should 
be no change or a reduction in TReg levels.   
Analysis 
Plan 
A one-tailed t-test will be conducted to assess whether the mean for the maximum 
change from vaccine 1 in percent TReg is significantly greater than 0.  If the mean 
change is less than 0.71 (i.e. near 0 or a decrease), we will infer that levels of 
percent TReg do not increase after vaccine 1.  Rather, they are either stable or 
decrease.   
Power 
Analyses 
In ATTAC-GM, the mean change in the percent TReg of CD4+ T cells between 
leukapheresis and vaccine 1, a period during which DI-TMZ is administered, was 
5.77 (SD=4.52).  Between vaccine 1 and the next cycle of DI-TMZ, the peak 
mean increase in percent TReg from vaccine 1 was -1.104 (SD=1.65).  Assuming 
48 patients and SD=1.65 for the mean change from vaccine 1 within I-ATTAC, 
there will be 90% power to detect an increase from vaccine 1 in the percent of 
TReg if the true mean increase is 0.71 or greater assuming a one-tailed test 
(α=0.05). 
Additional 
Analyses 
If the primary analysis that examines the maximum increase from vaccine 1 
percent TReg of CD4+ T cells observed at any point after the initial vaccine detects 
such an increase, the timing of that increase is of interest.  Hence, we will also 
examine the change from vaccine 1 to each of the follow-up assessments 
(leukapheresis 2, vaccine 4, vaccine 5, and vaccine 6). A generalized linear model 
may be used to explore these longitudinal changes.  We may also use one-tailed 
t-tests to describe these changes.  The change between vaccine 1 to leukapheresis 
2 reflects the impact of vaccine 1, 2 and 3; whereas the change at vaccine 4 
reflects the effect of the first 3 vaccines as well as Td pre-conditioning.   
 


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We will also conduct a t-test to examine the change in TReg levels observed before 
DI-TMZ and vaccine 1 to confirm that TReg levels are elevated with DI-TMZ, as 
well as the change between leukapheresis 1 and initiation of DI-TMZ.    
 
15.6.5 Secondary Objective #5:  Toxicity 
 
Objective 
Assess the safety of DI-TMZ treatment followed by DC vaccination. 
Analysis Set All patients who initiate DI-TMZ treatment. 
Outcome 
Proportion of patients with unacceptable toxicity as defined in Section 9.1.1. 
Hypothesis 
The toxicity profile associated with DI-TMZ followed by DC vaccination is safe. 
Monitoring 
Plan 
 
Table A: Toxicity Monitoring Rules 
Number of patients accrued 
Number of patients with unacceptable 
toxicity requiring accrual suspension 
≤5 
≥2 
6-11 
≥3 
12-17 
≥4 
18-23 
≥5 
24-29 
≥6 
30-34 
≥7 
35-39 
≥8 
≥40 
≥9 
 
Given that both long- and short-term toxicities are of interest in this study, it is 
not feasible to suspend accrual while toxicity is assessed as is often done in phase 
I trials.  If the criteria in Table A are satisfied or there are other reasons for 
concern about the safety of patient treatment (e.g., treatment-related toxic death), 
accrual will be suspended and data will be carefully reviewed to determine if 
accrual should be permanently terminated or the protocol modified.  Table A 
provides conditions under which accrual will be temporarily suspended and data 
carefully reviewed to determine the appropriate action, including permanent 
study termination, continuation with patient accrual after appropriate 
amendment, or continuation with patient accrual with no modification of the 
protocol. These guidelines have not been adjusted for differential length of 
follow-up of accrued patients.   
 
If a death occurs within 30 days of DC vaccine administration, that is not 
attributable to progressive disease or other obvious non-study related cause (i.e. 
motor vehicle accident), enrollment of new subjects and all vaccinations will be 
suspended until review by the PI and IND sponsor has been completed and the 
FDA notified. If attribution of death is determined by the PI, IND sponsor or 
FDA to be possibly, probably, or definitely related to trial drug, then continued 
treatment of all enrolled subjects with trial drug will be suspended until the 
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issued and approved by FDA. Any death attributed to trial drug, regardless of the 
timeline with respect to last treatment, will result in suspension of enrollment and 
suspension of continued treatment for enrolled subjects until review by FDA is 
complete and recommendations for study continuation issued and approved. 
 
TABLE B: Probability of Accrual Suspension 
Underlying unacceptable toxicity Rate 
Probability of accrual suspension 
0.05 
0.036 
0.10 
0.200 
0.15 
0.502 
0.20 
0.784 
0.25 
0.936 
0.30 
0.987 
 
The probability of accrual suspension as a function of the true unacceptable 
toxicity rate is provided in Table B.  These statistics were generated assuming 
toxicity outcome was known at the time of accrual, and ignored issues such as 
time to toxicity, accrual rate, and length of follow-up. 
 
Every 6 months from the time the first patient received vaccine #1, the toxicity 
experienced by patients accrued to this study will be summarized and reviewed 
regardless of the number of patients accrued to determine whether the overall 
toxicity profile of treatment is unacceptable or not. 
 
Analysis 
Plan 
The proportion of patients who experience an unacceptable toxicity as defined in 
Section 9.1.1 
Additional 
Analyses 
Adverse events will be summarized in several other forms to satisfy scientific 
and monitoring needs, as well as various regulatory reporting needs (e.g. FDA, 
DCI Safety Oversight Committee, and ClinicalTrials.gov).  For these summaries, 
the frequency of adverse events will be tabulated by the maximum grade 
experienced. 
15.7 Exploratory Objectives 
This study has 3 exploratory objectives:  (1) To assess the relationship between migration and 
serum CCL3, (2) To assess the relationship between pp65-specific polyfunctional CD8+ T cells 
and migration, and (3) To assess the relationship between pp65-specific polyfunctional CD8+ T 
cells and serum levels of CCL3. 
 
15.7.1 Exploratory Objective #1:  Migration and CCL3 
 
Objective 
To assess the relationship between serum CCL3 levels and increased DC 
migration. 
Analysis Set All patients who undergo migration studies who have CCL3 levels measured. 
Outcome 
Correlation coefficient between fold change of CCL3 and migration. 
Hypothesis 
We hypothesize that fold change in CCL3 will be highly correlated with 
increased migration. 


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Analysis 
Plan 
A Spearman rank correlation coefficient will assess the association between 
CCL3 change from pre to post-vaccine 4, and migration.   
Additional 
Analyses 
As an additional exploratory analysis of CCL3, a one-sample t-test or a Wilcoxon 
rank sum test will assess whether fold change in CCL3 between pre- and post-
Td pre-conditioning differs from 0. 
 
15.7.2 Exploratory Objective #2:  Polyfunctionality and Migration 
 
Objective 
To assess the relationship between pp65-specific polyfunctional CD8+ T cells 
and DC migration. 
Analysis Set All patients who undergo migration studies who have polyfunctional levels 
measured. 
Outcome 
Correlation coefficient between fold change in polyfunctionality and migration 
Hypothesis 
We hypothesize that increased pp65-specific polyfunctionality CD8+ T cells will 
be highly correlated with increased DC migration. 
Analysis 
Plan 
A Spearman rank correlation coefficient will assess the association between fold 
change in polyfunctionality and migration. 
 
15.7.3 Exploratory Objective #3:  Polyfunctionality and CCL3 
 
Objective 
To assess the relationship between pp65-specific polyfunctional CD8+ T cells 
and serum CCL3. 
Analysis Set All patients who undergo polyfunctional studies and have CCL3 levels measured. 
Outcome 
Correlation coefficient between fold change in polyfunctionality and fold change 
in CCL3. 
Hypothesis 
We hypothesize that increased pp65-specific polyfunctionality CD8+ T cells will 
be highly correlated with increases in serum CCL3 after Td preconditioning. 
Analysis 
Plan 
A Spearman rank correlation coefficient will assess the association between fold 
change in polyfunctionality and fold change in CCL3. 
15.8 Interim Analysis 
As described in Section 15.9, we anticipate that at the time of the primary study analysis 33 of the 
patients treated with DI-TMZ will have died. An interim analysis for futility will be conducted 
after approximately 17 patients have died, assuming that occurs before accrual is completed. The 
α-spending function that approximately an O’Brien-Fleming boundary will defined critical values 
that will be used in making that assessment [133]. Based upon these analyses and available 
correlative data, a decision will be made concerning continued accrual without modification, 
continued accrual with modifications to the protocol, or termination of patient accrual. 
15.9 Sample Size Calculation 
Approximately 64 patients will be accrued to this study, with the goal of initiating DI-TMZ in 48 
of these patients.   
 
To evaluate the observed OS within this study of pp65 DC vaccination with tetanus pre-
conditioning and adjuvant GM-CSF, we will consider Gilbert’s study of dose-dense temozolomide 
for newly diagnosed glioblastoma.  Gilbert reports a median OS from initiation of adjuvant 


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treatment among methylated patients to be 14.0 months (95% CI: 12.9 to 14.7 months).   
 
Though the median OS from initiation of adjuvant DI-TMZ treatment in the ATTAC-GM pilot is 
37.7 months (95% CI: 18.2, ∞), we hypothesize for power calculations that the true median OS for 
our novel treatment regimen is approximately 24 months.  Assuming accrual of 64 patients over a 
period of 24 months, 28 months follow-up after the last patient initiates adjuvant treatment, and 
12 patients who initiate vaccine treatment, a two-sided, a one-sample logrank test has 80% power 
at a 0.05 significance level to detect a hazard ratio of 0.6125 (i.e. = 14.7 / 24) when the median 
survival of the historic control group is 14.7 months, the upper bound of the 95% confidence 
interval for median survival reported by Gilbert [124, 125, 131].  We anticipate that at the time of 
the primary study analysis 33 of these patients will have died.     
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
16.1 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. 
16.2 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 Principal Investigator has received written and 
dated approval from the CPC and IRB. 
 
The Principal Investigator 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 Principal Investigator must obtain protocol re-approval from the IRB within 1 year of the most 
recent IRB approval. The Principal Investigator 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. 
16.3 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 Principal Investigator or authorized key personnel will discuss with the potential subject 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 who cannot read or understand English or are visually impaired. 
Potential subjects will have the opportunity to contact the Principal investigator or authorized key 


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personnel with questions, and will be given as much time as needed to make an informed decision 
about participation in the study. 
 
Before conducting any study-specific procedures, the Principal Investigator must obtain written 
informed consent from the subject. 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. 
The Principal Investigator is responsible for asking the subject whether the subject wishes to notify 
his/her primary care physician about participation in the study. If the subject agrees to such 
notification, the Principal Investigator will inform the subject’s primary care physician about the 
subject’s participation in the clinical study. 
16.4 Privacy, Confidentiality, and Data Storage 
The Principal Investigator 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 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 Clinical 
database, which is housed by the DCI. Access to electronic databases will be limited to the 
Principal Investigator, key personnel, statisticians, the Radiolabeled Pharmacy personnel, and the 
PRTBTC data manager. Data stored on portable memory devices will be de-identified. 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 Human 
Research Protection Program (HRPP) policy.  
 
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
16.5 Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and Safety 
Monitoring Plan. For a more detailed description of the DSMP for this protocol, refer the separate 
upload in electronic IRB. 
16.6 Protocol Amendments 
All protocol amendments must be initiated by the Principal Investigator 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 Principal Investigator must inform 
the IRB and all other applicable regulatory agencies of such action immediately. 


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Though not yet required, 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, etc.). 
16.7 Records Retention 
The Principal Investigator will maintain study-related records for the longer of a period of: 
- 
at least two years after the date on which a New Drug Application is approved by the FDA 
- 
at least two years after formal withdrawal of the IND associated with this protocol 
- 
at least six years after study completion (Duke policy). 
16.8 Conflict of Interest 
The Principal Investigator and Sub-Investigators must comply with applicable federal, state, and 
local regulations regarding reporting and disclosure of conflict of interest. Conflicts of interest 
may arise from situations in which financial or other personal considerations have the potential to 
compromise or bias professional judgment and objectivity. Conflicts of interest include but are not 
limited to royalty or consulting fees, speaking honoraria, advisory board appointments, publicly-
traded or privately-held equities, stock options, intellectual property, and gifts. 
 
The Duke University School of Medicine’s RIO reviews and manages research-related conflicts 
of interest. The Principal Investigator and Sub-Investigators must report conflicts of interest 
annually and within 10 days of a change in status, and when applicable, must have a documented 
management plan that is developed in conjunction with the Duke RIO and approved by the 
IRB/IEC. 
16.9 Registration Procedure 
After patients have been enrolled, subject registration will be entered into the Duke eResearch 
system and the subject’s visits associated in the Duke Epic Maestro Care system with this protocol 
which is entered after Duke IRB approval. 
 
 
 


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Walker, M.D., et al., Randomized comparisons of radiotherapy and nitrosoureas for the treatment of 
malignant glioma after surgery. N Engl J Med, 1980. 303(23): p. 1323-1329. 
3. 
Stupp, R., et al., Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. N Engl J 
Med, 2005. 352(10): p. 987-996. 
4. 
Walker, M.D., et al., Evaluation of BCNU and/or radiotherapy in the treatment of anaplastic gliomas. A 
cooperative clinical trial. J Neurosurg, 1978. 49(3): p. 333-343. 
5. 
Shapiro, W.R., Therapy of adult malignant brain tumors: what have the clinical trials taught us?. Semin 
Oncol, 1986. 13(1): p. 38-45. 
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Salford, L.G., A. Brun, and S. Nirfalk, Ten-year survival among patients with supratentorial astrocytomas 
grade III and IV. J Neurosurg, 1988. 69(4): p. 506-509. 
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18 APPENDICES 
18.1 Standard Radiation Therapy 
Radiotherapy typically begins within ≤ 5 weeks of surgery. One treatment of 1.8-2.0 Gy/fraction 
will be given daily 5 days per week for a total of 59.4-60.0 Gy over <7 weeks. 3D conformal and 
intensity-modulated RT is permitted. All portals should be treated during each treatment session. 
Doses are specified as the target dose that shall be to the center of the target volume.  
 
The gross target volume (GTV) for both the initial volume (GTV1) and the conedown volume 
(GTV2) should be based on the postoperative CT/MRI (and preferably the MRI; the preoperative 
scans may be used if postoperative scans are not available). This initial target volume (GTV1) 
should include the contrast-enhancing lesion (and should include the surgical resection cavity) and 
surrounding edema (if it exists) demonstrated on CT/MRI plus a 2.0-cm margin (this 2.0-cm 
margin-extended volume will be considered the initial planning target volume, or PTV1). The 
initial target volume should be treated to 46 Gy at 2Gy/fraction or 45-50.4 Gy at 1.8Gy/fraction. 
If no surrounding edema is present, the initial planning target volume (PTV1) should include the 
contrast-enhancing lesion (and should include the surgical resection cavity) plus a 2.5-cm margin. 
Please note that clinical judgment may be used to modify PTV1 to exclude sensitive structures 
such as the optic chiasm, non-cranial contents, or anatomic regions in the brain where natural 
barriers would likely preclude microscopic tumor extension, such as the cerebellum, the 
contralateral hemisphere, directly across from the tentorium cerebri, the ventricles, etc. After 46 
Gy, the tumor volume (GTV2) for the conedown treatment should include the contrast-enhancing 
lesion (without edema) on the pre-surgery CT/MRI scan plus a 1.5-2-cm margin (PTV2). Treat to 
14 Gy at 2Gy/fraction or 14.4-9.0 Gy at 1.8Gy/fraction to a total of 60.0 or 59.4Gy, respectively. 
 
Dose is prescribed to the isodose line such that at least 95% of the target volume receives he 
prescribed dose.  The optic apparatus should be limited to a maximum of 54Gy and no more than 
5% of the volume of the brainstem should receive >54Gy. 
 
Radiation should be delayed or interrupted if the platelet count is < 20,000. Radiation should not 
begin or resume until the platelet count is ≥ 20,000. Hematologic toxicities should be rated on a 
scale of 0-5 as defined in the NCI Common Terminology Criteria for Adverse Events (CTCAE) 
version5.0.  If radiotherapy has to be temporarily interrupted for technical or medical reasons 
unrelated to the temozolomide administration, then treatment with daily temozolomide should 
continue. If radiotherapy has to be permanently interrupted then treatment with daily 
temozolomide should stop.  The following should be recorded at entry into this study: daily 
treatment record, all isodose distributions (in color), dose volume histograms including the 
cumulative dose to the target volumes, optic chiasm, optic nerves and brain stem, and the 
radiotherapy summary. 
18.2 Temozolomide Therapy 
Please refer to the current package insert for Temodar®. 

ICF_000.pdf: 
 
 
Official Title: 
I-ATTAC: Improved Anti-Tumor Immunotherapy Targeted Against 
Cytomegalovirus in Patients With Newly-Diagnosed WHO Grade IV Unmethylated 
Glioma 
 
NCT:  NCT03927222 
 
IRB Document Date:  May 06, 2022 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 1 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
TITLE: 
I-ATTAC:  Improved Anti-Tumor Immunotherapy Targeted Against 
Cytomegalovirus in Patients with Newly-Diagnosed WHO Grade IV 
Unmethylated Glioma 
 
PROTOCOL NO.: 
Pro00090683  
 
WCG IRB Protocol #20190629 
 
IRB00106436 
 
SPONSOR: 
Duke University Medical Center 
 
INVESTIGATOR: 
Mustafa Khasraw, MBChB, MD, FRCP, FRACP 
 
DUMC Box 3624 
  
Durham, North Carolina 27710 
 
United States 
 
STUDY-RELATED 
PHONE NUMBER(S): 
Mustafa Khasraw, MBChB, MD, FRCP, FRACP 
 
919-684-5301 
 
919-206-0493 (after hours) 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 2 of 30 
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RESEARCH CONSENT SUMMARY 
 
You are being asked for your consent to take part in a research study. This document provides a 
concise summary of this research. It describes the key information that we believe most people need 
to decide whether to take part in this research. Later sections of this document will provide all 
relevant details. 
 
WHAT SHOULD I KNOW ABOUT THIS RESEARCH? 
• Someone will explain this research to you. 
• Taking part in this research is voluntary. Whether you take part is up to you. 
• If you don’t take part, it won’t be held against you. 
• You can take part now and later drop out, and it won’t be held against you 
• If you don’t understand, ask questions. 
• Ask all the questions you want before you decide. 
 
HOW LONG WILL I BE IN THIS RESEARCH? 
We expect that your taking part in this research will last 10 to 12 months, as long as your tumor has 
not grown back and will involve up to 10 study vaccine treatments. 
 
WHY IS THIS RESEARCH BEING DONE? 
The purpose of this research is to learn if an experimental vaccine called CMV pp65 DC can help to 
activate your immune system and help your body fight off the tumor cells in your brain. The safety 
of this treatment will also be studied. 
 
WHAT HAPPENS TO ME IF I AGREE TO TAKE PART IN THIS 
RESEARCH? 
If you decide to take part in this research study, the general procedures include a physical exam, 
medical history, vital signs, blood tests, and MRI scan of the brain to see if you are eligible to be in 
the study. You will need to undergo a procedure called a leukapheresis at the Duke University 
Medical Center Apheresis Unit. The first 3 CMV pp65 DC vaccines will be given over 6 weeks. You 
will return to clinic every 2 weeks to receive these vaccines. 
 
After you have received your first three study vaccines, you will be asked to repeat the MRI of the 
brain and have another leukapheresis procedure. Vaccines will be given every 35 days for a total of 
10 vaccine injections unless tumor progression occurs. 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 3 of 30 
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You will also be given Temozolomide or Temodar® (TMZ), which is an FDA-approved anti-cancer 
drug by your doctor as part of your standard care. You will receive only 1 cycle of TMZ meaning 
you will take TMZ daily for 21 days. This cycle will be completed before your first vaccine. 
 
COULD BEING IN THIS RESEARCH HURT ME? 
The most important risks or discomforts that you may expect from taking part in this research 
include: 
• Injection of the study vaccine may cause an allergic reaction that can include redness or 
swelling at the injection site and itching. 
• The leukapheresis procedure may be similar to risks experienced during blood donation and 
include discomfort or a swollen bruise at the site of needle puncture, a slight risk of 
developing a local infection at the site, and development of a small scar. 
• The GM-CSF Injection may cause headache, bone pain, muscle and joint pains, fever, and 
chills. 
• Temozolomide (TMZ) may cause loss of appetite, nausea and vomiting, especially on the 
first day of each cycle, constipation, and decrease in blood cell counts. 
 
WILL BEING IN THIS RESEARCH BENEFIT ME? 
The most important benefits that you may expect from taking part in this research is that this 
immunotherapy will allow your immune system to better fight your tumor. The knowledge gained 
from your participation may benefit others.  
  
WHAT OTHER CHOICES DO I HAVE BESIDES TAKING PART IN THIS 
RESEARCH? 
Instead of being in this research, your choices may include further surgery, chemotherapy and 
radiation. 
 
WHAT ELSE SHOULD I KNOW ABOUT THIS RESEARCH? 
For women, being a part of this study while pregnant may expose the unborn child to significant 
risks, some of which may be currently unforeseeable. You must agree to use 2 forms of appropriate 
contraceptive measures at the same time for the duration of the study vaccine administration and for 
6 months afterwards. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 4 of 30 
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For men, participation in this research may damage your sperm, which could cause harm to a child 
that you may father while on this study.  You must agree to use a medically acceptable form of birth 
control, in order to be in this study and for 6 months after the study vaccine administration. 
 
DETAILED RESEARCH CONSENT 
You are being asked to participate in a research study because you have a malignant brain tumor, 
which is usually treated with surgery, radiation, and chemotherapy.  
 
Research studies are voluntary and include only people who choose to take part. Please read this 
consent form carefully and take your time making your decision.  As the doctor or the study staff 
discusses this consent form with you, please ask him/her to explain any words or information that 
you do not clearly understand.  We encourage you to talk with your family and friends before you 
decide to take part in this research study.  The nature of the study, risks, inconveniences, and 
discomforts, and other important information about the study are listed below. 
 
Please tell your doctor or the medical staff if you are taking part in another research study. 
 
A NIH grant will help sponsor this study.  Portions of Dr. Mustafa Khasraw’s and his research 
team’s salaries will be paid by this grant. The CMV (cytomegalovirus) vaccine technology used in 
this study was developed by Drs. John Sampson, Gary Archer, and others at Duke and, if successful, 
both Duke and the developers could benefit financially. This technology, including the use of the 
tetanus-diphtheria (Td) vaccine, has been licensed to a Duke start-up company, Annias 
Immunotherapeutics.  Duke University and Drs. Sampson and Archer have an equity interest (stocks 
and/or options) in the company. 
 
WHO WILL BE MY DOCTOR ON THIS STUDY? 
If you decide to participate, Dr. Mustafa Khasraw will be your doctor for the study; however, your 
treating neuro-oncologist will remain as your primary doctor for the treatment of your brain tumor 
and will be in contact with your regular health care provider throughout the time that you are in the 
study and afterwards, if needed. 
 
WHY IS THIS STUDY BEING DONE? 
The purpose of this research study is to learn if a vaccine can help to activate your immune system 
and help your body fight off the tumor cells in your brain. The safety of this treatment will also be 
studied.  The vaccine, called CMV pp65 DC, is investigational, which means that it is not approved 
by the US Food and Drug Administration (FDA) and is still being tested in research studies.  


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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It is believed that the body’s immune (protection) system can attack tumor cells and kill them. This 
is thought to be due to immune cells called T-lymphocytes (T-cells), which can recognize special 
proteins on the surface of tumors as a signal to attack and fight the cancer. In most patients with 
advanced cancer, the immune system does not adequately destroy the tumor because the white blood 
cells or T-cells are not stimulated enough. 
 
Before your T-cells can become active against tumor cells, they require strong stimulation. There are 
special “stimulator” cells in the body called Dendritic Cells (DCs) that can take up proteins released 
from cancer cells and present pieces of these proteins (called peptides) to T cells to create this strong 
stimulation. When the vaccine for this study is made, it will be “pulsed” or loaded with genetic 
material called RNA (ribonucleic acid), which stimulates the DC to change the RNA into a protein 
called pp65. This protein is produced by a common virus called Cytomegalovirus (CMV) that 70-
80% of us have been exposed to in our lifetime. Recently, we have found that this virus is present in 
many malignant brain tumors. Brain tumors are very aggressive and, for reasons we do not yet 
understand, are difficult for the body to attack. The CMV virus is an area to target in the tumor that, 
if attacked by your immune systems cells, may prevent your tumor from growing. 
 
It is hoped that, by injecting the CMV pp65 DC vaccine into your skin, your immune system will be 
activated against the protein. Once it is activated against this protein, your immune system may 
recognize this protein on the surface of the tumor cells and attack the tumor cells in your brain and 
not attack normal cells.  Use of a vaccine that stimulates your immune system is called 
immunotherapy. An additional drug called granulocyte macrophage-colony stimulating factor (GM-
CSF) will be mixed with the vaccine to help boost your immune system. 
 
Tetanus-diphtheria (Td) immunization is a drug composed of deactivated (dead) tetanus and 
diphtheria toxins.  It works by causing an immune response to produce antibodies, in order for the 
body to recognize antigens from tetanus and diphtheria toxins if they are ever introduced into the 
body.  We have discovered through previous studies that giving the Td immunization prior to 
immunotherapy may help improve the effectiveness of the study drug by activating the immune 
response against antigens in the study drug vaccine.  
 
Using Td in this way is considered investigational, since it has not been approved by the FDA for 
this use.   
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
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HOW MANY PEOPLE WILL TAKE PART IN THIS STUDY? 
Approximately 64 people will take part in this study at Duke.   
 
WHAT HAS BEEN THE PRIOR EXPERIENCE WITH CMV PP65 DC 
IMMUNOTHERAPY? 
Previous research studies using CMV pp65 DC vaccine have been conducted here at Duke for brain 
tumors. No serious side effects have been reported.  In addition, patients have received DCs loaded 
with other substances at this and other institutions for the treatment of a variety of different tumors. 
Again, no serious side effects have been reported. The effectiveness of this type of therapy in 
treating tumors is unknown at the present time and is the reason for research studies such as this one. 
 
WHAT IS INVOLVED IN THE STUDY? 
If you agree to be in this study, you will be asked to sign this consent form.  You have already 
undergone surgery to remove the brain tumor.  In order for you to be eligible for this study, the 
surgery has to be very successful; almost the entire brain tumor needs to have been removed during 
surgery. 
 
You will have the following tests and procedures to make sure that you are eligible:  
• Physical exam and medical history 
• Vital signs 
• Blood tests 
• MRI of the brain 
 
We will need to review your pathology report from your brain tumor surgery to confirm that the 
brain tumor you have is a Grade IV glioblastoma. As part of further molecular diagnostic analysis of 
your tumor, slides from your surgery at Duke will be sent for standard biomarker analysis, which 
includes MGMT promotor methylation status and IDH-1/2 mutation analysis. If you have this 
surgery done at another hospital, different than Duke, and if such institutions do not incorporate 
these initial molecular diagnostic assays, slides from surgical blocks will be ordered for subsequent 
MGMT promoter methylation status and IDH-1/2 mutation analysis, so as to ensure the baseline 
molecular diagnostics of the tumor that was removed. 
 
As an optional part of the study, we may sample a small amount of tissue from the original tumor 
that is removed and stored as a surgical specimen block at either Duke University or the external 
institution (depending on where the surgery was performed). This means obtaining histologic slides 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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from the original specimen block stored at Duke or the external institution to perform detection of 
CMV in the tumor. Obtained slides will then be stored in the Duke Brain Tumor Immunotherapy 
Program (DBTIP) lab. The sampling of this tumor specimen will not exceed 10% of the original 
block so as to preserve adequate tissue should you or your doctor want future diagnostic testing as 
part of your clinical care or other clinical trials. This process is optional should you allow us to 
sample the tumor tissue. 
 
Please initial next to one of the statements below to indicate whether or not you agree to allow the 
collection of slides from initial surgery specimen blocks for detection of CMV studies.  
 
 
 
Yes, I agree to allow histologic slides to be obtained from the initial resection 
Initials 
specimen for future research on CMV in tumor tissue. 
 
 
 
No, I do not agree to allow histologic slides to be obtained from the initial 
Initials 
resection specimen for future research on CMV in tumor tissue. 
 
Leukapheresis: You will need to undergo a procedure called a leukapheresis at the Duke University 
Medical Center Apheresis Unit.  Leukapheresis is a medical procedure by which large numbers of 
white blood cells can be removed from your circulation, however, it is not a standard procedure for 
brain tumor therapy. Leukapheresis requires the insertion of a needle into the veins of both arms, 
after which you lie on a bed for about 2-3 hours while blood is collected, much like in a standard 
blood donation. The main difference is that only the white blood cells are collected while the red 
blood cells are returned to your body. A portion of the removed white blood cells will be used to 
make the DCs for the vaccine. During leukapheresis, a medication called citrate is added to the blood 
while in the machine to keep the blood from clotting in the tubing.  If you do not have sufficient 
venous access for the leukapheresis, a temporary intravenous catheter may need to be inserted in a 
deeper vein (central venous catheter). 
 
If you underwent leukapheresis as part of another companion study titled “Preliminary Testing 
and/or Procedures for Potential Clinical Trial Participants of the Preston Robert Tisch Brain 
Tumor Center,” the vaccines manufactured as part of that study will be used in the current study and 
any unused vaccine products will be handled as you indicated in the companion study. 
 
Td:  You will receive the standard dose of Td as an intramuscular injection in your arm before your 
first vaccine.  It is thought that the Td immunization may help improve the effectiveness of the CMV 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
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Protocol ID: Pro00090683 
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pp65 DC vaccine.  In addition to receiving this Td booster immunization, you will receive Td mixed 
with saline (salt water) one day before your 4th CMV pp65 DC vaccine.    
 
Initial CMV pp65 DC Vaccinations: The first 3 CMV pp65 DC vaccines will be given over the 
course of 6 weeks.  You will return to clinic every 2 weeks to receive these vaccines. You will 
receive all vaccine injections with a thin needle that is about 1 ½ inches long, which will be placed 
just under the skin (intradermal) in the area below your groin in both the right and left side at each 
administration session. Each injection will only take a few seconds. After each vaccine, you will 
have vital signs taken. Because this is a study to assess the safety of this vaccine, it is possible you 
may receive a dose of the vaccine which only later demonstrated to be harmful. After you have 
received your first three study vaccines, you will be asked to repeat the MRI of the brain and have 
another leukapheresis procedure. The leukapheresis procedure will be to produce more DC vaccine 
for your continued vaccinations and to monitor the effect of the vaccine. It is possible that, if you 
continue to do well and your tumor does not come back, you may need to have an additional 
leukapheresis procedure to produce more DCs for more vaccines. 
 
Chemotherapy Treatment: Temozolomide or Temodar® (TMZ) is an FDA-approved anti-cancer 
drug and will be given by your doctor as part of your standard care. You will receive only 1 cycle of 
TMZ, meaning you will take TMZ daily for 21 days.  This cycle will be completed before your first 
vaccine.  TMZ is a capsule that you take by mouth; the actual number of capsules you take will vary 
depending upon your height and weight. It should be taken in a fasting state (nothing to eat for one 
hour prior to each dose and for two hours after each dose). It has been well tolerated by both adults 
and children.  
 
As part of your standard care at Duke, your tumor tissue will be tested for a biomarker that is 
referred to as O6-methylguanine-DNA methyltransferase (MGMT), if this test was not already 
performed on your tumor tissue.  MGMT is a gene that has been shown to be important in predicting 
the response of glioblastoma to the chemotherapy drug temozolomide (TMZ).  Tumors with 
unmethylated MGMT do not respond as well to TMZ as tumors with methylated MGMT.  We will 
obtain the results of the MGMT test result from your medical record, and only individuals with 
unmethylated MGMT will be enrolled in this trial.  Although individuals with unmethylated MGMT 
tumor appear to be less responsive to treatment with TMZ, there is a chance that treatment with 
TMZ could help shrink your tumor, and by signing this consent form you are choosing not to receive 
additional cycles of TMZ.  There is a risk that the MGMT tumor testing may incorrectly identify 
your tumor type as unmethylated and result in you forgoing available therapy with TMZ. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 9 of 30 
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Vaccinations: DC vaccines will be given every 35 days for a total of 10 vaccines unless tumor 
progression occurs.  
 
During the study vaccine appointments, you will be checked for any side effects and/or symptoms.  
You will have your blood drawn to check your kidney function, your white blood cells, and your 
immune system. Approximately 6 tablespoons of blood will be drawn at the following time points:  
before the 1st leukapheresis, at the post radiation therapy/TMZ visit, on the day of but before the 1st 
and 3rd vaccines, on the day of but prior to pre-conditioning, before the 2nd leukapheresis, on same 
day of but before the 4th vaccine, and then prior to but on the same day as each remaining vaccine.  If 
your tumor progresses, we would also like to draw approximately 6 tablespoons of blood at that 
time.   
 
MRIs: You will also have several MRIs (magnetic resonance image) to confirm that the tumor is not 
growing. MRI is done initially to confirm you are eligible for this study, then every 2 months for the 
first year, after which you will have an MRI every 2 to 6 months at the discretion of your treating 
oncologist.  To get better pictures with the MRI, we will inject a contrast agent into your veins when 
you get your MRI. MRI uses magnetic waves to make medical images of the body. MRI involves 
entering a large room in which a magnet is present. You will be placed on a narrow bed and then slid 
into a small tunnel approximately 6 feet in length and 25 inches in diameter. You will be asked to lie 
still for about one hour on this bed. You will hear a loud machine-like noise. You may be asked to 
have a harmless monitoring device applied during the study. During the study, you can have voice 
contact and physical contact with someone in attendance if you desire. 
 
Follow-up Visits:  After you complete the study vaccines, you will no longer be required to 
continue visits for this study, but you will continue to be examined periodically in accordance with 
routine care.  An attempt will be made to obtain blood for immunologic monitoring 2-3 times a year 
at standard Duke neuro-oncology visits. We would like to repeat this blood draw for immunologic 
testing if you are told you have tumor progression.  The study team may stay in contact with you 
through periodic phone calls to ask about your health and about any medications you have taken or 
are currently taking for the treatment of your tumor.    
 
OPTIONAL STORAGE OF BLOOD AND TUMOR FOR FUTURE 
RESEARCH 
As part of this study, you are also being asked to allow the study sponsor and PI to store your 
unused blood and tumor samples for future testing to learn more about how the study drug has 
worked and to further study the immunology of brain tumors.  From these unused samples, it might 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
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also be possible to learn more about how to better treat your type of primary brain tumor, and for 
purposes that are not yet known. 
 
The samples will be stored in the Duke Brain Tumor Immunotherapy Program (DBTIP) Laboratory 
where all other samples from this study are stored.  The samples being stored are ONLY for this 
study.  Our animal studies being done in the DBTIP Lab are identifying markers (like CMV) that 
may help the effectiveness of the study drug; therefore, as other markers are identified, we would 
like to investigate whether these same markers are found in your unused samples. 
 
If you do not agree to banking of your unused samples, you can still take part in the main research 
study. 
 
Withdrawing consent for the storage and future testing of your unused blood and tumor samples will 
result in destruction of the unused sample.  However, if you withdraw your consent after the unused 
sample has been tested, the test results and the research study/sample-related information will remain 
in any database(s) that was created for the research study.  The reason for this is to comply with 
regulations that require the sponsor to make data available for review by the FDA or other 
appropriate regulatory authorities, or if this research is used to support an application for FDA 
approval to market the study drug. 
 
If you withdraw consent for participation in the main study or are discontinued from the main study, 
the unused blood and/or tumor samples you provided will continue to be available for storage and 
future testing unless you also withdraw your consent for this purpose as stated above.  If you want to 
withdraw your permission for the storage of this blood and/or tumor for future testing, we ask that 
you contact Dr. Khasraw in writing and let him know you are withdrawing your permission for this 
unused portion of blood to be used for future research. His mailing address is: Dr. Mustafa Khasraw, 
The Preston Robert Tisch Brain Tumor Center, DUMC Box 3624, Durham, NC 27710. 
 
Please initial next to one of the statements below to indicate whether or not you agree to allow 
storage of your unused samples for possible future research as part of this study in the Duke Brain 
Tumor Immunotherapy Program (DBTIP) Laboratory. 
 
_____    Yes, I agree to allow my unused blood and/or tumor samples to be stored for future  
Initials   research in the DBTIP Lab. 
 
_____      No, I do not agree to allow my unused blood and/or tumor samples to be stored for  
Initials   future research in the DBTIP Lab. 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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WHAT IF MY TUMOR COMES BACK?  
Your tumor may need to be biopsied or removed to confirm that the tumor has in fact come back as 
part of your routine clinical care outside of this research study. You will be asked to sign a surgical 
consent form if you have a biopsy or resection (removal of the tumor).  If this is clinically indicated 
and performed here at Duke or an external institution, it is standard practice to contact you about 
consenting to provide tissue for research purposes.  If you agree to participate in this study called the 
Duke Brain Tumor Center Biorepository study, you will sign a separate research consent and we will 
use a portion of tissue from that surgery specimen as part of this research study to see how well the 
immunotherapy worked in that tissue. This sampling includes obtaining slides sampled from the 
biopsy/resection specimen block to perform immunohistochemistry detection of CMV. Obtained 
slides will then be stored in the DBTIP lab. The sampling of biopsy/resection specimens will not 
exceed 10% of the specimen block so as to preserve adequate tissue should you or your doctor want 
future diagnostic testing as part of your clinical care or other clinical trials. You do not need to agree 
to participate in the Biorepository to be in this study. 
 
Please initial next to one of the statements below to indicate whether or not you agree to allow the 
collection of slides from future biopsy/resection specimen blocks for detection of CMV studies.  
 
_____   Yes, I agree to allow histologic slides to be obtained from a future biopsy/resection  
Initials specimen for future research on CMV in tumor tissue. 
 
_____   No, I do not agree to allow histologic slides to be obtained from a future  
Initials biopsy/resection specimen for future research on CMV in tumor tissue. 
 
HOW LONG WILL I BE IN THIS STUDY? 
As long as the tumor has not grown back, you will receive the CMV pp65 DC vaccine monthly 
(following the first 3 bi-weekly vaccines) for a total of 10 vaccines (approximately 10 to 12 months 
from time of consent to vaccine #10).  If an MRI suggests that the tumor has grown back, you will 
be offered other therapy and removed from the study.  
 
After you complete the study vaccine treatments, we will continue to review your medical record for 
as long as you are followed at Duke and we may collect information about treatments you receive 
for your brain tumor.  We may request your medical records when you are seen at another hospital, 
and we will contact your local physician’s office to collect information about how you are doing. 
Whether or not your tumor progresses, we will continue to track the size of your tumor based on 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
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MRI scans taken at Duke or elsewhere.  The study team may continue to follow you through 
periodic phone calls to see how you are doing indefinitely.   
 
You can choose to stop participating at any time without penalty or loss of any benefits to which you 
are entitled. However, if you decide to stop participating in the study, we encourage you to talk to 
your doctor first. 
 
ARE THERE BENEFITS TO TAKING PART IN THE STUDY? 
Although we hope that this immunotherapy will allow your immune system to better fight your 
tumor, it is entirely possible that you will not benefit directly from this study. However, even if you 
do not benefit from the study directly, the knowledge gained as a result from your participation may 
benefit others. 
 
WHAT ARE THE RISKS OF THE STUDY? 
While in this study, you are at risk for possible side effects from the immunotherapy and the study 
procedures. You should discuss these with the study doctor. The known side effects are listed in this 
consent form, but they will vary from person to person. There may also be other side effects that you 
experience that were not predicted. Many side effects go away shortly after the study drug is 
stopped, but in some cases, side effects may be serious, long lasting, and/or permanent and may even 
cause death. Side effects that may be associated with some of the procedures used in this study are as 
follows: 
 
For those of Reproductive Potential 
Female 
Being a part of this study while pregnant may expose the unborn child to significant risks, some of 
which may be currently unforeseeable.  Therefore, pregnant women will be excluded from the study.  
If you are a woman of childbearing potential, a blood pregnancy test will be done (using 1 teaspoon 
of blood drawn from a vein by needle-stick), and it must be negative before you can continue in this 
study.  If sexually active, you must agree to use 2 forms of appropriate contraceptive measures at the 
same time for the duration of the study vaccine administration and for 6 months afterwards and for 6 
months following bevacizumab administration, if applicable. Medically acceptable contraceptives 
include: (1) surgical sterilization (such as a tubal ligation or hysterectomy), (2) approved hormonal 
contraceptives (such as birth control pills, patches, implants or injections), (3) barrier methods (such 
as a condom or diaphragm) used with a spermicide, or (4) an intrauterine device (IUD). 
Contraceptive measures such as Plan B(TM), sold for emergency use after unprotected sex, are not 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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acceptable methods for routine use.  If you do become pregnant during this study or if you have 
unprotected sex, you must inform your study physician immediately. 
 
Male 
Your participation in this research may damage your sperm, which could cause harm to a child that 
you may father while on this study.  Such harm may be currently unforeseeable.  If you are sexually 
active, you must agree to use a medically acceptable form of birth control for the duration of the 
study vaccine administration and for 6 months afterwards and for 6 months following bevacizumab 
administration, if applicable. Medically acceptable contraceptives include: (1) surgical sterilization 
(such as a vasectomy), or (2) a condom used with a spermicide.  Contraceptive measures such as 
Plan B(TM), sold for emergency use after unprotected sex, are not acceptable methods for routine use. 
You should inform your partner of the potential for harm to an unborn child.  She should know that 
if pregnancy occurs, you will need to report it to the study doctor, and she should promptly notify 
her doctor. 
 
Td Vaccine (Adult Tetanus & Diphtheria)  
With a vaccine, like any medicine, there is a chance of side effects. These are usually mild and go 
away on their own. Serious side effects are also possible, but are very rare. Most people who get Td 
vaccine do not have any problems with it. 
 
Mild Problems Following Td 
(Did not interfere with activities) 
Most Likely 
• 
Pain where the shot was given (about 8 people in 10)  
• 
Redness or swelling where the shot was given (about 1 person in 3) 
 
Likely 
• 
Mild fever (about 1 person in 15) 
 
Less Likely  
• 
Headache or Tiredness (uncommon) 
 
Moderate Problems Following Td 
(Interfered with activities, but did not require medical attention) 
• 
Fever over 102°F (rare) 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
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Severe Problems Following Td 
(Unable to perform usual activities; required medical attention) 
• 
Swelling, severe pain, bleeding and/or redness in the arm where the shot was given (rare).  
Problems that could happen after any vaccine: 
• 
Brief fainting spells can happen after any medical procedure, including vaccination. Sitting 
or lying down for about 15 minutes can help prevent fainting, and injuries caused by a fall. 
Tell your doctor if you feel dizzy, or have vision changes or ringing in the ears. 
• 
Severe shoulder pain and reduced range of motion in the arm where a shot was given can 
happen, very rarely, after a vaccination.  
• 
Severe allergic reactions from a vaccine are very rare, estimated at less than 1 in a million 
doses. If one were to occur, it would usually be within a few minutes to a few hours after the 
vaccination.  
 
CMV pp65 DC Vaccine  
Most likely 
• Pain, redness, or swelling at the injection site  
 
Less Likely 
• Allergic reaction- The study vaccine may cause an allergic reaction. Symptoms of an allergic 
reaction may include: 
o itching  
o hives  
o low blood pressure  
o difficulty breathing  
o in rare occasions, death  
• Swelling of the Brain (Cerebral Edema)- Cerebral Edema  may be caused by the tumor, a 
dramatic increase in the number of immune cells in the brain, or the killing of tumor cells. 
Symptoms of swelling of the brain include:  
o severe headaches  
o confusion  
o lack of energy  
o unconsciousness  
o coma  
o losses of movement, sensation, or function in certain areas of the body 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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May 06, 2022 
 
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• In the event that you experience brain swelling after you receive DC vaccines due to an 
inflammatory response, your physician may treat you with a reduced dose of bevacizumab 
(also known by the brand name Avastin) for the swelling. The dose of bevacizumab you 
would receive is less than the full dose that is given to treat patients for a recurrence of their 
brain tumor. Bevacizumab will be administered through the vein approximately every three 
weeks, and the effects of the bevacizumab on the swelling will be evaluated by your 
physician after each MRI you receive.  If your physician does not feel it is safe for you to 
receive bevacizumab, your physician may discuss other interventions with you, including steroids 
or surgery, to treat the inflammatory response to DC vaccination. 
• Delayed Autoimmune Disease- The CMV pp65 DC vaccine may activate the immune system 
to such a high degree that the immune system may start to attack normal brain tissue or other 
tissues in the body, although this is very unlikely this type of severe reaction can cause 
serious injury or death.  
• Infection- There may be a small risk of infection due to potential contamination of the 
injection during the manufacturing or mixing process. This may result in redness, swelling, 
and/or irritation at the injection site, and in extremely rare cases, a severe blood infection that 
could lead to death.  
 
There may also be risks with the use of this study drug with the standard of care medications and 
procedures that you will receive such as the chemotherapy that are not known. 
 
It is possible that additional side effects not previously seen or predicted may occur with the 
combination of the CMV pp65 DC vaccine and Td toxoid; these may be mild or very serious.  Please 
immediately tell the study doctor or study staff if you have any side effects or problems during the 
study. 
 
Sargramostim (GM-CSF) 
The GM-CSF Injection may cause some, all, or none of the side effects listed below: 
 
More likely 
• Headache 
• Bone pain 
• Muscle and joint pains 
• Fever and chills 
• Rash and itchiness 
• A feeling of discomfort or not feeling well and/or tiredness 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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Likely 
• Stomach or abdominal pain or cramps 
• Weakness 
• Loss of appetite 
• Nausea and/or vomiting 
• Diarrhea 
• Excessive sweating 
• Inflammation of a vein through which the vaccine was given 
• Redness and pain at the injection site 
• Weight gain 
• Fewer platelets in the blood. A low number of platelets may cause you to bruise and bleed 
more easily. 
• Increase in the blood of certain enzymes or bilirubin (a substance that comes from the liver 
where waste products are broken down), which could indicate liver irritation or damage 
• Elevation in the blood of creatinine, which normally is removed from the blood by the kidney 
and could indicate kidney damage 
• Fluid build-up in the tissues usually of the lower legs 
 
Less Likely 
• Severe allergic reaction, which can be life threatening with shortness of breath, low blood 
pressure, and a rapid heartbeat 
• A severe reaction, which can cause shortness of breath, a low blood pressure, a rapid heart 
rate, fever, a feeling of warmth and back pain and which may occur only with the first dose, 
but not with further doses 
• An abnormally rapid heartbeat 
• Leakage of fluid into the lungs which may result in shortness of breath and difficulty 
breathing and/or leakage of fluid into body tissues with puffiness of legs, arms or abdomen, 
weight gain and a drop in blood pressure 
• Inflammation of the lungs, which may lead to pain and shortness of breath 
• A build-up of fluid around the heart, which may be painful 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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Temozolomide 
 
 
Most Likely 
• loss of appetite  
• nausea and vomiting, especially on the first day of each cycle 
• constipation  
• decrease in blood cell counts   
 
A decrease in the number of white blood cells may increase your risk of infections.  A reduction in 
the number of platelets may increase the risk of bleeding and a reduced number of red blood cells 
may increase fatigue or shortness of breath.  Some people who have taken temozolomide also had 
the following side effects: 
 
Likely 
• back, abdominal and/or stomach pain, breast pain 
• diarrhea 
• hair loss 
• dry skin, skin redness, itching and/or rash 
• swelling of extremities 
• inflammation of the mouth, throat and/or sinuses 
• headache, confusion, loss of memory, dizziness, fatigue, fever, and/or weakness 
• anxiety, depression 
• joint and muscle pain 
• abnormal coordination, gait and/or feelings in extremities 
• trouble sleeping or sleepiness 
• change in sense of taste 
• visual changes such as double or blurred vision 
• coughing or shortness of breath, respiratory tract infection 
• urinary incontinence/frequency, urinary tract infection 
• weight increase 
• seizures, hemiparesis (weakness on one side of the body) 
• adrenal hypercorticisim (elevated hormone levels) 
• allergic reaction, sometimes severe 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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Less Likely 
• Rarely, unusual (“opportunistic”) infections have occurred.  Rare cases of erythema 
multiforme (skin condition) have been reported which resolved after discontinuation of 
temozolomide and, in some cases, recurred upon restarting treatment with temozolomide.  
Another, rare yet serious side effect is liver damage which may cause yellowing of eyes and 
skin, swelling and may result in liver failure. 
• Very rare side effects have included secondary cancers including leukemia and 
myelodysplastic syndrome (MDS).  MDS is a disorder of the bone marrow in which blood 
cells that do not function normally are produced. 
 
Reproductive studies have not been done with temozolomide.  Immature sperm and testicular 
atrophy occurred in studies with rats and dogs, using doses of temozolomide ¼ and 5/8 of the 
recommended human doses.  In animal studies, temozolomide caused death and multiple 
malformations in fetal rats and rabbits exposed during pregnancy. 
 
If applicable, risks of bevacizumab (a drug often used for the management of malignant 
glioma that blocks blood vessel growth; blood vessels “feeding” the tumor are required for the 
tumor to grow) include: (Note: The risks below are for the standard dose of bevacizumab used to 
treat recurrent malignant glioma. If your study doctor feels you require bevacizumab, you will 
receive a dose in the study that is half the standard dose.) 
 
Very Common (20% or more of patients)  
• High blood pressure (hypertension), which may cause headache or blurred vision  
• Abdominal Pain  
 
Common (4-20% of patients) 
• Numbness, tingling or pain in the fingers or toes (peripheral sensory neuropathy)  
• Low numbers of white blood cells (neutropenia, leucopenia and lymphopenia) potentially 
associated with fever. Low white cell count may increase the risk of infection.  
• Low numbers of platelets (thrombocytopenia)  
• Shortness of breath (dyspnea)  
• Diarrhea  
• Bleeding from the rectum (rectal hemorrhage)  
• Nausea and vomiting  
• Pain, including headache and joints pain (arthralgia)  


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
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• Alteration in speech (dysarthria)  
• Constipation  
• Mucosal inflammation or inflammation of the mouth (stomatitis)  
• Protein in the urine  
• Mucocutaneous bleeding, including nose bleed (epistaxis)  
• Lack of energy, weakness (asthenia, fatigue), or dizziness  
• Loss of appetite (anorexia), or heartburn  
• Body water loss (dehydration)  
• Fever (pyrexia)  
• Runny nose (rhinitis), stuffy nose, hoarseness, or cough  
• Dry skin, flaking and inflammation of the skin (exfoliative dermatitis), change in skin color 
(skin discoloration)  
• Change in the sense of taste (dysgeusia)  
• Problems with the eyes (eye disorder), tearing (lacrimation increased)  
• Low numbers of red blood cells (anemia) which may require blood transfusion  
• Abnormal heartbeat which may cause palpitations or fainting  
• Internal bleeding which may cause black tarry stool, vomit in blood, coughing up blood, or 
blood in urine  
• Delay in healing of wounds or spontaneous opening of wounds. Fatal outcomes have been 
reported.  
• Damage to jawbone which may cause loss of teeth  
• Allergic reaction during or after infusion of bevacizumab which may cause fever, chills, rash, 
itching, hives, low blood pressure, wheezing, shortness of breath, swelling of the face or 
throat  
• Blood clot in limbs or lungs which may cause swelling, pain or shortness of breath  
• Infection, presence of bacteria in the blood (sepsis), collection of pus in tissue or organs 
(abscess)  
 
Occasional (3% or less of patients) 
• A tear or a hole in the gut (perforation of the gastrointestinal tract)  
• Abnormal tube-like connection (fistula) between internal organs such as the nose, throat, 
lungs, esophagus, rectum or vagina that are not normally connected. These conditions may 
cause serious infections or bleeding and require surgery to repair.  
• Bleeding (hemorrhage), including bleeding associated with the tumor  
• Clogging of a vessel in the lung (pulmonary embolism)  


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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• Blocking of the arteries by a blood clot, including stroke (cerebral vascular accident) or heart 
attack. This risk is significantly increased in patients who are elderly or with a history of 
diabetes.  
• Heart failure (cardiac failure congestive), especially in patients who have taken certain 
chemotherapy treatments in the past (doxorubicin or mitoxantrone) and rapid beating of the 
heart (supraventricular tachycardia)  
• Rapid beating of the heart (supraventricular tachycardia)  
• Blood clots in the veins (deep vein thrombosis)  
• Abdominal pain  
• Blockage in the intestine (ileus, intestinal obstruction)  
• Pain, tenderness, or blistering on the fingers or feet (hand-foot syndrome, palmar-plantar 
erythrodysesthesia syndrome)  
• Reduced consciousness, sleepiness, feeling tired (somnolence, lethargy)  
• Low levels of oxygen in the blood (hypoxia)  
• Fainting (syncope)  
• Gastrointestinal disorder  
• Voice changes, hoarseness (dysphonia)  
• Muscular pain (myalgia) and muscular weakness  
• Flesh-eating bacteria syndrome, an infection in the deep layers of the skin  
• Kidney damage which may require dialysis  
 
Uncommon (0.1% to Less than 1% of patients)  
• Abnormal connection between the windpipe (trachea) and the esophagus (the tube that 
connects the mouth to the stomach) (tracheo-esophageal fistula)  
• A hole in the gut lining of the stomach or duodenum (gastro-intestinal ulcer)  
 
Rare (0.01% to Less than 0.1% of patients)  
• Reversible posterior leukoencephalopathy syndrome: this may include symptoms of impaired 
brain function (headaches, vision changes, confusion, or seizures), and often, high blood 
pressure  
 
Very Rare (Less than 0.01% of patients)  
• Hypertensive encephalopathy: this may include symptoms of impaired brain function 
(headaches, vision changes, confusion, or seizures), and often, high blood pressure  
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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May 06, 2022 
 
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Frequency Unknown  
• Lesion in the gums with an exposed jawbone that does not heal and may be associated with 
pain and inflammation of the surrounding tissue (osteonecrosis of the jaw) in particular when 
treated with “bisphosphonate drugs” in this trial or in the recent past.  
• A hole in the gallbladder (gallbladder perforation)  
• A hole in the nasal passage (nasal septum perforation)  
• Abnormalities to the fetus/unborn child when bevacizumab is given during pregnancy  
 
In trials for colorectal cancer using bevacizumab and chemotherapy, female subjects had a 32% 
higher incidence of ovarian failure with early menopause (loss of menstrual cycle) and sterility 
(inability to have children) than subjects using chemotherapy alone. 
 
Blood Draws:  Blood draws (venipuncture) are the taking of blood from a vein in your arm by 
needle stick.  The risks associated with drawing blood from your arm include momentary discomfort 
and/or bruising.  Infection, excess bleeding, clotting, or fainting is also possible, although unlikely. 
 
MRI:  If you take part in this research, you will have an MRI.  MRI uses a magnet and radio waves 
to make images (pictures) of the inside of the head.  There have been no ill effects reported from 
exposure to the magnetism or radio waves used for these studies; however, it is possible that harmful 
effects could be recognized in the future.  A known risk is that the magnet could attract certain kinds 
of metal that may cause injury to you.  You will be asked about metal within your body (this 
includes certain dyes used in tattoos and body piercings).  If there is any question about potentially 
hazardous metal within your body, you will not be able to participate in this research study.  The 
MRI room will be locked so that no one carrying metal objects enters the room while you are having 
this scan performed.  In addition, the MRI scanner makes a loud buzzing noise that could affect 
hearing ability.  You will be provided with earplugs and assistance in their use in order to protect 
your hearing.  You will be able to communicate with the scanner technologist using an intercom 
and/or signaling device.  The technologist will try to help you feel as comfortable as possible in the 
scanner.  You can ask to stop the scan and be removed from the scanner at any time by using the 
intercom or signaling device. 
 
As mentioned above, each of the MRIs will be conducted with a contrast agent to better see if any 
tumor is present.  Contrast is given routinely to obtain better MRI scans of the brain.  It is 
administered through a catheter placed in your vein.  The catheter placement is similar to drawing 
blood except that the catheter remains in the vein during the time the contrast agent is being 
delivered.  The risks of a blood draw and insertion of a catheter are similar.  There have been a few, 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
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rare cases of allergies to the agent used in MRI contrast enhanced scans.  Subjects with allergies 
(such as rash) may be given Tylenol (acetaminophen) and Benadryl (diphenhydramine) prior to 
injection of the contrast. 
 
A rare but serious adverse reaction has been observed in patients that received a gadolinium-based 
contrast material during MRI examinations, a reaction called nephrogenic systemic fibrosis (NSF).  
Patients with kidney disease are at increased risk of developing NSF.  NSF may cause skin 
thickening, joint pain and/or swelling.  In rare cases, NSF can lead to lung and heart problems and 
cause death.  To minimize the likelihood that you will be affected, you will have a blood test to 
measure your kidney function.  If your blood test is abnormal, you will not be permitted to receive 
gadolinium. 
 
Leukapheresis:  Some side effects associated with the leukapheresis procedure may be similar to 
those experienced during blood donation and include discomfort or a swollen bruise at the site of 
needle puncture, a slight risk of developing a local infection at the site, and development of a small 
scar. In addition, there can be light-headedness, fainting, vomiting, and rapid breathing. Some side 
effects that may occur are unique to the leukapheresis procedure and include chills caused by 
cooling of the blood when it is contained in the special machine, as well as tingling and nausea 
caused by the blood thinning medicine (citrate) that prevents the blood from clotting while in transit 
within the machine or its tubing. These side effects can be controlled by slowing the rate at which 
blood is withdrawn, by warm blankets, by changing the amount of the blood thinning medicine, by 
giving calcium supplements (tablets taken by mouth or liquid administered by vein), or by 
discontinuing the procedure. Rarely, the leukapheresis procedure may be associated with loss of 
blood, breakdown of the blood, clotting of the blood, allergic reactions, accidental addition of air to 
the blood going back to you, and fluid overload resulting in shortness of breath or fluid loss resulting 
in decreased blood pressure.  If you require a central venous catheter for the leukapheresis 
procedure, the risks associated with this deeper venous catheter are air in the chest (~6%) due to a 
punctured lung, bleeding or fluid in the chest (~2%), and bleeding under the skin (~0.3%) and 
infection (~3%). However, procedures have been developed that use ultrasound (US) devices and 
fluoroscopy to provide imaging of the central veins during catheter placement. The advantages 
associated with using US and fluoroscopy include detection of exact vessel location, avoiding veins 
with clots, and guiding the proper placement of the catheter. Only personnel trained in the use of US 
and fluoroscopy central line placement at Duke will perform these procedures.  These procedures 
involve a small amount of radiation. The radiation exposure from this procedure is about 200 
microsievert.  
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
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The side effects listed for leukapheresis can usually be controlled and treated by discontinuing the 
leukapheresis procedure and by providing appropriate and immediate medical care. In addition, there 
is always the risk that very uncommon or previously unknown side effects may occur or that life-
threatening side effects may occur and death may result. 
 
Other Risks:  Subclinical (no signs or symptoms) autoimmunity may occur, although it is extremely 
unlikely.  This could be secondary to the disease process itself, the surgical procedure, radiation, 
CMV pp65 DC vaccine, Td toxoid, chemotherapy, the immune response in the brain, or the 
destruction of tumor cells.  Symptoms vary according to the particular disorder and are many; 
however, some of the symptoms could include depression, fatigue, itching/rash, nausea and/or 
vomiting, diarrhea, cramping, eye irritation/pain/redness/swelling, or various symptoms from 
pituitary gland disturbances.  You will be monitored throughout the course of the study for any of 
these symptoms.  Symptoms that fail to respond to medical therapy may lead to permanent 
impairment or even death.  
 
Drug and Food Interactions: For your safety, you must tell the study doctor or study nurse about 
all the prescribed medical foods and drugs, herbal products, over-the-counter (OTC) drugs, vitamins, 
natural remedies and alcohol that you are taking before you start the study and before taking any of 
these products while you are on the study. 
 
Due to the possible interference by antihistamines (such as Benadryl, Zyrtec, etc.), you are requested 
to avoid taking any antihistamines 2 days before, on the day of the vaccine administration, and for 2 
days following the vaccine administration.  If you have a condition or allergy that requires that you 
continue using antihistamines, please alert the study team.  Dr. Khasraw and your doctors will decide 
if it is safe for you to hold the medication during vaccine administrations. 
 
There may be risks, discomforts, drug interactions or side effects that are not yet known. 
 
WHAT ALTERNATIVES ARE THERE TO PARTICIPATION IN THIS 
STUDY? 
Your decision to receive this vaccine study drug is completely voluntary.  You do not have to 
receive the CMV pp65 DC vaccine if you don’t want to, and you can change your mind at any time.  
There will be no penalty to you, and you won’t lose any benefits to which you are otherwise entitled.  
Your regular medical care will not change if you decide not to receive the CMV pp65 DC vaccine.  
If you want to stop receiving the vaccine, please tell your study doctor. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
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Alternative treatments for your cancer could include further surgery, chemotherapy, radiation, or 
participation in a different research study protocol.  You can also elect to have treatment of your 
symptoms only. This is frequently referred to as comfort care.  Comfort care includes pain 
medication and other support.  It aims to maintain your comfort and dignity rather than cure disease.  
Usually this care can be provided at home. 
 
Please discuss all of your choices with the study doctor. 
 
WILL MY INFORMATION BE KEPT CONFIDENTIAL? 
Participation in research involves some loss of privacy. We will do our best to make sure that 
information about you is kept confidential, but we cannot guarantee total confidentiality. Your 
personal information may be viewed by individuals involved in this research and may be seen by 
people including those collaborating, funding, and regulating the study. We will share only the 
minimum necessary information in order to conduct the research. Your personal information may 
also be given out if required by law.  Any human samples received and stored by the DBTIP will be 
assigned an All Laboratory Processed Specimen (ALPS) number (a unique number assigned only to 
samples) and will also include the assigned study number, type of sample, and date received. No 
stored samples have subject initials. 
 
Your study records will be kept in a secure area and only authorized personnel will have access. As 
part of the study, Dr. Mustafa Khasraw and his study team will have access to the study results and 
will report the results and dates of your study-related tests and procedures to those named below. 
Some of these tests and procedures would have been done as part of your regular care.  The PI will 
use these test results both to treat you and to complete this research. These test results will be 
recorded in your medical record and will be reported to the Department of Neurosurgery at Duke 
University Health System. Results of tests and studies done solely for this research study and not as 
part of your regular care will also be included in your medical record.  As a participant of this study, 
you have the right to access your personal health information at any time. 
 
Your records may be reviewed in order to meet federal or state regulations. Reviewers may include 
representatives from the Food and Drug Administration, the National Institute of Health, the 
National Cancer Institute, the Department of Neurosurgery at Duke, the Duke University Health 
System Institutional Review Board, the Duke Office of Audit, Risk and Compliance, the Duke 
Cancer Institute, the Institutional Review Board (IRB), Annias Immunotherapeutics, Inc., and others 
as appropriate. If your records are reviewed by any of these groups, they may also need to review 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 25 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
your entire medical record.  If this information is disclosed to outside reviewers for audit purposes, it 
may be further disclosed by them and may not be covered by the federal privacy regulations. 
 
The study results will be retained in your research record for at least six years after the study is 
completed. At that time, either the research information not already in your medical record may be 
destroyed or information identifying you will be removed from the study results at Duke University 
Health System. Any research information in your medical record will be kept indefinitely. 
 
Your study data will be provided to Annias Immunotherapeutics, Inc.; however, your identity will 
not be revealed. 
 
While the information and data resulting from this study may be presented at scientific meetings or 
published in a scientific journal, your name or other personal information will not be revealed. 
 
You will be told of any significant new findings developed during the course of this study that may 
relate to your willingness to continue your participation. 
 
Some information collected about you only for this research study may be kept in a research study 
record separate from your medical record, and some research information may also be part of your 
medical record.  You will not have access to this research information until the end of the study.  
However, it will be available to your physicians if needed for your care. 
 
Some people or groups who receive your health information might not have to follow the same 
privacy rules. Once your information is shared outside of DUHS, we cannot guarantee that it will 
remain private. If you decide to share private information with anyone not involved in the study, the 
federal law designed to protect your health information privacy may no longer apply to the 
information you have shared.  Other laws may or may not protect sharing of private health 
information. 
 
The Department of Health and Human Services (DHHS) has issued a Certificate of Confidentiality 
to further protect your privacy. With this Certificate, the investigators cannot be forced (for example 
by court subpoena) to disclose research information that may identify you in any Federal, State, or 
local civil, criminal, administrative, legislative, or other proceedings. Disclosure will be necessary, 
however, upon request of DHHS for audit or program evaluation purposes. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 26 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
You should understand that a Confidentiality Certificate does not prevent you or a member of your 
family from voluntarily releasing information about yourself or your involvement in this research. 
Note however, that if an insurer or employer learns about your participation, and obtains your 
consent to receive research information, then the investigator may not use the Certificate of 
Confidentiality to withhold this information. This means that you and your family must also actively 
protect your own privacy. 
 
Finally, you should understand that the investigator is not prevented from taking steps, including 
reporting to authorities, to prevent serious harm to yourself or others. 
 
WHAT ARE THE COSTS? 
You or your insurance provider will be responsible and billed for all costs related to your routine 
medical care, including copayments and deductibles. Routine medical care services are those that 
you would have received for your condition if you were not participating in this research study. Not 
all services are covered by insurance.  Some procedures or scans may require pre-authorization by 
your insurance plan. We will notify you if we learn that a service is not covered by your insurance 
plan as part of the pre-authorization process. If it is not covered, you will be responsible for paying 
for it.  The amount of your out-of-pocket expense will depend on your insurance plan.  For 
beneficiaries with Medicare Advantage Plans, traditional Medicare is billed for the routine cost of a 
research study.  You may have more or higher co-pays than with a Medicare Advantage plan.  Please 
discuss the costs of the study with your study doctor.  At your request, a Financial Counselor in the 
clinic may provide you with an estimate of costs for routine services. 
 
Taking part in this study may cost you and/or your insurance company more than the cost of getting 
regular medical treatment.   
 
Please talk with the study team about the specific services and procedures that the sponsor will pay 
for, and the ones for which you or your insurance will be responsible.  We will monitor your DUHS 
patient care charges to make sure that costs are directed appropriately.  If you have any questions or 
concerns about appropriate billing, contact your study team coordinator so that he/she can help find a 
resolution. 
 
Duke will provide the pp65 CMV DC vaccines free of charge to you while you are taking part in the 
study.  Other research-related costs that will be provided free of charge to you while you are taking  


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 27 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
part in the study are listed below.  The following are research-related costs that will not be charged 
to you or your insurance: 
• 
Leukapheresis 
• 
Td vaccine (Adult Tetanus & Diphtheria) used in preconditioning  
• 
DC vaccines 
• 
Immunologic testing on your blood samples 
 
WHAT ABOUT COMPENSATION? 
You will receive no compensation for participating in this study. There are also no plans to provide 
any compensation to you for any new products or discoveries that may result from your participation 
in this research or from the use of your blood or tumor samples. 
 
WHAT ABOUT RESEARCH RELATED INJURIES? 
Immediate necessary medical care is available at Duke University Medical Center in the event that 
you are injured as a result of your participation in this research study. However, there is no 
commitment by Duke University, Duke University Health System, Inc., or your Duke physicians to 
provide monetary compensation or free medical care to you in the event of a study-related injury. 
 
For questions about the study or research-related injury, contact Dr. Khasraw at 919-684-5301 
during regular business hours and at 919-206-0493 after hours and on weekends and holidays. 
 
WHAT ABOUT MY RIGHTS TO DECLINE PARTICIPATION OR 
WITHDRAW FROM THE STUDY? 
You may choose not to be in the study, or, if you agree to be in the study, you may withdraw from 
the study at any time. If you withdraw from the study, no new data about you will be collected for 
study purposes unless the data concern an adverse event (a bad effect) related to the study.  If such 
an adverse event occurs, we may need to review your entire medical record. 
 
Your decision not to participate or to withdraw from the study will not involve any penalty or loss of 
benefits to which you are entitled, and will not affect your access to health care at Duke. If you do 
decide to withdraw, we ask that you contact Dr. Mustafa Khasraw in writing and let him know that 
you are withdrawing from the study. His mailing address is: Dr. Mustafa Khasraw, The Preston 
Robert Tisch Brain Tumor Center, DUMC 3624, Durham, N.C. 27710. Dr. Khasraw may ask you to 
return for a check-up before you stop your study vaccinations if he thinks that stopping the 
vaccination suddenly may harm you. He may also ask you to complete the tests that would ordinarily 
occur when a person completes the study.  


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 28 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
 
We will tell you about new information that may affect your health, welfare, or willingness to stay in 
this study. 
 
Your doctor may decide to take you off this study if your condition gets worse, if you have serious 
side effects, or if your study doctor determines that it is no longer in your best interest to continue.  
The sponsor or regulatory agencies may stop this study at any time without your consent. Reasons 
why this might occur include:  if you need additional medication, don’t follow the study plan, 
experience a study-related injury, or for administrative reasons. If this occurs, you will be notified 
and your study doctor will discuss other options with you. 
 
Your samples and/or data may be stored and shared for future research without additional informed 
consent if identifiable private information, such as your name and medical record number, are 
removed. If your identifying information is removed from your samples or data, we will no longer be 
able to identify and destroy them. 
 
Please initial next to one of the statements below to indicate whether or not you agree to allow your 
samples and/or data to be stored and shared for future research without additional informed consent 
if identifiably private information is removed.  
 
_____    Yes, I agree to allow my samples and/or data to be stored and shared for future research 
Initials  without additional informed consent if identifiably private information is removed. 
 
_____    No, I do not agree to allow my samples and/or data to be stored and shared for future 
Initials  research without additional informed consent if identifiably private information is removed. 
 
If you agree to allow your tissue/blood/cells to be kept for future research with identifying 
information that could link your sample to you, you are free to change your mind at any time. We 
ask that you contact Dr. Khasraw in writing and let him know you are withdrawing your permission 
for your identifiable tissue/blood/cells to be used for future research. His mailing address is Dr. 
Mustafa Khasraw, The Preston Robert Tisch Brain Tumor Center, DUMC 3624, Durham, N.C. 
27710. At that time, we will ask you to indicate in writing if you want the unused identifiable 
tissue/blood/cells destroyed or if your samples (having all identifying information removed that 
would link the sample to you) could be used for other research. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 29 of 30 
Subject Initials: 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
The use of your data and samples may result in commercial profit. You will not be compensated for 
the use of your data and samples other than what is described in this consent form. 
 
A description of this clinical trial will be available on https://www.ClinicalTrials.gov, as required by 
U.S. Law.  This website will not include information that can identify you.  At most, the website will 
include a summary of the results.  You can search this website at any time. 
 
WHOM DO I CALL IF I HAVE QUESTIONS OR PROBLEMS? 
For questions about the study or a research-related injury, or if you have problems, concerns, 
questions, complaints or suggestions about the research, contact Dr. Khasraw at 919-684-5301 
during regular business hours and at 919-206-0493 after hours and on weekends and holidays. 
 
For questions about your rights as a research participant, or to discuss problems, concerns or 
suggestions related to the research, or to obtain information or offer input about the research, 
contact: 
 
WCG Institutional Review Board (WCG IRB)  
1019 39th Avenue SE Suite 120  
Puyallup, Washington 98374-2115  
Telephone: 855-818-2289 or 360-252-2500  
E-mail: researchquestions@wcgirb.com 
 
WCG IRB is a group of people who perform independent review of research.  
 
WCG IRB will not be able to answer some study-specific questions, such as questions about 
appointment times. However, you may contact WCG IRB if the research staff cannot be reached or if 
you wish to talk to someone other than the research staff.  
 
You may also contact the Duke University Health System Institutional Review Board (IRB) Office at 
(919) 668-5111. 
 


 
 
 
 
Consent To Participate In A Research Study 
I-ATTAC: Improved Anti-Tumor Immunotherapy  
Targeted Against Cytomegalovirus in Patients with  
Newly-Diagnosed WHO Grade IV Unmethylated Glioma  
 
 
IRB APPROVED 
AS MODIFIED 
May 06, 2022 
 
Protocol ID: Pro00090683 
Page 30 of 30 
 
DUKE UNIVERSITY HEALTH SYSTEM 
Form 
M0345 
STATEMENT OF CONSENT 
"The purpose of this study, procedures to be followed, risks and benefits have been explained to 
me.  I have been allowed to ask questions, and my questions have been answered to my 
satisfaction.  I have been told whom to contact if I have questions, to discuss problems, concerns, 
or suggestions related to the research, or to obtain information or offer input about the research.  
I have read this consent form and agree to be in this study, with the understanding that I may 
withdraw at any time.  I have been told that I will be given a signed and dated copy of this 
consent form." 
 
 
 
 
 
 
 
 
Signature of Subject 
Date 
Time 
 
 
 
 
 
 
 
 
Signature of Person Obtaining Consent 
Date 
Time