Clinical trial NCT02529072
Conditions: Malignant Glioma Astrocytoma Glioblastoma
Sponsor: Gary Archer Ph.D.
Start date: 2016-01-01; Completion date: 2019-12-30
Enrollment: 6 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Patients will be randomized to one of two treatment arms - Group I and Group II. Group I will receive nivolumab monotherapy until surgical resection, and Group II will receive nivolumab alone and with DC vaccine therapy until surgical resection. During surgical resection blood and tumor samples will be assessed and compared. Following surgery, both groups will continue to receive DC vaccines (total of 8) and nivolumab therapy until confirmed progression.

Detailed description:
This two-arm randomized trial will evaluate the safety of nivolumab in combination with DC vaccinations for the treatment of bevacizumab-naïve subjects with first or second recurrent, resectable World Health Organization (WHO) Grade III and IV malignant gliomas (MGs). Up to 66 patients will be enrolled and treated with the goal of accruing 30 patients (15 per arm) that will receive nivolumab and at least 3 vaccines. After enrollment, leukapheresis will be done for generation of DC vaccines and immunologic monitoring. All subjects will undergo standard of care tetanus booster vaccination with 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly (I.M.) into the deltoid muscle to ensure adequate immunity to the tetanus antigen. Subjects will initially return every 2 weeks and receive approximately 3 infusions of nivolumab 3 mg/kg IV while the DC vaccines are being prepared from the initial leukapheresis and will then be randomized 1:1 to one of 2 arms (Group I: nivolumab only pre-surgery; Group II: nivolumab with DC vaccines pre-surgery). Patients who are unable to tolerate nivolumab will be withdrawn from the study and replaced. Patients whose DCs or Peripheral Blood Lymphocytes (PBLs) fail to meet release criteria will continue to receive nivolumab only and will not undergo repeat leukapheresis. For patients whose leukapheresis yields less than 4 vaccines, a repeat leukapheresis may be obtained a minimum of 2 weeks from the previous leukapheresis (and may be repeated as needed) if stable. Peripheral blood will be drawn for immune monitoring prior to treatment with the 4th cycle of nivolumab (first post-randomization infusion of nivolumab).

Group I Treatment Plan (Nivolumab Only Pre-Surgery) After randomization, patients in Group I will receive nivolumab 3 mg/kg IV every 2 weeks x approximately 8 weeks. The subject will then undergo surgical resection of tumor within approximately 1-3 weeks. Approximately 2-4 weeks later, leukapheresis is repeated for generation of DC vaccines and immunologic monitoring. Approximately 1 day to 2 weeks after leukapheresis, the subject will resume nivolumab 3mg/kg IV every two weeks with DC vaccine administration intradermally (i.d.) for a total of 3 vaccines. At the time of the third DC vaccine, patients will receive vaccine site pre-conditioning. A single dose of Td toxoid (1 flocculation unit (Lf) in 0.4 milliliters (mL) of saline) will be administered to a single side of the groin i.d. (as described above for all vaccine administrations 12-24 hours prior to the third DC vaccine, which is always given bilaterally at the groin site. At the vaccine #3 visit, prior to vaccine # 3 administration, erythema and induration measurements will be taken of pre-conditioning site. Group I subjects will then receive monthly DC vaccine administrations intradermally for 5 months or until progression (whichever comes first).Total vaccines to be administered will be 8 post-surgery unless subject is removed. Nivolumab will continue until progression. At the clinic visit following the last vaccine (#8), subjects will have peripheral blood drawn for immune monitoring prior to infusion of nivolumab.

Group II Treatment Plan (Nivolumab with DC Vaccines Pre-Surgery) After randomization, patients in Group II will receive the fourth cycle of nivolumab then receive nivolumab 3 mg/kg IV along with DC vaccines intradermally every 2 weeks x approximately 6 weeks for a total of 3 vaccines. At the time of the third DC vaccine, patients will receive vaccine site pre-conditioning. A single dose of Td toxoid (1 Lf in 0.4 mL of saline) will be administered to a single side of the groin i.d. 12-24 hours before the third DC vaccine, which is always given bilaterally at the groin site. At the vaccine #3 visit, prior to vaccine #3 administration, erythema and induration measurements will be taken of pre-conditioning site.The subject will then undergo surgical resection of tumor within approximately 1-3 weeks. Approximately 2-4 weeks later, leukapheresis is repeated for generation of DC vaccines and immunologic monitoring. Approximately 1 day to 2 weeks after leukapheresis, the subject will resume nivolumab 3mg/kg IV every two weeks. When DC vaccines have completed processing and are available for administration, monthly DC vaccine administrations as described above will be administered for 5 months or until progression (whichever comes first). Total vaccines to be administered will be 8 (3 pre- and 5 post-surgery) unless subject is removed. Nivolumab will continue until progression. At the clinic visit following the last vaccine (#8), subjects will have peripheral blood drawn for immune monitoring prior to infusion of nivolumab.

Arms: arm 1: Patients will receive nivolumab 3 mg/kg IV every 2 weeks for 8 weeks followed by surgery. Following resection, nivolumab and DC vaccine will be administered every 2 weeks (± 1) for a total of 3 vaccines, followed by biweekly treatment with nivolumab and monthly DC vaccinations for a total of 5 more vaccines. Patients will continue to receive nivolumab every 2 weeks until progression. arm 2: Patients will initially receive the fourth cycle of nivolumab then receive nivolumab 3 mg/kg IV and DC vaccine every 2 weeks for a total of 3 vaccines, and then surgery. Subsequent to surgery, the patient will resume biweekly treatment with nivolumab and monthly DC vaccinations for a total of 5 more vaccines. Patients will continue to receive nivolumab every 2 weeks until progression.
Interventions: intervention 1: nivolumab intervention 2: DC
Intervention descriptions: intervention 1: Nivolumab is a fully human monoclonal antibody that targets the programmed death-1 (PD-1) cluster of differentiation 279 cell surface membrane receptor. PD-1 is a negative regulatory molecule expressed by activated T and B lymphocytes. Binding of PD-1 to its ligands, programmed death-ligands 1 and 2, results in the down-regulation of lymphocyte activation. Inhibition of the interaction between PD-1 and its ligands promotes immune responses and antigen-specific T cell responses to both foreign antigens as well as self-antigens. Nivolumab is expressed in Chinese hamster ovary cells and is produced using standard mammalian cell cultivation and chromatographic purification technologies. The clinical study product is a sterile solution for parenteral administration. intervention 2: DCs are potent immunostimulatory cells that continuously sample the antigenic environment of the host and specifically activate cluster of differentiation 4 positive (CD4+) and cluster of differentiation 8 positive (CD8+) T-cells and B-cells. They are at the crossroads of many of the elegant networks of the immune system, and DCs represent the most promising contemporary biologic entity for realizing the promise of immunotherapy. Potent immune responses and encouraging clinical results have been seen in Phase I and II human clinical trials in systemic cancers. Numerous animal studies and the investigator's institution's humans studies have demonstrated potent antitumor responses using DC-based immunotherapy against MGs.

Protocol documents:
Prot_SAP_000.pdf: 
CONFIDENTIAL 
The information contained in this document is regarded as confidential, and may not be disclosed to 
another party unless such disclosure is required to initiate the study, to conduct study-related activities, or 
to comply with national, state, or local laws and regulations.  Written authorization from the coordinating 
site and sponsor is required for disclosure otherwise. 
PT-PhI-II v05.22.12 
AVeRT: Anti-PD-1 monoclonal antibody (nivolumab) in combination with DC Vaccines for the Treatment 
of Recurrent Grade III and Grade IV Brain Tumors 
 
 
 
 
 
 
 
 
 
 
Sponsor: 
PI – Duke Cancer Institute 
 
 
 
Funding Source: 
BMS and Departmental Funds 
 
 
 
Protocol Source: 
PI - Duke Cancer Institute 
 
 
 
Duke IRB#: 
Pro00065241 
 
 
 
IND#: 
 
16301 
 
 
Principal Investigator 
Katherine Peters, M.D., 
Ph.D. 
katherine.peters @duke.edu 
 
 
Sub-Investigator(s) 
Annick Desjardins, M.D. 
annick.desjardins@dm.duke.edu 
 
Henry Friedman, M.D.  
henry.friedman@duke.edu 
 
Darell Bigner, M.D. 
darell.bigner@duke.edu 
 
Dina Randazzo, D.O. 
dina.randazzo@dm.duke.edu 
 
John Sampson, M.D., Ph.D. 
john.sampson@duke.edu 
 
Gary Archer, Ph.D. 
gary.archer@duke.edu 
 
Elizabeth Reap, Ph.D. 
elizabeth.reap@duke.edu 
 
Luis Sanchez-Perez, Ph.D. 
luis.sanchez-perez@duke.edu 
 
Peter Fecci, M.D., Ph.D. 
peter.fecci@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 
Rachel Hesler, Ph.D. 
rachel.hesler@duke.edu 
 
Data Manager 
Eric Lipp 
eric.lipp@duke.edu 
 
 
 
Original version: 
04/02/2015 
 
Amended version 1: 
BMS Modification 
Requests 
06/09/2015 
 
Amended version 2: 
PRTBTC PRC 
Modification 
Requests 
07/24/2015 
 


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Amended version 3: 
CPC Modifications 
and PI-initiated 
corrections 
08/24/2015 
.Added abbreviation (Section  4); Removed research summary 
(Section  5); Updated study schema (Section 6); Added more 
background on nivolumab (Section 7); Moved randomization 
(Sections 9, 12, 15); Moved surgery (Sections 9, 12); Removed 
specific Td manufacturer (Sections 9, 10, 12); Corrected dose of 
Td/Saline for pre-conditioning (Sections 9, 12); Corrected cell 
concentration error (Section 10); Corrected name of pharmacy 
dispensing Td (Section 10); Removed specific research nurse 
name (Section 10); Updated Tests & Procedures Table (Section 
12); Corrected # of vaccines for primary analysis (Section 15). 
Amended version 4: 
CPC Modifications 
Request 
9/8/2015 
Clarified rationale for surgery following nivolumab ± DC vaccine 
and subjects will be removed if surgery must be performed prior 
to plan (Sections 7, 9 and 12). 
Amended version 5: 
FDA Modification 
Requests 
9/16/2015 
Clinical benefit in Section 12.2.1 is clarified; tolerating study drug 
in same section also clarified; Time point as listed in the PRTBTC 
Imaging SOP is clarified in Section 18.3. 
Amended version 6: 
PI modification 
requests 
1/20/2016 
Included Apheresis blood work prior to each leukapheresis 
(Section 12); updated the blood work drawn prior to leukapheresis 
and each infusion of nivolumab (Section 12), corrected the total 
number of vaccines required for replacement of patients for 
primary endpoint evaluation (Section 15.7); and updated the 
documents in the appendices by removing them from protocol 
and uploading as separate attachments in eIRB (Section 18). 
Amended version 7: 
PI modification 
requests 
2/4/2016 
Use of antihistamines before, during and following vaccine 
`administration recommendation (Section 12.7.3). 
Amended version 8: 
PI modification 
requests 
5/12/2016 
Overall formatting change to meet FDA standards for electronic 
submission (entire document); Changed Primary Study 
Coordinator (Cover Page); Removed reference to “alone” from 
Group 2 description since giving nivolumab alone is done prior to 
randomization (Sections 7.3 & 9.1.6); Clarified blood draws 
(immune monitoring, and pre-nivo) timing, volume, and tubes, and 
removed CCL3 blood assessments (Sections 6, 8, 9.1, 12.1, 12.2, 
12.7.5 & 15.6); Clarified subjects whose cells fail to qualify will not 
undergo repeat leukapheresis (Section 9.1); Described how Td 
booster will be administered on all enrolled subjects, removed 
saline injections during Td pre-conditioning (Sections 9.1,12.2); 
Changed timing of study treatments to allow more approximation 
and flexibility (Section 9.1, 9.1.4, & 15); Changed listing Dr. John 
Sampson to simply Duke Neurosurgeon (Sections 9.1 & 12.2); 
Updated AE and SAE collection to starting at time of consent 
through to 30 days after study ends, and includes leukapheresis 
(Sections 9.1.3, 9.1.4, 12.6.2, 13.1, & 13.2.1); Added language 
regarding a deviation log for non-reportable events for missed 
appointments (Section 9.1.4); Clarified that research team will 
document concomitant medications, not just study coordinator 
(Section 9.1.5); Added more language on nivolumab infusion and 
timing (Section 9.2); Updated location of leukapheresis (Section 
10.1); Clarified eligibility criteria (Section 11);  Increased 
dexamethasone from 2 mg/day to 4 mg/day (Section 11.2); 
Updated the Schedule of Events table(s) (Section 12); Removed 
WHO Performance Status (Sections 12.1 & 18.6); Simplified 
description of standard of care MRI (Sections 12.1 & 12.2.1); 
Removed references to a medical monitor (Sections 12.2.2 & 
12.2.3); Changed nivolumab dose interruption timing from >6 
weeks to >8 weeks (Section 12.2.2); Removed steroid usage as a 


Duke Cancer Institute  
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criterion for early withdrawal and moved to own section (Sections 
12.6.1 & 12.7.4); Added description of vital sign monitoring with 
nivolumab and vaccine administrations (Section 12.7.2); Changed 
the wording from “bi-weekly” to every other week (QOW) 
(Sections 4, 6, and 15); Clarified that TUMS are given three times 
a day instead of “with meals” (Section 12.2); Updated consenting 
by PI to include her designee (Section 16.3); and fixed minor edits 
and formatting errors (throughout). 
Amended version 9: 
PI modification 
requests 
8/5/2016 
Use of bevacizumab in subjects that develop inflammation was 
added (Section 9.1.6);  Use of Antihistamines (Section 9.1.4) and 
Use of Corticosteroids (Section 9.1.5) were moved from Study 
Assessments (Section 12.7) to Study Design (Section 9.1);  
added definition of WBRT to the List of Abbreviations; changed 
Primary Regulatory Coordinator to Rachel Hesler; clarification of 
the timing of MRIs and tests and procedures at Nivolumab 
infusion #1 (Section 12); Removed reference to BMS Guidelines 
in appendices in inclusion criteria (Section 11.1) and fixed minor 
formatting errors (throughout). 
Amended version 
10: PI modification 
requests 
8/29/2016 
Due to a change in the quality control timeline, the vaccine 
schedule for Group II subjects was changed to 3 vaccines prior to 
surgery and 5 vaccines after surgery, keeping the total number of 
vaccines at 8 (Section 6, Section 9.1, Section 12, Section 15); 
Changes were made to Group I vaccine timeline to keep 
consistent (Section 6, Section 9.1, Section 12); As a result of 
these changes to the vaccine timeline, the immune monitoring 
timeline was updated (Section 6, Section 9.1, Section 12) and the 
primary safety analyses will now focus on all subjects who 
complete at least 3, not 4, vaccinations or terminate early due to 
unacceptable toxicity (Section 9.1, Section 15); Clarified that 
Nivolumab will be infused over 30-60 minutes (Section 9.2); 
Clarified that the surgery is for de-bulking purposes (Section 7.3); 
fixed minor formatting errors (throughout). 
Amended version 
11: PI modification 
requests 
02/13/2017 
At leukapheresis #2, subjects (group 1 and group 2) do not need 
to have a physical exam, neuro exam, KPS, O2 saturation 
(Section 12). Subjects will continue to receive these exams at the 
Nivolumab infusion after leukapheresis #2 (1 day to 2 weeks 
later); Corrected Table 3 to indicate that patients who have 
completed at least 3 (not 4) vaccinations will be included in safety 
analysis (Section 15.7); For subjects whose first leukapheresis 
yields 8 or more vaccines, the duration of the second 
leukapheresis can be 2 hours instead of 4 hours (Section 12.2); 
Complications following resection added to definition of 
unacceptable toxicity (Section 9.1);  Vital signs will be taken right 
after injection of DC vaccine, not 30-60 minutes post vaccine 
(Section 12.7.2, Section 12);  In the Criteria for Early Withdrawal 
section (Section 12.6.1), correction of reference to section 
defining unacceptable toxicity (Section 9.1.1) instead of 
nivolumab discontinuation criteria (Section 12.2.2); added that a 
subject can continue on one study drug if the other study drug is 
discontinued due to unacceptable toxicity; Removed restatement 
of the definition of unacceptable toxicity and instead referred to 
definition in Section 9.1.1 (Section 15.4.1);  Correction of 
reference #112 (Section 17) 
Amended version 12 
03/15/2017 
PI changed (title page and Section 13.2.1) 
 
 
 


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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 
7.2 
Study Agent ................................................................................................................................. 13 
7.2.1 
Nivolumab ........................................................................................................................... 13 
7.2.2 
DC Vaccine ......................................................................................................................... 14 
7.2.3 
Tetanus-Diphtheria Toxoid (Td) .......................................................................................... 15 
7.2.4 
Pre-Clinical Experience ....................................................................................................... 16 
7.2.5 
Clinical Experience .............................................................................................................. 18 
7.3 
Study Purpose/Rationale ............................................................................................................ 20 
8 
OBJECTIVES AND ENDPOINTS ...................................................................................................... 22 
9 
INVESTIGATIONAL PLAN ................................................................................................................. 22 
9.1 
Study Design ............................................................................................................................... 22 
9.1.1 
Definition of Unacceptable Toxicity ..................................................................................... 24 
9.1.2 
Dose Modification ................................................................................................................ 24 
9.1.3 
Safety Considerations ......................................................................................................... 24 
9.1.4 
Use of Antihistamines ......................................................................................................... 24 
9.1.5 
Use of Corticosteroids ......................................................................................................... 25 
9.1.6 
Use of Bevacizumab ........................................................................................................... 25 
9.1.7 
Missed Doses ...................................................................................................................... 25 
9.1.8 
Concomitant Medications .................................................................................................... 25 
9.1.9 
Randomization .................................................................................................................... 25 
9.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration .......................................... 25 
9.3 
Rationale for Correlative Studies ................................................................................................ 26 
9.4 
Definition of Evaluable Subjects, On Study, and End of Study ................................................... 26 
9.5 
Early Study Termination .............................................................................................................. 26 
10 
STUDY DRUG .................................................................................................................................... 26 
10.1 
Names, Classification, and Mechanism of Action ....................................................................... 26 
10.2 
Packaging and Labeling .............................................................................................................. 28 
10.3 
Supply, Receipt, and Storage ..................................................................................................... 28 
10.4 
Dispensing and Preparation ........................................................................................................ 28 
10.5 
Compliance and Accountability ................................................................................................... 28 


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10.6 
Disposal and Destruction ............................................................................................................ 29 
11 
SUBJECT ELIGIBILITY ...................................................................................................................... 30 
11.1 
Inclusion Criteria ......................................................................................................................... 30 
11.2 
Exclusion Criteria ........................................................................................................................ 30 
12 
SCREENING AND ON-STUDY TESTS AND PROCEDURES .......................................................... 31 
12.1 
Screening Examination ............................................................................................................... 34 
12.2 
Treatment Period ........................................................................................................................ 34 
12.2.1 
Rationale for Continued Treatment in Cases of Suspected Progressive Disease ............. 35 
12.2.2 
Discontinuation Criteria for Nivolumab ................................................................................ 36 
12.2.3 
Treatment of Nivolumab-Related Infusion Reactions ......................................................... 36 
12.3 
End of Treatment ........................................................................................................................ 37 
12.4 
Follow-up Period ......................................................................................................................... 37 
12.5 
End of Study ................................................................................................................................ 37 
12.6 
Early Withdrawal of Subject(s) .................................................................................................... 38 
12.6.1 
Criteria for Early Withdrawal ............................................................................................... 38 
12.6.2 
Follow-up Requirements for Early Withdrawal .................................................................... 38 
12.6.3 
Replacement of Early Withdrawal(s) ................................................................................... 38 
12.7 
Study Assessments ..................................................................................................................... 38 
12.7.1 
Medical History .................................................................................................................... 38 
12.7.2 
Physical Exam ..................................................................................................................... 38 
12.7.3 
Immunologic Assessments ................................................................................................. 39 
12.7.4 
Correlative Assessments .................................................................................................... 39 
13 
SAFETY MONITORING AND REPORTING ...................................................................................... 39 
13.1 
Adverse Events ........................................................................................................................... 39 
13.1.1 
Reporting of AEs ................................................................................................................. 40 
13.2 
Serious Adverse Events .............................................................................................................. 40 
13.2.1 
Reporting of SAEs ............................................................................................................... 40 
13.3 
Safety Oversight Committee (SOC) ............................................................................................ 41 
13.4 
External Data and Safety Monitoring Board (DSMB) .................................................................. 41 
14 
QUALITY CONTROL AND QUALITY ASSURANCE ......................................................................... 42 
14.1 
Monitoring .................................................................................................................................... 42 
14.2 
Audits .......................................................................................................................................... 42 
14.3 
Data Management and Processing ............................................................................................. 42 
14.3.1 
Study Documentation .......................................................................................................... 42 
14.3.2 
Case Report Forms (CRFs) ................................................................................................ 43 
14.3.3 
Data Management Procedures and Data Verification......................................................... 43 
14.3.4 
Coding ................................................................................................................................. 43 
14.3.5 
Study Closure ...................................................................................................................... 43 
15 
STATISTICAL METHODS AND DATA ANALYSIS ............................................................................ 45 


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15.1 
Analysis Sets ............................................................................................................................... 45 
15.2 
Patient Demographics and Other Baseline Characteristics ........................................................ 45 
15.3 
Treatments .................................................................................................................................. 45 
15.4 
Primary Objective ........................................................................................................................ 45 
15.4.1 
Variable ............................................................................................................................... 45 
15.4.2 
Statistical Hypothesis, Model, and Method of Analysis ....................................................... 46 
15.4.3 
Handling of Missing Values, Censoring, and Discontinuations ........................................... 46 
15.5 
Secondary Objective ................................................................................................................... 46 
15.6 
Exploratory Objectives ................................................................................................................ 47 
15.6.1 
Exploratory Objective #1:  Immunologic Response to pp65 ............................................... 47 
15.6.2 
Exploratory Objectives #2: T-cell Costimulatory Markers ................................................... 47 
15.6.3 
Exploratory Objectives #3: Cytokines and Other Soluble Factors ...................................... 47 
15.6.4 
Exploratory Objectives #4: Gene Expression ..................................................................... 47 
15.6.5 
Exploratory Objectives #5: Epitope Spreading ................................................................... 47 
15.7 
Interim Safety Analysis ................................................................................................................ 48 
15.8 
Sample Size Calculation ............................................................................................................. 49 
16 
ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ................................................................... 49 
16.1 
Regulatory and Ethical Compliance ............................................................................................ 49 
16.2 
DUHS Institutional Review Board and DCI Cancer Protocol Committee.................................... 49 
16.3 
Informed Consent ........................................................................................................................ 50 
16.4 
Study Documentation .................................................................................................................. 50 
16.5 
Privacy, Confidentiality, and Data Storage ................................................................................. 50 
16.6 
Data and Safety Monitoring ......................................................................................................... 51 
16.7 
Protocol Amendments ................................................................................................................. 51 
16.8 
Records Retention ...................................................................................................................... 51 
16.9 
Conflict of Interest ....................................................................................................................... 51 
16.10 
Registration Procedure ........................................................................................................... 52 
17 
REFERENCES ................................................................................................................................... 53 
18 
APPENDICES .................................................................................................................................... 60 
18.1 
Nivolumab Investigator Brochure(s) ............................................................................................ 60 
18.2 
I-O Safety Algorithms .................................................................................................................. 61 
18.3 
PRTBTC Imaging SOP ............................................................................................................... 62 
18.4 
CTRAF ........................................................................................................................................ 63 
18.5 
MMSE .......................................................................................................................................... 64 
18.6 
PRTBTC DSMBplus Charter ....................................................................................................... 65 
 
 
 


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2 LIST OF FIGURES 
Figure 1. Study Schema .............................................................................................................................. 12 
 
3 LIST OF TABLES 
Table 1: Objectives and Endpoints ............................................................................................................. 22 
Table 2. Screening and On-Study Tests and Procedures .......................................................................... 31 
Table 3. Conditions for Temporary Accrual Suspension ............................................................................ 48 
Table 4. Probability of Accrual Suspension as a Function of the True Unacceptable Toxicity Rate .......... 48 
 
 
 


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4 
LIST OF ABBREVIATIONS 
Ab 
Antibody 
ABC 
Automated Blood Count 
ACD 
Acid Citrate Dextrose 
ACLS 
Advanced Cardiac Life Support 
ACTH 
Adrenocorticotropic Hormone 
AE 
Adverse Event 
AIDS 
Acquired Immune Deficiency Syndrome 
APAAP 
Alkaline Phosphatase Antialkaline Phosphatase Complex 
AST 
Aspartate Aminotransferase 
AT 
Ambient Temperature 
AUC 
Area Under the Curve 
β-HCG 
Beta-Human Chorionic Gonadotropin 
BMS 
Bristol-Myers Squibb 
BMT 
Bone Marrow Transplant 
BTSC 
Brain Tumor Stem Cells 
Ca++ 
Calcium 
CCL3 
C-C motif Chemokine Ligand 3 
cDNA 
Complimentary Deoxyribonucleic Acid 
CFA 
Complete Freund’s Adjuvant 
CFC 
Cytokine Flow Cytometry 
CLIA 
Clinical Laboratory Improvement Act 
CLN 
Cervical lymph Nodes 
Cmax 
Maximum Concentration of Drug in Plasma or Serum 
CMP 
Comprehensive Metabolic Panel 
CMV 
Cytomegalovirus 
CNC 
Clinical Neurologic Change 
CNS 
Central Nervous System 
Con-A 
Concanavalin A 
CPC 
Cancer Protocol Committee 
CRFs 
Case Report Forms 
CT 
Computed Tomography 
CTL 
Cytotoxic T-Lymphocyte 
CTQA 
Clinical Trials Quality Assurance 
DAR 
Drug Accountability Record 
DC 
Dendritic Cell 
DCI 
Duke Cancer Institute 
DILI 
Drug Induced Liver Injury 
DLT 
Dose Limiting Toxicity 
DNA 
Deoxyribonucleic Acid 
DSMB 
Data Safety and Monitoring Board 
DSMP 
Data Safety and Monitoring Plan 
DTH 
Delayed-type Hypersensitivity 
DUMC 
Duke University Medical Center 
EAE 
Experimental Autoimmune Encephalomyelitis 
EBRT 
External Beam Radiation Therapy 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-linked Immunospot 
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII 
Epidermal Growth Factor Receptor variant type III 
EGFRvIII-KLH 
EGFRvIII conjugated to Keyhole Limpet Hemocyanin 
FACS 
Fluorescence Activated Cell Sorting 
FFPE 
Formalin-Fixed, Paraffin-Embedded 


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FDA 
Federal Drug Administration 
FEV 
Forced Expiratory Volume 
GBM 
Glioblastoma  
GM-CSF 
Granulocyte Macrophage Colony Stimulating Factor 
HAMA 
Human Anti-Murine Antibody  
H&E 
Hematoxylin and Eosin  
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
HMO 
Health Maintenance Organization 
I.C. 
Intracerebral 
ICS 
Intracellular Cytokine Staining 
I.D. 
Intradermal 
IFN-γ 
Interferon-gamma 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IHC 
Immunohistochemistry 
IL-4 
Interleukin-4 
IL-12 
Interleukin-12 
IL-13 
Interleukin-13 
I.M. 
Intramuscular 
IRB 
Institutional Review Board 
ISH 
In Situ Hybridization  
I.V. 
Intravenous 
KLH 
Keyhole Limpet Hemocyanin 
KPS 
Karnofsky Performance Status 
LAMP 
Lysosomal-associated Membrane Protein 
Lf 
 Flocculation unit 
MAb 
Monoclonal Antibody 
MG 
Malignant Glioma 
MGMT 
Methylguanine Methyltransferase 
MHC 
Major Histocompatibility Complex 
mL 
MilliLiter 
MLE 
Maximum Likelihood Estimator 
MMSE 
Mini-Mental Status Examination 
MRI 
Magnetic Resonance Imaging 
mRNA 
Messenger Ribonucleic Acid 
MTD 
Maximally Tolerated Dose 
NA 
Non-adherent 
NCI CTC 
National Cancer Institute Common Toxicity Criteria 
ng 
NanoGram 
NIH 
National Institutes of Health 
NK 
Natural Killer 
ORR 
Overall Response Rate 
OS 
Overall Survival 
OVA 
Ovalbumin 
PBLs 
Peripheral Blood Lymphocytes 
PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline 
PCR 
Polymerase Chain Reactions 
PD 
Progressive Disease 
PHA 
Phytohemagglutinin 
PFS 
Progression Free Survival 
PI 
Principle Investigator 


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PO 
By Mouth 
PRTBTC 
Preston Robert Tisch Brain Tumor Center 
PT 
Prothrombin Time 
PTT 
Partial Thromboblastin Time 
QOW 
Every Other Week 
qPCR 
Real-time PCR 
RANO 
Response Assessment in Neuro-Oncology 
RECIST 
Response Evaluation Criteria in Solid Tumors 
RIO 
Research Integrity Office 
RNA 
Ribonucleic Acid 
RT 
Radiation Therapy 
RT-PCR 
Reverse Transcriptase Polymerase Chain Reaction 
SAE 
Severe Adverse Event 
S.C. 
Subcutaneous 
SFC 
Spot-Forming Cells  
SOC 
Standard of Care; Safety Oversight Committee 
TAA 
Tumor-Associated Antigens 
TCR 
T cell Receptor 
TD 
Tetanus-Diphtheria  
TGF-β 
Transforming Growth Factor-β 
TH2 
T helper type 2 
TMZ 
Temozolomide 
TNF-α 
Tumor Necrosis Factor-α 
TRegs 
Regulatory T cells 
TTP 
Time to Progression 
TTRNA 
Total Tumor mRNA 
ULN 
Upper Limit of Normal 
VDLNs 
Vaccine-site Draining Lymph Nodes 
WBI 
Whole Body Irradiation 
WBRT 
Whole Brain Radiation Therapy 
XRT 
External Radiation Therapy 
 
 
 


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5 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
Please see eIRB for separate document uploaded in Section 6. 
 
 


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6 
STUDY SCHEMA 
 
 
Figure 1. Study Schema 
 
 
 
 


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7 
BACKGROUND AND SIGNIFICANCE 
7.1 Study Disease 
 
Malignant primary brain tumors are more common than Hodgkin’s disease and account for more human 
deaths than melanoma or than cancer of the bladder or kidney. Despite aggressive, computer-guided 
tumor resection, high-dose external beam RT or brachytherapy, and multi-mechanistic chemotherapy 
delivered at toxic doses, most patients with malignant primary brain tumors live <15 months from the time 
of diagnosis, and patients with recurrent tumors usually survive <12 weeks[1-6]. The estimated cost of 
treatment for each patient with a malignant brain tumor is between $30,000 and several hundred 
thousand dollars annually. Thus, the annual treatment cost alone for these patients, not mentioning the 
lost earning potential of afflicted individuals, is greater than the entire annual budget of the National 
Institute of Neurological Diseases and Stroke. In fact, conventional therapy for patients with malignant 
brain tumor is the most expensive medical therapy per quality-adjusted life-year saved currently provided 
in the United States[7, 8]. Moreover, the non-specific nature of conventional therapy for brain tumors 
often results in incapacitating damage to surrounding normal brain and systemic tissues[9, 10]. Thus, in 
order to be more effective, therapeutic strategies will have to precisely target tumor cells while minimizing 
collateral damage to neighboring eloquent cerebral cortex. The rationale for employing the immune 
system to target brain tumors is based on the premise that the inherent biologic specificity of immunologic 
reactivity could meet the clear need for more specific and precise therapy. 
7.2 Study Agent 
7.2.1 
Nivolumab 
Programmed cell death 1 (PD-1) is a key immune receptor expressed by activated T cells that mediates 
immunosuppression.  Blockade of PD-1 may help overcome immune resistance. Nivolumab is a fully 
human monoclonal immunoglobulin (Ig) G4 antibody that binds to the PD-1 cell surface membrane 
receptor, a negative regulatory molecule expressed by activated T and B lymphocytes. Inhibition of the 
interaction between PD-1 and its ligands promote immune responses and antigen-specific T cell 
responses to both foreign and self-antigens. PD-1 receptor blockade by nivolumab is a new approach for 
immunotherapy of tumors. Results from a Phase I/II study CA209003 indicate that nivolumab is active in 
multiple tumor types. Nivolumab 3 mg/kg monotherapy is currently being studied in phase 3 clinical trials 
in advanced melanoma, renal cell carcinoma (RCC) and non-small cell lung carcinoma (NSCLC). 
 
GBM, the most common primary brain tumor in adults, has an aggressive clinical course and a median 
survival of 12–15 months post first-line therapy with maximal surgical resection, radiation, and 
temozolomide. Bevacizumab is approved in the United States for patients with progressive disease 
following therapy; no data has shown durable improvement of disease-related symptoms or OS. With 
limited efficacy of current therapy, more effective treatments to extend survival and preserve quality of life 
are needed. Ipilimumab, a cytotoxic T-lymphocyte antigen-4 receptor blocking antibody, has shown 
clinical activity in advanced melanoma patients with brain metastases; preclinical studies demonstrate a 
benefit from combining a PD-1 pathway inhibitor with radiation in a mouse glioma model. Duke has a 
phase IIb, randomized, open-label study to evaluate the efficacy and safety of nivolumab alone or with 
ipilimumab, versus bevacizumab in patients with recurrent GBM. Patients with KPS ≥70, grade IV 
malignant glioma treated with radiotherapy and temozolomide, and documented first GBM recurrence 
within 28 days of randomization are eligible. Patients with >1 recurrence of GBM, extracranial disease, 
autoimmune conditions, or previous VEGF inhibitor or anti-angiogenic treatment are ineligible. Safety 
cohort 1; nivolumab 3 mg/kg [n=10; every 2 weeks x 4] and nivolumab 1 mg/kg + ipilimumab 3 mg/kg 
[n=10; every 3 weeks x 4 followed by nivolumab 3 mg/kg every 2 weeks] will establish safety and 
tolerability in GBM patients. Upon successful completion of cohort 1, efficacy cohort 2 will enroll up to 240 
pts with recurrent GBM, randomized 1:1:1 to receive nivolumab, nivolumab + ipilimumab (dosed as cohort 
1), or bevacizumab (10 mg/kg every 2 weeks). The primary objectives are to evaluate safety in cohort 1 
and OS in cohort 2, vs bevacizumab, with secondary objectives of PFS and ORR. Responses will be 


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assessed (Response Assessment Neuro-Oncology criteria) at the end of weeks 6 and 12, and every 8 
weeks until progression or treatment discontinuation. Clinical trial information: NCT02017717. 
7.2.2 
DC Vaccine 
DC vaccine: Human Cytomegalovirus pp65-lysosomal-associated membrane protein mRNA-pulsed 
autologous dendritic cells with granulocyte macrophage-colony stimulating factor. 
 
DCs are potent immunostimulatory cells that continuously sample the antigenic environment of the host 
and specifically activate CD4+ and CD8+ T-cells and B-cells [11, 12]. They are at the crossroads of many 
of the elegant networks of the immune system, and DCs represent the most promising contemporary 
biologic entity for realizing the promise of immunotherapy. Potent immune responses and encouraging 
clinical results have been seen in Phase I and II human clinical trials in systemic cancers[13-29]. 
 
Human CMV is an endemic β-Herpesvirus that does not usually cause significant clinical disease[30]. 
During primary maternal infection, however, human CMV can cause severe encephalitis in fetuses and 
lead to congenital brain defects. Human CMV disease is also a significant problem in 
immunocompromised adults such as organ transplant recipients or patients with AIDS[30]. Herpesviruses 
have also been implicated in a number of human malignancies including lymphoma, nasopharyngeal 
cancer, cervical cancer, and Kaposi’s sarcoma[31, 32]. Recently, expression of proteins unique to human 
CMV has been reported within a large proportion of malignant tumors including colorectal carcinoma, 
prostate cancer, and malignant astrocytomas[33-35]. Universal detection of the human CMV 
immunodominant protein pp65, immediate early gene 1 protein (IE1), and several other early antigens 
was demonstrated using IHC in Grade II-IV astrocytomas[35]. Presence of the virus in these samples was 
confirmed with ISH, PCR for human CMV-specific glycoprotein B (UL55), electron microscopic detection 
of intact virions[35], and direct detection of the virus from fresh operative samples in the shell vial assay  
(unpublished data). Human CMV antigens were not detected in surrounding normal brain samples, 
meningiomas, or brains affected by ischemia, Alzheimer’s disease, paraneoplastic encephalitis, or 
Cryptococcal cerebritis. 
 
The presence of highly-immunogenic human CMV antigens within MGs affords a unique opportunity to 
target these tumors immunologically. There is a vast amount of experience with both the safety and 
efficacy of immunotherapy targeting human CMV, and the presence of this virus within brain tumors may 
allow this experience to be leveraged toward the effective eradication of MG expressing human CMV 
antigens. Adoptive T-cell therapy has been used to safely and successfully protect against CMV 
reactivation in myelodepleted BMT patients[36-39]. In addition, T-cell mediated immunotherapy has 
proven highly effective in the treatment of CMV-associated disease within the CNS and in the treatment 
of acute CMV infections[37, 40]. Tumors associated with other human Herpesviruses, such as Epstein-
Barr virus-associated lymphoma, including tumors within the CNS, have also been effectively treated and 
even large tumors have been cured by immunotherapy[41-46]. More recently, a vaccine directed against 
the potent viral antigens of human papilloma virus has also been shown to reduce the incidence of human 
papilloma virus-related cervical intraepithelial neoplasia in a prospective, randomized, double-blind trial. 
 
The potential for non-specific targeting of normal tissues is thought to be minimal in seropositive patients. 
After initial infection, CMV establishes lifelong latency in the infected individual, with cells of the myeloid 
lineage constituting a major reservoir for persistence of the virus. Virus can be detected within myeloid 
progenitors in the bone marrow, with a small portion of these cells demonstrating viral DNA replication 
without any detectable gene expression[47, 48]. Also a small proportion (typically 1 in 1,000 to 1 in 
10,000) of peripheral blood monocytes can be found to contain CMV DNA, while detection of viral RNA 
(gene expression) is not detected[49-51].   
 
Vaccination specifically against CMV [52-56] has effectively reduced the risk of viral infection and 
transmission to fetuses in animal models [57-59] and in clinical trials[52, 56, 60-63]. Human clinical trials 
have also demonstrated some benefit of administering neutralizing antibodies in the treatment of human 
CMV infection[64-67], highlighting the importance of the development of vaccination strategies that elicit 
both cellular and humoral immune responses. DCs strongly activate both T-cell and B-cell responses in 


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vivo, and DCs pulsed in vitro with CMV antigens have been shown to be potent inducers of CMV-specific 
CTL responses in several studies[68-72], in addition to our own work which is outlined below. 
 
The use of RNA to encode tumor antigens for DCs was pioneered at Duke University in Dr. Gilboa’s 
laboratory, but the ability of RNA-loaded DCs to stimulate potent antitumor immunity has been 
independently confirmed in murine and human systems [73-78]. In fact, there is accumulating evidence 
that RNA transfection represents a superior method for loading antigens onto DCs [75, 79]. This novel 
and innovative approach to DC antigen loading has multiple conceptual advantages over other forms of 
antigen delivery as well. RNA-based antigen loading does not require knowledge of major MHC 
restriction, and responses are not restricted to single MHC haplotypes or to a narrow B- or T-cell 
repertoire. This diversity increases the likelihood of inducing effective and sustained antitumor immune 
responses by simultaneous activation of both CTLs and helper T-cells [80-82]. Furthermore, in direct 
comparisons, RNA-loaded DCs have been found to be better stimulators of antigen-specific T-cells than 
other approaches [79]. Finally, RNA also carries a significant safety advantage, not possessed by other 
nucleic acid or viral vectors, in that it cannot be integrated permanently into the host genome. In addition 
to the preliminary data we present below, Kobayashi et al., have demonstrated that tumor mRNA-loaded 
DCs can elicit a specific CD8+ CTL response against autologous tumor cells in patients with MG. 
 
7.2.3 
Tetanus-Diphtheria Toxoid (Td) 
The current use of Td toxoid is for active immunization in children and adults against infection with the 
bacteria Clostridium tetani and Corynebacterium diphtheria. Tetanus infection is manifested primarily by 
neuromuscular dysfunction caused by a potent exotoxin released by C. tetani. Diphtheria is an acute 
toxin-mediated infectious disease caused by toxigenic strains of C. diphtheriae. Protection against 
disease is due to the development of neutralizing antibodies to the diphtheria toxin. Td toxoids adsorbed 
are readily available as several approved administrations [i.e. Daptacel (DTaP), Infanrix (DTap), Tenivac 
(Td adult), Boostrix (Tdap)] [83, 84]. Protection against disease is due to the development of neutralizing 
antibodies to the tetanus toxin. A serum tetanus antitoxin level of at least 0.01 IU/mL, measured by 
neutralization assays, is considered the minimum protective level. A level ≥0.1 IU/mL by ELISA has been 
considered as protective [85]. A serum diphtheria antitoxin level of 0.01 IU/mL, measured by 
neutralization assays, is the lowest level giving some degree of protection; a level of 0.1 IU/mL by ELISA 
is regarded as protective. Diphtheria antitoxin levels ≥1.0 IU/mL by ELISA have been associated with 
long-term protection. 
 
Following deep s.c./i.m. administration of the tetanus toxoid vaccine, toxoid molecules are taken up at the 
vaccination site by immature DCs, which are professional antigen-presenting cells. Within these cells, 
they are processed through the endosomal pathway (involving the phagolysosome) where they are bound 
to MHC type II molecules on the surface of DCs. The MHC II:toxoid complex then migrates to the cell 
surface. While this process is happening within the cell, the now activated mature DC at the vaccine site 
migrates along lymph channels to the draining lymph node where they encounter naive TH2 cells, each 
with their own unique TCR. Identifying and then binding of the MHC II:toxoid to the specific TH2 receptor 
then activates the naive T cell, causing it to proliferate. Simultaneously, toxoid molecules not taken up by 
DCs pass along lymph channels to the same draining lymph nodes where they come into contact with B 
cells, each with their own unique B-cell receptor (BCR). Binding to the B cell through the specific 
immunoglobulin receptor that recognizes tetanus toxoid results in the internalization of toxoid, processing 
through the endosomal pathway and presentation on the cell surface as an MHC II:toxoid complex, 
similarly to DCs undergoing the same process.  
 
These two processes occur in the same part of the lymph node with the result that the B cell with the 
MHC II:toxoid complex on its surface now comes into contact with the activated TH2 whose receptors are 
specific for this complex. The process, termed linked recognition, results in the TH2 activating the B cell to 
become a plasma cell with the production initially of IgM, with a later switch to IgG antibodies produced. 
Additionally, a subset of these B cells becomes memory cells. 
 
The novelty of using Td toxoid vaccination lies in the ability of this potent recall antigen to enhance 
antitumor responses as part of a cancer vaccination protocol. Td toxoid induces an inflammatory milieu 


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within the intradermal vaccine site, thereby promoting the migration of injected tumor-specific DCs. 
Additionally, in the context of vaccinating the host with tumor-derived peptides, conditioning the vaccine 
site with Td toxoid has demonstrated enhanced immunogenicity with these peptides.  
 
Our data from the ATTAC clinical trial (Duke IRB Protocol # Pro00003877) demonstrating the capacity to 
enhance DC migration to VDLNs via Td pre-conditioning of the vaccine site offer potential therapeutic 
interventions whereby we can enhance the immunologic responses to ultimately overcome the inherent 
challenges in faithfully eradicating established tumors[86]. In a completed randomized clinical trial, we 
found that migration of injected DCs to VDLNs following vaccine site pre-conditioning with Td toxoid was 
significantly increased compared to controls and that the efficiency of DC migration was strongly 
associated with clinical outcomes of patients with newly-diagnosed GBM, the most fatal type of malignant 
brain tumors. To address this observation, we took our Td pre-conditioning platform back into the 
preclinical setting using transgenic mouse models and were able to corroborate the effects of Td pre-
conditioning on increasing the lymph node homing of intradermally administered DCs. Moreover, Td 
administration at a single vaccine site increases the migration of a bilateral DC vaccine to both inguinal 
lymph nodes. Regardless of the side of the Td intradermal skin prep, DC migration to bilateral inguinal 
VDLNs was equally increased, supporting a systemic response to recruit peripherally administered DCs.  
 
Our Td pre-conditioning platform in the context of DC vaccination also elicited superior anti-tumor 
responses compared to controls receiving DC vaccines without Td pre-conditioning. In our clinical trial, 
patients with newly-diagnosed GBM who were administered the Td skin prep before DC vaccination 
revealed significantly longer progression-free and overall survival rates compared to the control cohort. In 
evaluating the relationship between DC migration and clinical responses, we observed a modest positive 
correlation between levels of DC migration and survival. In our preclinical model, Td pre-conditioning prior 
to vaccination with tumor antigen-specific DCs dramatically suppressed the growth of established and 
highly aggressive B16-F10/OVA tumors. The use of Td with a DC vaccine increased antitumor responses 
in an antigen-specific manner, as non-specific DC vaccines were not potentiated with Td pre-conditioning. 
Furthermore, in a challenge setting, where mice are administered the treatment platform prior to 
challenge with tumor inoculation, Td pre-conditioning at the vaccine site induced a significant survival 
benefit compared to controls. 
7.2.4 
Pre-Clinical Experience 
7.2.4.1 
Nivolumab 
Preclinical animal models of tumors have shown that blockade by PD-1 by monoclonal antibodies (mAbs) 
can enhance the anti-tumor immune response and result in tumor rejection. Antitumor activity by PD-1 
blockade functions in PD-L1-positive tumors as well as in tumors that are negative for the expression of 
PD-L1. This suggests that host mechanisms (ie, expression of PD-L1 in antigen-presenting cells) limit the 
antitumor response. Consequently, both PD-L1 positive and negative tumors may be targeted using this 
approach. In humans, constitutive PD-L1 expression is normally limited to macrophage-lineage cells, 
although expression of PD-L1 can be induced on other hematologic cells as well, including activated T 
cells. However, aberrant expression of PD-L1 by tumor cells has been reported in a number of human 
malignancies. PD-L1 expressed by tumor cells has been shown to enhance apoptosis of activated tumor-
specific T cells in vitro. Moreover, the expression of PD-L1 may protect the tumor cells from the induction 
of apoptosis by effector T cells.  
 
Preclinical data indicate that the combination of PD-1 and CTLA-4 receptor blockade may improve 
antitumor activity. In vitro combinations of nivolumab plus ipilimumab increase IFN-γ production 2- to 7-
fold over either agent alone in a mixed lymphocyte reaction. Increased antitumor activity of the 
combination was also observed in 3 of 5 syngeneic murine cancer models. In a murine melanoma 
vaccine model, blockade with either CTLA-4 or PD-1 antibodies increased the proportion of CTLA-4 and 
PD-1-expressing CD4/CD8 tumor infiltrating T effector cells, and dual blockade increased tumor 
infiltration of T effector cells and decreased intratumoral T regulatory cells, as compared to either agent 
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In a 4-week toxicity study of nivolumab in combination with ipilimumab conducted in cynomolgus 
monkeys demonstrated that the combination of nivolumab and ipilimumab resulted in dose-dependent 
gastrointestinal (GI) toxicity. Histologic findings included inflammatory changes in the large intestine, 
which increased in incidence and severity in a dose-dependent manner. GI toxicity/colitis was not 
observed in cynomolgus monkeys administered nivolumab alone, but was observed in monkeys receiving 
ipilimumab. Nivolumab in combination with ipilimumab was also associated with lymphoid hypocellularity 
of the cortex and/or medulla of the thymus and with acinar cell degranulation in the pancreas. Additional 
findings included interstitial mononuclear cell infiltrates in the kidneys, portal mononuclear cell infiltrates in 
the liver and myeloid hypercellularity in the bone marrow. Nivolumab in combination with ipilimumab at 
the high-dose level (ie, 50 mg/kg and 10 mg/kg, respectively) was associated with the death of 1 animal, 
attributed to acute gastric dilatation without histopathological evidence of colitis upon pathology 
evaluation of the GI tract. Please see nivolumab IB[88] for more information. 
7.2.4.2 
DC Vaccines 
In our laboratories and those of others, systemic immunization using DCs co-cultured with 
uncharacterized tumor homogenate[89], whole tumor RNA[90], unidentified peptides eluted from tumor 
cells by gentle acid washing[91], or a distinct peptide encompassing the tumor-specific EGFRvIII 
mutation[92] have been shown to induce humoral and cell mediated systemic immune responses and to 
prolong the survival of rodents with brain tumors. 
 
In our laboratory, inbred VM/Dk mice received three or four weekly intraperitoneal injections of autologous 
bone marrow-derived DCs transiently co-cultured with tumor homogenate. The homogenate was derived 
from a syngeneic murine astrocytoma cell line derived from a spontaneously occurring astrocytoma in the 
inbred VM/Dk mouse strain. Splenocytes from mice immunized in this way were able, in vitro, to lyse the 
astrocytoma cell line that was used to generate the tumor homogenate. They were also able to lyse other 
astrocytoma cell lines derived from the same inbred mouse strain, but they had no effect against 
syngeneic fibroblasts. Similarly, these immunized mice also demonstrated a significantly increased 
antibody titer against the astrocytoma cell line used to generate the homogenate. In addition, mice 
immunized with DCs transiently co-cultured with tumor homogenate that were subsequently challenged 
with a lethal dose of this astrocytoma cell line intracerebrally were found to have a median survival 
>160% longer than those immunized with DCs cultured without tumor homogenate (P=0.016). In addition, 
50% of the mice treated with the tumor homogenate-supplemented DCs survived long-term without any 
evidence of tumor growth and also survived a rechallenge of tumor cells indicating that a sustained 
antitumor immune response had been established. These findings are especially significant in light of the 
fact that the astrocytoma cell line used is known to secrete the immunosuppressive agent TGF-β which is 
secreted by most human gliomas[93-97]. 
 
In another report from our laboratory[90], C57BL/6 mice received three weekly intraperitoneal injections of 
autologous bone-marrow derived DCs co-cultured with tumor homogenate or whole tumor RNA derived 
from the poorly immunogenic B16F10 melanoma cell line.  Standard in vitro cytotoxicity assays again 
revealed that splenocytes harvested from mice immunized with DCs transiently co-cultured with either 
tumor-derived homogenate or whole tumor RNA were able to lyse B16F10 melanoma cells but not 
unrelated tumor cells from the same MHC background. In this experiment, mice immunized with 
autologous bone-marrow derived DCs co-cultured with tumor homogenate or whole tumor RNA increased 
median survival by >233% (P=0.0006) and 48% (P=0.0001), respectively, relative to mice immunized with 
DCs co-cultured with tumor homogenate or whole tumor RNA derived from an unrelated tumor with the 
same MHC background. In addition, 8/13 (61.5%) in the specific homogenate group and 4/10 (40%) in 
the specific RNA group survived beyond the endpoint of the study without evidence of tumor. 
Immunization of mice with pre-existing tumors with specific tumor homogenate also demonstrated the 
potency of this immunization approach by increasing survival by 62.5% relative to controls. In these mice 
an inflammatory infiltrate composed of mononuclear cells and polymorphonuclear leukocytes was 
identified only in mice treated with DCs co-cultured with tumor homogenate that matched the intracerebral 
tumor challenge. 
 
In a recent report from another laboratory, the survival of tumor-bearing rats injected subcutaneously with 
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wash was significantly prolonged compared to tumor-bearing rats receiving equivalent numbers of DCs 
co-cultured with peptides acid-eluted from normal astrocytes (P< 0.05).  Median survivals in these groups 
were 35 and 22 days respectively. In addition, three of the twelve rats (25%) treated with DCs co-cultured 
with acid-eluted tumor peptides remained alive at the end of the experiment. In addition, 
immunohistochemical analysis of five animals from each group in this experiment documented an 
increased peritumoral and intratumoral infiltration of CD8+T-cells, and to a lesser extent CD4+ T-cells and 
macrophages, in the group treated with DCs co-cultured with peptides acid-eluted from tumor cells when 
compared to controls. 
7.2.5 
Clinical Experience 
7.2.5.1 
Nivolumab 
CA209003 is an ongoing Phase 1 open label, multiple dose escalation study in 306 subjects with select 
previously treated advanced solid tumors, including melanoma, RCC, NSCLC, colorectal cancer, and 
hormone-refractory prostate cancer. Subjects received nivolumab at doses of 0.1, 0.3, 1, 3, or 10 mg/kg 
intravenously every 2 weeks, up to a maximum of 2 years of total therapy. As of 18-Mar-2013, a total of 
306 melanoma subjects were treated with nivolumab in the dose range of 0.1 - 10 mg/kg. 
 
No maximal tolerated dose was identified in CA209003. The incidence, severity and relationship of AEs 
were generally similar across dose levels and tumor types. Nivolumab related AEs of any grade occurred 
in 75.2% of subjects. Of the 306 treated subjects, 303 (99.0%) subjects have at least 1 reported AE 
regardless of causality.  The most frequently reported   AEs were fatigue (54.9%), decreased appetite 
(35.0%), diarrhea (34.3%), nausea (30.1%), and cough (29.4%). Treatment-related AEs were reported in 
230 (75.2%) of the 306 subjects. The most frequently reported treatment-related AEs were fatigue 
(28.1%), rash (14.7%), diarrhea (13.4%), and pruritus (10.5%). Most treatment-related AEs were low 
grade. Treatment-related high grade (Grade 3-4) AEs were reported in 52 (17.0%) of subjects. The most 
common treatment-related high grade AEs were fatigue (2.3%) and diarrhea (1%). 
 
Drug-related SAEs occurred in 11.5% of subjects. Grade 3-4 drug-related SAEs reported in at least 2 
subjects included: diarrhea (3 subjects, 1.0%), pneumonitis (3 subjects, 1.0%), pneumonia (2 subjects, 
0.7%) and lipase increased (2 subjects, 0.7%). 
 
Select AE categories (events with a potential inflammatory mechanism requiring more frequent monitoring 
and/or unique intervention such as immunosuppressants and/or endocrine replacement therapy) include: 
GI AEs, pulmonary AEs, renal AEs, hepatic AEs, skin AEs, and endocrinopathies. In addition, select AEs 
include a category for infusion reactions. Each category is composed of a discrete set of preferred terms, 
including those of greatest clinical relevance. These select AEs are considered events of interest based 
on the mechanism of action and were previously referred to as immune-related AEs or immune-mediated 
AEs. 
 
The 10 mg/kg cohort had numerically greater frequency of high-grade select AEs including the 
subcategories of endocrinopathies, GI, pulmonary, and infusion reactions. Most high grade events 
resolved following the treatment guidelines for the treatment of pulmonary events, GI events, hepatic 
events, renal events, and endocrine events, respectively. 
 
In CA209003 (MDX1106-03), the clinical activity of nivolumab was demonstrated in a variety of tumor 
types and across a range of doses (0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg). As of the clinical 
cut-off date of 05-Mar-2013, a total of 306 subjects with melanoma, RCC, and NSCLC have been treated 
with nivolumab. All subjects initiated treatment at least one year prior to analysis. A response of either CR 
or PR, as determined by investigator assessed tumor evaluations based on modified RECIST 1.0, has 
been reported at all dose levels. 
 
Among 107 patients with advanced melanoma who received nivolumab, the preliminary objective 
response rate was 33/107 (31%). Responses occurred at each dose level, with 6/17 (35%), 5/18 (28%), 
11/35 (31%), 7/17 (41%), and 4/20 (20%) melanoma subjects responding at  0.1, 0.3, 1, 3, and 10 mg/kg, 
respectively.  Duration of response ranged from 24.1 to 48.7+, 18.4 to 66.3+, 32.4 to 108.1+, 40.1+ to 


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115.4+, and 73.9 to 117.0+ months in melanoma subjects treated at 0.1, 0.3, 1, 3, and 10 mg/kg, 
respectively. An additional 7% of melanoma subjects had stable disease for 24 weeks or longer. Across 
dose levels, melanoma subjects achieved a median overall survival of 16.8 months (95% CI: 12.5, 31.6), 
with a 2-year overall survival rate of 43%. 
 
Here at Duke, we are currently enrolling in CA209143, “A Randomized Phase 3 Open Label Study of 
Nivolumab Versus Bevacizumab and a Safety Study of Nivolumab or Nivolumab in Combination With 
Ipilimumab in Adult Subjects With Recurrent GBM” where we evaluate the efficacy and safety of 
nivolumab alone or with ipilimumab, vs bevacizumab in subjects with recurrent GBM. Subjects with KPS 
≥70, grade IV malignant glioma treated with radiotherapy and temozolomide, and documented first GBM 
recurrence within 28 days of randomization are eligible. Those with >1 recurrence of GBM, extracranial 
disease, autoimmune conditions, or previous VEGF inhibitor or anti-angiogenic treatment are ineligible. 
Safety cohort 1; nivolumab 3 mg/kg [n=10; Q2W x 4] and nivolumab 1 mg/kg + ipilimumab 3 mg/kg [n=10; 
Q3W x 4 followed by nivolumab 3 mg/kg Q2W]) will establish safety and tolerability in GBM pts. Upon 
successful completion of cohort 1, efficacy cohort 2 will enroll up to 240 pts with recurrent GBM, 
randomized 1:1:1 to receive nivolumab, nivolumab + ipilimumab (dosed as cohort 1), or bevacizumab (10 
mg/kg Q2W). The primary objectives are to evaluate safety in cohort 1 and OS in cohort 2, vs 
bevacizumab, with secondary objectives of PFS and ORR. Responses will be assessed (RANO criteria) 
at the end of weeks 6 and 12, and every 8 weeks until progression or treatment discontinuation. Clinical 
trial information: NCT02017717.  To date, nivolumab has been well tolerated in this patient population – 
20 subjects have been treated on the nivolumab monotherapy arm. 
 
Please see nivolumab IB[88] for more information. 
7.2.5.2 
DC Vaccines 
The occurrence of human DCs in the peripheral blood is low (0.15% of circulating mononuclear cells), 
and procedures to isolate circulating DCs are cumbersome, relying on negative selection techniques to 
deplete the mononuclear cell fraction of contaminating monocytes and lymphocytes. Furthermore, brain 
tumor patients are characteristically immunosuppressed either from the use of steroids or due to the fact 
that malignant brain tumors secrete immunosuppressive agents like TGF-y. We have been using a simple 
method described previously[117], to generate human DCs by culturing PBMCs in media supplemented 
with GM-CSF and IL-4. We have compared the ability to generate DCs from patients with malignant brain 
tumors and patients undergoing craniotomy for non-tumor related procedures. The phenotype of DCs 
from both tumor and normal populations were identical and were characterized as being highly positive 
for HLA-ABC and HLA-DR, the co-stimulatory molecules CD80 and CD86, and the DC/monocyte marker 
CD11c, but negative for the monocyte marker CD14. The cells were negative for the B and NK cell 
lineage markers, CD19 and CD56, respectively, which is consistent with published DC phenotypes. 
 
DC immunotherapy in patients with MGs has been evaluated in only a few studies. In the published study 
by Yu et al. [118], patients received biweekly intradermal injections of peripheral blood derived DCs 
pulsed with uncharacterized peptides eluted from the surface of autologous glioma cells by gentle acid 
washings. All patients were required to complete a course of RT and were off steroids at the time of 
immunization. Toxicity was minimal and included only mild fever and lymphadenopathy. There was no 
clinical or radiographic evidence of autoimmune encephalomyelitis in any patient and no serious adverse 
events occurred. The immunization resulted in enhanced CTL activity in 4/7 patients and both cytotoxic 
and memory T-cells were found to have infiltrated the patient’s tumors whom underwent reoperation after 
immunization. Although this study was performed in a selected population of patients, the median survival 
of 455 days in the treated group compared very favorably with an institutional control group where median 
survival was only 257 days. Similarly, when immunized patients were compared to expected outcome per 
Curran’s recursive partition analysis, which controls for known prognostic factors (Karnofsky Performance 
Status, histology, surgery, mental status, etc.), the results still appeared quite favorable. Unfortunately, no 
clinical responses were seen and any antigen-specific immune response could not be characterized 
because the immunizing antigens were not characterized. 
 


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A study by Kikuchi et al. (2001)[119] used autologous DCs fused with autologous tumor cells as an 
immunogen in an 8 patient trial. The immunization schedule consisted of 3 to 7 vaccinations 3 weeks 
apart given intradermally. All vaccinations were well tolerated.  
 
In another Phase I/II trial, tumor lysate pulsed DCs were given to ten patients who received 
immunizations every three weeks for a minimum of one and a maximum of 10 immunizations. There were 
only two minor clinical responses seen. Of five patients evaluated by ELISPOT before and after 
vaccination T-cells reactive against tumor lysate-pulsed DCs were increased in two patients[120]. In a 
more recently published study, patients with GBM were treated with 1 x 106 to 1 x 107 DCs pulsed with 
acid eluted autologous tumor peptides[121]. There was no evidence of DLT or serious adverse events. 
One patient had an objective clinical response documented by magnetic resonance imaging, and six 
patients developed measurable systemic antitumor CTL responses. 
 
In a more recent study, Kikuchi et al.[98] investigated the safety and clinical response to immunotherapy 
using fusions of DCs and glioma cells combined with recombinant human IL-12 for the treatment of MG. 
Fifteen patients with MG participated in this study. Cultured autologous glioma cells were established 
from surgical specimens in each case. Fusion cells were prepared from DCs and glioma cells using 
polyethylene glycol. All patients received fusion cells intradermally on day 1. IL-12 was injected 
subcutaneously at the same site on days 3 and 7. No serious adverse effects were observed. In four 
patients, magnetic resonance imaging showed a greater than 50% reduction in tumor size.  One patient 
had a mixed response. In our ongoing Phase I/II clinical trial (BB IND 9944) patients with newly-
diagnosed MGs are vaccinated with mature DCs loaded with a peptide spanning the fusion junction of 
EGFRvIII conjugated to keyhole limpet hemocyanin (PEPvIII-KLH) (500 mcg/immunization), mixed with 
GM-CSF (approx. 150 mcg/immunization). EGFRvIII is a tumor specific antigen, which is expressed on 
approximately 47% of all MGs. The vaccination protocol consists of 3 vaccines 2 weeks apart of PEPvIII-
KLH loaded, mature DCs beginning 2 weeks following completion of post-resection RT. To date, 19 
patients have been enrolled with 16 completing vaccination with no adverse events. No patient showed a 
positive delayed-type hypersensitivity reaction to KLH or PEPvIII before vaccination and of the evaluable 
patients after vaccination, 14/15 (93.3%) patients reacted to KLH and 11/15 (73.3%) reacted to PEPvIII. 
In vitro proliferation in response to PEPvIII was seen in 11/12 (92%) and to KLH in 9/12 (75%) of patients 
tested. Two patients, one with anaplastic astrocytoma and one with GBM with residual radiographic 
disease after resection, and radiation, have had a nearly complete response in our prior vaccination study 
which also included the use of chemotherapy agents such as TMZ. These patients have remained stable 
for 174.9 and 217.3 weeks. Of the 14 patients without radiographically evident disease, 4/14 (28.6%) 
have not progressed at 102.7, 171.3, 180.7, 430.7 weeks with a median overall time to progression of 
10.4 months comparing favorably with a historical unvaccinated cohort (EGFRvIII positive and gross total 
resection) that had a median TTP of 7.1 months (n=39).   For patients with GBM, the median survival time 
was 20.0 months which compares favorably with recently published trials evaluating newly-diagnosed 
patients with GBM treated with GLIADEL®(13.9 months)[99]; radiation and concurrent TMZ (14.6 
months); or radiolabeled anti-tenascin monoclonal antibodies performed at Duke University  (18.3 
months)[100]. 
 
7.3 Study Purpose/Rationale 
 
Immune checkpoint blockade is a rapidly advancing therapeutic approach in the field of immuno-oncology 
and treatment with investigational agents targeting this mechanism has induced regressions in several 
types of cancer. Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) 
receptor are two important cellular targets that play complementary roles in regulating adaptive immunity. 
Whereas PD-1 contributes to T cell exhaustion in peripheral tissues, CTLA-4 inhibits at earlier points in T 
cell activation. In preclinical  models,  combined  blockade  of  PD-1  and  CTLA-4  achieved  more  
pronounced antitumor activity than blockade of either pathway alone[87]. 
 
CMV antigens have been identified in GBM and may make excellent antitumor immunotherapeutic 
targets. Vaccination and adoptive T cell strategies targeting CMV in humans in other contexts have been 
safe and effective. DC vaccinations targeting other antigens in GBM have been safe and effective. Other 


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peptide vaccines given to patients with GBM during recovery from TMZ-induced lymphopenia have 
produced potent tumor-specific immune responses. 
 
We have shown that immunosuppressive TRegs are disproportionately represented in the blood and 
tumors of patients with malignant gliomas (MGs). This finding is accompanied by significant T cell 
dysfunction resulting in a profound immunosuppression[101, 102] that likely significantly limits 
endogenous antitumor immune responses.  Programmed cell death 1 (PD-1) is a key immune receptor 
expressed by activated T cells that mediates immunosuppression.  Blockade of PD-1 may help overcome 
immune resistance. Nivolumab is a fully human monoclonal immunoglobulin (Ig) G4 antibody that binds to 
the PD-1 cell surface membrane receptor, a negative regulatory molecule expressed by activated T and B 
lymphocytes. Inhibition of the interaction between PD-1 and its ligands promote immune responses and 
antigen-specific T cell responses to both foreign and self-antigens. PD-1 receptor blockade by nivolumab 
is a new approach for immunotherapy of tumors. 
 
We believe the use of serial vaccination in conjunction with an anti-PD-1 monoclonal antibody, is a novel 
platform for enhancing the efficacy of tumor-specific vaccines.  In addition, immune checkpoint receptors 
such as program death- ligand (PD-L)1/B7-H1/CD274, a transmembrane receptor ligand and negative 
regulator of T cell signaling, have been reported to be upregulated in gliomas. Studies have also 
suggested an association   between   malignancy   grade   of   astrocytic   tumors   and   tumor   cell   PD-
L1 expression.  Additionally, use of an inhibitor of PD-L1 in mouse glioma models suggests benefit in 
combination with radiation therapy. 
 
We have demonstrated in murine models that DCs loaded with tumor-specific antigens in the form of 
mRNA can induce potent and specific humoral and cell-mediated immune responses that are effective 
against murine i.c. tumors, including a syngeneic murine astrocytoma, without inducing autoimmunity[89, 
92, 103]. Our previous clinical experience has also shown that DC vaccines in combination with standard 
of care radiation therapy and chemotherapy are capable of generating potent, tumor-specific immune 
responses and clinical radiographic responses in patients with MGs. We and others have also shown that 
antigens derived from CMV are contained within MGs and may serve as potent and specific 
immunotherapy targets. Vaccination and adoptive T-cell strategies targeting CMV in humans in other 
contexts, including the targeting of lesions within the CNS, have been safe and effective. We have also 
shown that DCs generated from patients with GBM and loaded with pp65-LAMP mRNA are capable of 
generating CD4+ and CD8+ T-cells that produce IFN-γ and kill malignant astrocytes infected with CMV in 
an antigen-specific fashion. We have found that TILs isolated from these patients are significantly 
enriched for T-cells that specifically recognize CMV antigens, suggesting that this response may be 
important in the biology of these tumors.  
 
Surgery alone has not been established as the standard of care treatment in the recurrent brain tumor 
population, rather, it is used in combination with medical treatment. Therefore, it is our belief that 
providing nivolumab treatment ± DC vaccine prior to surgery will be safe and well tolerated.  However, 
subjects will be closely monitored for any indication that surgical resection should be performed sooner 
than the planned schedule. Therefore, we would like to evaluate the safety of nivolumab in combination 
with DC vaccinations for the treatment of bevacizumab-naïve subjects with first or second recurrent, 
resectable WHO Grade III and IV MGs and to assess the immunologic changes from nivolumab therapy 
with and without DC.  In order to do that, we will randomize subjects to two groups: Group I will receive 
nivolumab monotherapy until surgical resection, and Group II will receive nivolumab with DC vaccine 
therapy until surgical resection.  During surgical resection blood and tumor samples will be assessed and 
compared.  Following surgery, both groups will continue to receive DC vaccines (total of 8) and nivolumab 
therapy until confirmed progression. The purpose of the surgical resection is de-bulking of the tumor.  The 
subject will continue on study treatment following surgery, at the discretion of the treating physician, until 
they have radiographic or clinical progression (as described in Section 12.2.1), regardless of the 
pathology results from surgery. 
 
 


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8 
OBJECTIVES AND ENDPOINTS 
 
Table 1: Objectives and Endpoints 
  
Objective 
Endpoint 
Analysis 
Primary 
To evaluate the safety of 
nivolumab in combination 
with DC vaccinations for 
the treatment of 
bevacizumab-naïve 
subjects with first or 
second recurrent, 
resectable WHO Grade III 
and IV MGs. 
Proportion of patients with 
unacceptable toxicity. 
See Section 15.4 
Secondary 
To describe overall survival 
and progression free 
survival. 
Median overall and 
progression-free survival 
from initiation of protocol 
treatment. 
See Section  15.5 
Exploratory    
To evaluate PBMCs for 
immunologic response to 
pp65 in the presence of 
nivolumab.  
Mean or median change from 
baseline at each follow-up 
assessment in PBMC-based 
immune measures 
See Section 15.6.1 
Exploratory 
To assess whether 
treatment modulates T cell 
costimulatory markers 
Mean or median change from 
baseline in the expression of 
costimulatory markers. 
See Section 15.6.2 
Exploratory 
To assess the effect of 
treatment on cytokines and 
other soluble factors 
Mean or median change from 
baseline in the expression of 
cytokines and other soluble 
factors 
See Section 15.6.3 
Exploratory 
To assess the impact of 
pre-surgical treatment on 
gene expression 
Mean or median change in 
the level of gene expression 
in PBMCs, and mean or 
median level of gene 
expression in tumor 
See Section 15.6.4 
Exploratory 
To determine if evidence 
exists for epitope 
spreading intratumorally or 
systemically after 
treatment. 
Changes in TCR repertoire 
diversity  TILs, and PBLs. 
See Section 15.6.5 
 
 
9 
INVESTIGATIONAL PLAN 
9.1 Study Design 
 
This two-arm randomized trial (please see section 9.1.9 for details) will evaluate the safety of nivolumab 
in combination with DC vaccinations for the treatment of bevacizumab-naïve subjects with first or second 
recurrent, resectable WHO Grade III and IV MGs. Up to 66 patients will be enrolled and treated with the 
goal of accruing 30 patients (15 per arm) that will receive nivolumab and at least 3 vaccines. After 
enrollment, leukapheresis will be done for generation of DC vaccines and immunologic monitoring (please 
see section 12.7.5 for details). Following consent, all subjects will undergo standard of care tetanus 


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booster vaccination with 0.5 mL of Td (tetanus and diphtheria toxoids adsorbed) intramuscularly (I.M.) 
into the deltoid muscle to ensure adequate immunity to the tetanus antigen. All subjects will initially return 
every 2 weeks and receive approximately 3 infusions of nivolumab 3 mg/kg IV while the DC vaccines are 
being prepared from the initial leukapheresis, Nivolumab infusions will continue until the DCs are ready 
for vaccinations. All subjects will then be randomized 1:1 to one of 2 arms (Group I: nivolumab only pre-
surgery; Group II: nivolumab with DC vaccines pre-surgery). Patients who are unable to tolerate 
nivolumab will be withdrawn from the study and replaced. Patients whose DCs or PBLs fail to meet 
release criteria will continue to receive nivolumab only and will not undergo repeat leukapheresis. For 
patients whose leukapheresis yields less than 4 vaccines, repeat leukapheresis may be obtained a 
minimum of 2 weeks from the previous leukapheresis (and may be repeated as needed) if pre-pheresis 
blood work is within the Apheresis Center’s parameters and as long as this will not cause a significant 
delay in treatment for the patient.  For patients that need to come off study due to adverse events, they 
will be eligible for surgery and other treatment modalities. Patients will be monitored closely and those 
patients that have clinical indications for surgical resection prior to the timeframe indicated below will be 
withdrawn from the study and undergo surgical resection. Peripheral blood will be drawn for immune 
monitoring prior to treatment with the 5th cycle of nivolumab (second post-randomization infusion of 
nivolumab).  
 
All vaccines will be administered intradermally near each groin, divided into 2-4 injections in each side as 
tolerated in order to inject the full amount without leakage.  It is recommended that the injections be given 
approximately 10 cm below the inguinal ligament. Vaccine injections must be performed using an 
appropriate needle for intradermal administration.  A 25-gauge needle is recommended.  
 
Group I Treatment Plan (Nivolumab Only Pre-Surgery) 
After randomization, patients in Group I will receive nivolumab 3 mg/kg IV every 2 weeks x approximately 
8 weeks.  The subject will then undergo surgical resection of tumor within approximately 1-3 weeks by a 
Duke neurosurgeon. Approximately 2–4 weeks later, leukapheresis is repeated for generation of DC 
vaccines and immunologic monitoring. Approximately 1 day to 2 weeks after leukapheresis, the subject 
will resume nivolumab 3mg/kg IV every two weeks with DC vaccine administration intradermally for a total 
of 3 vaccines.  At the time of the third DC vaccine, patients will receive vaccine site pre-conditioning. A 
single dose of Td toxoid (1 flocculation unit, Lf, in 0.4 mLs of saline) will be administered to a single side 
of the groin i.d. (as described above for all vaccine administrations)12-24 hours prior to the third DC 
vaccine, which is always given bilaterally at the groin site.  At the vaccine #3 visit, prior to vaccine # 3 
administration, erythema and induration measurements will be taken of pre-conditioning site. Group I 
subjects will then receive monthly DC vaccine administrations intradermally for 5 months or until 
progression (whichever comes first). Total vaccines to be administered will be 8 post-surgery unless 
subject is removed.  Nivolumab will continue until progression. At the clinic visit following the last vaccine 
(#8), subjects will have peripheral blood drawn for immune monitoring prior to infusion of nivolumab. 
 
Group II Treatment Plan (Nivolumab with DC Vaccines Pre-Surgery) 
After randomization, patients in Group II will receive the fourth cycle of nivolumab then receive nivolumab 
3 mg/kg IV along with DC vaccines intradermally every 2 weeks x approximately 6 weeks for a total of 3 
vaccines.  At the time of the third DC vaccine, patients will receive vaccine site pre-conditioning. A single 
dose of Td toxoid (1 flocculation unit, Lf, in 0.4 mLs of saline) will be administered to a single side of the 
groin i.d. (as described above for all vaccine administrations)12-24 hours prior to the third DC vaccine, 
which is always given bilaterally at the groin site.  At the vaccine #3 visit, prior to vaccine #3 
administration, erythema and induration measurements will be taken of pre-conditioning site.  The subject 
will then undergo surgical resection of tumor within approximately 1-3 weeks by a Duke neurosurgeon. 
Approximately 2-4 weeks later, leukapheresis is repeated for generation of DC vaccines and immunologic 
monitoring.  Approximately 1 day to 2 weeks after leukapheresis, the subject will resume nivolumab 
3mg/kg IV every two weeks. When DC vaccines have completed processing and are available for 
administration, DC vaccine administrations as described above will resume monthly for 5 months or until 
progression (whichever comes first). Total vaccines to be administered will be 8 (3 pre- and 5 post-
surgery) unless subject is removed.  Nivolumab will continue until progression.  At the clinic visit following 
the last vaccine (#8), subjects will have peripheral blood drawn for immune monitoring prior to infusion of 
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9.1.1 
Definition of Unacceptable Toxicity 
Toxicities will be graded according to the NCI CTCAE version 4 criteria. An unacceptable toxicity is 
defined as any grade 3, 4, or 5 adverse event that is possibly, probably, or definitely related to either 
nivolumab or DC vaccination treatment during concurrent treatment, or any Grade 2 drug-related uveitis 
or eye pain or blurred vision that does not respond to topical therapy and does not improve to Grade 1 
severity within the re-treatment period OR requires systemic treatment.  In addition, any complication 
following resection (ex. excessive intracranial bleeding, delays in wound healing) that are prolonged 
longer than 4-6 weeks post-surgery will be considered an unacceptable toxicity.   
9.1.2 
Dose Modification 
The DC vaccine and nivolumab dose will not be modified in this trial.  Please see the nivolumab IB and 
Section 12.2.2 for discontinuation of nivolumab related to adverse events. 
9.1.3 
Safety Considerations 
Management of Toxicities 
If a Grade 3 NCI CTC or greater toxicity is seen that is not attributable to a concomitant medication (other 
than nivolumab or vaccine), co-morbid event, or disease progression that has been documented 
radiographically or clinically, the next scheduled study-specific treatment for that patient (nivolumab 
and/or DC vaccine) will be withheld for up to 2 months or until the NCI CTC toxicity improves to a Grade 2 
or until the KPS score returns to within 10 points of baseline.  
 
Long Term Survivors 
For subjects still actively receiving study treatment ≥ 3 years from study initiation, planned procedures 
requiring hospitalization, or long-term clinical decline that is now seen in patients years from WBRT, 
which are clearly not related to study drug, but are the natural development common in this patient 
population, will not be considered a toxicity attributable to vaccine or nivolumab treatment nor will have 
nivolumab held or delayed. 
 
Adverse Event Reporting and Documentation 
An “Adverse Event” will be defined as any adverse change from the subject’s pre-treatment baseline 
condition, including any clinical or laboratory test abnormality that occurs during the course of research 
after initiation of nivolumab.  Adverse events will be categorized and graded in accordance with the NCI 
CTC (Version 4).  AEs and SAEs will be collected starting at the time of consent. 
 
A “Serious Adverse Event” will be defined as an undesirable sign, symptom or medical condition which 1) 
is fatal or life threatening; 2) requires inpatient hospitalization or a prolongation of existing hospitalization; 
3) results in persistent or significant disability/incapacity; 4) constitutes a congenital anomaly or a birth 
defect and/or; 5) medically significant such that it may jeopardize the subject, and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
Please see section 11 for further information. 
 
Collection and reporting of SAEs will be done through 30 days post end of study visit.  Please see the 
BMS Guidelines and the management algorithms provided by BMS in the appendices of this protocol. 
9.1.4 
Use of Antihistamines 
Subjects will be advised to avoid antihistamine use 48 hours prior to each vaccine administration, the day 
of vaccine administration, and for 48 hours following each vaccine administration.  If the subject has a 
pre-existing condition that requires antihistamine usage, the PI and the treating oncologist will decide if it 
is safe and appropriate for the subject’s antihistamines to be held before and following vaccine 
administrations. 
 


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9.1.5 
Use of Corticosteroids 
Once vaccinations have been initiated, if patients subsequently require increased steroids, they will still 
be permitted to remain on the study, but every effort will be made to minimize steroid requirements. 
9.1.6 
Use of Bevacizumab 
In the event that a patient demonstrates neurologic or radiographic signs suggestive of an inflammatory 
reaction that requires steroid management, patients will be initiated on bevacizumab at the discretion of 
the treating physician. Bevacizumab treatment will begin anytime four weeks or more post-resection.  
Bevacizumab will be administered at 5mg/kg IV QOW following nivolumab infusion and can be increased 
to 10mg/kg IV QOW if needed.  Bevacizumab will not be provided by the study. 
 
9.1.7 
Missed Doses 
To ensure that repetitive infusions of nivolumab and DC vaccines will be given to patients, both will be 
given within approximately ± 2 day window.  A margin of 2 days surrounding each treatment will make 
scheduling more feasible. Nivolumab infusions will continue until progression, however, DC vaccinations 
will only be given for a maximum of 8.  At the discretion of the study PI, patients who miss either the 
nivolumab infusion or the vaccine administration for whatever reason will have their appointment re-
scheduled to administer treatment as soon as possible and this will be considered a non-reportable 
deviation will be and will be added to the deviation log. For continued non-compliance with the scheduled 
nivolumab or vaccine appointments, the subject may be removed from the trial at the discretion of the 
study PI.  Please see the nivolumab IB[88] on management of missed doses of nivolumab. 
9.1.8 
Concomitant Medications 
Concomitant medications will be managed by the treating oncologist and recorded at each study visit by 
the research team. 
9.1.9 
Randomization 
Permuted block randomization will be used to assign patients to one of two treatment arms (Group I: 
nivolumab monotherapy until surgery and Group II: nivolumab with DC vaccine therapy until surgery).  
The randomization module for the Duke electronic database system (Title 21 CFR Part 11 Compliant) will 
be used for this purpose. 
9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
 
Nivolumab 
Nivolumab will be infused at a dosage of 3 mg/kg IV every other week (+/- approximately 2 days) over 
approximately 30-60 minutes.  Each infusion of nivolumab will be considered one cycle.  On the days 
where patients receive both nivolumab and DC vaccine, the order of the administration does not matter 
(i.e., DC vaccine can be administered before or after nivolumab and does not need to be given in the 
same order at follow-up visits). 
 
Single-dose pharmacokinetics (PK) of nivolumab was studied in 39 subjects with cancer. The single-dose 
PK of nivolumab was linear and dose-proportional in the range of 0.3 mg/kg to 10 mg/kg. The mean 
terminal T-HALF of nivolumab ranged between 17 and 25 days across the dose range of 0.3 mg/kg to 10 
mg/kg.  Geometric mean total clearance varied from 0.13 mL/h/kg to 0.19 mL/h/kg, while mean volume of 
distribution varied between 83 mL/kg and 113 mL/kg across doses. The clearance and half-life of 
nivolumab are consistent with that of IgG4.  
 
The multiple-dose PK of nivolumab given every 2 weeks was determined from MDX1106-03 study as well 
as by opulation  PK  using  data  from  669  subjects  across  nivolumab  studies. Multiple-dose PK of 
nivolumab following every 2 week dosing was linear with dose-proportional increase in Cmax and AUC 
(TAU) in the studied range of 0.1 mg/kg to 10 mg/kg.  Nivolumab accumulation with every 2 week dosing 


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frequency was in the range of 2.9 to 3.3 based on AUC (TAU), 2.0 to 2.4 based on Cmax, and 3.1 to 4.8 
based on Cmin. 
 
The PK, clinical activity, and safety of  nivolumab  have  been  assessed  in  approximately 32 clinical 
studies  sponsored  by  BMS  or  Ono  Pharmaceutical  Co.,  Ltd.  (ONO):  11 Phase 1 studies, 13 Phase 
2 studies, and 8 Phase 3 studies. Approximately 4,000 subjects have received nivolumab monotherapy in 
single- or multiple-dose Phase 1/2/3 studies or studies with nivolumab in combination with other 
therapeutics (ipilimumab, cytotoxic chemotherapy, anti-angiogenics, and targeted therapies). The 
description and status of all studies are outlined briefly in the nivolumab IB[88]. Results from the ongoing 
studies are preliminary and are subject to change. 
 
DC Vaccine 
Only 1 dose level of DCs (2 x 107) will be given.   
 
Vaccination and adoptive T-cell strategies in humans in other contexts have been safe and effective. 
Therapeutic TMZ induces a profound lymphopenia that may enhance anti-tumor vaccination responses 
when given during the homeostatic T-cell proliferation that occurs in response to lymphodepletion. Other 
DC vaccines given to patients with high-grade gliomas during recovery from TMZ-induced lymphopenia 
have produced potent tumor-specific immune responses and have been well tolerated. 
 
In one of our dendritic cell immunotherapy trials (Pro00003877 ATTAC), we evaluated the impact of 
vaccine site pre-conditioning with Td toxoid. Patients randomized to Td showed increased dendritic cell 
migration bilaterally and significantly improved progression-free and overall survival. Furthermore, we 
observed a modest association between effective DC migration to VDLNs and clinical outcomes[86]. 
9.3 
Rationale for Correlative Studies 
 
Peripheral blood and tumor specimens are collected in this study for use in assessing the impact of 
nivolumab and DC vaccination treatment on levels of immune function.  Although this Phase I study is not 
designed to be definitive towards this end, these analyses will provide the initial data necessary to 
adequately power future studies.  
9.4 
Definition of Evaluable Subjects, On Study, and End of Study 
 
Please refer to section 15 for detail concerning which patients will be included in which analyses.    
 
Once the patient signs an ICF, that subject will be considered “on study” and the patient will be monitored 
for all AEs and SAEs. Rationale for taking patient off protocol treatment will be documented. 
9.5 
Early Study Termination 
 
This study can be terminated at any time for any reason by the PI-sponsor. If this occurs, all subjects on 
study should be notified as soon as possible. Additional procedures and/or follow up should occur in 
accordance with Section 12.7. Section 12.6 describes procedures and process for prematurely withdrawn 
patients. 
 
10 STUDY DRUG 
10.1 
Names, Classification, and Mechanism of Action 
 
Nivolumab 
Nivolumab (also referred to as BMS-936558 or MDX1106) is a fully human monoclonal antibody (HuMAb; 
immunoglobulin G4 [IgG4]-S228P) that targets the programmed death-1 (PD-1) cluster of differentiation 
279 (CD279) cell surface membrane receptor. PD-1 is a negative regulatory molecule expressed by 


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activated T and B lymphocytes.  Binding of PD-1 to its ligands, programmed death–ligands 1 (PD-L1) and 
2 (PD-L2), results in the down-regulation of lymphocyte activation. Inhibition of the interaction between 
PD-1 and its ligands promotes immune responses and antigen-specific T cell responses to both foreign 
antigens as well as self-antigens. Nivolumab is expressed in Chinese hamster ovary (CHO) cells and is 
produced using standard mammalian cell cultivation and chromatographic purification technologies. The 
clinical study product is a sterile solution for parenteral administration.  Please see nivolumab IB[88] for 
more information. 
 
DC Vaccine 
Human CMV pp65-LAMP mRNA-pulsed autologous DCs is the name of the study drug given with every 
vaccine. The class of action for the DC vaccine is a biological. 
 
Tetanus-diphtheria toxoid (Td toxoid adsorbed)  
Td is indicated for active booster immunization against tetanus, diphtheria, and pertussis as a single 
dose; substitute 1-time dose of Tdap for Td booster, then standardly boost with Td every 5-10 years. 
Please refer to section 7.2 on the use of Td in this protocol. The class of action for the tetanus toxoid is an 
antitoxin. 
 
Leukapheresis and dendritic cell vaccine generation  
Two leukapheresis procedures will be performed in this protocol. The leukapheresis will be used for DC 
generation and immune monitoring. Leukaphereses will be approximately a 4-hour leukapheresis. It is 
estimated that 10-12 L of blood will be processed during this leukapheresis.  
 
DCs will be generated from leukapheresis in vitro by 7-day culture with GM-CSF and IL-4. PBMC for in 
vitro generation of DCs will be obtained by leukapheresis at either the Duke Apheresis Unit or Duke Bone 
Marrow Transplant Unit and transported to the Cell Processing facility. For patients without sufficient 
venous access for leukapheresis a temporary intravenous catheter may be inserted. 
 
At the end of the 7 day incubation for generation of DC a sample of the media is taken for mycoplasma 
testing, the cells are then harvested and electroporated with pp65-LAMP mRNA. The DCs are placed in a 
flask with AIM V media GM-CSF + IL-4 + TNF-α + IL-6 + IL-1β at 37OC, 5% CO2 for 18-20 hours for 
maturation. The cells are washed twice with PBS and frozen at 2-4 x 107 cells/mL in 90% autologous 
human AB serum (Valley Biomedical, Winchester, VA 22602), 10% DMSO and 5% glucose in a 
controlled-rate freezer at a rate of 1oC/minute.  
 
The DCs are then stored until needed at –135oC.  After freezing, an aliquot of cells is thawed for QA/QC.  
This testing will look at viability, (>70%) endotoxin content, (<5 E.U. /Kg B.W.) mycoplasma contamination 
(negative) and sterility testing for aerobic and anaerobic bacterial cultures (1 x 106 DCs) and fungal 
cultures (1 x 106 DCs). 
 
For each vaccination, cells that have passed QA/QC will be rapidly thawed at 37oC, washed three times 
with PBS and counted. The cell concentration will be adjusted to 5 x 107 cells/mL and DCs will be 
resuspended in preservative free saline and placed into a sterile tuberculin syringe with a 27 gauge 
needle. 
 
From the final preparation a sample of cells will be sent for Gram stain and endotoxin testing prior to 
administration. DC vaccination will not be given until endotoxin testing has been passed (< 5.0 E.U/Kg) 
and the Gram stain has been found to be negative. An aliquot of cells will also be sent for aerobic and 
anaerobic bacterial cultures (1 x 106 DCs) and fungal cultures (1 x 106 DCs). 
 
In the event of a positive sterility or mycoplasma test, the Principal Investigator or his or her designee 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 >38.5oC or clinical evidence of infection at the injection site (drainage, erythema or edema) 
or systemically, the patient will have swabs taken from the injection sites (if possible), along with blood, 
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The patient 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 immunization samples will be sent for sent for bacterial, 
fungal, and mycoplasmal culture and sensitivity testing and endotoxin testing, and immunizations will 
proceed only if the patient fully recovers and subsequent samples are found to be sterile. 
10.2 
Packaging and Labeling 
 
• 
For nivolumab: (Please see Appendices for BMS Guidelines for packaging and labeling) 
Name 
MRN 
DOB  
Drug: nivolumab 
Lot #: Lot XXX 
Caution New Drug Limited By Federal Law To Investigational Use 
 
• 
For CMV pp65-LAMP mRNA-pulsed DCs (DC vaccine): 
Name 
MRN 
DOB  
Drug: pp65DCs 
Lot #: Lot 001 
Caution New Drug Limited By Federal Law To Investigational Use 
 
• 
For tetanus diphtheria toxoid  
Name 
MRN 
DOB  
Drug: Td  
Lot #: Lot 001 
10.3 
Supply, Receipt, and Storage 
 
The DCs will be stored in a locked liquid nitrogen freezer in the FDA-approved Clinical Processing Suite 
in the DBTIP Laboratory. The Nautilus LIMS (Laboratory Information Management System) database will 
track receipt and storage location. 
 
These study agents are supplied and stored by the Duke Investigational Chemotherapy Services (ICS): 
nivolumab is supplied in a single use glass vial, Bristol-Myers Squibb, NJ; and Tetanus Diphtheria Toxoid 
adsorbed, stored at 4oC.  
 
10.4 
Dispensing and Preparation 
 
The DC products will be delivered from the DBTIP Laboratory directly to the bedside under the 
supervision of the research nurse, or his/her designee. DCs will be administered according to protocol. 
The patient’s name, Study ID, DOB, and Duke history number will be double verified prior to DC 
administration as is standard Duke BMT transfusion procedure. The nivolumab will be dispensed from the 
Duke Investigational Chemotherapy Service to the Duke Cancer Center’s Oncology Treatment Room 
where infusion will take place per standard operating procedures for that clinic. The tetanus will be 
dispensed from the Duke Investigational Chemotherapy Services (ICS) Pharmacy. 
10.5 
Compliance and Accountability 
 


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The DC vaccines will be stored in the BTIP in a temperature controlled, locked access controlled storage 
unit. A drug log sheet will be used to track and document the drug. The products will be signed out and 
distributed by the 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 ordered, received, stored, and dispensed for BTIP protocols that are approved 
by the DUHS IRB. Investigational drugs are stored separately from other drugs in an area of limited 
access and in accordance with special storage requirements. They are clearly labeled with the identity of 
the study drug and other control numbers. All drug accountability, transfers, receipts, and disposal are 
recorded in the Duke Nautilus system. 
 
Tetanus compliance and accountability will be managed by the Duke ICS Pharmacy. 
10.6 
Disposal and Destruction 
 
Unused study drugs will be destroyed per Duke Policy and institutional guidelines. 
 
 


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11 SUBJECT ELIGIBILITY 
11.1 
Inclusion Criteria 
• 
Age 18-80 years  
• 
First or second recurrence of MG (WHO Grade III or IV glioma or astrocytoma) in surgically 
accessible areas with prior histologic diagnosis of MG 
• 
Bevacizumab-naïve – no prior exposure to Bevacizumab 
• 
KPS of ≥ 70% 
• 
RT with ≥ 45 Gy tumor dose, completed ≥ 8 weeks prior to study entry 
• 
Laboratory values must meet the following criteria (using CTCAE v4): 
1) WBC ≥ 2000/uL 
2) Neutrophils ≥ 1500/uL 
3) Platelets ≥ 100x103/uL 
4) Hemoglobin ≥ 9.0 g/dL 
5) Serum creatinine ≤ 1.5x ULN or creatinine clearance (CrCl)≥ 40 mL/min (using the 
Cockcroft-Gault formula) 
a. Female CrCl = (140 - age in years) x weight in kg x 0.85 /72 x serum creatinine in 
mg/dL 
b. Male CrCl = (140 - age in years) x weight in kg x 1.00/72 x serum creatinine in mg/dL 
6) AST ≤ 3x ULN 
7) ALT ≤ 3x ULN 
8) Bilirubin≤ 1.5x ULN (except subjects with Gilbert Syndrome, who can have total 
bilirubin < 3.0 mg/dL) 
9) Subjects must have resting baseline O2 saturation by pulse oximetry of ≥ 92% at 
rest. 
• 
Patients of child bearing potential or with partners of child-bearing potential must agree to 
practice recommended contraceptive methods to prevent pregnancy during treatment and for 5 
months after the last dose of nivolumab for women, 7 months after the last dose of nivolumab for 
men, and 6 months after the last dose of bevacizumab for subjects receiving bevacizumab as 
stated in the informed consent. 
11.2 
Exclusion Criteria 
• 
Contrast-enhancing tumor component crossing the midline, multi-focal tumor, or tumor 
dissemination (subependymal or leptomeningeal) 
• 
Clinically significant increased intracranial pressure (e.g., impending herniation), uncontrolled 
seizures, or requirement for immediate palliative treatment 
• 
Pregnant or need to breast feed during the study period (Negative β-HCG test required), or 
unable to maintain use of contraception while on study and for 31 weeks after the last dose of 
nivolumab 
• 
Active infection requiring treatment or an unexplained febrile (> 101.5o F) illness 
• 
Known immunosuppressive disease, autoimmune disease or human immunodeficiency virus 
infection, Hepatitis B or Hepatitis C  
• 
Known allergy or hypersensitivity to tetanus, or any other tetanus or diphtheria toxoid-containing 
vaccine, or any component of this vaccine (i.e., aluminum phosphate, formaldehyde) 
• 
Known severe (Grade 3 or 4) infusion-related allergy or hypersensitivity to any monoclonal 
antibody 
• 
Previous radiation therapy with anything other than standard radiation therapy (such as previous 
stereotactic radiosurgery) or previous treatment with an immune checkpoint inhibitor (i.e., 
nivolumab, pembrolizumab, ipilimumab) 
• 
Unstable or severe intercurrent medical conditions such as severe heart (New York Association 
Class 3 or 4) or lung (FEV1 < 50%) disease, uncontrolled diabetes mellitus. 
• 
Corticosteroid use > 4mg/day at time of consent 
• 
Prior inguinal lymph node dissection. 
 
 


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12 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
Table 2. Screening and On-Study Tests and Procedures 
Prior to Randomization: 
 
1Serum pregnancy testing is required at screening, prior to each leukapheresis, and prior to the first nivolumab infusion.  Additional 
testing can be performed as the treating physician feels is necessary and a urine pregnancy test is allowed at any additional time points. 
2Labs for leukapheresis should be drawn within 48 hours prior to leukapheresis. 
3Thyroid profile and Free T3 should be drawn at screening.  These tests will be repeated prior to cycle 4 and may be drawn sooner at the 
discretion of the treating physician. 
4CMV screen will be drawn prior to 1st leukapheresis.   One red top tube will be drawn and taken to the DBTIP Lab for processing.  The 
CMV screen only needs to be drawn once and may be batch processed.   
5Autoimmunity testing is not required and will be drawn at the discretion of the treating physician based on clinical symptoms at any 
time point deemed necessary by the treating physician. 
6Immune monitoring prior to leukapheresis will consist of one red top tube. These tubes will be sent to the DBTIP lab for processing.   
7Immune monitoring will be drawn at the time of progression, if possible. 
8Baseline MRI can be performed prior to consent since this is a standard of care test.  The baseline MRI is not required to be performed 
within a certain period of time before screening.  A new baseline MRI can be performed prior to leukapheresis at the discretion of the 
treating physician.  Additional MRIs can be performed at the discretion of the treating physician.  If a patient is removed from the study 
due to clinical progression, a MRI is not required. 
9O2 saturation only required at screening.  O2 saturation will be rechecked at the discretion of the treating physician based on clinical 
symptoms. 
10AEs and SAEs to be captured starting at the time of consent. 
11Tumor pathology must be confirmed by at least outside pathology report prior to enrollment.  It is preferable that pathology be 
confirmed by Duke Pathology Department prior to enrollment, but this is not required.  Tissue may be obtained at the time of 
progression based on the discretion of the treating physician. 
12Randomization will occur after cycle 3 of nivolumab and prior to cycle 4 of nivolumab in order for the DBTIP lab to prepare for the first 
DC vaccination. 
13Labs and clinic visit (physical exam, neuro exam, KPS) do not need to be repeated prior to Nivolumab #1 if previously performed for 
screening with the same week. 
 
 
 
 
 
 
 
Screening 
Leukapheresis #1 
Nivolumab IV 
 Cycle 1  
Nivolumab IV 
Cycle 2 
Nivolumab IV  
Cycle 3 
Progression 
Beta HCG 
X1 
X2 
X1,13 
 
 
 
CBC w/diff 
X 
X2 
X13 
X 
X 
 
CMP 
X 
X2 
X13 
X 
X 
 
Ionized Calcium 
 
X2 
 
 
 
 
LDH 
X 
 
X13 
X 
X 
 
Mg 
X 
 
X13 
X 
X 
 
Amylase/Lipase 
X 
 
X13 
X 
X 
 
Thyroid Profile and Free 
T3 
X3 
 
 
 
 
 
CMV 
 
X4 
 
 
 
 
Autoimmunity Testing 
X5 
X5 
X13 
X5 
X5 
X5 
Blood for Immunologic 
Monitoring 
X4 
X6 
 
 
 
X7 
MRI 
X8 
 
 
 
 
X 
Physical Exam, Neuro 
Exam, KPS, O2 
Saturation9 
X 
 
X13 
X 
X 
 
Con Meds 
X 
 
X 
X 
X 
 
Baseline symptoms and 
medical history 
X 
 
 
 
 
 
AE/SAE monitoring10 
X 
X 
X 
X 
X 
X 
Tumor Pathology 
X11 
 
 
 
 
X11 
MMSE 
X 
 
 
 
 
 
Nivolumab 
 
 
X 
X 
X 
 
Randomization 
 
 
 
 
X12 
 


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Post-Randomization, Group 1: 
 
 
1Serum pregnancy testing can be performed as the PI feels is necessary and a urine pregnancy test is allowed.  Serum pregnancy test is 
required prior to leukapheresis. 
2Labs for leukapheresis should be drawn within 48 hours prior to leukapheresis. 
3Thyroid profile and free T3 should be drawn prior to cycle 4 and every 4 cycles thereafter.  These tests can be repeated as necessary per 
the discretion of the treating physician. 
4Autoimmunity testing to be done at the discretion of the treating physician based on clinical symptoms at any time point deemed 
necessary by the treating physician. 
5Immune monitoring will be done prior to both leukapheresis procedures and will consist of one red top tube. This tube will be sent to 
the DBTIP lab for processing.   
6Immune monitoring will consist of 10 yellow top ACD tubes and 1 red top tube.  Immune monitoring will be drawn at Nivolumab cycles 
5, 12, 18 and 21 (or the first cycle of Nivolumab two weeks after vaccine #8 is administered), and prior to surgery Immune monitoring 
will be drawn at the time of progression, if possible.  After a year, an attempt will be made to obtain blood for immunologic monitoring 2-
3 times a year at standard Duke neuro-oncology visits (10 yellow, 1 red). 
7MRI to be done prior to cycle 4, before and after surgery per the discretion of the neurosurgeon, and prior to every 4th cycle of 
nivolumab for one year starting with cycle 12 (i.e., prior to cycles 12, 16, 20, etc.).  Additional MRIs can be performed at the discretion of 
the treating physician.  If a patient is removed from the study due to clinical progression, a MRI is not required. 
8O2 saturation only required at screening.  O2 saturation will be rechecked at the discretion of the treating physician based on clinical 
symptoms. 
9Tissue will be collected at the time of surgery.  Tissue may also be obtained at the time of progression at the discretion of the treating 
physician. 
10Td preconditioning to be administered 12-24 hours prior to DC vaccine #3. 
11DC Vaccines to be administered QOW x 3 (Nivo cycles 8-10).  Vitals will be checked on the day of the DC vaccine administration at any 
time prior to administration as well as after DC vaccine administration. 
12DC Vaccines to be administered monthly x 5 (Nivo cycles 12, 14, 16, 18 20).  Vitals will be checked on the day of the DC vaccine 
administration at any time prior to administration as well as after DC vaccine administration. 
 
 
 
 
 
 
 
 
 
Post-Randomization, Group 2: 
 
Nivolumab QOW 
(Cycles 4-7) 
Surgery 
Leukapheresis 
#2 
Nivolumab QOW 
(Cycles 8-11) with 
DC Vaccine QOW 
(x 3) 
Nivolumab QOW 
(Cycles 12-20) 
with DC Vaccine 
Monthly (x 5) 
Nivolumab 
QOW 
(Cycles 21+) 
Progression 
Beta HCG 
 
 
X2 
 
 
 
 
CBC w/diff 
X 
X 
X2 
X 
X 
X 
 
CMP 
X 
X 
X2 
X 
X 
X 
 
Ionized Calcium 
 
 
X2 
 
 
 
 
LDH 
X 
 
 
X 
X 
X 
 
Mg 
X 
 
 
X 
X 
X 
 
Amylase/Lipase 
X 
 
 
X 
X 
X 
 
Thyroid Profile and Free 
T3 
X3 
 
 
X3 
X3 
X3 
 
Autoimmunity Testing 
X4 
X4 
X4 
X4 
X4 
X4 
X4 
Blood for Immunologic 
Monitoring 
X6 
X6 
X5 
X6 
X6 
 
X6 
MRI 
X7 
X7 
 
X7 
X7 
X7 
X7 
Physical Exam, Neuro 
Exam, KPS, O2 
Saturation8 
X 
 
 
X 
X 
X 
 
Con Meds 
X 
 
X 
X 
X 
X 
 
AE/SAE monitoring 
X 
X 
X 
X 
X 
X 
X 
Tumor Pathology 
 
X9 
 
 
 
 
X9 
Nivolumab 
X 
 
 
X 
X 
X 
 
Td Pre-conditioning 
 
 
 
X6,10 
 
 
 
DC Vaccine with vitals 
 
 
 
X11 
X12 
 
 


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1Serum pregnancy testing can be performed as the PI feels is necessary and a urine pregnancy test is allowed.  Serum pregnancy test is 
required prior to leukapheresis. 
2Labs for leukapheresis should be drawn within 48 hours prior to leukapheresis. 
3Thyroid profile and Free T3 should be drawn prior to cycle 4 and every 4 cycles thereafter.  These tests can be repeated as necessary per 
the discretion of the treating physician. 
4Autoimmunity testing to be done at the discretion of the treating physician based on clinical symptoms at any time point deemed 
necessary by the treating physician. 
5Immune monitoring will be done prior to both leukapheresis procedures and will consist of one red top tube. This tube will be sent to 
the DBTIP lab for processing.   
6Immune monitoring will consist of 10 yellow top ACD tubes and 1 red top tube.  Immune monitoring will be drawn prior to surgery, 
prior to vaccines # 1, and 7, and prior to Nivolumab cycle 17 (or the first cycle of Nivolumab two weeks after vaccine #8 is administered).  
Immune monitoring will be drawn at the time of progression, if possible. After a year, an attempt will be made to obtain blood for 
immunologic monitoring 2-3 times a year at standard Duke neuro-oncology visits (10 yellow, 1 red). 
7MRI to be done prior to cycle 4, before and after surgery per the discretion of the neurosurgeon, and prior to every 4th cycle of 
nivolumab for one year (i.e., prior to cycles 8, 12, 16, 20, etc.).  Additional MRIs can be performed at the discretion of the treating 
physician.  If a patient is removed from the study due to clinical progression, a MRI is not required. 
8O2 saturation only required at screening.  O2 saturation will be rechecked at the discretion of the treating physician based on clinical 
symptoms. 
9Tissue will be collected at the time of surgery.  Tissue may also be obtained at the time of progression at the discretion of the treating 
physician. 
10Td preconditioning to be administered 12-24 hours prior to DC vaccine #3. 
11DC Vaccines to be administered QOW x 3 (Nivo cycles 5-7).  Vitals will be checked on the day of the DC vaccine administration at any 
time prior to administration as well as after DC vaccination administration. 
12DC Vaccines to be administered monthly x 5 (Nivo cycles 8, 10, 12, 14, 16).  Vitals will be checked on the day of the DC vaccine 
administration at any time prior to administration as well as after DC vaccination administration. 
 
 
 
 
 
 
 
 
 
 
 
Nivolumab 
IV (Cycle 4) 
Nivolumab QOW 
(Cycles 5-7) with DC 
Vaccine QOW  
Surgery 
Leukapheresis #2 
Nivolumab QOW  
(Cycles 8-15) with  
DC Vaccine 
Monthly (x 5) 
Nivolumab 
QOW 
 (Cycles 16+) 
Progression 
Beta HCG 
X1 
X1 
 
X2 
X1 
X1 
 
CBC w/diff 
X 
X 
X 
X2 
X 
X 
 
CMP 
X 
X 
X 
X2 
X 
X 
 
Ionized Calcium 
 
 
 
X2 
 
 
 
LDH 
X 
X 
 
 
X 
X 
 
Mg 
X 
X 
 
 
X 
X 
 
Amylase/Lipase 
X 
X 
 
 
X 
X 
 
Thyroid Profile and Free 
T3 
X3 
X3 
 
 
X3 
X3 
 
Autoimmunity Testing 
X4 
X4 
X4 
X4 
X4 
X4 
X4 
Blood for Immunologic 
Monitoring 
 
X6 
X6 
X5 
X6 
X6 
X6 
MRI 
X7 
 
X7 
 
X7 
X7 
X7 
Physical Exam, Neuro 
Exam, KPS, O2 
Saturation8 
X 
X 
 
 
X 
X 
 
Con Meds 
X 
X 
 
X 
X 
X 
 
AE/SAE monitoring 
X 
X 
X 
X 
X 
X 
X 
Tumor Pathology 
 
 
X9 
 
 
 
X9 
Nivolumab 
X 
X 
 
 
X 
X 
 
Td Pre-conditioning 
 
X6,10 
 
 
 
 
 
DC Vaccine with vitals 
 
X11 
 
 
X12 
 
 


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12.1 
Screening Examination 
 
The screening examination will take place at the Duke PRTBTC clinic visit. An informed consent must be 
signed by the patient before any study-specific treatment is initiated. The baseline physical and 
neurologic examination with KPS score along with standard of care blood work will be performed and 
documented by the neuro-oncology team and verified by the study team during this PRTBTC clinic visit. 
All subject data is standard of care evaluation that occurs for all patients being seen in the PRTBTC. If the 
subject is considered a screen failure prior to vaccine treatment, the source documents for electronic data 
entry will be stored in a locked cabinet in a locked room in the PRTBTC Offices. 
 
CBC w/diff, complete metabolic panel, magnesium, LDH, amylase, lipase, thyroid profile, free T3, and 
beta HCG (if necessary) will be drawn at screening.  The serum CMV screen (1 red top tube) will be 
drawn prior to the 1st leukapheresis during that visit.  The CMV screen will be sent back to the DBTIP Lab 
where the test will be performed. These tests may be batched tested and results delayed. 
 
Initial clinical evaluations will also include a baseline MMSE.  
 
A baseline MRI of the brain (with and without gadolinium enhancement) will be obtained as per standard 
of care. 
 
After patients have been consented, they will be entered into the Velos eResearch system. 
12.2 
Treatment Period 
 
After enrollment, leukapheresis will be done for generation of DC vaccines and immunologic monitoring. 
Within 48 hours of leukapheresis, patients will have blood samples taken for the following tests as 
required by the Duke Apheresis Center: CBC w/diff, CMP, ionized Calcium, and β-HCG (for females of 
child-bearing potential). Total estimated blood volume required for these evaluations is 12-15 mLs. For 
patients without sufficient venous access for leukapheresis, a temporary central intravenous catheter may 
be inserted. To prevent the development of hypocalcemia from the citrate used for leukapheresis, all 
patients will be instructed to take oral Tums, 2 tablets three times a day and at bedtime the day before 
and the day of the leukapheresis procedure. Patients who have lower levels of calcium will be treated per 
Apheresis lab standard protocols under the direction of apheresis attending physician. This first 
leukapheresis will be approximately a 4-hour leukapheresis, and it is estimated that 10-12 L of blood will 
be processed during this leukapheresis.  
 
Following consent, all subjects will undergo standard of care vaccination with 0.5 mL of Td (tetanus and 
diphtheria toxoids adsorbed) I.M. into the deltoid muscle to ensure adequate immunity to the tetanus 
antigen. Each subject will initially return every 2 weeks and receive nivolumab 3 mg/kg IV while the DC 
vaccines are being prepared from the initial leukapheresis and then randomized to two treatment arms 
(Group I and Group II).  Peripheral blood will be drawn for immune monitoring prior to treatment with the 
5th cycle of nivolumab (second post-randomization infusion of nivolumab).  Group I will receive nivolumab 
alone I.V. every 2 weeks x approximately 8 weeks and Group II will receive the 4th cycle of nivolumab 
then receive nivolumab 3 mg/kg IV every 2 weeks with DC vaccines administered intradermally and 
divided equally to both inguinal regions for a total of 3vaccines x approximately 6 weeks. At the time of 
the third DC vaccine, patients from both groups will receive vaccine site pre-conditioning. A single dose of 
Td toxoid (1 flocculation unit, Lf, in 0.4 mLs) will be administered to a single side of the groin 12-24 hours 
prior to the third DC vaccine, which is always given bilaterally at the groin site.  At the vaccine #3 visit, 
prior to vaccine # 3 administration, erythema and induration measurements will be taken of pre-
conditioning site.  The subject will then undergo surgical resection of tumor within approximately 1-3 
weeks by a Duke neurosurgeon. Approximately 2-4 weeks after surgery, leukapheresis is repeated for 
generation of DC vaccines and immunologic monitoring.  For subjects whose first leukapheresis yields 8 
or more vaccines, the duration of the second leukapheresis can be 2 hours instead of 4 hours.  
Approximately 1 day to 2 weeks after leukapheresis, the subject will resume nivolumab 3mg/kg IV every 


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two weeks along with DC vaccine administrations as described in section 7.1 (Group I and Group II differ) 
for a total of 8 vaccines or until progression (whichever comes first). Nivolumab will continue until 
progression.  See section 12.7.5 for additional immunologic assessments. 
 
If clinically indicated, patients will be assessed before initial infusion of nivolumab and as needed at the 
discretion of the treating oncologist, by a panel of clinical laboratory analyses to screen for the 
development of autoimmunity. The most common manifestations of autoimmunity seen in related 
trials[104-108] have included enterocolitis, dermatitis, uveitis, hepatitis, and hypophysitis. 
 
12.2.1 Rationale for Continued Treatment in Cases of Suspected Progressive Disease 
Accumulating clinical evidence indicates some subjects treated with immune system stimulating agents 
may develop progression of disease (by conventional response criteria) before demonstrating clinical 
objective responses and/or stable disease. This phenomenon was observed in approximately 10% of 
subjects in the Phase 1 study of nivolumab[109]. Two hypotheses have been put forth to explain this 
phenomenon. First, enhanced inflammation within tumors could lead to an increase in tumor size which 
would appear as enlarged index lesions and as newly visible small non-index lesions. Over time, both the 
malignant and inflammatory portions of the mass may then decrease leading to overt signs of clinical 
improvement. Alternatively, in some individuals, the kinetics of tumor growth may initially outpace anti-
tumor immune activity. With sufficient time, the anti-tumor activity will dominate and become clinically 
apparent. Therefore, subjects initially meeting radiologic criteria for disease progression (see Section 
12.1) will be allowed to continue study therapy until a second radiologic confirmation of progression 
performed approximately 8 weeks later as long as the following criteria are met: 1) the subject 
experiences investigator-assessed clinical benefit (i.e., no new clinical symptoms contributed to disease 
progression) and 2) the subject is tolerating the study treatment (i.e., no expected toxicities higher than 
grade 3 [with the exception of Grade 2 uveitis – see Section 12.2.2 below] or unexpected toxicities). 
 
Any evidence of tumor response will be determined according to the Duke PRTBTC SOP (see 
APPENDICES). RANO criteria[110] will be used for assessment of pseudoprogression. 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. Patients will be followed until death. 
 
Standard treatment for glioblastomas (including radiation therapy and temozolomide) may result in a 
transient increase in tumor enhancement (pseudoprogression) in a subset of subjects that eventually 
subsides without any change in therapy. Pseudoprogression may be difficult to differentiate from true 
tumor progression and may have important implications for patient management.  Accumulating evidence 
also indicates that some subjects treated with immune system stimulating agents may also develop 
apparent progression of disease (by conventional response criteria) before demonstrating clinical 
objective responses and/or stable disease[109].  This phenomenon was observed in approximately 10% 
of subjects in the Phase 1 study of nivolumab and has also been reported for ipilimumab 
monotherapy[111].  
 
In order to minimize premature discontinuation of study medication and distinguish pseudoprogression 
from progressive disease, subjects initially meeting radiologic criteria for disease progression may 
continue receiving study medication until confirmation of progression with an MRI performed 
approximately 8 weeks later. 
In order to continue study treatment after assessment of initial radiological progression, the following 
criteria must be met: 
1. The subject is believed to demonstrate clinical benefit as determined by the investigator. 
2. The subject is tolerating study medication. 
 
Subjects with confirmed progression (approximately 8 weeks after initially assessed progression) will 
discontinue study medication and enter the follow up/survival phase of the study. If progression is 
confirmed, then the date of disease progression will be the first date the subject met the criteria for 
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12.2.2 Discontinuation Criteria for Nivolumab 
• 
Any Grade 2 drug-related uveitis or eye pain or blurred vision that does not respond to 
topical therapy and does not improve to Grade 1 severity within the re-treatment period OR 
requires systemic treatment. 
• 
Any Grade 3 non-skin, drug-related adverse event lasting > 7 days, with the following 
exceptions for drug-related laboratory abnormalities: uveitis, pneumonitis, bronchospasm, 
diarrhea, colitis, neurologic toxicity, hypersensitivity reactions, and infusion reactions: 
- 
Grade 3 drug-related uveitis, pneumonitis, bronchospasm, diarrhea, colitis, neurologic 
toxicity, hypersensitivity reaction, or infusion reaction of any duration requires 
discontinuation. 
- 
Grade 3 drug-related laboratory abnormalities do not require treatment discontinuation 
except: 
♦ Grade 3 drug-related thrombocytopenia > 7 days or associated with bleeding requires 
discontinuation. 
♦ Any drug-related liver function test  (LFT)  abnormality that  meets  the  following criteria 
require discontinuation: 
o 
AST or ALT > 8 x ULN 
o 
Total bilirubin > 5 x ULN 
o 
Concurrent AST or ALT > 3 x ULN and total bilirubin > 2 x ULN. 
• 
Any Grade 4 drug-related adverse event or laboratory abnormality, except for the following 
events which do not require discontinuation: 
- 
Grade 4 amylase or lipase abnormalities that are not associated with symptoms or clinical 
manifestations of pancreatitis. It is recommended to consult with the PI or treating physician 
for Grade 4 amylase or lipase abnormalities. 
- 
Isolated Grade 4 electrolyte imbalances/abnormalities that are not associated with clinical 
sequelae and are corrected with supplementation/appropriate management within 72 hours of 
their onset. 
• 
Any dosing interruption lasting > 8 weeks from the last dose with the following exceptions: 
- 
Dosing interruptions to allow for prolonged steroid tapers to manage drug-related adverse 
events are allowed. Prior to re-initiating treatment in a subject with a dosing interruption 
lasting > 8 weeks, the PI or treating physician must be consulted. Tumor assessments 
should continue as per protocol even if dosing is interrupted. 
- 
Dosing interruptions > 8 weeks from the last dose that occur for non-drug-related reasons 
may be allowed if approved by the PI or treating physician. Prior to re-initiating treatment in a 
subject with a dosing interruption lasting > 8 weeks, the PI or treating physician must be 
consulted. Tumor assessments should continue as per protocol even if dosing is 
interrupted. 
• 
Any adverse event, laboratory abnormality, or intercurrent illness which, in the judgment of the 
Investigator, presents a substantial clinical risk to the subject with continued nivolumab a n d  
D C  dosing. 
 
12.2.3 Treatment of Nivolumab-Related Infusion Reactions 
Since nivolumab contains only human immunoglobulin protein sequences, it is unlikely to be 
immunogenic and induce infusion or hypersensitivity reactions. However, if such a reaction were to occur, 
it might manifest with fever, chills, rigors, headache, rash, pruritus, arthralgias, hypo- or hypertension, 
bronchospasm, or other symptoms. All Grade 3 or 4 infusion reactions should be reported within 24 hours 
to the PI and reported as an SAE if criteria are met. Infusion reactions should be graded according to NCI 
CTCAE (version 4) guidelines. 
 
Treatment recommendations are provided below and may be modified based on local treatment 
standards and guidelines as appropriate: 
 
For Grade 1 symptoms: (Mild reaction; infusion interruption not indicated; intervention not indicated) 


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Remain at bedside and monitor subject until recovery from symptoms. The following prophylactic 
premedications are recommended for future infusions: diphenhydramine 50 mg (or equivalent) and/or 
paracetamol 325 to 1000 mg (acetaminophen) at least 30 minutes before additional nivolumab 
administrations. 
 
For Grade 2 symptoms: (Moderate reaction requires therapy or infusion interruption but responds 
promptly to symptomatic treatment [e.g., antihistamines, non-steroidal anti-inflammatory drugs, narcotics, 
corticosteroids, bronchodilators, IV fluids]; prophylactic medications indicated for ≤ 24 hours). 
 
Stop the nivolumab infusion, begin an IV infusion of normal saline, and treat the subject with 
diphenhydramine 50 mg IV (or equivalent) and/or paracetamol 325 to 1000 mg (acetaminophen); remain 
at bedside and monitor subject until resolution of symptoms. Corticosteroid or bronchodilator therapy may 
also be administered as appropriate. If the infusion is interrupted, then restart the infusion at 50% of the 
original infusion rate when symptoms resolve; if no further complications ensue after 30 minutes, the rate 
may be increased to 100% of the original infusion rate. Monitor subject closely. If symptoms recur then no 
further nivolumab will be administered at that visit. Administer diphenhydramine 50 mg IV, and remain at 
bedside and monitor the subject until resolution of symptoms. The amount of study drug infused must be 
recorded on the case report form (CRF). The following prophylactic pre-medications are recommended 
for future infusions: diphenhydramine 50 mg (or equivalent) and/or paracetamol 325 to 1000 mg 
(acetaminophen) should be administered at least 30 minutes before additional nivolumab administrations. 
If necessary, corticosteroids (recommended dose: up to 25 mg of IV hydrocortisone or equivalent) may be 
used. 
 
For Grade 3 or Grade 4 symptoms: (Severe reaction, Grade 3: prolonged [i.e., not rapidly responsive to 
symptomatic medication and/or brief interruption of infusion]; recurrence of symptoms following initial 
improvement; hospitalization indicated for other clinical sequelae [e.g., renal impairment, pulmonary 
infiltrates]). Grade 4: (life-threatening; pressor or ventilatory support indicated). 
 
Immediately discontinue infusion of nivolumab. Begin an IV infusion of normal saline, and treat the 
subject as follows. Recommend bronchodilators, epinephrine 0.2 to 1 mg of a 1:1,000 solution for 
subcutaneous administration or 0.1 to 0.25 mg of a 1:10,000 solution injected slowly for IV administration, 
and/or diphenhydramine 50 mg IV with methylprednisolone 100 mg IV (or equivalent), as needed. Subject 
should be monitored until the investigator is comfortable that the symptoms will not recur. Nivolumab will 
be permanently discontinued. Investigators should follow their institutional guidelines for the treatment of 
anaphylaxis. Remain at bedside and monitor subject until recovery from symptoms. In the case of late-
occurring hypersensitivity symptoms (e.g., appearance of a localized or generalized pruritus within 1 
week after treatment), symptomatic treatment may be given (e.g., oral antihistamine, or corticosteroids). 
12.3 
End of Treatment 
 
Nivolumab and DC vaccines are given as described above for a total of 8 vaccines and nivolumab until 
progression.  Once all nivolumab doses and DC vaccinations have been administered, the treatment 
phase of the study will be over and the follow-up period will begin.  Please see Section 12.2.1 above for 
details in treating subjects beyond suspected progression. 
12.4 
Follow-up Period 
 
All patients will be followed for survival and data recorded by the study team. 
12.5 
End of Study 
 
Rationale for taking a patient off study will be documented (see section 12.2.2). All patients will be 
followed for survival and recorded by the study team. 


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12.6 
Early Withdrawal of Subject(s) 
12.6.1 Criteria for Early Withdrawal 
 
Subjects may voluntarily withdraw from the study at any time. The PI may also withdraw a subject from 
the study at any time based on his/her discretion. Reasons for PI-initiated withdrawal may include, but are 
not limited to the following: 
• 
Patients with an active infection requiring treatment or having an unexplained febrile illness 
(Tmax > 99.5 F). 
• 
Adverse events 
• 
Abnormal laboratory values 
• 
Unacceptable toxicity (see section 9.1.1). If a subject discontinues from one study drug 
(nivolumab or DC vaccine) due to unacceptable toxicity attributed to that study drug, the subject 
can continue on the other study drug at the discretion of the investigator. If in the investigator’s 
opinion the toxicity is related to both study drugs, the investigator can hold both study drugs.  
• 
Protocol deviation 
• 
Administrative issues 
• 
Disease progression 
• 
Pregnancy 
• 
Clinical indications for surgical resection prior to treatment plan. 
 
12.6.2 Follow-up Requirements for Early Withdrawal 
 
Subjects that received any nivolumab and/or vaccine therapy will be assessed and followed for serious 
adverse event monitoring/safety analysis for 30 days following the last infusion and/or vaccine (please 
see section 15.1 for safety analyses of the lead-in phase). 
12.6.3 Replacement of Early Withdrawal(s) 
 
Patients who terminate protocol treatment without experiencing an unacceptable adverse event or 
completing 3 vaccinations will be replaced. 
12.7 
Study Assessments 
12.7.1 Medical History 
 
Medical history will be obtained from the Duke electronic system and from the subject and/or family at the 
screening visit and reviewed at each study visit. This data may include the following: 
• 
All past medical and surgical history 
• 
Current medications  
• 
Changes in physical or neurologic symptoms 
• 
Any adverse events. 
12.7.2 Physical Exam 
 
Vital signs and physical and neurologic examinations will be assessed and recorded along with a KPS 
score prior to enrollment and at each visit. Vital signs will also be assessed prior to nivolumab and DC 
vaccines.  Lastly, vital signs will also be assessed after DC vaccine administration.   
 


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12.7.3 Immunologic Assessments 
Blood for serum CMV screen (1 red top tube) will be drawn prior to the 1st leukapheresis. Immunological 
response evaluations for baseline values will be conducted on the leukapheresis sample used to 
generate the DCs and on the second leukapheresis sample as well as from 1 red top tube done prior to 
each leukapheresis.  Peripheral blood drawn for immunologic monitoring (10 yellow and 1 red top tubes) 
will be drawn before surgery, before the fifth nivolumab infusion, and at progression (if feasible) in BOTH 
groups.  Group I will have blood drawn prior to the 12th and 18th infusions of nivolumab and Group 2 will 
have the blood drawn prior to the 7th vaccine. The total amount of blood required for 10 yellow/1 red top 
tubes will be about 100 mLs. Plasma from the yellow top tubes will be collected at each time point listed 
where immunologic monitoring is done.  Lastly, 10 yellow/1 red top tubes for immune monitoring will be 
drawn following the last vaccine (#8) at the next clinic visit prior to infusion of nivolumab monotherapy in 
both groups. After a year, an attempt will be made to obtain blood for immunologic monitoring 2-3 times a 
year at standard Duke neuro-oncology visits (10 yellow and 1 red top tubes). 
12.7.4 Correlative Assessments 
 
Testing will be performed by 'optimal batch testing'  in  which  samples  will be  thawed,  rested  overnight,  
and tested according to established/validated SOPs on file at Duke. All samples from each study 
participant will be analyzed together. The antigen to be tested will be pp65 with the CEF peptide and 
mitogen controls. 
 
The proportion of specific lymphocyte subsets and expression levels of T cell co-stimulatory markers in 
PBMC preparations and TILS will be quantified by flow cytometry. Analyses may include, but not 
necessarily be limited to, the proportion of T, B, and NK cells, granulocytes, the proportion of memory and 
effector T cell subsets, and expression levels of PD-1, PD-L1, other B7 family members, ICOS, and Ki67. 
 
Fresh tumor tissue will be collected and frozen for gene expression and T cell receptor sequencing.  The 
expression level of genes in TILs and whole blood samples that are associated with response to 
nivolumab will be assessed by quantitative molecular methods such as microarray and/or quantitative RT-
PCR analysis. Analysis may include, but will not necessarily be limited to, genes associated with immune-
related pathways, such as T cell activation, antigen processing and presentation, and apoptosis. 
 
As samples permit but not limited to soluble factors, such as cytokines, chemokines, soluble receptors, 
and antibodies to tumor antigens, will be characterized and quantified by immunoassays in serum. 
Analyses may include, but will not necessarily be limited to, soluble CD25, soluble PD-1, soluble LAG-3, 
CCL3, and CXCL-9. Collected serum samples will also be used for the assessment of tumor antigen-
specific responses elicited following treatment with and the combination therapy to explore which 
antitumor antibodies are most associated with clinical response. 
 
13 SAFETY MONITORING AND REPORTING 
 
The PI is ultimately responsible for the identification and documentation of adverse events and serious 
adverse events, 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. 
 
13.1 
Adverse Events 
 
An AE is any untoward medical occurrence in a subject receiving study drug (for purposes of this study, 
this is defined as nivolumab or DC vaccine) 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 


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abnormal laboratory finding), symptom, or disease temporally associated with the use of the nivolumab 
and/or DC vaccines, whether or not related. All abnormal laboratory findings that can be graded by the 
CTCAE version 4.0 will be collected as adverse events.  
 
From the time the subject signs consent through the End of Study visit (as defined in Section 12.6), all 
AEs must be recorded in the subject medical record and adverse events case report form. All SAEs will 
be recorded from the time consent is signed until 30 days from the end of study visit. 
 
AEs will be assessed according to the CTCAE version 4. 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 the study drugs) will be kept which 
will categorize the event by organ system, relationship to which treatment, its grade of severity, and 
resolution. Periodic review by the PI and monthly review at the PRTBTC Adverse Event meeting of the 
collective adverse events will occur with the intention of identifying any trends or patterns in toxicity. If any 
such trends are identified, depending on their severity and frequency, a protocol amendment will be 
considered. 
13.2 
Serious Adverse Events 
 
An AE is considered “serious” if in the opinion of the investigator it is one of the following outcomes 
following the initiation of study intervention (nivolumab and DC vaccine): 
 
- 
Fatal 
- 
Life-threatening 
- 
Constitutes a congenital anomaly or birth defect 
- 
A medically significant condition (defined as an event that compromises subject safety or may 
require medical or surgical intervention to prevent one of the three outcomes above). 
- 
Requires inpatient hospitalization or prolongation of existing hospitalization 
- 
Results in persistent or significant incapacity or substantial disruption to conduct normal life 
functions. 
13.2.1 Reporting of SAEs 
 
All SAEs that are thought to be possibly, probably, or definitely related to study treatment (including 
leukapheresis) should be reported immediately to Dr. Katherine Peters (Pager: 919-970-7591) or her 
designee (919-684-8111) and to the FDA. Fatal or life-threatening, unexpected adverse events will be 
reported to the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 7 calendar 
days after first knowledge by the sponsor followed by as complete a report as possible within 8 additional 
calendar days. Serious, unexpected adverse events that are not fatal or life-threatening will be reported to 
the FDA by telephone, facsimile, or in writing as soon as possible, but no later than 15 calendar days 
after first knowledge by the sponsor. 
 
All Serious Adverse Events must be reported to BMS Worldwide Safety. The sponsor/investigator will be 
required to reconcile SAEs reported in the clinical database with SAE cases transmitted to BMS Global 
Pharmacovigilance (GPV&E); worldwide.safety@bms. BMS requests this is initiated by the sponsor 


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investigator up to quarterly and prior to the database lock or final data summary. The process will be 
further defined. During reconciliation, any events found to not be reported previously to BMS must be sent 
to Worldwide.Safety@BMS.com.   
 
Potential drug induced liver injury (DILI) is also considered an important medical event. Wherever 
possible, timely confirmation of initial liver-related laboratory abnormalities should occur prior to the 
reporting of a potential DILI event.  All occurrences of potential DILIs, meeting the defined criteria, must 
be reported as SAEs. Potential drug induced liver injury is defined as: 
1. ALT or AST elevation > 3 times upper limit of normal (ULN)  
AND 
2. Total bilirubin > 2 times ULN, without initial findings of cholestasis (elevated serum alkaline 
phosphatase) 
AND 
No other immediately apparent possible causes of AST/ALT elevation and hyperbilirubinemia, including, 
but not limited to, viral hepatitis, pre-existing chronic or acute liver disease, or the administration of other 
drug(s) known to be hepatotoxic. 
 
Any overdose or pregnancy is considered a SAE regardless of CTCAE grade level and will be 
documented and reported. 
  
All adverse events that are considered serious, unanticipated, and related or possibly related to the 
research (as defined by 21CRF312.32[a]) will be reported to the Duke University Medical Center IRB and 
FDA using the appropriate SAE reporting process. At the time of the annual progress report to the Duke 
University Medical Center IRB and the FDA, a summary of the overall toxicity experience will be provided, 
including SAEs that are felt to be unlikely or unrelated to study treatment. 
13.3 
Safety Oversight Committee (SOC) 
 
The DCI SOC 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 SOC is review of safety data, toxicities and new information that may affect subject safety or efficacy. 
Annual safety reviews include but may not be limited to review of safety data, enrollment status, stopping 
rules if applicable, accrual, toxicities, reference literature, and interim analyses as provided by the 
sponsor-investigator. The SOC in concert with the DCI Monitoring Team (see Section 14.1 for Monitoring 
Team description) oversees the conduct of DUHS cancer-related, sponsor-investigator therapeutic 
intervention and prevention intervention studies that do not have an external monitoring plan, ensuring 
subject safety and that the protocol is conducted, recorded and reported in accordance with the protocol, 
SOPs, GCP, and applicable regulatory requirements. 
 
13.4 
External Data and Safety Monitoring Board (DSMB) 
 
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. 
 


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Due to potential for COI in relation to proprietary interest in the pp65CMV DC vaccine, a Data Safety and 
Monitoring Board (DSMBplus) has been established.  Please see Appendix – Section 18.6 for detail on 
the Duke PRTBTC DSMBplus Charter. 
 
14 QUALITY CONTROL AND QUALITY ASSURANCE 
14.1 Monitoring 
 
The DCI Monitoring Team will conduct monitoring visits to ensure subject safety and to ensure that the 
protocol is conducted, recorded, and reported in accordance with the protocol, standard operating 
procedures, good clinical practice, and applicable regulatory requirements.  As specified in the DCI Data 
and Safety Monitoring Plan, the DCI Monitoring Team will conduct routine monitoring after the third 
subject is enrolled, followed by annual monitoring of 1 – 3 subjects until the study is closed to enrollment 
and subjects are no longer receiving study interventions that are more than minimal risk.   
 
Additional monitoring may be prompted by findings from monitoring visits, unexpected frequency of 
serious and/or unexpected toxicities, or other concerns and may be initiated upon request of DUHS and 
DCI leadership, the DCI Cancer Protocol Committee, the SOC, the sponsor, the Principal Investigator, or 
the IRB.  All study documents must be made available upon request to the DCI Monitoring Team and 
other authorized regulatory authorities, including but not limited to the National Institute of Health, 
National Cancer Institute, and the FDA.  Every reasonable effort will be made to maintain confidentiality 
during study monitoring. 
 
14.2 
Audits 
 
The Duke School of Medicine CTQA office may conduct audits to evaluate compliance with the protocol 
and the principles of GCP.  The PI agrees to allow the CTQA auditor(s) direct access to all relevant 
documents and to allocate his/her time and the time of the study team to the CTQA auditor(s) in order to 
discuss findings and any relevant issues. 
 
CTQA audits are designed to protect the rights and well-being of human research subjects. CTQA 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. 
 
CTQA 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 


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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 will be the primary source document for the study. Source documents 
include all information in original records and certified copies of original records of clinical findings, 
observations, or other activities in a clinical investigation used for reconstructing and evaluating the 
investigation.1 Source documentations may also include paper eligibility checklists, data flowsheets, 
patient reported outcomes and other paper documents. The PI, study coordinator, study research nurse, 
data management team and all associated study key personnel, are permitted to make entries, changes, 
or corrections in the source documents or database per the study delegation of authority log. 
 
Errors on the source documents will be crossed out with a single line, and this line will not obscure the 
original entry. Changes or corrections will be dated, signed, initialed, and explained (if necessary). 
Database changes will be tracked via electronic trail automatically. 
14.3.3 Data Management Procedures and Data Verification 
 
The DCI IT Shared Resource has developed Title 21 CFR Part 11 compliant databases for cancer clinical 
trials. DCI IT has extensive expertise in database quality assurance, data standards, and use of 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 
 
                                                     
1 In 21 CFR 312.62(b), reference is made to records that are part of case histories as “supporting data;” the 
ICH guidance for industry E6 Good Clinical Practice: Consolidated Guidance (the ICH E6 guidance) 
(available 
at 
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm) 
uses 
the term “source data/documents.” For the purpose of this guidance, these terms describe the same 
information and have been used interchangeably. 


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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. 
 
Patients will be randomized to one of two treatment arms – Group I and Group II.  In Group I, patients will 
receive nivolumab every 2 weeks for approximately 8 weeks followed by surgery.  Following resection, 
nivolumab and DC vaccine will be administered every 2 weeks followed by QOW treatment with 
nivolumab and monthly DC vaccinations. In Group II, patients will initially receive the 4th cycle of 
nivolumab followed by nivolumab and DC vaccine every 2 weeks for a total of 3 vaccines for 
approximately 6 weeks, and then resection.  Subsequent to resection, the patient will resume QOW 
treatment with nivolumab and monthly DC vaccinations.  In both arms, a total of 8 DC vaccinations will be 
administered after which the administration of nivolumab will be continued QOW.   
 
15.1 
Analysis Sets 
 
The primary safety analyses will focus on all patients, regardless of treatment arm, who complete at least 
3 vaccinations in combination with nivolumab treatment, or terminate combination protocol treatment 
earlier after having experienced an unacceptable toxicity.  Additional secondary safety analyses will 
include any patient who receives any DC vaccination. 
 
Efficacy analyses will be conducted among patients who receive any nivolumab treatment and are able to 
tolerate nivolumab. 
 
Correlative analyses will primarily focus on patients who undergo resection and have DCs or PBLs that 
meet release criteria.  A patient within Group I who withdraws from protocol treatment after surgery will be 
included in the correlative analyses if the DC vaccine met the release criteria whether or not it was 
administered. 
15.2 
Patient Demographics and Other Baseline Characteristics 
 
The socio-demographic and clinical characteristics of subjects randomized to each of the two treatment 
arms will be summarized using descriptive statistics (e.g. means/standard deviations/percentiles or 
frequencies).  
15.3 
Treatments 
 
For each treatment arm, the number of doses of nivolumab and the number of patient vaccinations 
administered will be summarized with a frequency distribution. 
15.4 
Primary Objective 
 
The primary objective of this study is to assess the safety of administering DC vaccines with nivolumab 
for the treatment of bevacizumab-naïve subjects with first or second recurrent, resectable WHO Grade III 
and IV malignant gliomas. 
15.4.1 Variable 
To assess the safety of the combination of DC vaccination and nivolumab, the percentage of patients who 
experience unacceptable toxicity during combination treatment (i.e. DC vaccination + nivolumab) will be 
tabulated.   
 
Unacceptable toxicity is defined in Section 9.1.1.  


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15.4.2 Statistical Hypothesis, Model, and Method of Analysis 
15.4.2.1 
Statistical Hypothesis, Model, and Method of Analysis 
As described in section 15.8, the goal of the study is to determine whether the addition of the DC vaccine 
to nivolumab treatment increases the prevalence of unacceptable toxicity.  Topalian et al.[112] reports 
that 14% (approximate 95% confidence interval:  10%, 18%) of patients treated with nivolumab alone 
experienced a grade 3 or 4 adverse event.  If the true unacceptable toxicity rate with the combination 
treatment is approximately 10%, then the treatment is considered safe.  However, if the true unacceptable 
toxicity rate is 35% or greater, the treatment combination as prescribed in this protocol is not safe.  A one-
sample binomial test will be used to differentiate between an unacceptable toxicity rate of 10% and 35%.  
Further details are provided in section 15.7.  In addition, a 90% exact binomial confidence interval for the 
unacceptable toxicity rate will be generated. 
15.4.2.2 
Related Statistical Analyses 
Adverse events experienced by protocol subjects during combination treatment (nivolumab + DC vaccine) 
will also be summarized in several other forms to satisfy scientific and monitoring needs, as well as 
various regulatory reporting needs (e.g. FDA, DCI SOC, and ClinicalTrials.gov): 
 
• 
Among all patients who receive any combination protocol treatment, the frequency of adverse 
events that are possibly, probably, or definitely related to protocol treatment will be tabulated by 
the maximum grade for each type of adverse event. 
 
• 
Among all patients who receive any combination protocol treatment, the frequency of adverse 
events regardless of attribution will be tabulated by the maximum grade for each type of adverse 
event. 
 
• 
Among patients who terminate combination treatment due to an unacceptable toxicity or receive 
at least 3 vaccinations, the frequency of adverse events that are possibly, probably, or definitely 
related to protocol treatment during the 8 vaccinations will be tabulated by the maximum grade for 
each type of adverse event.   
 
• 
Among patients who terminate combination treatment due to an unacceptable toxicity or receive 
at least 3 vaccinations, the frequency of adverse events regardless of attribution to protocol 
treatment during the 8 vaccinations will be tabulated by the maximum grade for each type of 
adverse event. 
 
Given that nivolumab remains an investigational agent too, there will be a need to summarize adverse 
events experienced during the period of time when nivolumab is administered alone. Tabulations that 
summarize all adverse events as well as tabulations that summarize those that are possibly, probably, or 
definitely related to nivolumab treatment will be generated. 
15.4.3 Handling of Missing Values, Censoring, and Discontinuations 
If a patient terminates protocol treatment without experiencing an unacceptable toxicity and without 
receiving 3 vaccinations, the patient will be replaced for the primary analyses. 
15.5 
Secondary Objective 
 
The secondary objective of this study is to describe the survival and PFS of subjects treated with any 
protocol treatment (i.e. nivolumab or DC vaccines).  Survival is defined as the time between first initiation 
of nivolumab treatment and death, or last follow-up if the patient remains alive.  PFS is defined as the 
time between treatment initiation and initial progression or death, or date of last follow-up if the patient 
remains alive without disease progression.  The Kaplan-Meier estimator will be used to describe the OS 
and PFS experience of all patients.  Median OS and PFS will also be calculated.  Patients who are not 
able to tolerate nivolumab and are removed from the study will not be included in these analyses.  
Patients for whom DC vaccines cannot be manufactured will not be included in the survival analyses. 


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Though the primary analysis for this secondary objective will include all patients, additional exploratory 
analyses may be conducted.  OS and PFS will be examined within each assigned treatment group using 
an intent-to-treat approach.  Given the small sample size, any comparison of the treatment arms will have 
limited power to detect clinically meaningful differences.  Additional descriptive analyses may also be 
conducted, including an examination of OS/PFS from resection. 
15.6 
Exploratory Objectives 
 
Given the rapidly advancing field of check-point block inhibitors, additional exploratory statistical analyses 
related to immunologic function may be conducted in conjunction with the analyses described below. 
 
Many of these analyses will primarily focus on patients who undergo resection and have DCs or PBLs 
that meet release criteria.   
15.6.1 Exploratory Objective #1:  Immunologic Response to pp65 
Within each group, changes between baseline and a pre-resection assessment for various measures of 
immune response derived from PBMC plasma/serum will be described.  When appropriate, these 
descriptive statistics may include mean and standard deviation, or medians and quantiles.   
 
Within each group, a paired t-test will be conducted to assess the impact of nivolumab (Group I) or 
nivolomab with DC vaccination (Group II) on these changes in immune function.  Assuming normality is 
not appropriate, a Wilcoxon signed-rank test will be conducted. 
 
Analysis of covariance will be used to compare treatment groups with respect to change, with the 
baseline measure serving as a covariate.  If normality is not an appropriate assumption, a Wilcoxon rank 
sum test will compare treatment groups with respect to the change in the various measures of immune 
response that are of interest. 
15.6.2 Exploratory Objectives #2: T-cell Costimulatory Markers 
Analyses similar to those described in section 15.6.1 will be conducted to assess the impact of nivolumab 
and DC vaccination on T-cell costimulatory markers. 
15.6.3 Exploratory Objectives #3: Cytokines and Other Soluble Factors 
Analyses similar to those described in section 15.6.1 will be conducted to assess the impact of nivolumab 
and DC vaccination on cytokines and other soluble factors. 
15.6.4 Exploratory Objectives #4: Gene Expression 
Gene expression will be measured in PBMC samples collected serially from patients, as well as from 
resected tumor.  Analyses similar to those described in section 15.6.1 will be used to assess the impact of 
treatment on changes in gene expression as measured from PBMC samples.  A two-sample t-test or 
Wilcoxon rank sum test will compare groups with respect to gene expression as measured within tumors. 
15.6.5 Exploratory Objectives #5: Epitope Spreading 
The goal of this objective is to assess whether evidence exists for epitope spreading intratumorally or 
systemically after vaccine and anti-PD-1 treatment. Among all patients with pre- and post-treatment 
collection of PBMCs, TCR repertoires of PBLs (pre- and post-treatment) will be assessed. The post-
treatment collection occurs before resection.  For each sample, we will compute the total number of 
unique clones. Within each group, a comparison of TCR repertoires pre- and post-treatment is of interest. 
To test whether the paired samples have different variances with respect to the number of unique TCR 
clones, the Fligner-Killeen median test for homogeneity of variance will be used. This test is robust 
against departures from normality. To assess the distribution of T cell clonal frequency, Shannon’s 
entropy normalized by log2 (# of TCR unique clones) will be computed. Wilcoxon signed-rank test will 
compare the paired measures of entropy. The proportion of TCR overlap between matched specimens 


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will be calculated as the ratio of the number of TCR sequencing reads from clones found in both samples 
and the number of total TCR sequencing reads from both samples.  Wilcoxon rank sum test will be used 
to compare groups with respect to post-treatment measures of entropy for PBLs.  
 
The TCR repertoire of TILs obtained from the resection tissue will be assessed, and Shannon’s entropy 
will be computed as described above.  Wilcoxon rank sum test will be used to compare groups with 
respect to post-treatment measures of entropy for TILs.   
15.7 
Interim Safety Analysis 
Rigorous monitoring of toxicities in a manner similar to that found in many Phase I dose-escalation 
studies would require accrual suspension between stages of patient accrual while relevant data matures. 
Accrual will not be suspended to formally assess the toxicity profile unless the following guidelines are 
satisfied. Rather, the study will be monitored continuously for the occurrence of unacceptable adverse 
events, with the primary focus being on adverse events occurring during combination treatment (i.e. DC 
vaccination + nivolumab).  Included in these analyses are patients who have completed at least 3 
vaccinations in combination with nivolumab or terminated combination treatment earlier after having 
experienced an unacceptable adverse event. 
 
Aggregate summaries of adverse experiences will be generated and reviewed by the clinical team every 
3-6 months for the first year, and then every 6-12 months thereafter. 
 
Tabulated below are the conditions under which accrual will be temporarily suspended and data carefully 
reviewed to determine the appropriate action, including permanent study termination, continuation with 
patient accrual after appropriate amendment (including possibly a change in monitoring rules), or 
continuation with patient accrual with no modification of the protocol. Accrual will also be suspended 
whenever a death occurs that is possibly, probably, or definitely related to treatment with vaccine and 
nivolumab. 
 
Table 3. Conditions for Temporary Accrual Suspension 
Number of patients who 
have received 3 DC 
vaccinations or 
experienced an 
unacceptable adverse 
event during combination 
treatment 
Number of 
patients with 
unacceptable 
toxicity requiring 
accrual 
suspension 
6-7 
≥3 
8-10 
≥4 
11-14 
≥5 
15-20 
≥6 
21-24 
≥7 
≥25 
≥8 
 
These guidelines have not been adjusted for differential length of follow-up of accrued patients. The 
probability of accrual suspension as a function of the true unacceptable toxicity rate is tabulated below 
based upon simulation studies. These statistics were generated assuming toxicity outcome was known at 
the time of accrual, and ignored issues such as time to toxicity, accrual rate, and length of follow-up. 
 
Table 4. Probability of Accrual Suspension as a Function of the True Unacceptable Toxicity Rate 
Underlying 
unacceptable 
toxicity rate 
Probability of 
accrual 
suspension 
Expected # of 
Patients 
0.01 
<0.0001 
30 


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0.05 
0.004 
29.9 
0.1 
0.039 
29.1 
0.15 
0.15 
27.4 
0.2 
0.35 
24.2 
0.25 
0.58 
20.6 
0.3 
0.79 
16.5 
0.35 
0.91 
13.1 
0.4 
0.97 
10.7 
0.45 
0.99 
9.0 
0.5 
>0.99 
7.9 
 
Patients who terminate combination protocol treatment without experiencing an unacceptable toxicity 
during combination treatment and without receiving 3 vaccinations will be replaced for the evaluation of 
the primary endpoint. 
15.8 
Sample Size Calculation 
 
Topalian, et al.,[112] reports that 14% (approximate 95% confidence interval:  10%, 18%) of patients 
treated with nivolumab alone experienced a grade 3 or 4 adverse event. If the true unacceptable toxicity 
rate with the combination treatment is approximately 10%, then the treatment is considered safe.  
However, if the true unacceptable toxicity rate is 35% or greater, the treatment combination is not safe as 
prescribed in this protocol.  Based upon simulation studies with 30 subjects using the decision rules 
described in section 15.7, the probability of terminating accrual assuming a true unacceptable toxicity rate 
of 10% and 35% is 0.039 and 0.91, respectively. 
 
With the 6-month progression-free survival rate of GBM patients standardly treated after one recurrence 
being 42.6% (Friedman, JCO, 2009), we anticipate that approximately 50% of patients will drop out 
between enrollment and the 3rd vaccination.  In order to attain the goal of accruing 30 patients that will be 
included in the primary analyses, approximately 66 patients will be enrolled onto the study. 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
16.1 Regulatory and Ethical Compliance 
 
This protocol was designed and will be conducted and reported in accordance with the International 
Conference on Harmonization 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 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. 


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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 or her designee must obtain 
written informed consent from the subject or a legally acceptable representative.  The original informed 
consent form will be stored with the subject’s study records, and a copy of the informed consent form will 
be provided to the subject.  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 Study Documentation 
 
Study documentation includes but is not limited to source documents, CRFs, 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. 
 
A CRF will be the primary data collection document for the study.  The CRFs will be updated within two 
weeks of acquisition of new source data.  Only the Principal Investigator and study coordinator, research 
nurses, and investigators, are permitted to make entries, changes, or corrections in the CRF.  For paper 
CRFs, errors 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).  The Principal Investigator 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 utilized at Duke. 
16.5 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 


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research staff, the patient and his family, if desired. For all future visits, interactions with research staff 
(study doctor and study coordinators) regarding research activities will take place in a private exam room. 
All research related interactions with the participant will be conducted by qualified research staff who are 
directly involved in the conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock and key 
accessible only by the research staff. Subjects will be identified only by a unique study number and 
subject initials. Electronic records of subject data will be maintained using a Title 21 CFR Part 11 
Compliant Clinical database, which is housed by the DCI. Access to electronic databases will be limited to 
the Principal Investigator, key personnel, statisticians, the Radiolabeled Pharmacy personnel, and the 
PRTBTC data manager. Data stored on portable memory devices will be de-identified. The security and 
viability of the IT infrastructure will be managed by the DCI and/or Duke Medicine.  
 
Upon completion of the study, research records will be archived and handled per DUHS HRPP policy.  
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
 
16.6 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 Section 13.4 and Section 14. 
16.7 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. 
16.8 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 (if an 
IND is involved) 
- 
at least two years after formal withdrawal of the IND associated with this protocol (if an IND is 
involved) 
- 
at least six years after study completion (Duke policy). 
16.9 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 Research Integrity Office (RIO) reviews and manages 
research-related conflicts of interest.  The Principal Investigator and Sub-Investigators must report 
conflicts of interest annually and within 10 days of a change in status, and when applicable, must have a 
documented management plan that is developed in conjunction with the Duke RIO and approved by the 
IRB/IEC. 


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16.10 Registration Procedure 
 
After patients have been enrolled, subject registration will be entered into the Duke Velos eResearch 
system and the subject’s visits associated in the Duke Epic Maestro Care system with this protocol which 
is entered after Duke IRB approval. 
 
 
 


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18 APPENDICES 
 
18.1 
Nivolumab Investigator Brochure(s) 
Please see separate upload in eIRB. 


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18.2 
I-O Safety Algorithms 
Please see separate upload in eIRB. 
 


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18.3 
PRTBTC Imaging SOP 
 
Please see separate upload in eIRB. 
 
 
 


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18.4 
CTRAF 
Please see separate upload in eIRB. 
 


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18.5 
MMSE 
Please see separate upload in eIRB. 
 


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18.6 
PRTBTC DSMBplus Charter 
Please see separate upload in eIRB.