ACTION Trial: Adoptive Cellular Therapy Following Dose-Intensified Temozolomide in Newly-diagnosed Pediatric High-grade Gliomas (Phase I).
Phase: PHASE1
Conditions: Malignant Glioma, High Grade Glioma
Sponsor: University of Florida

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MCC Protocol #: 18752 
 
ClinicalTrials.gov Identifier: NCT03334305 
 
Title: ACTION Trial 
Adoptive Cellular Therapy following Dose-Intensified Temozolomide in Newly-diagnosed Pediatric High-
grade Gliomas (Phase I). 
 
Corresponding Organization: 
Moffitt Cancer Center 
12902 Magnolia Drive 
Tampa, FL 33612 
T: 813.742.7412 
F: 813.745.5666 
Email: SunshineProject@moffitt.org 
 
Coordinating Centers: 
 
Moffitt Clinical Research Network (MCRN) 
Moffitt Cancer Center  
12902 Magnolia Drive 
Tampa, FL 33612 
T: 813.745.6250 
F: 813.745.5666 
Email: SunshineProject@moffitt.org 
 
Preston A. Wells, Jr. Center for Brain Tumor Therapy  
University of Florida Brain Tumor Immunotherapy Program 
Lillian S. Wells Department of Neurosurgery  
Department of Neurosurgery 
Gainesville, FL  
T: 352-273-9000 
Email: duane.mitchell@neurosurgery.ufl.edu 
 
Protocol Chair: 
 
Duane A. Mitchell, MD, PhD, Immunotherapy Chair 
Preston A. Wells, Jr. Center for Brain Tumor Therapy  
University of Florida Brain Tumor Immunotherapy Program  
Department of Neurosurgery 
 
Site Principal Investigators 
 
Elias Sayour, MD, PhD 
University of Florida  
Gainesville, FL 
 
Girish Dhall, MD 
University of Alabama 
Birmingham, AL 
 
Eugene Hwang, MD 
Children’s National Medical Center 
Washington, DC 
 
Co- Investigators 
 
Sunshine Project Members 

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Statisticians: 
 
 
 
 
Jun (Vivien) Yin, PhD 
  
 
Moffitt Cancer Center 
  
 
 
12902 Magnolia Dr., MRC-Bio2  
  
 
Tampa, FL, 33612 
  
 
T: 813-745-2679 
 
  
 
Vivien.Yin@moffitt.org 
  
Responsible Research Nurse: 
N/A 
 
Study Coordinator: 
 
Sunshine Project Coordinator 
 
 
12902 Magnolia Dr. 
Tampa, FL 33612 
T: 813.745.6250 
SunshineProject@moffitt.org 
 
Responsible Data Manager: 
 
Sunshine Project Coordinator 
 
Moffitt Clinical Research Network  
12902 Magnolia Drive 
Tampa, FL 33612 
T: 813.745.6250 
SunshineProject@moffitt.org 
 
Sponsor: 
National Pediatric Cancer Foundation (NPCF) 
 
 
 
NCI-Supplied Agent(s):  N/A Other  
 
Agent(s):  Temozolomide (TMZ) 
 
Total tumor mRNA-pulsed autologous Dendritic Cells (DCs) (TTRNA-DCs) and tumor-
specific ex vivo expanded autologous lymphocyte transfer (TTRNA-xALT) with or without 
autologous hematopoietic stem cells (HSCs) 
 
IND#: 17298 
 
IND Sponsor: Mitchell 
 
Protocol Type / Version # / Version Date: 
Original/ Version 1/Version Date: September 21, 2015 
 
Amendment/Version 2/Version Date: May 31, 2016 
 
Amendment/version 3/ Version Date: July 8, 2016 
 
Amendment/Version 4/Version Date: September 22, 2016 
 
Amendment/Version 5/Version Date: December 9, 2016 
 
Amendment/Version 6/Version Date: February 16, 2017 
 
Amendment/Version 7/Version Date: May 10, 2017 
 
Amendment/Version 8/Version Date: January 5, 2018 
 
Amendment /Version 9/Version Date: July 26. 2018 
 
Amendment /Version 10/Version Date: January 7, 2019 

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Amendment/Version 11/Version Date: August 27, 2020 
 
Amendment/Version 12/Version Date: November 30, 2021 
Amendment/Version 13/Version Date: May 5, 2022 
Amendment/Version 14/Version Date: September 23, 2022 
Amendment/Version 14.1/Version Date: October 24, 2022 
Amendment/Version 15/Version Date: April 25, 2025 
 
 
 
 

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SCHEMA 
 
 
 
 
 
 
Screening consent will be obtained prior to surgical resection for tumor preservation. If pathology is appropriate and tumor 
tissue available with screening consent, treatment consent will be obtained . 
 
 

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CONTENTS 
1. 
BACKGROUND ..................................................................................................................................... 20 
1.1 
Study Disease(s) ...................................................................................................................................... 20 
1.2 
Investigational Agent(s) .......................................................................................................................... 20 
1.3 
Summary of Background and Supporting Data ...................................................................................... 21 
1.4 
Pre-clinical Experience ........................................................................................................................... 25 
1.5 
Clinical Experience ................................................................................................................................. 27 
1.5.1 Lymphopenia and Homeostatic Proliferation ............................................................................. 27 
1.5.2 RNA-Loaded Dendritic Cells ...................................................................................................... 29 
1.6 
DC + ALT Therapy ................................................................................................................................. 29 
1.6.1 RNA loaded DC vaccines and amplified tumor RNA (preclinical and clinical studies) ............. 29 
1.6.2 Amplified Total Tumor RNA from human malignant brain tumors ........................................... 30 
1.7 
Autoimmune Encephalomyelitis ............................................................................................................. 30 
1.8 
Rationale.................................................................................................................................................. 31 
1.9 
Re-MATCH phase 1 clinical trial of adoptive cellular therapy in recurrent medulloblastoma (reMB) 
and PNETs............................................................................................................................................... 33 
1.10 
Improved T cell Expansion ..................................................................................................................... 33 
1.11 
Study Design ........................................................................................................................................... 33 
1.12 
On-Study Tests and Procedures .............................................................................................................. 34 
1.13 
Correlative Studies Background .............................................................................................................. 36 
2. 
PATIENT SELECTION ......................................................................................................................... 36 
2.1 
Inclusion Criteria Screening .................................................................................................................... 36 
2.2 
Exclusion Criteria .................................................................................................................................... 37 
2.3 
Inclusion of Women and Minorities ........................................................................................................ 37 
2.4 
Consent/Assent Procedures ..................................................................................................................... 37 
3. 
REGISTRATION PROCEDURES ......................................................................................................... 38 
3.1 
Site and Investigator Registration with Moffitt Cancer Center .............................................................. 38 
3.2 
Patient Registration ................................................................................................................................. 38 

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3.3 
General Guidelines .................................................................................................................................. 39 
4. 
TREATMENT PLAN ............................................................................................................................. 39 
4.1 
Agent Administration .............................................................................................................................. 39 
4.1.1 Radiation Guidelines ................................................................................................................... 40 
4.1.2 Dose Escalation for Temozolomide Cycles 1-6 following radiation .......................................... 41 
4.1.3 Criteria for Starting Subsequent Cycles ...................................................................................... 42 
4.1.4 Immune Monitoring .................................................................................................................... 43 
4.1.5 Follow-up .................................................................................................................................... 43 
4.2 
General Concomitant Medication and Supportive Care Guidelines ....................................................... 44 
4.2.1 Treatment of pseudo-progression ................................................................................................ 44 
4.2.2 PCP Prophylaxis .......................................................................................................................... 44 
4.2.3 Neurosurgical Procedures ........................................................................................................... 44 
4.3 
Duration of Therapy ................................................................................................................................ 44 
4.3.1 Subject Withdrawal ..................................................................................................................... 45 
4.3.2 Subject Death .............................................................................................................................. 45 
4.3.3 Off Treatment Criteria ................................................................................................................. 46 
4.3.4 Off Study Criteria ........................................................................................................................ 46 
4.4 
Duration of Follow Up ............................................................................................................................ 46 
4.5 
Criteria for Removal from Study ............................................................................................................ 46 
4.6 
Criteria for Evaluable Subjects ................................................................................................................ 46 
4.7 
Replacement ............................................................................................................................................ 47 
5. 
DOSING DELAYS/DOSE MODIFICATIONS ..................................................................................... 47 
5.1 
Definition of Dose-Limiting Toxicity (DLT) .......................................................................................... 47 
5.2 
Dose Modification ................................................................................................................................... 48 
5.3 
Adverse Events and Management ........................................................................................................... 48 
6. 
SAFETY CONSIDERATIONS .............................................................................................................. 50 
6.1 
Allergic Reactions to DC Immunization or TTRNA-xALT ................................................................... 50 
6.2 
Cerebral Edema ....................................................................................................................................... 50 
6.3 
Infection .................................................................................................................................................. 51 
6.4 
Delayed Autoimmune Diseases .............................................................................................................. 51 
6.5 
Leukapheresis .......................................................................................................................................... 51 
6.5.1 Central Venous Catheter Placement ............................................................................................ 52 
7. 
ADVERSE EVENTS: DEFINITIONS AND REPORTING REQUIREMENTS .................................. 52 

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7.1 
Adverse Event Characteristics ................................................................................................................ 52 
7.2 
Expedited Adverse Event Reporting (Serious Adverse Event) ............................................................... 53 
7.2.1 Determination of Reporting Requirements: ................................................................................ 53 
7.2.2 Steps to Determine if an Adverse Event is to be reported in an Expedited Manner ................... 53 
7.2.3 Serious Adverse Event Reporting ............................................................................................... 54 
7.2.4 Distribution of Adverse Event and Serious Adverse Event Reports ........................................... 55 
7.2.5 Expedited Reporting Guidelines - Death .................................................................................... 55 
7.3 
Routine Adverse Event Reporting .......................................................................................................... 55 
7.4 
Secondary Malignancy ............................................................................................................................ 56 
8. 
PHARMACEUTICAL INFORMATION ............................................................................................... 57 
8.1 
Names, Classification, and Mechanism of Action .................................................................................. 57 
8.2 
Packaging and Labeling .......................................................................................................................... 57 
8.3 
Dispensing and Preparation ..................................................................................................................... 57 
8.4 
Commercial Agent(s) Non-investigational ............................................................................................. 58 
8.5 
GCSF, GMCSF, and Td Vaccine Risks .................................................................................................. 59 
9. 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES .............................................................. 61 
10. STUDY CALENDAR ............................................................................................................................. 63 
10.1 
Study Calendar Descriptions ................................................................................................................... 66 
10.2 
Laboratory Evaluations ........................................................................................................................... 67 
10.3 
Radiologic Evaluations ............................................................................................................................ 67 
11. MEASUREMENT OF EFFECT ............................................................................................................. 67 
11.1 
Immunologic confounders of radiographic response .............................................................................. 67 
11.2 
Antitumor Effect – Solid Tumors............................................................................................................ 68 
11.2.1 Definitions ................................................................................................................................... 68 
11.3 
General Methodology for Determining Tumor Measurements............................................................... 68 
11.4 
Tumor Response Criteria ........................................................................................................................ 69 
11.4.1 Evaluation of Best Overall Response .......................................................................................... 71 
11.4.2 Progression-Free Survival ........................................................................................................... 71 
12. DATA REPORTING / REGULATORY REQUIREMENTS ................................................................ 72 
12.1 
Data Reporting ........................................................................................................................................ 72 
12.1.1 Method for Data Reporting and Monitoring ............................................................................... 72 
12.1.2 Responsibility for Data Submission ............................................................................................ 74 

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12.2 
Moffitt Cancer Center Guidelines ........................................................................................................... 74 
12.2.1 Suspension/Termination .............................................................................................................. 74 
12.2.2 Trial Discontinuation................................................................................................................... 74 
12.2.3 Protocol Modifications ................................................................................................................ 74 
13. STATISTICAL CONSIDERATIONS .................................................................................................... 75 
13.1 
Study Design/Endpoints .......................................................................................................................... 75 
13.1.1 Analysis Sets ............................................................................................................................... 75 
13.1.2 Patient Demographics and other Baseline Characteristics .......................................................... 75 
13.1.3 Treatments ................................................................................................................................... 75 
13.1.4 Primary Objective........................................................................................................................ 75 
13.1.5 Secondary Objectives .................................................................................................................. 75 
13.1.6 Interim Analysis .......................................................................................................................... 76 
13.1.7 Safety endpoint statistical analysis and stopping criteria ............................................................ 76 
13.1.8 Handling of missing values, censoring, and discontinuations ..................................................... 76 
13.2 
Sample Size/Accrual Rate ....................................................................................................................... 76 
13.3 
Stratification Factors ............................................................................................................................... 77 
14. REFERENCES ........................................................................................................................................ 78 
15. APPENDIX A ......................................................................................................................................... 86 
16. APPENDIX B ......................................................................................................................................... 87 
 
 
 
 
 

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LIST OF POSSIBLE ABBREVIATIONS 
 
Ab
Antibody
AB
Alpha Beta/ Antibody
ABC
Automated Blood Count
ABMT 
Autologous Bone Marrow Transplantation 
ACTB
Beta Actin
ACD
Acid Citrate Dextrose
ACLS
Advanced Cardiac Life Support
ACTH
Adrenocorticotropic Hormone
AE
Adverse Event
AIDS 
Acquired Immunodeficiency Syndrome 
ALL
Acute Lymphoblastic Leukemia
ALK PHOS
Alkaline Phosphatase
ANOVA
Analysis Of Variance
Anti-HB
Anti- Hepatitis B
ANC
Absolute Neutrophil Count
ANLL 
Acute Non-Lymphatic Leukemia 
AML 
Acute Myelogenous Leukemia 
APAAP
Alkaline Phosphatase Antialkaline Phosphatase Complex
APC
Allophycocyanin
ALPS
Autoimmune LymphoProliferative Syndrome
ALT
Autologous Lymphocyte Transfer
nALT
Naïve Autologous Lymphocyte Transfer
xALT 
Ex vivo Activated Autologous Lymphocyte Transfer 
αCD3
anti-cell differentiation antigen 3
αIFN-γ
anti-interferon alpha
αTNF-α
anti-tumor necrosis factor-alpha
αIL2
anti-interleukin 2
AST
Aspartate Aminotransferase
ACTIVATE 
A Complementary Trial of an Immunotherapy Vaccine Against  Tumor-
Specific EGFRvIII 
AT
Ambient Temperature
AUC
Area under the concentration time curve
BD-FACS 
Becton-Dickinson Fluorescence Activated Cell Sorting 
β-HCG
Beta-Human Chorionic Gonadotropin
BMT
Bone Marrow Transplant
BMTU 
Bone Marrow Transplant Unit 
BSA
Bovine Serum Albumin
BSN
Bachelors of Science in Nursing
BTSC
Brain Tumor Stem Cells
BTIP
Brain Tumor Immunotherapy Program
BU
Busulfan
BUN 
Blood Urea Nitrogen 

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C
Chemotherapy
Ca
Calcium
Cc
Cubic Centimeters
C3D 
Cancer Clinical Database 
CA
California
CaBIG
Cancer Biomedical Informatics Grid
cDNA
Complimentary Deoxyribonucleic Acid
CDPP
Cisplatin
CEA
Carcinoembryonic Antigen
caAEARS 
Cancer Adverse Event Reporting System 
CD133
Cell Differentiation 133
CEF
Cytomegalovirus, Epstein Barr virus, Flu virus
CDE
Common Data Elements
CFA
Complete Freund’s Adjuvant
CFC
Cytokine Flow Cytometry
CLIA 
Clinical Laboratory Improvement Act 
CMP
Comprehensive Metabolic Panel
CMV
Cytomegalovirus
CNC
Clinical Neurologic Change
CNS
Central Nervous System
CO2 
Carbon Dioxide
Con-A
Concanavalin A
Co-PI 
Co-Primary Investigator 
CPC
Cancer Protocol Committee
Cr
Serum Creatinine
CR
Complete Response
CRF
Conformance Review Checklist
CSF
Cerebro Spinal Fluid
CSI 
Craniospinal Irradiation 
CT
Computed Tomography
CTCAE
Common Terminology Criteria for Adverse Events
CTL
Cytotoxic T-Lymphocyte
CTLA4
Cytotoxic T-Lymphocyte Antigen 4
CTX
Cyclophosphamide
Cy/Flu 
Cyclophosphamide and Fludarabine 
DC 
Dendritic Cell 
dL
Deciliter
DLT
Dose Limiting Toxicity
DLCO
Diffusion Capacity of Carbon Dioxide
DMSO
Dimethyl Sulfoxide
DMZ
Data Management Zone
DNA 
Deoxyribonucleic Acid 
DNase
Deoxyribonuclease
D. Sci.
Doctor of Science
DSMB
Data Safety Monitoring Board

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dT
Diphtheria and Tetanus
DTH
Delayed-type Hypersensitivity
DTRI
Duke Translational Research Institute
DUMC 
Duke University Medical Center 
EAE
Experimental Autoimmune Encephalomyelitis
EBRT
External Beam Radiation Therapy
ECHO
Echocardiography
EDTA
Ethylenediaminetetraacetic Acid
EKG
Electrocardiogram
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII-KLH
EGFRvIII conjugated to Keyhole Limpet Hemocyanin
ELISA
Enzyme-Linked ImmunoSorbent Assay
ELISPOT
Enzyme-linked Immunospot
ERADICATe
Evaluation of Recovery from Drug-Induced lymphopenia using
Cytomegalovirus-specific T-cell Adoptive Transfer 
FACS
Fluorescence Activated Cell Sorting
FACT-Br 
Functional Assessment of Cancer Therapy for Brain Tumors 
FACT-accredited
Foundation for the Accreditation of Cellular Therapy
FDA
Food and Drug Administration
FEC
Forced Expiratory Capacity
FEV
Forced Expiratory Volume
FEV1
Forced Expiratory Volume at 1 second
FITC
Fluorescein Isothiocyanate
FOXP3 
Forkhead Box P3 
FTP
File Transfer Protocol
GADPH
Glyceraldehyde-3-phosphate dehydrogenase
GBM
Glioblastoma Multiforme
GFP
Green Fluorescent Protein
GFR
Glomerular Filtration Rate
GLP 
Good Laboratory Practice 
G-CSF
Granulocyte Colony Stimulating Factor
G-Tube
Gastrostomy Tube
GM-CSF
Granulocyte Macrophage Colony Stimulating Factor
H&E
Hematoxylin and Eosin
HABS
Human Monoclonal Antibodies
HB 
Hepatitis B 
HbsAg 
Hepatitis B Surface Antigen 
HD
High Dose
HDC
High Dose Chemotherapy
hCD133
Human Cell Differentiation 133
HER2
Human Epidermal growth factor Receptor 2
hGADPH
Human glyceraldehyde-3-phosphate dehydrogenase
hHPRT 
Human Hypoxanthine Phosphoribosyltransferase 
HIPPA
The Health Insurance Portability and Accountability Act
HIV
Human Immunodeficiency Virus

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HPRT
Hypoxanthine Phosphoribosyltransferase
HLA
Human Leukocyte Antigens
HMO
Health Management Organization
HSC 
Hematopoietic Stem Cells 
HVTN
HIV Vaccine Trials Network
IBC
Institutional Biosafety Committee
ICCC
International Classification of Childhood Cancer
IFN
Interferon
IFN-
Interferon-gamma
IgG
Immunoglobulin G
IHC
Immunohistochemistry
IL-1β
Interleukin 1B
IL-2 
Interleukin-2 
IL-4
Interleukin-4
IL-6
Interleukin-6
IL-12
Interleukin-12
IL-10
Interleukin-10
IL-13
Interleukin-13
IL-15 
Interleukin-15 
IND
Investigational New Drug
IP10
Human interferon-inducible protein 10
IRB
Institutional Review Board
IV
Intravenous
K
Potassium
Kg
Kilogram
KLH 
Keyhole Limpet Hemocyanin 
KLEB
Poorly Differentiated Endometrial Cell Line
KPS
Karnofsky Performance Status
L
Liters
LIMS
Laboratory Information Management System
LMD
Leptomeningeal Disease
LPS 
Lansky Performance Score 
M²
Meters Squared
MA
Myeloablative
MAb
Monoclonal Antibody
MAbs aCD28 
Monoclonal Antibodies anti-CD28 
MB
Medulloblastoma
MAD
Maximally Achievable Dose
MAGE 
Melanoma Antigens 
M.D.
Medical Doctor
MEL
Melphalan
METS
Metastasis
Mcg
Micrograms
MG
Malignant Glioma
Mg 
Magnesium 

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Mg
Milligrams
MHC
Major Histocompatibility Complex
MIN
Minutes
Ml 
Milliliters 
mIU/ml
Million International Units/ Milliliter
MMSE
Mini-Mental Status Examination
MRCP
Member of the Royal College of Physicians
MRI
Magnetic Resonance Imaging
mRNA
Messenger Ribonucleic Acid
MTD 
Maximal Tolerated Dose 
MTIC
Maximal Tolerated Inhibitory Concentration
Na
Sodium
NA
Non-adherent
NC
North Carolina
NCC
Nucleated Cell Count
NDC 
National Documentation Center 
NEJM
New England Journal of Medicine
NEPSY
Neuropsychological Assessment
NCI
National Cancer Institute
NCI CTC
National Cancer Institute Common Toxicity Criteria
NG-Tube
Nasogastric Tube
NJ
New Jersey
NIAID 
National Institute for Allergy and Infectious Diseases 
NIH
National Institutes of Health
NINDS
National Institutes of Neurological Diseases and Strokes
NK
Natural Killer
NMA
Non-Myeloablative
NR
Not Recorded
NSC 
Cancer Chemotherapy National Service Center 
NY
New York
OS
Overall Survival
OVA
Ovalbumin
PALS
Pediatric Advanced Life Support
PBLs
Peripheral Blood Lymphocytes
PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PBSC
Peripheral Blood Stem Cell
PBSCT
Peripheral Blood Stem Cell Transplant
PBTC
Pediatric Brian Tumor Consortium
PBTFI
Pediatric Brain Tumor Foundation Institute
PCP
Pneumocystis Carinii Pneumonia
PCR 
Polymerase Chain Reaction 
PD
Progressive Disease
PE
Phycoerythrin
PEPvIII
Peptide variant III

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PFS
Progression Free Survival
PFS-12
Progression Free Survival, 12 Months
PHA
Phytohemagglutinin
Ph.D. 
Doctorate of Philosophy 
PHI
Private Health Information
PI
Principal Investigator
PNETS
Primitive Neuro Ectodermal Tumors
PO
Per Os (by mouth)
polyAAA
Poly Adenosine Tail
PR 
Partial Response 
PRTBC
Preston Robert Tisch Brain Tumor Center
PT
Prothrombin Time
PTT
Partial Thromboplastin Time
QA/QC
Quality Assessment/ Quality Control
Qdot
Quantum dot
QOL
Quality of Life
®
Registered 
RAC
Recombinant DNA Advisory Committee
RE-MATCH
Recurrent Medulloblastoma and Primitive Neuroectodermal Tumor 
Adoptive T Cell Therapy during Recovery from Myeloablative 
Chemotherapy and Hematopoietic Stem Cell Transplantation 
reMB/PNETS
Recurrent Medulloblastoma/Primitive Neuro Ectodermal Tumors
RECIST
Response Evaluation Criteria in Solid Tumors
Re-START
REcurrent GBM Stem cell Tumor Amplified RNA 
immunotherapy Trial
RDC 
Remote Data Capture 
RN
Registered Nurse
RNA
Ribonucleic Acid
RPA
Ribonuclease Protection Assay
RPMI
Roswell Park Memorial Institute
RT-PCR
Reverse Transcriptase Polymerase Chain Reaction
Rx 
Treatment 
S
Surgery
SAE
Serious Adverse Event
SEB 
Staphylococcal Enterotoxin B 
SEER
Surveillance, Epidemiology, and End Results
SD
Standard Deviation
SD
Stable Disease
SGOT 
Serum Glutamic Oxaloacetic Transaminase 
SGPT
Serum Glutamate Pyruvate Transaminase
SH
Subtractive Hybridization
SIADH
Syndrome of Inappropriate Antidiuretic Hormone
SOP
Standard Operating Procedure
SOX2
Sex Determining Region Y Box 2

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SPORE
Specialized Programs of Research Excellence
SPICE
Specialized Program for Incredibly Complex Evaluations
SQ
Subcutaneous
TAA-40 
Top 40 Tumor Associated Antigens 
TAA
Tumor Associated Antigens
TBI
Total Body Irradiation
TCR
T-cell Receptor
Td
Tetanus and Diphtheria Toxoids
TERT
Telomerase
TGF-β1-2 
Transforming Growth Factor Beta1-2 
TILs
Tumor Infiltrating Lymphocytes
TLR
Toll Like Receptor
TM
Trademark
TMZ
Temozolomide
TNF
Tumor Necrosis Factor
TNF-α 
Tumor Necrosis Factor- Alpha 
Tregs 
Regulatory T-cells
TRP-2
Tyrosinase Related Protein 2
TTP
Time to Progression
TTRNA
Total Tumor mRNA
UF
University of Florida
µg
Micrograms
µl 
Micro liters 
US
United States
USP
United States Pharmacopeia
UV
Ultraviolet
Vβ
V Beta
VCR
Vincristine
VICTORI 
Dose-Finding and Safety Study of Autologous, Tumor-Specific Antigen-
Pulsed Dendritic Cell Immunotherapy for Malignant Brain Tumors 
VP 
VePesid® 
VP-16
Etoposide
WA
Washington

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STUDY SYNOPSIS 
 
Title 
Adoptive Cellular Therapy following Dose-Intensified Temozolomide in Newly- 
diagnosed Pediatric High-grade Gliomas (ACTION trial) 
Study Drug 
Total tumor mRNA-pulsed autologous Dendritic Cells (DCs) (TTRNA-DCs) and tumor- 
specific ex vivo expanded autologous lymphocyte transfer (TTRNA-xALT) with or 
without autologous hematopoietic stem cells (HSCs) 
Primary 
Objective 
To determine the safety of adoptive cellular therapy in pediatric patients with HGG 
receiving dose intensified TMZ and DC+xALT therapy with and without HSCs. 
Secondary 
Objectives 
1) Examine feasibility of completing treatment  in enrolled subjects 
2) Comparison of baseline to post-immunotherapy functional anti-tumor immune 
responses 
3) Analysis of progression-free survival and overall survival after treatment with DC + 
xALT therapy with and without HSCs 
Rationale 
Pilot clinical trial of adoptive immunotherapy for pediatric high-grade gliomas. 
DC vaccination and adoptive T cell strategies targeting unfractionated tumor antigens in 
children with MBs and other brain tumors have been shown to be feasible and safe with 
and without HSC transfer. The feasibility and safety of ACT using amplified tumor 
RNA-pulsed DCs to expand tumor-specific lymphocytes has been established in patients 
with recurrent MB/PNETs during our recently completed phase I trial (Re-MATCH trial, 
FDA IND BB-14058; PI: Duane A. Mitchell).  We have demonstrated in preclinical 
studies that HSCs may act synergistically to enhance the efficacy of adoptive cellular 
therapy. Therefore, we will enroll subjects with a diagnosis of pediatric High-grade 
Gliomas (HGG) to either receive DCs plus ex vivo expanded autologous lymphocyte 
transfer (DC/xALT) (Group A) or DCs/xALT plus autologous HSCs (Group B). 
 
This phase 1 study design will be performed in 12-18 subjects (6-12 Group A subjects 
followed by 6 Group B subjects) using a fixed dose of autologous T cells and DC 
vaccines.   The primary objective will be to demonstrate the safety of the DC + xALT  + 
HSCs in pediatric subjects with HGGs. We will also determine feasibility of successfully 
completing treatment in enrolled subjects and estimate the effect size and variation on 
immunologic and clinical response in order to determine the sample size required to 
appropriately power a phase II study design. 
 
Although standard treatment of primary HGG includes maximal surgical resection, and 
external beam radiotherapy, various adjuvant chemotherapy regimens including high-
dose chemotherapy and peripheral blood stem cell transplant (HDC+PBSCT) have been 
utilized; however, outcomes have remained poor despite this intensive therapy.  While 
chemotherapy induces a profound lymphopenia that would be predicted to prevent the 
induction of an effective immune response to anti-tumor vaccination, recent studies have 
shown, somewhat counterintuitively, that vaccination during recovery from profound but 
transient lymphopenia or the adoptive transfer of tumor-specific lymphocytes into 
lymphodepleted hosts leads to dramatic in vivo T cell expansion and potent immunologic 
and clinical responses to immunotherapy. Therefore, we expect that tumor-specific 
lymphocytes, expanded ex vivo with the use of TTRNA-pulsed DCs may provide a 
source of lymphocytes that preferentially expand in this lymphopenic environment and 
serve as a source of responder cells to subsequent DC vaccination. 

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We and others have successfully employed the use of DCs loaded with total tumor RNA 
as an innovative strategy to induce cellular immune responses against the repertoire of, as 
yet, largely uncharacterized antigens present in malignant brain tumors. Tumor cells from 
HGGs are often limited and cannot be reliably isolated or propagated in sufficient 
quantity to serve directly as a source of antigen for use in human vaccination protocols. 
We have, however, been able to reproducibly amplify the RNA content from as few as 
500 isolated tumor cells using RT-PCR (Reverse Transcriptase-Polymerase Chain 
Reaction) and in vitro transcription from amplified cDNA (complimentary 
Deoxyribonucleic Acid) templates to generate RNA libraries in sufficient quality and 
quantity for clinical-scale immunotherapy trials. 
 
We have established the feasibility and safety of adoptive cellular therapy (ACT) using 
TTRNA-pulsed DCs as a platform for expansion of tumor-specific lymphocytes and 
delivered after lymphodepletive conditioning in pediatric patients with recurrent 
medulloblastoma (MB) and primitive neuroectodermal tumors (PNETs) in an ongoing 
phase I/II trial (Re-MATCH protocol, FDA IND BB-14,058, UF IRB 128-2013; PI: 
Duane A. Mitchell, MD, PhD).  ACT and TTRNA-pulsed DCs will be given in 
conjunction with the adjuvants GM-CSF and tetanus-diphtheria toxoid vaccine (Td), 
which we have demonstrated can potentiate immunologic response to DC vaccination. 
 
GM-CSF is a powerful adjuvant capable of stimulating macrophage function, inducing 
proliferation and maturation of DCs, and is able to enhance T-lymphocyte stimulatory 
function. Intradermal administration of GM-CSF enhances the immunization efficacy at 
the site of administration in a dose dependent fashion. Significant anti-tumor immunity 
has been demonstrated in preclinical murine studies in which irradiated, stably 
transfected tumor cell lines secrete GM-CSF. 
 
Tetanus-diphtheria toxoid (Td) is a routinely used vaccine in the normal human 
population that we have shown in pilot studies may function as a potent adjuvant to 
enhance DC trafficking to vaccine-site draining lymph nodes (VDLNs). Our previous 
studies have shown that successful DC migration to VDLNs may be a requisite for 
clinical activity of RNA- pulsed DCs and administration of Td prior to vaccination may 
improve the effectiveness of DC vaccines in patients with GBM. 
 
The main objectives of this clinical trial are to assess the safety, immunologic effects, and 
estimate the clinical efficacy of DC vaccination during recovery from cycles of dose-
intensified temozolomide (TMZ) followed by adoptive transfer of ex vivo expanded 
tumor-specific lymphocytes with and without HSCs in pediatric subjects with newly-
diagnosed HGGs. The in vivo expansion, persistence, and function of tumor-specific 
lymphocytes will be followed serially in treated subjects using TCR sequencing and 
functional immunologic analysis. 

ACTION
 
18 
 
Inclusion 
Criteria 
Screening Eligibility 
 Age 3-21 years of age 
 Has undergone or will undergo surgical resection of suspected HGG  
Post-Surgical Resection Eligibility 
 Histologically confirmed WHO Grade III or IV malignant glioma 
  Karnofsky Performance Status (KPS) of > 60% (KPS for > 16 years of age) or 
Lansky performance Score (LPS) of ≥ 60 (LPS for < 16 years of age) assessed 
within 2 weeks prior to registration 
 Bone Marrow: 
-  ANC (Absolute neutrophil count) ≥ 1000/µl (unsupported) 
-  Platelets ≥ 100,000/µl (unsupported for at least 3 days) 
-  Hemoglobin > 8 g/dL (may be supported) 
 Renal: 
- Serum creatinine ≤ upper limit of institutional normal 
 Hepatic: 
-  Bilirubin ≤ 1.5 times upper limit of institutional normal for age. 
- SGPT (ALT) ≤ 3 times upper limit of institutional normal for age. 
-  SGOT (AST) ≤ 3 times upper limit of institutional normal for age. 
 Signed informed consent according to institutional guidelines. 
 Patient or patient guardian consent to PBSC harvest following registration. 
 Subjects of childbearing or child-fathering potential must be willing to use 
medically acceptable forms of birth control while being treated on study and for a 
minimum of six months after receiving study therapy (last date of DC or 
ALT/HSC administration). 
 Subjects with post-surgical neurological deficits should have deficits that are 
stable for a minimum of 1 week prior to leukapheresis. 
 
Exclusion 
Criteria 
 Pregnant or need to breast feed during the study period (Negative serum 
pregnancy test required). 
 Known autoimmune or immunosuppressive disease or human immunodeficiency 
virus infection. 
 Subjects with significant renal, cardiac (congestive cardiac failure, myocardial 
infarction, myocarditis), pulmonary, hepatic or other organ dysfunction. 
 Severe or unstable concurrent medical conditions. 

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 Prior allergic reaction to TMZ, GM-CSF, or Td 
 Subjects who are unwilling or unable to receive treatment and undergo follow-up 
evaluations at the enrolled National Pediatric Cancer Foundation (NPCF) 
treatment site. 
Study 
Design 
This multi-institutional clinical trial will consist of three parts:  Surgery, Chemoradiation, and 
adjuvant TMZ with Immunotherapy (Five TMZ cycles with TTRNA-DCs and TMZ Cycle 6 
followed by DCs + xALT +/- HSCs). Potentially eligible subjects can be screened and provide 
screening consent for tumor tissue collection at any NPCF site.  If potential subjects are referred 
from outside physicians, and it is not feasible for the subject to travel to a NPCF site, consent 
may be obtained by designated staff from a NPCF treating center via telephone from the 
subject/LAR in accordance with institutional policies and procedures.  Once screening consent is 
obtained, the Coordinating Center will interact with site to obtain tumor tissue in a manner 
suitable for RNA extraction per UF SOP. If standard-of-care sample collection occurred within 
12 weeks  
prior to enrollment and frozen sample remains, a subject may enroll using previously collected 
samples. Samples must additionally have been snap frozen within 20 minutes  
of collection and kept in a -70/-80 freezer. 
 
Once it has been determined that an adequate amount of tumor tissue was collected and a 
pathological diagnosis of HGG has been confirmed, the subject must be evaluated at a NPCF 
treatment site (UF Health Shands Children’s Hospital, Childrens of Alabama or Children’s 
National Medical Center).  The NPCF treatment site will confirm eligibility, obtain consent for 
the treatment phase of the study, and administer all subsequent therapy. 
 
Up to 24 subjects will be screened to treat 12-18 evaluable subjects. Since HSCs may act 
synergistically to enhance the efficacy of cellular therapeutics in pediatric patients, subjects will 
be grouped into cohorts that receive either TTRNA-DC and TTRNA-xALT alone (Group A) or 
TTRNA-DC and TTRNA-xALT with HSCs (Group B). 
 
All enrolled subjects will undergo a mobilized leukapheresis prior to chemoradiation for 
collection of PBSCs and PBMCs for generation of DCs. G-CSF mobilization of CD34+ HSCs 
will occur for both groups and leukapheresis for autologous peripheral blood stem cells (PBSCs) 
may be collected for up to 3 days for Group B subjects to obtain a recommended dose of > 2 x 
106 CD34+ HSCs/kg. If unable to reach the recommended dose, all available stem cells will be 
infused. 
 
After chemoradiation subjects will receive the first cycle of dose-intensified TMZ followed by 
three biweekly TTRNA-DC vaccines with GM-CSF. Fourteen days (+/- 3 days) after the third 
biweekly DC vaccine, subjects will undergo a second leukapheresis (non-mobilized) to collect 
PBMCs for generation of DCs and xALT. Subjects will then receive an additional five cycles of 
dose-intensified TMZ with concurrent monthly DC vaccinations on days 22-24 of each cycle. 
 
During the 6th TMZ cycle, subjects in Group A will receive TTRNA-DCs and TTRNA-xALT 
48-96 hrs after completion of 21-day TMZ.   Group B subjects will receive HSCs 24-72 hrs after 
completion of TMZ and then TTRNA-DCs and TTRNA-xALT will be administered12-36 hrs 
after HSC infusion. Both groups will receive an additional two bi-weekly TTRNA-DC vaccines 
during Cycle 6. 
 
If a subject cannot tolerate diTMZ, the subject will remain on study and evaluable for the primary 
endpoint. Immunologic endpoint analyses would describe any differences observed in subjects 
that did not received diTMZ but not be powered to detect any differences.   

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20 
 
 
After tumor progression, subjects will be followed for survival status only.  Subjects will be 
followed until death due to any cause. As part of standard care for these subjects, upon tumor 
progression, participants may undergo stereotactic biopsy or resection. As this is not a research 
procedure, consent will be obtained separately.  If tissue is obtained, it will be used to confirm 
tumor progression histologically and to assess immunologic cell infiltration and antigen 
expression profile. 
 
 
1. BACKGROUND 
 
1.1 
Study Disease(s) 
 
Pediatric brain tumors are now the leading cause of cancer-related deaths in children in the United States 
due to the advances in the treatment of more common hematologic malignancies. Standard and intensive 
multi-modality therapies employed in the treatment of pediatric brain cancers encompass maximal surgical 
resection, brain and spinal irradiation, and adjuvant chemotherapeutic regimens including high-dose 
chemotherapy coupled with autologous hematopoietic stem cell (HDC+HSC) rescue. While these regimens 
can cure up to 70% of children with medulloblastoma, the most common pediatric brain tumor, the 
prognosis for children with high-grade gliomas (HGGs) remains abysmally poor even after HDC+HSC 
rescue.6,7  Therefore, there is a clear and urgent need for the development of targeted therapeutics that do not 
add significant toxicity to current treatment regimens for HGGs. Despite considerable advancements and 
promising clinical results observed in immunotherapy trials at our center and others directed against adult 
malignant brain tumors, efforts in the immunologic treatment of pediatric brain tumors have been limited to 
relatively few notable studies. This is often due, at least in part, to the limited amount of viable tumor tissue 
available for tumor cell- based vaccine preparations, and the lack of identification of consistently expressed 
tumor-specific antigens within these cancers. 
 
1.2 
Investigational Agent(s) 
 
The use of total tumor RNA-loaded dendritic cells (DCs) was pioneered as a novel platform for inducing 
potent immunologic responses against the variety of uncharacterized and patient- specific antigens present 
within malignant tumor cells. We have demonstrated that sufficient RNA for clinical vaccine 
preparations can be amplified with high fidelity using existing molecular technologies from as few 
as 500 isolated pediatric and adult brain tumor cells, thus allowing vaccine preparation from surgical 
biopsies and even microdissected archival tumor specimens. We are currently exploring adoptive cellular 
therapy using amplified tumor RNA- pulsed DCs and autologous lymphocyte transfer (DC + xALT 
therapy) in pediatric patients with recurrent MB and PNETs (Re-MATCH protocol, FDA IND BB-
14,058, UF IRB 128-2013; PI: Duane A. Mitchell, MD, PhD). In this proposal, we aim to extend 
evaluation of this novel platform to the treatment of pediatric HGGs. 
 
Immunotherapy administered during recovery from HDC and salvage chemotherapy may have 
tremendous advantages, as adoptive cellular therapy following lymphodepletive conditioning 
regimens has emerged as the most effective treatment strategy for advanced and refractory melanoma. 
Remarkable objective clinical responses in up to 75% of treated refractory melanoma patients have been 
observed, including durable complete regressions of metastatic lesions within the CNS. Although the 
mechanisms by which lymphodepletion enhances immunotherapy in humans are not well elucidated, 
elegant studies in murine tumor models have highlighted some key catalysts for induction of potent anti-
tumor immunity. Depletion of immunosuppressive Tregs, increased bioavailability of inflammatory 

ACTION
 
21 
 
cytokines and homeostatic proliferative cytokines (most notably IL-7 and IL-15) after removal of host 
lymphocytes and NK cells that compete with transferred tumor-specific T cells, and increased toll-
receptor agonistic signals during myeloablative therapy play significant roles. 
 
1.3 
Summary of Background and Supporting Data 
 
Adoptive cellular therapy, involving the ex vivo expansion and intravenous transfer of autologous tumor-
specific lymphocytes after non-myeloablative (NMA) and myeloablative (MA) conditioning regimens, has 
emerged as a remarkably effective treatment modality for advanced malignant disease.8 This success has 
been most notably in the setting of metastatic melanoma; with objective clinical responses observed in 30-
50% of patients treated with adoptive cellular therapy after NMA conditioning and in up to 75% of patients 
after receiving MA regimens9,10 Importantly, these responses have also included a greater than 40% durable 
complete response rate (>3 years) of lesions within the central nervous system (CNS), demonstrating that 
the brain is not refractory to effective treatment by cellular immunotherapy approaches.11 Therefore, despite 
its somewhat complex and highly-specialized nature, adoptive cellular therapy has demonstrated 
significant curative potential in patients with advanced, invasive, and refractory malignant disease. 
A major limitation in the extension of these successes to cancers other than melanoma, however, has been 
the inability to reliably expand polyclonal tumor-infiltrating lymphocytes (TILs) from the majority of other 
solid tumors. To circumvent these limitations, genetically engineered T cells or chimeric antigen receptor 
(CAR) modified T cells have emerged as a favorable treatment strategy.12,13 However, this approach is 
significantly hampered by the very few tumor-specific antigens and receptors identified to date, and 
toxicities that have emerged when engineered T cells with high-affinity receptors have targeted antigens 
with shared expression in normal tissues.14 
 
Through prior phase I and II clinical trials (FDA IND BB-9939, 12839, 13240, 13560, 14058; PI: Duane A. 
Mitchell, M.D., Ph.D.), we have gained considerable experience in evaluating the safety and potential 
efficacy of cellular immunotherapy targeting tumor-specific antigens in adult patients with glioblastoma.15-19 
These trials have shown the capacity to induce potent tumor specific immune responses in vaccinated 
patients, elicit complete radiographic responses in patients with residual disease, and achieve patient 
survival that significantly exceeds that of historical controls (Figs. 1-2). The advent of myelosuppressive 
alkylator-based chemotherapy with TMZ as standard-of-care treatment in GBM patients would be predicted 
to curtail the capacity to induce effective immune responses in these patients. However, somewhat counter-
intuitively, we have shown TMZ to function as a potent adjuvant for enhancing responses to vaccination in 
humans (Figs. 2-3) and to antigen-specific vaccines and adoptive T cell therapy in experimental mice (Figs. 
4-6).16 These observations are consistent with recent reports demonstrating that treatment-induced 
lymphopenia creates a homeostatic environment in which ex vivo activated and adoptively transferred  or 
vaccine-induce tumor specific lymphocytes can gain a  proliferative advantage over host T cells and achieve 
markedly high precursor frequencies.  
 
The identification and validation of tumor-specific antigens in pediatric brain tumors is limited at present but 
we have shown that DCs pulsed with the total antigenic content of tumor cells in the form of RNA can 
induce potent anti-tumor immunity against the largely uncharacterized antigens present in murine and human 
tumors. We have shown that despite increased levels of immunosuppressive CD4+CD25+CD127-FOXP3+ 
Tregs in patients with GBM, DC vaccination coupled with adoptive transfer of naïve autologous lymphocytes 
(DC + nALT therapy) during hematopoietic recovery from chemotherapy-induced lymphopenia can safely 
elicit cellular and humoral immune responses in patients with GBM and achieve promising clinical responses 
(ATTAC Protocol, Anti-Tumor Immunotherapy Targeted Against Cytomegalovirus in Patients with Newly 
Diagnosed Glioblastoma Multiforme during Recovery from Therapeutic Temozolomide-induced 
Lymphopenia; FDA IND-BB-12839, Duke IRB 3877; PI: Duane A. Mitchell).19 
 

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Fig. 1: Lymphocyte counts of patients with newly-
diagnosed GBM receiving standard dose adjuvant TMZ 
(200 
mg/m2/5d) 
and 
dose-intensified 
TMZ 
(100 
mg/m2/21d). Standard dose TMZ 
induces Grade 2 
lymphopenia in 100% of patients (<800 cells/uL; blue line) 
and transient Grade 3 lymphopenia in 40% of patients. 
Sustained Grade 3 lymphopenia (<500 cells/uL; red line) 
was induced by dose-intensified TMZ cycle #3 in all 
patients. 
 
 
0
100000
200000
300000
400000
500000
Standard TMZ Dose
(200 mg/m2/5d)
Vac 3
Vac 4
Vac 5
Vac 6
Vac 7
Vac 8
Lymphodepletive TMZ
(100 mg/m2/21d)
Anti-PEPvIII Titers (1/x)
 
 
Fig. 2: Antibody titers in two representative patients during standard-
dose TMZ (200 mg/m2/5d) and dose-intensified TMZ (100 
mg/m2/21d) are shown. All patients receiving the 21 day dose 
regimen developed EGFRvIII-specific antibodies with titers 
detectable at greater than 1:100,000 dilution.  Humoral and cellular 
immune responses have been dramatically better in patients receiving 
the higher TMZ dose. 
Vaccine 1
Vaccine 2
Vaccine 3
Pre-TMZ
TMZ Cycle-1
TMZ Cycle-2
TMZ Cycle-3
TMZ Cycle-4
TMZ Cycle-5
TMZ Cycle-6
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
Standard Dose TMZ (200mg/m2/5d)
Dose-Intesified TMZ (100mg/m2/21d)
Grade 3 Lymphopenia
<500 cells/L
Grade 2 Lymphopenia
<800 cells/L
Absolute Lymphocyte Count
(cells /L blood)

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23 
 
 
 
Fig. 4: Mice were given increasing doses of TMZ (50 mg/kg 
-200 mg/kg x 1) prior to transfer of OT-I OVA-specific T 
cells as responder cells (1x10 cells/mouse) and a single 
vaccination with OVA RNA-loaded DCs. Expansion of 
OVA-specific T cells was monitored in peripheral blood 
using an OVA-specific tetramer and anti- CD8 monoclonal 
antibody. Acute immunologic responses showed a dose-
dependent increase in mice pre-treated with TMZ.  Five day 
course of lymphodepletive TMZ (60mg/kg/5d) also leads to 
a significant enhancement of vaccine response to OVA RNA-
loaded DCs compared to untreated animals (not shown). 
Fig. 5: Untreated mice revealed a normal acute phase 
expansion of OVA-specific T cells in response to RNA pulsed 
DCs that peaked at 7-10 days followed by a contraction phase 
of OVA-specific T cells in which 90-95% of cells undergo 
apoptosis. TMZ-pretreated 
animals (60 mg/kg x 5 days), 
however, did not exhibit contraction of the stimulated T cells 
even a month after primary stimulation 
and maintained 
elevated levels of effector/memory OVA-specific 
T cells 
(IFNg+CD44hi/CD43low) in the blood. 
 
 
Vaccine DTH Response
Pre Vaccine
Dose 1 TMZ
Dose 3 TMZ
Dose 5 TMZ
0
20,000
40,000
60,000
80,000
100,000
Standard  200mg/m2/5d
Undectable
Undectable
Dose-Intensified 100mg/m 2/21d
mm squared
Candida DTH Response
Pre Vaccine
Dose 1 TMZ
Dose 3 TMZ
Dose 5 TMZ
0
2,000
4,000
6,000
8,000
Standard  200mg/m2/5d
Undetectable
Dose-Intensified 100mg/m 2/21d
mm squared
Fig. 3: DTH responses to EGFRvIII were undetectable prior to vaccination 
in patients receiving non-lymphodepletive TMZ (200 mg/m2/5d) and 
lymphodepletive TMZ (100 mg/m2/21d) cycles and increased during 
vaccination in all treated patients. DTH response to vaccine was 
progressively greater in patients receiving lymphodepletive TMZ (left; 
p=0.0037, Two-way ANOVA, repeated measures for interaction), while pre-
existing DTH responses against Candida were maintained during non-
lymphodepletive TMZ but abolished by the third cycle of TMZ in all patients 
receiving lymphodepletive TMZ (right; p=0.035, Two-way ANOVA, 
repeated measures for interaction). Results demonstrate a differential 
response to TMZ cycles of vaccine-induced immunity compared to pre-
existent Candida immunity.  
0
10
20
30
40
50
60
70
No Vaccine Untreated 
TMZ
(50mg/kg)
TMZ
(75mg/kg)
TMZ
(200mg/kg)
0
10
20
30
40
50
60
70
No Vaccine Untreated 
TMZ
(50mg/kg)
TMZ
(75mg/kg)
TMZ
(200mg/kg)
%  O V A - s p e c i f i c  C D 8 +  T  c e l l s

ACTION
 
24 
 
 
 
Fig. 6: C57BL/6 mice (n=5) were treated with TMZ (60 mg/kg/5d) 
or vehicle or given TBI (500 rad). 48 hours later mice were 
vaccinated with DC electroporated with OVA RNA (5 x 105 DC 
i.d.). Peripheral blood was sampled bi-weekly for precursor 
frequency of OVA-specific T cells and after 12 weeks, lymph nodes, 
bone marrow, spleen and peripheral blood were examined for 
memory OVA T cells (CD44hi x CD43lo x OVA tetramer pos). 
Results demonstrated that TMZ pre-treatment dramatically 
enhanced the expansion of OVA-specific memory T cells in all 
compartments (P<0.005). 
0
10
20
30
40
50
60
70
Vehicle + no
vaccine  
Vehicle + DC-OVA TMZ + DC-OVA
TBI + DC-OVA
% of OVA specific CD8+ T c
0
10
20
30
40
50
60
70
Vehicle + no
vaccine  
Vehicle + DC-OVA TMZ + DC-OVA
TBI + DC-OVA
% of OVA specific CD8+ T c
Lymph nodes
PBLs
Spleen
Bone marrow
Lymph nodes
PBLs
Spleen
Bone marrow
Percent OVA-Specific T-cells
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
DC therapy led to an overall increase in antigen-specific T cells producing IFNor expressing cytolytic 
function (CD107 expression) in treated patients, but was unable to increase antigen-specific polyfunctional 
immune responses (T cells simultaneously secreting IFN, IL-2, TNF, and expressing surface CD107) 
which have recently been shown to be important in mediating anti-viral and anti-tumor immunity (Figs. 7-
8).20-24 However, polyfunctional immune responses were restored in these patients cells using ex vivo 
activation with RNA-pulsed DCs (Figs. 7-8) demonstrating that autologous lymphocyte transfer of ex vivo 
activated T cells (xALT) may be a mechanism for overcoming functional deficits that persist in vivo in 
patients with malignant brain tumors. 
 
Based on these observations, we have initiated a clinical trial exploring DC + xALT therapy during recovery 
from NMA and MA conditioning in children with reMB/PNET (Re-MATCH Protocol, FDA IND-BB 
14058, UF IRB 128-2013; PI: Duane A. Mitchell). We have successfully expanded T cells to clinical-scale 
(>3 x 107 cells/Kg, dose level 2 of 3) using tumor RNA-pulsed DCs from 10 out of 11 subjects in our phase I 
portion of the trial. 
 
The capacity to amplify the tumor RNA for pulsing dendritic cells has been demonstrated in prior 
published studies from freshly resected tumor specimens and micro-dissected archival histologic tumor 
sections for loading of DCs from a variety of tumor types including colorectal tumors (Fig. 9), renal 
carcinomas, melanomas, and pediatric and adult brain tumors (Fig 10). We have successfully amplified 
tumor RNA to clinical scale (>1 mg of polyAAA+ RNA) from as few as 500 sorted glioma tumor cells, 
including CD133+ putative ‘brain tumor stem cells’ (BTSC). We have demonstrated the use of 
unfractionated tumor RNA in an EAE-susceptible preclinical glioma model to be efficacious and without 
toxicity,25,26 and clinical studies using tumor RNA pulsed DCs in pediatric patients with malignant brain 
tumors have been well-tolerated and shown to be capable of inducing anti-tumor immunity.27-29 We 
anticipate, however, that with increasingly potent anti-tumor immunity the risk of induction of intolerable 
autoimmunity will likely also increase. We have therefore explored substractive hybridization (SH) of 
normal brain antigens from total tumor RNA preparations as a means to enrich amplified tumor RNA for 
tumor-specific transcripts. This provides a potential added safety measure should use of unfractionated 
total tumor RNA preparations prove to be toxic in treated patients. 
 
 

ACTION
 
25 
 
Fig. 7:  Induction of cytotoxic T cell responses in vitro using 
DC pulsed with amplified tumor RNA:   Microdissection of a 
colorectal metastatic nodule from a frozen section. A liver 
metastasis was resected for curative intent from a patient with 
CEA- positive colorectal cancer. Frozen sections were cut at 
20 nm in a cryostat mounted on glass slides and stained with 
H&E. A single tumor nodule can be seen in A, surrounded by 
normal tissue. 
Microdissection was performed using an inverted microscope 
and 
an 
attached 
mechanical 
micromanipulator 
for 
manipulating a glass capillary tube, which is used as a cutting 
tool. Under x40-x400 magnification, tumor cells are separated 
from benign tissue by first removing the normal cells adjacent 
to the tumor nodule (B) and then collecting the tumor tissue 
in the middle (C). Thirty-five tumor nodules were isolated and 
pooled from two slides, and RNA was extracted using 
phenol/chloroform and ethanol precipitation. RNA yield, 
determined by UV absorption, was 2.24 µg. The integrity of 
the RNA was confirmed by agarose gel electrophoresis under 
denaturing conditions and ethidium bromide staining (data not 
shown). 
 
 
Fig. 8: Bottom panel: Induction of a primary, CEA-specific 
CTL response in vitro using DCs transfected with RNA 
amplified from microdissected colorectal tumor cells. PBMCs 
from a healthy volunteer were stimulated with autologous 
DCs transfected with various RNA preparations and tested for 
the presence of CEA- specific CTL. DCs transfected with in 
vitro synthesized CEA RNA or GFP RNA were used as 
specific 
and 
nonspecific 
targets, 
respectively. 
For 
stimulations, DCs were transfected with in vitro transcribed 
CEA RNA, RNA isolated from a CEA+ cell line (SW1463), 
a CEA- cell line (KLEB), RNA amplified from SW1463 and 
KLEB cells, and RNA amplified from the microdissected 
tumor cells. These results are representative of two 
experiments; bars, SD. Figure taken from (Boczkowski and 
Nair et al., Cancer Res. 2000; 60(4):1028-34) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1.4 
Pre-clinical Experience 
 
Pre-Clinical Results Against Intracerebral Tumors 
 
In our laboratories and those of others, systemic immunization using DCs co-cultured with uncharacterized 
tumor homogenate,30 whole tumor RNA,31 unidentified peptides eluted from tumor cells by gentle acid 
washing,32 or a distinct peptide encompassing the tumor-specific EGFRvIII (Epidermal Growth Factor 
Receptor v. III) mutation33 have been shown to induce humoral and cell-mediated systemic immune 
responses and to prolong the survival of mice with intracranial brain tumors. We have used a strain of mice 
(VMDk) that is susceptible to experimental autoimmune encephalitis (EAE) to demonstrate the safety and 
efficacy of total tumor RNA pulsed DCs in mediating potent antitumor immune responses and regression of 
established tumor that has prolonged survival in treated animals.25,26 Preclinical efficacy and toxicity data in 
this strain of mice has been used in support of several FDA and IRB approved IND applications investigating 
DC based immunotherapy in patients with malignant brain tumors (FDA INDs 9944, 12839, 13240, 13630 
and Duke IRB protocols 4040, 3877, 0580, 0581, 0947, 3108, and 6677; PI: Duane A. Mitchell, M.D., Ph.D.). 
 
 
 

ACTION
 
26 
 
 
 
 
50bp Ladder
18S RNA
-actin
hGADPH
hHPRT
hCD133
18S RNA
hGADPH
hHPRT
hCD133
-actin
26cycles
50 cycles
250bp
4.4kb
2.4kb
0.25-9.5kb ladder
GBM-1  500 cells
GBM-1 1000 cells
GBM-2  500 cells
GBM-2 1000 cells
Fig. 9: Amplification of tumor RNA to 
clinical scale from freshly resected glioma 
specimens.  RNA was extracted from 500 and 
1000 isolated tumor cells from a resected 
pediatric glioma and subjected to reverse 
transcriptase cDNA synthesis using oligo dT 
primers and a cap-switch 5’ for 2nd strand 
cDNA synthesis.  Full-length cDNA libraries 
were amplified by PCR using a 3’ oligo dT 
primer and 5’ cap-switch oligo containing a T7 
polymerase 
initiate 
site. 
Presence 
of 
housekeeping genes (18S RNA, b-actin, 
GAPDH, and HPRT) was analyzed using 5’ 
primers (exon 1) as well as presence of the 
brain tumor stem cell marker CD133.  in vitro 
transcribed mRNA (right panel) showed wide 
range of RNA transcripts (250bp to > 4.0 Kb). 
 
Fig. 10: High fidelity maintenance of gene expression 
in total tumor RNA after amplification.  In order to 
test 
the fidelity of RNA amplification, real-time 
comparative PCR was conducted on tumor RNA from 
a resected glioblastoma specimen using four separate 
RNA extractions from aliquoted tumor cells and four 
separate amplification reactions. The expression levels 
of tumor associated transcripts (EGFR, Musashi-1, 
nestin, SOX-2) were normalized against GAPDH.   
Experimental results demonstrated a high degree of 
reproducibility in maintenance of gene expression 
profile after amplification (30 PCR cycles).  We have 
found that 20-25 cycles of cDNA library amplification 
is sufficient to produce clinical-scale quantities of 
tumor RNA from 5 out of 5 brain tumor specimens 
evaluated (4 adult GBM and 1 pediatric malignant 
glioma). 

ACTION
 
27 
 
 
1.5 
Clinical Experience 
 
1.5.1 
Lymphopenia and Homeostatic Proliferation 
 
We have gained considerable experience in the induction of anti-tumor immunity in adult patients with 
newly-diagnosed GBM receiving cycles of TMZ as adjuvant chemotherapy. Prolonged therapy with TMZ 
is also associated with an increased incidence of Pneumocystis carinii pneumonia, and as a result, patients 
treated with TMZ are widely believed to be profoundly immunosuppressed.34,35 Our preliminary data, 
however, demonstrates that while TMZ administration abolishes pre-existing immunity to recall antigens 
in patients with GBM, it does not prevent induction of de novo protective immunity against tumor antigens 
during vaccination. 
 
We believe that the potent and specific anti-tumor immune responses generated in our patients are the 
result of a direct competitive advantage enjoyed by B- and T-cells recognizing unique antigens contained 
within a vaccine during the hematopoietic recovery from the profound lymphopenia induced by 
myeloreductive chemotherapy. Although counterintuitive, our murine models, and those of others, 
confirm that vaccine-induced immune responses can be significantly enhanced when given during the 
recovery from lymphodepletive treatment regimens. 
 
Additionally, our preliminary data demonstrate that in mice and humans, increasing lymphodepletive 
doses of TMZ led to progressively higher antibody titers and antigen-specific T cell frequencies in 
response to vaccination that can ultimately exceed a precursor frequency of 80% and are capable of 
inducing superior antitumor responses. Similarly, as we dose-intensified the chemotherapy treatment 
regimen in patients with GBM and reached Grade III lymphopenia routinely, tumor-specific 
immunological responses were dramatically enhanced compared to less myelosuppressive chemotherapy 
doses. While vaccine-induced responses were dramatically enhanced during lymphodepleting 
chemotherapy administration in patients with GBM, immunologic recall responses, for example to 
Candida, were rapidly abolished during treatment cycles in these patients, demonstrating that the 
immunosuppressive effects of chemotherapy can be dramatically overridden by repetitive vaccination in 
patients with malignant brain tumors. 
 
Although this proposal argues that the profound lymphopenia induced by escalating doses of myeloablative 
chemotherapy may actually enhance promising immunotherapeutic approaches like the one described 
above in adult patients with GBM, there is already a strong rationale for use of high-dose alkylator based 
therapy against solid tumors. The steep dose- response relationships demonstrated by other alkylating 
agents such as carmustine, cyclophosphamide, or melphalan suggests that efforts to maximize drug levels 
may result in improvement in therapeutic outcome.36 In fact, response rates can exceed 65% in patients 
with MG with carmustine when dose escalation is supported by hematopoietic stem cells (HSCs).37,38 This 
rationale has supported the routine use of HDC+HSC in pediatric patients with recurrent and high-risk 
brain tumors. Thus immunotherapy targeting HGGs conducted in children during hematopoietic recovery 
from diTMZ in order to enhance the efficacy of a novel immunotherapeutic strategy. Interestingly, an 
independent benefit on the immune response might be conferred by the HSC transplant itself. HSC support 
has been shown in a murine model to cause an independent and robust expansion of tumor-specific effector 
CD8+ T cells after adoptive transfer into myeloablated hosts.39 Our preclinical studies have demonstrated 
that HSCs can enhance T cell trafficking to intracranial tumors and facilitate immunologic tumor rejection 
(Flores et al. Oncoimmunology 2015). 
 

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Following periods of lymphopenia, there is a homeostatic proliferation of the host’s remaining 
lymphocytes which is designed to recover normal lymphocyte counts.40 Probably as a result of a surge 
in cytokines (IL-7, IL-15) in response to lymphopenia, lymphocytes undergoing homeostatic 
proliferation enjoy a reduced activation threshold40,41 and differentiate directly into effector memory 
T cells capable of rapid and intense response to antigen.42 Still, lymphocytes must encounter their 
cognate antigen and compete for limiting amounts of these homeostatic cytokines to proliferate  even 
under these conditions.40 Thus, B- or T cells specific for antigens that predominate during this recovery 
period, like those provided in the form of a vaccine, have a competitive advantage and become 
disproportionately over-represented in the recovering lymphocyte population, both in murine 
models43,44 and in humans.45 These skewed homeostatic responses have been shown to enhance 
antitumor immunity43,44,46 but can also increase the risk of autoimmunity.47,48 
 
Leveraging this principle, Rosenberg and colleagues used intentional non-myeloablative lymphodepletion 
to enhance the preferential expansion and maintenance of adoptively transferred, tumor-specific T cells. 
This has resulted in dramatic clinical responses49-53 along with some autoimmune toxicity in patients with 
advanced malignant melanoma.54,55 These studies have shown that under these conditions, transferred T 
cells can expand dramatically in the lymphopenic host to constitute up to 90% of the host’s T cell 
repertoire and can be maintained for months following transfer.54,56 These studies demonstrated that 
clinical regression of systemic disease correlates with the frequency of tumor-specific T cells achieved in 
the peripheral blood and persistence of these cells in vivo.49-53,57  Subsequent studies in murine models 
demonstrated this anti-tumor effect was also significantly enhanced by myeloablative conditioning 
regimens coupled with autologous HSC support. Interestingly, HSCs were found to confer an enhancing 
effect on the in vivo expansion and persistence of tumor-specific lymphocytes transferred simultaneously 
with HSCs independent of the effects of lymphopenia.39 Pursuant to these findings, Rosenberg and 
colleagues proceeded to evaluate myeloablative conditioning regimens coupled with PBSCT in patients 
with malignant melanoma receiving adoptive cellular therapy. Adoptive lymphocyte transfer was also 
conducted in the peri- transplant period within 24hrs of PBSCT demonstrating feasibility and safety in 
humans of this approach.10 As demonstrated in murine studies, this conditioning regimen enhanced anti-
tumor responses in patients with refractory metastatic disease, resulting in increased objective clinical 
responses from 30-50% of patients with nonmyeloablative regimens to over 70% in patients receiving 
myeloablative conditioning coupled with PBSCT. 
 
The mechanisms by which lymphodepletion leads to an enhancement of immune responses in humans are 
not well elucidated but elegant murine studies have implicated the following important processes: 
 Increased in production of homeostatic cytokines such as IL-7 and IL-15 that drive lymphocyte 
proliferation;58 
 Decreased competition with adoptively transferred tumor-specific lymphocytes through removal of 
“cytokine sinks” consisting of host lymphocytes and NK cells that decrease the bioavailability of 
growth factors;59 
 Removal of CD4+CD25+FOXP3+ Tregs that attenuate anti-tumor immunity;60 
 Increased toll-like receptor agonistic signals and inflammatory cytokines through release of gut 
microbial antigens such as endotoxin during damage to gut endothelium by myeloablative therapy;61 
 Direct enhancing effects of hematopoietic stem cell transplant on the in vivo expansion and function 
of adoptively transferred lymphocytes.62 
 

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The source of anti-tumor lymphocytes for human studies in melanoma have been polyclonal populations 
of autologous CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) expanded from resected melanoma 
deposits. While TILs are a readily expandable source of lymphocytes from accessible melanoma lesions, 
no published studies have demonstrated capacity to reliably expand TILs with anti-tumor activity from 
malignant brain tumors. We have demonstrated in several published studies, however, the capacity to 
induce anti-tumor lymphocytes in vitro and in vivo using tumor RNA-pulsed DCs from a variety of human 
and murine tumors, including malignant brain tumors.25,26,31,63-71 
 
1.5.2 
RNA-Loaded Dendritic Cells 
 
In this proposal, we will investigate the potential of DCs loaded with a broad range of antigens derived 
directly from HGG tumor tissue as a strategy to effectively expand tumor-specific lymphocytes in vitro for 
adoptive transfer after non-myeloablative chemotherapy coupled with HSCs. Continued expansion of anti-
tumor T cells in vivo will be attempted with intradermal total tumor RNA-loaded DC vaccines throughout 
hematopoietic recovery in enrolled subjects. 
 
The use of RNA to encode tumor antigens for DCs was pioneered in Dr. Nair’s and Gilboa’s laboratory, 
but the ability of mRNA-loaded DCs to stimulate potent anti-tumor immunity has been independently 
confirmed in murine and human systems.66,72-76 In fact, there is accumulating evidence that RNA 
transfection represents a superior method for loading antigens onto DCs.74,77 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 Histocompatability 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 anti-tumor immune 
responses by simultaneous activation of both CTLs and helper T cells.78-80 Furthermore, in direct 
comparisons, mRNA-loaded DCs have been found to be better stimulators of antigen-specific T cells than 
other approaches.77 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. 
 
1.6 
DC + ALT Therapy 
 
1.6.1 
RNA loaded DC vaccines and amplified tumor RNA (preclinical and clinical studies) 
We have shown use of RNA-pulsed DCs to be a versatile platform for activating tumor-specific T cells in 
vitro and in vivo in several murine and human systems. Several clinical trials have been conducted by our 
team demonstrating the feasibility and safety of tumor RNA- pulsed DCs in human patients. While 
specific tumor-associated antigens such as carcinoembryonic antigen (CEA), telomerase (TERT), 
melanoma antigens (MAGE, TRP-2), and human CMV pp65 have all been utilized as RNA-encoded 
antigens in our DC trials, studies have demonstrated that the majority of endogenous anti-tumor immune 
responses in patients with malignancy are against unidentified, patient-specific antigens.81 The use of total 
tumor RNA pulsed DCs to expand tumor-specific lymphocytes allows for these patient-specific antigens 
to be targeted, however, sufficient tumor tissue for clinical-scale vaccination is not always readily 
available. Our laboratory has utilized amplification of total tumor RNA with reverse-transcriptase primed 
PCR to generate cDNA library templates encoding for the antigenic content of tumor cells from as few as 
500 starting tumor cells. Through inclusion of a T7 RNA polymerase binding site in the 5’ primer used for 
amplification, total tumor RNA can be readily generated through in vitro transcription after cDNA 
 
 

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amplification. Using such techniques we have been able to generate sufficient RNA for clinical DC 
vaccine preparations from colorectal tumors, renal carcinoma, and pediatric and adult brain tumor 
specimens using excess tumor material harvested during surgical resection.65,70,82 
 
1.6.2 
Amplified Total Tumor RNA from human malignant brain tumors 
 
While expansion of tumor cells using in vitro culture is feasible, primary brain tumor cells are often 
difficult to propagate and gene expression microarray analysis has demonstrated that the majority of 
tumor specific genes expressed in vivo are not recapitulated within in vitro propagated tumor cells. 
Furthermore, we have demonstrated in murine intracranial glioma models that a significant shift in brain 
tumor gene expression is induced in response to host anti- tumor immunity.83 This strongly suggests that 
the antigenic content of tumor cells propagated in vitro will be significantly different than in vivo 
propagated tumors, and thus the relevance of in vitro propagated tumors as an antigenic source for 
immunotherapy is questionable. This observation prompted us to investigate the capacity to amplify the 
RNA content of tumor cells isolated directly from surgically resected MG specimens in order to utilize an 
antigenic source more representative of the antigens expressed within patients’ tumor cells in vivo. Our 
laboratory has successfully amplified tumor RNA from murine and human carcinomas and demonstrated 
the capacity to elicit effective cytotoxic anti-tumor responses using DC pulsed with amplified tumor 
RNA.65,82 Based on current clinical protocols at our institution utilizing total tumor RNA pulsed DCs,84 
we have determined a production requirement for clinical use of up to 750ug of amplified tumor mRNA 
per patient. We have successfully amplified tumor mRNA to clinical scale (over 1mg) from as few as 500 
astrocytoma cells from resected human glioma specimens from adult and pediatric brain tumors 
Verification of full-length cDNA templates was conducted using real-time PCR analysis of housekeeping 
genes (-actin, HRPT, GADPH, 18S RNA) and stem cell markers (nestin, SOX2, musashi-1, EGFR), 5’ 
gene specific primers spanning exon- intron splice junctions, gel electrophoresis demonstrating mRNAs 
spanning from 0.2Kb to over 5Kb in length, and Northern blot analysis demonstrating full-length -actin 
mRNA in amplified tumor samples. Enrichment of tumor antigens was performed using subtractive 
hybridization of excess pooled normal brain RNA from tumor RNA prior to amplification and in vitro 
RNA synthesis and verification of enrichment of tumor-associated genes by comparative real-time PCR. 
 
1.7 
Autoimmune Encephalomyelitis 
 
While DC vaccinations administered to patients with GBM have been safe in our clinical trial experience 
and others,85-87 they are also quite capable of initiating significant autoimmune responses in mice and 
humans.88,85 In light of the expression of normal and fetal brain antigens on brain tumor tissue89-92 and 
human glioma cell lines,93 active immunization of patients with malignant brain tumors risks inducing an 
uncontrolled autoimmune response against normal CNS antigens that is similar to experimental 
autoimmune encephalomyelitis (EAE). The susceptibility of humans to the induction of EAE was 
demonstrated accidentally when patients were vaccinated against rabies with spinal cords from rabbits that 
were infected with the rabies virus.94-98 Lethal EAE has also been induced in non-human primates after 
vaccination with unrefined human GBM tissue.99 It is likely, then, that with increasingly potent 
immunotherapies that target antigens shared by tumor and normal tissue, that the risk for autoimmunity 
against normal brain tissue will increase. 
 
To focus the immunologic response against tumor-specific antigens and potentially reduce the risk for 
autoimmunity, subtractive hybridization can be conducted to generate a template for mRNA synthesis that 
consists of cDNAs expressed preferentially in malignant tumor cells. Such approaches can also aid in future 
 
 

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efforts in antigen discovery should the targeting of tumor cells prove advantageous. This is important 
because most well-characterized tumor antigens are over- expressed normal proteins which have triggered 
immunologic tolerance to some degree. This compromises their effectiveness as tumor rejection antigens 
and poses a risk of autoimmunity if effectively targeted.81,100 Conversely, tumor-specific antigens derived 
from mutations in somatic genes are less prejudiced by central tolerance and less likely to be associated 
with autoimmunity. Some studies also suggest that the autonomous immune response to human tumors is 
dominated by such neoantigens.101 The stringency of SH techniques including suppression subtractive 
PCR amplification techniques can be utilized to enrich cDNA libraries for uniquely expressed and 
mutated transcripts from tumor cells using normal brain RNA as a subtractor substrate.102 Such methods 
are under active development in our laboratory and may be utilized to enhance either the safety profile or 
tumor-specific immune response in subsequent clinical studies. 
 
1.8 
Rationale 
 
Despite brain tumors being the leading cause of cancer deaths in children and the considerable advances in 
immunologic targeting of adult brain tumors, studies examining immunotherapy for pediatric brain tumors 
are markedly lacking except for a notable few reports. Efforts in targeting pediatric brain tumors are 
hindered somewhat by the relatively small number of clinical cases compared to adult cancers, the lack of 
syngeneic murine models of common pediatric brain tumors in which immunotherapy efforts can be 
evaluated in preclinical studies, and the significant regulatory hurdles in conducting experimental 
therapies in the pediatric setting. 
 
Despite these limitations, the use of NMA TMZ chemotherapy in pediatric patients with HGGs affords an 
ideal setting in which to leverage the recent findings by our laboratory and others in the benefits of 
treatment-induced lymphopenia in enhancing immune responses to tumor immunotherapy. 
 
Three notable studies have demonstrated the safety and potential efficacy of immunotherapy in children 
with malignant brain tumors. 
 
Ashley and colleagues conducted a phase I study of 9 pediatric patients with recurrent brain tumors using 
monocyte-derived DCs pulsed with tumor RNA isolated from resected tumor specimens.27 DCs were 
derived from monocytes after 7 days of culture with IL-4 and granulocyte-macrophage colony-stimulating 
factor (GM-CSF), incubated with RNA for 45 minutes in serum-free media, washed and then 
cyropreserved until administration. DC vaccines were administered intradermally and intravenously in 
treated patients. The feasibility and safety of tumor RNA pulsed DC vaccines was demonstrated in this 
study with 7 of the 9 enrolled patients receiving RNA-pulsed DC vaccines. 1 patient demonstrated a partial 
response to therapy as assessed by MRI and 2 patients showed disease stabilization. Tumor-specific 
immune responses were demonstrated after vaccination in 2 of 7 patients. 
 
A subsequent study by the same group in 11 pediatric patients with newly-diagnosed, Stage 4 
neuroblastoma, evaluated tumor RNA-pulsed DC vaccines after HDC+PBSCT.28 The patients received 
two courses of carboplatin induction therapy followed by standard chemotherapy, surgery, radiation, and 
HDC + stem cell rescue. DC vaccines were administered intravenously and intradermally beginning 6 
weeks after stem cell transplantation and were prepared as described in the previous study. The 
investigators noted immunologic impairment in recall responses and proliferative responses in patients at 6 
weeks post HDC+PBCT. Seven of 11 enrolled patients were successfully treated during this study. The 
safety and feasibility of the total tumor RNA DC vaccines after HDC + PBSCT was demonstrated by this 
study but no clinical or radiographic responses were observed. 
 

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Importantly, Ashley and colleagues demonstrated the safety and feasibility of total tumor RNA- pulsed 
DCs in pediatric patients with recurrent and primary brain tumors in these studies. Recent advances, 
however, in our understanding of DC biology and their use in anti-tumor immunotherapy have made even 
the limited clinical responses observed in the first study somewhat encouraging. These significant 
differences in the previously published reports and the methods we will use in this study are as follows: 
 
Ashley and colleagues used immature DCs in their study which have now been shown to be vastly inferior 
in the induction of immunologic responses. Our DCs are matured with a combination of IL-1, TNF, 
IFN, and IL-6. 
 
Route of injection comparisons have shown that intravenous injection of DCs in humans is in many cases 
tolerogenic and the preferred route for induction of immunity is intradermal. Our vaccines will be 
administered through intradermal injection only. 
 
We have shown electroporation of RNA into DCs to be 100 to 1000 times more efficient than 
co-incubation of DCs with “naked RNA.” The labile nature of RNA results in degradation with a half-life 
of seconds to minutes, thus electroporation to deliver RNA directly into DCs is far superior in mediating 
gene expression and anti-tumor immunity. 
 
We and others have shown that the early recovery period after lymphodepletion is the most effective 
period for enhancing immunity to vaccination and adoptive T cell transfer and thus we will administer 
DC+xALT therapy during the post-transplant period (24 hours after PBSCT) as has been done in studies 
at the NCI using myeloablative therapy and adoptive cellular therapy in patients with melanoma. 
 
Ashley and colleagues administered 3 to 5 vaccines to their patients, while in this trial, we will 
continually 
vaccinate patients throughout the recovery period of lymphopenia and normalization of T cell function as 
we have shown capacity to increase immunologic responses dramatically over time in vaccinated patients 
with newly-diagnosed GBM. In fact, significant anti-tumor immune responses have often occurred in our 
patients only after repetitive monthly vaccinations. 
 
We believe these advances will significantly enhance the potential clinical impact of our proposed 
therapeutic approach. Admittedly, as more potent vaccination platforms are advanced, the risks of 
inducing intolerable autoimmunity will also likely increase, and thus careful evaluation of the safety of 
these potent vaccination platforms will be necessary. Our proposed phase I study will establish the safety 
of DC + xALT therapy using amplified total tumor RNA pulsed DC vaccines to expand tumor-specific 
lymphocytes in vitro for adoptive transfer and maintenance in vivo with DC vaccination. 
 
Improving survival in pediatric patients with HGG will require combined therapy with resection, chemo-
radiotherapy, followed by adjuvant chemotherapy (Temozolomide) and potentially novel approaches 
including immunotherapy.  Based on the preclinical and clinical experience with adoptive cellular therapy 
summarized above, we believe there is the potential to induce immune responses (anti-tumor immunity) 
in children with brain tumors (high-grade gliomas) utilizing the immunotherapy strategy proposed in this 
trial.  We have developed the use of total tumor RNA (TTRNA) loaded DCs as an effective platform for 
induction of tumor-specific lymphocytes and will use this approach to expand lymphocytes specific for 
HGG in these children undergoing primary resection, radiation and adjuvant chemotherapy.  
Administering DC vaccine following temozolomide as standard treatment in children with HGG is also 
expected to augment tumor-specific immune responses during the hematopoietic recovery phase of each 
cycle of chemotherapy.  In order to maintain T cell expansion and function, serial vaccinations with 
TTRNA-DCs will be administered. Based on durable remissions achieved using adoptive cellular therapy 

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after lymphodepletive regimens in adults with advanced stage melanoma, we believe this treatment 
platform may also improve outcomes for children with HGG.  
 
1.9 
Re-MATCH phase 1 clinical trial of adoptive cellular therapy in recurrent medulloblastoma 
(reMB) and PNETs 
 
Nine subjects with reMB/PNET were treated during the phase 1 trial.  Seven were evaluable for safety 
evaluation.  Of the two determined non-evaluable, one subject failed to generate T cells and was treated 
with DC vaccine only and one subject suffered disease progression prior to the DLT evaluation period.  
There were no immunotherapy related DLTs.  A single patient experienced a grade III asymptomatic 
elevation in liver alkaline phosphatase detected during routine clinical follow-up at nine months post 
immunotherapy treatment. The etiology was not determined and resolved spontaneously without medical 
intervention.  This AE was ruled as possibly related to study drug since no attributable cause was 
determined.  Overall, the treatment was deemed feasible and safe for pediatric patients with recurrent 
MB/PNET.  A maximum achievable dose of T cells (3 x 10^7 cells/Kg) was reached in treated subjects. 
 
Recent analysis of the progression-free survival outcomes for patients enrolled on the phase 1 trial and 
treated with chemotherapy followed by adoptive cellular therapy compared to a historical cohort of 
patients with recurrent medulloblastoma treated with chemotherapy only demonstrated promising clinical 
efficacy for the cellular therapy regimen.   Median progression-free survival on Re-MATCH phase 1 
patients is 21 months vs 10.5 months historical control (P=0.07) and 12-month PFS is 67% vs 33%, 24-
month PFS is 33% vs 0%.  A single-arm phase 2 trial is underway using the maximal achievable dose of T 
cells to assess PFS in 35 subjects. 
 
1.10 Improved T cell Expansion 
 
Under support of a research grant from the Pediatric Cancer Foundation, we have explored the capacity to 
enhance the expansion of antigen-specific T cells using the RNA-pulsed DC platform through employing a 
rapid expansion protocol (REP) developed at the National Cancer Institute for expansion of melanoma-
reactive tumor-infiltrating lymphocytes.   The REP employs irradiated allogeneic PBMCs as feeder cells 
and low-dose anti-CD3 monoclonal antibodies (OKT3) and IL-2 for the rapid expansion of activated 
lymphocytes in culture.  We have validated the capacity to expand lymphocytes stimulated by RNA-
pulsed DCs 200-500 fold using the REP protocol and reliably achieve greater than 1 x 10^10 T cells with 
an input of 1 x 10^8 activated lymphocytes.  We have additionally demonstrated that the utilization of IL-7 
and IL-21 during co-culture of T cells with RNA-pulsed DCs prior to REP leads to a greater generation of 
central memory antigen-reactive T cells which have been shown to be superior in anti-tumor efficacy.  
Further investigation in our lab demonstrated that IL-21 alone can promote memory cell generation that was 
equal to that of the combination of IL-7 and IL-21, thus IL-21 will be used alone for initial T cell 
stimulation. This study will employ xALT utilizing our improved stimulation and expansion protocol at a 
target dose of 3 x 10^8 T cells/Kg. 
 
1.11 Study Design 
 
Primary and Secondary Objectives -The primary purpose of this study is to explore the safety of DC 
vaccines and ACT in patients with primary HGGs.  Secondary objectives will include feasibility assessment 
as a proportion of enrolled subjects in which immunotherapy is successfully delivered, and immunologic 
responses to estimate the mean difference and the variation in INF gamma secretion between baseline and 
2 weeks post-completion of immunotherapy in Group A and Group B. OS and PFS will be secondary 
clinical outcomes measures. We do not expect the sample size of the current study to be large enough to 
allow definitive conclusions, but we will use a t-test to compare gamma secretion differences (baseline – 2 

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weeks post-immunotherapy) between Group A and B, and Kaplan-Meier methods to compare progression-
free and overall survival. 
 
Enrolled subjects will be pediatric patients with newly-diagnosed Grade III or Grade IV HGG’s. This study 
will establish the safety, feasibility, and immunologic effects of DC + xALT therapy with or without HSC 
transfer in pediatric subjects receiving dose-intensified adjuvant TMZ. 
 
This multi-institutional clinical trial will consist of three parts: Surgery, Chemoradiation, and adjuvant 
dose-intensified TMZ with Immunotherapy (five TMZ cycles with TTRNA-DC and TMZ, cycle 6 
followed by DCs and xALT +/- HSCs). Potentially eligible subjects will be enrolled on a screening 
consent for the sterile collection of tumor material in a manner suitable for RNA extraction, amplification, 
and loading of autologous DCs. Following surgical resection with confirmatory pathologic diagnosis, 
subjects will be enrolled in the trial after informed consent has been obtained. 
 
Potential subjects can be screened and provide consent for tumor tissue collection at any NPCF site.  If 
potential subjects are referred from outside physicians, and it is not feasible for the subject to travel to a 
NPCF site, tissue consent may be obtained by designated staff from a NPCF treating center via telephone 
from the subject/LAR in accordance with institutional policies and procedures.  Once screening consent is 
obtained, the Coordinating Center will interact with site to obtain tumor tissue in a manner suitable for 
RNA extraction per UF SOP. If standard-of-care sample collection occurred within 12 weeks  
prior to enrollment and frozen sample remains, a subject may enroll using previously collected samples. 
Samples must additionally have been snap frozen within 20 minutes  
of collection and kept in a -70/-80 freezer. 
 
Once it has been determined that an adequate amount of tumor tissue was collected and a pathological 
diagnosis of HGG has been confirmed, the subject must be evaluated at a NPCF treatment site (UF Health 
Shands Children’s Hospital, Childrens of Alabama or Children’s National Medical Center). The NPCF 
treatment site will confirm eligibility, obtain consent for the treatment phase of the study, and administer 
all subsequent therapy. 
 
Up to 24 subjects will be screened to treat 12-18 evaluable subjects, six subjects into Group A followed by 
six subjects into Group B. The first six evaluable subjects enrolled will be for Group A evaluating the 
safety of TTRNA- DC vaccines and TTRNA-xALT at a target dose of 3 x 108/kg.  If 2 of 6 patients 
experience immunotherapy related DLT following administration of 3 x108/kg x-ALT, an additional 6 
patients will be enrolled on Group A to receive x-ALT at a dose of 3 x107/kg. The trial will stop if 2 of 6 
patients develop DLT at this dose level. Once MTD is identified in Group A patients, 6 patients will be 
treated in Group B using this MTD for x-ALT infusion.  
 
1.12 On-Study Tests and Procedures 
Timepoint 
Date 
Study Requirements 
Screening
Tissue screening consent 
Surgery
Resection  
Tumor tissue for extraction of total tumor RNA 
MRI brain (and spine if applicable) with & without contrast 
post-surgery 
Post-Surgical Resection 
 
Inclusion & exclusion criteria 
Treatment consent 
Medical history, physical & neurological exam 

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Performance Status: KPS or LPS 
CBC w/ differential, PT/INR, PTT, comprehensive 
metabolic panel 
β-HCG pregnancy test (serum) 
Mobilized Leukapheresis 
CBC w/ differential, CD4/CD8 ratio, comprehensive 
metabolic panel, research labs within 24 hrs prior to 
leukapheresis 
β-HCG pregnancy test (serum or urine) 
Infectious disease testing to include, at a 
minimum: HIV, Hepatitis B, Hepatitis C & 
CMV 
Medical history, physical & neurological 
exam 
Chemoradiation 
TMZ 90mg/m2 daily for up to 49 days. Taken 1 hr prior to 
radiation and each morning on non-xRT days 
Prior toTMZ Cycle 1 and 
on day of Dendritic Cell 
Vaccine
Medical history, physical & neurological exam 
CBC w/ differential, CD4/CD 8 ratio, comprehensive 
metabolic panel
β-HCG pregnancy test (serum or urine) 
Within 24 hours prior: Research labs 
Performance Status: KPS or LPS 
Non-Mobilized     
Leukapheresis 
CBC w/ differential, CD4/CD 8 ratio, comprehensive 
metabolic panel, research labs within 24hrs prior to 
leukapheresis 
β-HCG pregnancy test (serum or urine) 
Infectious disease testing 
Medical history, physical & neurological exam 
TMZ Cycles 2-5 and on 
day of Dendritic Cell 
Vaccine 
Medical history, physical & neurological exam 
Within 24 hours prior to vaccine and within 1 week of 
starting TMZ cycles: CBC w/ differential, CD4/CD 8 ratio, 
comprehensive metabolic panel 
β-HCG pregnancy test (serum or urine) 
Within 24 hours prior: Research labs 
Performance Status: KPS or LPS
Brain (and spine if applicable) MRI with and without 
contrast (within 2 weeks prior to C1, C2 and C4) 
TMZ Cycle  6 and on day 
of DC Vaccine and xALT – 
Group A and B
Medical history, physical & neurological exam 

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Within 24 hours prior to vaccine and within 1 week of 
starting TMZ cycle: CBC w/ differential, CD4/CD 8 ratio, 
comprehensive metabolic panel 
β-HCG pregnancy test (serum or urine) 
Within 24 hours prior: Research labs 
Performance Status: KPS or LPS 
Brain (and spine if applicable) MRI with and without 
contrast (within 2 weeks prior to C6) 
Post ALT infusion
 
Day 1, Day 4 (+/- 1 day):  CBC w/ differential, CD4/CD 8 
ratio, comprehensive metabolic panel and research labs (if 
feasible)
TMZ Cycle 6 – Group B 
Only 
HSC infusion (12-36-hours prior to DC vaccine and xALT) 
Bi-Monthly follow-up
+/- 1 month for one year 
and then per PI discretion 
(minimum of every 6 
months) until tumor 
progression or death due to 
any cause 
Medical history, physical & neurological exam 
Contrast-enhanced MRI scan 
CBC w/ diff, CD4/CD 8 ratio, research labs (if feasible) 
Brain (and spine if applicable) MRI with and without 
contrast  
 
1.13 Correlative Studies Background 
 
Our preclinical studies have demonstrated that adoptive cellular therapy in tumor-bearing hosts results in 
the in vivo expansion and persistence of specific TCR V beta families that are associated with tumor 
rejection.  Additionally, the transfer of HSCs leads to a greater expansion and altered diversity of T cells 
in the treated hosts. We will examine the TCR V beta gene usage in ex vivo expanded lymphocytes prior 
to adoptive transfer as well as in the peripheral blood of treated subjects prior to leukapheresis, during 
cycles of TMZ and each DC vaccination, and prior to DC + xALT therapy and at follow-up visits until 
progression to determine if clonal expansion of T cells are observed during immunotherapy and if HSC 
transfer alters the kinetics of expansion and the diversity of TCRs observed in treated subjects. 
 
2. PATIENT SELECTION 
 
2.1 
Inclusion Criteria Screening 
 
 Age 3-21 years of age. 
 Has undergone or will undergo surgical resection of suspected HGG  
 
Post-Surgical Resection 
 Histologically confirmed WHO Grade III or IV malignant glioma. 

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 Karnofsky Performance Status (KPS) of > 60% (KPS for > 16 years of age) or Lansky performance 
Score (LPS) of ≥ 60 (LPS for < 16 years of age) assessed within 2 weeks prior to registration. 
 Bone Marrow: 
- ANC (Absolute neutrophil count) ≥ 1000/µl (unsupported - no growth factors within 3 days) 
- Platelets ≥ 100,000/µl (unsupported- defined as no platelet transfusion within 3 days) 
- Hemoglobin > 8 g/dL (may be supported) 
  Renal: 
- Serum creatinine ≤ upper limit of institutional normal 
 Hepatic: 
- Bilirubin ≤ 1.5 times upper limit of institutional normal for age. 
- SGPT (ALT) ≤ 3 times upper limit of institutional normal for age. 
- SGOT (AST) ≤ 3 times upper limit of institutional normal for age. 
 Signed informed consent according to institutional guidelines. 
 Patient or patient guardian consent to PBSC harvest following registration  
 Subjects of childbearing or child-fathering potential must be willing to use medically acceptable 
forms of birth control while being treated on study and for a minimum of six months after 
receiving study therapy (last date of DC or ALT/HSC administration). 
Pregnancy: The effects of Temozolomide on the developing human fetus are unknown.  For this 
reason and because antineoplastic agents as well as other therapeutic agents used in this trial are 
known to be teratogenic, women of child- bearing potential and men must agree to use adequate 
contraception (hormonal or barrier method of birth control; abstinence) prior to study entry and for 
the duration of study participation. Should a woman become pregnant or suspect she is pregnant 
while she or her partner is participating in this study, she should inform her treating physician 
immediately.  Men treated or enrolled on this protocol must also agree to use adequate 
contraception prior to the study, for the duration of study participation, and 4 months after 
completion of DRUG administration. 
 Subjects with post-surgical neurological deficits should have deficits that are stable for a minimum of 
1 week prior to leukapheresis.   
 
2.2 
Exclusion Criteria 
 
 Pregnant or need to breast feed during the study period (Negative serum pregnancy test required). 
 Known autoimmune or immunosuppressive disease or human immunodeficiency virus infection. 
 Subjects with significant renal, cardiac (congestive cardiac failure, myocardial infarction, 
myocarditis), pulmonary, hepatic or other organ dysfunction. 
 Severe or unstable concurrent medical conditions. 
 Patients scheduled to receive any other concurrent anticancer or investigational drug therapy. 
 Prior allergic reaction to TMZ, GM-CSF, or Td 
 Patients who are unwilling or unable to receive treatment and undergo follow-up evaluations at a 
Sunshine Project Consortium treatment site. 
 
2.3 
Inclusion of Women and Minorities 
 
Our brain tumor studies are open to both males and females, and varying ethnicities and races.  Patients, 
regardless of gender, race or ethnicity, meeting eligibility criteria may be enrolled in this trial if consent to 
participate is provided.    
  
 
2.4 
Consent/Assent Procedures 

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Provisions for seeking, obtaining and documenting consent for adult subjects and assent for children and 
permission of their parents or guardians will be in accordance with the determinations and requirements 
of the IRB per 21 CFR 50.  The treatment portion of this clinical trial involves greater than minimal risk 
but holds out the prospect of direct benefit to individual subjects (21 CFR 50.52) as described in section 
1.8.  In addition to parental permission, the assent of minor subjects will be obtained, when feasible, as 
determined by the IRB.  Potential subjects can be screened and provide consent for tumor tissue 
collection at any NPCF site.  If potential subjects are referred from outside physicians and it is not 
feasible for the subject to travel to a NPCF site, tissue consent may be obtained by designated staff from 
a NPCF treating center via telephone from the subject/LAR in accordance with institutional policies and 
procedures. Once screening consent is obtained, the Coordinating Center will interact with site to obtain 
tumor tissue in a manner suitable for RNA extraction per UF SOP. If standard-of-care sample collection 
occurred within 12 weeks prior to enrollment and frozen sample remains, a subject may enroll using 
previously collected samples. Samples must additionally have been snap frozen within 20 minutes  
of collection and kept in a -70/-80 freezer. 
 
Once it has been determined that an adequate amount of tumor tissue was collected and a pathological 
diagnosis of HGG has been confirmed, the subject must be evaluated at a NPCF treatment site (UF 
Health Shands Children’s Hospital, Children’s Hospital Los Angeles, or Nationwide Children’s 
Hospital). The Sunshine Project Consortium treatment site will confirm eligibility, obtain consent for the 
treatment phase of the study, and administer all subsequent trial-related therapy.  
 
A multi-disciplinary team at the site (at minimum a radiation oncologist or medical oncologist) will 
evaluate potential subjects. During their visit, a physician-investigator and/or a research coordinator will 
explain the study to the subject and/or parents/guardian, using the IRB-approved consent document to 
guide the discussion. The informed consent document will be provided to the subject, who will be given 
time to read over and ask questions of the study team. If the subject agrees to participate, a signed 
consent form will be obtained prior to initiation of any trial-related interventions and a copy of the signed 
consent will be provided to the subject.  Those subjects that turn 18 while on-study will be re-consented. 
 
 
3. REGISTRATION PROCEDURES 
 
3.1 
Site and Investigator Registration with Moffitt Cancer Center 
 
Only those members of the Sunshine Project Consortium registered with Moffitt Cancer Center may 
participate and enroll subjects on this trial. Sunshine Project Consortium Member sites are defined as 
those centers who have established financial agreements with Moffitt Cancer Center and who have 
designated a local Principal Investigator who will assume the responsibility of conduct of the trial 
according to Moffitt Clinical Research Network (MCRN) procedure and Federal and State regulations. 
 
All subjects to be enrolled on this trial must be registered with the MCRN office and the Sunshine 
Project Coordinator prior to being enrolled at the local level. Subjects may not be treated with any agent 
without registration with Moffitt staff and an associated Sequence number confirmed in the Moffitt 
Cancer Center electronic database OnCore. 
 
3.2 
Patient Registration 
 
When a potential subject presents to a participating site, the local investigator will confirm candidacy for 
screening by reviewing known inclusion and exclusion criteria. The participating site must notify the 

ACTION
 
39 
 
coordinating center to confirm slots are available for participation, and then discuss the protocol with the 
potential subject. Before a subject can be enrolled, it is the responsibility of the local investigator to verify, 
sign, and date a completed Moffitt Cancer Center Eligibility Checklist and Moffitt Cancer Center 
Registration sheet. 
 
The local investigator will confirm all screening eligibility criteria are met. A signed copy of the Checklist 
and Registration sheet must be e-mailed, with all supporting source documentation and signed consent 
form, to the Sunshine Project Coordinator prior to a patient being enrolled and initiation of trial-related 
procedures. 
 
The source documentation and registration sheets must be sent to the Sunshine Project Coordinator at 
Moffitt Cancer Center via email at SunshineProject@moffitt.org.. 
Note: No study therapy may be initiated before confirmation that the subject is eligible for screening.   
 
3.3 
General Guidelines 
 
Following registration, subject information must be placed in the OnCore system within 24 hours of 
receipt of Subject Sequence Number (OnCore CRF pages: demographics, consent, eligibility, on-
study, and treatment). 
 
4.  TREATMENT PLAN 
 
Chemo-Radiation on this protocol can be administered on an outpatient basis or per institutional 
guidelines. Reported adverse events and potential risks are described in Section 7. No investigational or 
commercial agents or therapies other than those described as acceptable within the protocol may be 
administered with the intent to treat the patient's tumor. 
 
4.1 
Agent Administration 
 
Drug Name 
Route 
Dose 
Schedule 
Instructions for administration 
Temozolomide 
(TMZ) during 
radiation 
PO 
90 mg/m2/day  
Day 1 of  xRT- max of 
49 days 
PO 1 hour prior to radiation and in 
AM on non-xRT days. 
Dose-intensified 
(DI) Temozolomide 
PO 
See dose 
escalation table 
Days 1-21 of 28 (+7) 
day cycle 
PO in AM 
TTRNA-loaded DCs 
(10 vaccines-V1-
V10) 
Intra- 
dermal 
(ID) 
1x107cells 
V1: Cycle 1 Day 22-24 
V2: Cycle 1 Day 36-38 
V3: Cycle 1 Day 50-52 
V4: Cycle 2 Day 22-24 
V5: Cycle 3 Day 22-24 
V6: Cycle 4 Day 22-24 
V7: Cycle 5 Day 22-24 
V8: Cycle 6 Day 23-25 
V9: Cycle 6 Day 37-39 
V10: Cycle 6 Day 51-53 
Each immunization will be 
administered according to SOP-
UFBTIP-128. DC immunizations will be 
divided equally between both inguinal 
regions. A total volume of 200 µL per 
side will be delivered, with no more 
than 0.1 ml in each injection site 
intradermal after preparation of skin 
with ELMA anesthetic cream.  
May be administered up to 72 hours 
after dose-intensified TMZ. If dose-
intensified TMZ is delayed within 
the allowable +7 day window, the 
vaccine administration day should 
be adjusted accordingly. 

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*Adoptive Tumor- 
specific T cells 
(xALT) 
IV 
Target dose of 
3x108cells/kg 
or all available 
cells 
Day 23-25 
Cycle 6 only 
xALT will be administered according to 
SOP-UFBTIP-2132.  Cells will be 
given IV over 10-60 minutes through 
venous catheter.  Pre-medicate 30–60 
minutes prior to infusion with 
acetaminophen (10-15 mg/kg/dose) and 
Benadryl (1 mg/kg) to reduce infusion-
related reactions. Vital signs will be 
assessed prior to and immediately 
following infusion, then every 15 
minutes for 1 hour after completion of 
infusion.   
Hematopoietic Stem 
Cells (Group B 
Only) 
IV 
Target dose of 
2x106 CD34+ 
cells/kg or all 
available cells 
Day 22-24 
Cycle 6 
only 
IV through venous catheter. 
Premedication, administration and 
monitoring will be done in 
accordance with institutional 
policies. 
Sargramostim (GM- 
CSF) 
Intra- 
dermal 
(ID) 
150ug 
Embedded 
within each 
vaccination of 
TTRNA- 
loaded DCs 
Concurrent with each DC 
vaccine (V1-V10) as 
above 
 
Tetanus/diphtheria 
(Td) vaccination 
V1: IM 
V3, V6, V8
DC site 
pre-
treatment: 
ID 
V1: 5 Lf 
V3, V6, V8 
DC site pre-
treatment:  1 Lf 
V1:  5 Lf 
V3, V6, V8 DC site pre-
treatment:  1 Lf 
Td vaccine #1 to be given IM. 
Td given as vaccine site pre-treatment 
will be given ID 4-24 hours prior to 
DC vaccine #3, #6, and #8 
Filgrastim (G-CSF) 
 
IV/SQ 
10 
micrograms/k 
g/day (max 
dose of 480 
mcg/day) 
 
Prior to mobilized 
leukapheresis and PRN  
until count recovery 
following NMA chemo 
(Group B) per PI and 
institutional policy. 
 
 
 
* If 2 of 6 patients experience immunotherapy related DLT following administration of 3 x108cells/kg x-ALT, an additional 6 patients will be 
enrolled on Group A to receive x-ALT at a dose of 3 x107cells/kg. The trial will stop if 2 of 6 patients develop DLT at this dose level. Once 
MTD is identified in Group A patients, 6 patients will be treated in Group B using this MTD for x-ALT infusion.  
 
4.1.1 
Radiation Guidelines 
 
Standard external beam RT will be administered concomitantly with TMZ. Institutional practices for 
administration of external beam RT for subjects with HGG may be followed. Otherwise the following 
guidelines should be used:  
 
Target volume definition: 
 Gross Tumor Volume (GTV): The GTV includes all gross residual tumor and/or the tumor bed as 
defined by MR imaging and operative report. The GTV in many cases will involve a contracted or 
collapsed tumor bed. Tissue defects resulting from surgical approaches will not be included as part of 
the GTV when not previously involved by tumor. 

ACTION
 
41 
 
 
 Clinical Target Volume (CTV): The CTV is meant to treat subclinical microscopic disease and will 
be an anatomically constrained 5-10 mm margin on the GTV, with additional expansion as necessary to 
encompass areas of T2/FLAIR change suspicious for tumor involvement prior to surgery. The CTV is 
limited to the confines of the bony calvarium, falx and tentorium where applicable and extends up to but 
not beyond neuroanatomic structures through which tumor extension or invasion is certain not to have 
occurred. When the GTV approaches the boundary of an anatomic compartment, the CTV will extend up 
to and include the boundary. 
 
 Planning target volume 1 (PTV1): CTV + 3 mm 
 
 Planning target volume 2 (PTV2): GTV + 3 mm 
 
Total dose:  
PTV1: 50.4 Gy 
PTV2: 59.4 Gy (Note: 54 Gy in infants who have undergone a gross total resection) 
 
Dose per fraction:  
1.8 Gy/fx daily, 5 days per week. PTV1 and PTV2 should be delivered in sequential phases over 33 
fractions. 
 
Temozolomide during radiation 
 
TMZ should be administered continuously from day 1 of radiotherapy to the last day of radiation at a daily 
oral dose of 90 mg/m2 radiation for a maximum of 49 days.  
 
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. 
 
TMZ administration and any modifications during radiation should be at the discretion of the treating 
physician or radiation oncologist.  The total number of days and total dose as well as any missed doses 
will be recorded in the data management system.  
 
4.1.2 
Dose Escalation for Temozolomide Cycles 1-6 following radiation 
 
Dose Level 
Dose 
Timing 
-2
50 mg/m2/day
If dose level -1 not tolerated
-1
75mg/m2/day
If Dose level 0 not tolerated
0
85mg/m2/day
Starting dose C1 and C2
+1 
100mg/m2/day 
One time dose escalation for 
C3-6
 
Cycle 1: TMZ will be started at a dose of 85mg/m2/day for 21 days of a 28 (+7) day cycle.   
 
Cycle 2: TMZ will be given at a dose of 85mg/m2/day if ANC ≥ 1000 and platelets ≥ 100,000 within 4 
weeks of last dose TMZ in C1. If counts do not meet criteria to start C2 within 4 weeks, wait for recovery 
and then decrease one dose level to -1 for C2 and continue treatment.  
 

ACTION
 
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Cycle 3:   If dose level 0 of TMZ is well tolerated in previous cycles, increase TMZ to 100mg/m2/day. If 
dose has been reduced to -1 in C2, continue the rest of cycles at 75 mg/m2/day. If counts do not meet 
criteria to start C3 within 4 weeks, wait for full recovery and then decrease one dose level to -2 for C3 
and continue treatment.  TMZ may be held at current dose and not escalated or stopped if Principal 
Investigator deems necessary in view of patient safety.   
 
Cycles 4 - 6: Continue at same dose as tolerated in previous cycles. If severe myelosuppression is 
experienced during these 3 cycles, dose reductions will be instituted as stated above.  DI TMZ will be 
complete with the cycle preceding the ALT infusion (Cycle 6). 
 
Prior to chemoradiation, enrolled subjects will undergo a mobilized leukapheresis for collection of PBSCs 
and PBMCs for generation of DCs. G-CSF mobilization of CD34+ HSCs will occur for both groups and  
leukapheresis for autologous peripheral blood stem cells (PBSCs) may be collected for up to 3 days for 
Group B subjects to obtain a recommended dose of > 2 x 106 CD34+ HSCs/kg. If unable to reach the 
recommended dose, all available stem cells will be infused.  
 
Chemoradiotherapy should begin within 5 weeks +/- 1 week of surgery. Standard external beam RT will be 
administered concomitant with TMZ. Institutional practices for administration of external beam RT for 
subjects with HGG may be followed. Otherwise the guidelines from section 4.1.1 should be used. 
Temozolomide 90mg/m2/day will be given continuously during radiation for up to 49 days to account for 
delays in radiation treatment. Modifications to dosing may be necessary due to ongoing clinical and 
laboratory assessment and tolerability of concomitant TMZ.  These modifications may be made at treating 
investigator’s discretion, but must be reported in database and justification for dose-modification 
documented.  
 
Five weeks (+/- 1) after the completion of chemoradiotherapy, the first cycle of adjuvant TMZ will be 
given at a dosage of 85 mg/m2/day orally for 21 days followed by three biweekly TTRNA-DC vaccines 
(1x107 cells) with GM-CSF(150µg per injection embedded within the DC vaccine) beginning on Days 22-
24 of cycle 1. Study vaccine will be given intradermally at day 22-24 after the first TMZ cycle and divided 
equally between both inguinal regions in accordance with SOP-UFBTIP-128. All subjects will receive Td 
booster IM (5 Lf) at Vaccine #1 regardless of booster history.  
 
Vaccine #2 and #3 will occur at 2 week intervals. Fourteen days (+/- 3 days) after the third biweekly DC 
vaccine, subjects will undergo a second leukapheresis (non-mobilized) to collect PBMCs for generation of 
further TTRNA-DC vaccines and for the expansion of vaccine-stimulated T cells using TTRNA-pulsed 
DCs (TTRNA-xALT). The collection goal for TTRNA- xALT and TTRNA-pulsed Dendritic Cell 
vaccine is a minimum of 5x109 mononuclear cells. 
 
4.1.3 
Criteria for Starting Subsequent Cycles  
 
A cycle may be repeated every 28 days if platelets are greater than ≥100,000 (unsupported for at least 3 
days) and ANC is ≥1,000 and the patient has at least stable disease.  All other non-hematologic toxicities 
must have resolved to baseline to move onto cycles 2 and beyond. 
 
The second cycle of TMZ beginning after the second leukapheresis will also be administered at a dose of 
85 mg/m2/day orally for 21 days of a 28 (+7) day cycle. If no dose modifications are required, starting 
with cycle 3, subjects will be escalated to a dose of 100 mg/m2/day of TMZ for 21 days of 28 (+7) day 
cycle. TMZ cycles 2-5 will be followed by a single monthly TTRNA-DC vaccine given on day 22-24 (+ 
7).  
 

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During Cycle 6, both Groups A and B will receive an intravenous infusion of the TTRNA-xALT (3x108 
cells/kg) along with the DC vaccine between days 23-25 and an additional two biweekly TTRNA-DC 
vaccines.  All DC vaccines will be given intradermal and divided equally to both inguinal regions. 
Autologous HSCs will be administered intravenously 12-36 hours prior to DC vaccines/xALT to Group B 
only. 
 
Since HSCs may act synergistically to enhance the efficacy of cellular therapeutics in pediatric patients, 
subjects will be grouped into cohorts that receive either TTRNA-xALT alone (Group A) or TTRNA-
xALT and HSCs (Group B). Subjects in Group A will receive only TTRNA-DCs and TTRNA-xALT 
between day 23-25 after completion of 21-day TMZ during the 6th cycle.  During the 6th TMZ cycle, 
subjects in Group B will receive HSCs between days 22-24 after completion of TMZ. TTRNA-DCs and 
TTRNA-xALT will be administered 12-36 hrs after HSC infusion. Both groups will receive an additional 
two bi-weekly TTRNA-DC vaccines during Cycle 6. In addition to receiving a full Td booster vaccine at 
Vaccine #1, all subjects will undergo vaccine site pretreatment with a one-fifth dose of Td (1 Lf) 
intradermally 4-24 hours prior to Vaccine #3, #6, and #8. While receiving TMZ, subjects should receive 
PCP prophylaxis with pentamidine. The subjects will then be observed with serial MRIs for follow up until 
progression. After immunotherapy is completed, subjects should not receive any additional anti-tumor 
treatment until documented tumor progression.  
 
If either DCs or xALT are generated but do not meet release criteria, the subject may remain on study and 
receive the qualified product (either DCs or xALT) but will be replaced for the purposes of safety 
assessment. Subjects that are unable to tolerate TMZ will continue on study and receive cellular therapy 
per protocol.  If either DCs, xALT or PBSC are generated but do not meet targeted dose, all available 
cells will be administered and dose documented. Subjects who progress prior to receiving DC vaccination 
can receive immunotherapy treatment if cellular products have been generated and meet release criteria. 
For subjects that meet iRANO criteria for progression after the initiation of study drug, the subject will be 
replaced for evaluability and safety but may continue to receive cellular therapy if cellular products have 
been generated and meet release criteria. Once progression is determined, the subject will be managed by 
salvage therapy under the direction of the Protocol Chair until immunotherapy treatment is available for 
administration.  
 
4.1.4 
Immune Monitoring  
 
Peripheral blood will be drawn  within 24 hours of leukapheresis, within 24 hours prior to the start of 
each TMZ cycle, if feasible, within 24 hours prior to each vaccination (1-10),  1 day post xALT infusion, 
and 4 days +/1 day post xALT infusion (if feasible).  Research blood will be drawn at each bi-monthly 
follow-up visit, if feasible.  
 
Will conduct assays to test the ability to expand T cells against tumor antigens, exploring 
immunoreactivity against neoantigens and tumor-associated antigens (TAAs) in vitro. Assays will be 
conducted to test whether T cell responses are driven predominately by neoantigens or TAAa through 
analysis of RNAs encoding the individual antigens in T cell stimulation assays. 
 
 
4.1.5 
Follow-up 
 
Progression free survival.  Subjects will be followed bi-monthly (+/- 1 month) for one year and then per 
PI discretion (minimum of every 6 months) until tumor progression or death due to any cause.  Subjects 
will undergo medical history, neurological and physical exam, MRI, and CBC with differential, CD4/CD8 
ratio, and research labs.   

ACTION
 
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Upon progression, subjects will be followed for overall survival only. This contact for follow-up will be 
made every 3 to 6 months and may be via phone or in-person. 
 
Subjects will be followed until death due to any cause. As part of standard care for these subjects, upon 
tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research 
procedure, consent will be obtained separately.  If tissue is obtained, it will be used to confirm tumor 
progression histologically and to assess immunologic cell infiltration and antigen expression profile in 
recurrent lesions. 
 
4.2 
General Concomitant Medication and Supportive Care Guidelines 
 
There is a potential for interaction of immunotherapy with other concomitantly administered drugs. The 
following medications will be recorded in the data management system with start date, stop date, and 
indication: 
Steroids  
Anticonvulsants  
Any medication used in the treatment of a VACCINE/STUDY DRUG related AE/SAE 
Any medication deemed to possibly be interactive with vaccine or study drug by PI or designee.  
 
The site investigator will determine if medications given while on treatment constitute significant drug-drug 
interactions that require reporting of this medication to the Principal Investigator. 
 
4.2.1 
Treatment of pseudo-progression 
 
For signs and symptoms of pseudo-progression, treatment with bevacizumab is recommended to minimize 
swelling; would caution use of corticosteroids, as these agents may induce lymphopenia post-
immunotherapy.  Treatment options should be discussed with the Protocol Chair prior to initiation of 
therapy. 
 
Treatment of pseudoprogression or inflammation 
 
bevacizumab
7.5 mg/kg q 3 weeks.
 
 
4.2.2 
PCP Prophylaxis 
 
pentamidine 
300 mg IH or 4 mg/kg IV monthly starting with 
chemoradiation 
 
4.2.3 
Neurosurgical Procedures 
 
In uncertain cases where there is significant clinical decline and possible pseudo-progression is suspected, 
if biopsy or resection is feasible, pathological assessment should be strongly considered.   
 
4.3 
Duration of Therapy 
 
The duration of treatment following the initial non-mobilized leukapheresis is shown in the table below. 
Subjects may have up to 4 weeks between cycles for ANC and platelets to recover or for unacceptable 
toxicity recovery therefore treatment may last 9 - 14 months.   

ACTION
 
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Length of Treatment 
Weeks 1-6
Chemoradiation
Weeks 7-12
Rest
Weeks 13-15
C1 TMZ
Weeks 16-21 
DC Vaccine 1,2,3 and 2nd non-mobilized leukapheresis 
Weeks 22-25
C2 + DC 4
Weeks 26-29
C3 + DC 5
Weeks 30-33
C4 + DC 6
Weeks 34-37
C5 + DC 7
Weeks 38-40
C6 TMZ
Weeks 41-46
DC 8 + xALT +/- HSC, DC 9, DC 10
 
4.3.1 
Subject Withdrawal 
 
Subjects may withdraw at any time or be dropped from the study at the discretion of investigator should 
any untoward effects occur. In addition, a patient may be withdrawn by the investigator if he/she violates 
the study plan or for administrative and/or other safety reasons. The investigator or study coordinator 
must notify Moffitt immediately when a patient has been discontinued/withdrawn due to a serious adverse 
experience. All trial treatment-related toxicities and SAEs at the time of discontinuation/withdrawal 
should be followed until resolution or stabilization. Subjects who discontinue/withdrawal from the study 
will continue to be followed for disease progression. 
 
Subjects who discontinue study participation for reasons unrelated to the study (e.g., personal reasons, or 
adverse events after registration but prior to receiving study therapy) may be replaced as required for the 
study to meet its objective. The replacement will generally receive the same treatment or treatment 
sequence (as appropriate) as the allocation number replaced. 
 
Subjects may be removed from the study if any of the following criteria applies: 
 
 Unacceptable treatment related toxicity, NCI CTC AE version 4.0 Grade 3 or 4 that fails to 
recover to baseline or < Grade 3 in the absence of treatment within 4 weeks 
 Grade 3 neurologic toxicity that does not recover in 5 days. 
 Any toxicity or other issue that causes a delay of study drug administration by more than 4  
 weeks 
 Intercurrent illness that prevents further administration of treatment 
 An adverse event which requires discontinuation of the trial medication or results in inability to 
continue to comply with trial procedures 
 Subject decides to withdraw from the study 
 General or specific changes in the subject's condition render the subject unacceptable for further 
treatment in the judgment of the investigator 
 Subject non-compliance with treatment or trial requirements: defined as any deviation from the 
protocol without prior agreement of the principal investigator 
 Investigator non-compliance: defined as any significant medical or non-medical deviation 
from the protocol without agreement of the principal investigator 
 Loss to follow-up 
 
4.3.2 
 Subject Death 
 

ACTION
 
46 
 
Any deaths that occur within the trial treatment period, beginning with leukapheresis through 30 days 
after administration of the last dose of trial drug, regardless of relatedness, must be reported to the 
Moffitt Coordinating Center via the OnCore system for the purposes of serious adverse event (SAE) 
reporting.  Deaths will be reviewed and reported in accordance with protocol section 7.2.5. Any deaths 
outside of these reporting timeframes thought to be related or possibly related to trial drug must also be 
reported. 
 
4.3.3 
 Off Treatment Criteria 
 
For the purposes of this trial, “Off treatment” is defined as the day the last dose of investigational agent 
is administered.  
 
4.3.4 
 Off Study Criteria 
 
A subject is considered “Off study” if the subject withdraws consent, or if the subject dies.  The 
“off study” date is considered the last day of Follow Up. 
 
4.4 
Duration of Follow Up 
 
Duration of Follow Up is defined as follows: 
 
Subjects will be followed until death due to any cause. MRI and clinical evaluation for assessment of disease 
progression will be conducted bi-monthly +/- 1 month for one year and then per PI discretion (minimum of 
every 6 months) until tumor progression or death due to any cause. Radiographic disease progression will 
be assessed.  As part of standard care for these subjects, upon tumor progression, participants may undergo 
stereotactic biopsy or resection. As this is not a research procedure, consent will be obtained separately.  If 
tissue is obtained, it will be used to confirm tumor progression histologically and to assess immunologic cell 
infiltration and antigen expression profile in recurrent lesions. 
 
Upon progression, subjects will be followed for overall survival only.  
 
 
Follow-up Requirements for Early Withdrawal: 
 
Subjects treated on this study that are withdrawn by the PI for any of the aforementioned reasons will continue 
to be followed for survival by the study coordinator until death or are lost to follow up. Subjects that are 
withdrawn prior to any protocol treatment for any of the aforementioned reasons as well as for failure of 
T cells to qualify or inability to undergo leukapheresis will be considered eligibility failures and thus will 
not be followed for survival. 
 
4.5 
Criteria for Removal from Study 
 
Subjects will be removed from study when any of the criteria listed in Section 4.3.1 applies. The reason and 
date for the removal from treatment and study must be captured in the OnCore eCRF. 
 
4.6 
Criteria for Evaluable Subjects 
 
Evaluable subjects are those that have received 10 vaccines with a minimum of 2 weeks follow up.  
Once potential subject signs the informed consent form, that subject will be considered “on study”. Rationale 
for taking subject off protocol treatment will be documented. 

ACTION
 
47 
 
 
4.7 
Replacement 
 
All subjects enrolled on the study, even if taken off study prior to treatment, will be included in the data 
analysis on an intent to treat basis. However, subjects will be replaced for safety assessments for the 
following reasons:  
 
 If either DCs and/or xALT are generated but do not meet release criteria or targeted dose then the patient 
may remain on study and receive the qualified product but will be replaced for the purposes of safety 
assessment. 
 Subjects 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 physiologic levels, may remain in the study but 
will be replaced for assessment of safety as increased steroid usage may mask risks of autoimmune 
toxicity.  For the purposes of this study, physiologic dose will be defined as > 4 mg of 
dexamethasone/day. 
 Subjects receiving less than 10 vaccines without toxicity will be replaced for safety assessments. 
 If subject cannot tolerate DI TMZ the subject will remain on study and evaluable for primary endpoint. 
Immunologic endpoint analyses would describe any differences observed in subjects that did not 
received DI TMZ but not be powered to detect any differences.   
 
 
Subjects receiving or requiring any other concurrent anticancer or investigational drug therapy will be excluded 
and replaced.   
 
If a subject withdraws from the study before receiving immunotherapy, then that subject will be replaced.  
PFS and OS will be reported for all subjects on an intent-to-treat basis. 
 
5. DOSING DELAYS/DOSE MODIFICATIONS 
 
5.1 
Definition of Dose-Limiting Toxicity (DLT) 
 
A DLT will be defined as any immunotherapy-related: 1) Grade III or greater non-neurologic toxicity; 
2) Grade III neurologic toxicity that does not improve to Grade II or better within 5 days; or 3) Grade 
IV neurologic toxicity.  If neurologic toxicity returns to baseline prior to next vaccine, the next vaccine 
can be administered on schedule.  Vaccine can be withheld for up to four weeks if neurologic symptoms 
have improved to grade II or below within 5 days (thus not a DLT) but have not returned to baseline by 
time of next scheduled vaccine. If the event cannot be reversed within 5 days of its onset (improving to 
Grade II or better), a DLT will be declared for that patient and no further vaccinations or study-related 
procedures will be performed. If the event is reversed, but Grade III neurologic toxicity is again seen 
with subsequent vaccinations, all further vaccinations and study-related procedures will be withheld and 
a DLT will be declared. Medical therapy may be used to reverse any toxicity if necessary, but any 
episode of a toxicity requiring surgical intervention will still be considered a DLT even if reversible by 
surgery. All patients meeting DLT criteria will no longer receive immunotherapy and be taken off study.  
 
Cerebral edema toxicity exception: 
 
NCI CTCAE criteria categorize all cerebral edema as grade 4.  Cerebral edema normally presents in 
patients with malignant gliomas as part of the disease process and can be exacerbated by standard of care 
chemotherapy and radiation. Furthermore, an effective anti-tumor immune response may involve 

ACTION
 
48 
 
inflammatory response and edema in infiltrative tumor cells. Therefore cerebral edema toxicity, although 
ranked grade IV by NCI CTCAE criteria, will be not be considered a DLT if patient is stable or improved 
clinically.   If a cerebral edema is observed in a patient in clinical decline, the event will be considered a 
DLT if it is clearly attributable to the investigational drug and patient does not show improvement within 5 
days of clinical management. 
 
Grade III or greater toxicities associated with DC-based immunotherapy have been rare. Adoptive T cell 
therapy using tumor-infiltrating lymphocytes and high-dose IL-2 in patients with melanoma and treatment 
with anti-CLTA-4 monoclonal antibody blockade have been the immunotherapy regimens most often 
associated with treatment related toxicities. To take the most conservative approach to assessing possible 
toxicities associated with this treatment, investigators should be vigilant for any similar toxicity associated 
with this therapy, in addition to autoimmune toxicity specific to the CNS. Possible immune-mediated 
disorders that have been observed in patients who have received immunotherapy in early phase trials have 
involved the skin (vitiligo and cutaneous leukocytoclastic vasculitis), the thyroid gland (autoimmune 
thyroiditis), the liver (autoimmune hepatitis) and the pituitary (hypophysitis). Abnormal lab results, which 
may be immune-mediated, include elevations of serum lipase and amylase and liver function tests. If a 
patient has an AE that is thought to be possibly related to autoimmune antibodies (e.g., thyroiditis, hepatitis, 
thrombocytopenia) the PI will send a blood sample for appropriate autoimmune antibody testing. If specific 
autoantibodies are present, the serum sample taken at baseline will be tested for the presence of those 
autoantibodies.  
 
Continuation of DC + xALT therapy in the presence of immune-mediated events may proceed only after 
discussion with the Protocol Chair, DSMB and patient on a case-by-case basis with consideration to risk-
benefit analysis. The IRB will be notified promptly and review the event and determine whether the 
problem does or does not represent an unanticipated risk to the study participants. If review determines that 
unanticipated increased risk did occur a revised consent form stating such a risk will be drafted, approved 
by the IRB and prior to continuing therapy will require the patient to be re-consented with the revised 
consent. 
 
5.2 
Dose Modification 
 
If 2 of 6 patients experience immunotherapy related DLT following administration of 3 x108/kg x-ALT, an 
additional 6 patients will be enrolled on group A to receive x-ALT at a dose of 3 x107/kg. The trial will stop 
if 2 of 6 patients develop DLT at this dose level. Once MTD is identified in group A patients, 6 patients 
will be treated in group B using this MTD for x-ALT infusion.  There is no dose modification for DC 
vaccine in this study. 
 
5.3 
Adverse Events and Management 
 
Hematological AE related to Temozolomide on Day 1 of Each Cycle (within the prior 48 hours 
before Day 1) 
AE 
Delay 
ANC< 1.5 x 109/L and/or Platelet 
count < 100 x 109/L  
Delay up to 4 weeks until all resolved. If 
unresolved after 4 weeks then stop. If 
resolved, dose delay/reductions based 
on hematologic and non-hematologic 
AEs in table below are applicable. 
 
Worst Treatment-Related Hematologic AE during the Previous Cycles 

ACTION
 
49 
 
Worst AE 
 
Platelets 
≥100 x 109/L 
< 100 x 109/L 
 
ANC 
 
≥ 1.5 x 109/L   
Dose unchanged  
Reduce by 1 
dose level
≥1 & <1.5 x 
109/L 
Dose unchanged  
Reduce by 1 
dose level
< 1 x 109/L   
Reduce by 1 dose 
level 
Reduce by 1 
dose level
 
Non-Hematologic Temozolomide related AE (except for alopecia, nausea and vomiting if controlled 
by maximal antiemetic therapy) On the day 1 of Each Cycle (within the prior 72 hours) 
Grade 
Delay 
2-3  
Delay up to 4 weeks until all resolved (to grade ≤ 
1).  If unresolved after 4 weeks then stop. If 
resolved, 
dose 
delay/reductions 
based 
on 
hematologic and non-hematologic AEs are 
applicable. 
4 
Stop (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy)
 
Summary of Dose Modification or Discontinuation During Post-Radiation Temozolomide Worst 
Non-Hematologic AE (except for alopecia, nausea and vomiting if controlled by maximal antiemetic 
therapy) During the Previous Cycles 
 
Grade 
Dose Modification 
0-2  
No dose modifications for non-hematologic AEs. Dose escalations (only for 
cycle 3) or reductions based on ANC and platelet counts are applicable. 
3  
Reduce by one dose level (except for alopecia, nausea and vomiting if controlled 
by maximal antiemetic therapy). Dose modifications (escalations or reductions) 
based on ANC and platelet counts are not applicable. No further escalation is 
possible. If the same non-hematologic grade 3 AE recurs (except for alopecia, 
nausea and vomiting if controlled by maximal antiemetic therapy) after reduction 
for that AE, then stop. 
4  
Stop (except for alopecia, nausea and vomiting if controlled by maximal 
antiemetic therapy). Dose modifications (escalations or reductions) based on 
ANC and platelet counts are not applicable. 
 
In the case of hypersensitivity reactions, treatment measures deemed medically appropriate will be 
initiated and the PI notified of the event. The following treatment recommendations may be applicable 
and can be adopted at the judgment of the supervising pediatrician: 
 
CTCAE (Common Terminology Criteria for Adverse Events) v.4.0 Grade I Allergy (transient flushing or 
rash, drug fever <38°C): 
 
 Supervise at the bedside. 
 
CTCAE v.4.0 Grade II Allergy (urticaria, drug fever=38°C, and/or asymptomatic bronchospasm): 

ACTION
 
50 
 
 
 Interrupt the infusion of xALT and disconnect infusion tubing from patient. Do not inject any 
remaining DCs. 
 Administer IV antihistamines (diphenhydramine 1mg/kg and ranitidine 3 mg/kg). 
 After recovery of symptoms, resume the infusion at half the initial infusion rate. If no further 
symptoms appear, complete the administration of the xALT. If symptoms reappear, stop infusion 
and discontinue patient from the study. 
 
CTCAE v.4.0 Grade III or IV Allergy (symptomatic bronchospasm requiring parenteral medication(s) 
with or without urticaria; allergy-related edema/angioedema; hypotension; anaphylaxis): 
 
 Stop the infusion of xALT and disconnect infusion tubing from patient. Do not inject any 
remaining DCs 
 Administer epinephrine (1:10,000) in 3.5 to 5 mL IV boluses (no more than 6 doses). 
 Administer IV antihistamine (diphenhydramine 1mg/kg (up to 50 mg) IV push). 
 If wheezing persists: 0.35 mL of inhaled albuterol or other bronchodilators. 
 Consider methylprednisolone (1-2mg/kg or 30-60 mg IV push), which may prevent recurrent or 
ongoing reactions. 
 Anaphylaxis will be treated promptly with standard of care treatment procedures. 
 Patient must be taken off treatment. 
 
All toxicities should be graded according to the Common Terminology Criteria for Adverse Events 
(version 4.0). 
 
The CTCAE version 4.0 is identified and located on the CTEP website at http://ctep.cancer.gov. 
All appropriate treatment areas should have access to a copy of the CTCAE version 4.0. 
 
6. SAFETY CONSIDERATIONS 
 
6.1 
Allergic Reactions to DC Immunization or TTRNA-xALT 
 
Injection of antigen presenting cells may result in an allergic reaction, which could include redness and 
swelling at the injection site, itching, hives, low blood pressure, difficulty breathing, or in the most 
extreme circumstances, death. In addition, if the immune system becomes overly activated, potential 
discomforts may include pain, redness and swelling at the injection site. The risks associated with the 
injection of autologous lymphocytes for immunotherapy in humans are currently unknown. The best 
current assessment of potential risks of TTRNA-xALT can be gathered from the extensive experience 
gained at our institution and others in autologous PBSCT using mobilized progenitors. We have 
experienced no grade III or IV toxicities associated with infusion of autologous lymphocytes in patients 
who have received mobilized PBSCT. A recent review describing management of toxicities associated 
with this procedure describes only complications associated with the consolidation and conditioning 
regimens utilized in stem cell transplantation, as well as the post-transplant period where patients exhibit 
profound neutropenia. Subjects may experience an allergic reaction to preserved cells or other transfusion 
associated side effects such as pain at the injection site, mild swelling or edema, hypotension, or shortness 
of breath. 
 
6.2 
Cerebral Edema 
 
Cerebral edema may be secondary to the disease process itself, the surgical procedure, necrosis from 
previous radiation, or inflammation due to immune infiltration of the brain or destruction of tumor cells. 

ACTION
 
51 
 
Symptoms may include, but are not limited to, severe headache, confusion, lethargy, unresponsiveness, 
coma, or focal neurological deficits. Subjects will be monitored throughout the course of the study and 
those subjects with any signs or symptoms of cerebral edema may need their steroid doses increased, 
treatment with an osmotic diuretic, or surgical decompression. 
 
Edema that fails to respond to aggressive therapy may lead to permanent neurological impairment. The 
probability of this risk can be predicted to some degree based upon tumor size, location, pre-operative 
neurological impairment, and post-operative course prior to DC injections. Subjects will be monitored 
throughout the course of the study. 
 
6.3 
Infection 
 
The DC injections or the TTRNA-xALT may include the risk of infection due to potential contamination of 
the DCs or leukocyte product in the laboratory. This may result in localized redness, swelling, or induration 
at the injection site. In the most extreme situation, this may lead to systemic bacterial/fungal sepsis and 
possibly death. The probability of this risk is relatively low, given the small injection volume (1 mL divided 
between >2 intradermal locations) and the fact that the DCs will be strictly tested for sterility prior to each 
injection. The risk of infection due to potential contamination of the DCs in the laboratory will be 
minimized by biosafety quality assurance and testing. All cell cultures will be handled under sterile 
conditions in a core tissue culture facility dedicated to the processing of human cells. Prior to injection into 
subjects, DCs must pass sterility tests in thiglycolate broth, tryptic soy blood agar, and inhibitory 
Sabouraud agar. Following injections, subjects will be monitored throughout the course of the study for any 
signs and symptoms of infection.  
 
 
6.4 
Delayed Autoimmune Diseases 
 
It is possible that delayed autoimmune disease(s) may develop as a result of injection with DCs or 
TTRNA-xALT. This means that the immune system may be stimulated to attack natural tissue in the body. 
Animal studies have reported the development of autoimmunity in the context of vaccination and recovery 
from lymphopenia. However, our current experience with DC vaccination in glioma patients has not 
demonstrated evidence of autoimmunity in treated patients. It therefore, is unknown what the risk of 
delayed autoimmune disease is for this study. 
 
6.5 
Leukapheresis 
 
Autologous hematopoietic stem cells (HSCs) for administration to Group B patients will be processed and 
stored under FACT-accredited processes by the clinical bone marrow transplant team at the participating 
treatment center.  The mobilized pheresis will be collected by the clinical Bone Marrow Transplant team 
and stored at ambient temperature until processing following FACT-accredited guidelines for 
hematopoietic stem cell harvest and processing. Subjects will undergo pheresis collection until a target 
dose of 2 x 10^6 CD34+ cells/Kg has been collected or maximal dose achieved on first day of collection.  
HSCs will be processed and stored by BMT clinical team following institutional clinical SOPs and stored 
according to FACT -accredited guidelines. Following pheresis for PBSCs, subjects will undergo 
leukapheresis to collect PBMCs for DC generation, which will proceed to target of 5 x 10^9 mononuclear 
cells or until completion of second day of pheresis. The second pheresis collection will be transported to 
the cGMP laboratory for DC generation following SOP-UFBTIP-121 (for cells collected at UF Health) or 
SOP-UFBTIP-127 (for cells collected at external sites). 
 

ACTION
 
52 
 
As with any donation of blood, a variety of minor reactions may occur with leukapheresis, which include 
fainting, dizziness, or nausea. Uncommon but serious complications may also result, which include 
bleeding, infection, an adverse reaction to the anticoagulant or replacement fluids, hypocalcemia, 
hypotension, shock, convulsions, air emboli, heart failure, or the inability to transfuse blood back into the 
patient. These risks are reduced by the fact that the procedure will be performed by qualified staff at a 
specialized clinical hemapheresis unit. Subjects will be carefully monitored throughout the procedure by 
trained nursing and medical staff and managed per institutional policy. Blood will be routinely screened for 
HIV, hepatitis, and syphilis to minimize the risk of transmitting infection. 
 
6.5.1 Central Venous Catheter Placement 
 
Patients may require placement of a central venous catheter, using conscious sedation or anesthesia, for 
the leukapheresis procedure. Separate operative/procedural consent will be obtained from the 
surgeon/proceduralist placing the central line. The surgeon/proceduralist may use fluoroscopy to place the 
central venous catheter and ensure that it is in a satisfactory position. Fluoroscopy involves low dose 
radiation and is low risk.  Risk of central line placement include (but not limited to): bruising, bleeding, 
injury to the vessel which may be permanent, possible injury to adjacent structures/organs, pneumothorax, 
infection and postoperative pain/discomfort. Removal of the central line is low risk but does include 
possible thrombosis or stenosis of the vessel. This may prevent future use of this vessel for additional 
leukapheresis. Risks of conscious sedation include pain from the initial needle stick when placing the IV. 
Possible bruising from where the needle went into the skin may occur, as well as rarely, an allergic reaction 
to the medication. Vomiting or inhaling food contents, drowsiness, amnesia, hypotension, respiratory 
depression or rarely, death, may result from conscious sedation.  Risks of anesthesia may include 
complications such as breathing difficulties, low blood pressure, or an irregular heartbeat, which can 
rarely lead to death. 
 
 
7. ADVERSE EVENTS: DEFINITIONS AND REPORTING REQUIREMENTS 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are done to 
ensure the safety of subjects enrolled in the studies as well as those who will enroll in future studies using 
similar agents. Adverse events are reported in a routine manner at scheduled times during a trial (See 
section 7.3). 
 
Additionally, certain adverse events must be reported in an expedited manner for more timely monitoring 
of patient safety and care. The following sections provide information about expedited reporting. The 
characteristics of an observed AE will determine whether the event requires expedited reporting via a 
Medwatch form and OnCore SAE eCRF to the Coordinating center in addition to routine reporting. 
 
7.1 
Adverse Event Characteristics 
 
Characteristics of an Adverse Event must be characterized as follows: 
 CTCAE term (AE description) and grade:  The descriptions and grading scales found in the revised 
NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
4.0 will be utilized for AE reporting.  All appropriate treatment areas should have access to a copy of the 
CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. 
 
 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the investigational study treatment. 

ACTION
 
53 
 
- 
Probable – The AE is likely related to the investigational study treatment. 
- 
Possible – The AE may be related to the investigational study treatment. 
- 
Unlikely – The AE is doubtfully related to the investigational study treatment. 
- 
Unrelated – The AE is clearly NOT related to the investigational study treatment. 
 
7.2 
Expedited Adverse Event Reporting (Serious Adverse Event) 
 
7.2.1 
Determination of Reporting Requirements: 
 
Reporting requirements should include the following considerations: 1) whether the patient has received 
the investigational or commercial agent; 2) the characteristics of the adverse event including the grade 
(severity), the relationship to the study immunotherapy (attribution), and the prior experience 
(expectedness) of the adverse event. 3) the reporting window: This study will include two adverse event 
collection periods:  leukapheresis and immunotherapy (DC + xALT +/- HSC). The leukapheresis collection 
periods will begin at initiation of leukapheresis and continue until 24 hours after the completion of the 
procedure. The immunotherapy collection period will begin with first DC vaccine and continue through 30 
days after administration of the last dose of trial drug or subject death, whichever comes first. 
 
 Events occurring outside the reporting windows that have a possible relationship to vaccine therapy 
must also be reported. All AEs must be recorded in the subject’s medical and/or record and case report 
form. 
 
7.2.2 
Steps to Determine if an Adverse Event is to be reported in an Expedited Manner 
 
Step 1: Identify the type of event: The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0. 
 
Step 2: Grade the event using the NCI CTCAE version 4.0. 
 
Step 3: Determine whether the adverse event is related to the protocol therapy (investigational or 
commercial). Attribution categories are as follows: Unrelated, Unlikely, Possible, Probable, and Definite. 
All AE’s, regardless of grade, deemed by the PI as possibly, probably or definitely related to 
Immunotherapy should be reported to the MCRN Coordinating Office and Protocol Chairs (Immunotherapy 
and Neuro-Oncology) within 24 hours of knowledge of the event via e-mail.   
 
Step 4: Determine the prior experience of the adverse event. Expected events are those that have been 
previously identified as resulting from administration of the agent. An adverse event is considered 
unexpected, for expedited reporting purposes only, when either the type of event or the severity of the 
event is NOT listed in the investigational brochure or package insert for the investigational agent. 
 
If any of the following criteria are met, the Adverse Event should be reported in an expedited 
manner and will qualify as a Serious Adverse Event on this trial. 
 
A Serious Adverse Event is defined as any untoward medical occurrence that, at any dose, meets any of 
the following criteria: 
 
Results in death. 
 
Is life-threatening: The term ‘life-threatening’ in the definition refers to an event in which the 
patient was at risk of death at the time of the event, it does not refer to an event which 
hypothetically might have caused death if it were more severe. 

ACTION
 
54 
 
 
Requires inpatient hospitalization or prolongation of existing hospitalization: A hospitalization or 
prolongation of hospitalization will not be regarded as an SAE if at least one of the following 
exceptions is met: 
o The admission results in a hospital stay of less than 24 hours. 
o The admission is for observation or minor treatments such as hydration and released in 24 hrs.  
o The admission is pre-planned (e.g. elective or scheduled surgery arranged prior to the start 
of the study). 
o The admission is not associated with an AE. (e.g. social hospitalization for purposes of 
respite care). 
 
Disability or Permanent Damage 
 
Congenital Anomaly/Birth Defect 
 
Other Serious (Important Medical Events) 
 
Please see the FDA website: http://www.fda.gov/Safety/MedWatch/HowToReport/ucm053087.htm for 
more information regarding the definition of a Serious Adverse Event 
 
7.2.3 
Serious Adverse Event Reporting 
 
Serious Adverse Event (SAE) reporting for this study must follow a two-step reporting process to comply 
with Moffitt AE reporting guidelines (Policy standard CTO.65). 
 
Step 1: All SAEs will be reported to the MCRN Coordinating office within 24 hours of learning of the 
event via the OnCore eCRF. In addition, an email notification should be sent to the Sunshine Project 
coordinator (affiliate.research@moffitt.org) indicating that an SAE has been entered in the system. 
 
Step 2: Within 24 hours of learning of the event, the reporting investigator should download a Medwatch 
form, Form FDA 3500A – Mandatory Reporting from the FDA website 
http://www.fda.gov/safety/medwatch/howtoreport/downloadforms/default.htm 
 
Fill out required information and 
 
Email or fax the completed Medwatch form to Sunshineproject@moffitt.org. 
 
A follow up Medwatch form will be required to update all information captured on the original form. 
 
Serious Adverse Event Reporting at Site Level: 
 
All SAE’s should be reported to the treating institutions IRB or Ethics board per local IRB policy. All 
SAEs must also be reported to the Moffitt Cancer Center Coordinating Center for review per standard 
operating procedures.  The Protocol Chair and IND holder, Dr. Mitchell, Preston A. Wells, Jr. Center for 
Brain Tumor Therapy at University of Florida, will establish FDA reporting system in collaboration with 
Moffitt Coordinating Center.  
 
Supporting source documentation for serious adverse events will be monitored and the following should be 
available to support reported information: 
 
Laboratory reports 
 
Emergency room notes 
 
Hospital discharge summary 
 
Death certificate (if available) 
 
Autopsy report (if available) 
 

ACTION
 
55 
 
REMINDER: All Deaths occurring during the trial treatment period beginning with leukapheresis 
through 30 days post administration of the trial drug must be reported as a Serious Adverse Event. 
 
7.2.4 
Distribution of Adverse Event and Serious Adverse Event Reports 
 
Routine Adverse Events for each patient will be discussed on the bi-weekly calls held by the Coordinating 
Center. The calls are a consistent bi-weekly check-in with PIs and site coordinators to ensure the continued 
safety of all subjects receiving treatment, and for the distribution of information regarding dosing and drug 
toxicity seen in real time with enrolled subjects. 
 
The Sunshine Project Coordinators are responsible for the distribution of Serious Adverse Event reports 
(via Medwatch forms) to all enrolling sites participating on the trial. Sites are responsible for the reporting 
of those Serious Adverse Event reports to the local PI and IRB according to the site’s reporting policy. 
 
7.2.5 
Expedited Reporting Guidelines - Death 
 
A death on study requires both routine and expedited reporting, regardless of causality.  
Attribution to treatment or other cause must be provided. 
 
If a death occurs within the trial treatment AE reporting periods, enrollment of new subjects will be 
suspended until review by the Protocol Chair/IND sponsor has been completed and the FDA notified.  
Causes of death definitely not related to study participation (i.e., motor vehicle accident) will result in 
notification of FDA but will not result in suspension of new subject enrollment or continued treatment 
for enrolled subjects.  Given the high degree of expected mortality in patients with HGG, treatment for 
enrolled subjects may continue on schedule unless the cause of death is assessed to be possibly, probably 
or definitely related to the trial drug.  If attribution of death is determined by the Protocol Chair/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 review is completed and recommendations 
for study continuation have been 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.  The IRB will be notified of subject deaths 
per local policy.  Reporting to and review by the Moffitt Protocol Monitoring Committee and Clinical 
Trials Oversight Committee will be in accordance with standard procedures.  Deaths or life-threatening 
adverse events will be reported to FDA by the IND sponsor as soon as possible but no later than 7 
calendar days after the sponsor's initial receipt of the information (21 CFR 312.32). 
 
Death due to progressive disease should be reported as Grade 5 “Neoplasms benign, malignant and 
unspecified (incl cysts and polyps) - Other (Progressive Disease)” under the system organ class 
(SOC) of the same name. Evidence that the death was a manifestation of underlying disease (e.g., 
radiological changes suggesting tumor growth or progression: clinical deterioration associated with a 
disease process) should be submitted. 
 
 
7.3 
Routine Adverse Event Reporting 
 
All adverse events (AE) will be monitored throughout the course of the study. The National Cancer 
Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) v 4.0 will be utilized for AE 
reporting. 
 

ACTION
 
56 
 
Adverse event data collection and reporting, which are required as part of every clinical trial, are done to 
ensure the safety of subjects enrolled in the studies as well as those who will enroll in future studies using 
similar agents.  AEs are reported in a routine manner and must be collected at every assigned or 
unintended study visit and/or contact with the patient (phone call, email, etc.) 
 
An adverse experience is defined as: 
 Any unfavorable and unintended sign, symptom, or disease temporally associated with the use of an 
investigational drug, whether or not related to the investigational drug. 
 
An adverse event can result from use of the drug as stipulated in the protocol or labeling, as well as from 
accidental or intentional overdose, drug abuse, or drug withdrawal. 
 
 Any worsening of a pre-existing condition or illness is considered an adverse event. 
 
CTCAE Grade 1 or 2 adverse events will be recorded if the attribution is at least possibly related to the 
DC or xALT vaccines.  
 
CTCAE Grade 3 or higher adverse events will be recorded regardless of relatedness to trial drug or 
protocol procedures.  
 
Laboratory abnormalities and changes in vital signs or test results are considered clinically significant 
and are adverse events if it results in discontinuation from the study, necessitates change in 
therapeutic medical intervention, or is considered a dose limiting toxicity. 
 
The investigator will evaluate all adverse experiences as to their severity and relationship to the trial 
drug or protocol procedures. 
 
Adverse Events will be reported in routine study data submissions via the clinical data management 
system. All recorded toxicities and adverse events will be followed until resolution, or return to baseline 
during the 30 days following the last dose of investigational drug or subject death whichever comes first.  
If the subject has progression of disease and begins another line of therapy or another therapeutic trial the 
adverse events collection will be discontinued. 
 
Please note: All adverse events must be entered into the clinical trial management system within required 
timelines as set forth in the current MCC standards and policies.  
 
7.4 
Secondary Malignancy 
 
A secondary malignancy is defined as a cancer caused by treatment for a previous malignancy (e.g., 
treatment with investigational agent/intervention, radiation or chemotherapy). A secondary malignancy is 
not considered a metastasis of the initial neoplasm. Second malignancies require ONLY routine AE 
reporting unless otherwise specified. 
 
CTEP requires all secondary malignancies that occur following treatment with an agent under an NCI 
IND/IDE be reported expeditiously via CTEP-AERS. Three options are available to describe the event: 
 
 Leukemia secondary to oncology chemotherapy (e.g., acute myelocytic leukemia [AML]) 
 Myelodysplastic syndrome (MDS) 
 Treatment-related secondary malignancy 

ACTION
 
57 
 
 
Any malignancy possibly related to cancer treatment (including AML/MDS) should also be reported via 
the routine reporting mechanisms outlined in each protocol. 
 
 
8. PHARMACEUTICAL INFORMATION 
 
8.1 
Names, Classification, and Mechanism of Action 
 
The active immunotherapy components are TTRNA-DCs and TTRNA-xALT. 
 
TTRNA-DCs are autologous dendritic cells loaded with total tumor messenger ribonucleic acid (mRNA) 
(TTRNA) derived from malignant tumor. 
 
TTRNA-xALT is autologous lymphocytes stimulated ex vivo against tumor antigens using TTRNA-DCs 
for autologous lymphocyte transfer.   
 
The classification is a biologic.  
 
8.2 
Packaging and Labeling 
 
All products will be appropriately labeled with subject identifiers, contents, lot number, manufacture date, 
and the “Caution: New Drug-Limited by Federal Law to Investigational Use” statement. 
 
8.3 
Dispensing and Preparation 
 
Study personnel at external sites will be provided with SOP  on the transport of tumor tissue and 
leukapheresis product as well as the preparation and administration of experimental therapeutic agents.  
Tumor tissue and leukapheresis product will be collected, prepared, and shipped to the University of 
Florida in accordance with SOP-UFBTIP-120 or SOP-UFBTIP-130 for tumor tissue and SOP-UFBTIP-
127 for leukapheresis product. External sites will be supplied with kits that will include all materials 
necessary to prepare and transport tumor tissue and leukapheresis product, including shipping labels. 
 
TTRNA DC and TTRNA-xALT products prepared by the University of Florida will be shipped to external 
sites in a liquid nitrogen dry-shipper under controlled conditions in accordance with SOP-UFBTIP-129.  
External sites will store products in accordance with standard operating procedures/written instructions 
until time of formulation.  When the subject is present and both the subject and site staff are ready for 
administration, products will be formulated in accordance with SOP-UFBTIP-124 (Procedure for DC 
Vaccine Preparation) and SOP-UFBTIP-125 (Procedure for Preparation of TTRNA-ALT for 
Adoptive Transfer), and administered in accordance with SOP-UFBTIP-128 (DC Vaccine) and SOP-
UFBTIP-2132 (ALT infusion).  
 
Products will be labeled with, at a minimum, the subject’s name, medical record number, study ID code 
and Lot number. Individuals transporting samples must be certified to ship biological products, diagnostic 
or infectious materials.  All packaging must comply with the Department of Transportation (DOT), the 
Federal Aviation Authority (FAA), and the International Air Transport Association (IATA) dangerous 
goods regulations. 
 
The final TTRNA-DC product is a subject-specific single-dose syringe containing a target dose of 1x107 
cells formulated in 400 µL of preservative free saline.  The administered volume is 0.4 mL.   

ACTION
 
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The final targeted dose of TTRNA-xALT product is a subject-specific single-dose syringe or infusion 
bag containing a total dose goal of 3x108 cells/kg formulated as 2.5x107 cells/mL in preservative free 
saline with 1% HSA.  The administered volume is dependent on the cell dose required for each subject. 
 
8.4 
Commercial Agent(s) Non-investigational 
 
Temozolomide: Temozolomide (Temodar™) NSC # 362856 Source and Pharmacology: An orally 
administered alkylating agent, a second generation imadazotetrazine. A prodrug of MTIC, temozolomide 
spontaneously decomposes to MTIC at physiologic pH.  Temozolomide exerts its effect by cross-linking 
DNA. This is likely a site specific alkylation at the O6 -position of guanine with some effect at the N7 
position. Temozolomide reaches its peak concentration in 1 hour. Food reduces the rate and extent of 
absorption. It has an elimination half-life of 1.13hr (intraperitoneally) and 1.29hr (orally) with an oral 
bioavailability of 0.98. Total apparent body clearance is 100ml/min/m2 and plasma elimination half-life is 
~100 minutes. 
 
Risks and side effects related to Temozolomide include those which are: 
Likely 
In 100 people receiving 
Temozolomide, more than 
20 and up to 100 may 
have: 
 
Less Likely 
In 100 people receiving 
Temozolomide, from 4 
to 20 may have: 
Rare, but serious 
In 100 people receiving 
Temozolomide, 3 or fewer may 
have: 
 Constipation, nausea, 
vomiting, diarrhea 
 Dizziness 
 Muscle weakness, 
paralysis, difficulty 
walking 
 Trouble with memory 
 Tiredness 
 Difficulty sleeping 
 Hair loss 
 
 Headache, seizure 
 Infection, 
especially when 
white blood cell 
count is low 
 Anemia which may 
cause tiredness 
 Bruising, bleeding 
 Damage to the bone 
marrow 
(irreversible) which 
may cause 
infection, bleeding, 
may require blood 
transfusions 
 
 Allergic reaction which 
may cause rash, low blood 
pressure, wheezing, 
shortness of breath, 
swelling of the face or 
throat 
 Cancer of bone marrow 
caused by chemotherapy 
 Rash 
 Severe skin rash with 
blisters and can involve 
inside of mouth and other 
parts of the body 
 Liver damage which may 
cause yellowing of eyes and 
skin, swelling and may 
result in liver failure. 
 
 
 

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8.5 
GCSF, GMCSF, and Td Vaccine Risks 
 
Risks and side effects related to the Filgrastim (G-CSF) include those which are: 
 
Likely 
(“Likely” refers to a side effect 
that is expected to occur in more 
than 20% of patients.) 
 
Less Likely 
(“Less likely” refers to a side 
effect that is expected to occur in 
20% or fewer patients.) 
Rare, but Serious 
(These possible risks have been 
reported in rare occurrences, 
typically less than 2% of 
patients.  They may be serious if 
they occur.) 
 Local irritation (skin) at 
injection site 
 Ache or pain inside the 
bones, increased levels of 
liver enzymes and uric acid 
in the blood, low number 
of platelets in the blood 
 Allergic reaction, low 
fever 
 Enlargement or rupture 
of the spleen 
 Worsening of pre- 
existing skin rashes 
 Temporary hair loss 
 Inflammation of a blood 
vessel in the skin 
 
 
 
 

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Risks and side effects related to Sargramostim (GM-CSF) include those which are: 
Likely: 
Less Likely: 
Rare, but Serious: 
 Headache 
 Bone pain 
 Muscle and joint pains 
 Fever and Chills 
 Rash and itchiness 
 A feeling of discomfort or 
not feeling well and/or 
tiredness  
 Stomach or abdominal pain or 
cramps 
 Weakness 
 Loss of appetite 
 Nausea and/or vomiting 
 Diarrhea 
 Excessive sweating 
 Inflammation of a vein 
through which the drug was 
given 
 Redness and pain at the 
injection site 
 Weight gain 
 Fewer platelets in the blood. A 
low number of platelets causes 
you to bruise and bleed more 
easily 
 Increase in the blood of certain 
enzymes or bilirubin (a 
substance that comes from the 
liver breaking down waste 
products) 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 
 Severe allergic reaction which 
can be life threatening with 
shortness of breath, low blood 
pressure, and a rapid heart beat 
 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 which 
may occur only with the first 
dose and not with further doses 
 An abnormally rapid heat 
beat 
 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 

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Risks and side effects related to the Td (Tetanus, Diphtheria)Vaccine include those which are: 
 
Mild Problems 
(Did not interfere with activities) 
 
Moderate Problems 
(Interfered with activities, but 
did not require medical 
attention) 
 
Severe Problems 
(Unable to perform usual 
activities; required medical 
attention) 
 
Pain where the shot was 
given (about 8 people in 10) 
 
Redness or swelling where 
the shot was given (about 1 
person in 4) 
 
Mild fever (rare) 
 
Headache (about 1 person in 
4) 
 
Tiredness (about 1 person in 
4) 
 Fever over 102°F (rare) 
 
Swelling, severe pain, 
bleeding and/or redness in the 
arm where the shot was given 
(rare). 
 
Risks Associated with Non-investigational Agents 
 
Complete information on FDA-approved drug products included in this clinical trial can be found on the 
Drugs@FDA website.   Each drug product label will include: 
 description of the drug 
 clinical pharmacology 
 indications (uses for the drug) 
 contraindications (who should not take the drug) 
 warnings 
 precautions 
 adverse events (side effect) 
 drug abuse and dependence 
 dosage and administration 
 use in pregnancy, use in nursing mothers 
 use in children and older patients 
 how the drug is supplied 
 safety Information for the patient 
 
9. BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
Immune Monitoring: 
Peripheral blood will be drawn within 24 hrs of leukapheresis, within 24 hours prior to the start of each 
TMZ cycle if feasible, within 24 hours prior to each vaccination (1-10),  Day 1 post xALT infusion, and 
4 days +/1 day post xALT infusion (if feasible).  Research blood will be drawn at each bi-monthly 
follow-up visit, if feasible, until progression for T cell kinetics, to test the ability to expand T cells against 
tumor antigens, exploring immunoreactivity against neoantigens and tumor-associated antigens (TAAs) in 
vitro. Assays will be conducted to test whether T cell responses are driven predominately by neoantigens 
or TAAa through analysis of RNAs encoding the individual antigens in T cell stimulation assays.  Blood 
will be processed in accordance with SOP-UFBTIP-126.   
 

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Peripheral blood for immune monitoring will require approximately 3 ml of whole blood in SST tube and 
8-10 ml in 2 ACD-A tubes. Research blood may remain at room temperature and must be processed 
within 6 hours of collection.  

ACTION Trial 
10. STUDY CALENDAR 
 
 
Study Requirements 
 
 
Surgery 
 
 
Enrollment 
 
Mobilized 
Leukapheresis 
 
Chemo- 
radiation17 
 
Non -Mobilized 
Leukapheresis 
Adjuvant TMZ Cycles 
 
DC 
Vaccine 
Bi Monthly 
Follow-
Up21 
#1 
#2-#5 
#6 
Inclusion & exclusion criteria 
 
X 
 
 
 
 
 
 
 
 
Informed consent to collect 
tumor
X 
 
 
 
 
 
 
 
 
 
Consent to treatment 
 
X 
 
 
 
 
 
 
 
 
Brain MRI (and spine if 
applicable)  
X1 
 
 
 
 
X7 
X7 
X7 
 
X7 
Performance Status 
 
X 
 
 
 
X 
X 
X 
X 
 
Serum/urine pregnancy test 
 
X4 
X 
 
X 
X4 
X 4 
X4 
 
 
Tumor tissue for extraction of 
total tumor RNA
X2 
 
 
 
 
 
 
 
 
 
CBC with differential & 
CD4+/CD8+ T cell ratio
 
X 
X 
 
X 
X8 
X8 
X8 
X 
X8 
PT/INR, PTT 
 
X 
 
 
 
 
 
 
 
 
Comprehensive metabolic 
panel
 
X 
X 
 
X 
X8 
X8 
X8 
X 
X8 
Infectious disease testing 
 
 
X3 
 
 X3 
 
 
 
 
 
Leukapheresis 
 
 
X19 
 
 X19 
 
 
 
 
 
Temozolomide 
 
 
 
X5 
 
X9 
X9 
X9 
 
 
External Beam RT 
 
 
 
X6 
 
 
 
 
 
 
History, physical & 
neurological exam 
 
X 
X 
 
X 
X10 
X10 
X10 
 
X10 
TTRNA -DCs 
 
 
 
 
 
X11 
X11 
X11 
X11 
 
TTRNA-xALT 
 
 
 
 
 
 
 
X13 
 
 
HSC infusion 
 
 
 
 
 
 
 
X12 
 
 
Peripheral blood for immune 
monitoring 
 
 
X14 
 
 X14 
 
 
 
X14 
X14 
Td booster 
 
 
 
 
 
 
 
 
X15 
 
Td Skin Prep 
 
 
 
 
 
 
 
 
X16 
 
Concomitant medications 
 
 
X20 
 
X 
X 
X 
X 
X 
 
Adverse event assessment 
 
 
X18 
 
 X18 
X18 
X18 
X1,8 
X18 
X18 

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Footnotes for Schedule of Study Assessments: 
1 
A diagnostic contrast-enhanced MRI of the brain (and spine if applicable) must be performed pre- and postoperatively within 
28 days prior to study registration.  
2 
Potentially eligible subjects will be enrolled on a screening consent for the sterile collection of tumor material in a 
manner suitable for RNA extraction, amplification, and loading of autologous DCs. 
3 
Pre-leukapheresis infectious disease testing per institutional policy to include at a minimum HIV, Hepatitis B, Hepatitis C,  and 
CMV. 
4 
Negative serum pregnancy test at enrollment and negative serum/urine pregnancy test within 72 hours prior to starting each 
TMZ cycle. 
5 
Administer TMZ continuously from Day 1 of RT to the last day of RT at a daily oral dose of 90 mg/m2 for a maximum of 49 
days. 
6 
Per section 4.1.1 or institutional guidelines for HGG.  
7 
MRI imaging within 2 week prior to C1, C2, C4, C6, then bi-monthly +/- 1 month. 
8 
CBC with differential and CD4+/CD8+ T cell ratio at each vaccine visit and, if clinically indicated, at routine clinic 
visits. 
9 
Cycle #1 should be started 5 (+/- 1) weeks after completing chemoradiation. Administer once per day for 21 consecutive 
days (days 1-21) for a 28 (+7) day cycle. The starting dose for the Cycle 1 and 2 will be 85mg/m2/day, with a single dose 
escalation to 100 mg/m2/day in subsequent cycles if no dose modifying adverse events are noted.  
10 
Within 14 days prior to initial vaccine therapy and prior to subsequent TMZ cycles.  
11 
Administer vaccine #1 intradermal at day 22-24 (+7) after the first TMZ cycle. Vaccines #2 and #3 will occur at 2 (+/- 1 
day) week intervals. Monthly vaccines #4-7 will be given on day 22-24 (+7) of each TMZ cycle. Vaccine #8 will be given on 
day 23-25 of TMZ cycle 6. Vaccines #9 and #10 will occur at 2 (+/- 1 day) week intervals until a total of 10 vaccines have 
been administered or until tumor progression (whichever comes first).   
12 
Peripheral blood stem cell infusion to be completed on day 22-24 of TMZ cycle 6 (Group B only). Autologous HSCs 
will be administered 12-36 hrs prior to DC vaccine/xALT. 
13 
TTRNA-xALT on day 23-25 of TMZ cycle 6. 
14 
2  tubes (ACD-A) and 1 tube (SST) Peripheral blood will be drawn  within 24 hrs of leukapheresis, within 24 hours prior to 
the start of each TMZ cycle if feasible, within 24 hours prior to each vaccination (1-10),  Day 1 post xALT infusion, and 4 
days +/1 day post xALT infusion (if feasible).  Research blood will be drawn at each bi-monthly follow-up visit, if feasible. 
Peripheral blood will be processed in accordance with SOP-UFBTIP-126. 
15 
All subjects will receive IM Td booster (5 Lf ) at Vaccine #1. 
16 
All subjects will undergo vaccine site pretreatment with Td (1 Lf ) 4-24 hours prior to Vaccine #3, #6, and #8. 
17 
Within 7 weeks of surgery. 
18 
CTCAE Grade 1 or 2 adverse events will be recorded if the attribution is at least possibly related to the DC or xALT 
vaccines.  CTCAE Grade 3 or higher adverse events will be recorded regardless of relatedness.  
Adverse events will be collected during two periods: leukapheresis and immunotherapy (DC + xALT +/- HSC) as 
described in Section 10.1.   
19 
Enrolled subjects will undergo a mobilized leukapheresis prior to chemoradiation for collection of PBMCs for generation 
of DCs and HSCs for Group B subjects. Fourteen days (+/- 3 days) after the third biweekly DC vaccine, subjects will 
undergo a second  leukapheresis (non-mobilized) to collect PBMCs for generation of DCs and xALT. 
20 
Concomitant medications should be recorded from Day 1 Cycle 1 through 30 days post last dose of Immunotherapy 
21 
Subjects will undergo bi-monthly follow-up +/- 1 month for one year and then at the PI discretion (at least every 6 months) 
until tumor progression or death due to any cause 
 

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10.1 Study Calendar Descriptions 
Demographics 
Subject’s gender, race, ethnicity and date of birth will be collected at screening. 
 
Medical History 
Medical history findings (i.e. previous diagnoses, diseases or surgeries) and treatments considered 
relevant to the study (including planned interventions) will be collected prior to the first dose of study 
drug. 
 
Physical Measurements 
The screening examination should include physical measurements (height, weight, performance 
status). Weight and performance status should be collected at each study visit. 
 
Concomitant Medications 
All concomitant medications (including first dose / last dose and if medication was given for 
Immunotherapy related AE) must be recorded from 4 weeks prior to immunotherapy through 30 days 
after the last dose of immunotherapy. 
 
Physical Exam 
In addition to physical measurements, signs and symptoms should be assessed at each study visit by 
the appropriate staff. 
 
Performance Status 
Lansky or Karnofsky status will be evaluated and documented at screening and visits per study 
calendar. 
 
Adverse Events 
Adverse events Grade 3 and higher  and all adverse events regardless of grade that are at least 
possibly related to DC vaccine or xALT will be collected during two periods: leukapheresis and 
immunotherapy (DC + xALT +/-HSC).  
 
The leukapheresis collection periods will begin at initiation of leukapheresis and continue until 24 hours 
after the completion of the procedure.  
 
The immunotherapy collection period will begin with first DC vaccine and continue through 30 days 
after administration of the last dose of trial drug or subject death, whichever comes first. 
 
Events occurring outside the reporting windows, regardless of grade, that have a possible relationship to 
vaccine therapy must also be reported. All AEs Grade 3 or higher must be recorded in the subject’s 
medical and/or research record and case report form. 
 
Adverse events will be graded according to the NCI-CTCAE version 4.0. 
 
 

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10.2 Laboratory Evaluations 
Safety labs, including chemistry, hematology and coagulation tests will be measured by the local lab 
for clinical assessment. 
 
 Hematology: A complete blood count (CBC) with differential will include the following tests: 
hemoglobin, hematocrit, white cell count (WBC) with differential as percentage or absolute value, red 
cell count (RBC), reticulocyte count and platelet count. 
 
 Coagulation: Coagulation tests are as follows: Prothrombin time (PT), activated partial 
thromboplastin time (PTT), and international normalized ratio (INR). 
 
 Blood Chemistry: A blood chemistry will include the following: albumin, alkaline phosphatase, total 
bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, potassium, total protein, SGOT 
[AST], SGPT [ALT], sodium. 
 
 Pregnancy Test: Women of child bearing potential are required to have a serum pregnancy test (B-
HCG) performed prior enrollment and serum or urine test prior to the start of study drug. 
 
The definition of a woman of childbearing potential is defined as: a woman is considered of childbearing 
potential (WOCBP) i.e., fertile, following menarche and until becoming post-menopausal unless 
permanently sterile. Permanent sterilization methods include hysterectomy, bilateral salpingectomy, and 
bilateral oophorectomy. 
 
10.3 Radiologic Evaluations 
MRI is the best currently available and reproducible method to measure lesions selected for response 
assessment. Measurability of lesions on MRI scan is based on the assumption that MRI slice thickness is 5 
mm or less. When MRI  scans have slice thickness greater than 5 mm, the minimum size for a measurable 
lesion should be twice the slice thickness. In the interest of consistent measurements, the same method of 
evaluation used at baseline must be utilized throughout the subjects’ time on study. 
 
Study evaluations will take place in accordance with the Study Calendar in section 10. All radiographic 
evaluations must be performed within 28 days of start of therapy utilizing MRI. Unusual circumstances 
related to >28 day old scans not involving target lesions may be discussed with the Protocol Chair. Tumor 
response will be assessed by the investigator using the iRANO (immunotherapy Response Assessment in 
Neuro-oncology). 
 
11. MEASUREMENT OF EFFECT 
 
11.1 Immunologic confounders of radiographic response 
 
Inflammatory responses induced by immunologic treatment may confound radiographic assessment of 
tumor progression versus treatment response.  PFS and OS are not primary endpoints of this study and 
iRANO criteria will be applied to assess progression defined below.  However, if treatment response vs 
progression is unclear after administration of i mmunotherapy but criteria for radiographic progression are 
met, then subjects may remain on study without change in therapy and subsequent evaluation by MRI can 
be reassessed.  If continued neurologic and/or radiographic evaluation determines tumor progression then 
the date of PFS will be back-dated to the original MRI evaluation meeting criteria. 

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11.2 Antitumor Effect – Solid Tumors 
 
For the purposes of this study, patients should be re-evaluated for response every 8-12 weeks.  In addition to 
a baseline scan, confirmatory scans should also be obtained 8 -12 (not less than 4) weeks following initial 
documentation of objective response. 
 
Response and progression will be evaluated in this study using the Immunotherapy Response Assessment in 
Neuro-Oncology (iRANO) criteria as proposed by “Immunotherapy response assessment in neuro-
oncology: a report of the RANO working Group” Lancet Oncology 2015 Nov;16: e534-42.  
 
11.2.1 Definitions 
 
Evaluable for toxicity.  All subjects will be evaluable for toxicity from the time of their first treatment with 
DC vaccine.  
 
Evaluable for objective response.  For those subjects who have had a complete resection or measurable 
disease present at baseline (post-surgery), have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for response. (Note:  Subjects who exhibit objective 
disease progression prior to the end of cycle 1 will also be considered evaluable.). 
 
Of note, patients who need increased corticosteroid use within 2 weeks of MRI assessment relative to the 
dose taken at the time of the previous assessment, cannot be classified as having a complete response, 
partial response, or stable disease and should be classified as non-evaluable at that timepoint. Conversely, 
patients who decrease corticosteroid use within 2 weeks of MRI assessment relative to the dose taken at 
the time of the previous assessment cannot be classified as having progressive disease and should be 
classified as non-evaluable.103  
 
11.3  General Methodology for Determining Tumor Measurements 
 
All radiographic responses will be based on MRI scans of the brain and spine (if applicable) obtained with 
and without contrast. Tumor size will be based on the product of the maximal diameters in 2 different 
planes. Response will be assessed as a percentage change in tumor size from baseline. Tumor 
measurements in the follow-up scans will be compared to those of the pre-treatment (baseline - post 
surgery) scan to document response. Determination of progressive disease will be based on comparison of 
the current tumor dimensions with the smallest measurements of the tumor recorded since the start of 
treatment. However, based on the iRANO criteria, if the initial radiological progression is within the 
first 6 months, this will serve as the new reference scan if treatment is continued. The assessment of 
steroid dose as “stable or tapering” will be based on the dose the patient was receiving on the day of the 
follow-up scan with that on the day of the pre-treatment scan. Residual disease will be defined as 
contiguous contrast enhancement ≥ 1 cm2 (product of measurement obtained in 2 perpendicular planes (i.e., 
axial and coronal)).  
 
 
 

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11.4 Tumor Response Criteria 
iRANO criteria for Malignant glioma 
Complete response 
Disappearance of all enhancing disease for ≥ 4 weeks; no new 
lesions; stable or improved T2/FLAIR; no more than physiological 
steroids; clinically stable or improved
Partial response 
≥ 50% decrease in the sum of biperpendicular diameters of 
enhancing disease for ≥ 4 weeks; no new lesions’   stable or 
improved T2/FLAIR; stable or decreased steroid dose; clinically 
stable or improved
Stable disease 
Does not qualify for complete response, partial response, or 
progressive disease; no new lesions; stable or improved T2/FLAIR; 
stable or decreased steroid dose; clinically stable or improved
Progressive disease 
≥ 25% increase in the sum of biperpendicular diameters of 
enhancing disease; or new lesions; or substantial worsened 
T2/FLAIR; or substantial clinical decline
 
The iRANO criteria integrate into the existing RANO criteria for malignant glioma, low-grade glioma, and 
brain metastases by providing recommendations for the interpretation of progressive imaging changes. 
Specifically, iRANO recommends confirmation of disease progression on follow-up imaging 3 months after 
initial radiographic progression if there is no new or substantially worsened neurological deficits that are not 
due to comorbid events or concurrent medication, and it is 6 months or less from starting immunotherapy. If 
follow-up imaging confirms disease progression, the date of actual progression should be back-dated to the 
date of initial radiographic progression. The appearance of new lesions 6 months or less from the initiation 
of immunotherapy alone does not define progressive disease. FLAIR=fluid-attenuated inversion recovery. 
iRANO=immunotherapy Response Assessment in Neuro-Oncology. N/A=not applicable.  
The algorithm below indicates the iRANO treatment algorithm for the assessment of progressive imaging 
findings in patients with neuro-oncological malignancies undergoing immunotherapy:  
 

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Progressive Disease (PD) 
 
Progressive neurologic abnormalities or worsening neurologic status not explained by causes unrelated 
to tumor progression (e.g., anticonvulsant or corticosteroid toxicity wean, electrolyte disturbances, 
sepsis, hyperglycemia, hydrocephalus etc.), OR ≥25% increase in the bi-dimensional measurement of 
enhancing disease, OR the appearance of a new tumor lesion, OR substantial worsened T2/FLAIR. 
Increasing doses of corticosteroids required to maintain stable neurological status should be strongly 
considered as a sign of clinical progression unless in the context of recent wean or transient neurologic 
change. 
 
Patients with early progressive imaging findings, including patients who develop new lesions but who do 
not have substantial neurological decline, confirmation of radiographic progression by follow-up imaging 
should be sought 3 months after initial radiographic evidence of progressive disease to decrease the 
likelihood of prematurely declaring progressive disease in patients with pseudoprogression or delayed 
response. Imaging within the 3-month follow-up can be done as medically appropriate at the discretion of 
the treating clinician. 
 
Patients who develop substantial new or worsened neurological deficits not due to comorbid events or a 
change in co-administered medication at any time within the 3-month follow-up window should be 
designated as non-responsive to treatment and should discontinue immunotherapy. For these patients, the 
date of actual tumour progression should also be back-dated to the date when radiographic progressive 
disease was initially identified.103 
 
Pseudoprogression 
 
For subjects showing possible radiographic evidence of tumor progression on MRI (on required MRI 
studies performed after courses 3, 6 and 9), during the first 6 months after immunotherapy, the treating 
physician in consultation with the Protocol Chair will have the option of allowing the patient to remain on 
study or holding protocol therapy, and repeating disease reassessment in 3 months. Symptomatic patients 
should be treated with BVZ (7.5 mg/kg q 3 weeks) and MRI repeated in 3 months. If SD or resolution of 
neuroimaging changes and clinical stability or improvement, patient should continue DC vaccine along 
with BVZ.  If the repeat MRI(s) or clinical status shows continue to worsen, patients should be taken off 
protocol therapy. Clinical worsening or new/worsening changes on neuroimaging more than 6 months 
following start of vaccine therapy should be considered true progression and patient should discontinue 
protocol therapy and be replaced.  If follow-up imaging or clinical evaluation confirms disease progression, 
rather than pseudoprogression, then the time of progression will be the date of the initial MRI 
demonstrating progression, not the follow up scan(s). 
 
11.4.1 Evaluation of Best Overall Response 
 
The best overall response is the best response recorded from the start of the treatment until disease 
progression/recurrence. The patient's best response assignment will depend on the achievement of both 
measurement and confirmation criteria. 
 
11.4.2 Progression-Free Survival 
 
PFS is defined as the duration of time from start of treatment to time of progression or death, whichever 
occurs first. 
 
 

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12. DATA REPORTING / REGULATORY REQUIREMENTS 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 7.0 (Adverse 
Events: Definition and Reporting Requirements). 
 
12.1 Data Reporting 
 
Data collection for this study will be done exclusively through OnCore.  All members of the study team 
are granted access to the trial in OnCore by the MCRN External Research Regulatory Coordinator. See 
instructions in Moffitt Clinical Research Network Handbook for OnCore access. 
 
Regulatory documents and case report forms will be monitored by the Sunshine Project. Monitoring will 
be performed regularly for accuracy, completeness, and source verification of data entry, validation of 
appropriate informed consent process, reporting of SAEs, and adherence to the protocol, Good Clinical 
Practice (GCP) guidelines, and applicable regulatory requirements. 
 
Study documents should be retained for a minimum of 10 years after the last approval of a marketing 
application in an International Conference on Harmonisation (ICH) region and until there are no pending 
or contemplated marketing applications in an ICH region or until at least 10 years have elapsed since the 
formal discontinuation of clinical development of the study intervention. These documents should be 
retained for a longer period, however, if required by local regulations. No records will be destroyed 
without the written consent of the sponsor, if applicable. It is the responsibility of the sponsor to inform 
the investigator when there is no longer a need for these documents to be retained. Permission must be 
acquired from the State of Florida for document destruction after the 10-year minimum record-retention 
period described above has elapsed. 
 
12.1.1 Method for Data Reporting and Monitoring 
 
Research Monitoring Core 
Data will be captured in OnCore, Moffitt’s Clinical Trials Database. Regulatory documents and case 
report forms will be reviewed routinely for accuracy, completeness and source verification of data entry, 
validation of appropriate informed consent process, adherence to study procedures, and reporting of SAEs 
and protocol deviations according to Moffitt’s Monitoring Policies. Monitoring Visits will include a 
review of data entry to source documentation, regulatory documents, and when appropriate, investigational 
pharmacy or clinical laboratory. 
 
Audit and Inspection 
Inspections by regulatory health authority representatives i.e. FDA and IEC(s)/IRB(s) are possible. 
Investigator responsibilities are set out in the ICH guideline for Good Clinical Practice (GCP), Form FDA 
1572 and in the US Code of Federal Regulations. 
 
Investigators must enter study data into Moffitt’s electronic database, OnCore. The investigator will permit 
study related audit visit by a sponsor or it’s representatives, IRB/EC review, and regulatory inspection(s), 
providing direct access to the facilities where the study took place, to source documents, to CRF’s and to 
other study documents. 
 
Essential documents shall be archived safely and securely in such a way that ensures that they are readily 
available upon authorities’ request. 
 

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Subject (hospital) files will be archived according to local regulations and in accordance with the 
maximum period of time permitted by the hospital, institution or private practice. 
 
Records pertaining to this trial must be retained for a period of 2 years following the date a marketing 
application is approved for the drug for the indication for which it is being investigated or, if no 
application is to be filed or if the application is not approved for such indication, until 2 years after the 
investigation is discontinued and FDA is notified.  Research records should also be retained in accordance 
with institutional policy. 
 
Sunshine Project Clinical Trials Oversight Committee (CTOC) 
The CTOC is a group of pediatric oncologists who specialize in phase 1 clinical trials. This group of 
physicians review and evaluate the conduct of each trial the Sunshine Project conducts. Every quarter, the 
CTOC meets to ensure all clinical research conducted or coordinated by NPCF is scientifically well 
designed, responsibly managed, appropriately reported, and protects the rights and welfare of human 
participants. 
 
The CTOC provides an independent review of the interim results of each trial and offers objective 
guidance regarding unforeseen events that may occur during the course of a study. The CTOC will help to 
evaluate and ensure events that may have an impact on patient safety are appropriately addressed and 
managed on an on-going basis. At the quarterly meetings, discussions will include adverse events, accrual, 
and protocol deviations. Adverse event and deviation collection begins when a patient is first placed on 
trial. 
 
All events are classified using the NCI Common Toxicity Criteria and are summarized and presented to all 
members at each safety meeting. The CTOC will also help to evaluate and ensure events that may have an 
impact on patient safety are appropriately addressed and managed. The CTOC will make recommendations 
regarding the conduct and safety of the trial and a public report will be made available to all participating 
member sites. The CTOC will be convened in the event of safety concerns brought to the attention of 
investigators or the study chair. 
 
Protocol Monitoring Committee 
The Moffitt Cancer Center reviews investigator initiated trials that take place at Moffitt through the 
Protocol Support Office (PSO) at Protocol Monitoring Committee meetings. The PSO reviews all 
deviations, AEs, and SAEs on all Sunshine trials. Reports for phase I trials are created and reviewed when 
a trial increases a dose level or when a maximized tolerated dose is met. Because Moffitt acts as the main 
coordinating center for the Sunshine Trials, all Sunshine Project trials will undergo review by the PSO 
when a dose level is increased. 
 
The PMC meets once a month, and reviews and evaluates safety and/or efficacy data for all physician 
authored clinical intervention trials.  The PMC ensures the safety of subjects and the validity and integrity 
of data. PMC reviews SAEs, deviations, Interim analysis, interim and final reports from the external Data 
Monitoring Committee (DMC) as well as audits both internally and externally. The PMC can make the 
following determinations, Accepted, Acceptable with Corrective Action and Tabled. 
 
Scientific Review Committee (SRC) 
The two Therapeutic boards of the SRC meet every other week one on the first Wednesday and the second 
one meets on the third Thursday of every month. 
 
Each SRC conducts a formal internal peer review of all clinical protocols and general scientific oversight 
of interventional clinical research. Protocols are reviewed for scientific merit, adequate study design, 

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safety, availability of targeted study population, and feasibility of timely completion of all proposed 
research projects to be conducted by its assigned programs at the Cancer Center.  The SRC is responsible 
for evaluating the risk/benefit assessment and corresponding data and safety monitoring plan as part of the 
scientific review and approval process. 
 
Sunshine Project Bi-Weekly Teleconference 
Starting with the first patient enrollment, a safety call consisting of principle investigators, a 
biostatistician, and study coordinators will occur biweekly. In addition, all AE’s will be collected and 
shared with drug suppliers for input prior to escalating or de-escalating to the next dose level. 
 
Moffitt Cancer Center Scientific Review Committee and Institutional Review Board All Sunshine 
Project trials undergo an initial review at the center’s Scientific Review Committee and Central 
Institutional Review Board. 
 
 
12.1.2 Responsibility for Data Submission 
 
It is the responsibility of the PI(s) at the site to ensure that all study team members at the site understand 
the procedures for data submission, and that protocol specified data is entered accurately and in a timely 
manner into the OnCore system. 
 
All eCRF entries into OnCore will be verified via source documentation during on-site or remote 
monitoring visits. 
 
 
12.2 Moffitt Cancer Center Guidelines 
 
This protocol will adhere to the policies and requirements of the Moffitt Cancer Center Guidelines. 
 
The Moffitt Cancer Center is responsible for distributing all IND Action Letters or Safety Reports 
received to all participating institutions for submission to their individual IRBs for action as required. 
 
12.2.1 Suspension/Termination 
The PMC and/or the IRB may vote to suspend or terminate approval of a research study not being 
conducted in accordance with the IRB, the Cancer Center and/or regulatory requirements or that has been 
associated with unexpected problems or serious harm to subjects.  The PMC/IRB will notify the PI in 
writing of such suspension or terminations.  It is the responsibility of the PMC/IRB Chairperson to ensure 
prompt written notification of any suspensions or terminations of PMC/IRB approval to the relevant 
Federal Agencies, including OHRP, FDA, the study sponsor/funding source and if applicable, the Affiliate 
Program. 
 
12.2.2 Trial Discontinuation 
For reasonable cause the Investigator and/or sponsor may terminate this study prematurely. Conditions that 
may warrant termination include, but are not limited to: the discovery of an unexpected, significant, or 
unacceptable risk to the subjects enrolled in the study or if the accrual goals are met. A written notification 
of termination will be issued. 
 
12.2.3 Protocol Modifications 
No modifications will be made to the protocol without the agreement of the investigators. Changes that 
significantly affect the safety of the subjects, the scope of the investigation, or the scientific quality of the 

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study will require Scientific Review Committee and Institutional Review Board approval prior to 
implementation, except where the modification is necessary to eliminate apparent immediate hazard to 
human subjects.  Any departures from the protocol must be fully documented in the case report form and 
the source documentation. 
 
13. STATISTICAL CONSIDERATIONS 
 
13.1 Study Design/Endpoints 
 
13.1.1 Analysis Sets 
The populations that may be used for analysis are defined as: 
 
All subjects who receive any protocol treatment will be included in safety analyses. 
 
 If either DCs and/or xALT do not meet release criteria or targeted dose then the patient may remain on 
study and receive the qualified product but will be replaced for the purposes of safety and efficacy 
assessment. 
 Subjects 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 physiologic levels, may remain in the study but 
will be replaced for assessment of safety as increased steroid usage may mask risks of autoimmune 
toxicity.  For the purposes of this study, physiologic dose will be defined as > 4 mg of 
dexamethasone/day. 
 Subjects receiving less than 10 vaccines without toxicity will be replaced for safety assessments. 
 
All statistical analyses 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. 
 
13.1.2 Patient Demographics and other Baseline Characteristics 
Summaries of clinical and socio-demographic characteristics will be generated for all subjects as well as 
patient subgroups defined by assigned treatment sequence. Categorical descriptors will be summarized 
using frequency distributions; whereas, interval variables will be summarized using percentiles, as well as 
means and standard deviations. 
 
13.1.3 Treatments 
 
The number of subjects who receive ACT treatment will be summarized. 
 
13.1.4 Primary Objective 
 
The primary purpose of this study is to explore the safety, feasibility and tolerability of DC vaccines and 
ACT in subjects with primary HGGs. 
 
13.1.5 Secondary Objectives 
 
Secondary objectives will be to estimate the mean difference and the variation in INF gamma secretion 
between baseline and 2 weeks post-completion of immunotherapy in Group A and Group B. OS and PFS 
will be secondary clinical outcomes measures If the effect of HSCs appears promising, we will use this 
information to determine the sample size required to appropriately power a phase II study. We do not 

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76 
expect the sample size of the current study to be large enough to allow definitive conclusions, but we will 
use a t-test to compare gamma secretion differences (baseline – 2 weeks post-immunotherapy) between the 
two groups, and Kaplan-Meier methods to compare progression-free and overall survival. 
 
13.1.6 Interim Analysis 
 
No interim efficacy analysis is planned. 
 
The safety of ACT using DC+xALT and MTD of tumor-specific lymphocytes was established in our 
ongoing phase I/II trial (Re-MATCH). Should differential dose-limiting toxicity be observed in subjects 
enrolled on this trial, a redesign to assess the MTD and DLT will be performed following our current phase 
I trial design in subjects with reMB/PNETs. 
 
 
13.1.7 Safety endpoint statistical analysis and stopping criteria  
 
No interim efficacy analysis is planned. 
 
Any deaths within the treatment period will be reported as described in 7.2.5. 
 
If 2 of 6 patients experience immunotherapy related DLT following administration of 3 x108/kg x-ALT, 
an additional 6 patients will be enrolled on group A to receive x-ALT at a dose of 3 x107/kg. The trial will 
stop if 2 of 6 patients develop DLT at this dose level. Once MTD is identified in group A patients, 6 
patients will be treated in group B using this MTD for x-ALT infusion.  
 
An overall toxicity rate within the 12 subjects will also be monitored to ensure that unacceptable toxicity 
is not observed during the treatment of both cohorts.  We will use a dynamic sequential testing method 
(Kramar and Bascoul-Mollevi, 2009) to determine if the sequential proportion of enrolled and treated 
subjects who have experienced DLTs that have exceeded a maximum acceptable rate  10.0% This method 
employs a concave alpha-spending function that allows a greater chance of detecting an unacceptably 
high toxicity rate earlier in the study while maintaining a nominal one-sided Type I error rate of 10% over 
the entire sequence of tests performed(Kramar A and C Bascoul-Mollevi, 2009. Trials based on sequential 
monitoring of serious adverse events. Med Decis Making 29:343-350). Thus, the trial will be stopped if 
any of the following occur after dose modification of xALT to the lower dose of 3 x 107/kg: 
 
1. 2 of the first 2, 3, 4, 5,6 subjects treated experience  DLTs 
2. 3 of the first 7 subjects treated experience DLTs 
3. 4 subjects experience DLTs at any point during the trial 
 
13.1.8 Handling of missing values, censoring, and discontinuations 
 
If a patient drops out of the study before receiving immunotherapy, then that patient will be replaced.  PFS 
and OS will be reported for all subjects on an intent to treat basis. 
 
13.2 Sample Size/Accrual Rate 
 
This pilot study design will be performed in 12-18 subjects (6-12 Group A subjects and 6 Group B 
subjects) where we will demonstrate feasibility and safety in pediatric subjects with HGGs and estimate 
the effect of size and variation in order to determine the sample size required to appropriately power a 
phase II study design. 

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13.3 Stratification Factors 
 
Up to twenty-four subjects will be enrolled to treat 12-18 evaluable subjects, 6-12 subjects into 
Group A followed by 6 subjects into Group B. 

 
78 
 
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86 
 
15. APPENDIX A  
Performance Scales 
 
Lansky Performance Scale 
(subjects < 16 yo) 
Karnofsky Performance Scale  
(subjects ≥ 16 yo) 
Percent
Percent
100 
Fully active. 
100 
Normal, no complaints, no 
evidence of disease.
90 
Minor restriction in physically 
strenuous play. 
90 
Able to carry on normal 
activity; minor signs or 
symptoms of disease.
80 
Restricted in strenuous play, 
tires more easily, otherwise 
active.
80 
Normal activity with effort; 
some signs or symptoms of 
disease.
70 
Both greater restrictions of, and 
less time spent in active play. 
70 
Cares for self, unable to carry 
on normal activity or to do 
active work.
60 
Ambulatory up to 50% of time, 
limited active play with 
assistance/supervision.
60 
Requires occasional 
assistance, but is able to care 
for most of his/her needs.
50 
Considerable assistance 
required for any active play, 
fully able to engage in quiet 
play
50 
Requires considerable 
assistance and frequent 
medical care. 
40 
Able to initiate quite activities.  
40 
Disabled, requires special care 
and assistance.
30 
Needs considerable assistance 
for quiet activity.  
30 
Severely disabled, 
hospitalization indicated.  
Death not imminent.
20 
Limited to very passive activity 
initiated by others (e.g. TV). 
20 
Very sick, hospitalization 
indicated. Death not 
imminent.
10 
Completely disabled, not even 
passive play. 
10 
Moribund, fatal processes 
progressing rapidly.
0
Dead.
0
Dead.
 
 
 

 
87 
 
16. APPENDIX B 
 
 
 
TEMOZOLOMIDE PILL DIARY 
 
 
Name:  ______________________ 
 
Study ID: ____________________ 
 
Dose:  _______ 
Cycle: _____ 
Course: 
Date 
Time 
Dose 
Comments 
Day 1 
 
 
 
 
Day 2 
 
 
 
 
Day 3 
 
 
 
 
Day 4 
 
 
 
 
Day 5 
 
 
 
 
Day 6 
 
 
 
 
Day 7 
 
 
 
 
Day 8 
 
 
 
 
Day 9 
 
 
 
 
Day 10 
 
 
 
 
Day 11 
 
 
 
 
Day 12 
 
 
 
 
Day 13 
 
 
 
 
Day 14 
 
 
 
 
Day 15 
 
 
 
 
Day 16 
 
 
 
 
Day 17 
 
 
 
 
Day 18 
 
 
 
 
Day 19 
 
 
 
 
Day 20 
 
 
 
 
Day 21