Conditions: Glioblastoma Astrocytoma, Grade IV Giant Cell Glioblastoma Glioblastoma Multiforme
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 64
Healthy volunteers: no

Brief summary:
This randomized phase II study will assess the impact of pre-conditioning on migration and survival among newly diagnosed glioblastoma (GBM) patients who have undergone definitive resection and completed standard temozolomide (TMZ) and radiation treatment, as well as the impact of tetanus pre-conditioning and basiliximab together on survival. After completing standard of care radiotherapy with concurrent TMZ, patients will be randomized to 1 of 3 treatment arms: 1). receive cytomegalovirus (CMV)-specific dendritic cell (DC) vaccines with unpulsed (not loaded) DC pre-conditioning prior to the 4th vaccine; 2). receive CMV-specific DC vaccines with Tetanus-Diphtheria Toxoid (Td) pre-conditioning prior to the 4th vaccine; 3). receive basiliximab infusions prior to the 1st and 2nd DC vaccines along with Td pre-conditioning prior to the 4th vaccine. A permuted block randomization algorithm using a 1:1:1 allocation ratio will be used to assign patients to a treatment arm. Randomization will be stratified by CMV status (positive, negative), with the assignment to arms I and II being double-blinded. Effective March 2017, randomization to Group III has been terminated.

Detailed description:
A maximum of 100 patients with resected, newly-diagnosed World Health Organization (WHO) Grade IV GBM will be enrolled in this study with the expectation that approximately 79 patients will be randomized to subsequent treatment after completion of radiation treatment with concurrent temozolomide. Effective March 2017, randomization to Group III has been terminated. All consented patients will undergo a leukapheresis after resection for harvest of Peripheral Blood Lymphocytes (PBLs) for generation of DCs. Patients will then receive Radiation Therapy (RT) and concurrent TMZ at a standard targeted dose of 75 mg/m\^2/d. Patients should start RT within approximately 6 weeks of surgery. Patients who experience progressive disease during radiation, are dependent on steroid supplements above physiologic levels at time of first vaccination, are unable to tolerate TMZ, or whose DCs or PBLs fail to meet release criteria will be withdrawn from the study and replaced and will not undergo repeat leukapheresis. For patients whose initial leukapheresis yields less than 3 vaccines, repeat leukapheresis may be obtained a minimum of 2 weeks from the previous leukapheresis (and may be repeated as needed) if pre-pheresis blood work is within the Apheresis Center's parameters and as long as this does not cause a significant delay in treatment for the patient.

After RT and concurrent TMZ, patients will then be randomized and begin the initial cycle of TMZ at a standard targeted dose of 150-200mg/m\^2/d for 5 days at the discretion of the treating oncologist 4 (± 2) weeks after completing RT. The study cycle of TMZ comprises a targeted dose of 150-200mg/m\^2/d for 5 days every 5 (± 1) weeks. All patients will receive up to a total of 10 DC vaccines given bilaterally at the groin site unless progression occurs. DC vaccines will be given intradermally (i.d.) and divided equally to both inguinal regions. DC vaccines #1-3 will be given every two weeks, thus delaying the initiation of TMZ cycle 2. Patients will then be vaccinated in conjunction with subsequent TMZ cycles every 5 (± 1) weeks for a total of 6 to 12 cycles after RT at the discretion of the treating oncologist. DCs will be given on day 21 ± 2 days of each TMZ cycle. DC vaccinations will continue during TMZ cycles up to a total of 10 unless progression occurs.

Before the first DC vaccination, all patients will receive immunization with 0.5 mL of Td intramuscularly into the deltoid muscle to ensure adequate immunity to the tetanus antigen. Those assigned to Group III will receive basiliximab 20 mg infusions 1 week before the 1st and 1 week before the 2nd vaccine. At the time of the fourth DC vaccine, patients will receive pre-conditioning per the assigned group (Group I-unpulsed DCs i.d.; Group II- Td i.d.; Group III-Td i.d.). A single dose of Td toxoid (1 flocculation unit, in 0.3 milliliters (mLs) of saline for a total volume of 0.4 mLs) or 0.4 mLs of 1 x 10\^6 autologous unpulsed DCs in saline will be administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral side 12-24 hours prior to the fourth DC vaccine, which is always given bilaterally at the groin site. Patients in Groups I and II will then receive 111In-labeled DCs to compare the effects of different skin preparations on DC migration followed by Single-Photon Emission Computed Tomography and Computed Tomography (SPECT/CT) imaging immediately and at 1 and 2 days after injection. Group III will not undergo migration studies. Groups I and II will be double blinded. Group III will not be blinded.

All patients will undergo leukapheresis again for immunologic monitoring with specific assessment of baseline antigen-specific cellular and humoral immune responses and further DC generations 4 (± 2) weeks after vaccine #3. Patients will be imaged bimonthly without receiving any other prescribed anti-tumor therapy. Patients will undergo an additional leukapheresis for generation of DCs if needed to continue vaccinations.

As part of standard care for these patients, upon tumor progression, participants may undergo stereotactic biopsy or resection. As this is not a research procedure consent will be obtained separately. However, if tissue is obtained, it will be used to confirm tumor progression histologically and to assess immunologic cell infiltration and pp65 antigen escape at the tumor site.

Arms:
arm 1: 0.4 mLs of 1 x 10\^6 autologous unpulsed DCs in saline will be administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral side 1 day prior to the 4th human CMV pp65-LAMP mRNA-pulsed autologous DCs vaccine. pp65 DC Vaccine #4 is 111In-labeled DCs for migration studies. arm 2: Tetanus diptheria toxoid (Td) (1 flocculation unit) will be administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral side 1 day prior to the 4th human CMV pp65-LAMP mRNA-pulsed autologous DCs vaccine. pp65 DC Vaccine #4 is 111In-labeled DCs for migration studies. arm 3: Basiliximab infusions prior to human CMV pp65-LAMP mRNA-pulsed autologous DCs vaccines #1 and #2 with Td pre-conditioning (1 flocculation unit) will be administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral side 1 day prior to the 4th human CMV pp65-LAMP mRNA-pulsed autologous DCs vaccine.

Interventions: intervention 1: Unpulsed DCs intervention 2: Td intervention 3: Human CMV pp65-LAMP mRNA-pulsed autologous DCs intervention 4: 111In-labeled DCs intervention 5: Temozolomide intervention 6: Saline intervention 7: Basiliximab
Intervention descriptions: intervention 1: Patients in Group I will receive 1 x 10\^6 autologous unpulsed DCs in saline administered to a single side of the groin intradermally 1 day before the fourth vaccine. intervention 2: Patients in Groups II and III will receive a single dose of Td toxoid (1 flocculation unit, Lf, in 0.4 mLs) administered to a single side of the groin given intradermally 1 day before the fourth vaccine. intervention 3: 2x10\^7 human CMV pp65-LAMP mRNA-pulsed autologous DCs are given intradermally and bilaterally at the groin site (divided equally to both inguinal regions). Patients will receive up to a total of 10 DC vaccines. intervention 4: 111In-labeled DCs are 2 x 10\^7 pp65-LAMP mRNA loaded mature DCs will be labeled with 111In (50 μCi / 5 x 10\^7 DCs) and given i.d. as fourth vaccine for Groups I and II only. intervention 5: Temozolomide is a standard chemotherapy given to all enrolled patients at a targeted dose of 150-200mg/m2/d for 5 days every 5 (± 1) weeks for a total of 6 to 12 cycles at the discretion of the treating oncologist. intervention 6: 0.4mL of saline given in the opposite groin 1 day before the fourth vaccine in all groups intervention 7: Group III will receive basiliximab infusions (20 mg I.V) 1 week before the first vaccine and 1 week before the second vaccine.

Protocol document:
Prot_SAP_000.pdf: 
NCT02366728 
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 
 
 
 
ELEVATE 
Evaluation of overcoming Limited migration and Enhancing 
Cytomegalovirus-specific dendritic cell Vaccines with 
Adjuvant TEtanus pre-conditioning in patients with newly-
diagnosed glioblastoma  
 
 
 
 
 
 
 
Sponsor: 
PI – Duke Cancer Institute 
 
 
Funding Source: 
NIH Grant 
 
 
Protocol Source: 
PI - Duke Cancer Institute 
 
 
Duke IRB#: 
Pro00054740 
 
 
IND#: 
 
16301 
 
 
Principal Investigator 
Dina Randazzo, DO 
dina.randazzo@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., Ph.D. 
darell.bigner@duke.edu 
Katherine Peters, M.D. 
katherine.peters@duke.edu 
Kristen Batich 
kristen.batich@dm.duke.edu 
John Sampson, M.D., Ph.D. 
john.sampson@duke.edu 
Elizabeth Reap, Ph.D. 
elizabeth.reap@duke.edu 
 
Statistician 
James E. Herndon II, Ph.D. 
james.herndon@duke.edu 
Lead Study Coordinator 
Jordan Parker, MS 
jordan.parker@duke.edu 
Regulatory Coordinator 
Rachel Hesler, Ph.D. 
rachel.hesler@duke.edu 
Therapeutic Material 
Manufacture Manager 
Gary Archer, Ph.D. 
gary.archer@duke.edu 
Data Manager 
Eric Lipp 
eric.lipp@duke.edu 
 
 
Original version: 
20140722 
 
Amended version 1: 
Date: 20140818 (CPC 
Mods) 
 
Amended version 2: 
Date: 20150109 (FDA 
Mods) 
 
Amended version 3: 
Date:20150506 (IRB Mods) 
 


ELEVATE 
Version: 20181120 
 
Amended version 4 
(AMD 001): 
Date: 20150518 (PI-
initiated) 
Randomization moved up; Blinding of pre-conditioning; tissue 
testing; updated information on where to send samples; 
Hepatitis C and autoimmune disease exclusion criteria added; 
and clarification on RT/TMZ therapy requirements for 
participation. 
Amended version 5 
(AMD 005): 
Date: 20150702 (PI-
initiated) 
Added a 3rd arm to provide basiliximab (Sections: 6, 7.2, 7.3, 8, 
9.1, 9.4, 10, 12.2, 12.6, 12.7, 13.1, 15, and 18.7) 
Added Curran Group Status to eligibility criteria (Section 11 
and 12.7.2) 
Updated language on source docs (Section 12.1) 
Updated withdrawal section to match TMZ treatment plan 
(Section 12.6) 
Updated emergency unblinding language (Section 13.3) 
Updated objectives with additional arm (Sections 8, 9, 12, and 
15) 
Made all subjects receive Td booster regardless of prior 
immunization history to mirror previous DC migration study 
(Section 9) 
Amended version 6 
(AMD 007) 
Date: 20150821 (PI-
initiated) 
To correct minor error on the cover page & update funding; to 
remove the company name of Td (Sections 9 & 10); correct the 
name of the pharmacy providing the Td (Sections 4, 9 & 10); 
correct cell concentration error (Section 10); correct wording on 
randomization (Section 9); and clarified treatment summary 
table by adding rows for leukapheresis and correcting blood 
draw errors (Section 12). 
Amended version 7 
(AMD 012) 
Date: 20151016 (PI-
initiated)  
To include 1 red top tube prior to each leukapheresis for 
immune monitoring (Section 12); to increase the enrollment and 
chemoradiation timeframe (Section 9, 12, & 18); corrected 
reference errors (Section 15). 
Amended version 8 
(AMD 014) 
Date: 20160210 (PI-
initiated) 
Clarified allowance of prevention of prior cancer treatment in 
exclusion criterion (Section 11); Use of antihistamines before, 
during and following vaccine administration recommendation 
(Section 12.7.3). 
Amended version 9 
(AMD 020) 
Date: 20160518 (PI-
initiated) 
Included blood to evaluate CCL3 levels prior to the Td booster 
given IM (Sections 12.2 & 12.7.5 ); allowed repeated 
leukapheresis if less than 3 vaccines generated from initial 
pheresis and again as needed for continued vaccinations, 
clarified that Tums is taken 3 times a day, and that a central line 
may be needed for the leukapheresis (Sections 9.1, 10.1 & 12.2 
), reduced the total number of vaccines from 20 to 10 (Sections 
6, 9.1, 12.2, and 12.3).  
Amended version 10 
(AMD 026) 
Date: 20160727 (PI-
initiated) 
Removed LSQ lab test from study (Section 12.2) and included 
the already planned CMP with removal of LSQ (Section 
18.4.2); added IHC assays to tumor samples already being 
obtained (Section 12.1). 
Amended version 11 
(AMD 027) 
Date: 20160806 (PI-
initiated) 
To update the location of the cell processing suite with 
inclusion of associated abbreviations (Sections 4, 10.3, and 
10.4). 
Amended version 12 
(AMD 029) 
Date: 20160819 (PI-
initiated) 
To allow consenting and RT/TMZ to occur within 6weeks of 
OR (Section 12.1). 
Amended version 13 
(AMD 32) 
Date: 20160915 (PI-
initiated) 
To remove reference to appendices with TMZ drug dosing 
information and removed section in appendices.  Instead, 
referred the reader to the TMZ package insert uploaded in eIRB 
(Sections 9.1 and 18.4). 
Amended version 14 
(AMD 34) 
Date: 20161017 (PI-
initiated) 
To include tumor analysis up front and at recurrence and add 
abbreviation for IDH (Sections 4 and 12). 
Amended version 16 
(AMD 45) 
Date:20170223 (PI- 
initiated) 
Changed the PI (title page, Sections 9.1.4, 12.1, 13.2.1, and 
13.3); changed the primary study coordinator and primary 
regulatory coordinator (title page); 
Amended version 15 
(AMD 49) 
Date: 20170324 (PI-
initiated) 
Randomization to Group III is closed due to results from a 
related study (Sections 9.1, 9.1.1, and 15); Due to the end to 


ELEVATE 
Version: 20181120 
 
enrollment in Group III, adjusted the projected number of 
subjects in the study from 102 to 79.  Up to 100 people may 
sign the consent to reach this goal (Sections 9.1, 9.1.1, and 15); 
Fixed inconsistencies in timing of Td Booster, which must 
occur before the DC vaccine #1 but can occur the same day as 
DC vaccine #1 (Sections 6, 9.1, and 12.7.5); Clarified the 
timing of blood draws for immune monitoring and subclinical 
autoimmunity at DC Vaccine #1 (Sections 12.2 and 12.7.5); 
Removed the blood draws for CCL3 levels pre-/ post-Td 
Booster, CCL3 is still measured from the IM blood taken at 
vaccine #1 and the SPECT/CT imaging visits (Sections 12.2 
and 12.7.5); Clarified that after cycle 1, all remaining TMZ 
cycles will be 5 (±1) weeks (Section 9.1); Clarified the Follow-
up Period for subjects (Sections 12.4 and 12.5); Clarified the 
recording of AEs (Section 13.1); Clarified MMSE and immune 
monitoring in Table 4 (Section 12.2); For baseline MRI, 
changed “standard 5mm slices with 2.5mm spacing” to “per 
standard of care” (Sections 12.1 and 12.2); Clarified inclusion 
criteria regarding MRI at time of consent (Section 11 and 12.1);   
Added “Patients should start RT within approximately 6 weeks 
of surgery” (Section 9.1) 
Amended version 16 
(AMD 58) 
Date: 20170523 (PI-
initiated) 
Made inclusion criteria for pregnancy more clear by clarifying 
that the test will be performed within 48 hours of leukapheresis, 
the first study-related procedure (Section 11);  Removed Curran 
as eligibility criteria but kept it as study assessment performed 
at time of consent (Sections 11 and 12.7.2); Clarified immune 
monitoring blood draws time points after completion of study 
vaccines (Sections 12, 12.4, and 12.7.5) 
Amended version 17 
(AMD 65) 
Date: 20170914 (PI-
initiated) 
Clarified language in inclusion criterion #1 regarding definitive 
resection (Section 11); Corrected error in Section 9.1.4 
regarding timing of recording AEs, which was missed in 
previous amendment (AMD 49); Clarified the information 
about subsequent therapies that needs to be recorded during 
follow-up period (Section 12.4); Clarified length of radiation is 
approximately 6 weeks to make Section 9 and Section 12.6.1 
consistent; Updated description of person delivering vaccine 
from MPACT to clinic and added that each vaccine will have a 
separate cooler (Section 10.4): Clarified dose adjustment of 
TMZ (Section 9.1.2) 
Amended version 18 
(AMD 73) 
Date: 20180406 (PI-
initiated) 
Study closed to enrollment (Section 9); Analysis plan updated 
(Section 15) 
Amended version 19 
(AMD 80) 
Date: 20181120 (PI-
initiated) 
Added that patient’s cells from the leukapheresis procedure can 
be transferred to the INTERCEPT trial (Pro00083828) if they 
become progressive and are eligible (Sections 9.1, 10.1, and 
12.3). 
 


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2 
 
 
1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ........................................................................................................ 2 
2 
LIST OF FIGURES ................................................................................................................ 6 
3 
LIST OF TABLES .................................................................................................................. 6 
4 
LIST OF ABBREVIATIONS ................................................................................................. 7 
5 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY ................................................ 10 
6 
STUDY SCHEMA................................................................................................................ 11 
7 
BACKGROUND AND SIGNIFICANCE ............................................................................ 12 
7.1 
Study Disease ................................................................................................................. 12 
7.2 
Study Agent .................................................................................................................... 12 
7.2.1 
Pre-Clinical Experience .......................................................................................... 18 
7.2.2 
Clinical Experience ................................................................................................. 20 
7.3 
Study Purpose/Rationale ................................................................................................ 24 
8 
OBJECTIVES AND ENDPOINTS ...................................................................................... 26 
9 
INVESTIGATIONAL PLAN ............................................................................................... 26 
9.1 
Study Design .................................................................................................................. 26 
9.1.1 
Randomization of Vaccine Site Pre-Conditioning Strategies ................................. 29 
9.1.2 
Definition of Unacceptable Toxicities .................................................................... 29 
9.1.3 
Dose Modification .................................................................................................. 29 
9.1.4 
Safety Considerations ............................................................................................. 29 
9.1.5 
Missed Doses .......................................................................................................... 30 
9.1.6 
Concomitant Medications ....................................................................................... 31 
9.1.7 
Study Drug(s) Blinding ........................................................................................... 31 
9.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration ............................. 31 
9.3 
Rationale for Correlative Studies ................................................................................... 31 
9.4 
Definition of Evaluable Subjects, On Study, and End of Study .................................... 32 
9.5 
Early Study Termination ................................................................................................ 32 
10 STUDY DRUG ..................................................................................................................... 32 
10.1 
Names, Classification, and Mechanism of Action ..................................................... 32 
10.2 
Packaging and Labeling of Study Agents................................................................... 35 
10.3 
Supply, Receipt, and Storage ...................................................................................... 35 


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10.4 
Dispensing and Preparation ........................................................................................ 36 
10.5 
Compliance and Accountability ................................................................................. 37 
10.6 
Disposal and Destruction ............................................................................................ 37 
11 SUBJECT ELIGIBILITY ..................................................................................................... 37 
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES........................................ 39 
12.1 
Screening Examination ............................................................................................... 39 
12.2 
Treatment Period ........................................................................................................ 40 
12.3 
End of Treatment ........................................................................................................ 42 
12.4 
Follow-up Period ........................................................................................................ 42 
12.5 
End of Study ............................................................................................................... 43 
12.6 
Early Withdrawal of Subject(s) .................................................................................. 43 
12.6.1 Criteria for Early Withdrawal ................................................................................. 43 
12.6.2 Follow-up Requirements for Early Withdrawal ..................................................... 43 
12.6.3 Replacement of Early Withdrawal(s) ...................................................................... 44 
12.7 
Study Assessments ..................................................................................................... 44 
12.7.1 Medical History ...................................................................................................... 44 
12.7.2 Physical Exam ......................................................................................................... 44 
12.7.3 Use of Antihistamines ............................................................................................. 44 
12.7.4 Radiologic Evaluations ........................................................................................... 44 
12.7.5 Immunologic Assessments...................................................................................... 45 
13 SAFETY MONITORING AND REPORTING .................................................................... 47 
13.1 
Adverse Events ........................................................................................................... 47 
13.1.1 Reporting of AEs .................................................................................................... 47 
13.2 
Serious Adverse Events .............................................................................................. 47 
13.2.1 Reporting of SAEs .................................................................................................. 48 
13.3 
Emergency Unblinding of Investigational Treatment ................................................ 48 
13.4 
Safety Oversight Committee (SOC) ........................................................................... 48 
13.5 
External Data and Safety Monitoring Board (DSMB) ............................................... 49 
14 QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 49 
14.1 
Monitoring .................................................................................................................. 49 
14.2 
Audits.......................................................................................................................... 49 
14.3 
Data Management and Processing .............................................................................. 50 
14.3.1 Study Documentation.............................................................................................. 50 
14.3.2 Data Management ................................................................................................... 50 


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14.3.3 Data Management Procedures and Data Verification ............................................. 51 
14.3.4 Coding ..................................................................................................................... 51 
14.3.5 Study Closure .......................................................................................................... 51 
15 STATISTICAL METHODS AND DATA ANALYSIS ...................................................... 51 
15.1 
Study Design Overview .............................................................................................. 52 
15.2 
Analysis Sets .............................................................................................................. 52 
15.3 
Patient Demographics and Other Baseline Characteristics ........................................ 53 
15.4 
Treatments .................................................................................................................. 53 
15.5 
Primary Objectives ..................................................................................................... 53 
15.5.1 Key Primary Objective:  Overall Survival .............................................................. 53 
15.5.2 Other Primary Objective:  Migration ...................................................................... 53 
15.5.3 Other Primary Objective:  Survival Among CMV Positive Patients ...................... 53 
15.5.4 Other Primary Objective:  Survival Among CMV Negative Patients .................... 54 
15.6 
Secondary Objectives ................................................................................................. 54 
15.6.1 Key Secondary Objective:  PFS .............................................................................. 54 
15.6.2 Other Secondary Objective:  PFS Among CMV Positive Patients ......................... 54 
15.6.3 Other Secondary Objective:  PFS Among CMV Negative Patients ....................... 54 
15.7 
Exploratory Objectives ............................................................................................... 54 
15.8 
Interim Analysis ......................................................................................................... 55 
15.9 
Sample Size Calculation ............................................................................................. 55 
15.9.1 Overview of Sample Size Requirements ................................................................ 56 
15.9.2 Key Primary Objective:  Survival ........................................................................... 56 
15.9.3 Other Primary Objective:  Migration ...................................................................... 57 
15.9.4 Other Primary Objective:  Survival Among CMV Positive Patients ...................... 58 
15.9.5 Other Primary Objective:  Survival Among CMV Negative Patients .................... 59 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ............................................. 59 
16.1 
Regulatory and Ethical Compliance ........................................................................... 59 
16.2 
DUHS Institutional Review Board and DCI Cancer Protocol Committee ................. 59 
16.3 
Informed Consent ....................................................................................................... 60 
16.4 
Privacy, Confidentiality, and Data Storage ................................................................ 60 
16.5 
Data and Safety Monitoring ....................................................................................... 61 
16.6 
Protocol Amendments ................................................................................................ 61 
16.7 
Records Retention....................................................................................................... 61 
16.8 
Conflict of Interest ...................................................................................................... 61 


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16.9 
Registration Procedure ............................................................................................... 61 
17 REFERENCES ..................................................................................................................... 63 
18 APPENDICES ...................................................................................................................... 69 
18.1 
Research Summary ..................................................................................................... 69 
18.2 
SOPs and FORMS ...................................................................................................... 70 
18.3 
Standard Radiation Therapy ....................................................................................... 71 
18.4 
Standard Temozolomide Therapy .............................................................................. 72 
18.5 
DCI Data and Safety Monitoring Plan ....................................................................... 73 
18.6 
Duke PRTBTC DSMBplus Charter ........................................................................... 74 
18.7 
Basiliximab Package Insert ........................................................................................ 75 
 
 
 


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2 LIST OF FIGURES 
Figure 1. Study Schema ................................................................................................................ 11 
Figure 2. Homeostatic lymphodepletion induced by temozolomide enhances antigen-specific 
immune responses. ........................................................................................................................ 15 
Figure 3. PC61 in vivo extends survival and permits tumor rejection in a murine model of 
intracranial glioma. ....................................................................................................................... 20 
Figure 4. Patient from prior vaccination study with a nearly complete response. ........................ 22 
Figure 5. TTP comparison between VICTORI Trial, ACTIVATE Trial, and a historical control.
....................................................................................................................................................... 22 
Figure 6. Basiliximab reduces TRegs in patients with GBM receiving DC vaccination. ............ 23 
Figure 7. Survival comparison of patients with GBM treated with PEPvIII-KLH loaded DCs 
(VICTORI Trial) with patients treated at Duke University with 81C6 anti-tenascin 131I labeled 
MAb. ............................................................................................................................................. 24 
 
3 LIST OF TABLES 
Table 1. Objectives and Endpoints ............................................................................................... 26 
Table 2. Storage of Study Agents ................................................................................................. 36 
Table 3. Dispensing of Study Agents ........................................................................................... 37 
Table 4. Schedule of Events .......................................................................................................... 41 
Table 5. Anticipated Accrual and Randomization Rates .............................................................. 57 
Table 6. Anticipated Accrual and Randomization Rate for CMV Positive Patients .................... 58 
 
 


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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 
BMT 
Bone Marrow Transplant 
BTSC 
Brain Tumor Stem Cells 
Ca++ 
Calcium 
CCL3 
Chemokine (C-C motif) ligand 3 
cDNA 
Complimentary Deoxyribonucleic Acid 
CFA 
Complete Freund’s Adjuvant 
CFC 
Cytokine Flow Cytometry 
cGMP 
Current Good Manufacturing Practice 
CLIA 
Clinical Laboratory Improvement Act 
CLN 
Cervical lymph Nodes 
Cmax 
Maximum Concentration of Drug in Plasma or Serum 
CMP 
Comprehensive Metabolic Panel 
CMV 
Cytomegalovirus 
CNC 
Clinical Neurologic Change 
CNS 
Central Nervous System 
Con-A 
Concanavalin A 
CPC 
Cancer Protocol Committee 
CT 
Computed Tomography 
CTL 
Cytotoxic T-Lymphocyte 
CTQA 
Clinical Trials Quality Assurance 
DAR 
Drug Accountability Record 
DC 
Dendritic Cell 
DCI 
Duke Cancer Institute 
DLT 
Dose Limiting Toxicity 
DNA 
Deoxyribonucleic Acid 
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 
FDA 
Federal Drug Administration 


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8 
 
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 
Investigational Chemotherapy Services 
I.D. 
Intradermal 
IDH 
Isocitrate Dehydrogenase 
IFN- 
Interferon-gamma 
IgG 
Immunoglobulin G 
IgM 
Immunoglobulin M 
IHC 
Immunohistochemistry 
IL-2 
Interleukin-2 
IL-4 
Interleukin-4 
IL-12 
Interleukin-12 
IL-13 
Interleukin-13 
I.M. 
Intramuscular 
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 
MMSE 
Mini-Mental Status Examination 
MPACT 
Molecular Products and Cellular Therapies 
MRI 
Magnetic Resonance Imaging 
mRNA 
Messenger Ribonucleic Acid 
MTD 
Maximally Tolerated Dose 
NA 
Non-adherent 
NCI CTC 
National Cancer Institute Common Toxicity Criteria 
ng 
NanoGram 
NIH 
National Institutes of Health 
NK 
Natural Killer 
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 
RANO 
Response Assessment in Neuro-Oncology 
RECIST 
Response Evaluation Criteria in Solid Tumors 
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 
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 
VDLNs 
Vaccine-site Draining Lymph Nodes 
WBI 
Whole Body Irradiation 
 
 


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5 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
Please see Research Summary section in eIRB. 
 
 


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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 [3], 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 [4-9]. 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 [10, 11]. Moreover, the non-specific nature of conventional therapy for brain 
tumors often results in incapacitating damage to surrounding normal brain and systemic tissues 
[12, 13]. 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 
 
Human Cytomegalovirus pp65-lysosomal-associated membrane protein (pp65-LAMP) 
mRNA-pulsed autologous dendritic cells  
 
DCs are potent immunostimulatory cells that continuously sample the antigenic environment of 
the host and specifically activate CD4+ and CD8+ T cells and B-cells [14, 15]. 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[16-32]. 
 
Human CMV is an endemic -Herpesvirus that does not usually cause significant clinical 
disease[33]. 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[33]. Herpesviruses have also been implicated in a number of human malignancies 
including lymphoma, nasopharyngeal cancer, cervical cancer, and Kaposi’s sarcoma[34, 35]. 
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[36-38]. 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[38]. Presence of the virus in these samples was confirmed with 
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intact virions[38], 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[33], 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 [39-
42]. In addition, T cell mediated immunotherapy has proven highly effective in the treatment of 
CMV-associated disease within the CNS[43] and in the treatment of acute CMV infections[40, 
44]. 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[45-50]. 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[51]. 
 
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[52, 53]. 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[54-
56]   
 
Vaccination specifically against CMV [57-61] has effectively reduced the risk of viral infection 
and transmission to fetuses in animal models [62-64] and in clinical trials[57, 61, 65-68]. Human 
clinical trials have also demonstrated some benefit of administering neutralizing antibodies in the 
treatment of human CMV infection[69-72], highlighting the importance of the development of 
vaccination strategies that elicit both cellular and humoral immune responses. DCs strongly 
activate both T cell and B-cell responses in vivo[15], and DCs pulsed in vitro with CMV antigens 
have been shown to be potent inducers of CMV-specific CTL responses in several studies[73-
77], 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 [78-83]. In fact, there is 
accumulating evidence that RNA transfection represents a superior method for loading antigens 
onto DCs [80, 84]. 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 
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inducing effective and sustained antitumor immune responses by simultaneous activation of both 
CTLs and helper T cells [85-87]. Furthermore, in direct comparisons, RNA-loaded DCs have 
been found to be better stimulators of antigen-specific T cells than other approaches [84]. 
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. [88] have demonstrated that tumor mRNA-
loaded DCs can elicit a specific CD8+ CTL response against autologous tumor cells in patients 
with MG. 
 
Temozolomide 
 
TMZ, a methylating agent with good blood-brain barrier penetration, has recently been shown to 
increase survival by a small, but statistically significant, 2.5 months in a subset of patients with 
newly-diagnosed GBM if given in conjunction with RT following initial resection of the 
tumor[2, 89, 90]. Leukopenia is essentially the only known human toxicity of TMZ. Although 
initially counter-intuitive, this TMZ-induced leukopenia may actually be advantageous in 
treating patients with immunotherapy due to the subsequent homeostatic proliferation it induces. 
In this protocol patients will receive standard of care doses of TMZ concurrent with RT 
following initial resection of the tumor. We believe that the myelosuppression induced by 
therapeutic TMZ treatment, if carefully timed before vaccination will actually enhance the 
proliferation and maintenance of these tumor-specific T cells through the natural forces that 
drive T cell homeostatic proliferative recovery. In addition, TMZ has also been shown to 
preferentially deplete TRegs[91]. Thus, this combination strategy will uniquely exploit the toxicity 
of one effective therapy for GBMs, TMZ, to enhance another already promising therapy, 
immunotherapy. In preparation for this protocol, we have evaluated, in animal models, TMZ and 
sublethal WBI, as a positive control, as methods for induction of treatment-induced lymphopenia 
in order to determine the ability of TMZ-induced lymphodepletion to enhance active and 
adoptive immunotherapy. These studies were initially performed in a murine T cell receptor 
transgenic model in which the antigen-specific T cells can be followed in vivo in mice receiving 
adoptive transfer of lymphocytes. In this model system, we found that adoptive lymphocyte 
transfer coupled with DC vaccination (DC + ALT) is a potent mechanism for inducing antigen-
specific T cell expansion after TMZ treatment. TMZ was found to be an effective agent for 
inducing homeostatic proliferation of transferred CD4+ and CD8+ T cells and for enhancing DC 
or peptide vaccinations with or without ALT (Figure 2). 
 
 
Markedly elevated levels of antigen-specific T cells could be achieved and maintained in mice 
receiving ALT after therapeutic TMZ-induced lymphodepletion compared to normal hosts 
receiving ALT. The precursor frequency of OVA-specific CD8+ T cells in the peripheral blood 
of untreated mice receiving OVA peptide vaccine after transfer of OT-I transgenic T cells was 
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Figure 2. Homeostatic lymphodepletion induced by temozolomide enhances antigen-specific immune 
responses. Untreated mice or mice receiving non-myeloablative high-dose temozolomide treatment (5 days x 60 
mg/kg) were administered a mixture of OVA-specific and normal lymphocytes intravenously and received either no 
vaccine, vaccination with DCs transfected with OVA RNA, or OVA peptide vaccination in complete Freund’s 
adjuvant.  The peripheral blood of treated animals was collected and analyzed 6 days after vaccination for the 
expansion of OVA-specific T cells in the circulation using αCD8 monoclonal antibodies and a specific tetramer.  As 
shown above, mice treated with temozolomide prior to ALT demonstrated markedly increased T cell expansion after 
vaccination compared to untreated hosts receiving ALT. 
 
 frequency of 54%, exhibiting markedly increased levels of OVA-specific T cells in the blood 
(P<0.0001). Similar results were found in mice receiving DC vaccination, with untreated animals 
having an average precursor frequency of 3.6% and TMZ pretreated animals achieving an 
average of 11.9% OVA-specific CD8+ T cells after a single vaccination. These results 
demonstrate that active and adoptive immunotherapy administered during hematopoeitic 
recovery from TMZ treatment may be a very effective way to enhance the efficacy of 
immunotherapy. 
 
Basiliximab (Simulect®) 
 
Basiliximab (Simulect®) is a chimeric (murine/human) monoclonal antibody (IgG1K), produced 
by recombinant DNA technology, that functions as an immunosuppressive agent, specifically 
binding to and blocking the interleukin-2 receptor α-chain (IL-2Rα, also known as CD25 
antigen) on the surface of activated T-lymphocytes.  
Basiliximab has been most frequently used as an immunosuppressant in combination with other 
broadly active immunosuppressants, such as cyclosporine and corticosteroids that may target the 
redundancy pathways for effector T cell development[92]. As an immunosuppressant, it is 
administered within 2 hours prior to transplantation surgery (Day 0) and the second dose 
administered on Day 4 post-transplantation. The regimen of basiliximab was chosen to provide 
No Vaccine
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102
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104
CD8 APC
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30-45 days of IL-2Rα saturation during which time effector T cells may be expected to up-
regulate IL-2Rα expression and be inhibited. Thus, we believe, and our preclinical animal studies 
support, that the activity of IL-2Rα blockade is entirely dependent on the context in which it’s 
given. We believe that blocking IL-2Rα signaling during recovery from TMZ-induced 
lymphopenia will predominantly impact the recovery, function, and survival of TRegs without 
having negative effects on the recovery, function and survival of tumor-specific T cells activated 
through vaccination or through cross-priming at the tumor site where the role of other cytokines 
will predominate.  Please see package insert in the appendices (see Section 18.7). 
 
111Indium-labeling of Cells for in vivo Trafficking Studies 
 
The in vivo distribution of DCs will be evaluated using 111Indium (111In)-labeled pp65-LAMP 
mRNA loaded mature DCs. 111In-labeled DCs have been used extensively for evaluation of 
adoptively transferred tumor infiltrating lymphocytes, natural killer cells, granulocytes, dendritic 
cells, and whole blood leukocytes, for in vivo localization studies in humans[93-95].  DCs will be 
labeled at the Duke Radiopharmacy Lab according to standardized protocols. Briefly, 2 x 107 
pp65-LAMP mRNA loaded mature DCs will be labeled with 111In (50 μCi / 5 x 107 DCs) 
(Nycomed-Amersham, Chicago, IL) prior to injection. Intradermal DC injections sites will be 
carefully marked at 10 cm from the groin crease.  Gamma camera (dual-headed) images will then 
be taken immediately after injections, and at 24 and 48 hours after injections to compare DC 
migration from the inguinal intradermal injection sites to the inguinal lymph nodes (SOP-
DBTIP-078 “Administration of 111In-labeled Dendritic Cells for Trafficking”).  Regions of 
interest will be analyzed using decay corrected counts.  These correlative studies are designed to 
determine the effects of skin site preparation have on DC migration. 
 
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)] [96, 97]. 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 
[98]. 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 [99]. 
 
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 
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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 [100].  
 
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 
[100]. 
 
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 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. 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 


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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. 
     
7.2.1 Pre-Clinical Experience 
In our laboratories and those of others, systemic immunization using DCs co-cultured with 
uncharacterized tumor homogenate[101], whole tumor RNA[102], unidentified peptides eluted 
from tumor cells by gentle acid washing[103], or a distinct peptide encompassing the tumor-
specific EGFRvIII mutation[104] 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[101], 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- which is secreted by most 
human gliomas[105-109]. 
 
In another report from our laboratory[102], 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 


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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[103], the survival of tumor-bearing rats injected 
subcutaneously with autologous bone marrow-derived DCs co-cultured with peptides eluted 
from tumor cells with a gentle acid wash was significantly prolonged compared to tumor-bearing 
rats receiving equivalent numbers of DCs co-cultured with peptides acid-eluted from normal 
astrocytes (P< 0.05).  Median survivals in these groups were 35 and 22 days respectively. In 
addition, three of the twelve rats (25%) treated with DCs co-cultured with acid-eluted tumor 
peptides remained alive at the end of the experiment. In addition, immunohistochemical analysis 
of five animals from each group in this experiment documented an increased peritumoral and 
intratumoral infiltration of CD8+T cells, and to a lesser extent CD4+ T cells and macrophages, in 
the group treated with DCs co-cultured with peptides acid-eluted from tumor cells when 
compared to controls.  
 
Inhibition of TReg Function with Anti-CD25-specific Monoclonal Antibodies 
We have reported that our murine model of glioma indeed recapitulates MG-induced changes to 
the human peripheral blood CD4 and TReg compartments. Specifically, tumor-bearing mice 
exhibit CD4 lymphopenia, while CD4+CD25+Foxp3+GITR+ TRegs come to represent an increased 
fraction of the peripheral blood CD4+ T cells that remain, despite themselves being reduced in 
number. Extending study to other sites, similar phenomena are observed in the spleens and CLN, 
while the reverse scenario emerges in bone marrow.  
 
We employed this model to investigate the in vivo effects of TReg removal on anti-glioma 
immune responses. The current doctrine is that effective depletion of TRegs may be achieved 
simply by systemic administration of anti-CD25 mAb[110, 111]. Likewise, in peripheral, non-
CNS tumor models, the administration of anti-CD25 mAb has been employed in attempts to 
remove TRegs and has effectively elicited prolonged survival to subcutaneous tumor 
challenge[112-114]. These studies, however, predated our ability to examine Foxp3 expression 
in TRegs with antibody staining.     
 
 Following in vivo administration of anti-CD25 mAb (PC61), we discovered that 
CD4+Foxp3+GITR+ cells failed to entirely disappear, despite the present thinking. Instead, they 
persisted at significant levels in all sites tested. When isolated based on CD4 and GITR 
expression, however, these cells demonstrated none of the typical suppressive capacities of 
CD4+CD25+GITR+ TRegs in vitro. Accordingly, systemic anti-CD25 mAb proved capable of 
enhancing T cell proliferation, IFN-γ production and glioma-specific CTL responses in treated 
mice. These effects translate to spontaneous tumor rejection in a murine model of established 
intracranial glioma (Figure 3). 
 
Furthermore, when combined with a DC-based immunization strategy, anti-CD25 mAb elicited 
glioma rejection in 100% of challenged mice without attendant induction of EAE[115, 116]. 
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TRegs without comprehensively eliminating the cells in vivo. This activity proves permissive for 
potent antitumor immunity in a murine glioma model that aptly recapitulates tumor-induced 
changes to the CD4 and TReg compartments. 
 
 
Figure 3. PC61 in vivo extends survival and permits tumor rejection in a murine model of intracranial 
glioma.  A. VM/Dk mice (n=10 per group) received i.p. PC61 or isotype control on Day -4. On Day 0, all mice were 
challenged intracranially with 10,000 syngeneic SMA-560 astrocytoma cells. Anti-CD25 produced 50% long-term 
survivors with no evidence of tumor. Differences in survival curves are significant (p=0.0002). B. Effects of PC61 
in a therapeutic model. Treated mice (n=7) received i.p. PC61 three days following tumor implantation, when tumor 
was established. PC61 significantly extended median survival (p=0.0003) and produced nearly 15% long-term 
survivors with no evidence of tumor. C. Combination of anti-CD25 and DC vaccine produce 100% survival 
following intracranial tumor challenge. VM/Dk mice (n=5 per group) were adminisitered 0.5 mg PC61 (anti-CD25) 
or isotype control antibody i.p. on Day -10. On Day -7, mice were vaccinated with 2.5x105 DC electroporated with 
total tumor RNA from the SMA-560 glioma cell line or injected with an equivalent volume of PBS. Injections were 
delivered s.c. at the base of each ear. On Day 0, all mice were challenged i.c. with 10,000 syngeneic SMA-560 cells. 
Kaplan-Meier survival data are presented as the percentage of mice surviving in each group. Differences in survival 
were determined by log rank test. Anti-CD25 (p=0.0198) and DC vaccine (p=0.0153) alone each produced 
significant survival benefits, but the combination of the two elicited 100% long-term survival (p=0.0018). 
 
 
7.2.2 Clinical Experience 
Prior Experience in Patients with Intracerebral Tumors 
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 immnosuppressed either 
from the use of steroids or due to the fact that malignant brain tumors secrete 
immunosuppressive agents like TGF-. We have been using a simple method described 
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20
25
30
35
40
45
50
Days
Non-Depleted
        Depleted
Percent Surviving
Treg-
0
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20
30
40
50
60
70
80
90
100
0
5
10
15
20
25
30
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40
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50
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Non-Depleted
        Depleted
Percent Surviving
Treg -
A
B
0
10
20
30
40
50
60
70
80
90
100
0
5
10
15 20
25 30
35
40
45 50
55 60
Days
Vaccine only
Vaccine + Anti-CD25
PBS
Anti-CD25 only
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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 natural killer (NK) cell lineage markers, CD19 and CD56, respectively, 
which is consistent with published DC phenotypes. 
 
DC immunotherapy in patients with MGs has been evaluated 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. 
 
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.[122] 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 
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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 
 
Figure 4. Patient from prior vaccination study with a nearly complete response.  Progressive MG after radiation 
and temozolomide (top) with nearly complete response, 3 months after PEPvIII-pulsed DC vaccine (bottom). 
 
0
50
100
150
200
250
0
10
20
30
40
50
60
70
80
90
100
ACTIVATE Peptide Trial
VICTORI DC Trial
Historical Controls
Percent of Patients with Progression
28.52 weeks
64.5 weeks
46.9 weeks
Weeks
 
Figure 5. TTP comparison between VICTORI Trial, ACTIVATE Trial, and a historical control.  TTP comparison in 
patients treated with PEPvIII-KLH loaded DCs (VICTORI Trial), patients treated with PEPvIII-KLH alone 
(ACTIVATE Trial), and a historical control group that had a 95% resection of an EGFRvIII-positive tumor.  There 


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was no significant difference in the TTP between the patients in the VICTORI and ACTIVATE trials, but both were 
significantly better than historical controls (P<0.001). 
radiation, have had a nearly complete response in our prior vaccination study which also 
included the use of chemotherapy agents such as TMZ  (Figure 4). 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).  
(Figure 5)  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)[1]; radiation and concurrent TMZ (14.6 months)[2]; or 
radiolabeled anti-tenascin monoclonal antibodies performed at Duke University  (18.3 
months)[123] (Figure 7). This Phase I study has been completed. No toxicity has been seen 
(Figure 6). PBMC were isolated and analyzed by flow cytometry.  CD4+ is shown as percent of 
lymphocytes and TRegs are shown as percent of CD4.  TRegs defined as CD4+CD25+Foxp3+.  
Based on these results, the dose for this study will be 20 mg. 
 
 
Figure 6. Basiliximab reduces TRegs in patients with GBM receiving DC vaccination.  (B=Basiliximab, 
V=vaccine) 


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0
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100
150
200
250
300
350
0
10
20
30
40
50
60
70
80
90
100
VICTORI DC Trial
Percent Surviving
79 Weeks
110.8 Weeks
81C6 MAb Trial
Weeks
 
Figure 7. Survival comparison of patients with GBM treated with PEPvIII-KLH loaded DCs (VICTORI Trial) 
with patients treated at Duke University with 81C6 anti-tenascin 131I labeled MAb.  Both patient populations had 
similar eligibility criteria. Published median survival for such patients treated with Gliadel™ is 59.6 weeks [1] and 
with concurrent RT and temozolomide is 58.4 weeks [2] 
7.3 Study Purpose/Rationale 
 
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 [101, 104, 124]. 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- 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.  
 
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 peptide vaccines given to patients with GBM during 
recovery from TMZ-induced lymphopenia have produced potent tumor-specific immune 
responses. TMZ has recently shown modest efficacy, but is not curative, in some patients with 
newly-diagnosed GBM and is now frequently given to these patients during and after RT. 
Preclinical studies demonstrate that vaccine responses are enhanced during the recovery from 
TMZ-induced lymphopenia, but a preferential recovery of immunosuppressive TRegs during this 
homeostatic event likely serves to limit tumor-antigen directed immune responses. Preclinical 
studies have shown that unarmed antibodies specific for the high affinity IL-2 receptor 
constitutively expressed on TRegs abrogate their immunosuppressive phenotype and indirectly 
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redundant function of IL-2 at physiologic levels is the maintenance of TRegs. This may be 
especially true within the context of recovery from lymphodepletion when high levels of 
homeostatic cytokines such as IL-7 and IL-15 may drive activated T cell division in the absence 
of IL-2.  Basiliximab a chimeric (murine/human) monoclonal antibody (IgG1K), produced by 
recombinant DNA technology, functions as an immunosuppressive agent, specifically binding to 
and blocking the interleukin-2 receptor α-chain (IL-2Rα, also known as CD25 antigen) on the 
surface of activated T-lymphocytes that may abrogate the functional recovery of TRegs in patients 
with GBM recovering from TMZ-induced lymphopenia and enhance tumor-specific immune 
responses. 
 
DC vaccines have shown considerable promise in the treatment of cancer. However, generally 
<5% of injected DCs actually reach the vaccine-site draining lymph nodes, limiting the induction 
of anti-tumor immune responses. We conducted a clinical trial with newly-diagnosed GBM 
patients who were randomized to pre-immunization with Td toxoid or unpulsed DCs before 
vaccination with CMV pp65 RNA-pulsed DCs  (ATTAC Trial: FDA-IND-BB-12839). Strikingly, 
Td-treated patients had systemically enhanced DC migration and significantly improved PFS and 
OS compared to controls (PFS >36.6 vs 4.4 months, OS >36.6 vs 11.6 months). Our murine 
studies corroborated that Td pretreatment systemically enhanced DC migration and suppressed 
tumor growth. In the proposed clinical study, we aim to: 1) enhance the migration of CMV pp65 
RNA-pulsed DCs with Td, and 2) to evaluate the effect of basiliximab and Td on survival 
outcomes when combined with DCs. 
 
 


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8 OBJECTIVES AND ENDPOINTS 
 
Table 1. Objectives and Endpoints 
 
Objective 
Endpoint 
Analysis 
Key Primary 
To examine the impact of Td pre-
conditioning with or without 
basiliximab on survival 
Median survival from randomization 
See Section 15.5.1 
Other Primary 
To determine whether pre-
conditioning of the vaccine site with 
Td toxoid systemically increases the 
migration of CMV pp65 RNA-
pulsed DCs to site-draining inguinal 
lymph nodes 
The percentage of 111In-labeled DCs 
reaching inguinal nodes will be 
calculated from the initial signal at the 
injection site in the groin to 48 hours 
after the 4th vaccination 
See Section 15.5.2 
Other Primary 
To examine the impact to Td pre-
conditioning on survival among 
patients who are CMV positive 
Median survival from randomization 
See Section 15.5.3 
Other Primary 
To assess the impact of Td pre-
conditioning on survival among 
patients who are CMV negative 
Median survival from randomization 
See Section 15.5.4 
Key Secondary 
To examine the impact of Td pre-
conditioning with and without 
basiliximab on progression-free 
survival 
Median progression-free survival from 
randomization 
See Section 15.6.1 
Other 
Secondary 
To examine the impact of Td pre-
conditioning at the vaccine site on 
progression-free survival among 
patients who are CMV positive 
Median progression-free survival from 
randomization 
See Section 15.6.2 
Other 
Secondary 
To examine the impact of Td pre-
conditioning on progression-free 
survival among patients who are 
CMV negative 
Median progression-free survival from 
randomization 
See Section 15.6.3 
Exploratory 
To describe changes from baseline 
in immune response as measured by 
IFNγ ELISpot 
Median change from baseline in 
ELISpot at each follow-up assessment 
See Section 15.7 
Exploratory 
To assess T cell polyfunctionality 
when ELISpot results are positive 
Proportion of patients with 1, 2 and 3 
functions at each assessment 
See Section 15.7 
Exploratory 
 
To describe changes in TRegs over 
time 
Median  change from baseline in  TReg 
levels  at each follow-up assessment 
See Section 15.7 
Exploratory 
To measure levels of CCL3 and 
other inflammatory soluble factors 
Median change from baseline in CCL3 
and other inflammatory soluble factors. 
See Section 15.7 
9 INVESTIGATIONAL PLAN 
9.1 Study Design 
 
A maximum of 100 patients with resected, newly-diagnosed WHO Grade IV glioma will be 
enrolled in this study with the expectation that approximately 79 patients will be randomized to 
subsequent treatment after completion of radiation treatment with concurrent temozolomide.   At 
the time that accrual to Groups I and II are complete an interim analysis will be conducted in 
order to make a decision concerning the accrual of additional patients to address efficacy 
objectives within patients subgroups defined by CMV status (see Section 15.8).  The study was 


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closed to enrollment in April 2018 (Amendment 73). All patients previously enrolled will 
continue on study as planned. 
 
All enrolled patients will undergo a leukapheresis after resection for harvest of PBLs for 
generation of DCs. Patients will then receive approximately 6  weeks of standard of care RT and 
concurrent TMZ at a standard targeted dose of 75 mg/m2/d (please see Package Insert uploaded 
in eIRB for standard therapy). Patients should start RT within approximately 6 weeks of surgery.  
Patients who either experience progressive disease during radiation, are dependent on steroid 
supplements above physiologic levels at time of first vaccination, are unable to tolerate TMZ, or 
whose DCs or PBLs fail to meet release criteria will be withdrawn from the study and replaced 
and will not undergo repeat leukapheresis. For patients whose initial leukapheresis yields less 
than 3 vaccines, repeat leukapheresis may be obtained a minimum of 2 weeks from the previous 
leukapheresis (and may be repeated as needed) if pre-pheresis blood work is within the 
Apheresis Center’s parameters and as long as this does not cause a significant delay in treatment 
for the patient. At the post-RT clinic visit where patient’s MRI and blood work is evaluated prior 
to ordering the first post-RT cycle of TMZ, patients will be randomized to receive DC 
vaccination with one of three treating regimens: Group I: unpulsed DC pre-conditioning prior to 
vaccination #4; Group II: Td pre-conditioning prior to vaccination #4; or Group III: basiliximab 
infusions at vaccination #1 and #2 with Td pre-conditioning prior to vaccination #4.  As of 
March 2017 (AMD 49), randomization of patients to Group III has been terminated.  Please see 
Section 9.1.1 for a detailed outline of the randomization strategy for vaccine site pre-
conditioning and Section 9.1.7 for a detailed description of the double blinding process.  The 
study cycle of TMZ comprises a targeted dose of 150-200mg/m2/d for 5 days every 5 (± 1) 
weeks. All patients will receive up to a total of 10 DC vaccines given bilaterally at the groin site 
unless progression occurs. DC vaccines will be given i.d. and divided equally to both inguinal 
regions. DC vaccines #1-3 will be given every two weeks, thus delaying the initiation of TMZ 
cycle 2 for all patients.  All remaining TMZ cycles will continue every 5 (± 1) weeks. Before the 
first DC vaccination, patients will receive 0.5 mL of Td (tetanus and diphtheria toxoids 
adsorbed) intramuscularly into the deltoid muscle to ensure adequate immunity to the tetanus 
antigen. The Td vaccine will be obtained through Duke Investigational Chemotherapy Services 
(ICS) Pharmacy. All patients will undergo leukapheresis again for immunologic monitoring with 
specific assessment of baseline antigen-specific cellular and humoral immune responses and 
further DC generations 4 ± 2 weeks after vaccine #3.  
 
If the patient is determined to have progressive disease, the patient may be considered for 
eligibility on the INTERCEPT study (INTracerebral EGFR-vIII Chimeric Antigen Receptor 
Gene-Modified T Cells for PaTients with Recurrent GBM, Pro00083828).  If the patient is 
determined to be eligible for the INTERCEPT study (Pro00083828), the cells extracted from 
leukapheresis while they were on this study, ELEVATE, will be used to create the CAR T cells 
for administration in the INTERCEPT study.      
 
Group I Treatment Plan (DC Vaccinations with pre-conditioning with Unpulsed DCs prior to the 
4th DC vaccine) Patients in Group I will receive 1 x 106 autologous unpulsed DCs in saline 
administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral 
side one day prior to the fourth DC vaccine, which is always given bilaterally at the groin site.  
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skin preparations on DC migration followed by SPECT/CT imaging immediately and at 1 and 2 
days after injections. 
 
Group II Treatment Plan (DC Vaccinations with pre-conditioning with Td prior to the 4th DC 
vaccine) 
Patients in Group II will receive a single dose of Td toxoid (1 flocculation unit, Lf, in 0.4 mLs) 
administered to a single side of the groin, and 0.4 mLs of saline administered to the contralateral 
side one day prior to the fourth DC vaccine, which is always given bilaterally at the groin site. 
Patients will then receive 111In-labeled DCs as the 4th vaccine to compare the effects of different 
skin preparations on DC migration followed by SPECT/CT imaging immediately and at 1 and 2 
days after injection. 
 
Group III Treatment Plan (DC Vaccinations with basiliximab infusions prior to 1st and 2nd DC 
vaccine with Td pre-conditioning prior to 4th DC vaccine) 
Patients in Group III will receive the first 3 DC vaccines every 2 weeks same as Groups I and II 
only they will also receive basiliximab 20 mg I.V. 7 days before DC vaccine # 1 and 7 days 
before DC vaccine # 2.  Prior to the 4th vaccine, patients will receive a single dose of Td toxoid 
(1 flocculation unit, Lf, in 0.4 mLs) administered to a single side of the groin, and 0.4 mLs of 
saline administered to the contralateral side one day prior to the fourth DC vaccine, which is 
always given bilaterally at the groin site.  This group will not undergo migration studies. 
 
Effective March 2017 (AMD 49), randomization of patients to Group III has been terminated 
due to results from the related REGULATE study (IRB # Pro00000581), a study that evaluated 
the safety, toxicity, and efficacy of T Reg inhibition with basiliximab.  REGULATE was used as 
the model for Group III in ELEVATE.  Recent analyses of REGULATE data have shown that 
although the regulatory T cells were depleted, the cells did not show pp65 reactivity and their 
activation status was reduced.  Patients previously randomized to Group III will continue on 
study as planned.   
 
With AMD49, approximately 79 patients will ultimately be randomized to subsequent treatment 
after completion of radiation treatment with concurrent temozolomide, including 9 patients 
previously randomized to Group III.  As originally planned, an interim analysis will be 
conducted after approximately 35 patients have been  randomized to Groups I and II each in 
order to make a decision concerning the accrual of additional patients to address efficacy 
objectives within patient subgroups defined by CMV status (see Section 15.8). 
 
All Groups 
All patients will then be vaccinated in conjunction with subsequent TMZ cycles every 5 ± 1 
weeks for a total of 6 to 12 cycles after RT at the discretion of the treating oncologist. DCs will 
be given on day 21 ± 2 days of each TMZ cycle. DC vaccinations will continue during TMZ 
cycles up to a total of 10 vaccines unless progression occurs. Patients will be imaged bimonthly 
at the discretion of the treating oncologist without receiving any other prescribed anti-tumor 
therapy. Patients will undergo an additional leukapheresis for generation of DCs if needed to 
continue vaccinations. As part of standard care for these patients, upon tumor progression, 
participants may undergo stereotactic biopsy or resection. As this is not a research procedure 
consent will be obtained separately. However, if tissue is obtained, it will be used to confirm 


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tumor progression histologically and to assess immunologic cell infiltration and pp65 antigen 
escape at the tumor site. 
 
9.1.1 Randomization of Vaccine Site Pre-Conditioning Strategies 
A randomization will be performed to assign patients to one of the three treatment strategies after 
completion of radiation with concurrent temozolomide. A permuted block randomization scheme 
stratified by CMV serology will be used to assign patients to treatment groups: unpulsed DCs 
pre-conditioning prior to vaccination #4, Td pre-conditioning prior to vaccination #4, or 
basiliximab infusions at vaccination #1 and #2 and Td pre-conditioning prior to vaccination #4.  
 
As previously noted, the randomization of subjects to Group III has been terminated as of AMD 
49.  With this amendment, the randomization scheme stratified by CMV serology has been 
modified to assign patients to one of two treatment groups. 
 
The administration of pre-conditioning agent (Td or unpulsed DCs) will be blinded for Groups I 
and II, however, the administration of the pre-conditioning agent (Td) and basiliximab for Group 
III will not be.  Hence, patients and providers will know when a patient has been assigned to the 
third treatment group (please see randomization and blinding SOP 18.2 in appendices). 
 
9.1.2 Definition of Unacceptable Toxicities 
Toxicities will be graded according to the NCI CTCAE version 4 criteria. An unacceptable 
toxicity is defined as any Grade 3 toxicity that is possibly, probably, or definitely attributed to a 
study agent (basiliximab, pre-conditioning agent [Td or blinded Td/unpulsed DC], or pp65 DC 
vaccine) that does not resolve to baseline in 2-3 weeks, any Grade 3 hypersensitivity reactions or 
autoimmune toxicity requiring steroids or hormone replacement, any Grade 4 toxicity, including 
neurologic events not due to progressive disease, or any life–threatening event not attributable to 
concomitant medication, co-morbid event, or disease progression. A Grade 3 or greater toxicity 
that is clearly related to TMZ (an expected and known side effect) that does not resolve and 
requires an interruption, dose adjustment to metronomic TMZ schedule, or stoppage will result 
in subject removal from this protocol.  
 
9.1.3 Dose Modification 
TMZ will be administered concomitant with standard external beam RT under the direction of 
the oncologist.  Please see TMZ package insert uploaded in eIRB for standard of care treatment 
guidelines.  The dose may be adjusted at the discretion of the treating oncologist. 
 
The study drug (CMV pp65 RNA-pulsed DCs) dose will not be modified in this trial.  Those 
subjects whose cells fail to meet release criteria will be removed and replaced. 
 
9.1.4 Safety Considerations 
Management of Toxicities 
If a Grade 3 NCI CTC or greater toxicity is seen that is not attributable to a concomitant 
medication, co-morbid event, or disease progression that has been documented radiographically 
or clinically, the next immunization for that patient will be withheld for up to 2 months or until 
the NCI CTC toxicity improves to a Grade 2 or until the KPS score returns to within 10 points of 
baseline. However, 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 


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are the natural development common in this patient population, will not be considered an 
unacceptable toxicity nor will have immunizations withheld. 
 
Adverse Event Reporting and Documentation 
An “Adverse Event” will be defined as any adverse change from the subject’s pre-treatment 
baseline condition (which is assessed at the post-RT/TMZ clinic visit), including any clinical or 
laboratory test abnormality that occurs during the course of research after the time of 
randomization. Adverse events will be categorized and graded in accordance with the NCI CTC 
(Version 4). 
 
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. 
 
A summary of all adverse events (not just those considered related to study drug) that occur 
following randomization will be kept which will categorize the event by organ system, 
relationship to 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. 
 
All adverse events which are serious and unexpected should be reported immediately to Dr. Dina 
Randazzo (Pager: 919-970-9692) 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 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 using the appropriate SAE report form. At the time of the annual progress report to 
the Duke University Medical Center IRB, a summary of the overall toxicity experience will be 
provided. 
 
9.1.5 Missed Doses 
To ensure that repetitive DC vaccines will be given to patients, DCs will be given on day 21 ± 2 
days of each TMZ cycle.  Therefore, with a margin of 2 days surrounding each TMZ cycle Day 
21, all patients will receive up to 10 DC vaccines or until progression, whichever comes first. At 
the discretion of the study PI, patients who miss vaccine administration for whatever reason will 
have their appointment re-scheduled to administer the vaccine as soon as possible and a 
deviation will be filed with the Duke IRB. For continued non-compliance with the scheduled 
vaccine appointments, the subject may be removed from the trial at the discretion of the study PI. 


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9.1.6 Concomitant Medications 
Concomitant medications will be managed by the treating oncologist and recorded at each study 
visit by the study coordinator. 
 
9.1.7 Study Drug(s) Blinding 
The Duke ICS Pharmacy will be responsible for the preparation of the Td and the saline used for 
the pre-conditioning done prior to the fourth vaccine.  The unpulsed DCs used for pre-
conditioning will be prepared in the DBTIP Laboratory and delivered to the ICS for the labeling 
and distribution of the blinded pre-conditioning drugs (i.e., Td, unpulsed DCs, and saline).  Only 
those key personnel needed for the preparation and distribution of the pre-conditioning drugs 
will be unblinded (i.e., ICS pharmacy staff and DBTIP Lab study drug manufacturing personnel, 
as well as the statisticians).  The patient and the remaining key personnel (including the PI and 
research nurses administering the pre-conditioning drugs) will be blinded to the pre-conditioning 
regimen.  Emergency unblinding procedures are described in 13.3. 
 
The administration of pre-conditioning agent (Td or unpulsed DCs) will be blinded for Groups I 
and II, however, the administration of the pre-conditioning agent (Td) and basiliximab for Group 
III will not be.  Hence, patients and providers will know when a patient has been assigned to the 
third treatment group (please see randomization and blinding SOP 18.2 in appendices). 
 
9.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
 
In our previous randomized clinical trial of 12 patients with newly-diagnosed GBM, we 
evaluated the impact of vaccine site pre-conditioning with Td toxoid[125]. Patients randomized 
to Td showed increased dendritic cell migration bilaterally and significantly improved 
progression-free and overall survival. Furthermore, we observed a modest association between 
effective DC migration to VDLNs and clinical outcomes. Based on the small sample size of the 
initial study, we would like to conduct a validation study with Td pre-conditioning that is 
powered sufficiently to evaluate the impact of DC migration on clinical outcomes. For that 
reason, we will conduct this validation trial with identical Td formulation, doses, and DC vaccine 
treatment schedules as in the original study.  The 4th vaccine is the first monthly vaccine and we 
believe that the influence of the skin preconditioning would be most isolated at that time. In the 
original study, Td was used based on our mouse studies which revealed a memory CD4 T cell 
response to tetanus and unpulsed DCs were used based on research[126] that showed injection of 
DCs 24 hours before DC vaccine increased migration. Therefore, 2 conditioning regimens were 
tested: Td versus unpulsed DCs with saline in the contralateral side as control. 
  
9.3 Rationale for Correlative Studies 
 
Please see Section 7.2 111Indium-labeling of Cells for in vivo Trafficking Studies. 
 
 
 


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9.4 Definition of Evaluable Subjects, On Study, and End of Study 
 
Subjects evaluable for the analysis of the primary endpoints of DC migration will include all 
randomized patients in Groups I and II who receive the 4th DC vaccine.  Survival analyses will 
include all randomized patients.  
 
Once the patient signs an ICF, that subject will be considered “on study”. Rationale for taking 
patient off protocol treatment will be documented. 
9.5 Early Study Termination 
 
This study can be terminated at any time for any reason by the PI-sponsor. If this occurs, all 
subjects on study should be notified as soon as possible. Additional procedures and/or follow up 
should occur in accordance with Section 12.7. Section 12.6 describes procedures and process for 
prematurely withdrawn patients. 
 
10 STUDY DRUG 
10.1 Names, Classification, and Mechanism of Action 
 
Tetanus-diphtheria toxoid (Td toxoid adsorbed).  
 
Td is indicated for active booster immunization against tetanus, diphtheria, and pertussis as a 
single dose; substitute 1-time dose of Tdap for Td booster, then standardly boost with Td every 
10 years. Please refer to section 7.2 on the use of Td in this protocol. 
 
DC vaccine 
 
Human CMV pp65-LAMP mRNA-pulsed autologous DCs is the name of the study drug given 
with every vaccine. The name of the study drug at vaccine #4 will be 1 x 106 autologous 
unpulsed DCs or 1 Lf Td toxoid (depending on randomization). This vaccination will then 
consist of administration of 2 x 107 CMV pp65-LAMP mRNA loaded mature DCs as described 
above except that these DCs will be labeled with 111In (50 μCi / 5 x 107 DCs) (Nycomed-
Amersham, Chicago, IL) prior to injection.  The class of action for all study drugs is a biological 
with the exception of the tetanus toxoid, which is an antitoxin. 
 
Leukapheresis and dendritic cell vaccine generation  
 
At least two leukaphereses will be performed on each patient enrolled on this protocol. The 
leukapheresis will be used for DC generation and immunologic monitoring. All leukaphereses 
will be approximately a 4-hour leukapheresis. It is estimated that 10-12 L of blood will be 
processed during this leukapheresis. An additional 4-hour leukapheresis will be requested from 
patients with positive immunological responses to the therapy for additional immunologic 
monitoring studies or for patients who need additional DCs generated to ensure a total of 10 
vaccines. 
 


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DCs will be generated from the first 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 the Duke 
Apheresis Unit and transported to the Cell Processing facility. For patients without sufficient 
venous access for leukapheresis a temporary intravenous catheter may be inserted. 
 
At the end of the 7 day incubation for generation of DC a sample of the media is taken for 
mycoplasma testing, the cells are then harvested and electroporated with pp65-LAMP mRNA. 
The DCs are placed in a flask with AIM V media GM-CSF + IL-4 + TNF-α + IL-6 + IL-1β at 
37OC, 5% CO2 for 18-20 hours for maturation. The cells are washed twice with PBS and frozen 
at 2-4 x 107cells/mL in 90% autologous human AB serum (Valley Biomedical, Winchester, VA 
22602), 10% DMSO and 5% 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. 
 
For all DC preparations, including unlabeled DCs for vaccination and 111In-labeled DCs, from 
the final preparation a sample of cells will be sent for Gram stain and endotoxin testing prior to 
administration. DC vaccination will not be 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 >38.5 oC 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, urine and sputum (if possible) sent 
for bacterial, fungal, and mycoplasmal culture and sensitivity testing.  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. 
 
If the patient is determined to have progressive disease, the patient may be considered for 
eligibility on the INTERCEPT study (INTracerebral EGFR-vIII Chimeric Antigen Receptor 
Gene-Modified T Cells for PaTients with Recurrent GBM, Pro00083828).  If the patient is 
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leukapheresis while they were on this study, ELEVATE, will be used to create the CAR T cells 
for administration in the INTERCEPT study. 
 
Basiliximab 
 
Because our Phase I study showed no difference between 20 and 40 mg of basiliximab in TReg 
depletion, patients will receive basiliximab 20 mg 7 days before vaccines # 1 and # 2 in the 
Infusion Center of the Duke Cancer Center.   
 
A maximum tolerated dose of basiliximab has not been determined in brain cancer patients. 
During the course of clinical studies, basiliximab has been administered to adult renal 
transplantation patients in single doses of up to 60 mg, or in divided doses over 3-5 days of up to 
120 mg, without any associated serious adverse events. 
Basiliximab is contraindicated in patients with known hypersensitivity to basiliximab or any 
other component of the formulation. 
 
Basiliximab functions as an IL-2 receptor antagonist by binding with high affinity (Ka = 1 x 1010 
M-1) to the alpha chain of the high affinity IL-2 receptor complex and inhibiting IL-2 binding. 
Basiliximab is specifically targeted against IL-2Rα, which is selectively expressed on the surface 
of activated T-lymphocytes. This specific high affinity binding of basiliximab to IL-2Rα 
competitively inhibits IL-2-mediated activation of lymphocytes, a critical pathway in the cellular 
immune response involved in allograft rejection. While in the circulation, basiliximab impairs 
the response of the immune system to antigenic challenges. Whether the ability to respond to 
repeated or ongoing challenges with those antigens returns to normal after basiliximab is cleared 
is unknown. 
 
Single-dose and multiple-dose pharmacokinetic studies have been conducted in patients 
undergoing first kidney transplantation. Cumulative doses ranged from 15 mg up to 150 mg. 
Peak mean ± SD serum concentration following intravenous infusion of 20 mg over 30 minutes 
is 7.1 ± 5.1 mg/L. There is a dose-proportional increase in Cmax and AUC up to the highest 
tested single dose of 60 mg. The volume of distribution at steady state is 8.6 ± 4.1 L. The extent 
and degree of distribution to various body compartments have not been fully studied. The 
terminal half-life is 7.2 ± 3.2 days. Total body clearance is 41 ± 19 mL/h. No clinically relevant 
influence of body weight or gender on distribution volume or clearance has been observed in 
adult patients. Elimination half-life was not influenced by age (20-69 years), gender or race.   
 
All patients will receive basiliximab mixed with 50-100 mLs of sterile 0.9% sodium chloride or 
dextrose 5% solution.  Basiliximab solution will be administered in a peripheral or central vein 
over a 20-60-minute period.  When mixing the solution, gently invert the bag in order to avoid 
foaming: do not shake. Infusion should be administered intravenously immediately after 
preparation.  If it needs to be held longer, it should be refrigerated between 2° and 8°C (36° to 
46°F) for up to 24 hours or at room temperature for 4 hours after which it should be discarded.  
Medications for the treatment of severe hypersensitivity reactions including anaphylaxis should 
be available for immediate use.  Patients will be monitored for anaphylactic events for 1hour. 
Please see drug fact sheet in the Appendix – Section 18.7. 


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10.2 Packaging and Labeling of Study Agents 
 
 For CMV pp65-LAMP mRNA-pulsed DCs: 
Name 
MRN 
DOB  
Drug: pp65DCs 
Lot #: Lot 001 
Caution New Drug Limited By Federal Law To Investigational Use 
 For autologous unpulsed DCs: 
Name 
MRN 
DOB  
Drug: unpulsed DCs (blinded to personnel administering drug) 
Lot #: Lot 001 
Caution New Drug Limited By Federal Law To Investigational Use 
 For tetanus diphtheria toxoid used in pre-conditioning 
Name 
MRN 
DOB  
Drug: Td (blinded to personnel administering drug) 
Lot #: Lot 001 
 For CMV pp65-LAMP mRNA loaded mature DCs labeled with 111In (50 μCi / 5 x 107 
DCs) 
Name 
MRN 
DOB  
Drug: Radiolabeled pp65DCs 
Lot #: Lot 001 
Caution New Drug Limited By Federal Law To Investigational Use 
 For basiliximab: 
Name 
MRN 
DOB 
Drug: basiliximab 
Lot #: Lot 001 
 Td™ (Tetanus diphtheria toxoid adsorbed); Stored at 4oC used as Td booster vaccine 
Drug: Td 
Lot #  
Expiration Date 
10.3 Supply, Receipt, and Storage 
 
The DCs will be stored in a locked liquid nitrogen freezer in the Molecular Products and Cellular 
Therapies (MPACT) cGMP facility. The Nautilus LIMS (Laboratory Information Management 
System) database will track receipt and storage location. 


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Table 2. Storage of Study Agents 
MPACT Facility 
Duke ICS Pharmacy 
Duke Radiopharmacy 
CMV pp65-LAMP 
mRNA-pulsed DCs 
Td used for booster and for 
pre-conditioning 
CMV pp65-LAMP 
mRNA loaded mature 
DCs labeled with 111In 
Unpulsed DCs 
Saline used for pre-
conditioning 
 
 
Basiliximab 
 
 
10.4 Dispensing and Preparation 
 
The unblinded DC products (pp65 DC vaccines) will be delivered from the MPACT facility 
directly to the clinic under the supervision of the trained research staff. Each labeled patient 
vaccine will be transported to the clinic in a separate cooler.  DCs will be administered according 
to protocol. The patient’s name, Study ID, DOB, and Duke history number will be double 
verified prior to DC administration as is standard Duke transfusion procedure.  
 
The radiolabeled DC products used with the migration studies within Groups I and II will be 
prepared from the pp65 DC vaccines sent from the MPACT facility to the Radiopharmacy and 
dispensed radiolabeled from the Duke Radiopharmacy.  
 
 
Please see Section 9.1.7 for blinding, preparing and dispensing the pre-conditioning study drugs 
(i.e., saline, Td, and unpulsed DCs). 
 
Patients in Group III will receive basiliximab from the Duke ICS Pharmacy.   
 
 


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Table 3. Dispensing of Study Agents 
MPACT Facility 
Duke ICS Pharmacy 
Duke Radiopharmacy 
CMV pp65-LAMP 
mRNA-pulsed DCs 
Td used for booster and for 
pre-conditioning (blinded 
for Groups I and II only) 
CMV pp65-LAMP 
mRNA loaded mature 
DCs labeled with 111In 
 
Saline used for pre-
conditioning (blinded) 
 
 
Unpulsed DCs for pre-
conditioning (blinded) 
 
 
Basiliximab 
 
 
10.5 Compliance and Accountability 
 
All 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 transfers, receipts, and disposal are recorded in the Duke Nautilus system. 
10.6 Disposal and Destruction 
 
Radioactively labeled drug will be disposed of according to standard Duke radiation safety 
practices. Un-labeled drug will be autoclaved. 
11 SUBJECT ELIGIBILITY 
 
Inclusion Criteria  
 Age ≥18 years of age. 
 WHO Grade IV Glioma with definitive resection prior to consent, with residual 
radiographic contrast enhancing disease on the post-operative CT or MRI of <1 cm in 
maximal diameter in any axial plane.  
 MRI post RT does not show progressive disease at time of randomization 
 Karnofsky Performance Status (KPS) of > 80%. 
 Hemoglobin ≥ 9.0 g/dl, ANC ≥ 1,500 cells/µl, platelets ≥ 125,000 cells/µl. 
 Serum creatinine ≤ 1.5 mg/dl, serum SGOT and bilirubin ≤ 1.5 times upper limit of normal. 
 Signed informed consent approved by the Institutional Review Board.  
 Female patients must not be pregnant or breast-feeding. Female patients of childbearing 
potential (defined as < 2 years after last menstruation or not surgically sterile) must use a 
highly effective contraceptive method (allowed methods of birth control, [i.e. with a failure 
rate of < 1% per year] are implants, injectables, combined oral contraceptives, intra-uterine 
device [IUD; only hormonal], sexual abstinence or vasectomized partner) during the trial 


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and for a period of > 6 months following the last administration of trial drug(s). Female 
patients with an intact uterus (unless amenorrhea for the last 24 months) must have a 
negative serum pregnancy test within 48 hours prior to first study procedure 
(leukapheresis). 
 Fertile male patients must agree to use a highly effective contraceptive method (allowed 
methods of birth control [i.e. with a failure rate of < 1% per year] include a female partner 
using implants, injectables, combined oral contraceptives, IUDs [only hormonal], sexual 
abstinence or prior vasectomy) during the trial and for a period of > 6 months following 
the last administration of trial drugs. 
 
Exclusion Criteria: 
 Pregnant or breast-feeding. 
 Women of childbearing potential and men who are sexually active and not willing/able to 
use medically acceptable forms of contraception. 
 Patients with known potentially anaphylactic allergic reactions to gadolinium-DTPA. 
 Patients who cannot undergo MRI or SPECT due to obesity or to having certain metal in 
their bodies (specifically pacemakers, infusion pumps, metal aneurysm clips, metal 
prostheses, joints, rods, or plates). 
 Patients with evidence of tumor in the brainstem, cerebellum, or spinal cord, radiological 
evidence of multifocal disease, or leptomeningeal disease. 
 Severe, active comorbidity, including any of the following: 
‐ Unstable angina and/or congestive heart failure requiring hospitalization; 
‐ Transmural myocardial infarction within the last 6 months; 
‐ Acute bacterial or fungal infection requiring intravenous antibiotics at the time of 
study initiation; 
‐ Chronic obstructive pulmonary disease exacerbation or other respiratory illness 
requiring hospitalization or precluding study therapy; 
‐ Known hepatic insufficiency resulting in clinical jaundice and/or coagulation 
defects; 
‐ Known HIV and Hepatitis C positive status; 
‐ Major medical illnesses or psychiatric impairments that, in the investigator's 
opinion, will prevent administration or completion of protocol therapy; 
‐ Active connective tissue disorders, such as lupus or scleroderma that, in the opinion 
of the treating physician, may put the patient at high risk for radiation toxicity. 
 Co-medication that may interfere with study results; e.g. immuno-suppressive agents other 
than corticosteroids. 
 Prior, unrelated malignancy requiring current active treatment with the exception of 
cervical carcinoma in situ and adequately treated basal cell or squamous cell carcinoma of 
the skin. (Treatment with tamoxifen or aromatase inhibitors or other hormonal therapy that 
may be indicated in prevention of prior cancer disease recurrence, are not considered 
current active treatment.) 
 Patients are not permitted to have had any other conventional therapeutic intervention other 
than steroids prior to enrollment outside of standard of care chemotherapy and radiation 
therapy.  Patients who receive previous inguinal lymph node dissection, radiosurgery, 
brachytherapy, or radiolabeled monoclonal antibodies will be excluded. 


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 Current, recent (within 4 weeks of the administration of this study agent), or planned 
participation in an experimental drug study. 
 Known history of autoimmune disease (with the exceptions of medically-controlled 
hypothyroidism and Type I Diabetes Mellitus). 
 
12 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
12.1 Screening Examination 
 
The screening examination will take place at the Duke PRTBTC clinic visit. An informed 
consent must be signed by the patient before any screening procedure takes place. 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 obtained from the Duke 
electronic medical record. 
 
CMP and CBC with auto differential are drawn at clinic visits and prior to surgical procedures 
per standard of care.  A CMV immune screen will also need to be drawn at one of these clinic 
visits following consent and prior to initiation of vaccine therapy. 
 
Initial clinical evaluations will also include a baseline MMSE testing. A baseline and study 
eligibility MRI (with and without gadolinium enhancement) of the brain per standard of care for 
comparison to subsequent MRI images will also be obtained. 
 
The patient must have had a definitive resection.  Residual radiographic contrast enhancement on 
post-resection CT or MRI must not exceed 1 cm in diameter in any axial planes at time of 
consent.  Patients with progressive disease after radiation will not be a candidate for the vaccine 
despite being previously consented and will be removed from the study and replaced (please see 
Section 12.7.4 for radiologic evaluations).  
 
During collection of tumor sample at resection at Duke University, primary tissue will be banked 
and stored for IHC assays. Tissue will be stored in formalin-fixed blocks for subsequent 
partitioning of pathology slides. For initial resection at Duke, slides will be sent for MGMT 
promotor methylation status and IDH-1/2 mutation analysis as part of the routine molecular 
diagnostic assay performed at Duke. If the surgery is performed outside of Duke and if such 
institutions do not incorporate these initial molecular diagnostic assays, slides from surgical 
blocks will be ordered for subsequent MGMT promoter methylation status and IDH-1/2 mutation 
analysis, so as to ensure baseline molecular diagnostics of the tumor for each patient.  At the 
discretion of the study investigator, for surgical blocks containing initial resection and biopsy at 
recurrence (if applicable) tissue, formalin-fixed paraffin-embedded slides will be requested for 
immunohistochemistry assays for the detection of CMV. These slides for CMV detection will 
not exceed 10% of the original specimen block, so as to preserve adequate tissue should patients 
prefer future diagnostic studies. Patient consent will be obtained for obtaining these slides in 
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If resection is done outside of Duke University Hospital, formalin-fixed paraffin-embedded 
slides sectioned from outside blocks, not to exceed 10% of surgical specimen block tissue, 
prepared on Fischer Plus glass or Histostix coated slides when available will be mailed to: 
 
Dina Randazzo, DO  
Duke Brain Tumor Immunotherapy Program 
Department of Neurosurgery  
The Preston Robert Tisch Brain Tumor Center at Duke 
Box 3050 
220 Sands Building  
Duke University Medical Center 
Durham, North Carolina 27710, USA 
 
After patients have been consented, they will be entered into the Velos eResearch system.  
12.2 Treatment Period 
 
All enrolled patients will then undergo a leukapheresis for generation of DCs and for baseline 
immunological monitoring. Within 48 hours of leukapheresis, patients will have blood samples 
taken for the following tests as required by the Duke Apheresis Center: CBC, 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. One red top tube will be obtained prior to each 
leukapheresis for immune monitoring (approximately 4 mLs). 
 
 


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Table 4. Schedule of Events 
Appointment 
Time and 
Range 
Tumor 
Pathology1  
Physical and 
Neurologic 
Exam 
KPS, MMSE2 
MRI3 
 
CMV 
Screen
4 
CMP 
and  
CBC5 
Blood for 
immunologic 
monitoring and 
Sub-clinical 
Autoimmunity6 
Screening and  
Pre-enrollment 
Prior to 
Consent 
X 
X 
X 
 
X 
 
Consent 
Within 6 
weeks of OR 
 
 
 
X 
X 
 
Leukapheresis7 
After Consent 
 
 
 
 
X 
X 
RT/TMZ 
Per SOC 
(starts within 
6  weeks of 
OR) 
 
 
 
 
X 
 
Post-RT TMZ 
cycle 1 
Within 4 (±2) 
weeks of RT 
 
 
X 
 
X 
 
Basiliximab 
Infusion (Group 
III only) 
Day 14 (± 2 
days) 
 
X 
 
 
X 
X 
Td Booster IM 
Day 20 (± 2 
days) so long 
as before V#1 
 
 
 
 
 
 
Vaccine #1  
Day 21 (± 2 
days) 
 
X 
 
 
 
X 
Basiliximab 
Infusion (Group 
III only) 
Day 28  (± 2 
days) 
 
X 
 
 
X 
X 
Vaccine #2 
Day 35  (± 2 
days) 
 
X 
 
 
 
 
Vaccine #3 
Day 49  (± 2 
days) 
 
X 
 
 
 
 
Leukapheresis 
4 (± 2) weeks 
after V#3 
 
 
 
 
X 
X 
                                                 
1 WHO Grade IV Glioma with definitive resection prior to enrollment, with residual radiographic contrast enhancement on most 
recent CT or MRI of <1 cm in maximal diameter in any axial plane. 
2 Clinical evaluations with each vaccine will include a general physical examination, complete neurologic examination, and KPS 
rating.  MMSE will be performed at baseline only.   
3 A baseline and study eligibility MRI (with and without gadolinium enhancement) of the brain per standard of care for comparison 
to subsequent MRI images will also be obtained. MRI or contrasted CT, every 8 ± 4 weeks per PRTBTC standard of care (may be 
adjusted by the treating neuro-oncologist).  
4 A CMV immune screen will also need to be drawn at one of these clinic visits following consent and prior to initiation of vaccine 
therapy. 
5 CMP and CBC (with auto differential) to confirm eligibility, and as per standard of care prior to and following surgery, and 
during SOC monthly and study cycles of TMZ and RT (if applicable). 
6 Blood work for immunologic monitoring will be drawn before each leukapheresis (1 red top only prior to phereses), basiliximab 
(Group III), on the day of vaccine #1 (9 yellow/2 red top only at this visit), and then prior to each vaccine after vaccine #3 (9 
yellow/2 red top tubes) and/or at progression (whichever comes first). For subjects who complete all 10 study vaccines without 
progressing, an attempt will be made to obtain blood for immunologic monitoring 2-3 times a year at standard Duke Preston Robert 
Tisch Brain Tumor Center visits and at progression (whichever comes first). Peripheral blood for immunologic immune monitoring 
will be placed in 9 yellow tops and 2 red top tubes for a total of about 90 mLs and delivered to the BTIP lab for processing. Plasma 
from the yellow top tubes will be collected at each time point listed where immunologic monitoring is done. 2 yellow and 1 red top 
tubes will be obtained at the pre-conditioning visit (all Groups) and at the SPEC/CT imaging visits (Groups I and II only).  Blood 
work for sub-clinical autoimmunity will be drawn before basiliximab (Group III), or vaccine #1 (all other groups), and repeated 
only if clinically indicated.  Laboratory evaluations to detect subclinical disease will be obtained as follows: serum thyroglobulin 
Ab (Lab Code 1802105), rheumatoid factor (Lab Code 5560195), antinuclear Ab (Lab Code 5560214), Thyroid Profile (Lab Code 
1850277), and CMP (Lab Code 1004261). Total volume for sub-clinical autoimmune testing is < 20 mLs. 
7 Initial leukapheresis will be scheduled following consent.  Per standard Duke Apheresis Protocol, all subjects will have CBC, 
CMP, ionized Calcium, and βHCG within 48 hours prior to the procedure(s). Follow up leukapheresis will occur approximately 4 
± 2 weeks after third immunization to obtain PBMCs for Immunologic Monitoring and generation of additional DCs for continued 
vaccinations. Leukapheresis can be obtained up to every 2 weeks if needed according to the Duke Apheresis Center protocol, but 
these will likely be needed less than every 2 months throughout the study to generate enough DCs to continue vaccinations. Blood 
for immune monitoring will be drawn prior to each leukapheresis (1 red top tube – approximately 4 mLs). 


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Appointment 
Time and 
Range 
Tumor 
Pathology1  
Physical and 
Neurologic 
Exam 
KPS, MMSE2 
MRI3 
 
CMV 
Screen
4 
CMP 
and  
CBC5 
Blood for 
immunologic 
monitoring and 
Sub-clinical 
Autoimmunity6 
TMZ Cycle 2 
(slight delay start) 
To start 
following V#3 
 
X 
X 
 
X 
 
Pre-conditioning8 
1 day before 
V#4 
 
X 
 
 
X 
X 
Vaccine # 4 9 
(111In-labeled 
DCs) (Groups I & 
II only) 
Day 21(± 2 
days) of 
second cycle of 
TMZ 
 
 
 
 
 
X 
Migration 
studies10  
1 day and 2 
days after  
V#4 
 
 
 
 
 
X 
Vaccines with 
Cycles of TMZ11 
On day 21 (± 2 
days) of each 
cycle of TMZ 
 
X 
X 
 
X 
X 
Monthly Vaccines 
After TMZ not to 
exceed 10 
Every month 
(± 2 weeks) 
 
X 
X 
 
X 
X 
Progression12 
 
X 
 
X 
 
 
X 
 
12.3 End of Treatment 
 
Vaccines are given as described above for a total of 10 or until progression (whichever comes 
first).  Once the vaccinations are complete, the treatment phase of the study will be over and the 
follow-up period will begin. 
 
If the patient is determined to have progressive disease, the patient may be considered for 
eligibility on the INTERCEPT study (INTracerebral EGFR-vIII Chimeric Antigen Receptor 
Gene-Modified T Cells for PaTients with Recurrent GBM, Pro00083828).  If the patient is 
determined to be eligible for the INTERCEPT study (Pro00083828), the cells extracted from 
leukapheresis while they were on this study, ELEVATE, will be used to create the CAR T cells 
for administration in the INTERCEPT study. 
12.4 Follow-up Period 
 
Patients will be followed for survival, progression, and subsequent therapies only, and this data 
will be recorded by the research team.  For recording of subsequent therapies, the type of 
therapy(ies) that the subject receives will be recorded with approximate start and stop date, if 
                                                 
8  On day before the fourth DC vaccine, patients will receive vaccine site pre-conditioning strategy (Group I-unpulsed DCs i.d.; 
Group II and III- Td i.d.). A single dose of Td toxoid (1 flocculation unit, Lf) or 1 x 106 autologous unpulsed DCs will be 
administered to a single side of the groin 12-24 hours prior to the fourth DC vaccine, which is always given bilaterally at the groin 
site. Standard of care blood for CBC and CMP will be drawn during the pre-conditioning visit. 2 yellow and 1 red top tubes for 
immune monitoring will be obtained during the pre-conditioning visit.  
9 At the day of the 4th vaccine (prior to administration), 9 yellow and 2 red top tubes obtained for immune monitoring. Patients in 
Groups I and II will then receive 111In-labeled DCs to compare the effects of different skin preparations on DC migration followed 
by SPECT/CT imaging immediately.  
10 SPECT/CT imaging will be performed at 1 and 2 days after injection of Vaccine #4 for patients in Groups I and II. At the 
SPECT/CT imaging visits 1 and 2 days after Vaccine #4, 2 yellow and 1 red top tubes for immune monitoring will be obtained 
only on patients randomized to Groups I and II (not Group III). 
11 Monthly cycles of TMZ will be initiated at the discretion of the treating neuro-oncologist according to pathology.  
12 As part of standard care for these patients, upon tumor progression, participants may undergo stereotactic biopsy or resection. 
As this is not a research procedure consent will be obtained separately. However, if tissue is obtained, it will be used to confirm 
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possible. For subjects who complete all 10 study vaccines without progressing, an attempt will 
be made to obtain blood for immunologic monitoring 2-3 times a year at standard Duke Preston 
Robert Tisch Brain Tumor Center visits.  If the subject progresses in the follow-up period, blood 
for immunological monitoring will be drawn at the time of progression, if possible.     
12.5 End of Study 
 
Rationale for taking a patient off study will be documented (see section 12.6.1). In the Follow-up 
Period, patients will be followed for survival, progression, and subsequent therapies only, and 
this data will be recorded by the research team. 
12.6 Early Withdrawal of Subject(s) 
 
12.6.1 Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time. Subjects may be withdrawn by 
the PI and considered screen failures prior to vaccine therapy and the PI may also withdraw a 
subject from the study at any time based on his/her discretion. Reasons for PI-initiated 
withdrawal may include, but are not limited to the following: 
 
Inability to complete approximately 6 weeks of RT with TMZ as per standard of care. 
 
Inability to tolerate TMZ (either requiring a delay in cycles of TMZ, dose adjustment, or 
discontinuation). 
 
Patients with an active infection requiring treatment or having an unexplained febrile 
illness (Tmax > 99.5° F). 
 
Patients requiring an increase in corticosteroids, with the exception of nasal or inhaled 
steroid, such that at the time of first vaccination they require a dose above physiologic 
levels, will be removed from the study and replaced.  For the purposes of this study, 
physiologic dose will be defined as <2 mg of dexamethasone / day. Once vaccinations have 
been initiated, if patients subsequently require increased steroids, they will still be 
permitted to remain on the study, but every effort will be made to minimize steroid 
requirements. 
 
Adverse events 
 
Abnormal laboratory values 
 
Abnormal test procedure results (DCs fail to meet release criteria; inability to tolerate 
leukapheresis) 
 
Protocol deviation 
 
Administrative issues 
 
Disease progression 
 
Pregnancy 
 
12.6.2 Follow-up Requirements for Early Withdrawal 
Consented subjects that are withdrawn prior to randomization for any of the aforementioned 
reasons will be considered eligibility failures and thus will not be followed for survival.  All 
other subjects will be followed by the study coordinator until death or are lost to follow-up. 
 


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Subjects that received any basiliximab (Group III only) or vaccine therapy will be assessed and 
followed or adverse event monitoring/safety analysis for at least 1 month following the last 
vaccine (or basiliximab infusion if in Group III).   
 
12.6.3 Replacement of Early Withdrawal(s) 
Subjects who voluntarily withdraw prematurely or who are withdrawn by the PI prior to 
randomization 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.  Curran Group Status will be performed at time 
of consent. Please see appendices – Section 18.2 for Curran Group Status Form. 
 
12.7.3 Use of Antihistamines 
Subjects will be advised to avoid antihistamine use 48 hours prior to each vaccine 
administration, the day of vaccine administration, and for 48 hours following each vaccine 
administration.  If the subject has a pre-existing condition that requires antihistamine usage, the 
PI and the treating oncologist will decide if it is safe and appropriate for the subject’s 
antihistamines to be held before and following vaccine administrations. 
 
12.7.4 Radiologic Evaluations 
Patients with newly-diagnosed GBM will be imaged by MRI as per standard of care for 
eligibility and baseline measurements, and to assess progression prior to vaccine therapy; 
although the purpose of this study is not to detect tumor responses, any evidence of tumor 
response will be determined according to the Duke PRTBTC SOP (see Appendices – Section 
18.2). RANO criteria[127] will be used for overall assessment of tumor response and 
pseudoprogression.  Pseudoprogression by the RANO response criteria is defined as follows:  1) 
≥ 25% increase in the sum of the products of perpendicular diameters of enhancing lesions 
compared with the smallest tumor measurement obtained either at baseline (if no decrease) or 
best response; 2) significant increase in T2/FLAIR non-enhancing lesion compared with baseline 
scan or best response after initiation of therapy ‐ not caused by comorbid events (eg, radiation 
therapy, demyelination, ischemic injury, infection, seizures, postoperative changes, or other 
treatment effects); 3) any new lesion; clear clinical deterioration not attributable to other causes 
apart from the tumor (eg, seizures, medication adverse effects, complications of therapy, 
cerebrovascular events, infection, and so on); 4) failure to return for evaluation as a result of 
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vaccine therapy for a minimum of 2-3 subsequent months so long as subject remains clinically 
and radiographically stable compared to the MRI showing suspected pseudoprogression.  If the 
subject continues to do well beyond the 3 months, they will continue on study as planned. 
Subjects demonstrating definitive progression will be removed from study.  Tumor progression 
will need to be documented histologically, unless there are clinical contraindications, to exclude 
inflammatory responses presenting as radiographic or clinical changes, which could indicate 
potentially toxic or therapeutic responses and not tumor progression. If tissue is obtained through 
the Duke Brain Tumor Biorepository, it will be used to confirm tumor progression histologically 
and to assess immunologic cell infiltration and antigen escape using IHC. Upon progression, 
patients may be treated on other therapies as directed by the treating Oncologist. 
 
12.7.5 Immunologic Assessments 
Immunological response evaluations for baseline values will be conducted on the leukapheresis 
sample used to generate the DCs and on a second leukapheresis sample obtained 4 ± 2 weeks 
after the third immunization. Blood work for immunologic monitoring (9 yellow and 2 red top 
tubes) will be drawn before basiliximab (Group III only), vaccine #1, and then as described 
above after vaccine #3 and/or at progression (whichever comes first).  For subjects who complete 
all 10 study vaccines without progressing, an attempt will be made to obtain blood for 
immunologic monitoring 2-3 times a year at standard Duke neuro-oncology visits and at 
progression (whichever comes first). The total amount of blood required for this purpose will be 
about 90 mLs. The immune monitoring blood drawn at vaccine #1 will be analyzed for baseline 
CCL3 level. Plasma from the yellow top tubes will be collected at each time point listed where 
immunologic monitoring is done.  Blood work for sub-clinical autoimmunity will be drawn 
before basiliximab (Group III only), or vaccine #1 (all other groups), and repeated only if 
clinically indicated. Laboratory evaluations to detect subclinical disease will be obtained as 
follows: serum thyroglobulin Ab (Lab Code 1802105), rheumatoid factor (Lab Code 5560195), 
antinuclear Ab (Lab Code 5560214), Thyroid Profile (Lab Code 1850277), and CMP (Lab Code 
1004261). A comparison of pre-therapy lymphocyte functions to those at intervals after each 
immunization will be made. These tests may provide evidence for the development of immune 
responses following DCs immunization and will play an important role in the design of future 
DC-based clinical trials.  Cellular immune responses to pp65 will be measured in a tiered 
approach. First, PBMCs will be assessed at pre-treatment (leukapheresis) and during treatment 
for responses to pp65 using a qualified IFN-y ELISpot.   If positive in the ELISPOT assay, pp65 
antigen-specific T cell response will be measured by polyfunctional T cell assays using 8-9 color 
panels previously optimized in our laboratory which includes detection of CCL3 on T cells. The 
effect of treatment on cytokines and other soluble factors will be assayed in the serum/plasma. 
Serum/plasma will be measured to determine levels of CCL3 from the blood drawn at the 
vaccine #1 visit (in ALL Groups), and repeated at the SPECT/CT imaging visits (2 yellow and 1 
red top tubes, in Groups I and II only). TReg kinetics will be measured by using a constellation of 
markers specific for TRegs such as foxp3, CD25 and CD4. 
 
In addition, blood and tumor samples will be stored for possible future research in the Duke 
Brain Tumor Immunotherapy Program (DBTIP) Laboratory where all other samples from this 
study are stored.  The samples being stored are ONLY for this study.  Our mouse studies being 
done in the DBTIP Lab are identifying markers that may influence migration, therefore, as other 
markers are identified, we would like to investigate whether these same markers are identified in 
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Gamma interferon-enzyme-linked immunospot (IFN-γ ELISpot) assay 
Using a tiered approach as described above, 90 mLs of peripheral blood will be drawn into (9 
yellow and 2 reds) vaccutainer tubes containing ACD.  PBMC will be separated by density 
gradient centrifugation using Leukosep tubes.  Antigen specific T cell responses to pp65 will be 
measured ex vivo by direct IFN-γ ELISpot assay. PBMCs at 2.5x105 per well will be stimulated 
overnight with a pool of synthetic peptide (15-mers overlapping by 11 amino acids, JPT Peptide 
Technologies, Germany). 
 
Single-use aliquots of the lyophilized pp65 peptide pool will be resuspended to a final 
concentration of 1 μg/mL. Each assay will include test PBMC cultured with no peptide, pp65 
peptide pool, PHA (2.5 μg/mL), and positive and negative control PBMC wells for pp65 antigen. 
PBMCs from different visits for each patient will be batch analyzed on one plate.  PBMC added 
to duplicate wells of ELISpot assay plates coated with mouse IgG1 anti-human IFN-γ mAb will 
be incubated overnight at 37°C, 5% CO2, washed with PBS/Tween-20, incubated with 
biotinylated mouse IgG1 anti-human IFN-γ for 1 hour at room temperature, washed with PBS, 
incubated with avidin-peroxidase complex for 1 hour at room temperature, washed, incubated 
with substrate (3-amino-9-ethylcarbazole) for 4 minutes at room temperature and spot 
development will be stopped by distilled water rinse.  Spot enumeration by automated analysis 
with a Zeiss KS ELISpot system will be performed.  Results will be expressed as the mean 
SFC/106 PBMC after subtraction of counts from cells cultured with no peptide.   
 
Polyfunctional Flow cytometry 
 
Polyfunctional T cell analysis will be done in Dr. Reap’s immunology laboratory on samples 
positive for secretion of IFN by ELISpot after stimulation with pp65. Dr. Reap’s immunology 
laboratory conducts all the clinical immune monitoring for the Duke Brain Tumor Center. 
Polyfunctional T cell panels have been developed in our laboratory and optimized for detection 
of CCL3 in T cells. This process involves the rapid early detection and analysis of the production 
of TNF-a CCL3, IFN- and IL-2 prior to cellular secretion following antigen-specific stimulation 
in vitro. The functional CD4 and CD8 immune response of the patients will be monitored using 
an 8 color assay. 
 
Isolated PBMC will be stimulated for 6 hours with the pool of 138 peptides spanning the entire 
pp65 gene. The cells will be stained for the surface markers CD3 (to identify it as a T cell), CD4 
(to define as specific helper cell), CD8 (to define antigen specific cytotoxic T cells), CD27, 
CD28, CD45RO (to identify maturation and activation state) and the intracellular cytokines IL-2, 
TNF-a and IFN-. This procedure allows for the highly sensitive detection of IFN-γ, TNF-a and 
IL-2-producing cells which are assumed to be specifically responding to the stimulating antigen. 
This will also allow us to quantitate the change in antigen specific effector, central memory, and 
effector memory cells following each vaccination and monitor polyfunctional T cell responses.  
 
Serology/Multiplex Platform 
 
CCL3 and levels of other inflammatory soluble factors may be assayed by multiplex platform. 
Antibody levels to the immunizing antigen may be measured. 


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TReg Kinetics 
 
TRegs will be analyzed by flow cytometry using a constellation of markers specific for TRegs 
included CD4, CD25 and foxp3.  
 
13 SAFETY MONITORING AND REPORTING 
13.1 Adverse Events 
 
An AE is any untoward medical occurrence in a subject receiving any of the study drugs 
(basiliximab, Td or unpulsed DCs for pre-conditioning, 111In-labeled DCs, and pp65 DC 
vaccines) and which does not necessarily have a causal relationship with this treatment. For this 
protocol, the definition of AE also includes worsening of any pre-existing medical condition. An 
AE can therefore be any unfavorable and unintended or worsening sign  (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of the DCs, whether 
or not related to use of the DCs. Abnormal laboratory findings without clinical significance  
(based on the PI’s judgment) should not be recorded as AEs. But laboratory value changes that 
require therapy or adjustment in prior therapy are considered adverse events. 
 
From the time the subject is randomized through 1 month after the subject’s last vaccine, all AEs 
must be recorded in the subject’s medical record and adverse events case report form. 
 
AEs will be assessed according to the CTCAE version 4.0. If CTCAE grading does not exist for 
an AE, the severity of the AE will be graded as mild (1), moderate (2), severe (3), life-
threatening (4), or fatal (5). 
 
Attribution of AEs will be indicated as follows: 
- 
Definite:  The AE is clearly related to the study drug 
- 
Probably:  The AE is likely related to the study drug 
- 
Possible:  The AE may be related to the study drug 
- 
Unlikely:  The AE is doubtfully related to the study drug 
- 
Unrelated:  The AE is clearly NOT related to the study drug 
 
13.1.1 Reporting of AEs 
A summary of all adverse events (not just those considered related to 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 
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 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 should be reported immediately to Dr. Dina Randazzo (Pager: 919-970-9692) 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 adverse events that are considered serious, unanticipated, and related or possibly related to 
the research (as defined by 21CRF312.32[a]) will be reported to the Duke University Medical 
Center IRB and the FDA using the appropriate SAE reporting process. At the time of the annual 
progress report to the Duke University Medical Center IRB and the FDA, a summary of the 
overall toxicity experience will be provided. 
13.3 
Emergency Unblinding of Investigational Treatment 
 
In the case of an allergic reaction following the pre-conditioning part of the study, the research 
nurse administering the study drug(s) will alert the PI (or her designate) for PI notification and 
approval to initiate unblinding and the ICS will be contacted to obtain information on the pre-
conditioning regimen the subject received.   Dr. Randazzo (study PI) can be reached at 919-684-
5301 during regular business hours and at 919-970-9692 (or 919-684-8111 for page office if 
designate) after hours and on weekends and holidays.  The ICS Pharmacy can be reached at 919-
668-0657.    
13.4 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 includes but may not be limited 
to review of safety data, enrollment status, stopping rules if applicable, accrual, toxicities, 
reference literature, and interim analyses as provided by the sponsor-investigator. The SOC in 
concert with the DCI Monitoring Team (see Section 14.1 for Monitoring Team description) 
oversees the conduct of DUHS cancer-related, sponsor-investigator therapeutic intervention and 
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safety and that the protocol is conducted, recorded and reported in accordance with the protocol, 
SOPs, GCP, and applicable regulatory requirements.  
13.5 
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. 
 
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 


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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 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 Data Management 
The subject’s medical records will be the primary source document for the study. Source 
documents include all information in original records and certified copies of original records of 
clinical findings, observations, or other activities in a clinical investigation used for 


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reconstructing and evaluating the investigation.13 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. 
 
14.3.4 Coding 
All medical terms will be coded using CTCAE (version 4). 
 
14.3.5 Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following 
activities: 
 
 Data clarification and/or resolution 
 Accounting, reconciliation, and destruction/return of used and unused study drugs 
 Review of site study records for completeness 
 Shipment of all remaining laboratory samples to the designated laboratories 
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. 
                                                 
13 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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15.1 
Study Design Overview 
 
This randomized phase II study will assess the impact of tetanus pre-conditioning on migration 
among newly diagnosed GBM patients who have undergone definitive resection and completed 
standard temozolomide and radiation treatment, as well as the impact of tetanus pre-conditioning 
and basiliximab together on survival.  After completing standard of care radiotherapy with 
concurrent temozolomide, patients will be randomized to 1 of 3 treatment arms: 
 
 Group I will receive DC vaccinations with unpulsed DC pre-conditioning prior to the 
4th vaccination;  
 Group II will receive DC vaccinations with Td toxoid pre-conditioning prior to the 4th 
vaccination; and 
 Group III will receive basiliximab infusions prior to the 1st and 2nd DC vaccinations 
along with Td toxoid pre-conditioning prior to the 4th vaccination.   
 
A permuted block randomization algorithm using a 1:1:1 allocation ratio will be used to assign 
patients to a treatment arm.  Randomization will be stratified by CMV status (positive, negative), 
with the assignment to arms I and II being double-blinded (See Sections 9.1.1 and 9.1.7 for 
further details).  
 
The study was initially activated with Groups I and II.  However, approximately 2 months the 
study’s initial activation, the study has been amended to include Group III.   
 
Once accrual to Groups I and II are complete, a decision will be made concerning the accrual of 
additional patients to the Groups I and II in order to assess the impact of Td toxoid pre-
conditioning within subgroups defined by CMV status.   Options include continued accrual to 
both strata, accrual to only one stratum, or termination of accrual.  Details are provided in 
Section 15.8.  
 
Per Section 9.1, the study has been amended to terminate randomization to Group III.  Rationale 
is provided in Section 9.1.  Though randomization to Group III has been terminated, the analyses 
proposed for Group III will still be done even though the power of planned analyses will be 
greatly reduced due to a sample size smaller than anticipated within Group III. 
 
The study was closed to enrollment in April 2018 (Amendment 73).  At the time the study was 
closed to new enrollment, a sufficient number of subjects have been enrolled to power the 
migration endpoint described in Section 15.9.3.  An interim analysis of survival was initially 
planned after 35 patients were randomized to each Arm A and Arm B (Section 15.8).  Survival 
analyses will still be conducted, but they may not be powered for the comparisons of survival 
described in Section 15.9.2.  Exploratory immune analyses described in Section 15.7 will be 
conducted as planned. 
15.2 Analysis Sets 
 
Subject to the availability of data, all randomized patients will be included in all analyses of 
correlative and efficacy endpoints.   


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15.3 Patient Demographics and Other Baseline Characteristics 
 
Summaries of clinical and socio-demographic characteristics will be generated for all enrolled 
patients, as well as a summary stratified by assigned treatment group for patients randomized.  
Categorical descriptors will be summarized using frequency distributions; whereas, interval 
variables will be summarized using percentiles, as well as means and standard deviations. 
15.4 Treatments 
 
A frequency distribution will be generated for the number of vaccines received among patients 
within each treatment arm.   
15.5 Primary Objectives 
 
This study has one key primary objective and three other primary objectives (See Section 8).  
The key primary objective is to assess the impact of Td pre-conditioning with or without 
basiliximab on survival. 
 
Other primary objectives include an examination of the effect of pre-conditioning of the vaccine 
site with Td toxoid as opposed to unpulsed DCs at the time of the 4th vaccination on the 
migration of CMV pp65 RNA-pulsed DCs to site draining inguinal lymph nodes, as well as an 
assessment of the impact of Td pre-conditioning within subgroups of patients defined by CMV 
status (negative/positive).   
 
15.5.1 Key Primary Objective:  Overall Survival 
The Kaplan-Meier estimator will be used to describe the survival within each of the 3 treatment 
arms.  Median survival will be estimated within each arm, with 95% confidence intervals.  OS 
will be defined as the time between randomization and death, or last follow-up if alive.  Two 
pairwise one-tailed log-rank tests will be conducted to assess the impact of Td and/or 
basiliximab.  One test will compare the survival experience of patients in the TD toxoid arm 
(Group II) and the unpulsed DC arm (Group I), and the other test will compare the survival 
experience within the basiliximab arm (Group III) and the unpulsed DC arm (Group I).  An 
intent-to-treat philosophy will be followed in these analyses.   
 
15.5.2 Other Primary Objective:  Migration  
Within Groups 1 and 2, the percentage of 111In-labeled DCs reaching inguinal nodes at 
vaccination #4 is calculated from the initial signal at the injection site in the groin to 48 hours 
post-vaccination.   A Wilcoxon rank-sum test will be used to compare assigned treatment groups 
(Groups I and II) with respect to migration.  This analysis will be based upon the first 12 patients 
within each arm (Groups I and II) that receive vaccination #4. 
 
15.5.3 Other Primary Objective:  Survival Among CMV Positive Patients   
To assess the impact of Td pre-conditioning on survival among patients who are CMV positive, 
an analysis similar to those described for the key primary objective will be generated within the 
subgroup of patients who are CMV positive.  Specifically, the Kaplan-Meier estimator will be 
used to describe the survival among CMV positive patients within Groups I and II.  Median 
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OS will be defined as the time between randomization and death, or last follow-up if alive.  A 
log-rank test will be conducted to assess the impact of pre-conditioning.  An intent-to-treat 
philosophy will be followed in these analyses. 
 
Subgroup analyses will be underpowered and descriptive unless a decision is made to continue 
accrual and treatment of CMV positive patients after the completion of accrual to Group III (See 
Section 15.8). 
 
15.5.4 Other Primary Objective:  Survival Among CMV Negative Patients   
Analyses similar to those described in section 15.5.3 for patients who are CMV positive will be 
conducted among patients who are CMV negative. 
15.6 Secondary Objectives 
 
Secondary objectives and endpoints that are presented in section 8 include an examination of the 
impact of Td pre-conditioning with and without basiliximab on progression-free survival, as well 
as the impact of Td pre-conditioning on progress-free survival within patient subgroups defined 
by CMV status. 
 
15.6.1 Key Secondary Objective:  PFS  
The Kaplan-Meier estimator will be used to describe progression-free survival (PFS) within each 
arm.  Median PFS will be estimated within each of the 3 treatment arms, with 95% confidence 
intervals.  PFS will be defined as the time between randomization and initial failure (disease 
progression or death).  If the patient remains alive without disease progression, PFS will be 
censored at the time of last follow-up.  Two pairwise log-rank tests will be conducted to assess 
the impact of Td and/or basiliximab.  One test will compare the PFS experience of patients in the 
TD toxoid arm (Group II) and the unpulsed DC arm (Group I), and the other test will compare 
the survival experience within the basiliximab arm (Group III) and the unpulsed DC arm (Group 
I).  An intent-to-treat philosophy will be followed in these analyses. 
 
15.6.2 Other Secondary Objective:  PFS Among CMV Positive Patients   
To assess the impact of Td pre-conditioning on PFS among patients who are CMV positive, 
analysis similar to those described for the key primary objective will be generated within the 
subgroup of patients who are CMV positive.  Methods similar to those described in Section 
15.5.3 will be used. 
 
15.6.3 Other Secondary Objective:  PFS Among CMV Negative Patients   
Analyses similar to those described in section 15.6.2 for patients who are CMV positive will be 
conducted among patients who are CMV negative. 
15.7 Exploratory Objectives 
 
Changes from baseline in immune response as measured by ELISpot will be explored at each 
follow-up assessment and within each treatment group.  Though analyses are exploratory, a 
Kruskal-Wallis will be used to compare treatment groups relative to maximum changes in 
ELISpot observed.  A similar analysis will be conducted for levels of TReg, CCL3, and other 


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inflammatory soluble factors.  Parametric analyses, such as an analysis of variance, may be used 
in lieu of a Wilcoxon test if underlying assumptions are satisfied.   
 
When ELISpot results are positive, the extent of T cell functionality will be assessed.  For each 
patient, the proportion of patients with 1, 2, and 3 functions will be determined.  A negative 
ELISpot will be assumed to have 0 functions.  Within each treatment group, changes from 
baseline in the percentage of patients with 0, 1, 2 and 3 functions will be explored Hotelling’s T-
test or a nonparametric equivalent.  Additional statistical analyses may be conducted to changes 
in polyfunctionality, as well as to address other exploratory goals 
15.8 Interim Analysis 
 
Once accrual to Groups I and II are complete, analyses will be conducted to help determine 
whether accrual of patients to Groups I and II should continue in order to assess the impact of Td 
toxoid pre-conditioning within subgroups defined by CMV status. Based upon these analyses 
and other factors, one of 3 decisions will be made: 1) Continue accrual to both CMV positive and 
CMV negative strata within Groups I and II; 2) Continue accrual to only one CMV stratum 
within Groups I and II; or 3) Terminate accrual to all arms.  It should be noted that there are no 
options to continue accrual to Group III once the initial sample size goals are attained. 
 
At the time that this interim analysis is conducted, the number of deaths required per Section 
15.9.2 for the overall comparison of survival within Groups I and II to have the desired power 
will not have yet been attained.  With the assumptions described in Section 15.9.2, we anticipate 
that a total of 31 deaths will have occurred in Groups I and II combined by the time of these 
interim analyses if the alternative hypothesis is true. 
 
Based upon data available at the time of the interim analysis, the conditional probability of 
observing a statistically significant result within each CMV subgroup will be generated.  That 
calculation will account for the accrual rate observed to date within each subgroup, which 
reflects both overall accrual and the prevalence of CMV positivity.  The sample size 
requirements provided in Sections 15.9.4 and 15.9.5 will also be adjusted to reflect subgroup 
accrual rates observed to date. 
 
Factors that will be considered in the decision to extend accrual to address subgroup efficacy 
questions include the conditional probability referenced about, as well as the feasibility of 
completing extended subgroup accrual in light of accrual rate and competing priorities. 
15.9 Sample Size Calculation 
 
The following sections concerning sample size and power reflect the study as it was originally 
designed.  Per Section 9.1, the randomization of patients to Group III has been terminated with 
AMD 49.  Otherwise, the overall design of the rest of the study remains. 
 
Our goal remains to randomize a total of 35 patients to Groups I and II each.  As described in 
Section 15.9.2, a 1-tailed logrank test will be conducted at the 0.15 level of significance to 
compare survival in Group I and II.  For this comparison to have 90% power to detect a hazard 
ratio of 0.5, 45 deaths need to be observed within the two arms being compared. 


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At this time, approximately 35 patients have been randomized to each of Groups I and II, the 
interim analysis described in section 15.8 will be conducted to determine whether accrual of 
patients to CMV subgroups should continue. 
 
Included in the following sample size and power calculations are estimates of the time required 
to complete accrual and observe the required number of events.  Though these estimates are no 
longer valid, re-estimates will not be generated due to the rate of randomizing patients to Groups 
I and II having varied over time due to the number of randomizing arms as well as early 
problems with vaccine contamination. 
 
15.9.1 Overview of Sample Size Requirements 
A maximum of 100 patients will be initially enrolled in this study over a period of 29 months 
with the expectation that approximately 79 patients will be randomized after completion of 
radiation with concurrent temozolomide.  Justification for the required number of randomized 
patients for the key primary outcome variables are provided below.  The sample size calculations 
described in Section 15.9.2 for the key primary objective and the primary subgroups objectives 
reflect the fact that accrual to Group III was initiated a few months after accrual was initiated on 
Groups I and II.   
 
At the time that accrual to Groups I and II are completed, a decision will be made concerning the 
accrual of additional patients to address efficacy questions within CMV subgroups (see Section 
15.8).  A priori sample size requirements for subgroups are described in Sections 15.9.4 and 
15.9.5; however, these calculations will be updated at the time of the interim analysis to reflect 
observed subgroup accrual rates.  At the time of writing this protocol, we anticipate that a 
maximum of 64 additional patients will be accrued during the extended accrual period so that 58 
additional patients will be randomized to Groups I or II. 
 
Hence, a maximum total of 180 patients will be accrued to this study so approximately 160 
patients will be randomized. 
 
Sample size requirements for the migration studies are provided in Section 15.9.3.  As described 
in that section, the first 12 patients in Groups I and II will undergo migration studies are be used 
in those analyses. 
 
15.9.2 Key Primary Objective:  Survival  
The key primary goal of this study is to determine whether the addition of either Td pre-
conditioning with vaccination #4 or basiliximab administration with vaccination #1 and #2 that is 
administered in conjunction with DC vaccination and temozolomide is worthy of investigation in 
a large phase III study.  Two pairwise comparisons of survival are of primary interest: 1) Td 
Toxoid arm (Group II) versus Unpulsed DC arm (Group I), and; 2) Basiliximab arm (Group III) 
versus Unpulsed DC arm (Group I). 
 
Though this phase II study is comparative, a false-positive rate of 0.15 will be used to test each 
hypothesis while maintaining reasonably high power[128-130].  This rate of false-positivity is 
used in lieu of a false positive rate of 0.2 as suggested in the literature in an attempt to partially 
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Within the ATTAC study, the median survival in the DC vaccine arm without pulsed DC pre-
conditioning was approximately 12 months; whereas, within the Td pre-conditioning arm median 
survival was estimated to be approximately 36 months.  Study results suggest a hazard ratio of 
approximately 0.33.  Due to the small sample sizes in the ATTAC study, estimates are all 
associated with wide confidence intervals.  Given these imprecise estimates and the potential for 
“regression to the mean” in this study replicating the ATTAC trial, we will target a smaller 
hazard ratio of 0.5 for a comparison of unpulsed DC (Group I) and Td (Group II).  With pilot 
data not being available for the comparison of basiliximab (Group III) and unpulsed DC (Group 
I), we will also target a hazard ratio of 0.5 for that comparison. 
 
For each pairwise comparison, a 1-tailed logrank test will be conducted at the 0.15 level of 
significance.  In order that each comparison will have 90% power to detect a hazard ratio of 0.5, 
45 deaths need to be observed within the two arms being compared[131].   
 
We anticipate that patients will be accrued at the rate of 4 patients per month, with a 
randomization rate of 3.5 patients per month.  As described earlier, the protocol was initially 
activated with only Groups I and II.  However, with a later amendment, patients will be 
randomized to one of 3 arms.  In the following calculations, we will assume that there is a period 
of 2 months during which patients are randomized to one of 2 arms after which patients will be 
randomized to one of 3 arms.  Tabulated below is the anticipated accrual and randomization rate 
within each arm during each period of accrual. 
 
Table 5. Anticipated Accrual and Randomization Rates 
# of Arms Accruing 
Accrual Rate 
(Patients / Month / Arm) 
Randomization Rate 
(Patients / Month / Arm) 
2 arms  
2 
1.75 
3 arms 
1.33 
1.17 
 
Approximately 32 patients will be randomized to Group III before accrual will be terminated to 
Group III and the interim analysis described in section 15.8 conducted.   Per Section 9.1, the 
randomization of patients to Group III has been terminated with AMD 49.  At the time 
approximately 35 patients have been randomized to each of Groups I and II, the interim analysis 
described in section 15.8 will be conducted to determine whether accrual of patients to CMV 
subgroups should continue.  At the time of this interim analysis, we anticipate that approximately 
35 patients will have been accrued to Groups I and II each and that a total of 31 patients within 
Groups I and II will be dead under the alternative hypothesis where the median survival in Group 
I is 12 months and the median survival in Group II is 24 months.  Approximately 12 months are 
needed after the last patient randomization to observe 45 events[132-134]. 
 
In addition to the comparison of Groups I and II, we plan to compare the survival of patients 
enrolled on Arms I and III.  Under the null hypothesis, approximately 13 month of follow-up 
after the last patient randomization is required to observe 45 events. 
 
15.9.3 Other Primary Objective:  Migration  
One goal of this study is to validate observations made in the recently completed ATTAC trial 
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the ATTAC trial in that patients with newly diagnosed GBM underwent surgery followed first by 
SOC treatment (temozolomide + XRT) and then by 3 vaccinations.  Prior to the 4th vaccination, 
patients were randomized to unpulsed DCs or Td tetanus pre-conditioning.  The percentage of 
111In-labeled DCs reaching inguinal nodes was calculated from the initial signal at the injection 
site in the groin to 48 hours after the 4th vaccination.   
 
In this study, the first 12 patients in Groups I and II will have their DCs labeled will undergo 
migrations studies to assess the migration objective. 
 
A bootstrap approach was used to determine the sample size required for a comparison of 
migration rates using the Wilcoxon rank-sum test (also the Mann-Whitney U test) to have 95% 
power to detect a difference similar to that observed in the ATTAC study.  Re-sampling with 
replacement from the pilot ATTAC study data was used in the 10,000 bootstrapping simulations. 
 
With 12 patients in the Td toxoid arm (Group I) and 12 patients in the unpulsed DC arm (Group 
II) who have undergone migration studies at vaccination #4, simulations show that there is 95% 
power to detect the difference observed in the ATTAC trial assuming α=0.05. 
 
15.9.4 Other Primary Objective:  Survival Among CMV Positive Patients   
One of the other primary goals of this study is to determine whether Td pre-conditioning at 
vaccination #4 in conjunction with DC vaccination and temozolomide is worthy of investigation 
in a large phase III study among patients who are CMV positive.  That assessment involves a 
comparison of survival in Groups I and II among patients who are CMV positive. 
 
The sample size requirements needed to address this subgroup question are similar to those 
described for the key primary objective (Section 15.9.2).  A 1-tailed logrank test will be 
conducted at the 0.15 level of significance.  To have 90% power to detect a hazard ratio of 0.5, 
45 deaths need to be observed within the two arms being compared[131].   
 
As described in section 15.9.2, we anticipate that patients will be accrued to the study at the rate 
of 4 patients per month, with a randomization rate of 3.5 patients per month.  We also anticipate 
that approximately 50% of patients will be CMV positive. 
 
As described in Section 15.9.2, the study will randomize patients to one of two arms, and after 
amendment, it will randomize patients to one of 3 arms.  If a decision is made to continue patient 
accrual (See Section 15.8), the study will randomize patients to either Group I or II.  Tabulated 
below is the anticipated accrual and randomization rate for CMV positive patients within each 
arm during each period of accrual. 
 
Table 6. Anticipated Accrual and Randomization Rate for CMV Positive Patients 
# of Arms Accruing 
Accrual Rate 
(Patients / Month / Arm) 
Randomization Rate 
(Patients / Month / Arm) 
2 arms  
1 
0.88 
3 arms 
0.67 
0.58 
 
At the time of the interim analysis, we anticipate that approximately 36 CMV positive patients 
will have been accrued, with half being assigned to Group I and half to Group II.  If accrual to 


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the CMV positive patient subgroup is continued for an additional 16 months, we expect to accrue 
32 additional patients to that subgroup of which 28 will be randomized to either Group I or II.  
After completion of extended accrual, an additional 12 months of follow-up are anticipated to 
observe approximately 45 events assuming the treatment-arm median survivals are 12 and 24 
months. 
 
If all the assumptions made in this sample size calculation remain valid during the conduct of the 
study and accrual is extended for CMV positive patients, approximately 57 months would be 
required to address this subgroup objective.  The 57 months assumes 2 months of 2-arm 
randomization, 27 months of 3-arm randomization, 16 months of expanded subgroup accrual, 
and 12 months of follow-up. 
 
15.9.5 Other Primary Objective:  Survival Among CMV Negative Patients   
The final other primary goals of this study is to determine whether Td pre-conditioning at 
vaccination #4 in conjunction with DC vaccination and temozolomide is worthy of investigation 
in a large phase III study among patients who are CMV negative.  That assessment involves a 
comparison of survival in Groups I and II among patients who are CMV negative.  The sample 
size requirements needed to address this subgroup question are identical to those described in 
section 15.9.4 given that the prevalence of CMV negative is anticipated to be 50%.   
 
16 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
16.1 Regulatory and Ethical Compliance 
 
This protocol was designed and will be conducted and reported in accordance with the ICH 
Harmonized Tripartite Guidelines for Good Clinical Practice, the Declaration of Helsinki, and 
applicable federal, state, and local regulations. 
16.2 DUHS Institutional Review Board and DCI Cancer Protocol Committee 
 
The protocol, informed consent form, advertising material, and additional protocol-related 
documents must be submitted to the DUHS IRB and DCI CPC for review. The study may be 
initiated only after the Principal Investigator has received written and dated approval from the 
CPC and IRB. 
 
The Principal Investigator must submit and obtain approval from the IRB for all subsequent 
protocol amendments and changes to the informed consent form. The CPC should be informed 
about 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 must obtain written 
informed consent from the subject. The original informed consent form will be stored with the 
subject’s study records, and a copy of the informed consent form will be provided to the subject. 
The Principal Investigator is responsible for asking the subject whether the subject wishes to 
notify his/her primary care physician about participation in the study. If the subject agrees to 
such notification, the Principal Investigator will inform the subject’s primary care physician 
about the subject’s participation in the clinical study. 
16.4 Privacy, Confidentiality, and Data Storage 
 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s 
data will be maintained. RDSPs will be approved by the appropriate institutional Site Based 
Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects 
during the course of the study. Prospective participants will be consented in an exam room where 
it is just the research staff, the patient and his family, if desired. For all future visits, interactions 
with research staff (study doctor and study coordinators) regarding research activities will take 
place in a private exam room. All research related interactions with the participant will be 
conducted by qualified research staff who are directly involved in the conduct of the research 
study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under 
lock and key accessible only by the research staff. Subjects will be identified only by a unique 
study number and subject initials. Electronic records of subject data will be maintained using an 
Oracle Clinical database, which is housed by the DCI. Access to electronic databases will be 
limited to the Principal Investigator, key personnel, statisticians, the Radiolabeled Pharmacy 
personnel, and the 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.  


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Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
16.5 Data and Safety Monitoring 
 
Data and Safety Monitoring will be performed in accordance with the DCI Data and Safety 
Monitoring Plan. For a more detailed description of the DSMP for this protocol, refer to attached 
plan in Appendices (Section 18.5). 
16.6 Protocol Amendments 
 
All protocol amendments must be initiated by the Principal Investigator and approved by the IRB 
prior to implementation. IRB approval is not required for protocol changes that occur to protect 
the safety of a subject from an immediate hazard. However, the Principal Investigator must 
inform the IRB and all other applicable regulatory agencies of such action immediately. 
 
Though not yet required, the CPC should be informed about any protocol amendments that 
potentially affect research design or data analysis (i.e., amendments affecting subject population, 
inclusion/exclusion criteria, agent administration, etc.). 
16.7 Records Retention 
 
The Principal Investigator will maintain study-related records for the longer of a period of: 
‐ 
at least two years after the date on which a New Drug Application is approved by the 
FDA 
‐ 
at least two years after formal withdrawal of the IND associated with this protocol 
‐ 
at least six years after study completion (Duke policy). 
16.8 Conflict of Interest 
 
The Principal Investigator and Sub-Investigators must comply with applicable federal, state, and 
local regulations regarding reporting and disclosure of conflict of interest. Conflicts of interest 
may arise from situations in which financial or other personal considerations have the potential 
to compromise or bias professional judgment and objectivity. Conflicts of interest include but are 
not limited to royalty or consulting fees, speaking honoraria, advisory board appointments, 
publicly-traded or privately-held equities, stock options, intellectual property, and gifts. 
 
The Duke University School of Medicine’s RIO reviews and manages research-related conflicts 
of interest. The Principal Investigator and Sub-Investigators must report conflicts of interest 
annually and within 10 days of a change in status, and when applicable, must have a documented 
management plan that is developed in conjunction with the Duke RIO and approved by the 
IRB/IEC. 
 
16.9 Registration Procedure 
 


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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 
Research Summary 
See eIRB. 
 


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18.2 
SOPs and FORMS 
Separate upload in the protocol section in eIRB. 
 
 


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18.3 
 
 
Standard Radiation Therapy 
Radiotherapy typically begins within ≤ 5 weeks of surgery. One treatment of 1.8-2.0 Gy/fraction 
will be given daily 5 days per week for a total of 59.4-60.0 Gy over <7 weeks. 3D conformal and 
intensity-modulated RT is permitted. All portals should be treated during each treatment session. 
Doses are specified as the target dose that shall be to the center of the target volume.  
 
The gross target volume (GTV) for both the initial volume (GTV1) and the conedown volume 
(GTV2) should be based on the postoperative CT/MRI (and preferably the MRI; the preoperative 
scans may be used if postoperative scans are not available). This initial target volume (GTV1) 
should include the contrast-enhancing lesion (and should include the surgical resection cavity) 
and surrounding edema (if it exists) demonstrated on CT/MRI plus a 2.0-cm margin (this 2.0-cm 
margin-extended volume will be considered the initial planning target volume, or PTV1). The 
initial target volume should be treated to 46 Gy at 2Gy/fraction or 45-50.4 Gy at 1.8Gy/fraction. 
If no surrounding edema is present, the initial planning target volume (PTV1) should include the 
contrast-enhancing lesion (and should include the surgical resection cavity) plus a 2.5-cm 
margin. Please note that clinical judgment may be used to modify PTV1 to exclude sensitive 
structures such as the optic chiasm, non-cranial contents, or anatomic regions in the brain where 
natural barriers would likely preclude microscopic tumor extension, such as the cerebellum, the 
contralateral hemisphere, directly across from the tentorium cerebri, the ventricles, etc. After 46 
Gy, the tumor volume (GTV2) for the conedown treatment should include the contrast-
enhancing lesion (without edema) on the pre-surgery CT/MRI scan plus a 1.5-2-cm margin 
(PTV2). Treat to 14 Gy at 2Gy/fraction or 14.4-9.0 Gy at 1.8Gy/fraction to a total of 60.0 or 
59.4Gy, respectively. 
 
Dose is prescribed to the isodose line such that at least 95% of the target volume receives he 
prescribed dose. 
The optic apparatus should be limited to a maximum of 54Gy and no more than 5% of the 
volume of the brainstem should receive >54Gy. 
 
Radiation should be delayed or interrupted if the platelet count is < 20,000. Radiation should not 
begin or resume until the platelet count is ≥ 20,000. Hematologic toxicities should be rated on a 
scale of 0-5 as defined in the NCI Common Terminology Criteria for Adverse Events (CTCAE) 
version 3.0.  If radiotherapy has to be temporarily interrupted for technical or medical reasons 
unrelated to the temozolomide administration, then treatment with daily temozolomide should 
continue. If radiotherapy has to be permanently interrupted then treatment with daily 
temozolomide should stop.  The following should be recorded at entry into this study: daily 
treatment record, all isodose distributions (in color), dose volume histograms including the 
cumulative dose to the target volumes, optic chiasm, optic nerves and brain stem, and the 
radiotherapy summary. 
 
 


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18.4 
Standard Temozolomide Therapy 
Please refer to the package insert uploaded in the drug section in eIRB. 
 
 


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18.5 
DCI Data and Safety Monitoring Plan 
Separate upload in the protocol section in eIRB. 
 
 


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18.6 
Duke PRTBTC DSMBplus Charter 
Separate upload in the protocol section in eIRB. 
 
 


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18.7 
Basiliximab Package Insert 
Separate upload in the protocol section in eIRB.