EGFRvIII Chimeric Antigen Receptor (CAR) Gene-modified T Cells for Patients With Newly-Diagnosed GBM During Lymphopenia
Phase: PHASE1
Conditions: Glioblastoma, Gliosarcoma
Sponsor: Daniel Landi

 
 
 
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A Phase I Study 
 
ExCeL: EGFRvIII Chimeric Antigen Receptor (CAR) 
Gene-modified T cells for Patients with Newly-
Diagnosed GBM during Lymphopenia 
 
 
 
 
 
 
 
Sponsor 
PI - Duke Cancer Institute 
 
 
Funding Source: 
NIH Grant  
 
 
Protocol Source: 
PI - Duke Cancer Institute 
 
 
Duke IRB#: 
Pro00069444 
 
 
IND#: 
 
16,787 
 
 
Co-Principal Investigators 
David Ashley, MBBS, FRACP, PhD 
DUMC Box 3624 
Durham, NC 27710 
Pager: (919) 206‐3433 
Phone: (919) 681‐3824 
david.ashley@duke.edu 
 
Daniel Landi, MD 
DUMC Box 102382 
Durham, NC 27710 
Pager: (919) 206-3405 
Phone: (919) 613-7504 
daniel.landi@duke.edu 
 
Sub-Investigator(s) 
Roger McLendon, M.D. 
roger.mclendon@duke.edu 
 
Daniel Barboriak, M.D. 
daniel.barboriak@duke.edu 
 
Elizabeth Reap, Ph.D. 
elizabeth.reap@duke.edu 
 
Luis Sanchez-Perez, Ph.D. 
luis.sanchez-perez@duke.edu 
 
Sub-Investigator(s) 
Gary Archer, Ph.D. 
gary.archer@duke.edu 
 
Annick Desjardins, MD, FRCPC 
desja002@duke.edu 
 
Henry Friedman, M.D. 
henry.friedman@duke.edu 
 
Allan Friedman, M.D. 
allan.friedman@duke.edu 
 
Katherine Peters, M.D., Ph.D. 
katherine.peters@duke.edu 
 
Peter Fecci, M.D. 
peter.fecci@duke.edu 
 
John Sampson, M.D., Ph.D. 
john.sampson@duke.edu 
 
Dina Randazzo, D.O. 
dina.randazzo@duke.edu 
 
Margaret Johnson, M.D. 
margaret.o.johnson@duke.edu 
 
 
Statistician 
James E. Herndon II, Ph.D. 
james.herndon@duke.edu 
 
Lead Study Coordinator 
Stevie Threatt, C.R.C 
stevie.threatt@duke.edu 
 
Regulatory Coordinator 
Jennifer Jackman, PhD 
jennifer.gamboa@duke.edu 
 
Data Manager 
Eric Lipp, B.S. 
eric.lipp@duke.edu 
 
 
 
Original version: 
12/10/2015 
 
Amended version for FDA mods: 
01/15/2016 
Changed from CRM to 3+3 study design (Sections 
4, 9.1.1, & 15); Eliminated first 3 doses of CARs 
(Sections 7.3, 9.2, & 15.4); Revised DLT definition 

 
 
 
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(Section 9.1.2); Added language on CRS 
(Sections 4, 9.1, & 12); Clarified micro curie for 
Indium (Sections 9.1.1 & 10.4.3); Clarified storage 
of CARs & dosage concentration of labeled versus 
unlabeled CARs (Section 10.4.1); Inserted 
positive test result management plan (Section 
10.4.2); Clarified BactAlert testing for a total of 14 
days on CARs (Section 10.4.2); Clarified age 
range & removed reference to Gilbert’s Syndrome 
in Inclusion Criteria (Section 11); Clarified SOC 
clinic visit will occur during the pause between 
TMZ cycles 3 & 4 for restaging (Section 12); 
Added measurements of blood results to 
withdrawal criteria (Section 12.6); Included a 
maximum follow up time period for subjects 
treated with CARs (Section 12.4); Increased the 
monitoring time from 2 to 4 weeks for safety and 
DLT evaluation between cohort enrollment 
(Sections 9.1.2 & 15.1); Study assessments 
clarified to occur with each study/clinic visit 
(Section 12.7); Efficacy analysis with and without 
subjects whose products failed release criteria 
included (Sections 12.7.2, 15.1, & 15.6); AE 
language updated to match 21CFR312 (Section 
13).  
Amended version for missing FDA mod: 
01/27/2016 
Clarified that dosage of EGFRvIII CARs will be 
based on transduced cells/kg (Sections 9.1.1 & 
15.4). 
Amended version for IRB mods: 
3/11/2016 
Clarified blood volume and tubes for immune 
monitoring, RCR PCR and CRS (Sections 9 & 12); 
included low hematocrit as criterion for early 
withdrawal (Section 12.7.1). 
Amended version for PI Change: 
3/17/17 
PI changed to Annick Desjardins.  Lead Study 
Coordinator and Regulatory Coordinator updated.  
DBTIP laboratory changed to MPACT facility in 
sections 10.3, 10.4.1, and 12.2. 
Amended version for protocol changes: 
6/7/17 
Updated inclusion/exclusion criteria (Section 11).  
Updated TMZ dosing (Section 9.1 and 12).  
Updated scintigraphy timepoints to make 
consistent (Section 9.1.1, 9.3, 12, 12.5). Clarified 
tracking starting point of concomitant meds 
(Section 9.1.4). Clarified Eligibility confirmation 
(Section 9.4). Updated screening (Section 12.1) 
and Table 6.  Update EGFRvIII CARs 
administration (Section 12.2).  eBrowser changed 
to EPIC (Section 12.8.1).  Corrected biorepository 
IRB# (Section 9.1).  Changes CTQA to OARC 
(Section 14.2).  Clarified the period of time in 
between treatment cohorts (Section 9.1.1).  
Formatting changes to spacing and font. 
Amended version for protocol changes: 
8/8/17 
PI changed to co-PIs of David Ashley and Daniel 
Landi.  Removed Standard Radiation Therapy and 
Standard Temozolomide appendices.  Added 
decreasing steroid rule (Section 9.1 and 12).  
Changed TMZ dosing interval and clarified 
progressive disease vs. pseudoprogression 
(Section 9.1 and 12).  Updated eligibility to include 
gliosarcoma and remove residual disease 
requirement.  Removed steroid eligibility and 
removed multifocal disease exclusion criteria 
(Section 11).  Updated the Schedule of Events 
(Table 6).  Updated concomitant medication 

 
 
 
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tracking timeframe (Section 9.1.4).  Added 
“radiographic response” to the End of Study 
requirements (Section 12.6).  Changed RT and 
TMZ completion requirement to 5 weeks (Section 
12.7.1).  Added collection of survival status, 
treatment regimens, dose, dates, and radiographic 
responses to Follow-up Requirements for Early 
Withdrawal (Section 12.7.2).  Modified the AE and 
SAE collection windows (Section 13 and 13.1).  
Added Section 13.2: Adverse Events of Special 
Interest.  Removed redundant withdrawal criteria 
and updated the criteria (Section 12.7.1).   
Amended version for eligibility 
modifications 
11/14/17 
Removed the White Blood Cell (WBC) inclusion 
criteria (Inclusion #5) and re-worded Exclusion 
Criteria #10 (Section 11). Removed irrelevant 
footer text. 
Amended version for protocol changes 
3/7/18 
Clarified 111In-labeling and SPECT/CT and added 
whole body planar imaging as it is standard 
clinical practice prior to SPECT/CT (Sections 
7.2.6, 9.1.1, 9.3, 12, 12.2, 12.5, 15.6). Updated 
adjuvant TMZ requirements (Sections 9.1, 11, 12). 
Added that patient’s cells can be transferred to the 
INTERCEPT trial (Pro00083828) if they become 
recurrent before being treated on this study 
(Sections 9.1 and 12.7.1).  Clarified that all WHO 
Grade IV malignant gliomas are eligible, not 
exclusively GBM (Sections 9.1.1, 12, 15.4, 15.5, 
15.6).  Added the DLT observation period to 
Section 9.1.2.  Updated radiation risk language for 
111In-labeling (only valid for dose expansion) in 
Section 9.1.3.  Updated the definition of Evaluable 
Subject in Section 9.4.  Updated Table 6 with 
changes made throughout protocol and added 1 
gold top blood draw to immune monitoring for FDA 
required C-reactive protein test.  Removed TMZ 
Appendix given that the TMZ cycle maintenance 
will be at the discretion of the treating physician.  
Removed SOP and Safety Monitoring Plan 
Appendix given that they do not provide any 
useful information.  Throughout protocol made 
administrative changes and changed 1 month time 
point to 28 days. 
Amended version for protocol changes 
4/2/18 
Increased immune monitoring blood draws from 6 
yellow top tubes to 9 yellow top tubes (Sections 
9.1, 12.2, 12.5, and Table 6). 
 
 

 
 
 
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1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ............................................................................................... 4 
2 
LIST OF FIGURES ...................................................................................................... 7 
3 
LIST OF TABLES ........................................................................................................ 7 
4 
LIST OF ABBREVIATIONS ......................................................................................... 8 
5 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY ........................................... 11 
6 
STUDY SCHEMA ...................................................................................................... 12 
7 
BACKGROUND AND SIGNIFICANCE ...................................................................... 13 
7.1 
Study Disease ..................................................................................................... 13 
7.1.1 
Disease and Current Therapy ....................................................................... 13 
7.2 
Study Agent ......................................................................................................... 13 
7.2.1 
Rationale for Immunotherapy ........................................................................ 13 
7.2.2 
EGFRvIII and GBM ....................................................................................... 14 
7.2.3 
Preclinical Studies to Provide the Rationale for this Clinical Protocol ........... 15 
7.2.4 
Prior Trials of Cell Transfer Therapy Using Tumor Infiltrating Lymphocytes in 
Patients with Metastatic Melanoma ........................................................................... 16 
7.2.5 
Trials of Cell Transfer Therapy Using Transduction of Anti-TAATCR Genes 
into Non-reactive PBL ................................................................................................ 17 
7.2.6 
111Indium-labeling of Cells for in vivo Trafficking Studies .............................. 18 
7.2.7 
Safety Considerations ................................................................................... 18 
7.3 
Study Purpose/Rationale ..................................................................................... 19 
8 
OBJECTIVES AND ENDPOINTS .............................................................................. 19 
9 
INVESTIGATIONAL PLAN ........................................................................................ 20 
9.1 
Study Design ....................................................................................................... 20 
9.1.1 
Dose Escalation and Expansion.................................................................... 21 
9.1.2 
Definition of Dose-Limiting Toxicity (DLT) ..................................................... 22 
9.1.3 
Safety Considerations ................................................................................... 22 
9.1.4 
Concomitant Medications .............................................................................. 25 
9.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration ................... 25 
9.3 
Rationale for Correlative Studies ......................................................................... 25 
9.4 
Definition of Evaluable Subjects, On Study, and End of Study ............................ 25 
9.5 
Early Study Termination ...................................................................................... 26 
10 
STUDY DRUG ........................................................................................................ 26 
10.1 
Names, Classification, and Mechanism of Action ............................................. 26 
10.2 
Packaging and Labeling ................................................................................... 26 

 
 
 
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10.3 
Supply, Receipt, and Storage ........................................................................... 26 
10.4 
Dispensing and Preparation ............................................................................. 26 
10.4.1 
EGFRvIII CARs .......................................................................................... 26 
10.4.2 
Release Criteria for EGFRvIII CARs .......................................................... 26 
10.4.3 
111In-labeled T cells .................................................................................... 27 
10.5 
Compliance and Accountability ........................................................................ 28 
10.6 
Disposal and Destruction ................................................................................. 28 
11 
SUBJECT ELIGIBILITY .......................................................................................... 28 
12 
SCREENING AND ON-STUDY TESTS AND PROCEDURES ............................... 29 
12.1 
Screening Examination .................................................................................... 32 
12.2 
Treatment Period .............................................................................................. 32 
12.3 
CRS Management Plan .................................................................................... 33 
12.4 
End of Treatment .............................................................................................. 33 
12.5 
Follow-up Period .............................................................................................. 33 
12.6 
End of Study ..................................................................................................... 34 
12.7 
Early Withdrawal of Subject(s) ......................................................................... 34 
12.7.1 
Criteria for Early Withdrawal ...................................................................... 34 
12.7.2 
Follow-up Requirements for Early Withdrawal ........................................... 34 
12.7.3 
Replacement of Early Withdrawal(s) .......................................................... 34 
12.8 
Study Assessments .......................................................................................... 34 
12.8.1 
Medical History .......................................................................................... 34 
12.8.2 
Physical Exam ........................................................................................... 35 
12.8.3 
Correlative Assessments ........................................................................... 35 
13 
SAFETY MONITORING AND REPORTING ........................................................... 35 
13.1 
Adverse Events ................................................................................................ 36 
13.1.1 
Reporting of AEs ........................................................................................ 36 
13.2 
Adverse Events of Special Interest ................................................................... 36 
13.3 
Serious Adverse Events ................................................................................... 36 
13.3.1 
Reporting of SAEs ..................................................................................... 37 
13.4 
Safety Oversight Committee (SOCom) ............................................................ 37 
14 
QUALITY CONTROL AND QUALITY ASSURANCE .............................................. 37 
14.1 
Monitoring ......................................................................................................... 37 
14.2 
Audits ............................................................................................................... 38 
14.3 
Data Management and Processing .................................................................. 38 
14.3.1 
Study Documentation................................................................................. 38 

 
 
 
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14.3.2 
Case Report Forms (CRFs) ....................................................................... 38 
14.3.3 
Data Management Procedures and Data Verification ................................ 39 
14.3.4 
Coding ....................................................................................................... 39 
14.3.5 
Study Closure ............................................................................................ 39 
15 
STATISTICAL METHODS AND DATA ANALYSIS ................................................. 40 
15.1 
Analysis Sets .................................................................................................... 40 
15.2 
Patient Demographics and Other Baseline Characteristics .............................. 40 
15.3 
Treatments ....................................................................................................... 40 
15.4 
Primary Objective ............................................................................................. 40 
15.5 
Secondary Objectives ...................................................................................... 40 
15.6 
Exploratory Objectives ..................................................................................... 40 
15.7 
Sample Size Calculation................................................................................... 41 
16 
ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ........................................ 41 
16.1 
Regulatory and Ethical Compliance ................................................................. 41 
16.2 
DUHS Institutional Review Board and DCI Cancer Protocol Committee .......... 41 
16.3 
Informed Consent ............................................................................................. 42 
16.4 
Privacy, Confidentiality, and Data Storage ....................................................... 42 
16.5 
Data and Safety Monitoring .............................................................................. 42 
16.6 
Protocol Amendments ...................................................................................... 43 
16.7 
Records Retention ............................................................................................ 43 
16.8 
Conflict of Interest ............................................................................................ 43 
16.9 
Registration Procedure ..................................................................................... 43 
17 
REFERENCES ....................................................................................................... 44 
18 
APPENDICES ......................................................................................................... 50 
18.1 
Research Summary .......................................................................................... 50 
18.2 
Figures and Schemas (in order of reference in protocol) ................................. 51 
 
 
 

 
 
 
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2 LIST OF FIGURES 
Figure 1. Study Schema ................................................................................................... 12 
Figure 2. Highly Reactive Transferred Cells Traffic to Brain and Destroy Tumor Deposit in 
Melanoma Patients. .......................................................................................................... 51 
Figure 3. Design of 3rd Generation EGFRvIII CAR. ......................................................... 51 
Figure 4. EGFR Mutation .................................................................................................. 52 
Figure 5. CAR Transduced Murine T-Cells Specifically Recognize EGFRvIII Expressing 
GBM. ................................................................................................................................ 52 
Figure 6. Construction and Analysis of Anti- EGFRvIII CAR Vectors. ............................... 53 
Figure 7. Specific Lysis of U87 EGFRvIII by Anti-EGFRvIII CAR Transduced T Cells. .... 54 
Figure 8. Specific Lysis of U251 EGFRvIII by Anti‐EGFRvIII CAR Transduced T Cells. .. 55 
Figure 9. Expression of EGFRvIII in Glioma Tumor Stem Cell Lines. ............................... 56 
Figure 10. Correlative Studies Schema ............................................................................ 57 
 
3 LIST OF TABLES 
Table 1. Test of CARs Targeting EGFRvIII ....................................................................... 16 
Table 2. Objectives and Endpoints ................................................................................... 19 
Table 3. Dose Escalation and Expansion ......................................................................... 21 
Table 4. Dose Escalation Rules ........................................................................................ 21 
Table 5. Release Criteria for EGFRvIII CARs ................................................................... 26 
Table 6. Screening and On-Study Tests and Procedures ................................................. 31 
Table 7. Half Width of 95% Confidence Interval as a Function of the DLT Rate ............... 41 
Table 8. Frequency and Duration of Objective Responses ............................................... 58 
 
 
 

 
 
 
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4 LIST OF ABBREVIATIONS 
 
Ab 
Antibody 
ABC 
Automated Blood Count 
ADP 
Adenosine 5’-diphosphate 
AE 
Adverse Event 
ALT 
Autologous Lymphocyte Transfer 
AST 
Aspartate Aminotransferase 
BBB 
Blood Brain Barrier 
β-HCG 
Beta-Human Chorionic Gonadotropin 
BMP 
Basic Metabolic Panel 
BMT 
Bone Marrow Transplant 
BTC 
Brain Tumor Center 
Ca++ 
Calcium 
CAR 
Chimeric Antigen Receptor 
CBC 
Complete Blood Count 
CLIA 
Clinical Laboratory Improvement Act 
CMP 
Comprehensive Metabolic Panel 
CNC 
Clinical Neurologic Change 
CNS 
Central Nervous System 
CPC 
Cancer Protocol Committee 
CRP 
C-Reactive Protein 
CRS 
Cytokine Release Syndrome 
CSF 
Cerebral Spinal Fluid 
CT 
Computed Tomography 
CTLs 
Cytotoxic T lymphocytes 
DCI 
Duke Cancer Institute 
DI 
Dose Intensified 
DLT 
Dose Limiting Toxicity 
DUHS 
Duke University Health System 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-linked Immunospot 
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII 
EGFR variant III 
FACS 
Fluorescence Activated Cell Sorting 
Fc 
Fragment Crystallizable 
FDA 
Federal Drug Administration 
GBM 
Glioblastoma Multiforme 
GCP 
Good Clinical Practice 
Gd-DTPA 
Gadolinium-Diethylene Triamine Pentaacetic Acid 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HPLC 
High Pressure Liquid Chromatography 
IC 
Intracerebral 
ICH 
International Conference on Harmonisation 

 
 
 
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ICP 
Intracranial Pressure 
IFN 
Interferon-gamma 
IHC 
Immunohistochemistry 
124I-HSA 
124I-labeled Human Serum Albumin  
IL-2 
Interleukin-2 
IL-4 
Interleukin-4 
IM 
Immune Monitoring 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
KPS 
Karnofsky Performance Status 
LTR 
Long Terminal Repeat 
MAb 
Monoclonal Antibody 
MART-1 
Melanoma Antigen Recognized by T Cells - 1 
MG 
Malignant Glioma 
MHC 
Major Histocompatibility Complex 
MRI 
Magnetic Resonance Imaging 
MTD 
Maximally Tolerated Dose 
NCI CTCAE 
National Cancer Institute Common Terminology Criteria for 
Adverse Events 
NICU 
Neuroscience Intensive Care Unit 
NIH 
National Institutes of Health 
OARC 
Office of Audit, Risk and Compliance 
OS 
Overall Survival 
PBMCs 
Peripheral Blood Mononuclear Cells 
PBLs 
Peripheral Blood Lymphocytes 
PCR 
Polymerase Chain Reaction 
PD 
Progressive Disease 
PE-conjugated 
Phycoerythrin-conjugated 
PET 
Positron Emission Tomography 
PFS 
Progression Free Survival 
PI 
Principle Investigator 
PT 
Prothrombin Time 
PTT 
Partial Thromboblastin Time 
RCR 
Replication Competent Retrovirus 
RDSP 
Research Data Security Plan 
RECIST 
Response Evaluation Criteria in Solid Tumors 
REP 
Rapid Expansion Protocol 
RIO 
Research Integrity Office 
RT 
Radiation Therapy 
RT-PCR 
Reverse Transcriptase Polymerase Chain Reaction 
SAE 
Severe Adverse Event 
ScFv 
Single-chain Variable Fragment 
SOC 
Standard of Care 
SOCom 
Safety Oversight Committee 
SPORE 
Specialized Program of Research Excellence 

 
 
 
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TAA 
Tumor Associated Antigens  
TBI 
Total Body Irradiation 
TCR 
T cell Receptor 
TIL 
Tumor Infiltrating Lymphocyte 
TMZ 
Temozolomide 
TNFα 
Tumor Necrosis Factor alpha 
TSC 
Tumor Stem Cell 
WHO 
World Health Organization 
XRT/TMZ 
Temozolomide During Concomitant Radiation Therapy 
 
 
 

 
 
 
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5 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
 
Please see separate document (available upon request). 
 

 
 
 
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6 STUDY SCHEMA 
 
Figure 1. Study Schema 
 
 
 
 
 

 
 
 
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7 BACKGROUND AND SIGNIFICANCE 
7.1 
Study Disease 
7.1.1 Disease and Current Therapy 
The American Cancer Society estimates that approximately 20,500 new cases of primary 
brain and nervous system tumors will develop and approximately 12,740 patients will die from those 
tumors in the U.S. each year. Brain tumors account for approximately 85 to 90% of all central 
nervous system malignancies. Glioblastoma is the most aggressive and most common glioma, 
accounting for 51% of all gliomas, and it is uniformly lethal. Once the primary modalities of 
treatment, surgery, radiation, and chemotherapy have failed, the treatment options for patients with 
GBM are limited and median overall survival from diagnosis is only 14.6 months. 
Patients with GBM have no curative and limited therapeutic options. As mentioned, median 
OS for patients with newly diagnosed GBM is 14.6 months despite maximal resection, 
incapacitating radiation and systemic chemotherapy, which are inherently non-specific and 
damaging to surrounding eloquent normal tissue.1,2 Immunotherapeutic treatments serve as 
promising new alternatives due to the inherent specificity of the immune system. 
7.2 
Study Agent 
7.2.1 Rationale for Immunotherapy 
One emerging immunotherapeutic platform that shows potential success is ACT, the infusion 
of tumor reactive immune cells into a patient with cancer. The infusion of antitumor CTLs derived 
from the tumor itself (TILs) or T cells with TCRs of endogenous or genetically engineered anti-tumor 
specificity can cause regression in established human tumors such as lymphoma, nasopharyngeal 
cancer, synovial sarcoma and metastatic melanoma and has shown efficacy against lesions in the 
”immune-privileged” brain in the case of melanoma Figure 2).3-6 While ACT with TILs in the 
treatment of metastatic melanoma has shown objective response rates of 49%  to 72%,7 it has been 
limited by difficulties in obtaining tumor-reactive cells from patients, excluding many patients from 
treatment. Additionally, such therapies based on TCRs are reliant upon engagement of the 
endogenous or engineered TCR with the tumor-associated peptide as presented by HLA; however, 
a common mechanism of tumor escape is HLA down regulation8-10 which renders these malignant 
T cells invisible to TCR-based immunotherapy. Clearly, more efficacious methodologies that can 
be applied to a larger pool of patients are still required. 
CARs are cell surface receptors engineered to be expressed on T cells and generated 
through combining the antibody Fc variable region of an antibody with intracellular T cell signaling 
moieties (Figure 2)11. Engagement of the incredibly high avidity CAR Fc variable region with antigen 
triggers T cell activation similar to that observed through the native TCR. Furthermore, as the Fc 
variable region is antibody-based, it is not only higher avidity than a TCR but it binds antigen directly 
rather than in the context of a peptide-HLA complex as a TCR does. Therefore, a single CAR can 
be used with any patient regardless of HLA type, making it more widely applicable than other HLA 
dependent platforms such as TCR-based ACT or active immunotherapeutic vaccination with tumor-
associated peptide that can only be bound in the context of a specific HLA.12,13 As CAR-based 
therapy does not rely on HLA presentation of antigen, it can additionally subvert the common 
immune-evasion of HLA down-regulation by malignant cells,14,15 rendering CARs a potentially more 
efficacious therapy than TCR-based modalities. 
One of the critical barriers to CAR-based ACT has been the physical and functional 
persistence of transduced T cells in the host.16-18 Initial 1st generation CAR vectors generally 
included a single CD3 intracellular domain and successfully redirected cellular cytotoxicity but 
could not persist in vivo. Second generation CARs included an additional intracellular signaling 
domain (frequently CD28) to promote survival and proliferation after repeated antigen exposure;19-
21 however, 3rd generation CARs incorporating OX40 or 41BB domains further enhanced survival 
and showed greater anti-tumor efficacy  than 2nd generation CARs,22-24 bringing the total intracellular 
signaling moieties to three. Both the humanized EGFRvIII-targeted CAR vector for clinical studies 

 
 
 
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and the preclinical murine vector outlined in this proposal are 3rd generation constructs with CD3, 
CD28 and 41BB intracellular signaling domains; these domains in total have been shown to be 
superior in inducing anti-tumor efficacy in comparison to vectors containing any combination of the 
two.24 In recent murine preclinical models, newer generation CARs have shown tremendous anti-
tumor efficacy when redirected against ovarian cancer,25 lymphoma,26 breast cancer,23 and as an 
anti-angiogenic agent against vascular endothelial growth factor receptor 2 (VEGF-R2). 27 Clinical 
trials utilizing CARs have targeted a variety of antigens and malignancies, including GD2 in 
neuroblastoma,16 carbonic anhydrase IX in renal cell carcinoma,28 ERBB2+ metastatic cancer,29 and 
CD19 in B cell malignancies.30 Targeting GD2 showed promising tumor necrosis or regression in 
half of the tested subjects and targeting CD19 resulted in 6 of 8 patients with progressive 
malignancy obtaining remission; however; in both of the other aforementioned trials, severe adverse 
events were reported, including the death of a patient in the trial targeting ERBB2. These events 
were due to the expression of the targeted antigen on normal tissues and the subsequent potent 
cytotoxicity of the CAR bearing T cells at these off-target sites. This is another critical barrier to 
CAR-based immunotherapy, the target antigen must be carefully selected to reduce the risk of life-
threatening toxicity from activated CAR-transduced T cells. 
An ideal target for a CAR must be tumor-specific to prevent the redirected cytotoxicity from 
engendering life-threatening on-target toxicity and should be located on the cell surface to enable 
binding of the Fc variable region. Additionally, the length of the antigen must be optimized with the 
vector design to facilitate binding between the tumor epitope and the CAR for productive signaling. 
Variations on CAR design to include or exclude extracellular spacer regions have shown that the 
cytotoxicity of the CAR can vary based upon how well the CAR can access the targeted epitope31,32. 
7.2.2 EGFRvIII and GBM 
The EGFR is a 170-kDa transmembrane tyrosine kinase. Activation of EGFR launches a host 
of downstream signaling cascades leading to cell proliferation, invasion, motility and adhesion. 
EGFR overexpression leads to unregulated cell growth and malignant transformation, and is 
frequently implicated in a wide range of malignancies. 
The EGFRvIII mutation (Figure 3) consists of an in-frame deletion of 801 base pairs from the 
extracellular domain of the EGFR that splits a codon and produces a novel glycine at the fusion 
junction33 and is most frequently seen in patients with GBM, but has been found in a broad array of 
other common cancers including breast, lung, head and neck.34-37 EGFRvIII is an absolutely tumor-
specific antigen not expressed on normal tissues and present on the cellular surface of one-third of 
GBMs38 with a largely truncated extracellular motif leaving the tumor-specific epitope easily 
accessible.39  
As previous CAR clinical trials have engendered severe adverse events due to specific off-
site but on-target toxicity, EGFRvIII as the targeted antigen overcomes a critical barrier in current 
CAR-mediated immunotherapy as it is both tumor-specific and physically well-suited for CAR-based 
therapy. EGFRvIII is also a validated tumor antigen as recent work from Dr. Sampson, lead PI on 
this grant, demonstrates that humoral responses induced by peptide vaccination with EGFRvIII in 
patients with GBM leads to significant improvement in both PFS and OS.38 In collaboration with Dr. 
Rosenberg at the NCI, we have developed 3rd generation human and murine (CD3/CD28/4-1BB) 
EGFRvIII-targeted CAR vectors that specifically recognize the EGFRvIII tumor mutation as 
demonstrated by in vitro IFN secretion (Figure 4). It is also worth noting that EGFRvIII promotes 
tumor growth through the secretion of cytokines40 and exosomes,41 is expressed on CD133+ brain 
tumor cells,42 and serves as a negative prognostic indicator.43 Therefore, EGFRvIII is not only a 
tumor-specific target, but inhibition of this protein should also be efficacious in a mixed setting of 
EGFRvIII+ and EGFRvIII- tumor cells. Importantly, it has also been shown that the “immunologically 
privileged” location of brain tumors behind the BBB is not an impediment to ACT as activated T 
cells have been shown to enter the CNS24,44 and furthermore, that patients with melanoma brain 
metastases showed regression to immunotherapy with T cells bearing TCRs of engineered 
specificity to the melanoma antigens gp100 or DMF5 5 as well as the necrosis and regression 
showed by CAR-based therapy targeting GD2 in neuroblastoma.16 The ongoing collaboration 
between Drs. Johnson and Sampson represents a unique opportunity to combine expertise in the 
fields of genetically engineered tumor reactive T cells and the immunotherapeutic targeting of 

 
 
 
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EGFRvIII. Furthermore, examining the use of EGFRvIII-specific CARs will serve as an important 
proof-of-concept by testing CARs against a validated target and will furthermore overcome any 
limitations inherent to a humoral-biased anti-tumor response through incorporating the T cell arm 
of adaptive immunity as well as any subsequent bystander effects45-47 of CTL function. Translation 
of these findings to clinical practice as outlined in AIM 3 will be broadly applicable to cancer 
immunotherapy as EGFRvIII is present in numerous malignancies. Furthermore, successful 
EGFRvIII-targeted CAR based therapy could significantly alter critical barriers in clinical practice by 
replacing non-specific and collaterally damaging current standard-of-care treatments with a tumor-
specific modality possessing dramatically improved therapeutic efficacy. 
7.2.3 Preclinical Studies to Provide the Rationale for this Clinical Protocol 
Based on a review of scientific literature and publically available databases, the amino acid 
sequences for seven monoclonal antibodies (4 murine and 3 human) specific for EGFRvIII were 
obtained and used to assemble scFv genes. These scFv genes were inserted into gamma-retroviral 
vector MSGV1 using T cell signaling domains from CD28-CD3zeta (28Z) or, CD8-CD28-41BB-
CD3zeta (28BBZ) to produce CAR expression vectors ( 
Figure 5).  Gamma-retroviral vectors were used to transduce human PBL and tested for 
reactivity by co-culture assays using as targets a variety of cell lines engineered to express 
EGFRvIII (there are no established cell lines that express EGFRvIII).  Initial characterization of the 
seven different anti-EGFRvIII CARs suggested that a CAR based human mAb 139 yielded high 
specific reactivity against EGFRvIII expressing targets, and was thus chosen for development as a 
clinical reagent. Human mAb-based CARs may also have an advantage that they may not elicit an 
anti-CAR immune response as has been seen in some patients being treated with murine-based 
CARs. Anti-EGFRvIII mAb 139-CAR vectors were used to transduce PBL and CAR expression 
determined by FACS ( 
Figure 5) and biological activity tested by co-culture (Table 1). While detection of the 28BBZ 
construction by FACS was less than the 28Z construct, the transduced T cells were equally reactive 
against EGFRvIII expressing targets. We and others48 have previously determined the ability of 
EGFRvIII CAR engineered T cells to lyse target cells in a standard 51Cr-release assay ( 
Figure 6 and Figure 7) and mediate effective regression of intracranial established high grade 
gliomas.  As shown in figures 6 and 7, both vectors specifically lysed only cell lines engineered to 
express the mutant EGFRvIII and not control or wild-type EGFR engineered cell lines. Based on 
our experience and in publications reported by others22,48, the presence of signaling domains from 
the 4-1BB protein is associated with a better survival of CAR engineered cells in animal models. 
Thus, the construct containing the 4-1BB signaling elements was chosen for clinical production. 
By detailed molecular analysis of many different classes of cancer cell lines, it has now been 
demonstrated that established cancer cell lines often do not mirror the molecular characteristics of 
primary human cancers and this is the case for glioma lines. An alternative to the use of establish 
glioma cell lines is the analysis of TSC lines. The TSC paradigm proposes that a subpopulation of 
cells exist in cancer that give rise to all the cells in a differentiated tumor. The Neuro-Oncology 
Branch NCI has demonstrated that in situ glioma cells share properties not found in glioma cell 
lines, and harbor features consistent with tumor stem cells. It was further demonstrated that marked 
phenotypic and genotypic differences exist between primary human tumor-derived TSCs and their 
matched glioma cell lines. TSCs derived directly from primary glioblastomas harbor extensive 
similarities to normal neural stem cells and recapitulate the genotype, gene expression patterns, 
and in vivo biology of human glioblastomas. These findings suggest that glioma-derived TSCs may 
be a more reliable model than many commonly utilized glioma cell lines for understanding the 
biology of primary human tumors. They therefore analyzed three TSC lines for the presence of 
EGFRvIII and demonstrated by RT-PCR that EGFRvIII is expressed in these lines (Figure 8).  PBL 
from two donors were then engineered with the EGFRvIII CAR (28BBZ) and co-culture with glioma 
TSC lines and control EGFRvIII expressing cell lines48. Previous published studies demonstrate 
that EGFRvIII CAR engineered cells from both donors produced IFN-g following co-culture with the 
TSC lines48. These results further support the use of EGFRvIII CAR engineered T cells as a 
potential immunotherapy for patients with GBM. 
 

 
 
 
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Table 1. Test of CARs Targeting EGFRvIII 
 
7.2.4 Prior Trials of Cell Transfer Therapy Using Tumor Infiltrating 
Lymphocytes in Patients with Metastatic Melanoma 
In the great majority of murine models demonstrating the therapeutic effectiveness of the 
adoptive transfer of lymphocytes mediating tumor regression, immunosuppression of the host prior 
to the adoptive transfer of lymphocytes was required49-52, thus NCI incorporated a lymphodepleting 
chemotherapy into their human cell transfer studies. A protocol was developed to rapidly expand 
heterogeneous TILs for adoptive transfer. TILs were expanded using the REP in the presence of 
OKT3, irradiated allogeneic feeder cells and IL-2. These REP’ed TILs retained highly specific in 
vitro anti-tumor activity, often contained reactivities against several antigenic epitopes and 
contained both CD8+ and CD4+ lymphocytes. These autologous bulk TILs were re-infused to 
patients following a non-myeloablative chemotherapy with cyclophosphamide and fludarabine. 
These patients subsequently received high-dose IL-2 (protocol 99-C-0158). Forty-three patients 
received this treatment. This regimen resulted in objective cancer regressions in 49% of patients 
(21 of 43) with metastatic melanoma (Table 8)53.  
Murine models predicted that increasing the extent of lymphodepletion could increase the 
effectiveness of the cell transfer therapy. Thus, they performed two additional sequential trials of 
ACT with autologous anti-TILs in patients with metastatic melanoma. Increasing intensity of host 
preparative lymphodepletion consisting of cyclophosphamide and fludarabine with either 200cGy 
(25 patients) or 1200 cGy (25 patients) TBI was administered prior to cell transfer. While non-
myeloablative chemotherapy alone showed an objective response rate of 49%, when 200cGy or 
1200cGy TBI was added the response rates were 52% and 72% respectively (Table 8). Complete 
response rates for the three trials were 13%, 20% and 40%. TBI appeared to result in increased 
patient survival.  Nineteen of the 20 patients that experienced a complete response are ongoing 
from 3 to 5 years. Responses were seen in all visceral sites including brain. 
Host lymphodepletion was associated with increased serum levels of the lymphocyte 
homeostatic cytokines IL-7 and IL-15. Objective responses were correlated with the telomere length 
of the transferred cells. Patients exhibited the expected hematological toxicities associated with the 
cyclophosphamide, fludarabine and TBI preparative regimens. Patients recovered marrow function 
rapidly after cell infusion with absolute neutrophil counts greater than 500 per mm3 by day 12 and 
Two donor PBL (Effector I and II) were transduced with anti-EGFRvIII CAR vectors 
containing CD28-CD3zeta (139-28Z) or CD8-CD28-41BB-CD3zeta (139-28BBZ) signaling 
domains. 5-6 post-transduction PBL were co- cultured with glioma cell lines U251 and U87 
that had been engineered to express wild type EGFR, EGFRvIII, or GFP. An anti- ERBB2 
CAR served as a positive control in all co-cultures. Shown is resultant IFN-g production 
(pg/ml) following 18hr incubation. 
 
Effector I 
U251 
U251-EGFR 
U251-vIII 
UnTd 
0 
0 
0 
GFP 
0 
0 
0 
Anti-ERBB2 
1195 
2201 
2692 
139-28Z 
0 
0 
2743 
139-28BBZ 
0 
0 
1820 
 
Effector II 
U87 
U87-GFP 
U87-EGFR 
U87-vIII 
GFP 
203 
389 
236 
339 
Anti-ERBB2 
1959 
1061 
671 
932 
139-28Z 
759 
451 
561 
1797 
139-28BBZ 
605 
460 
499 
2217 
 

 
 
 
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sustained platelet counts above 20,000 per mm3 by day 14 (except 4 patients on the TBI 1200 
protocol with platelet recovery on days 16, 17, 20, and 22). 
7.2.5 Trials of Cell Transfer Therapy Using Transduction of Anti-TAATCR 
Genes into Non-reactive PBL 
It is often not possible to isolate sufficient tumor samples from melanoma patients and even 
when tumor is available, only about 60 to 70% generate melanoma reactive TIL cultures. As a 
potential alternative to obtain tumor reactive T cells, TCR gene transfer with anti-TAA properties 
have been developed and utilized in clinical studies52,54,55. Retroviral vector mediated gene transfer 
have been used to engineer human T cells with high efficiency and mediate regression of melanoma 
metastasis in patients with disseminated disease 5,52.  
Studies in the NCI have identified genes that encode melanoma TAA recognized by TILs in 
the context of multiple MHC class I molecules 32-34. These TAA appeared to be clinically relevant 
antigens responsible for mediating tumor regression in patients with advanced melanoma since the 
TIL used to identify these antigens were often capable of mediating in vivo anti-tumor regression. 
Two antigens, which were present in virtually all fresh and cultured melanomas, were called MART-
1 and gp100. The genes encoding these two antigens have been cloned and sequenced. The 
MART-1 gene encodes a 118 amino acid protein of 13 kd. The gp100 gene encodes a protein 
identical to that recognized by monoclonal antibody HMB-45. These antigens were thus the original 
targets of their gene therapy cell transfer protocols in patients with metastatic melanoma. 
They then isolated TCR genes from both gp100 and MART-1 reactive T cells. These studies 
have recently been published and are presented briefly here52,5. To test the in vivo efficacy of these 
F4 MART-1 TCR engineered T cells, 31 HLA-A*0201 patients with progressive metastatic 
melanoma were treated. Results in the first 17 patients were published in 36. All patients were 
refractory to prior therapy with IL-2. T cell cultures from all patients were biologically reactive, with 
specific secretion of interferon-γ following co-culture with either MART-1 peptide pulsed T2 cells 
and or melanoma cell lines expressing the MART-1 antigen. Gene transfer efficiencies measured 
by staining for V 12 expression in these lymphocytes ranged from 17% to 67% (mean value 42%). 
Four of the 31 patients demonstrated a sustained objective regression of their metastatic melanoma 
assessed by standard RECIST criteria. There were no toxicities in any patient attributed to the gene- 
marked cells. The NCI then demonstrated for the first time in humans, that normal autologous T 
lymphocytes, transduced ex vivo with anti-TAA TCR genes and reinfused in cancer patients can 
persist and express the transgene long-term in vivo and mediate the durable regression of large 
established tumors5,52. A similar study was conducted using gp100 TCR gene marked cells, 
however this retroviral vector had a low titer when produced under GMP conditions. Fourteen 
patients were treated on this study. No antitumor responses have been seen. There have been no 
grade 5 toxicities observed on this study, and all grade 3 and 4 toxicities observed were expected 
toxicities associated with the non-myeloabalative chemotherapy regimen or IL-2. 
The low response rate in the prior MART-1 TCR gene transfer protocol led to identification of 
F5 MART-1 reactive TCR with higher avidity than the MART-1 F4 TCR used in the prior gene 
therapy clinical trial5, and additionally to generate a TCR that recognizes the gp100:154-162 This 
TCR was raised in an HLA-A2 transgenic mouse immunized with this peptide37. NCI has now 
treated 24 patients with metastatic melanoma using autologous PBL transduced with an improved 
MART-1 F5 TCR following a non- myeolablative chemotherapy. Six patients (25%) have achieved 
an objective partial response. Toxicities observed were expected toxicities associated with the non-
myeloabalative chemotherapy regimen or IL-2, and were similar to those seen in the prior TCR 
gene therapy trial except that 15 patients developed a transient mild anterior uveitis easily reversed 
by steroid eye drops and ten patients developed decreased hearing reversed by middle ear steroid 
injections. Transient rashes have also been seen. There were no treatment related deaths. They 
have now treated 21 patients with metastatic melanoma using autologous PBL transduced with this 
improved gp100 TCR following a non-myeloablative chemotherapy. Four patients (19%) have 
achieved an objective partial response. Seven patients developed a transient mild anterior uveitis 
reversed by steroid eye drops and ten patients developed decreased hearing reversed by middle 
ear steroid injections. There were no treatment related deaths. 
In addition to the studies listed above in patients with metastatic melanoma, the NCI has 
recently initiated several studies for patients with other metastatic cancers using PBL transduced 

 
 
 
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with TCR genes or CAR genes. They have studied PBL transduced with TCR genes targeting p53, 
CEA, NY-ESO-1 and TRAIL bound to the DR4 Receptor, and PBL transduced with CAR targeting 
Her-2 and CD19. Patients on these studies also received a non-myeloablative chemotherapy 
regimen consisting of cyclophosphamide and fludarabine, and high dose IL-2. In two studies, 08-
C-0121 (anti- ESO-1 TCR) and 09-C-0082 (anti-CD19 CAR), they have seen impressive clinical 
responses. The clinical responses observed in seven of the fifteen evaluable patient receiving anti-
ESO TCR-engineered peripheral blood lymphocytes have been very encouraging. Of the six 
synovial cell sarcoma, four patients have confirmed PRs. Of the nine patients with highly ESO 
expressing melanoma, there have been 2 confirmed complete responses and 2 confirmed partial 
responses. In study 09-C-0082, an impressive ongoing partial remission at 7 months post treatment 
of the lymphoma occurred in the first patient treated on this study56. This patient had heavily pre-
treated, progressive follicular lymphoma that involved all major lymph node areas. Only three small 
lesions that were consistent with lymphoma persisted on PET imaging post-treatment. Most 
toxicities observed in these studies were expected toxicities of the chemotherapy and aldesleukin 
administration. However, in 2 studies, they have observed serious adverse events related to the 
transduced cells. In 09-C-0051 (anti-Her2 CAR transduced PBL), the first patient, with Her-2 
expressing metastatic colorectal cancer, was treated with 1010 autologous T cells transduced with 
the retrovirus encoding an anti-Her-2 CAR. This patient developed respiratory distress and died 
four days later29. This toxicity was apparently due to a previously unrecognized ability of this CAR 
to recognize Her-2 expressed on lung epithelial cells although the exact explanation for the toxicity 
is not clear. In 09-C-0047 (anti-CEA TCR transduced PBL) all three patients treated experienced a 
variety of gastrointestinal events which were attributed to the gene/cell therapy including diarrhea, 
and colitis. All gastrointestinal events have since resolved in these patients, and all of the patient’s 
colonic mucosa has returned to normal, and the patients have normal bowel function. Grade 3 
diarrhea lasting longer than 72 hours is considered a DLT per protocol and this event was observed 
in two of three patients enrolled in protocol 09-C-0047, meeting the criteria for stopping protocol 
accrual.  One of the patients experienced an objective regression of liver metastases. 
7.2.6 111Indium-labeling of Cells for in vivo Trafficking Studies 
The in vivo distribution of adoptively transferred CAR cells will be evaluated using 111In-
labeled autologous lymphocytes.  111In-labeled leukocytes have been used extensively for 
evaluation of adoptively transferred tumor infiltrating lymphocytes, natural killer cells, granulocytes, 
and whole blood leukocytes, for in vivo localization studies in humans57-62.  EGFRvIII CARs will be 
labeled at the Duke Radiopharmacy Lab according to standardized protocols.  Briefly, CARs will be 
counted and re-suspended in PBS.  4-6x108 cells will be labeled with 500 µCi of 111In. The cells will 
be washed and mixed with cold CARs to achieve the desired cell dose with a total of 500 µCi of 
111In.  The labeled CARs will be infused into patients through intravenous catheter within the 
Ambulatory Bone Marrow Transplant Unit.  Distribution of 111In-labeled EGFRvIII CARs will be 
evaluated at 1, 2 and 3 days post-infusion using SPECT/CT. 
7.2.7 Safety Considerations 
Several safety concerns regarding the infusion of large numbers of retrovirally-modified tumor 
reactive T cells have been addressed in previous clinical studies.  
CAR transduced human T cells have been administered to humans in several clinical trials. 
Pule, et al. engineered human T cells to express a CAR directed to the disialoganglioside GD2, a 
tumor associated antigen expressed by human neuroblastoma cells16. These CAR transduced cells 
were administered to eleven children between the ages of 3 and 10, all of whom had metastatic 
neuroblastoma. There were no adverse effects attributable to the genetically modified CAR T cells 
in the eleven subjects that were followed for up to two years following cell infusion. Two patients 
exhibited an objective response. Persistence of the transduced cells was greater when the CAR 
were inserted into EBV specific cytolytic T lymphocytes than when the CAR were inserted into T 
cells that were activated by an anti- CD3 monoclonal antibody. 
The NCI treated patients with B cell lymphomas utilizing lymphocytes transduced with a 
retrovirus encoding a CAR that recognizes the CD19 molecule30,63-65. As expected by the targeting 
of CD19, the protocol was associated with elimination of normal B cells. One patient required ICU 

 
 
 
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support for toxicity similar to tumor lysis syndrome and a second patient treated on this study died 
prior to response evaluation from H1N1 influenza infection. This death is possibly related to the 
research, but is not related to the anti-CD19 CAR transduced cells. 
A patient with metastatic colorectal cancer was treated with 1010 autologous T cells 
transduced with the retrovirus encoding an anti-Her-2 CAR. This patient developed respiratory 
distress and died four days later29. This toxicity was apparently due to a previously unrecognized 
ability of this CAR to recognize Her-2 expressed on lung epithelial cells although the exact 
explanation for the toxicity is not clear.  As the EGFRvIII protein is tumor specific normal cell 
recognition is not expected. In addition, as previously described, multiple EGFRvIII vaccine trials 
have been reported and no significant vaccine-related toxicities have been observed66-68. 
The expansion of tumor reactive cells is a desirable outcome following the infusion of antigen 
reactive T cells and this expansion could mimic the clonal expansion of a transformed lymphocyte. 
The transfer of gene modified cells has a significant risk for malignant transformation in this patient 
population. While the risk of insertional mutagenesis is a known possibility using retroviral vectors, 
this has only been observed in the setting of patients with primary immunodeficiencies using 
retroviral vector-mediated gene transfer into CD34+ bone marrow cells. In the case of retroviral 
vector-mediated gene transfer into mature T cells, there has been no evidence of long-term 
toxicities associated with these procedures since the first NCI sponsored gene transfer study in 
1989.  Although continued follow-up of all gene therapy patients will be required, data suggest that 
the introduction of retroviral vectors transduced into mature T cells is a safe procedure. The NCI 
risk of insertional mutagenesis is extremely low, and the proposed protocol follows all current FDA 
guidelines regarding testing and follow up of patients receiving gene transduced cells. 
 
7.3 
Study Purpose/Rationale 
The goal of this protocol is to transfer PBL transduced with genes encoding a CAR that recognizes 
an EGFRvIII, tumor-specific antigen into patients with WHO Grade IV malignant glioma.  The CAR used 
will be targeted to a tumor-specific mutation, EGFRvIII, which is expressed on a subset of patients.  
Normal PBLs derived from patients with WHO Grade IV malignant glioma will be genetically engineered 
with a viral vector encoding the CAR and infused into newly diagnosed malignant glioma patients with the 
aim of mediating regression of their tumors. In a recent trial, EGFRvIII CARs were administered to 
recurrent malignant glioma patients.  The starting EGFRvIII CAR dose for this study (ExCeL) was shown 
to be safe in that recent study in patients with recurrent malignant glioma.   
Correlative studies are also proposed to document the expansion, functional persistence,19,69 and 
trafficking70-72 to tumor of adoptively transferred EGFRvIII CARs as these have been the correlates of 
successful therapy for other investigators. 
8 OBJECTIVES AND ENDPOINTS 
 
Table 2. Objectives and Endpoints 
 
Objective 
Endpoint 
Analysis 
Primary 
Determine the MTD of a single IV 
infusion of EGFRvIII CAR T cells 
in patients with newly-diagnosed 
WHO Grade IV malignant glioma. 
MTD  
 
See Section 15.4 
Key Secondary 
To determine the DLT of a single 
IV infusion EGFRvIII CAR T cells 
in patients with newly-diagnosed 
WHO Grade IV malignant glioma. 
DLT 
See Section 15.5 
 
Exploratory 
To determine if EGFRvIII CARs 
transferred into patients with WHO 
Grade IV malignant glioma 
expand.  
Median number of EGFRvIII CARs in 
peripheral blood and KI67 
See Section 15.6 
Exploratory 
To determine if EGFRvIII CARS 
persist and remain functional in 
the periphery. 
Median number of  EGFRVIII CARs by 
tetramer and functional assays 
See Section 15.6 

 
 
 
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Exploratory 
To assess whether EGFRvIII 
CARs traffic to tumor site. 
Median proportion of injected 111In-labeled 
EGFRvIII CARs present in tumor 
See Section 15.6 
Exploratory 
To describe the survival 
experience of patients treated with 
EGFRvIII CAR T cells. 
Median survival 
See Section 15.6 
Exploratory 
To describe the progression-free 
survival experience of newly-
diagnosed, EGFRvIII-expressing 
WHO Grade IV malignant glioma 
patients treated with EGFRvIII 
CAR T cells. 
Proportion of patients alive without disease 
progression 6 months after EGFRvIII CAR 
treatment 
See Section 15.6 
9 INVESTIGATIONAL PLAN 
9.1 
Study Design 
 
Treatment Plan 
 
Following consent, subjects who are part of the dose escalation cohort will be assigned into the 
appropriate dose-escalation cohorts. Patients will undergo a 2-4 hour leukapheresis to harvest PBMCs 
for the generation of EGFRvIII CAR T cells prior to beginning RT and concurrent TMZ.  T cells will be 
isolated from the patient’s PBMCs and transduced to express CARs.  Briefly, PBMC will be stimulated 
with OKT3 (anti-CD3 mAb) and transduced on RetroNectin® coated plates.  Transduced cells will be 
expanded in IL-2 for 14 days.  
Patients will then complete approximately 6 weeks of standard of care RT and concurrent TMZ at a 
targeted dose of 75 mg/m2/d. Patients whose blood counts do not meet eligibility within 6 weeks of 
completing standard of care RT and concurrent TMZ will be withdrawn from the study before initiation of 
TMZ treatment and replaced.  Patients who appear to have progressive disease following radiation may 
actually have pseudoprogression due to the radiation and will be allowed to continue to cycle 1 of (dose 
intensified) DI TMZ in order to not be excluded from the study prematurely, at the discretion of the treating 
physician.  If the patient is determined to have progressive disease, the patient may be eligible for the 
INTERCEPT study (INTracerebral EGFR-vIII Chimeric Antigen Receptor Gene-Modified T Cells for 
PaTients with Recurrent GBM, Pro00083828).  If the patient is determined to be eligible for the 
INTERCEPT study (Pro00083828), the cells extracted from leukapheresis while they were on this study, 
ExCeL, will be used to create the CAR T cells for administration in the INTERCEPT study.      
Patients without definitive disease progression who remain eligible will then receive up to 3 cycles of 
dose-intensified (DI) TMZ at 50-100 mg/m2/day.  Each full cycle of DI TMZ is 50-100 mg/m2/day for 21 
days every 28 days, at the discretion of the treating physician.  The purpose of the DI TMZ cycles is to 
achieve grade 3 lymphopenia.  If the patient is able to tolerate the TMZ, they will receive 3 full cycles of 
DI TMZ prior to receiving the CAR T cells.  If the patient is unable to complete the 3 cycles of DI TMZ, but 
has achieved grade 3 lymphopenia, they may receive CAR treatment at the discretion of the study PI 
and/or treating oncologist. If the patient’s grade 3 lymphopenia subsides during the ~2 weeks it takes to 
produce the CAR T cells and before the scheduled receipt of CAR T cells, the treating physician has the 
discretion to re-initiate TMZ to re-establish grade 3 lymphopenia. If the patient is unable to tolerate the 3 
full cycles of TMZ and does not reach grade 3 lymphopenia, or if the CAR-specific T cells do not meet 
release criteria, the patient will be withdrawn before CAR treatment and replaced.   
Two weeks prior to CAR T cell administration, the patient must be on stable or decreasing steroids (≤ 
4 mg/day).  If patients are decreasing steroid use, once they are at 2 mg/day, they may be supplemented 
with hydrocortisone, at the discretion of the treating oncologist.  
At least 48 hours after the patient’s last dose of TMZ, the total dose of EGFRvIII CAR T cells will be 
delivered intravenously. If sufficient CAR-specific T cells cannot be generated to meet the targeted 
assigned dose within the dose-escalation portion of the study, the patient will be treated at a lower pre-
defined dose level using available CAR-specific T cells and replaced in the assigned higher dose.  The 
administered dose will be the highest defined dose level for which there are sufficient CAR-specific T cells 
available.  Within the expanded cohort, if sufficient CAR-specific T cells can’t be generated to meet the 
MTD dose, all available T cells will be administered.  
Blood samples for immune monitoring (9 yellow, 1 red) will be drawn prior to EGFRvIII CAR infusion, 
1, 5, 10, and 28 days after the infusion, then 3 and 6 months, then yearly until progression (or death or 
lost to contact). The return visits for immune monitoring at the 3 months, ~6 months, and ~yearly will 

 
 
 
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coincide with standard of care clinic visits. A portion of the immune monitoring blood sample will be used 
for RCR PCR per the FDA prior to EGFRvIII CAR infusion, and at 3, 6 and 12 months during standard of 
care clinic visits. Lastly, blood for evaluation of CRS (1 red, 1 purple top – 3mLs each) will be drawn prior 
to cell infusion, 1 and 4 hours after infusion, and on days 1, 2, 5, 10, and at approximately 28 days. 
Measurements for CRS include IL-2, IL-6, TNFα, IFN, GM-CSF, and C-reactive protein (CRP). 
Patients will return to clinic approximately 28 days following the EGFRvIII CAR T cell infusion to be 
evaluated for cycles of standard of care 5-day TMZ at 150-200 mg/m2/day for the first 5-day cycle, followed 
by 200 mg/m2/day (if tolerated by the patient) for 5-days every 28 days at the discretion of the treating 
oncologist. This will result in an approximately > 30-day delay between the last TMZ administration prior 
to EGFRvIII CAR T cell infusion and the TMZ cycle initiated after the study drug infusion. This delay in 
the TMZ cycle after the CAR T cell infusion is done in an attempt to decrease the chance of study drug 
washout by TMZ.  
Tumor progression will be documented histologically, unless there are clinical contraindications, to 
exclude inflammatory responses presenting as radiographic or clinical changes, which could indicate a 
potentially toxic or therapeutic responses and not tumor progression.  If tissue is obtained through the 
Duke Brain Tumor Center Biorepository IRB# Pro00007434, it will be used to confirm tumor progression 
histologically, and to assess immunologic cell infiltration and EGFRvIII antigen escape at the tumor site.  
Patients will be eligible for additional adjuvant therapy at the time of tumor progression. 
9.1.1 Dose Escalation and Expansion 
A classical “3+3” study design will be used to estimate the MTD for CAR-specific T cells 
treatment among patients with newly-diagnosed WHO grade IV malignant glioma.  As shown below 
in Table 3, 4 dose levels will be considered based on transduced cells/kg:  #1:  4.5 x 106/kg, #2: 1.5 
x 107/kg, #3: 4.5 x 107/kg, and #4: 1.5 x 108/kg.  
 
Table 3. Dose Escalation and Expansion 
 
Dose Escalation and Expansion 
Cohort 
Dose of CAR-specific T 
cells 
# of Subjects 
Schema 
1 
4.5 x 106/kg 
3-6 
Dose escalation:  “Standard 3+3” 
2 
1.5 x 107/kg 
3-6 
3 
4.5 x 107/kg 
3-6 
4 
1.5 x 108/kg * 
3-6 
* If the patient’s targeted dose is 1.5 x 108/kg (dose level #4), the calculated dose will be capped 
at 1.0 x 1010 total cells.   
 
Starting with the lowest dose level, cohorts of 3-6 subjects will be accrued at each dose level 
according to the following dose escalation rules: 
 
Table 4. Dose Escalation Rules 
Number of patients with DLT 
observed during the first 4 weeks 
after CAR treatment 
Escalation Decision Rule 
0 out of 3 
Enter 3 patients at the next dose level 
1 out of 3 
Enter 3 more patients at this dose level. 
 
If 0 of these 3 patients experience DLT, proceed to the 
next dose level 
 
If 1 or more of this group suffer DLT, then dose 
escalation is stopped.  Three (3) additional patients 
will be entered at the next lowest dose level if only 3 
patients were treated previously at that dose. 

 
 
 
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≥ 2 
Dose escalation will be stopped.  Three (3) additional 
patients will be entered at the next lowest dose level if only 
3 patients were treated previously at that dose. 
DLT = dose limiting toxicity; MTD = maximum tolerated dose 
 
If a patient terminates the infusion of EGFRvIII CAR T cells prematurely or is lost to follow-up 
during the first 4 weeks after CAR treatment without experiencing a DLT, then the patient will not 
be evaluable for the determination of the MTD and will be replaced.  The MTD is the highest dose 
level at which ≤1 of 6 patients experiences DLT during the 4 week observation period after CAR 
treatment. The last patient in a given cohort, receiving that dose level, should be observed for DLTs 
for at least 4 weeks prior to moving on to the next cohort/higher dose.  Section 9.1.2 provides 
information about what constitutes a DLT. 
An expanded cohort of a total of 12 patients will be enrolled at the MTD of EGFRvIII CAR T 
cells in order to obtain a more precise estimate of the probability of unacceptable toxicity.  This 
cohort will also have the cells radiolabeled with 111In to track their distribution.  Briefly, CARs will be 
counted and re-suspended in PBS.  4-6x108 cells will be labeled with 500 micro Ci of 111In. The 
cells will be washed and mixed with cold CARs to achieve the desired cell dose with a total of 500 
µCi of 111In. The labeled CARs will be infused into the patient through an intravenous catheter within 
the Ambulatory Bone Marrow Transplant (BMT) Unit.  Distribution of 111In-labeled EGFRvIII CARs 
will be evaluated at 1, 2, and 3 days post-infusion using whole body planar imaging followed by 
SPECT/CT, when indicated. 
In the rare event that a sufficient amount of CAR-specific T cells cannot be generated for a 
patient’s intended dose within the dose-escalation portion of this study, the patient will be treated 
at a lower pre-defined dose level using available CAR-specific T cells.  If this patient experiences a 
DLT, data will be carefully reviewed to determine its impact upon dose escalation and future patient 
treatment.  If this situation occurs with the expanded cohort, all available cells will be administered.   
9.1.2 Definition of Dose-Limiting Toxicity (DLT) 
The DLT observation period is the 4 weeks after EGFRvIII CAR T cell infusion.  Toxicities will 
be graded according to the NCI CTCAE version 4 criteria.  A DLT will be defined as any Grade IV 
event of any duration that is at least (possibly, probably, or definitely) attributable to EGFRvIII CARs, 
or any Grade III toxicity that is at least (possibly, probably, or definitely) attributable to EGFRvIII 
CARs that is not reversible within 4 weeks. DLT definition includes and will be reported to FDA all 
of the following: 
a. Any treatment-emergent CTCAE Grade 4 or 5 CRS. 
b. Any treatment-emergent CTCAE Grade 3 CRS that do not resolve to ≤ Grade 2 
within 7 days. 
c. Any treatment-emergent autoimmune toxicity ≥ Grade 3. 
d. CTCAE grade 3-5 allergic reactions related to the study cell infusion. 
e. CTCAE grades 3 and greater organ toxicity (cardiac, dermatologic, 
gastrointestinal, hepatic, pulmonary, renal/genitourinary, or neurologic) not pre- 
existing or due to the underlying malignancy and occurring within 30 days of 
study product infusion. 
9.1.3 Safety Considerations 
Potential Benefits 
Based on experience in our previous EGFRvIII vaccine trials, immunotherapy may be of 
benefit to patients with MGs.  Of course, because individuals respond differently to therapy, no one 
can know in advance if it will be beneficial in an individual case.  The potential benefits may include 
reduction and/or remission of the patient’s brain cancer.  Because this procedure is experimental, 
it cannot be guaranteed that patients will receive any benefit as a result of participating in this 
research study.  The information collected in this research may help scientists better understand 
the mechanisms involved in the immune system’s ability to fight cancer.  If such an understanding 
comes from this research, then it may benefit society by furthering the development of improved 
treatment methods for human malignant brain tumors in the future. 

 
 
 
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Potential Risks 
Cytokine Release Syndrome 
Cytokine release syndrome (CRS) is a potentially life-threatening toxicity that has been 
observed following administration of natural and bispecific antibodies and, more recently, following 
adoptive T cell therapies for cancer73. CRS is associated with elevated circulating levels of cytokines 
including IL-6 and IFN. Commonly referred to as an infusion reaction, it results from the release of 
cytokines from cells targeted by the antibody as well as immune effector cells recruited to the area. 
When cytokines are released into the circulation, systemic symptoms such as fever, nausea, chills, 
hypotension, tachycardia, asthenia, headache, rash, scratchy throat, and dyspnea can result. In 
most patients, the symptoms are mild to moderate in severity and are managed easily. However, 
some patients may experience severe, life-threatening reactions that result from massive release 
of cytokines. Severe reactions occur more commonly during the first infusion in patients with 
hematologic malignancies who have not received prior chemotherapy; severe reactions are marked 
by their rapid onset and the acuity of associated symptoms. Massive cytokine release is an 
oncologic emergency, and special precautions must be taken to prevent life-threatening 
complications (please see CRS Management Plan). 
Tumor Lysis Syndrome 
The infusion of antigen reactive T cells may cause tumor lysis syndrome, a group of 
complications from release of large amounts of potassium, phosphate, and nucleic acid caused by 
the breakdown of tumor cells after cancer treatment. Tumor lysis syndrome may cause kidney 
failure, abnormal heart rhythm, and may even lead to death.  
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.  Symptoms may include, but are not limited to, severe headache, 
confusion, lethargy, unresponsiveness, coma, or focal neurological deficits.  Patients will be 
monitored throughout the course of the study and those patients with any signs or symptoms of 
cerebral edema may need their steroid doses increased, treatment with an osmotic diuretic, or 
surgical decompression.  Edema that fails to respond to aggressive therapy may lead to permanent 
neurological impairment.  The probability of this risk can be predicted to some degree based upon 
tumor size, location, pre-operative neurological impairment, and post-operative course prior to 
EGFRvIII CAR T cell infusion. Patients will be monitored throughout the course of the study. 
Infection 
The EGFRvIII CAR T cells may include the risk of infection due to potential contamination of 
the cells in the laboratory.  This may result in localized redness, swelling, or induration at the infusion 
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 fact that the cells will be strictly tested 
for sterility prior to each infusion.  The risk of infection due to potential contamination of the cells 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 infusion into patients, EGFRvIII CAR T cells must pass sterility tests.  Following 
infusions, patients will be monitored throughout the course of the study for any signs and symptoms 
of infection. If an active infection is suspected, patients will be cultured and treated with appropriate 
antibiotics. 
RCR 
The infusion of antigen reactive T cells and this expansion could mimic the clonal expansion 
of a transformed lymphocyte.  We do not believe the transfer of these gene modified cells has a 
significant risk for malignant transformation in this patient population. While the risk of insertional 
mutagenesis is a known possibility using retroviral vectors, this has only been observed in the 
setting of patients with primary immunodeficiencies using retroviral vector-mediated gene transfer 
into CD34+ bone marrow cells. In the case of retroviral vector-mediated gene transfer into mature 
T cells, there has been no evidence of long-term toxicities associated with these procedures since 
the first NCI sponsored gene transfer study in 1989.  Although continued follow-up of all gene 
therapy patients will be required, data suggest that the introduction of retroviral vectors transduced 
into mature T cells is a safe procedure. While we believe the risk of insertional mutagenesis is 

 
 
 
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extremely low, the proposed protocol follows all current FDA guidelines regarding testing and follow 
up of patients receiving gene transduced cells. 
Delayed Autoimmune Diseases 
It is possible that delayed autoimmune disease(s) may develop as a result of infusion with 
EGFRvIII CAR T cells.  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 T cell infusion and recovery from lymphopenia.  However, our current experience with autologous 
lymphocyte transfer in patients with GBM has not demonstrated evidence of autoimmunity in treated 
patients.  Furthermore, the doses of TMZ used in this study for induction of lymphopenia are 
standard doses administered to patients with GBM.  It therefore, is unknown what the risk of delayed 
autoimmune disease is for this study. 
Phlebotomy 
Drawing blood or inserting an intravenous catheter into an arm vein may result in bruising or 
swelling in the area of the insertion, bleeding at the site of the needle puncture, light headedness, 
fainting and very rarely, local infection, which may be severe.  These risks are reduced by the fact 
that the blood will be drawn by a qualified physician, nurse or phlebotomist (a professional trained 
to draw blood). 
MRI 
The risks and/or discomforts associated with the performance of MRI include the anxiety 
produced from being in a tight, enclosed space (claustrophobia).  In addition, the machine operates 
using a large and powerful magnet.  The magnetism of the machine attracts certain metals: 
therefore, people with these metals in their bodies (specifically pacemakers, infusion pumps, metal 
aneurysm clips, metal prostheses, joints, rods or plates) will be excluded from the study. Patients 
will also be checked to make sure that they do not bring any metal objects into the MRI facility.  
Dental fillings are less affected by the magnetic fields generated and are therefore permitted.  It will 
be asked that patients let the physicians conducting this study know of any metal in their bodies 
other than dental fillings. 
Allergic Reactions to Contrast Agents 
During the MRI, patients will be given a contrast agent.  The agent is given routinely to obtain 
enhanced MRI scans of the brain.  The agent is administered through the vein and requires the 
placement of an IV catheter.  The catheter placement is similar to drawing blood except that the 
catheter remains in the vein during the time the agent is actively delivered.  The risks of a blood 
draw and insertion of a catheter are similar.  There have been a few, rare cases of allergies to the 
agent used in MRI contrast enhanced scans. Patients with any known severe allergies to contrast 
agents will be excluded from the study.  Patients with mild allergies (i.e., rash only) will be pretreated 
with Tylenol and Benadryl prior to injection of the contrast agent. 
Temozolomide 
TMZ has been well tolerated by both adults and children with the most common toxicity being 
mild myelosuppression.  Other, less likely, potential toxicities include nausea and vomiting, 
constipation, headache, alopecia, rash, burning sensation of skin, esophagitis, pain, diarrhea, 
lethargy, hepatotoxicity, anorexia, fatigue and hyperglycemia.  Hypersensitivity reactions have not 
yet been noted with TMZ.  As in the case with many anti-cancer drugs, TMZ may be carcinogenic.  
Rats given TMZ have developed breast cancer.  The significance of this finding for human is not 
presently known. TMZ therapy will be followed but given as standard of care. If toxicities occur, the 
Principle investigator and primary physician will titrate therapy based on standard clinical guidelines 
as outlined above. 
111Indium Labeled EGFRvIII CARs 
Patients’ CARs will be radiolabeled with 111In for correlative studies in the expanded cohort. 
The radiation exposure to the patient who receives an 111In-labeled EGFRvIII CARs will be minimal 
at proposed doses and is roughly equivalent to natural radiation that people experience living in 
higher altitudes. This can be compared to the same amount of radiation one would get from living 
in a high altitude city such as Denver for 48 weeks or taking 110 airplane flights from New York to 
Los Angeles. The tests or treatments will include a whole body planar imaging scan and a white 
cell localization scan.  
Unknown Risks 

 
 
 
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The overall risk classification of this research is unknown.  Clinical trials using immunotherapy 
on brain tumor patients only recently published in the literature. From our experience in ongoing 
and previous trials we have not seen any toxicities or serious unexpected adverse events. 
Confidentiality 
Participation in research investigations may result in a loss of confidentiality.  However, all 
data from preoperative and postoperative evaluations will be coded to protect the patient’s identity.  
The coding, and the results of these studies will be available only to the individuals involved with 
the study, the clinical staff administering the study, representatives of the National Institutes of 
Health, and representatives of the U.S. Food and Drug Administration.  Any publications resulting 
from this study will not use patient identifying data. 
Treatment Alternatives & Financial Reimbursement 
Alternative treatments for newly-diagnosed malignant brain tumors include additional surgery, 
radiation, and/or chemotherapy.  If the patient chooses not to participate in this trial, they certainly 
may seek alternative treatment.  If the patient fails treatment through this trial, these alternatives 
may still be available to the patient.  There will be no financial reimbursement to patients for study 
participation. 
9.1.4 Concomitant Medications 
Concomitant medications will be managed by the treating neuro-oncologist and recorded at 
each study visit, beginning at pre-CAR T cell infusion visit, with the exception of steroids, which may 
be tracked earlier by the study coordinator/research nurse. 
9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
In a recent trial, EGFRvIII CARs were administered to recurrent malignant glioma patients.  The 
starting EGFRvIII CAR dose for this study (ExCeL) was shown to be safe in that recent study in patients 
with recurrent malignant glioma. The EGFRvIII CAR infusion will be given after chemoradiation and up to 
3 cycles of dose-intensified (DI) post-radiation temozolomide to leverage the homeostatic effects of 
chemotherapy-induced lymphodepletion. 
9.3 
Rationale for Correlative Studies 
Correlative studies are also proposed to document the expansion, functional persistence,19,69 and 
trafficking70-72 to tumor of adoptively transferred CARs as these have been the correlates of successful 
therapy for other investigators.  
Peripheral blood for IM will be drawn leukapheresis, pre-infusion, 1, 5, 10, and 28 days after CARs 
treatment, then 3 and 6 months post-infusion, and then yearly (until progression, death, or lost to contact).  
Whole body planar imaging and SPECT/CT on the expanded cohort of 12 patients enrolled at the MTD 
on days 1, 2, and 3 post-infusion will be used to determine the IC localization of indium-labeled T cells.  If 
there is no signal on whole body planar imaging, the radiologist may decide not to perform SPECT/CT. 
9.4 
Definition of Evaluable Subjects, On Study, and End of Study 
While all patients who received any EGFRvIII CAR T cells will be included in safety summaries for 
the purposes of monitoring and reporting of adverse events to appropriate agencies, patients evaluable 
for determination of the MTD will include:   
 
Patients who receive the entire CARs infusion (i.e. the infusion is not stopped prematurely), and 2) 
have 4 weeks of follow-up for adverse events after the infusion 
 
Patients who receive the entire CARS infusion (i.e. the infusion is not stopped prematurely) and 
terminated follow-up prematurely during the 4 week follow-up period after having experienced a DLT. 
 
Patients who terminate infusion before completion due to a DLT 
 For the efficacy analyses, evaluable patients will include two groups of patients:  all patients, and the 
subgroup of patients who receive EGFRvIII CAR. Once a patient’s eligibility is confirmed after completion 
of XRT/TMZ, that subject will be considered “on study”.  The rationale for taking a patient off study will be 
documented.  Please see Section 12.7 for information on patient withdrawals. 

 
 
 
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9.5 
Early Study Termination 
This study can be terminated at any time for any reason by the PI-sponsor.  If this occurs, all 
subjects on study will be notified as soon as possible.  Additional procedures and/or follow up should 
occur in accordance with Section 12.7, which describes procedures and processes for prematurely 
withdrawn patients. 
10 STUDY DRUG 
10.1 
Names, Classification, and Mechanism of Action 
The name of the drug is CAR gene-modified T cells or abbreviated as EGFRvIII CARs.  The class 
of action is a biological and the mechanism of action is cytotoxicity.  The drug substance is autologous T 
cells transduced with a retroviral vector encoding for a chimeric antigen receptor (CAR) directed against 
the tumor specific antigen, EGFRvIII.  EGFRvIII CARs are genetically engineered T cells that have been 
taken from patients with WHO Grade IV malignant glioma ex vivo to express a CAR recognizing the GBM 
tumor-specific antigen EGFRvIII, which is expressed on a subset of GBMs but not in normal human 
tissues with the aim of mediating regression of their tumors. 
10.2 
Packaging and Labeling 
 
 
 
 
 
 
 
 
 
10.3 
Supply, Receipt, and Storage 
The drug will be manufactured in the Molecular Products and Cellular Therapies (MPACT) cGMP 
facility.  All drug accountability, transfers, receipts, and disposals are recorded in the Duke Nautilus 
system. 
10.4 
Dispensing and Preparation 
10.4.1 
EGFRvIII CARs 
The CAR product will be held at room temperature (21-25OC) until the gram stain and endotoxin 
tests are completed.  The cells are delivered from the MPACT facility directly to the bedside under the 
supervision of the research nurse or her designee. EGFRvIII CARs will be administered according to 
protocol.  The patient’s name, Study ID, DOB, and Duke history number will be verified by two separate 
people prior to the CAR administration as is standard Duke BMT transfusion procedure. For 111In labeling 
a portion of the cells will be labeled with 111In and then mixed with unlabeled cells to achieve the final 
dose.  The unlabeled cells will be held at 37OC until final mixing.   The radiolabeled EGFRvIII CARs will 
be dispensed from the Duke Radiopharmacy. 
10.4.2 
Release Criteria for EGFRvIII CARs 
Tests performed on the final product: 
 
Table 5. Release Criteria for EGFRvIII CARs 
Test 
Method 
Limits 
Result 
Initials/Date 
Pt. Name/MRN 
EGFRvIII CAR T Cells 
Dose  
Administration Date 
For Autologous use only 
Not evaluated for infectious substances 
Caution New Drug Limited by Federal Law to Investigational Use  

 
 
 
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Cell viability1 
Trypan 
blue 
exclusion 
>70% 
 
 
Total viable cell 
number1 
Visual microscopic 
count 
>1 x 107 
 
 
Tumor reactivity2 
 ƴ-IFN release vs. 
EGFRvIII antigen 
>200 pg/ml 
 
 
CAR expression2 
FACS analysis of 
the 
transduced 
cells 
PBL >10% 
 
 
Microbiological 
studies 
Gram stain1,3 
No micro-
organisms seen 
 
 
 
Aerobic culture3,4 
No growth 
 
 
 
Fungal culture3,4 
No growth 
 
 
 
Anaerobic 
culture3,4 
No growth 
 
 
 
Mycoplasma test5 
Negative 
 
 
Endotoxin 
Limulus assay1 
<5 E.U./Kg 
 
 
RCR 
S+L-Assay4 
 RCR-PCR6 
Negative 
 
 
 
In the event of a positive sterility test, after the product has been infused, the Principal Investigator 
or his or her designate will notify the treating physician and the patient.  The FDA and IRB will be notified 
within 15 calendar days.  The patient will be asked to be evaluated by a physician within 24 hours.  If the 
patient has or develops a temperature >38.5 oC or clinical evidence of infection at the injection site 
(erythema or edema) or systemically, the patient will have samples of from the injection sites, along with 
blood, urine and sputum (if possible) sent for bacterial, fungal, culture and sensitivity testing and will be 
treated expectantly with antibiotics based on the sensitivities of the organisms identified from the 
immunization product and an independent infectious disease consultation will be obtained to guide further 
therapy.  Any remaining lymphocytes will be sent for bacterial, and fungal, culture and sensitivity testing 
and endotoxin testing. 
10.4.3 
111In-labeled T cells 
CARs are washed three times with PBS, assessed for viability, and counted.  CARs are re-
suspended in PBS.  4-6x108 cells will be labeled with 500 micro Ci of 111In. The cells will be washed and 
mixed with cold CARs to achieve the desired cell dose containing 500 micro Ci of 111In. The cell 
concentration is adjusted to 2.5 x107 cells per mL and resuspended in preservative free saline with 1% 
human serum albumin (Baxter Healthcare Corp., US License No 140 NDC0944-0490-0L) and placed into 
the appropriate size syringe for infusion.   
From this final preparation, a sample of cells will be sent for Gram stain and endotoxin testing prior 
to administration.  111In-labeled T cells will not be given until endotoxin testing has been passed (< 5.0 
E.U./Kg and the Gram stain has been found to be negative.  An aliquot of cells will also be sent for aerobic 
and anaerobic bacterial cultures (1 x 106 PBLs) and fungal cultures (1 x 106 PBLs). The final formulated 
                                            
1 Performed on sample of the final product immediately prior to infusion.  Results are available at the time of 
infusion. 
2 Performed 2-10 days post transduction.  Results are available at the time of infusion. 
3 Performed 2-4 days prior to infusion.  Results are available at the time of infusion but may not be definitive. 
BactAlert bottles will be incubated for 14 days.   
4 Sample collected from the final product prior to infusion.  Results will not be available before cells are 
infused into the patient. BactAlert bottles will be incubated for 14 days.   
5 Performed 2-10 days prior to infusion.  Results are available at the time of infusion. 
6 Performed on sample approximately 1-4 days prior to infusion.  Results are available at the time of infusion. 

 
 
 
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T cells will be stored at 2-8°C for up to 6 hours. After this time, the material will be disposed of if not given 
to the patient. 
In the event of a positive sterility test, the Principal Investigator or his or her designate will notify the 
treating physician and the patient.  The FDA and IRB will be notified within 15 calendar days.  The patient 
will be asked to be evaluated by a physician within 24 hours.  If the patient has or develops a temperature 
>38.5 oC or clinical evidence of infection at the injection site (erythema or edema) or systemically, the 
patient will have samples of from the injection sites, along with blood, urine and sputum (if possible) sent 
for bacterial, fungal, culture and sensitivity testing and will be treated expectantly with antibiotics based 
on the sensitivities of the organisms identified from the immunization product and an independent 
infectious disease consultation will be obtained to guide further therapy.  Any remaining lymphocytes will 
be sent for bacterial, and fungal, culture and sensitivity testing and endotoxin testing. 
10.5 
Compliance and Accountability 
The EGFRvIII CAR will be signed out and distributed by the BTIP laboratory manager.  The Duke 
BTIP personnel use safe medication practices to reduce the risk of medication errors and adverse events 
when setting up study drug procedures. Investigational drugs are stored separately from other drugs in 
an area of limited access and in accordance with special storage requirements.  They are clearly labeled 
with the identity of the study drug and other control numbers. Investigational drugs are accounted for 
using the Duke Nautilus System. All drug transfers, receipts, and disposal are recorded in Nautilus. 
10.6 
Disposal and Destruction 
 
Radioactively labeled drug will be disposed of according to standard radiation safety practices.  
Patient rooms and the patient themselves will be monitored before discharge by a member of the Duke 
Radiation safety committee.  Un-labeled cells will be autoclaved. 
11 SUBJECT ELIGIBILITY  
Inclusion Criteria 
1. Age 18-80 years of age 
2. Histopathologically proven newly-diagnosed, supratentorial glioblastoma or gliosarcoma (WHO 
Grade IV). 
3. KPS score ≥ 70. 
4. The presence of the target antigen, EGFRvIII, must be identified on tumor tissue by IHC or PCR. 
5. Hematology: 
 
ANC ≥ 1000/mm3 without the support of filgrastim 
 
Platelet count ≥ 100,000/mm3 
 
Hemoglobin ≥ 8.0 g/dl (eligibility level for hemoglobin may be reached by transfusion). 
6. Chemistry: 
 
ALT/AST ≤ 2.5 times the upper limit of normal 
 
Creatinine ≤ 1.6 mg/dl 
 
Total bilirubin ≤ 1.5 mg/dl. 
 
Exclusion Criteria 
1. Patients who are pregnant, breast-feeding, or unwilling to practice an effective method of birth control. 
2. Patients with known potentially anaphylactic allergic reactions to gadolinium-DTPA. 
3. Patients who cannot undergo MRI or SPECT for any reason including due to obesity or to having 
certain metal in their bodies (specifically pacemakers, infusion pumps, metal aneurysm clips, metal 
prostheses, joints, rods, or plates). 
4. Patients with evidence of tumor in the brainstem, cerebellum, or spinal cord, or with evidence of 
leptomeningeal disease. 
5. Active infection requiring treatment or an unexplained febrile (> 101.5o F) illness. 
6. Known autoimmune disease, immunosuppressive disease or human immunodeficiency virus 
infection (i.e., known HIV or Hepatitis C). 
7. Patients with unstable or severe intercurrent medical conditions such as severe heart or lung disease. 

 
 
 
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8. Patients with previous history of radiosurgery, brachytherapy, gliadel implantation, or radiolabeled 
monoclonal antibodies. 
9. Prior antitumor therapy for glioma (other than steroids). 
10. Allergic to TMZ.   
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
In brief, patients will be screened and evaluated for study entry (please see Table 6).  The screening 
examination must include a pathology report confirming the EGFRvIII+ WHO grade IV malignant glioma 
diagnosis.  Blood tests per Table 6 will be drawn as well as the post-surgical MRI.  The baseline physical and 
neurologic examination with KPS score will be performed and documented by the neuro-oncology team and 
verified by the study team during the Duke Brain Tumor Center clinic visit (of note, the standard of care clinic 
visit may be used to fulfill the initial screening visit requirement). Patients will be offered treatment options 
during this BTC clinic visit.   
Once the consent has been signed, patients will be scheduled to undergo a leukapheresis in order to 
obtain T cells for infusion (please see Table 6). Patients will then receive approximately 6 weeks of standard 
of care RT and concurrent TMZ at a standard targeted dose of 75 mg/m2/d. Patients whose blood counts do 
not meet eligibility within 6 weeks of completing standard of care RT and concurrent TMZ will be considered 
a screen failure.  Patients who appear to have progressive disease following radiation may actually have 
pseudoprogression due to the radiation and will be allowed to continue to cycle 1 of DI TMZ in order to not be 
excluded from the study prematurely, at the discretion of the treating physician. If the patient is determined to 
have progressive disease, the patient may be eligible for the INTERCEPT study (INTracerebral EGFR-vIII 
Chimeric Antigen Receptor Gene-Modified T Cells for PaTients with Recurrent GBM, Pro00083828).  If the 
patient is determined to be eligible for the INTERCEPT study (Pro00083828), the cells extracted from 
leukapheresis while they were on this study, ExCeL, will be used to create the CAR T cells for administration 
in the INTERCEPT study.      
Patients without definitive disease progression who remain eligible will then receive up to 3 cycles of DI 
TMZ at 50-100 mg/m2/day.  Each full cycle of DI TMZ is 50-100 mg/m2/day for 21 days every 28 days, at the 
discretion of the treating physician.  The purpose of the DI TMZ cycles is to achieve grade 3 lymphopenia. If 
the patient is able to tolerate the TMZ, they will receive 3 full cycles of DI TMZ prior to receiving the CAR T 
cells.  If the patient is unable to complete the 3 cycles of DI TMZ, but has achieved grade 3 lymphopenia, they 
may receive CAR treatment at the discretion of the study PI and/or treating oncologist. If the patient’s grade 
3 lymphopenia subsides during the ~2 weeks it takes to produce the CAR T cells and before their scheduled 
receipt of CAR T cells, the treating physician has the discretion to re-initiate TMZ to re-establish grade 3 
lymphopenia, at the discretion of the treating physician.  If the patient is unable to tolerate the 3 full cycles of 
TMZ and does not reach grade 3 lymphopenia, or if the CAR-specific T cells do not meet release criteria, the 
patient will be withdrawn and replaced.   
Two weeks prior to CAR T cell administration, the patient must be on a stable or decreasing steroid dose 
(≤ 4 mg/day).  If patients are decreasing steroid use, once they are at 2 mg/day, they may be supplemented 
with hydrocortisone, at the discretion of the treating physician.  
The patient will be scheduled for CAR T cell (or CAR T cell labeled with 111In) infusion at least 48 hours 
(+72 hours) after their last dose of DI TMZ. In the expanded cohort group, 3 days of whole body planar imaging 
and SPECT/CT imaging will be done following infusion on days 1, 2, and 3.  Note, if there is no signal on 
whole body planar imaging, the radiologist may decide not to perform SPECT/CT. Blood for immune 
monitoring will be taken on all patients at leukapheresis, pre-infusion, 1, 5, 10, and 28 days following infusion, 
and at 3 and 6 months post-infusion, and then yearly (until progression, death, or lost to contact). The return 
visits for immune monitoring at the 3, 6, and yearly will coincide with standard of care clinic visits. Blood for 
RCR PCR will be taken prior to cell infusion and at 3 and 6 months, and at one year as required by the FDA 
during routine clinic visits. Lastly, blood for evaluation of CRS will be drawn prior to cell infusion, 1 and 4 hours 
after infusion, and on days 1, 2, 5, 10, and at 28 days. Measurements for CRS include IL-2, IL-6, TNFα, IFN, 
GM-CSF, and CRP.  
Blood samples will be archived annually thereafter if all previous testing has been negative.  
Patients will return to clinic, as per standard of care, approximately 28 days after EGFRvIII CAR T cell 
infusion to be evaluated radiographically and clinically for cycles of standard of care 5-day TMZ at 150-200 
mg/m2/day per the treating oncologist.  This will result in a 28-day pause between the last cycle of TMZ 

 
 
 
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administered prior to EGFRvIII CARs and the first cycle administered after study drug infusion in order to 
decrease the chance of study drug washout from TMZ. 

 
 
 
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Table 6. Screening and On-Study Tests and Procedures 
 
Pre-
XRT/TMZ 
Screening 
Leukapheresis 
Post- 
XRT/TMZ 
screening 
DI TMZ 
Cycle 1 
Post DI 
TMZ 
Cycle 1 
 
DI TMZ 
Cycle 21 
DI TMZ 
Cycle 31 
Pre-
CAR T 
Cell 
Infusion 
CAR T 
Cell 
Infusion
2 
5-day 
TMZ 
Cycles 
4-12 
Progression 
 
 
 
Main study 
consent 
X 
 
 
 
 
 
 
 
 
 
 
History and 
Physical 
X 
 
X 
 
X3 
 
 
X4 
 
X5 
 
Neurological 
Exam 
X 
 
X 
 
X3 
 
 
X4 
 
 X5 
 
KPS 
X 
 
X 
 
X3 
 
 
X4 
 
X5 
 
Curran 
 
 
X 
 
 
 
 
 
 
 
 
CBC w/ 
differential  
X 
X 
X 
X6 
X3 
X6,3 
X6 
X4 
 
X7 
 
CMP  
X 
X 
X 
 
X3 
X3 
 
X4 
 
X8 
 
Beta HCG  
X 
X 
 
 
 
 
 
 
 
 
 
CRS 
 
 
 
 
 
 
 
 
X9 
X10 
 
Immune 
Monitoring 
 
X11 
 
 
 
 
 
 
X12 
X13 
X13 
CAR T Cells 
 
 
 
 
 
 
 
 
X14 
 
 
DI Temodar  
 
 
 
X15 
 
X15 
X15 
 
 
 
 
5-day 
Temodar 
 
 
 
 
 
 
 
 
 
X16 
 
Vitals 
X 
 
X 
 
X3 
 
 
X3 
X17 
X5 
 
MRI 
X 
 
X 
 
X3 
 
 
X3 
 
X5 
 
Pathology 
testing 
X 
 
 
 
 
 
 
 
 
 
X 
Adverse 
Events 
 
 
 
 
 
 
 
Ongoing 
Con Meds 
 
 
 
 
 
 
 
Ongoing 
                                            
1 Some patients may not receive DI TMZ cycle 3 or cycles 2 and 3, please see Section 9.1 for details about DI TMZ requirements. 
2 48 hours (+72 hours) after last dose of DI TMZ 
3 ± 7 days from the last dose of TMZ  
4 Within 2 days prior to CAR T Cell infusion. 
5 Prior to Cycle 4 and then prior to every other Cycle (Cycle 6, 8, etc), at the discretion of the treating physician. 
6 Day 14 (± 3 Days) of the TMZ cycle. 
7 Day 21 (± 3 days) and day 28 (± 7 days) of each 5-day TMZ cycle 
8 Day 28 (± 7 days) of each 5-day TMZ cycle 
9 Prior to CAR T Cell Infusion, and at 1 and 4 hours and days 1, 2, 5, and 10 post CAR T Cell infusion.  Draw 1 red top and 1 purple 
top (3 mL each) and 1 gold top at each time point. 
10 28 days post CAR T Cell Infusion.  Draw 1 red top and 1 purple top (3mL each) and 1 gold top. 
11 2 red tops (10 mL each). 
12 Prior to CAR T Cell Infusion on day 22 (+3 day window), and at 1, 5, and 10 days after the infusion (9 yellow tops (8.5mL each) 
and 1 red top (10mL) at each time point. 
13 1, 3, 6, and 12 months post CAR T Cell infusion and then yearly and at progression.  Draw 9 yellow tops (8.5mL each) and 1 red 
top (10mL) at each time point.  Draws will coincide with standard of care return clinic visits. 
14 At least 48 hours after last dose of DI TMZ.  Patients will be pre-medicated 30-60 minutes prior to infusion with acetaminophen 
(650 mg po) and Benadryl (25 mg by mouth) to reduce infusion-related reactions.  Patients must be on stable of decreasing steroid dose 
(≤ 4 mg/day) 2 weeks prior to CAR T cell infusion. 
15 Days 1-21 of a 28-day cycle, at the discretion of the treating physician. 
16 5 day TMZ to be taken on days 1-5 of a 28 day cycle.  The first cycle of 5-day TMZ will be initiated approximately 1 month after 
CAR T Cell infusion. 
17 Vital signs will be measured prior to the CAR T cell infusion and every 15 minutes for 1 hour post-infusion.  Measurements to be 
reported include temperature in Celsius or Fahrenheit, pulse (beats per minute), respiratory rate (breaths per minute) and blood pressure. 

 
 
 
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12.1 
Screening Examination 
A preliminary screening visit will take place prior to the patient undergoing leukapheresis and 
radiation.  In order to not have to repeat clinic visits on back to back days, the standard of care clinic 
visit can be used to fulfil the initial screening visit requirement.  Subject data to be collected at the initial 
screening examination includes a pathology report, MRI report, CMP and CBC with Differential (just 
prior to leukapheresis only), and a physical and neurologic examination with KPS score.  The patient 
will then return for the final screening visit after radiation has been completed.  Subject data to be 
collected at the Screening Examination includes CMP and CBC with Differential, a physical and 
neurologic examination with KPS score, and baseline Curran Status (Curran only performed at post 
XRT screening).  The blood test results from the post radiation screening visit will be the results used 
to confirm final eligibility.  If these test results do not meet the eligibility criteria, the patient can recheck 
these tests at a local lab up until 6 weeks post radiation in order to be considered eligible for the study.  
Patients whose lab results do not meet the eligibility criteria by 6 weeks post radiation will be considered 
a screen failure.  
Patients with newly-diagnosed EGFRvIII-positive WHO grade IV malignant glioma will be imaged 
by MRI for baseline measurements and to assess progression prior to receiving EGFRvIII CARs; 
although the purpose of this study is not to detect tumor responses, any evidence of tumor response 
will be determined by serial measures of the product of the 2 largest cross-sectional diameters.74,75  For 
patients with ≥ 25% increase in the product of the largest perpendicular diameters of any enhancing 
lesion or any new enhancing tumor on MRI scans or unequivocal and significant worsening of non-
measurable tumor that is not attributable to co-morbid event or concurrent medication, progression will 
be defined. Patients may also be classified as progressive disease with significant neurologic decline 
felt to be due to underlying tumor and not attributable to a co-morbid event or concurrent medication 
regardless of MRI findings. Note – If there has been a major reduction in steroid dosage in the interval 
and the patient is felt to be clinically stable or improved, the proper assessment may be “indeterminate” 
and the therapy could be continued pending the next evaluation. The RANO criteria76 will be used for 
overall assessment of tumor response and pseudoprogression (PRTBTC Imaging SOP and FORM is 
available upon request). Tumor progression will need to be documented histologically, unless there are 
clinical contraindications, to exclude inflammatory responses presenting as radiographic or clinical 
changes, which could indicate potentially toxic or therapeutic responses and not tumor progression. If 
tissue is obtained, it will be used to confirm tumor progression histologically and to assess immunologic 
cell infiltration and antigen escape. Upon progression, patients may be treated on other therapies as 
directed by the treating neuro-oncologist.  
12.2 
Treatment Period 
Patients that have consented onto this study must first undergo a 2 to 4-hour leukapheresis to 
obtain T cells. After completion of SOC chemoradiotherapy, the treatment period will commence with 
patients receiving the single infusion of EGFRvIII CARs.  The expanded cohort will receive the 111In-
labeled EGFRvIII CARs.  
Infusions will be administered in the BMT Clinic, monitored by an RN during and for 1 hour 
following the procedure. If a patient develops signs of infection or is febrile prior to the infusion, 
treatment will be postponed pending clinical work-up. The T cell product will be delivered from the 
MPACT facility directly to the bedside under the supervision of the research nurse or her designate. 
For subjects receiving CAR T cells in the expanded cohort, the T cell product that will be labeled with 
111In will be delivered from the Radiopharmacy.  T cells will be administered according to protocol. The 
patient’s name, Study ID, DOB, and MRN will be double verified prior to infusion as is standard Duke 
BMT transfusion procedure. 
The patient will be pre-medicated 30-60 minutes prior to infusion with acetaminophen (650 mg 
po) and Benadryl (25 mg by mouth) to reduce infusion-related reactions. An intravenous catheter (18-
20 G) will be placed for the procedure. The EGFRvIII CARs will be delivered to the patient’s bedside in 
a syringe and applied according to SOP BTC-00069444.01, or the most recent version of the SOP. 
Vital signs will be assessed prior to and every 15 minutes for 1 hour following the infusion. The infusion 

 
 
 
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will be documented as well as any adverse experiences with infusion. All Duke transfusion and cell 
infusion policies will be followed. 
Prior to infusion, approximately 65 mLs of blood for IM, RCR PCR assay, and CRS will be drawn 
(9 yellow top, 1 (10 mL) red top, 1 purple top, and 1 (3mL) red top). 
12.3 
CRS Management Plan 
Grade 1 toxicity  
1. 
Will implement symptomatic management only. 
Grade 2 toxicity  
1. 
Monitoring hypotension: we will have clearly established a baseline blood pressure, pre-
infusion of CAR T cells, for each patient; if hypotension develops give fluids or 1 low-dose 
vasopressor.  
2. 
Respiratory symptoms: will be managed by oxygen.   
Care for the patients with grade 2 toxicity will be individually tailored, depending on the patient 
age and medical co-morbidity, and therefore clinical judgement will be crucial prior to use of 
immunosuppression for Grade 2 toxicity.  Patients with Grade 2 toxicity would be monitored in 
step-down units, including very close cardiac monitoring.  
 
Grade 3 toxicity  
1. 
If grade 3 toxicity develops where fluid resuscitation and low dose vasopressor(s) are not 
sufficient to reverse the hypotension, or patients require increasing oxygen support or develop 
grade 3 organ toxicity, the patients will be transferred and monitored closely in intensive care 
unit. All patients with Grade 3 will receive immunosuppressive agents.  In severe cases 
Tocilizumab, antihuman IL-6R mAb, will be administered.  This drug is stocked in the Duke 
pharmacy.   
Grade 4  
1. 
All patients with immediate life threatening toxicity will be treated with full support including 
mechanical ventilation, immunosuppressive agents, Tocilizumab to prevent inflammatory 
cascade and with close comprehensive organ monitoring.  
12.4 
End of Treatment 
Once the EGFRvIII CARS infusion is complete, the treatment phase of the study will be over and 
the follow-up period will begin. 
12.5 
Follow-up Period 
Following the infusion, blood samples for IM (9 yellow, 1 red) will be drawn 1, 5, 10, and 28 days 
after the infusion, then 3 and 6 months, then yearly until progression (or death or lost to contact). 
Lymphocytes in patients’ peripheral blood will be analyzed by FACS and quantified for persistence. 
Isolated PBMCs will be stained with fluorescence-labeled antibodies against cell surface and 
intracellular cytokine markers to determine function including staining with our novel EGFRvIII tetramer 
reagent.   Whole planar imaging, followed by SPECT/CT, if appropriate, will be done on days 1, 2, and 
3 following infusions to determine the IC localization of 111In-labeled EGFRvIII CARs.  Quantification of 
radioactivity will be assessed by previously published methods that calculate counts from reconstructed 
images based on the percentage of injected dose. Blood will be taken for RCR PCR at 3, 6 and 12 
months and blood for evaluation of CRS (1 red, 1 purple top – 3mLs each) will be drawn pre-infusion, 
1 and 4 hours after CARs infusion, and on days 1, 2, 5, 10, and 28. 

 
 
 
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Patients treated with EGFRvIII CARs will be followed for survival and data recorded by the study 
coordinator/research nurse until death or for 15 years (whichever comes first). 
12.6 
End of Study 
Rationale for taking a patient off study will be documented (see section 12.7.1). All patients will 
be followed for survival, subsequent therapies, and radiographic response and this information will be 
recorded by the study team.  
12.7 
Early Withdrawal of Subject(s) 
12.7.1 Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time.  
Subjects may be withdrawn by the PI prior to infusion of EGFRvIII CARS for the following: 
 
Progressive disease following any of the cycles of DI TMZ, at the discretion of the 
treating physician.  *Note that patients who are removed from study due to progressive 
disease may be eligible for the INTERCEPT study (Pro00083828), this would result in the 
autologous T cells from the leukapheresis in this study being transferred to the 
INTERCEPT study (Pro00083828). 
The PI may also withdraw a subject from the study at any time based on his/her discretion. 
Reasons for PI-initiated withdrawal may include, but are not limited to the following: 
 
Pregnancy. 
 
Upon request of the subject. 
 
If, in the investigator’s medical judgment, further participation would be injurious to the 
subject’s health or wellbeing. 
 
Protocol deviation. 
 
Administrative reasons, such as a major violation of the clinical trial protocol. 
 
Non-compliance of the subject. 
12.7.2 Follow-up Requirements for Early Withdrawal 
Subjects who have received EGFRvIII CAR T cell infusion 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/research nurse until death or are lost to follow up.  This follow-up may include the collection 
of survival status and doses, dates, and final radiographic responses associated with future treatment 
regimens. 
12.7.3 Replacement of Early Withdrawal(s) 
Subjects who do not receive EGFRvIII CAR treatment will be replaced. Subjects who terminate 
infusion prematurely or are lost to follow-up before the completion of 4 weeks of follow-up observation 
and do not experience dose-limiting toxicity will also be replaced. 
12.8 
Study Assessments 
12.8.1 Medical History 
Medical history will be obtained from the Duke EPIC system and from the subject and/or family 
at the screening visit and reviewed at each study/clinic visit. This data will include the following: 
 
All past medical and surgical history 
 
Current medications  
 
Changes in physical or neurologic symptoms 
 
Any adverse events  

 
 
 
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12.8.2 Physical Exam 
Vital signs and physical and neurologic examinations will be assessed and recorded along with 
a KPS score prior to enrollment and at each study/clinic visit. 
12.8.3 Correlative Assessments 
To assess if EGFRvIII CARS transferred into patients with WHO Grade IV malignant glioma 
expand and remain functional in the periphery, 2 red top tubes will be drawn at the leukapheresis visit 
for immune monitoring.   Fifty mLs of whole blood in 6 ACD yellow top tubes and 1 red top tube (9 mLs) 
for immune monitoring will be drawn pre-infusion and 1, 5, 10, and 28 days after CARs treatment, then 
3 and 6 months post-infusion, then yearly until progression, death or lost to contact. Patient PBMCs will 
be obtained from whole blood by purification using centrifugation on a Ficoll cushion within 8 hours of 
draw using a standard SOP. Cells will be frozen to -70 ˚C at -1 ˚C/min and stored in liquid nitrogen. To 
quantify the kinetics of CARs in blood, the expression of EGFRvIII CARs in PBMCs recovered from 
patients will be determined using the novel EGFRvIII peptide tetramer reagent developed by our 
laboratory. The number of CARs will be compared to total numbers of CD4+ and CD8+ lymphocytes 
using FACs; Ki67 staining will be used to assess cell proliferation. Cell phenotype and function will be 
assessed using a validated polyfunctional T cell assay in collaboration with Kent Weinhold, Ph.D., using 
the following markers: anchor gates (CD3 AmCyan, CD4 PerCP-Cy5.5 & CD8 APC-Cy7), a multiplexed 
exclusion channel (CD14 Cascade Blue+ CD19 Cascade Blue+ vAmine all collected in a single 
channel), maturational markers (CD27 APC, CD57 FITC & CD45RO ECD) to identify naïve (CD45RO-
CD27+), central memory (CD45RO+CD27+), effector memory (CD45RO+CD27-), effector 
(CD45RO+CD57+), and terminal effector (CD45RO-CD57+) CD4+ and CD8+ lymphocytes, and 
functional markers (IL-2 PE, TNF-a Alexa700, CD107a PE-Cy5.5 & IFN-g PE-Cy7). We will include the 
EGFRvIII peptide tetramer reagent in this assay to evaluate CAR-specific polyfunctionality using a 
fluorochrome not already in the panel. Samples from the expanded cohort will be analyzed after 
radioactivity has decayed. Polyfunctional analysis will also be done on the cells transferred to the 
patients.  
Also following infusion of 111In-labeled CARs in the expanded cohort, imaging on days 1, 2, and 
3 by SPECT/CT will be used to determine the IC localization of 111In-labeled EGFRvIII CARs to assess 
if EGFRvIII CARS traffic to the tumor site. Quantification of radioactivity will be assessed by previously 
published methods that calculate counts from reconstructed images based on the percentage of 
injected dose. 
For monitoring the RCR PCR assays to detect the GaLV envelop gene, the RCR will be 
performed using a validated assay prior to cell infusion and at 3 and 6 months, and at one year as 
required by the FDA. Blood samples will be archived annually thereafter if all previous testing has been 
negative. If a patient dies or develops neoplasms during this trial, efforts will be made to obtain a biopsy 
sample for RCR. If any post-treatment samples are positive, further analysis of the RCR and more 
extensive patient follow-up will be undertaken, in consultation with the FDA.  
Please see Figure 10. Correlative Studies Schemain Appendices. 
Lastly, blood for evaluation of CRS will be drawn prior to cell infusion, 1 and 4 hours after infusion, 
and on days 1, 2, 5, 10, and at 28 days. Measurements for CRS include IL-2, IL-6, TNFα, IFN, GM-
CSF, and CRP.  
13 SAFETY MONITORING AND REPORTING 
The PI is responsible for the identification and documentation of AEs and SAEs that may occur following 
treatment with EGFRvIII CARs, as defined below.  At each study visit, the PI or designee must assess, 
through non-suggestive inquiries of the subject or evaluation of study assessments, whether an AE or SAE 
has occurred. AEs and/or SAEs will be collected starting at the CAR T cell infusion visit and will be  recorded 
for baseline assessments.  Only Special Interest AEs related to leukapheresis, as described in Section 13.2 
will be recorded prior to the CAR T cell infusion.  Following EGFRvIII CAR infusion, all AEs and SAEs will 
be recorded by the study team(see Section 13.1.1 below). 

 
 
 
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13.1 
Adverse Events 
An AE is any untoward medical occurrence in a subject receiving EGFRvIII CARs and which 
does not necessarily have a causal relationship with this treatment.  For this protocol, the definition of 
AE also includes worsening of any pre-existing medical condition.  An AE can therefore be any 
unfavorable and unintended or worsening sign (including an abnormal laboratory finding), symptom, or 
disease temporally associated with the use of EGFRvIII CAR T cells, whether or not related to use of 
the EGFRvIII CAR T cells.  All CTCAE-defined AEs, including grade 1 laboratory events, are recorded 
and reported as required in 21CFR312. 
From the time of CAR T cell infusion through the End of Study visit (as defined in Section 12.6), 
as well as any Special Interest AEs related to leukapheresis (as defined in Section 13.2), all AEs must 
be recorded in the subject medical record and adverse events case report form.  However, for reporting 
purposes, only AEs that occur at the time of the EGFRvIII CAR T cell infusion or following the infusion 
will be submitted to the appropriate agencies as indicated. 
AEs will be assessed according to the CTCAE version 4.0.  If CTCAE grading does not exist 
for an AE, the severity of the AE will be graded as mild (1), moderate (2), severe (3), life-threatening 
(4), or fatal (5). 
Attribution of AEs will be indicated as follows: 
- 
Definite:  The AE is clearly related to the study drug 
- 
Probably:  The AE is likely related to the study drug 
- 
Possible:  The AE may be related to the study drug 
- 
Unlikely:  The AE is doubtfully related to the study drug 
- 
Unrelated:  The AE is clearly NOT related to the study drug 
13.1.1 Reporting of AEs 
A summary of all adverse events (not just those considered related to EGFRvIII CARs) that 
occur following treatment with EGFRvIII CARs will be kept which will categorize the event by 
organ system, relationship to treatment, its grade of severity, and resolution.  The PI will 
periodically review the collective adverse events with the intention of identifying any trends or 
patterns in toxicity.  If any such trends are identified, depending on their severity and frequency, 
a protocol amendment will be considered. 
13.2 
Adverse Events of Special Interest 
Adverse events will not be collected until the patient has received CAR T cells, unless they are 
considered an Adverse Event of Special Interest related to the leukapheresis procedure.  Only these 
special interest adverse events will be collected prior to CAR T cell infusion, all other events 
occurring prior to CAR T cell administration will not be recorded or monitored.   
Special Interest Adverse Events that may occur during the leukapheresis procedure include: 
 
Allergic Reaction 
 
Anaphylaxis 
 
Presyncope 
 
Syncope 
 
Vasovagal reaction 
Special Interest Adverse Events that may occur after the procedure, but may still be related to 
leukapheresis include: 
 
Vascular Access Complications 
 
Venous Injury 
13.3 
Serious Adverse Events 
An AE is considered “serious” if in the view of either the investigator or sponsor it leads to 
one of five specific sequelae: 

 
 
 
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- 
Death 
- 
Life-threatening adverse event 
- 
Inpatient hospitalization of 24 hours or longer or prolongation of existing hospitalization 
- 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions 
- 
Constitutes a congenital anomaly or birth defect 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment,  they 
may jeopardize the patient or subject and may require medical or surgical intervention to prevent one 
of the outcomes listed in this definition. 
13.3.1 Reporting of SAEs 
All SAEs that occur following treatment with EGFRvIII CARs should be reported 
immediately to Dr. David Ashley (Pager: 919-206-3433), Dr. Dan Landi (Pager: 919-206-3405), 
or their designee (919-684-5301) and to the FDA and Duke University Medical Center IRB.  Fatal 
or life-threatening, unexpected adverse events will be reported to the FDA by telephone, 
facsimile, or in writing as soon as possible, but no later than 7 calendar days after first knowledge 
by the sponsor followed by as complete a report as possible within 8 additional calendar days.  
Serious, unexpected adverse events that are not fatal or life-threatening will be reported to the 
FDA by telephone, facsimile, or in writing as soon as possible, but no later than 15 calendar days 
after first knowledge by the sponsor. 
All adverse events that are considered serious, unanticipated, and related or possibly 
related to EGRvIII CARs (as defined by 21CRF312.32[a]) will be reported to the Duke University 
Medical Center IRB and the FDA using the appropriate SAE report form. At the time of the annual 
progress report to the Duke University Medical Center IRB and the FDA, a summary of the overall 
toxicity experience will be provided. 
13.4 
Safety Oversight Committee (SOCom) 
The Duke Cancer Institute SOCom is responsible for annual data and safety monitoring of DUHS 
sponsor-investigator phase I and II, therapeutic interventional studies that do not have an independent 
DSMB. The primary focus of the SOCom is review of safety data, toxicities and new information that 
may affect subject safety or efficacy. Annual safety reviews includes but may not be limited to review 
of safety data, enrollment status, stopping rules if applicable, accrual, toxicities, reference literature, 
and interim analyses as provided by the sponsor-investigator. The SOCom in concert with the DCI 
Monitoring Team (see Monitoring section for Monitoring Team description) oversees the conduct of 
DUHS cancer-related, sponsor-investigator therapeutic intervention and prevention intervention studies 
that do not have an external monitoring plan, ensuring subject safety and that the protocol is conducted, 
recorded and reported in accordance with the protocol, SOPs, GCP, and applicable regulatory 
requirements. 
14 QUALITY CONTROL AND QUALITY ASSURANCE 
14.1 
Monitoring 
The DCI Monitoring Team will conduct monitoring visits to ensure subject safety and to ensure 
that the protocol is conducted, recorded, and reported in accordance with the protocol, standard 
operating procedures, good clinical practice, and applicable regulatory requirements.  As specified in 
the DCI Data and Safety Monitoring Plan, the DCI Monitoring Team will conduct routine monitoring 
after the third subject is enrolled, followed by annual monitoring of 1 – 3 subjects until the study is 
closed to enrollment and subjects are no longer receiving study interventions that are more than 
minimal risk.   
Additional monitoring may be prompted by findings from monitoring visits, unexpected frequency 
of serious and/or unexpected toxicities, or other concerns and may be initiated upon request of DUHS 

 
 
 
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and DCI leadership, the DCI Cancer Protocol Committee, the SOCom, the sponsor, the Principal 
Investigator, or the IRB.  All study documents must be made available upon request to the DCI 
Monitoring Team and other authorized regulatory authorities, including but not limited to the National 
Institute of Health, National Cancer Institute, and the FDA.  Every reasonable effort will be made to 
maintain confidentiality during study monitoring. 
14.2 
Audits 
The Duke School of Medicine OARC office may conduct audits to evaluate compliance with the 
protocol and the principles of GCP.  The PI agrees to allow the OARC auditor(s) direct access to all 
relevant documents and to allocate his/her time and the time of the study team to the OARC auditor(s) 
in order to discuss findings and any relevant issues. 
OARC audits are designed to protect the rights and well-being of human research subjects. 
OARC audits may be routine or directed (for cause). Routine audits are selected based upon risk 
metrics generally geared towards high subject enrollment, studies with limited oversight or monitoring, 
Investigator initiated Investigational Drugs or Devices, federally-funded studies, high degree of risk 
(based upon adverse events, type of study, or vulnerable populations), Phase I studies, or studies that 
involve Medicare populations. Directed audits occur at the directive of the IRB or an authorized 
Institutional Official. 
OARC audits examine research studies/clinical trials methodology, processes and systems to 
assess whether the research is conducted according to the protocol approved by the DUHS IRB. The 
primary purpose of the audit/review is to verify that the standards for safety of human subjects in clinical 
trials and the quality of data produced by the clinical trial research are met. The audit/review will serve 
as a quality assurance measure, internal to the institution. Additional goals of such audits are to detect 
both random and systemic errors occurring during the conduct of clinical research and to emphasize 
“best practices” in the research/clinical trials environment. 
14.3 
Data Management and Processing 
14.3.1 Study Documentation 
Study documentation includes but is not limited to source documents, case report forms, 
monitoring logs, appointment schedules, study team correspondence with sponsors or regulatory 
bodies/committees, and regulatory documents that can be found in the DCI-mandated “Regulatory 
Binder”, which includes but is not limited to signed protocol and amendments, approved and signed 
informed consent forms, FDA Form 1572, CAP and CLIA laboratory certifications, and clinical supplies 
receipts and distribution records. 
Source documents are original records that contain source data, which is all information in 
original records of clinical findings, observations, or other activities in a clinical trial necessary for the 
reconstruction and evaluation of the trial. Source documents include but are not limited to hospital 
records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation 
checklists, pharmacy dispensing records, recorded data from automated instruments, copies or 
transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, 
microfilm or magnetic media, x-rays, subject files, and records kept at the pharmacy, at the laboratories 
and at medico-technical departments involved in the clinical trial. When possible, the original record 
should be retained as the source document. However, a photocopy is acceptable provided that it is a 
clear, legible, and an exact duplication of the original document. 
14.3.2 Case Report Forms (CRFs) 
The subject’s medical records and CRFs will be the primary source document for the study. This 
data will be transferred to the electronic database. Only the PI, key personnel, the Radiolabeled 
Pharmacy personnel, and the PRTBTC data manager are permitted to make entries, changes, or 
corrections in the source documents or database. 

 
 
 
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Errors on the source documents will be crossed out with a single line, and this line will not obscure 
the original entry. Changes or corrections will be dated, initialed, and explained (if necessary). 
Electronic database changes will be tracked automatically. The PI or authorized key personnel will 
maintain a record of the changes and corrections. 
For electronic CRFs, an audit trail will be maintained by the electronic CRF management system. 
14.3.3 Data Management Procedures and Data Verification 
The DCI IS Shared Resource has a track record of developing Title 21 CFR Part 11 compliant 
databases for cancer clinical trials. IS also has extensive expertise in database quality assurance, data 
standards, and use of caBIG tools to support cancer researchers.  
Data queries will be generated automatically by the eCRF system. These data queries signify the 
presence of data inconsistencies. The study and data management team will cross-reference the data 
to verify accuracy. Missing or implausible data will be highlighted for the PI requiring appropriate 
responses (i.e., confirmation of data, correction of data, completion or confirmation that data is not 
available, etc.). 
The database will be reviewed and discussed prior to database closure, and will be closed only 
after resolution of all remaining queries. An audit trail will be kept of all subsequent changes to the data. 
14.3.4 Coding 
All medical terms will be coded using CTCAE (version 4). 
14.3.5 Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following activities: 
- 
Data clarification and/or resolution 
- 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
- 
Review of site study records for completeness 
- 
Shipment of all remaining laboratory samples to the designated laboratories 
 
 

 
 
 
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15 STATISTICAL METHODS AND DATA ANALYSIS 
 
All statistical analysis will be performed under the direction of the statistician designated in key 
personnel.  Any data analysis carried out independently by the investigator must be approved by the 
statistician before publication or presentation. 
15.1 
Analysis Sets 
EGFRvIII CAR Efficacy Analysis Set:  Efficacy analyses will focus on two groups of patients:  all 
patients, and the subgroup of patients who receive EGFRvIII CAR. 
Overall EGFRvIII CAR Safety Analysis Set:  For monitoring purposes and for reporting to 
appropriate agencies, safety analyses will include all patients who receive any EGFRvIII CAR 
treatment.   
MTD Analysis Set:  For the determination of MTD, patients without DLT who do not complete 4 
weeks of follow-up after CAR infusion or do not complete the intended infusion will be excluded from 
the Overall EGFRvIII CAR Safety Analysis Set to create the MTD Analysis Set. 
15.2 
Patient Demographics and Other Baseline Characteristics 
Patient clinical and socio-demographic characteristics will be summarized for all patients enrolled 
on this study, as well as the subgroup of patients who receive EGFRvIII CAR treatment.  Categorical 
descriptors will be summarized using frequency distributions; whereas, interval variables will be 
summarized using percentiles, as well as means and standard deviations. 
15.3 
Treatments 
The number of patients who receive EGFRvIII CAR treatment will be summarized by dose level. 
15.4 
Primary Objective 
Within this “3+3” phase I study, the primary objective is to determine the MTD of a single IV 
infusion of EGFRvIII CAR T cells in patients with newly-diagnosed WHO grade IV malignant glioma.  
Four dose levels will be considered based on transduced cells/kg:  #1:  4.5 x 106/kg, #2: 1.5 x 107/kg, 
#3: 4.5 x 107/kg, and #4: 1.5 x 108/kg.  The MTD is the highest dose level at which ≤1 or 6 patients 
experience dose-limiting toxicity during the 4 weeks after CAR infusion.  Section 9.1.2 provides 
information about what constitutes a dose-limiting toxicity.  Section 9.1.1 provides details concerning 
the determination of the MTD. 
15.5 
Secondary Objectives 
To determine the DLT of a single IV infusion EGFRvIII CAR T cells in patients with newly-
diagnosed WHO grade IV malignant glioma.  The proportion of patients who experience DLT within 
each dose level will be tabulated. 
15.6 
Exploratory Objectives 
1. To determine if CARs transferred into patients with WHO grade IV malignant glioma being treated 
with TMZ expand. 
2. To determine if EGFRvIII CARS persist and remain functional in the periphery. 
3. To assess whether EGFRvIII CARs traffic to the site of EGFRvIII expressing WHO grade IV 
malignant glioma. 
4. To describe the survival experience of patients with newly diagnosed, EGFRvIII-expressing WHO 
grade IV malignant glioma treated with EGFRvIII CAR T cells. 
5. To describe the progression free survival experience of EGFRvIII-expressing newly-diagnosed 
WHO grade IV malignant glioma patients treated with EGFRvIII CAR T cells. 
The analyses that address the exploratory correlative study objectives will be exploratory and 
descriptive. Based upon PBMCs collected from the peripheral blood, the median number of EGFRvIII 
CARs, the median percentage of CD4+ and CD8+ lymphocytes that are CARs, and the median level 
of KI67 will be estimated at each assessment time.  Assuming laboratory analyses demonstrate 

 
 
 
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CARs are present in the periphery, functional analyses may be conducted focusing on specific 
functions or time points.  Generalized linear models may be used to explore how these phenotypes 
change over time as a function of dose. 
Among patients within the expansion cohort who have been treated with 111In-labeled EGFRvIII 
CARs, the median proportion of injected 111In-labeled EGFRvIII CARs present in tumors will be 
estimated at each assessment point.  If the planar image provides no signal and the SPECT/CT is not 
conducted, we will impute the number of 111In-labeled EGFRvIII CARs present in tumors as 0. 
Two sets of efficacy analyses will be conducted.  One set of analysis will focus on all patients who 
enroll in the study whether or not EGFRvIII CAR treatment is administered, and the other set of 
analyses will focus on patients who receive EGFRvIII CAR treatment.  Among all patients, Kaplan-
Meier methods will graphically describe their survival from the time of study enrollment or histologic 
diagnosis.  Among patients who receive CAR treatment, Kaplan-Meier methods will also be used to 
describe the distribution of overall survival and progression-free survival. In these analyses, OS and 
PFS will be computed from the date that CAR treatment was initiated.  OS is defined as the time 
between the landmark (e.g. histologic diagnosis or initiation of CAR treatment) and death or date of last 
follow-up.  PFS is defined as the time between the landmark and first progression, death, or last follow-
up if the patient remains alive without disease progression.   
15.7 
Sample Size Calculation 
After completion of the expansion cohort, we anticipate that 18 patients (6 without 111In-labeled 
EGFRvIII CARs and 12 with) will have been treated at the MTD.  One motivation for treating additional 
patients at the MTD is to more precisely estimate the rate of DLT at the MTD.  Tabulated below as a 
function of the DLT rate is the half width of the associated 95% confidence interval: 
 
Table 7. Half Width of 95% Confidence Interval as a Function of the DLT Rate 
DLT Rate 
Half Width of 
95% 
Confidence 
Interval 
0.05 
0.025 
0.1 
0.032 
0.15 
0.039 
0.2 
0.044 
0.25 
0.047 
 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
16.1 
Regulatory and Ethical Compliance 
 
This protocol was designed and will be conducted and reported in accordance with the ICH 
Harmonized Tripartite Guidelines for Good Clinical Practice, the Declaration of Helsinki, and 
applicable federal, state, and local regulations. 
16.2 
DUHS Institutional Review Board and DCI Cancer Protocol 
Committee 
The protocol, informed consent form, advertising material, and additional protocol-related 
documents must be submitted to the DUHS IRB and DCI CPC for review.  The study may be initiated 
only after the Principal Investigator has received written and dated approval from the CPC and IRB. 
The Principal Investigator must submit and obtain approval from the IRB for all subsequent 
protocol amendments and changes to the informed consent form.  The CPC should be informed about 

 
 
 
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any protocol amendments that potentially affect research design or data analysis (i.e. amendments 
affecting subject population, inclusion/exclusion criteria, agent administration, statistical analysis, etc.). 
The Principal Investigator must obtain protocol re-approval from the IRB within 1 year of the most 
recent IRB approval.  The Principal Investigator must also obtain protocol re-approval from the CPC 
within 1 year of the most recent IRB approval, for as long as the protocol remains open to subject 
enrollment. 
16.3 
Informed Consent 
The informed consent form must be written in a manner that is understandable to the subject 
population.  Prior to its use, the informed consent form must be approved by the IRB. 
The Principal Investigator or authorized key personnel will discuss with the potential subject the 
purpose of the research, methods, potential risks and benefits, subject concerns, and other study-
related matters.  This discussion will occur in a location that ensures subject privacy and in a manner 
that minimizes the possibility of coercion.  Appropriate accommodations will be made available for 
potential subjects who cannot read or understand English or are visually impaired.  Potential subjects 
will have the opportunity to contact the Principal investigator or authorized key personnel with 
questions, and will be given as much time as needed to make an informed decision about participation 
in the study. 
Before conducting any study-specific procedures, the Principal Investigator must obtain written 
informed consent from the subject.  The original informed consent form will be stored with the subject’s 
study records, and a copy of the informed consent form will be provided to the subject.  The Principal 
Investigator is responsible for asking the subject whether the subject wishes to notify his/her primary 
care physician about participation in the study.  If the subject agrees to such notification, the Principal 
Investigator will inform the subject’s primary care physician about the subject’s participation in the 
clinical study. 
16.4 
Privacy, Confidentiality, and Data Storage 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s data 
will be maintained. RDSPs will be approved by the appropriate institutional Site Based Research group. 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects 
during the course of the study. Prospective participants will be consented in an exam room where it is 
just the research staff, the patient and his family, if desired. For all future visits, interactions with 
research staff (study doctor and study coordinators) regarding research activities will take place in a 
private exam room. All research related interactions with the participant will be conducted by qualified 
research staff who are directly involved in the conduct of the research study. 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock 
and key accessible only by the research staff. Subjects will be identified only by a unique study number 
and subject initials. Electronic records of subject data will be maintained using an Oracle Clinical 
database, which is housed by the DCI. Access to electronic databases will be limited to the Principal 
Investigator, key personnel, statisticians, the Radiolabeled Pharmacy personnel, and the BTC data 
manager. Data stored on portable memory devices will be de-identified. The security and viability of the 
IT infrastructure will be managed by the DCI and/or Duke Medicine.  
Upon completion of the study, research records will be archived and handled per DUHS HRPP 
policy.  
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
 
16.5 
Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and Safety 
Monitoring Plan. For a more detailed description of the DSMP for this protocol, refer to Sections 11 and 
12 and to Section 12.1 in eIRB for the Research Data Security Plan (available upon request). 

 
 
 
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16.6 
Protocol Amendments 
All protocol amendments must be initiated by the Principal Investigator and approved by the IRB 
prior to implementation.  IRB approval is not required for protocol changes that occur to protect the 
safety of a subject from an immediate hazard.  However, the Principal Investigator must inform the IRB 
and all other applicable regulatory agencies of such action immediately. 
Though not yet required, the CPC should be informed about any protocol amendments that 
potentially affect research design or data analysis (i.e. amendments affecting subject population, 
inclusion/exclusion criteria, agent administration, etc.). 
16.7 
Records Retention 
The Principal Investigator will maintain study-related records for the longer of a period of: 
- at least two years after the date on which a New Drug Application is approved by the FDA 
- at least two years after formal withdrawal of the IND associated with this protocol 
- at least six years after study completion (Duke policy). 
16.8 
Conflict of Interest 
The Principal Investigator and Sub-Investigators must comply with applicable federal, state, and 
local regulations regarding reporting and disclosure of conflict of interest.  Conflicts of interest may arise 
from situations in which financial or other personal considerations have the potential to compromise or 
bias professional judgment and objectivity.  Conflicts of interest include but are not limited to royalty or 
consulting fees, speaking honoraria, advisory board appointments, publicly-traded or privately-held 
equities, stock options, intellectual property, and gifts. 
The Duke University School of Medicine’s RIO reviews and manages research-related conflicts 
of interest.  The Principal Investigator and Sub-Investigators must report conflicts of interest annually 
and within 10 days of a change in status, and when applicable, must have a documented management 
plan that is developed in conjunction with the Duke RIO and approved by the IRB/IEC. 
16.9 
Registration Procedure 
After patients have been enrolled, protocol-specific information and subject registration will be 
entered into the Velos e-Research software system.  This system is managed by the School of 
Medicine. 
 
 
 

 
 
 
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18  APPENDICES 
18.1 
Research Summary 
 
Please see this Duke-specific document as a separate upload in the Duke eIRB (available upon 
request). 
 
 

 
 
 
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18.2 
Figures and Schemas (in order of reference in protocol) 
 
 
Figure 2. Highly Reactive Transferred Cells Traffic to Brain and Destroy Tumor Deposit in 
Melanoma Patients. Highly reactive transferred cells traffic to and destroy brain metastases in melanoma 
patients. Arrows in melanoma patient represent location of melanoma metastases. 
 
 
 
Figure 3. Design of 3rd Generation EGFRvIII CAR. Third-generation CARs incorporate at least two co-
stimulatory molecule endodomains, such as the endodomains of CD28 and 41BB. VH and VL were 
derived from the anti-EGFRvIII-specific clone 139. 
 
 

 
 
 
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Figure 4. EGFR Mutation 
 
 
 
 
Figure 5. CAR Transduced Murine T-Cells Specifically Recognize EGFRvIII Expressing GBM. 
EGFRvIII CARs were incubated with EGFRvIII-positive and negative GBM and submitted to IFN ELISpot 
assay. (*P<0.0022, Two-way ANOVA) 
 
 
GBM
GBMvIII
0
200
400
600
800
EGFRvIII-CAR
Control-CAR
Tumor alone
IFN SFU/1x106 cells
*

 
 
 
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Figure 6. Construction and Analysis of Anti- EGFRvIII CAR Vectors.Shown on the top of the figure is 
a diagram of the two CAR vector designs showing the relative locations of the scFv and T cell signaling 
domains.  In the lower half of the figure, CAR vectors based on human mAb 139 were used to engineer 
human T cells.  One week post-transduction, cells were stained with goat anti-human Fab and analyzed 
by FACS.  The shift in the histogram versus untransduced cells (UnTd) demonstrated successful gene 
transfer and expression of the CD28-CD3zeta (28Z) and CD8-CD28-41BB-CD3zeta (28BBZ) based 
vectors. 
 
 
 

 
 
 
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Figure 7. Specific Lysis of U87 EGFRvIII by Anti-EGFRvIII CAR Transduced T Cells.  
Anti‐EGFRvIII CAR transduced human PBL were co‐ cultured for 4h with indicated 51Cr labeled target tumor 
cell lines (parent U87, GFP, wild type EGFR, or EGFRvIII engineered). Specific lysis of tumor cells was 
measured at the given E:T ratio using the formula: [(specific release‐spontaneous  release)/(total release‐
spontaneous release)]. Specific lysis of untransduced (UnTd), control GFP vector (GFP), 139‐28Z CAR (vIII‐
28Z), and 139‐28BBZ (vIII‐BBZ) human PBL are plotted on the graph as indicated. 

 
 
 
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Figure 8. Specific Lysis of U251 EGFRvIII by Anti‐EGFRvIII CAR Transduced T Cells. 
Anti‐EGFRvIII CAR transduced human PBL were co‐cultured for 4h with indicated 51Cr labeled 
target tumor cell lines (parent U251, GFP, wild type EGFR, or EGFRvIII engineered). Specific 
lysis of tumor cells was measured at the given E:T ratio using the formula: [(specific release‐
spontaneous release)/(total release‐spontaneous release)]. Specific lysis of untransduced 
(UnTd), control GFP vector (GFP), anti‐ERBB2 CAR (ERBB2),139‐28Z CAR (vIII‐28Z), and 
139‐28BBZ (vIII‐BBZ) human PBL are plotted on the graph as indicated. 

 
 
 
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RT‐PCR using vIII specific primers: 
 
Marker  U251wt U251vIII       308   308+               822    822+               1228  1228+ 
 
 
 
EGFRwt 
 
 
 
 
EGFRvIII 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 9. Expression of EGFRvIII in Glioma Tumor Stem Cell Lines. 
RNA from control U251 cells (wild type EGFR or vIII engineered),  TSC lines 308, 822, 1228, or these lines 
grown for 3 days in serum (+ symbol) were isolated and subject to RT‐PCR using vIII specific primers.  RT‐
PCR products were subject to gel electrophoresis, and visualized with cyber green staining. The location of 
the appropriate size product bands is as indicated on the right. 

 
 
 
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Figure 10. Correlative Studies Schema 

 
 
 
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Table 8. Frequency and Duration of Objective Responses 
 
 
 
 
 
 
 
 
Frequency and Duration of Objective Responses 
 
Total No. 
PR 
CR 
OR 
of 
TBI 
Patients 
No. 
% 
Duration (months) 
No. 
% 
Duration (months) 
No. 
% 
None* 
43 
17 
39.5 
64+, 32+, 20+, 29, 28, 14, 13, 11, 8, 8, 7, 4, 3, 3, 2, 2, 2 
4 
9.3 
63+,  58+,  48+,  47+ 
21 
48.8 
2 Gy 
25 
11 
44.0 
33+, 29+, 23+, 14, 10, 6, 5, 5, 4, 3, 3 
2 
8.0 
37+,  25+ 
13 
52.0 
12 Gy 
25 
14 
56.0 
14+, 13+, 10+, 7+, 7+, 7+, 6+, 6+, 4+, 7, 6, 6, 4, 3 
4 
16.0 
17+, 15+, 13+, 8+ 
18 
72.0 
 
NOTE. All patients received cyclophosphamide 60 mg/kg X 2 days + fludarabine 25 mg/m2 X 5 days. 
Abbreviations: TBI, total-body irradiation; PR, partial response; CR, complete response; OR, objective response; TIL, tumor-infiltrating lymphocytes.