Clinical trial NCT02465268
Conditions: Glioblastoma Multiforme Glioblastoma Malignant Glioma Astrocytoma, Grade IV GBM
Sponsor: University of Florida
Start date: 2016-08-09; Completion date: 2023-11-30
Enrollment: 175 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this research study is to determine if an investigational dendritic cell vaccine, called pp65 DC, is effective for the treatment of a specific type of brain tumor called glioblastoma (GBM) when given with stronger doses of routine chemotherapy.

Detailed description:
Dendritic cells (DC) are involved in activating, or turning-on, your body's immune system. Your immune system helps guard your body from germs, viruses, and other threats. Although dendritic cells are very strong, the number of them in the body is not high enough to cause a powerful immune response; therefore, more DC are made in a laboratory with cells collected from an individual's blood.

In this study, we will make a vaccine that we hope will educate immune cells to target the pp65 antigen, a type of immune marker in GBM, thus resulting in what we call the pp65 DC vaccine. Use of a vaccine that activates your immune system is a type of immunotherapy. It is hoped that by giving the pp65 DC vaccine as a shot under the skin, the immune system will be activated to attack tumor cells in the brain while leaving normal cells alone.

To see if the pp65 DC vaccine is effective for the treatment of GBM, subjects will be assigned to different treatment groups. Two groups of subjects will receive the pp65 DC vaccine and one group will receive a placebo.

Arms: arm 1: Given under the skin at day 22-24 after the first temozolomide cycle then at 2 week intervals. Doses 4-10 will be given on day 22-24 of each temozolomide cycle. Doses will continue until a total of 10 or until progression or unacceptable toxicity. arm 2: Given under the skin at day 22-24 after the first temozolomide cycle then at 2 week intervals. Doses 4-10 will be given on day 22-24 of each temozolomide cycle. Doses will continue until a total of 10 or until progression or unacceptable toxicity. arm 3: Given under the skin at day 22-24 after the first temozolomide cycle then at 2 week intervals. Doses 4-10 will be given on day 22-24 of each temozolomide cycle. Doses will continue until a total of 10 or until progression or unacceptable toxicity.
Interventions: intervention 1: pp65-shLAMP DC with GM-CSF intervention 2: unpulsed PBMC and saline intervention 3: Td intervention 4: Saline intervention 5: pp65-flLAMP DC with GM-CSF
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: All subjects will receive a Td booster before study drug dose #1. Subjects in the experimental arms will receive Td skin prep before study drug doses #3, #6, and #9. intervention 4: None intervention 5: None

Protocol documents:
Prot_SAP_001.pdf: 
ATTAC-II 
 
 
Preston A. Wells Jr. Center for Brain Tumor Therapy 
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 is required for disclosure otherwise. 
ATTAC-II:    
A Phase II Randomized, Blinded, and Placebo-controlled Trial of CMV RNA-
Pulsed Dendritic Cells with Tetanus-Diphtheria Toxoid Vaccine in Patients 
with Newly-Diagnosed Glioblastoma  
 
 
Sponsor: 
University of Florida 
 
Funding Source: 
University of Florida 
 
Protocol Source: 
Chair, Duane A. Mitchell, MD, PhD 
 
UF IRB#: 
201400697 
 
FDA IND#: 
 
BB-16530 
 
Coordinating 
Center: 
Preston A. Wells Jr. Center for Brain Tumor Therapy 
University of Florida Brain Tumor Immunotherapy 
Program 
 
 
Protocol Chair  
Duane A. Mitchell, MD, PhD 
University of Florida 
 
Site Co-Principal Investigators 
– University of Florida 
Maryam Rahman, MD 
Ashley Ghiaseddin, MD 
 
Site Principal Investigator - 
Duke University Medical 
Center 
Katy Peters, MD, PhD 
 
Site Principal Investigator - 
Orlando Health Cancer 
Institute 
Naren Raj Ramakrishna, MD 
 
 
 
 
UF Investigators and Key 
Personnel 
Brian Cleaver, PhD 
Anjelika Dechkovskaia, MD  
Jeffrey Drake 
Catherine Flores, PhD 
Jianping Huang, MD, PhD 
Tony Yachnis, MD 
Oleg Yegorov, PhD 
Tara Massini, MD 
Jesse Kresak, MD 
John Rees, MD 
 
Lead Study Coordinator 
Phuong Deleyrolle, RN 
University of Florida 
 
Regulatory Coordinator 
Kristine Wynne, RN, BSN, MSHS 
University of Florida 
 
 
 
 
 
 
 
 


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Summary of Protocol Changes 
Version Date 
Sections Revised 
Purpose 
20150525 1530 RB 
N/A 
Initial Protocol Release 
20151026 1100 NM 
Entire Document 
-Revised study title 
-Updated investigators and key personnel 
-Updated FDA IND# 
-Deleted reference to double blinding of the study 
20160506 1300 KW 
Title Page 
 
-Individuals updated to reflect current personnel. 
-Added ‘FDA’ to IND Number for clarity. 
Table of Contents 
-Updated for pagination. 
2, Study Synopsis 
-Based on pre-clinical data suggesting that the activation and 
expansion of CMV pp-65-specific T cells can be enhanced 
through modification of the RNA encoding pp65 with a full-
length LAMP fusion protein, influenza virus matrix M1 was 
replaced with full-length LAMP protein. 
-Saline skin prep was removed and all subjects randomized to 
receive DCs will receive Td skin prep based on clinical data 
showing prolonged survival. 
-To serve as a true control, GM-CSF was removed from the 
PBMC arm. 
-Reference to Nature paper was added. 
-The primary objective was modified to evaluate the impact of 
Td on overall survival. 
-The secondary objectives were modified to evaluate 
progression-free survival and immunologic effects of short 
versus full-length LAMP. 
-Modifications were made to clarify inclusion criteria and 
timing of eligibility assessments. 
-Since manufacturing processes are different among the arms, 
the study design was changed to “single-blind”.    
-Randomization was changed to “prior to leukapheresis” to 
allow for adequate manufacturing time for each arm. 
-MRI/MRS imaging of inguinal lymph nodes replaced 
SPECT/CT imaging as the method to assess the impact of DC 
vaccination and unpulsed PBMC vaccination at inguinal 
draining lymph nodes.  
-Immune monitoring time points were expanded to include 
collection around MRI/MRS. 
-nMR analysis of urine was added as a biological correlate. 
-Imaging time points were amended to align with clinical 
standards. 
Minor editorial changes for clarity.    
3, Study Schema 
-Study schema was modified to reflect new study design. 


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Summary of Protocol Changes 
Version Date 
Sections Revised 
Purpose 
4, Abstract 
-Reference to EGFRvIII data removed. 
-Data related LAMP fusion proteins added. 
Study aims modified to align with primary and secondary 
objectives. 
5, Hypothesis and 
Objectives 
-Sections modified to align with modified treatment arms.  
-New section added to outline exploratory objectives. 
6, Background 
and Significance 
-Minor editorial, formatting, and clarification changes.  
-Section modified to delineate between ATTAC- and 
ATTAC-GM trials. 
-Survival data added for ATTAC-GM trial. 
7, Study 
Rationale 
-Reference to in vitro full-length LAMP data added. 
8, Subject 
Eligibility 
-Modifications were made to clarify inclusion criteria and 
timing of eligibility assessments.  
9, Investigational 
Plan 
-Section replaced to correlate with study design.  
9.4.2, DC 
Generation, 
Storage, and 
Testing  
9.4.3, Preparation 
of DCs or 
PBMCs for 
Vaccination 
-Reference to short and full-length LAMP added to correlate 
with study design. 
9.7, MRI/MRS 
Imaging of DC 
Migration 
-Reference to SPECT/CT replaced with MRI/MRS imaging. 
-MRI/MRS imaging time points added. 
-Immune monitoring time points around MRI/MRS added. 
-nMR analysis of urine around MRI/MRS added.  
10, Study 
Requirements 
-Modified to correlate with study design and DC migration 
requirements. 
-Imaging time points were amended to align with clinical 
standards. 
12, 
Immunological 
Response 
Evaluations 
-Section modified to add immunologic endpoints and 
response evaluations. 
12.2, 
Polyfunctional T 
cell Responses, T 
cell Phenotype, 
and Tetramer 
Analysis 
-Section modified to clearly define T cell response and data 
analysis. 


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Version Date 
Sections Revised 
Purpose 
12.3, Cytokine 
Bead Array 
Analysis 
12.4, Antibody 
Titers 
-New section added to outline processes for testing and 
analysis. 
13.1, Dose-
limiting Toxicity 
-Section modified to clearly define dose-limiting toxicities 
and stopping criteria.  
13.2 Adverse 
Events 
-Section revised to denote the collection of all grade 3 AEs 
and greater. 
13.3, Unblinding 
-Section deleted to reflect change in study design. 
13.4, Reporting of 
SAEs 
-Section modified to delineate responsibilities for reporting 
SAEs. 
13.5.8, SPECT 
Scans and 
Radiation 
Exposure 
-Section deleted.   
14.1.1 DSMB 
-Section modified to reflect updated review responsibilities. 
14.2, Audits 
-Auditing plan included for Duke University and the 
University of Florida outlining reporting responsibilities to the 
University of Florida. 
15, Statistical 
Methods and Data 
Analysis 
-Statistical evaluation methods revised to reflect new study 
design. 
 
16.1, Regulatory 
and Ethical 
Compliance 
- Added reference to FDA regulations. 
 
20160720 
Title Page 
-Individuals updated to reflect current personnel. 
Table of Contents 
-Updated for pagination. 
2, Study 
Synopsis. Study 
Design 
- Section modified to correlate with study design. Subject will 
be followed after disease progression. 
8, Subject 
Eligibility 
-Modifications were made to clarify timing of eligibility 
assessments.  
8.1, Inclusion 
criteria 
-Steroids replacement dose has been updated to reflect revised 
protocol. 
9.1, Overview 
- Section modified to outline process for Recursive 
Partitioning Analysis class determination. 
- Section modified to correlate with study design. Subject will 
be followed after disease progression. 


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10.1, Enrollment 
- HPN added at Enrollment visit to correlate with study design 
- MMSE added to determine Recursive Partitioning Analysis 
class according to the protocol statistical method (Section 
15.1 Study Design Overview) 
- Infectious Disease panel added at Enrollment visit to assess 
subject eligibility prior to randomization. 
10.2, Prior to 
Leukapheresis 
- Modification was made to clarify timing of randomization 
10.4, Adjuvant 
TMZ Cycles 
- Section modified to correlate with study design:  
Urine pregnancy testing added prior to Inguinal MRI/MRS 
procedure for subject safety;  
Urine for immune monitoring will be collected prior to 
vaccine 1 for baseline comparison;  
Humoral response testing material was updated  
10.6, Schedule  
of Study 
Assessments 
- Schedule of procedures table layout was modified. 
- Footnotes section modified to reflect changes to the protocol 
15.1, Study 
Design Overview 
- Section modified to reflect changes with study design 
18.5 Appendix E 
- Section added to define performance status conversion 
method.   
20161129 
Title Page 
-Updated to include current personnel. 
2, Study Synopsis 
 
-Eligibility criteria modified to allow inclusion of patients 
with larger residual tumor and to define acceptable steroid 
maintenance dose to align with standards in brain tumor 
protocols. 
8.1, Inclusion 
Criteria 
-Eligibility criteria modified to allow inclusion of patients 
with larger residual tumor and to define acceptable steroid 
maintenance dose to align with standards in brain tumor 
protocols. 
10.5, Study 
Requirements at 
Progression 
-Updated for internal consistency. 
10.6, 
Concomitant 
Medications 
-Section added to define requirements for concomitant 
medication collection and documentation. 
10.7, Schedule of 
Study 
Assessments 
-Updated to add comprehensive metabolic panel prior to the 
initiation of adjuvant chemotherapy to determine ongoing 
eligibility. 
20160328 (should 
have been 20170328) 
Title Page 
- 
Updated to include current personnel. 


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20170410 
Overall Document - 
Footer updated to reflect version change. 
- 
Table of Contents updated for pagination. 
- 
Additional editorial, typographical and formatting changes 
have been made throughout the body of the protocol, 
along with minor changes for document consistency, all of 
which are tracked but not individually listed.  
Title Page 
- 
Roles and responsibilities for multi-site implementation 
delineated and investigators and key personnel updated. 
Protocol 
Signature Page 
- 
Documentation of investigator and sponsor agreement 
added to the protocol per ICH GCP.  
List of 
Abbreviations 
- 
Acronyms/Abbreviation List updated to more accurately 
reflect use in protocol. 
2, Study Synopsis 
 
- 
Serum pregnancy test inclusion criterion clarified.  
- 
Window for study procedure leukapheresis #2 was 
modified to avoid further delay in cycle 2 adjuvant 
chemotherapy. 
- 
Criteria for assessing progression of disease prior to start 
of TMZ clarified. 
- 
Criteria for assessing tumor response, progression, and 
pseudoprogression updated to utilize criteria developed for 
the evaluation of neuro-oncology patients undergoing 
immunotherapy. 
6.2, Dendritic Cell 
Immunotherapy 
6.3, Autoimmune 
Encephalomyelitis 
- 
References to EGFRvII deleted. Results were negative. 
8, Subject 
Eligibility 
- 
Serum pregnancy test inclusion criterion clarified. 
- 
Criteria for assessing progression of disease prior to start 
of TMZ clarified. 
9.1, Overview 
- 
Window for study procedure leukapheresis #2 was 
modified to avoid further delay in cycle 2 adjuvant 
chemotherapy. 
- 
Criteria for assessing tumor response, progression, and 
pseudoprogression updated to utilize criteria developed for 
the evaluation of neuro-oncology patients undergoing 
immunotherapy. 
9.2, Registration 
Procedures 
- 
Procedures for subject registration added to provide for 
consistent multisite implementation. 
9.3, 
Randomization 
Procedures 
- 
Procedures for subject randomization added to provide for 
consistent multisite implementation. 
9.4, Radiation 
Therapy 
- 
Provision for local RT added to reduced travel and 
financial burden on subjects. 


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9.5, 
Temozolomide 
Therapy 
- 
Section revised to provide additional guidelines for 
maintenance chemotherapy management. 
- 
Chemotherapy regimen was modified for patients 70 years 
and older. There is no safety data available on dose 
intensified Temozolomide for patients 70 years old and 
older.  
9.6, Removal of 
PBMCs by 
Leukapheresis 
- 
Window for study procedure leukapheresis #2 was 
modified to avoid further delay in cycle 2 adjuvant 
chemotherapy. 
 
9.6.1, PBMC 
Generation, 
Storage, and 
Testing 
- 
Reference to new PBMC clinical product Batch Record 
added and existing SOP reference corrected. 
9.6.2, DC 
Generation, 
Storage, and 
Testing 
- 
Reference to new Batch Record for DC manufacturing 
added. 
9.7, Tetanus and 
Diphteria Toxoid 
Booster 
- 
Information on site pre-treatment added for consistency 
with other protocol sections. 
10.1, Enrollment 
10.3, Chemo-
radiation 
- 
Existing study requirements relocated to better reflect 
timing of protocol activities. 
- 
Timing of post-operative MRI added for clarity and 
flexibility. 
- 
Pregnancy test added for consistency with other protocol 
sections. 
10.7, Schedule of 
Study 
Assessments  
10.7, Footnotes 
- 
Window for study procedure leukapheresis #2 was 
modified to avoid further delay in cycle 2 adjuvant 
chemotherapy. 
- 
Roles and responsibilities for assessing tumor progression 
clarified. 
- 
Blood volume requirement for Immune monitoring 
samples modified to limit venous puncture for patients.   
11, Response 
Evaluation 
- 
Guidelines for tumor response evaluation for patients 
receiving immunotherapy per the immune-related 
response criteria (irRC) and the response assessment in 
neuro-oncology (RANO) working group added. 
- 
References to RECIST and RTOG 0525 were removed 
from this section. 
11.1, General 
Methodology for 
Determining 
Tumor 
Measurements 
- 
Guidelines for determining tumor measurements added for 
consistent methodology across all participating sites. 


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11.2-11.5, 
Response 
definitions 
- 
Response and progression language revised to reflect 
iRANO definitions. 
11.5, Response 
Evaluation after a 
Complete 
Resection  
- 
Section removed since the protocol was revised (Version 
20161129) to include patients with partial tumor resection. 
In this study response will be evaluated by iRANO for all 
patients. 
13.1, Dose 
Limiting Toxicity 
- 
DLT exception added to address Cerebral Edema Adverse 
Event grading. 
13.2, Adverse 
Events 
- 
Definitions modified to align with FDA regulations. 
- 
Roles, responsibilities, timeframes, mechanism, and 
requirements for reporting AEs clarified.  
13.3 (including 
13.3.1-13.3.3), 
Adverse Event 
Reporting 
- 
Procedures for AE assessment and reporting delineated for 
multisite implementation. 
13.4.12, 
Confidentiality 
- 
Procedures to minimize risk to subject privacy and 
confidentiality of data expanded. 
14.1.1, Data 
Safety Monitoring 
Board 
- 
Name of UFHCC DSMB updated to DISC as a result of 
Cancer Center re-structure. 
- 
Requirement for reporting to local IRB added. 
14.3.1, Study 
Documentation 
- 
List of relevant study documents expanded for 
completeness. 
14.3.2, Site 
Activation 
- 
Procedures and requirements for site activation added for 
multisite implementation. 
14.3.3, Case 
Report Forms 
- 
Procedures for data capture, collection, correction and 
monitoring modified for completeness. 
16 (16.1-16.6), 
Regulatory 
Requirements and 
Ethical 
Considerations 
- 
Ethical and regulatory requirements associated with FDA-
regulated trials expanded. 
17, References 
- 
References updated. 
Appendices C and 
D 
- 
Complete blood count schedule as outlined in the 
appendices were updated to correlate with protocol 
requirements. 


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9.5 
Temozolomide 
Therapy 
- 
Standard of care Temozolomide for patients 70 and older 
is updated to reflect the approved conventional daily dose 
of 150 to 200 mg per square meter of body surface area 
for 5 days 
20170622 
2, Study Synopsis 
9.1, Overview 
9.6, Removal of 
PBMCs by 
Leukapheresis 
10.7, Schedule of 
study assessments 
 
- 
Provision added to repeat leukapheresis to generate the 
required amount of vaccines to complete the study (up to 
10 vaccines). 
9.8 DC or PBMC 
Vaccination 
- 
Section revised to specify that vaccination may proceed 
with lower dose of cells if there are less cells than the 
targeted dose at time of vaccination. 
13.4.7, MRI 
- 
Section revised for consistency with inclusion criteria. 
Patients unable to undergo MRI will not be excluded from 
the study but followed by CT scans. 


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1, Study Synopsis 
8.1, Inclusion 
Criteria 
 
- 
Inclusion criterion #2 revised to provide clarification of 
the histopathologic diagnosis eligible for participation in 
this clinical trial. Variants of Glioblastoma WHO Grade 
IV are eligible for enrollment. 
- 
Inclusion criterion #3 revised to remove Cytomegalovirus 
(CMV) seropositive status as a requirement for 
participation in this trial.  Serology testing remains in the 
protocol and will be used to stratify randomization. During 
the first year of study enrollment, we observed an 
unexpectedly low frequency of CMV seropositivity 
(patients with circulating antibodies to CMV) in the GBM 
patient population.  While not used as eligibility in Phase I 
trials (Mitchell et al., Nature 2015 and Batich et al., Clin 
Can Res 2017), CMV seropositivity was included as an 
inclusion criterion for this Phase II trial to ensure similar 
immunologic exposure to the CMV virus across enrolled 
subjects on the vaccine and control arms.  Based on 
published reports of the CMV exposure for individuals 
older than 50 years old, a frequency of 60-90% 
seropositivity would have been expected.   Thus far, we 
have detected circulating CMV antibodies in only 33% of 
our clinic/inpatient GBM patients which is unexpectedly 
low for older individuals, and suggests, a possible false 
negative rate in patients, possibly secondary to 
immunosuppression.  Supportive of this notion, a 
publication in OncoImmunology from a virology group at 
Karolinska Institute noted “Discordant humoral and 
cellular immune responses to Cytomegalovirus (CMV) in 
glioblastoma patients whose tumors are positive for 
CMV” (Rahbar et al., OncoImmunology 2015).  In the 
conclusion of this paper the authors noted, “In GBM 
patients, HCMV activity is higher than in healthy controls 
and serology is a poor test to define previous or active 
HCMV infection in these patients.”  We plan to analyze 
retrospectively whether CMV seropositivity impacts on 
immunologic response and clinical outcomes to 
vaccination. 
 
3, Study Schema  
 
- 
Modified for consistency with eligibility criteria. 
 
10.1, Enrollment 
- 
Modified for consistency with eligibility criteria. 
 
Summary of 
Protocol Changes 
- 
Previous version date corrected – changed from 20170621 
to 20170622. 


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Addendum to the 
consent form 
document – 
Section 3. Will 
you be paid for 
taking part in this 
study? 
 
- 
Patients will be offered the opportunity to have their travels 
covered for research visits during their participation in the 
study. The documents was revised to include details about 
travel arrangements.  
Title Page 
- 
Updated investigators and key personnel 
2. Study Synopsis 
- 
Section modified to correlate with revised protocol  
3. Study Schema 
- 
Pre-treatment with Td or saline were added to the study 
schema to correlate with study design 
8.1 Inclusion 
criteria & 
8.2 Exclusion 
criteria 
- 
Post-op MRI requirements updated for clarity 
- 
Eligibility criteria were updated for clarity regarding 
contraceptive methods requirements 
- 
Progression disease will be assessed using modified RANO 
criteria 
9.1 Overview 
- 
Section updated to specify randomization stratification by: 
- 
Age (69 y.o. and younger or 70 y.o. and older), 
receiving different treatment regimen 
- 
RPA class (II, IV or V) 
- 
CMV serology (seropositive or seronegative) to 
distribute patients equally across the 3 treatment arms. 
- 
Criteria for assessing tumor response or progression changed 
from “iRANO” to “Modified RANO”. iRANO was created to 
assess tumor progression and response in patient undergoing 
immunotherapy. Since this protocol involves patients not 
receiving immunotherapy, the last modified RANO provides 
a more accurate and efficient assessment tool across the 3 
treatment arms. 
10.2 Prior to 
Leukapheresis  
- 
Infectious disease testing requirements updated to correlate 
with institutional practices at all participating sites. 
10.7 Schedule of 
Study Assessments 
- 
Footnote 4 updated to reflect revised protocol 
- 
Footnote 8: Post therapy MRI requirements updated to 
correlate with SOC 
11 Response 
Evaluation 
- 
Section revised to include the Modified RANO assessment 
criteria 
- 
Guidelines for determining tumor measurements updated to 
outlined Modified RANO requirements 
- 
Response and Progression language revised to reflect 
Modified RANO definitions 
- 
Guidelines added to provide guidance on clinical status 
assessment 
- 
Modified radiographic response assessment diagram replaced 
with Modified RANO algorithm 
13.2 Adverse 
Events 
- 
Section updated to provide additional information on AE 
collection requirements 


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13.4.6 
Leukapheresis 
- 
Section modified for consistency with protocol revision 
14.1.1 Data 
Monitoring Plan 
- 
Data monitoring plan added 
15.1 Study Design 
Overview 
- 
Section updated to specify randomization stratification by 
age 69 y.o and younger or 70 y.o. and older; and CMV 
serology status 
15.1 Study Design 
Overview 
- 
Section updated to specify randomization stratification by 
Age group, RPA class and CMV serology 
15.2 Sample Size 
Justification for 
Vaccine 
Comparison 
- 
Section revised to correct randomization timepoint. 
Randomization occurs on average 4 to 6 weeks after surgery 
and prior to chemo-radiation. 1st vaccination occurs on 
average 15 weeks after randomization. Power and stopping 
rule calculations are slightly different but inconsequently. 
These numbers were updated. 
15.3.1 Primary 
Objective and 
Secondary Clinical 
Objectives 
- 
An intent to treat subanalysis is added to this section. 
17. References 
- 
References updated 
Appendice G 
- 
Guidance on contraception was added 
20180104 
Table of Contents 
- 
Updated for pagination. 
13.3 
- 
Section updated to clarify IND Safety Reporting procedures 
and procedures should a subject death occur per FDA during 
its review of Duke University’s IND submission. 
20180222 
2, Study Synopsis - 
Inclusion criteria listed in the synopsis section updated to 
correlate with inclusion as stated in the protocol. 
- 
Study Design section modified to correlate with protocol 
modification. 
8.1, Inclusion 
criteria to be 
assessed prior to 
adjuvant TMZ 
- 
Inclusion criterion #1 updated to clarify that radiation dose 
of 1.8-2.0 Gy/fraction given 5 days per week for a total of 
59.4-60.0 Gy over < 7 weeks may be given as part of this 
protocol per institutional practices for administration of 
external beam RT for patients with GBM. 
9.1, Overview 
- 
Language related to measure of response as it applies to 
patients with no measurable disease at baseline or best 
response was removed from the study design section. 
Assessment of response/progression for patients with no 
measurable disease at baseline is described in details in 
Section 11, Response Evaluation. 
9.5, 
Temozolomide 
Therapy 
- 
Post radiation chemo therapy guidelines for patients who 
experienced significant hematologic AEs during chemo-
radiation are added. 


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11.10, Clinical 
Judgement 
- 
Provision added to provide allowance for clinical 
judgement to decide whether to discontinue study 
treatment during the interval of assessment for resolving 
tumor progression. 
14.1.2, DSMB 
- 
Section modified to reflect that the UFHCC DISC will be 
the DSMB of record for this trial and that reports and 
correspondence from the DISC will be provided to 
participating investigators for distribution and review 
locally as required.  Duke University’s DSMB will defer 
to the UF DISC since UF will review safety data for all 
sites and provide applicable reports.   
17, References 
- 
Section modified to add the article by Wu, J. and X. Xiong 
(2014) which was cited in the protocol text but not listed 
in the Reference Section. 
Appendices C, D 
- 
Delay and dose modifications guidelines updated to reflect 
standard of practice in regards to absolute neutrophils 
count. 
20180504 
16.2, Informed 
Consent 
- 
Provision for obtaining consent from potential participants 
whose primary language is not English added. 
20180629 
2, Study Synopsis 
- Inclusion criteria and study design in the synopsis section 
updated to correlate with inclusion criteria and study design 
as stated in the protocol. 
8.1 Inclusion 
Criteria 
- Requirement to have pre-op and post-op scan of the same 
type (MRI vs. CT) is removed. Eligibility is assessed based 
on tumor residual measurement therefore a MRI (or CT for 
patients unable to undergo MRI) available within 28 days of 
study enrollment is sufficient to assess eligibility 
independently of type of scan obtained pre-op. 
8.1 Inclusion 
criteria to be 
assessed prior to 
adjuvant TMZ 
- Inclusion criterion #1 updated to clarify that dose of 59.4-
60.0 Gy over < 7 weeks is the targeted total dose. Radiation 
may be delayed or interrupted as outlined in protocol 
appendix A. Patients who receive less than 59.4-60 Gy due 
to treatment delayed or interruption as outlined in the 
appendix A will remain eligible for the study unless they 
experienced significant toxicity that persisted over 4 weeks. 
9.1, Overview 
- Language added to clarify that patients who have 
histopathologic confirmation of treatment effect after biopsy 
or surgery may remain in the study. 
9.5, 
Temozolomide 
Therapy 
- Provision added to resume treatment for patients who 
experienced delay at the discretion of the PI in consultation 
with the treating physician if they believe patient may 
benefit from ongoing treatment.  
9.8, DC or PBMC 
Vaccination 
- Provision added to continue vaccination with investigational 
drug for patients who cannot safely resume Temozolomide.   


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11.11, 
Confirmation of 
Tumor 
Progression by 
Histopathology 
- Section added to provide guidelines regarding confirmation 
of tumor progression and confirmation of treatment effect by 
histopathology 
 
Version Date 
Section 
Purpose 
 
 
 
20180912 
Synopsis 
- Updated to reflect protocol changes 
9.1 Overview, 
10.5 At 
Progression 
- Blood samples for immunologic monitoring will be obtained 
at progression if feasible.  
9.8 DC or PBMC 
Vaccination 
- Provision added to allow participants to received generated 
vaccines when they no longer meet protocol requirement 
other than progression. They will be replaced for purpose of 
study endpoints if they do not meet criteria of an evaluable 
subject. 
10.7 Schedule of 
Study 
Assessments 
- Footnotes updated for consistency with protocol 
requirements.  
13.1 Dose-
limiting Toxicity 
- Stopping rules revised to continuously monitor vaccine-
related toxicity at an acceptable toxicity rate of 1 patient in 
7.  
15.2 Sample Size 
Justification for 
Vaccine 
Comparison 
- Section revised to state that Interim analysis will occur after 
40 observed deaths. This correspond to a trial “information 
fraction” of 52%. Interim analysis was initially planned at 24 
months. However, due to accrual rate, these numbers events 
were not observed at 24 months. 
20181108 
Synopsis and 9.1, 
Overview 
- Statement added to clarify that the subgroup of patients who 
were disqualified prior to receiving the 1st vaccine, may be 
allowed to receive vaccines that are already generated. They 
will not count toward the study endpoints but will be 
monitored for toxicities.  
20190415 
Synopsis and 8.1 
Inclusion Criteria 
- Inclusion criteria language related to completion of radiation 
therapy was modified to remove target dose of radiation, to 
clarify that the intent is to include patients who complete 
standard dose therapy with allowance for routine clinical 
management decisions such as dose modification to allow 
safe standard-of-care radiation delivery and chemotherapy 
treatment. 
9.4, Radiation 
Therapy 
- Revised for internal consistency. 


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9.6 Removal of 
PBMCs by 
leukapheresis 
- Clarification about additional leukapheresis procedures 
timeframe was added to limit/avoid treatment 
interruption/delay. 
9.8 
- Clarified that missed vaccines during Cycles 1-8 may be 
given with subsequent cycles of Temozolomide (or monthly 
for patients off Temozolomide) until patients receive a total 
of 10 vaccines if there is no tumor progression. 
10.3, 
Chemoradiation 
- Revised for internal consistency. 
10.7 
- Footnote related to radiation therapy update for internal 
consistency. 
15.3, Analytic 
Methods 
- Clarified what constitutes an evaluable subject for safety and 
toxicity and primary and secondary endpoints. 
18.1, Appendix 
A: Radiation 
Therapy 
 
- Revised for internal consistency. 
20190621 
13.1 Dose-
Limiting Toxicity 
- Section revised to allow for a longer period of clinical 
management (changed from 48 hours to 4 weeks) of 
potential drug-related Grade III neurologic toxicity to more 
closely align with immunotherapy trials for other cancers as 
well as standard clinical management guidelines.  Immune-
related neuro toxicities are a recognized potential adverse 
event of immunotherapy. Symptoms may present throughout 
the course of treatment and involve the peripheral and 
central nervous system and usually respond well to steroids, 
if started early. Management of patients with immune-
related adverse events includes treatment with a 4-week 
steroid taper.  Resumption of therapy can occur if a 
neurotoxic AE is improved to Grade II or lower within 4 
weeks of clinical management.  However, if Grade III 
neurologic toxicity is observed again with subsequent study 
vaccines, a DLT will be declared and the patient will not 
receive additional study vaccines. 
- Surgical intervention to reverse neurological toxicity will 
not constitute DLT if the toxicity is reversed within 4 weeks. 
Immune response may induce inflammation of CNS tissue. 
Surgical intervention may be required for decompression 
and removal of necrotic tissue to reduce cerebral edema. 
 
13.2 Stopping 
Rules 
- Section title added for clarity. Language related to stopping 
rules are unchanged. 
20190820 
Title Page 
- UF key personnel updated for accuracy. 


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13.1 Dose-
Limiting Toxicity 
- NCI CTCAE for AE grading revised from Version 4.0 to 
Version 5.0 per FDA’s recommendation. 
- The definition of drug-related has been revised for clarity 
per FDA’s recommendation. 
- In consultation with FDA following agency review of 
protocol version 20190621, the dose-limiting toxicity (DLT) 
definition, criteria and management have been modified to 
address FDA recommendations related to potential toxicities 
that may result from a longer period of clinical management.  
Specifically, the protocol has been revised to include a two-
stage management plan for declaring a possible DLT: 1) a 7 
day period for improvement of neurologic symptoms after 
medical intervention to grade 2 or better and 2) a 21-day 
period for return to baseline or improvement to grade I or 
better. 
- In consultation with FDA following agency review of 
protocol version 20190621, criteria for resumption of 
treatment has been modified to allow continued treatment 
only if neurotoxicity is improved to grade 1 or better or to 
patient’s baseline status. 
13.3 Adverse 
Events 
18.1 Appendix A 
- CTCAE version updated to 5.0 for internal consistency. 
20191101 
Title Page 
- Updated sponsor for IND 16530 to Immunomic 
Therapeutics, Inc (ITI).  The ownership of IND 16530 was 
transferred from UF/Dr. Mitchell to ITI per agreement of the 
parties. 
20200323 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis, 
9.1 Overview, 
15.1 Study 
Design Overview 
 
- Sections revised to increase total enrollment number to 
reach the study target of 120 evaluable subjects. The study 
target of 120 evaluable patients (defined as having received 
at least one study vaccine) as described in section 15.2 
Sample Size Justification for Vaccine Comparison remained 
unchanged. However, total enrollment number is required to 
achieve this goal due to screen-failures, subject withdrawals 
prior to vaccination and early progression or AEs that 
preclude vaccination. Based on the enrollment and 
withdrawal numbers since study activation, we expect that 
up to 175 patients will be need to achieve the study goal of 
120 evaluable patients. 


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10.4 Adjuvant 
TMZ Cycles 
Section updated to clarify that clinical management of 
patients will be done by initial assessment of disease status 
by the treating neuro-oncologist/study investigators, upon 
review of surveillance brain MRI during routine clinic visit 
followed by study vaccine administration or chemo cycle 
initiation. Clinical assessment of disease status will be 
followed by formal measurements of enhancing lesions for 
modified RANO criteria. 
 
20200610 
Synopsis; 
8.1 Inclusion 
Criteria; 
References 
- In view of cIMPACT-NOW, Consortium 
to Inform Molecular and Practical Approaches to CNS 
Tumor Taxonomy, latest recommendation, based on survival 
data, to reclassify the designation of “Diffuse Astrocytic 
Glioma, IDH wildtype with molecular features of 
glioblastoma, WHO Grade IV” to “Glioblastoma, IDH-
wildtype” if histologically lower grade but supported by 
molecular profiling, (Louis et al.; Brain Pathology; 2020) 
inclusion Criteria #2 was revised to clarify that eligibility 
diagnosis of GBM may be made by histopathology or 
molecular studies.  
20201201 
15.1 Study 
Design Overview 
15.2 Sample Size 
Justification for 
Vaccine 
Comparison 
15.3.2 Secondary 
Biological 
Objectives  
 
- The planned interim analysis was designed to halt enrollment 
early if results demonstrated unlikely benefit and was 
anticipated after 2 years of enrollment with anticipated 40 
events (deaths) to power the analysis. To date after more than 
four years of enrollment and a total of 132 patients enrolled 
on the study, we have just crossed the threshold of events for 
an interim analysis and anticipate enrollment for only an 
additional 10-12 months. The factors that have contributed to 
such a long interval before reaching threshold of events are 
slower monthly accrual than original projected but also 
potentially longer overall survival in the treated population of 
GBM patients than modeled in the interim analysis plan. 
Additionally, the immunotherapy treatment has been well 
tolerated in patients with only 2  Grade 3 or greater AEs 
associated with immunotherapy treatment amongst all treated 
subjects. Therefore, the study team and sponsor have 
determined that completion of enrollment on the study is the 
appropriate research plan for study completion and will forgo 
the interim analysis. 
20210810 
Title Page 
- Funding updated from NCI to UF.  NIH/NCI funding for this 
project has ended. 
 
Title Page, 14.1.1, 
Data Monitoring 
Plan 
- Orlando Health name updated due to recent rebranding. 


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Synopsis, 
9.1 Overview, 
15.1 Study 
Design Overview 
 
- Sections revised to increase total enrollment number to reach 
the study target of 120 evaluable subjects. The study target of 
120 evaluable patients (defined as having received at least 
one study vaccine) as described in section 15.2 Sample Size 
Justification for Vaccine Comparison remains unchanged.  
- Based on current withdrawals due to early progression, AEs 
that preclude vaccination, subjects requests or screen-failure 
rate, we plan to enroll up to 200 subject to achieve the study 
goal of 120 evaluable patients. 
 
10.6, 
Concomitant 
Medications 
- Section revised to clarify the use the use of bevacizumab for 
patients experiencing symptomatic radiation necrosis. 
20211021 
9.6, Removal of 
PBMCs by 
Leukapheresis 
- Section revised to add language related to placement of 
central venous catheter and potential use of fluoroscopy.   
 
13.5.7 Central 
Venous Catheter 
- Section 13.5.7 Central Venous Catheter added to the 
potential risk section of the protocol to outline the potential 
risk associated with placement of a central venous catheter 
20220119 
Title Page 
- Updated to reflect change in personnel.  The UFH Cancer 
Center will provide ongoing statistical support. 
20230509 
Title Page 
- Updated to reflect change in personnel. 
10.1 Enrollment 
- Molecular diagnostics were added to the study requirements 
for data analysis. With recent advancements on tumor 
molecular aberrations significance for high-grade brain 
tumors diagnosis and the understanding of their impact on 
biological mechanism of treatment resistance and patients’ 
prognosis, MGMT-promoter and IDH-1/2 status have 
become a requisite for data analysis on glioblastoma studies. 
12. 
Immunological 
Response 
Evaluations 
- Section has been updated to include correlative studies that 
will be conducted for data analysis and reporting at the end of 
the study.  
20231122 
Title Page 
- IND Sponsor updated to reflect change from Immunomic 
Therapeutics back to UF. 
- Update study personnel to reflect current personnel. 
20241007 
15.3.2 Secondary 
Biological 
Objectives 
- Section updated to ensure consistency with the analysis 
outlined in section 12.3 Multiplex Cytokine Quantification. 
Cytokines array analysis results will be reported as part of the 
study’s secondary biological objectives.  
 
 
 


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ATTAC-II:    
A Phase II Randomized, Blinded, and Placebo-controlled Trial of CMV RNA-Pulsed 
Dendritic Cells with Tetanus-Diphtheria Toxoid Vaccine in Patients with Newly-Diagnosed 
Glioblastoma 
 
PROTOCOL SIGNATURE PAGE 
 
I have read this protocol and agree to adhere to the requirements. I will provide copies of this 
protocol and all pertinent information to the study personnel under my supervision. I will discuss 
this material with them and ensure they are fully informed regarding the investigational plan and 
the conduct of the study according to 21 CFR parts 50, 54, 56 and 312, 45 CFR part 46, ICH 
Good Clinical Practices Guidelines and Institutional Review Board (IRB) requirements. The 
signature below constitutes the approval of this protocol and any attachments, and provides the 
necessary assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements and applicable US federal regulations and ICH guidelines. 
 
Clinical Site:  
 
 
 
 
 
 
 
 
 
 
Site Principal Investigator Printed Name: 
 
 
 
 
 
 
 
 
Site Principal Investigator Signature and Date: 
 
 
 
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
 
LIST OF POSSIBLE ABBREVIATIONS ........................................................................... 24 
 
STUDY SYNOPSIS ............................................................................................................. 29 
 
STUDY SCHEMA................................................................................................................ 35 
 
ABSTRACT .......................................................................................................................... 36 
 
HYPOTHESIS AND OBJECTIVES .................................................................................... 37 
5.1 
Hypothesis..................................................................................................................... 37 
5.2 
Objectives ..................................................................................................................... 37 
5.2.1 Primary Objective ..................................................................................................... 37 
5.2.2 Secondary Objectives................................................................................................ 38 
5.2.3 Exploratory Objectives: ............................................................................................ 38 
 
BACKGROUND AND SIGNIFICANCE ............................................................................ 38 
6.1 
Disease and Current Therapy ........................................................................................ 38 
6.2 
Dendritic Cell Immunotherapy ..................................................................................... 38 
6.2.1 Pre-Clinical Results against Intracerebral Tumors ................................................... 39 
6.2.2 Prior Experience in Patients with Intracerebral Tumors ........................................... 41 
6.2.3 RNA Transfected DCs .............................................................................................. 43 
6.3 
Autoimmune Encephalomyelitis ................................................................................... 43 
6.4 
Presence of CMV in Malignant Astrocytomas ............................................................. 45 
6.5 
Immunotherapy of CMV............................................................................................... 46 
6.6 
Targeting CMV in Patients with Malignant Gliomas ................................................... 47 
6.6.1 DCs Electroporated with pp65-LAMP mRNA ......................................................... 47 
6.6.2 Recognition of Human CMV antigens by TILs ........................................................ 49 
6.7 
ATTAC and ATTAC-GM Clinical Trials .................................................................... 50 
6.8 
T Cell Homeostatic Proliferation .................................................................................. 51 
6.9 
Treatment-Induced Lymphopenia from TMZ .............................................................. 53 
 
STUDY RATIONALE ......................................................................................................... 54 
 
SUBJECT ELIGIBILITY ..................................................................................................... 55 
8.1 
Inclusion Criteria .......................................................................................................... 55 
8.2 
Exclusion Criteria ......................................................................................................... 57 
 
INVESTIGATIONAL PLAN ............................................................................................... 58 
9.1 
Overview ....................................................................................................................... 58 
9.2 
Registration Procedures ................................................................................................ 60 
9.3 
Randomization Procedures ........................................................................................... 60 
9.4 
Radiation Therapy ......................................................................................................... 60 
9.5 
Temozolomide Therapy ................................................................................................ 61 
9.6 
Removal of PBMCs by Leukapheresis ......................................................................... 61 
9.6.1 PBMC Generation, Storage, and Testing .................................................................. 62 
9.6.2 DC Generation, Storage, and Testing ....................................................................... 62 
9.6.3 Preparation of DCs or PBMCs for Vaccination ........................................................ 62 
9.7 
Tetanus and Diphtheria Toxoid Booster and site pre-treatment ................................... 62 
9.8 
DC or PBMC Vaccination ............................................................................................ 63 
9.9 
MRI/MRS Imaging of DC Migration ........................................................................... 64 


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 STUDY REQUIREMENTS ................................................................................................. 64 
10.1 
Enrollment..................................................................................................................... 64 
10.2 
Prior to Leukapheresis (Vaccine Cell Generation) ....................................................... 65 
10.3 
Chemoradiation ............................................................................................................. 65 
10.4 
Adjuvant TMZ Cycles .................................................................................................. 65 
10.5 
At Progression ............................................................................................................... 66 
10.6 
Concomitant Medication ............................................................................................... 66 
10.7 
Schedule of Study Assessments .................................................................................... 68 
 RESPONSE EVALUATION................................................................................................ 70 
11.1 
General Methodology for Determining Tumor Measurements .................................... 70 
11.2 
Complete Response (CR) .............................................................................................. 71 
11.3 
Partial Response (PR) ................................................................................................... 72 
11.4 
Stable Disease (SD) ...................................................................................................... 72 
11.5 
Progressive Disease ...................................................................................................... 72 
11.6 
Symptomatic Deterioration & Reporting Clinical Status ............................................. 73 
11.7 
Steroid Use and Dose .................................................................................................... 73 
11.8 
Overall Objective Status ............................................................................................... 74 
11.9 
Detailed Modified Radiographic Response Assessment .............................................. 76 
11.10 
Clinical Judgement.................................................................................................... 77 
11.11 
Confirmation of Tumor Progression by Histopathology .......................................... 78 
 IMMUNOLOGICAL RESPONSE EVALUATIONS .......................................................... 78 
12.1 
ELISPOT Assay ............................................................................................................ 79 
12.2 
Polyfunctional T cell Responses, T cell Phenotype, and Other Type of Immune Cell 
Phenotype .................................................................................................................................. 79 
12.3 
Multiplex Cytokine Quantification ............................................................................... 80 
12.1 
Single Cell Gene Expression and Immune Profiling For Monitoring Changes in 
Glioblastoma Patients with Sustained Radiographic Response to CMV pp64-LAMP RNA-
Pulsed Dendritic Cell-Based Vaccines. .................................................................................... 80 
 SAFETY MONITORING AND REPORTING.................................................................... 81 
13.1 
Dose-limiting Toxicity .................................................................................................. 81 
13.2 
Stopping Rules .............................................................................................................. 82 
13.3 
Adverse Events ............................................................................................................. 83 
13.4 
Adverse Event and Serious Adverse Event (SAE) Reporting ...................................... 85 
13.4.1 
Adverse Event Reporting to IRB .......................................................................... 86 
13.4.2 
Adverse Event Reporting to the DSMB ................................................................ 86 
13.4.3 
Adverse Event Reporting to the FDA ................................................................... 86 
13.4.4 
Adverse Event Reporting to Sites ......................................................................... 86 
13.5 
Potential Risks .............................................................................................................. 87 
13.5.1 
Allergic Reactions to DC Immunization............................................................... 87 
13.5.2 
Cerebral Edema ..................................................................................................... 87 
13.5.3 
Infection ................................................................................................................ 87 
13.5.4 
Delayed Autoimmune Diseases ............................................................................ 87 
13.5.5 
Phlebotomy ........................................................................................................... 87 
13.5.6 
Leukapheresis ....................................................................................................... 88 
13.5.7 
Central Venous Catheter ....................................................................................... 88 


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13.5.8 
MRI ....................................................................................................................... 88 
13.5.9 
GM-CSF ................................................................................................................ 88 
13.5.10 Td .......................................................................................................................... 88 
13.5.11 Allergic Reactions to Contrast Agents .................................................................. 89 
13.5.12 Temozolomide ...................................................................................................... 89 
13.5.13 Confidentiality ...................................................................................................... 89 
 QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 89 
14.1 
Data and Safety Monitoring Plan .................................................................................. 89 
14.1.1 
Data Monitoring Plan ............................................................................................ 89 
14.1.2 
Data Safety Monitoring Board .............................................................................. 90 
14.2 
Audits ............................................................................................................................ 90 
14.3 
Data Management and Processing ................................................................................ 91 
14.3.1 
Study Documentation............................................................................................ 91 
14.3.2 
Site Activation ...................................................................................................... 91 
14.3.3 
Case Report Forms ................................................................................................ 92 
14.3.4 
Data Management Procedures and Data Verification ........................................... 92 
 STATISTICAL METHODS AND DATA ANALYSIS ...................................................... 92 
15.1 
Study Design Overview ................................................................................................ 92 
15.2 
Sample Size Justification for Vaccine Comparison ...................................................... 94 
15.3 
Analytic Methods .......................................................................................................... 95 
15.3.1 
Primary Objective and Secondary Clinical Objectives ......................................... 95 
15.3.2 
Secondary Biological Objectives .......................................................................... 96 
 Regulatory requirements AND ETHICAL CONSIDERATIONS ....................................... 96 
16.1 
Institutional Review Board ........................................................................................... 96 
16.2 
Informed Consent.......................................................................................................... 97 
16.3 
Privacy, Confidentiality, and Data Storage................................................................... 97 
16.4 
Protocol Amendments ................................................................................................... 98 
16.5 
Records Retention ......................................................................................................... 98 
16.6 
Conflict of Interest ........................................................................................................ 98 
 REFERENCES ..................................................................................................................... 99 
 APPENDICES .................................................................................................................... 112 
18.1 
Appendix A: Radiation Therapy ................................................................................. 112 
18.2 
Appendix B: Temozolomide Therapy during Radiation ............................................ 113 
18.3 
Appendix C: Temozolomide Therapy after Radiation ................................................ 115 
18.4 
Appendix D: Alternative Temozolomide Therapy after Radiation ............................ 118 
18.5 
Appendix E: ECOG Performance Status to Karnofsky Performance Status Comparison 
Table 119 
18.6 
Appendix G: GUIDANCE ON CONTRACEPTION ................................................. 121 
 
 
 
 
 
 
 


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 LIST OF POSSIBLE ABBREVIATIONS 
 
Ab 
Antibody 
ACTIVATE 
A Complementary Trial of an Immunotherapy Vaccine Against 
Tumor-Specific EGFRvIII 
AE 
Adverse Event 
AIDS 
Acquired Immune Deficiency Syndrome 
AMRIS 
Advanced Magnetic Resonance Imaging and Spectroscopy 
ANOVA 
Analysis of Variance 
ANC 
Absolute Neutrophil Count 
APC 
Allophycocyanin 
ALT 
Adoptive Lymphocyte Transfer 
ALT 
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
AT 
Ambient Temperature 
ATTAC 
Anti-Tumor Immunotherapy Targeted Against Cytomegalovirus in 
Patients 
with Newly Diagnosed Glioblastoma Multiforme during 
Recovery from Therapeutic Temozolomide-induced Lymphopenia 
β-HCG 
Beta-Human Chorionic Gonadotropin 
BID 
Twice Daily 
BMT 
Bone Marrow Transplant 
BTIP 
Brain Tumor Immunotherapy Program 
BUN 
Blood Urea Nitrogen 
CA 
California 
CBC 
Complete Blood Count 
Cc 
Cubic Centimeters 
CDC 
Centers for Disease Control 
cDNA 
Complimentary Deoxyribonucleic Acid 
CEA 
Carcinoembryonic Antigen 
CFA 
Complete Freund’s Adjuvant 
CFC 
Cytokine Flow Cytometry 
CLIA 
Clinical Laboratory Improvement Act 
cm 
centimeter 
CMV 
Cytomegalovirus 
CNS 
Central Nervous System 
Co-PI 
Co-Primary Investigator 
Cr 
Serum Creatinine 
CR 
Complete Response 
CRF 
Case Report Form 
CSF 
Cerebro Spinal Fluid 
CT 
Computed Tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTL 
Cytotoxic T Lymphocyte 
CV 
Curriculum Vitae 
DC 
Dendritic Cell 


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DCI 
Duke Cancer Institute 
DISC 
Data Integrity and Safety Committee 
DLT 
Dose Limiting Toxicity 
DNA 
Deoxyribonucleic Acid 
DSMB 
Data Safety Monitoring Board 
DUMC 
Duke University Medical Center 
EAE 
Experimental Autoimmune Encephalomyelitis 
EBRT 
External Beam Radiation Therapy 
EBV 
Epstein-Barr Virus 
ECHO 
Echocardiography 
eCRF 
Electronic Case Report Form 
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII 
Epidermal Growth Factor Receptor Mutation III 
EGFRvIII-KLH 
EGFRvIII conjugated to Keyhole Limpet Hemocyanin 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-linked Immunospot 
EORTC 
European Organization for Research and Treatment of Cancer 
FACS 
Fluorescence Activated Cell Sorting 
FACT-accredited 
Foundation for the Accreditation of Cellular Therapy 
FDA 
Food and Drug Administration 
FDF 
Financial Disclosure Form 
FITC 
Fluorescein Isothiocyanate 
GBM 
Glioblastoma Multiforme 
G-CSF 
Granulocyte Colony Stimulating Factor 
GFP 
Green Fluorescent Protein 
GLP 
Good Laboratory Practice 
GM-CSF 
Granulocyte Macrophage Colony Stimulating Factor 
HABS 
Human Monoclonal Antibodies 
HB 
Hepatitis B 
HbsAg 
Hepatitis B Surface Antigen 
HD 
High Dose 
HDC 
High Dose Chemotherapy 
H&E 
Hematoxylin and Eosin 
HHS 
Health and Human Services 
HIPAA 
The Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigens 
HSC 
Hematopoietic Stem Cells 
IBC 
Institutional Biosafety Committee 
IFN 
Interferon 
ICH 
International Conference on Harmonization 
IFN- 
Interferon-gamma 
IHC 
Immunohistochemistry 
IL-1β 
Interleukin 1B 
IL-2 
Interleukin-2 


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IL-4 
Interleukin-4 
IL-6 
Interleukin-6 
IL-12 
Interleukin-12 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
ISH 
In Situ Hybridization 
IV 
Intravenous 
KLH 
Keyhole Limpet Hemocyanin 
KPS 
Karnofsky Performance Status 
L 
Liters 
LAMP 
Lysosomal-Associated Membrane Protein 
Lf 
Flocculation Units 
LMD 
Leptomeningeal Disease 
M² 
Meters Squared 
MA 
Myeloablative 
MAb 
Monoclonal Antibody 
MAGE 
Melanoma Antigens 
MD 
Medical Doctor 
MD 
Maryland 
Mcg 
Micrograms 
MG 
Malignant Glioma 
Mg 
Magnesium 
Mg 
Milligrams 
MHC 
Major Histocompatibility Complex 
mL 
Milliliters 
mm 
millimeter 
MMSE 
Mini-Mental Status Examination 
MRI 
Magnetic Resonance Imaging 
MRS 
Magnetic Resonance Spectroscopy 
mRNA 
Messenger Ribonucleic Acid 
MTD 
Maximal Tolerated Dose 
Na 
Sodium 
NA 
Non-adherent 
NC 
North Carolina 
NEJM 
New England Journal of Medicine 
NCI 
National Cancer Institute 
NCI CTC 
National Cancer Institute Common Toxicity Criteria 
NIH 
National Institutes of Health 
NK 
Natural Killer 
NMA 
Non-Myeloablative 
nMR 
Nuclear Magnetic Resonance 
NY 
New York 
OHRP 
Office for Human Research Protections 
OS 
Overall Survival 
PBLs 
Peripheral Blood Lymphocytes 


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PBMC 
Peripheral Blood Mononuclear Cells 
PBS 
Phosphate Buffered Saline  
PBSC 
Peripheral Blood Stem Cell 
PBSCT 
Peripheral Blood Stem Cell Transplant 
PCR 
Polymerase Chain Reaction 
PD 
Progressive Disease 
PEPvIII 
Peptide variant III 
PFS 
Progression Free Survival 
PFS-12 
Progression Free Survival, 12 Months 
PhD 
Doctorate of Philosophy 
PHI 
Private/Protected Health Information 
PI 
Principal Investigator 
PO 
Per Os (by mouth) 
PR 
Partial Response 
PRTBC 
Preston Robert Tisch Brain Tumor Center 
PS 
Performance Status 
® 
Registered 
RAC 
Recombinant DNA Advisory Committee 
RANO 
Response Assessment in Neuro-Oncology 
RDC 
Remote Data Capture 
RN 
Registered Nurse 
RNA 
Ribonucleic Acid 
RPA 
Recursive Partitioning Analysis 
RT 
Radiation Therapy 
RTOG 
Radiation Therapy Oncology Group 
RT-PCR 
Reverse Transcriptase Polymerase Chain Reaction 
SAE 
Serious Adverse Event 
SD 
Standard Deviation 
SD 
Stable Disease 
SH 
Subtractive Hybridization 
SOC 
Standard of Care 
SOP 
Standard Operating Procedure 
SPECT 
Single Photon Emission Computed Tomography 
SQ 
Subcutaneous 
TCR 
T-cell Receptor 
Td 
Tetanus and Diphtheria Toxoids 
TGF- 
Transforming Growth Factor- 
TILs 
Tumor Infiltrating Lymphocytes 
TMZ 
Temozolomide 
TNF 
Tumor Necrosis Factor 
TNF-α 
Tumor Necrosis Factor-Alpha 
Tregs 
Regulatory T-cells 
TTP 
Time to Progression 
UF 
University of Florida 
UFHCC 
University of Florida Health Cancer Center 


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µg 
Micrograms 
µl 
Microliters 
US 
United States 
USP 
United States Pharmacopeia 
UV 
Ultraviolet 
VDLN 
Vaccine-site Draining Lymph Nodes 
VICTORI 
Dose-Finding and Safety Study of Autologous, Tumor-Specific 
Antigen-Pulsed Dendritic Cell Immunotherapy for Malignant Brain 
Tumors 
WA 
Washington 
WBI 
Whole Body Irradiation  
WHO 
World Health Organization 
 
 


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 STUDY SYNOPSIS  
 
Title 
ATTAC-II:  A Phase II Randomized, Blinded, and Placebo-controlled Trial of CMV RNA-
Pulsed Dendritic Cells with Tetanus-Diphtheria Toxoid Vaccine in Patients with Newly-
Diagnosed Glioblastoma  
Study Drugs: 
Autologous dendritic cells (DCs) derived from PBMC loaded with RNA encoding the human 
cytomegalovirus (CMV) matrix protein pp65 as a fusion protein with the short targeting 
peptide from lysosomal membrane associated protein (pp65-shLAMP) plus GM-CSF and 
tetanus-diphtheria (Td) vaccine as adjuvants; autologous DCs derived from PBMC loaded 
with pp65 RNA as a fusion protein with full-length LAMP protein (pp65-flLAMP) plus GM-
CSF and Td as adjuvants; or autologous unpulsed PBMCs administered as a control vaccine. 
Rationale: 
CMV antigens have been identified in glioblastoma (GBM) and may make excellent anti-
tumor immunotherapeutic targets.  Vaccination and adoptive T-cell strategies targeting CMV 
in humans in other contexts have been safe and effective. Our prior studies examining patients 
with GBM have demonstrated the feasibility, safety, immunogenicity, and potential clinical 
efficacy of this approach (Mitchell and Batich et al., Nature 2015). Temozolomide (TMZ) has 
recently shown modest efficacy, but is not curative, in some patients with newly diagnosed 
GBM and is now routinely given to these patients during and after radiation therapy (RT). 
Therapeutic TMZ induces a profound lymphopenia, however, that may inhibit anti-tumor 
vaccination. We have demonstrated, however, that repeated vaccination during recovery from 
cycles of lymphodepletive temozolomide can induce potent humoral and cellular 
immunologic responses. 
 
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a powerful adjuvant capable 
of stimulating macrophage function, inducing proliferation and maturation of DCs, and is able 
to enhance T-lymphocyte stimulatory function.  Intradermal administration of GM-CSF 
enhances the immunization efficacy at the site of administration in a dose dependent fashion.  
Significant anti-tumor immunity has been demonstrated in preclinical murine studies in which 
irradiated, stably transfected tumor cell lines secreting GM-CSF has protected against 
subsequent tumor challenge, especially against intracerebral tumors. 
 
Tetanus toxoid (Td) is a routinely used vaccine in the normal human population that we have 
shown in pilot studies may function as a potent adjuvant to enhance DC trafficking to vaccine-
site draining lymph nodes (VDLNs). Our previous studies have shown that successful DC 
migration to VDLNs may be a requisite for clinical activity of RNA-pulsed DCs and 
administration of Td prior to vaccination may improve the effectiveness of DC vaccines in 
patients with GBM. 
Primary 
Objective: 
1. To determine whether the addition of pp65-LAMP mRNA DC vaccine plus GM-CSF and 
Td to dose-intensified TMZ treatment is worthy of investigation in a large phase III study 
based on impact on overall survival. 
Secondary 
Objectives: 
1. Evaluate the impact of CMV pp65-LAMP RNA-pulsed DC vaccines on progression-free 
survival in patients with newly-diagnosed GBM.  
2. Determine the immunologic effects of vaccination with pp65 RNA fusion constructs 
incorporating full-length LAMP vs short LAMP sequences. 
Inclusion 
Criteria: 
Enrollment and randomization must occur prior to subjects undergoing standard of care 
chemo-radiation.  


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All the following inclusion criteria need to be met in order to randomize the subject: 
1. Age ≥ 18 years. 
2. Confirmed diagnosis of de novo Glioblastoma (WHO Grade IV glioma) by histopathology 
or molecular studies. 
(Secondary GBM not eligible). 
3. The tumor must have a supratentorial component. 
4. Patient must have undergone surgical resection of tumor with less than approximately 3cm 
x 3cm (9cm2) residual enhancing tumor as product of longest perpendicular planes by 
MRI.   
5. Patients must have recovered from the effects of surgery, postoperative infection, and 
other complications. 
6. A diagnostic contrast-enhanced MRI or CT scan (if MRI is not available) of the brain must 
be performed preoperatively and postoperatively. Post-op MRI must be performed within 
28 days prior to study enrollment. If no post op MRI/CT within 28 days of enrollment is 
available, a new brain MRI (or CT) must be obtained in order to determine residual burden 
and eligibility prior to randomization.  
Patients unable to undergo MR imaging because of non-compatible devices can be enrolled, 
provided pre- and post-operative contrast-enhanced CT scans are obtained and are of 
sufficient quality. 
7. Karnofsky Performance Status (KPS) ≥ 70.  
8. Signed informed consent.  If the patient’s mental status precludes his/her giving informed 
consent, written informed consent may be given by the legally authorized representative. 
9. For females of childbearing potential, negative serum pregnancy test at enrollment (test 
will be repeated within 72 hours prior to starting TMZ). 
10. Women of childbearing potential (WOCBP) must be willing to use acceptable 
contraceptive methods to avoid pregnancy throughout the study and for at least 24 weeks 
after the last dose of study drug.    
Refer to Appendix G for definition of WOCBP and guidance on acceptable contraceptive 
methods. 
11. Males with female partners of childbearing potential must agree to use physician-approved 
contraceptive methods (e.g., abstinence, condoms, vasectomy) throughout the study and 
should avoid conceiving children for 24 weeks following the last dose of study drug. 
Refer to Appendix G for guidance on acceptable contraceptive methods 
 
 
To be assessed prior to initiation of adjuvant TMZ: 
1. Patients must have completed standard RT at the discretion of the treating Radiation 
Oncologist  and concomitant TMZ therapy at a targeted dose of 75mg/m2/d for < 49 days 
without significant toxicity that persisted over 4 weeks.  Significant toxicity is defined as 
one or more of the following: 
a. ANC < 0.5 x 109/L (Grade 4)  
b. Platelet count < 10 x 109/L (Grade 4)  
c. Grade 3 or 4 non-hematologic toxicity (except alopecia, nausea and vomiting unless 
the patient has failed maximal antiemetic therapy, and fatigue). 
2. History & physical with neurologic examination within 14 days prior to initiation of 
adjuvant TMZ. 


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3. For patients receiving steroids, daily dose must be < 4 mg. 
4. CBC with differential obtained within 14 days prior to initiation of adjuvant TMZ with 
adequate bone marrow function as defined below: 
a. Absolute neutrophil count (ANC) ≥ 1500 cells/mm3. 
b. Platelet count ≥ 100,000 cells/mm3. 
c. Hemoglobin ≥ 10 g/dl. (The use of transfusion or other intervention to achieve Hgb ≥ 
10 g/dl is acceptable.) 
5. Adequate renal function within 14 days prior to initiation of adjuvant TMZ as defined 
below: 
a. BUN ≤ 25 mg/dl 
b. Creatinine ≤ 1.7 mg/dl 
6. Adequate hepatic function within 14 days prior to initiation of adjuvant TMZ as defined 
below: 
a. Bilirubin ≤ 2.0 mg/dl 
b. ALT ≤ 5 times institutional upper limits of normal for age 
c. AST ≤ 5 times institutional upper limits of normal for age 
Exclusion 
Criteria: 
All the following exclusion criteria need to be verified in order to randomize the subject: 
1. Prior invasive malignancy (except for non-melanomatous skin cancer) unless disease free 
for ≥ 3 years. (For example, carcinoma in situ of the breast, oral cavity, and cervix are all 
permissible.)  
2. Metastases detected below the tentorium or beyond the cranial vault and leptomeningeal 
involvement.  
3. Recurrent or multifocal malignant gliomas. 
4. HIV, Hepatitis B, or Hepatitis C seropositive. 
5. Known active infection or immunosuppressive disease. 
6. Prior chemotherapy or radiosensitizers (including Gliadel wafers) for cancers of the head 
and neck region, other than TMZ prescribed during radiation for GBM (prior 
chemotherapy for a different cancer is allowable). 
7. Prior radiotherapy to the head or neck (except for T1 glottic cancer and that prescribed for 
GBM ≤60Gy), resulting in overlap of radiation fields.  Radiosurgery is not permitted. 
8. Severe, active co-morbidity, defined as follows: 
a. Unstable angina and/or congestive heart failure requiring hospitalization. 
b. Transmural myocardial infarction within the last 6 months. 
c. Acute bacterial or fungal infection requiring intravenous antibiotics at initiation of 
XRT/TMZ. 
d. Chronic Obstructive Pulmonary Disease exacerbation or other respiratory illness 
requiring hospitalization or precluding study therapy at initiation of XRT/TMZ. 
e. Hepatic insufficiency resulting in clinical jaundice and/or coagulation defects. 
f. Acquired Immune Deficiency Syndrome (AIDS) based upon current CDC definition. 
The need to exclude patients with AIDS from this protocol is necessary because the 
treatments involved in this protocol may be significantly immunosuppressive. 
g. Patients 
with 
autoimmune disease requiring medical 
management with 
immunosuppressants. 
h. Major medical illnesses or psychiatric impairments that, in the investigator's opinion, 
will prevent administration or completion of protocol therapy. 


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i. Active connective tissue disorders such as lupus or scleroderma that, in the 
investigator’s opinion, place the patient at high risk for radiation toxicity. 
9. Pregnancy or women of childbearing potential and men who are sexually active and who 
are unwilling or unable to use an acceptable method of contraception for the entire study 
period; this exclusion is necessary because the treatment involved in this study may be 
significantly teratogenic.  
10. Pregnant or lactating women, due to possible adverse effects on the developing fetus or 
infant. 
11. Prior allergic reaction to TMZ, GM-CSF, or Td. 
12. Prior history of brachial neuritis or Guillain-Barré syndrome. 
13. Patients treated on any other therapeutic clinical protocols within 30 days prior to study 
entry. 
 
To be assessed prior to initiation of adjuvant TMZ: 
• Patient did not start RT and TMZ within 7 weeks of surgery. 
• Progression of disease as defined by modified RANO criteria. 
• More than 45 days after completion of RT and TMZ. 
Study Design: 
The eligibility criteria for this trial will be similar to those used in the RTOG 0525 study 
(Aldape 2009).  Prior to the initiation of TMZ and external beam RT, patients will sign consent 
and undergo leukapheresis for PBMCs in accordance with SOP-UFBTIP-131.  
 
Up to 200 newly diagnosed WHO Grade IV glioma patients will be enrolled in the study. 
Eligible patients will be randomized, undergo leukapheresis and receive “standard-of-care” 
radiotherapy concurrent with TMZ under the assumption that approximately 120 patients will 
be vaccinated according to treatment arm in this placebo-controlled, single-blinded, 
randomized Phase II study.  
 
Prior to leukapheresis, patients will be randomized to receive one of three treatment regimens 
with dose-intensified TMZ treatment at 100 mg/m2/day for 21 days with an allocation ratio of 
1:1:1 into one of three arms to receive: 
 
1)  pp65-shLAMP mRNA DCs with GM-CSF 150 µg (Td skin prep) 
2)  pp65-flLAMP mRNA DCs with GM-CSF 150 µg (Td skin prep) 
3)  Unpulsed PBMCs (saline skin prep) 
 
Randomization will be stratified by: 
- 
Age 69 years old and younger, who will receive dose-intensified adjuvant 
temozolomide; or 70 years old and older who will receive standard dose adjuvant 
temozolomide.  
- 
Recursive Partitioning Analysis (RPA) class (III, IV, or V) 
- 
CMV seronegative or seropositive 
 
Within 7 weeks of surgery, patients will undergo standard RT with concurrent TMZ at a target 
dose of 75 mg/m2/day.   
 


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Patients will receive the initial cycle of TMZ 4 (+/- 1) weeks after completing RT. Study drugs 
will be given intradermally at day 22-24 after the first TMZ cycle and divided equally between 
both inguinal regions in accordance with SOP-UFBTIP-128. All patients will receive Td 
booster (5 Lf) with vaccine #1 regardless of booster history.  Vaccine #2 and #3 will occur at 
2 week intervals.  
 
All patients will undergo repeat leukapheresis 2 to 4 weeks after vaccine #3 for PBMCs to 
generate additional DC vaccines and for immunologic monitoring with specific assessment of 
baseline antigen-specific cellular and humoral immune responses.  TMZ cycle 2 will occur 24 
hours to 1 week after repeat leukapheresis. Patients may undergo additional leukapheresis if 
needed to generate up to 10 DC vaccines.  
 
Patients will then be vaccinated monthly in conjunction with subsequent TMZ cycles every 
5(+/- 1) weeks for a total of 6 to 12 cycles after RT as per guidelines defined in recent phase 
III clinical trial evaluating TMZ regimens in patients with GBM (RTOG 0525).  Vaccines will 
be given on day 22-24 of each TMZ cycle.  Patients will continue with monthly vaccinations 
until a total of 10 vaccines have been administered or until tumor progression (whichever 
comes first).   
 
All patients will undergo vaccine site pretreatment with Td (1 Lf) or saline (depending on 
randomization arm) 6-24 hours prior to Vaccine #3, #6, and #9.  At vaccine #3, patients at the 
UF site will undergo MRI/MRS imaging of inguinal vaccine-site draining lymph nodes before 
vaccine site pretreatment, 48hrs post vaccination, and 5 to 7 days post vaccination (optional). 
 
Patients who have been enrolled in the study and become ineligible to receive study treatment 
for reasons other than progression may be allowed to receive vaccines that have been 
generated at the discretion of the PI as outlined in section 9.8, DC or PBMC Vaccination. 
 
Blood for immune monitoring will be collected during initial leukapheresis, prior to vaccines 
1-3, during MRI/MRS imaging time points surrounding vaccine #3, during the second 
leukapheresis, prior to vaccine 4, 7, 10, and at progression if feasible.  Peripheral blood will 
be processed in accordance with SOP-UFBTIP-126.   Urine samples will be collected at 
MRI/MRS imaging time points surrounding vaccine #3 and transferred to the UFBTIP 
laboratory for nMR analysis.  
 
Up to twelve cycles of TMZ may be given if the patient demonstrates continued improvement 
or stability on radiologic scan, decreased corticosteroid requirement, improvement in 
performance status, or improvement in neurologic function. 
 
Patients will be imaged bimonthly during TMZ cycles and every 3 months after the 12th TMZ 
cycle (or 1 year post surgery if TMZ discontinued prior to 12 cycles) without receiving any 
other prescribed antitumor therapy unless progression occurs. 
 
The primary measure of response will be by serial measures of the product of the two largest 
cross-sectional diameters.  The modified RANO criteria (Ellingson, Wen, Cloughesy 2017) 
will be used for assessment of pseudoprogression, progression or response.   


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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. As this is not a research procedure, consent for biopsy will be obtained 
separately. If tissue is obtained, it will be used to confirm tumor progression histologically 
and evaluate cellular infiltration and CMV antigen expression at the tumor site.  
 
If histopathology confirms treatment effect vs tumor progression patient may resume study 
treatment at the discretion of the PI in consultation with the treating physician, if the 
PI believes that it is safe to continue therapy and that patients may benefit from the ongoing 
treatment regimen as described in section 11.11 Confirmation of Tumor Progression by 
Histopathology. 
 
Patients will be followed until death due to any cause. MRI and clinical evaluation for 
assessment of disease progression will be conducted bi-monthly for the first 12 months and 
then every 3 months thereafter. 
 


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 STUDY SCHEMA 
 
 
Newly 
Diagnosed 
GBM 
(WHO Grade 
IV Glioma) 
(120 patients) 
Resection 
Enrollment 
Standard of Care 
External Beam Radiation 
Therapy with Concurrent 
Temozolomide 
(6 weeks) 
Leukapheresis 
DC 
Randomization 1:1:1 
into 1 of 3 Arms 
1 
Pp65-short LAMP DCs 
 with GM-CSF + 
Td skin prep 
(40 patients) 
2 
Pp65-full-length LAMP 
DCs 
with GM-CSF + 
Td skin prep 
3 
Autologous unpulsed 
PBMCs           + 
Saline skin prep 
(40 patients) 
TMZ Cycle 1 
Day 22-24 
Td Booster & Vaccine #1 
Leukapheresis 
TMZ Cycle 2 to 6-12 
Vaccines on Day 22-24 of 
each cycle for a total of 
10 vaccines 
Site pre-treatment with Td 
or saline prior to vaccine #6 
Radiographic or Clinical 
Tumor Progression 
Stereotactic Biopsy or 
Resection 
Immunohistochemical 
Studies 
Vaccine 
Vaccine #2, #3  
every two weeks 
Site pre-treatment with Td 
or saline prior to vaccine #3 
MRI/MRS Inguinal 
Imaging 
 (UF Site Only) 


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 ABSTRACT 
 
Despite aggressive, computer-guided surgery, high-dose focused radiation, and toxic, multi-
mechanistic chemotherapy, malignant gliomas (MGs) remain almost universally fatal.  Moreover, 
these inherently non-specific conventional treatments incapacitate patients as a result of damage 
to surrounding normal brain and systemic tissues(Imperato, Paleologos et al. 1990).  The inherent 
biologic specificity of immunotherapy, however, offers the prospect of targeting neoplastic cells 
more precisely.  DCs are endowed with an extraordinary ability to activate CD4+ and CD8+ T-
cells(Banchereau and Steinman 1998; Steinman 2001), and DCs loaded with antigens derived from 
tumor cells have the potential to induce potent anti-tumor immunity.  Unfortunately, the inherent 
genetic instability of tumor cells severely limits the number of conserved, homogeneously-
expressed, and immunogenic tumor-specific antigens. 
 
The discovery that MGs, but not surrounding normal brain tissue, serve as a refuge for CMV 
reactivation(Cobbs, Harkins et al. 2002) provide an unparalleled opportunity to subvert, as tumor-
specific antigens, the highly immunogenic viral proteins expressed by human CMV.  The 
immunologic responses to CMV have been well-characterized and the immunodominant viral 
protein pp65 is highly-conserved.  Finally, CMV-specific immunotherapy has been previously 
shown in humans to be safe and efficacious in combating CMV related disease within the central 
nervous system (CNS)(Bigger, Tanigawa et al. 1999), and antitumor immunotherapy targeting 
viral proteins in human CNS tumors associated with Epstein-Barr virus (EBV), another 
Herpesvirus, have been curative(Emanuel 1991; Papadopoulos, Ladanyi et al. 1994; Emanuel, 
Lucas et al. 1997; Rooney, Smith et al. 1998; Lucas and Barrett 1999; Liu, Savoldo et al. 2002).  
Similarly, vaccinations directed against the highly immunogenic antigens of human papilloma 
virus have recently been shown to reduce the incidence of cervical intraepithelial neoplasia in a 
prospective, randomized, double-blind trial(Koutsky, Ault et al. 2002). 
 
Chemotherapeutic agents frequently used in cancer therapy, however, often induce a profound 
lymphopenia that may inhibit even the most potent anti-tumor immune responses.  TMZ, a 
methylating chemotherapeutic agent, has recently shown efficacy in some patients with newly 
diagnosed GBM(Stupp, Mason et al. 2005) and is now routinely given to these patients during and 
after RT.  Therapeutic TMZ, however, also induces a profound lymphopenia.  
 
Human CMV antigens have been shown by a number of laboratories to be expressed in a high 
proportion (>90%) of malignant gliomas. We have recently completed a phase I/II clinical trial 
exploring the safety, immunogenicity, and potential clinical efficacy of autologous pp65 RNA-
pulsed DC vaccines in patients with newly-diagnosed GBM.  This trial explored the capacity to 
enhance DC migration to VDLNs using inflammatory skin preparations administered prior to pp65 
RNA-loaded DC vaccines in a randomized pilot trial design (n=12 patients).  The results of these 
studies demonstrated the capacity to safely expand CMV-specific cellular and humoral immunity 
in patients with GBM using autologous pp65 RNA-pulsed DC vaccines and demonstrated a strong 
correlation with successful DC migration to VDLNs and clinical outcomes (R=0.73, P=0.007; 
Pearson correlation coefficient).  Strikingly, patients randomized to receive a tetanus toxoid 
booster as an inflammatory stimulus at the vaccine-site showed an increased migration of DCs to 
VDLNs (P=0.04) and a corresponding increased progression-free and overall survival (P=0.01 
Logrank analysis).  These results suggested that successful DC trafficking in vivo is associated 


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with improved outcomes in patients with GBM receiving pp65 RNA-pulsed DC vaccines and that 
inflammatory stimuli that enhance DC migration improve the efficacy of this treatment modality.  
In support of this hypothesis, we found increased levels of chemokines that facilitate DC migration 
in patients randomized to the tetanus group.  Additionally, we have corroborated these findings 
using a transgenic mouse model employing GFP+ mice to evaluate DC migration.  Pp65 RNA-
pulsed DCs are a novel and promising therapeutic modality for patients with GBM, and our studies 
indicate that DC migration to VDLNs constitutes a major biological axis for potential clinical 
intervention in order to enhance the efficacy of this treatment strategy.  
 
Studies within our laboratory have demonstrated that the activation and expansion of CMV pp65-
specific T cells can be enhanced through modification of the RNA encoding pp65 with a full-
length LAMP fusion protein.  The fusion of antigenic proteins to LAMP channels antigens 
synthesized within the cytosol into the class II presentation pathway leading to enhanced CD4+ T 
cell activation and CTL induction. In the previous ATTAC trial, the CMV pp65 RNA construct 
encoded for a short peptide signal sequence from the LAMP protein which has been shown to 
channel proteins into the lysosomal processing pathway.  We compared short LAMP signal 
sequence fusions of CMV pp65 to fusions with the full-length LAMP protein and found full-length 
LAMP led to superior T cell expansion.  
 
In this trial, we will evaluate in a randomized, single-blinded phase 2 clinical trial whether the 
clinical efficacy of pp65 RNA-pulsed DCs is worthy of investigation in a definitive phase III 
clinical trial.  We will also examine whether immunologic responses to the full-length LAMP and 
short sequence LAMP fusion constructs differ significantly in vaccinated patients.  
  
 HYPOTHESIS AND OBJECTIVES 
 
5.1 Hypothesis  
▪ Our primary hypothesis is that: 
o pp65-LAMP mRNA DC vaccine plus GM-CSF and Td in adult patients 
with newly-diagnosed GBM receiving dose-intensified TMZ will be worthy 
of investigation in a large phase III study based on impact on overall 
survival. 
▪ Secondary hypotheses are that: 
o There is a relationship between CMV pp65-LAMP RNA-pulsed DC 
vaccines and progression-free survival in vaccinated patients. 
o Full-length LAMP vs short LAMP fusions with CMV pp65 will induce potent 
immune responses that mediate the safe eradication of invasive malignant 
disease in adult patients with newly diagnosed GBM.               
 
5.2 Objectives 
5.2.1 Primary Objective  
• To determine whether the addition of pp65-LAMP mRNA DC vaccine 
plus GM-CSF and Td to dose-intensified TMZ treatment is worthy of 
investigation in a large phase III study based on impact on overall 
survival. 
 


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5.2.2 Secondary Objectives  
• Evaluate the impact of CMV pp65-LAMP RNA-pulsed DC vaccines on 
progression-free survival in patients with newly-diagnosed GBM.  
• Determine the immunologic effects of vaccination with pp65 RNA fusion 
constructs incorporating full-length LAMP vs short LAMP sequences.  
5.2.3 Exploratory Objectives: 
• Evaluate immunologic responses within vaccine-site draining lymph 
nodes using magnetic resonance spectroscopy 
• Examine CCL3 and other chemokines/cytokines for utility as serum 
biomarkers 
for 
predicting 
DC 
migration 
and 
response 
to 
immunotherapy 
• Examine urinary biomarkers of immune response using nMR 
spectroscopy 
 
 BACKGROUND AND SIGNIFICANCE 
 
6.1 Disease and Current Therapy 
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(Kelly 1992), 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(Walker, Alexander et al. 1978; Walker, Green et al. 1980; Shapiro 
1986; Salford, Brun et al. 1988; Dinapoli, Brown et al. 1993; Stupp, Mason et al. 2005). 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(Pickard, Bailey et al. 1990; Ekman and 
Westphal 2005). Moreover, the non-specific nature of conventional therapy for brain tumors often 
results in incapacitating damage to surrounding normal brain and systemic tissues(Imperato, 
Paleologos et al. 1990; Hall and Fodstad 1992). 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. 
6.2 Dendritic Cell Immunotherapy 
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 (Banchereau and Steinman 
1998; Steinman 2001). 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(Hsu, Benike et al. 1996; Murphy, 
Tjoa et al. 1996; Nestle, Alijagic et al. 1998; Salgaller, Lodge et al. 1998; Salgaller, Tjoa et al. 


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1998; Tjoa, Simmons et al. 1998; Fujii, Shimizu et al. 1999; Holtl, Rieser et al. 1999; Lim and 
Bailey-Wood 1999; Morse, Deng et al. 1999; Thurner, Haendle et al. 1999; Lodge, Jones et al. 
2000; Murphy, Tjoa et al. 2000; Rieser, Ramoner et al. 2000; Schuler-Thurner, Dieckmann et al. 
2000; Heiser, Coleman et al. 2002; Su, Dannull et al. 2003). 
 
Numerous animal studies(Liau, Black et al. 1999; Yu, Wheeler et al. 2001), including many of our 
own(Ashley, Faiola et al. 1997; Heimberger, Crotty et al. 2000; Heimberger, Archer et al. 2002), 
and a few clinical studies(Yu, Wheeler et al. 2001; Liau, Prins et al. 2005) have demonstrated 
potent antitumor responses using DC-based immunotherapy against MGs.  
 
Adjuvants frequently used with vaccination include Freund’s incomplete adjuvant, bacilli 
Calmette-Guerin, QS-21, and diphtheria toxoid.  Supplemental cytokines have been used as well 
for the adjuvant immunological effects (Rosenberg, Yang et al. 1998). GM-CSF has been 
commonly used, as it is commercially available and well tolerated. GM-CSF is capable of 
stimulating macrophage function, inducing proliferation and maturation of dendritic cells, and is 
able to enhance T-lymphocyte stimulatory function.  Intradermal administration of GM-CSF 
enhances the immunization efficacy at the site of administration in a dose dependent fashion at an 
optimal dose of 125 g (Kremer, Stevens et al. 2000).  Significant anti-tumor immunity has been 
demonstrated in preclinical murine studies in which irradiated, stably transfected tumor cell lines 
secreting GM-CSF have protected against subsequent tumor challenge, especially against 
intracerebral tumors(Sampson, Ashley et al. 1997; Mach, Gillessen et al. 2000).  Furthermore, the 
potency of GM-CSF has been demonstrated in a Phase I clinical trial in melanoma patients 
vaccinated with irradiated autologous melanoma cells engineered to secrete GM-CSF(Soiffer, 
Lynch et al. 1998).  The immunization sites were intensely infiltrated with T lymphocytes, 
dendritic cells, macrophages, and eosinophils in 100% of evaluable patients.  Extensive tumor 
destruction was seen in 11 of 16 patients.  Both cytotoxic T cells and antibody responses were 
associated with this tumor destruction. Hence, GM-CSF has an extensive track record both as a 
growth factor and an adjuvant, is commercially available and has an acceptable toxicity profile. 
The experience on the ACTIVATE study supports the use of GM-CSF and sets the precedent for 
this phase I study. 
 
Td is a routinely used vaccine in the normal human population that we have shown in pilot studies 
may function as a potent adjuvant to enhance DC trafficking to VDLNs. Our previous studies have 
shown that successful DC migration to VDLNs may be a requisite for clinical activity of RNA-
pulsed DCs and administration of Td prior to vaccination may improve the effectiveness of DC 
vaccines in patients with GBM. 
 
6.2.1 Pre-Clinical Results against Intracerebral Tumors 
In our laboratories and those of others, systemic immunization using DCs co-cultured with 
uncharacterized tumor homogenate(Heimberger, Crotty et al. 2000), whole tumor RNA(Ashley, 
Faiola et al. 1997), unidentified peptides eluted from tumor cells by gentle acid washing(Liau, 
Black et al. 1999), or a distinct peptide encompassing the tumor-specific EGFRvIII 
mutation(Heimberger, Archer et al. 2002) have been shown to induce humoral and cell mediated 
systemic immune responses and to prolong the survival of rodents with brain tumors. 
 


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In prior preclinical studies (Heimberger, Crotty et al. 2000), 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 anti-tumor immune response had been 
established. These findings are especially significant in light of the fact that the astrocytoma cell 
line used is known to secrete the immunosuppressive agent transforming growth factor- (TGF-
) which is secreted by most human gliomas(Kuppner, Hamou et al. 1988; Wahl, Hunt et al. 1988; 
Zuber, Kuppner et al. 1988; Bodmer, Strommer et al. 1989; Maxwell, Galanopoulos et al. 1992). 
 
In another report (Ashley, Faiola et al. 1997), 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 major histocompatibility 
complex (MHC) background. In this experiment, mice immunized with autologous bone-marrow 
derived DCs co-cultured with tumor homogenate or whole tumor RNA increased median survival 
by >233% (P=0.0006) and 48% (P=0.0001), respectively, relative to mice immunized with DCs 
co-cultured with tumor homogenate or whole tumor RNA derived from an unrelated tumor with 
the same MHC background. In addition, 8/13 (61.5%) in the specific homogenate group and 4/10 
(40%) in the specific RNA group survived beyond the endpoint of the study without evidence of 
tumor. Immunization of mice with pre-existing tumors with specific tumor homogenate also 
demonstrated the potency of this immunization approach by increasing survival by 62.5% relative 
to controls. In these mice an inflammatory infiltrate composed of mononuclear cells and 
polymorphonuclear leukocytes was identified only in mice treated with DCs co-cultured with 
tumor homogenate that matched the intracerebral tumor challenge. 
 
In a report from (Liau, Black et al. 1999), the survival of tumor-bearing rats injected 
subcutaneously with autologous bone marrow-derive 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 


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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. 
 
6.2.2 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 immunosuppressed 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 previously(Romani, Reider et al. 1996), to 
generate human DCs by culturing peripheral blood mononuclear cells (PBMCs) in media 
supplemented with GM-CSF and interleukin-4 (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 in a few studies.  In the 
published study by Yu et al.(Yu, Wheeler et al. 2001), 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 cytotoxic T lymphocyte (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 Curran’s recursive partition analysis, which controls for known 
prognostic factors, 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) 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 3 weeks for a minimum of one and a maximum of 10 immunizations.  There 
were only two minor clinical responses seen.  Of 5 patients evaluated by enzyme-linked 
immunospot (ELISPOT) before and after vaccination T-cells reactive against tumor lysate-pulsed 
DCs were increased in two patients(Yamanaka R, Abe T et al., 2003). In a more recently published 
study, patients with GBM were treated with 1 x 106 to 1 x 107 DCs pulsed with acid eluted 


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autologous tumor peptides. There was no evidence of 
dose-limiting toxicity (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. 
 
Kikuchi et al.(Kikuchi, Akasaki et al. 2004) 
investigated the safety and clinical response to 
immunotherapy using fusions of DCs and glioma 
cells combined with recombinant human IL-12 for 
the treatment of MG. Fifteen patients with MG 
participated in this study. Cultured autologous glioma 
cells were established from surgical specimens in 
each case. Fusion cells were prepared from DCs and 
glioma cells using polyethylene glycol. All patients 
received fusion cells intradermally on day 1. IL-12 
was injected subcutaneously at the same site on days 
3 and 7. No serious adverse effects were observed. In 
four patients, magnetic resonance imaging showed a 
greater than 50% reduction in tumor size. One patient had a mixed response. 
 
In a Phase I/II clinical trial (BB IND 9944) at Duke University, patients with newly-diagnosed 
MGs were 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 (approximately 150 mcg/immunization).  EGFRvIII is a tumor specific 
antigen, which is expressed on approximately 47% of all MGs.  The vaccination protocol consist 
of 3 vaccines 2 weeks apart of PEPvIII-
KLH loaded, mature DCs beginning 2 
weeks following completion of post-
resection RT.  19 patients were enrolled 
with 16 completing vaccination with no 
adverse events.  No patient showed a 
positive 
delayed-type 
hypersensitivity 
reaction to KLH or PEPvIII before 
vaccination and of the evaluable patients 
after vaccination, 14/15 (93.3%) patients 
reacted to KLH and 11/15 (73.3%) reacted 
to PEPvIII.  In vitro proliferation in 
response to PEPvIII was seen in 11/12 
(92%) and to KLH in 9/12 (75%) of patients 
tested.  Two patients, one with anaplastic 
astrocytoma and one with GBM with 
residual 
radiographic 
disease 
after 
resection, and radiation, had a nearly 
complete radiographic response (Fig. 1).  
 
 
Fig. 1 
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
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
 
Fig. 2: 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(Westphal, Hilt et al. 2003) and with 
concurrent RT and temozolomide is 58.4 weeks(Stupp, Mason et 
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These patients have remained stable for 174.9 and 217.3 weeks.  Of the 14 patients without 
radiographically evident disease, 4/14 (28.6%) have not progressed at 102.7, 171.3, 180.7, 430.7 
weeks with a median overall time to progression of 10.4 months comparing favorably with a 
historical unvaccinated cohort (EGFRvIII positive and gross total resection) that had a median 
TTP of 7.1 months (n=39).  For patients with GBM, the median survival time was 20.0 months 
which compares favorably with recently published trials evaluating newly-diagnosed patients with 
GBM treated with GLIADEL® (13.9 months)(Westphal, Hilt et al. 2003); radiation and concurrent 
TMZ (14.6 months)(Stupp, Mason et al. 2005); or radiolabeled anti-tenascin monoclonal 
antibodies performed at Duke University (18.3 months)(Boskovitz, Wikstrand et al. 2004) (Fig. 
2). 
 
6.2.3 RNA Transfected DCs 
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(Boczkowski, Nair et al. 1996; 
Van Tendeloo, Ponsaerts et al. 2001; Ponsaerts, Van den Bosch et al. 2002; Ponsaerts, Van 
Tendeloo et al. 2002; Van Meirvenne, Straetman et al. 2002; Morse, Nair et al. 2003). In fact, there 
is accumulating evidence that RNA transfection represents a superior method for loading antigens 
onto DCs(Strobel, Berchtold et al. 2000; Van Tendeloo, Ponsaerts et al. 2001). This novel and 
innovative approach to DC antigen loading has multiple conceptual advantages over other forms 
of antigen delivery as well. RNA-based antigen loading does not require knowledge of major MHC 
restriction, and responses are not restricted to single MHC haplotypes or to a narrow B- or T-cell 
repertoire. This diversity increases the likelihood of inducing effective and sustained antitumor 
immune responses by simultaneous activation of both CTLs and helper T-cells(Sercarz, Lehmann 
et al. 1993; Kundig, Bachmann et al. 1996; Kim, Trivedi et al. 1998). Furthermore, in direct 
comparisons, RNA-loaded DCs have been found to be better stimulators of antigen-specific T-
cells than other approaches(Strobel, Berchtold et al. 2000). 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.(Kobayashi, Yamanaka et al. 2003) have demonstrated that tumor mRNA-loaded 
DCs can elicit a specific CD8+ CTL response against autologous tumor cells in patients with MG. 
6.3 Autoimmune Encephalomyelitis 
DCs, however, have been shown to be quite capable of initiating significant autoimmune responses 
in murine models, and there has been one incident of a spontaneous generalized vitiligo that 
occurred after a second intravenous infusion of DCs in a patient with melanoma. Although our 
group and others have demonstrated that DCs loaded with unselected tumor-derived antigens 
induce potent, specific, and clinically effective immune responses against brain tumors in rodent 
models without the induction of autoimmune reactivity(Ashley, Faiola et al. 1997; Liau, Black et 
al. 1999; Heimberger, Crotty et al. 2000; Heimberger, Archer et al. 2002), and although no 
autoimmune reactions have been identified in human DC trials in patients with MGs(Yu, Wheeler 
et al. 2001; Kikuchi, Akasaki et al. 2004; Liau, Prins et al. 2005), immunization in preclinical 
studies has only been effective when given before tumor challenge or in the context of very small 
established tumors. These data suggest that for DC-based immunotherapy to be effective in the 
context of large human tumors, a very strong and sustained antitumor immune response will be 
required(Ochsenbein, Klenerman et al. 1999). In animal models, when such responses have been 


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generated against tumor-associated antigens that are shared with host cells, severe and clinically 
significant autoimmune disease has occasionally resulted(Ludewig, Ochsenbein et al. 2000). 
 
Despite the fact, that no clinical or radiological evidence of experimental autoimmune 
encephalomyelitis (EAE) was induced by the DC-based immunization therapies in any of the 
preclinical experiments or the clinical trials outlined above, it must be acknowledged that one 
potential complication of DC-based immunotherapy, especially once optimized, is the induction 
of clinically significant autoimmunity. In fact, many published manuscripts have demonstrated the 
induction of clinically significant autoimmunity, including EAE, in preclinical studies employing 
DC-based or other immunization strategies(Borrow, Cornell et al. 1995; Dittel, Visintin et al. 1999; 
Bourquin, Iglesias et al. 2000; Ludewig, Ochsenbein et al. 2000). Although this may even be a 
desirable outcome for nonessential tissues, such as the prostate, when infiltrated by tumor, an 
autoimmune encephalomyelitis could be a lethal consequence of any non-specific active 
immunotherapy approach for primary intracerebral tumors where the target antigens are ill-defined 
and not tumor-specific. 
 
In light of the documented expression of normal and fetal brain antigens on human glioma cell 
lines(Wahlstrom, Linder et al. 1973), and brain tumor tissue(Siris 1936; Slagel, Wilson et al. 1969; 
Wickremesinghe and Yates 1971), active, specific immunization strategies, including DC-based 
approaches, with uncharacterized brain tumor antigens that are not tumor-specific possess the 
theoretical risk of inducing an uncontrolled autoimmune response against normal CNS antigens. 
Such a response would be similar to EAE, which is an acute or chronic, autoimmune, inflammatory 
demyelinating disease mediated primarily by antigen-specific CD4+ T-cells.  Myelin basic protein 
is the most common known antigenic trigger, but myelin proteolipid protein(Waksman, Porter et 
al. 1954; Wikstrand and Bigner 1979), myelin oligodendrocyte glycoprotein(Tuohy, Lu et al. 
1988), glial fibrillary acidic protein, and S-100(Linington, Berger et al. 1993), are also sufficient 
antigens for the induction of EAE. 
 
The susceptibility of humans to the induction of EAE was discovered accidentally when patients 
were immunized against rabies with spinal cords from rabbits that were infected with the rabies 
virus(Pasteur 1885; Remlinger 1904; Remlinger 1905; Stuart and Krikorian 1930; Wekerle, 
Kojima et al. 1994).  The toxic component found in these immunizations was subsequently proven 
to be related to encephalitogenic components contained within the spinal cord preparation and not 
the virus(Stuart and Krikorian 1928). EAE has also been induced in monkeys after repeated 
injections of homogenized CNS tissue(Rivers and Schwentker 1935).  It was not until the use of 
strong adjuvants such as complete Freund’s adjuvant (CFA), that EAE could be easily and 
reproducibly created in animals in this manner(Kabat, Wolf et al. 1947).  Since that time, it has 
been shown that EAE can be readily induced in the various species of rats, guinea pigs, mice, 
sheep, and monkeys after a single injection of CFA and either autologous or heterologous CNS 
tissue homogenate(Bigner, Pitts et al. 1981). 
 
Given the range of protocols that routinely use immunization with CNS tissue for the production 
of lethal EAE in non-human primates(Bigner, Pitts et al. 1981), and the documented susceptibility 
of humans to EAE, the induction of such autoimmune responses is a legitimate concern.  Some 
apprehension regarding EAE is also warranted on the basis of previous active, specific 
immunotherapy trials in humans with brain tumors.  Although no cases of EAE were reported in 
some human studies(Ommaya 1976; Albright, Seab et al. 1977; Mahaley, Bigner et al. 1983; 


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Bullard, Thomas et al. 1985), and protocols have been developed for safe active, specific 
immunotherapy with glioma-derived cells in primates(Wikstrand and Bigner 1981), careful review 
of several other studies(Bloom, Peckham et al. 1973) and(Trouillas and Lapras 1970; Trouillas 
1973) reveal one possible case of EAE within each study.  Thus, the risk of EAE, or other similar 
and potentially lethal autoimmune responses, may severely limit the efficacy of active, non-
specific immunotherapy for malignant brain tumors.  Thus targeting tumor-specific antigens, such 
as the more recently discovered viral antigens associated with CMV that are present within MGs, 
would enhance the safety of early trials in this field. 
 
6.4 Presence of CMV in Malignant Astrocytomas 
Human CMV is an endemic -Herpesvirus that does not usually cause significant clinical 
disease(Vancikova and Dvorak 2001).  During primary maternal infection, however, human CMV 
can cause severe encephalitis in fetuses and lead to congenital brain defects.  Human CMV disease 
is also a significant problem in immunocompromised adults such as organ transplant recipients or 
patients with acquired immune deficiency syndrome (AIDS)(Vancikova and Dvorak 2001).  
Herpesviruses have also been implicated in a number of human malignancies including lymphoma, 
nasopharyngeal cancer, cervical cancer, and Kaposi’s sarcoma(Rafferty 1973; Kadow, Regueiro-
Ren et al. 2002).  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(Cobbs, Harkins et al. 2002; Harkins, Volk et al. 2002; Samanta, Harkins 
et al. 2003). Universal detection of the human CMV immunodominant protein pp65, immediate 
early gene 1 protein (IE1), and several other early antigens was demonstrated using 
immunohistochemistry (IHC) in Grade II-IV astrocytomas(Cobbs, Harkins et al. 2002).  Presence 
of the virus in these samples was confirmed with in situ hybridization (ISH), polymerase chain 
reactions (PCR) for human CMV-specific glycoprotein B (UL55), electron microscopic detection 
of intact virions(Cobbs, Harkins et al. 2002).  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 reliable detection of CMV with MGs requires sensitive detection modalities, but these results 
have now been confirmed by nine independent laboratories(Cobbs, Harkins et al. 2002; Mitchell, 
Xie et al. 2008; Scheurer, Bondy et al. 2008; Soderberg-Naucler 2008; Dziurzynski, Wei et al. 
2011; Lucas, Bao et al. 2011; Sampson and Mitchell 2011; Soroceanu, Matlaf et al. 2011; 
Dziurzynski, Chang et al. 2012; Ranganathan, Clark et al. 2012). This finding offers an 
unparalleled opportunity to leverage immunotherapeutic approaches to target the highly 
immunogenic viral antigens expressed by CMV as tumor-specific targets.   
 
 
 
 
 
 
 
 
 
 


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A 
biologically plausible explanation for this unexpected specificity was also provided when it was 
subsequently shown that the EGFR is also the cellular receptor for CMV because the EGFR is 
often dramatically up-regulated in MGs, but is essentially not expressed in normal brain(Wang, 
Huong et al. 2003).  Interestingly, astrocytic cell lines are some of the few cell lines that support 
CMV propagation in vitro(Poland, Costello et al. 1990).  The presence of CMV antigens in 
malignant astrocytomas has been confirmed in independent tissue samples from our laboratory as 
well.  It is not known whether CMV plays a role in the pathogenesis of MG, or whether tumor 
growth simply provides an environment supportive of local reactivation of the virus.  Detection of 
CMV antigens in MG specimens has been independently confirmed in our laboratory by IHC, ISH, 
PCR, and reverse transcriptase polymerase chain reaction (RT-PCR).   
6.5  Immunotherapy of CMV 
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(Vancikova and Dvorak 
2001), and the presence of this virus within brain tumors may allow this experience to be leveraged 
toward the effective eradication of MG expressing human CMV antigens.  Adoptive T-cell therapy 
has been used to safely and successfully protect against CMV reactivation in myelodepleted bone 
marrow transplant (BMT) patients(Riddell and Greenberg 1995; Walter, Greenberg et al. 1995; 
Paar and Pollard 1996; Dazzi and Goldman 1998).  In addition, T-cell mediated immunotherapy 
has proven highly effective in the treatment of CMV-associated disease within the CNS(Bigger, 
Tanigawa et al. 1999) and in the treatment of acute CMV infections(Walter, Greenberg et al. 1995; 
Numazaki, Ikehata et al. 1997).  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(Emanuel 1991; 
Papadopoulos, Ladanyi et al. 1994; Emanuel, Lucas et al. 1997; Rooney, Smith et al. 1998; Lucas 
and Barrett 1999; Liu, Savoldo et al. 2002).  More recently, a vaccine directed against the potent 
viral antigens of human papilloma virus has also been shown to reduce the incidence of human 
 
 
Expression of CMV antigens in primary GBM tumors by Western blot analysis. Single cell 
digest were prepared from freshly resected GBM specimens and washed with 2X in PBS to remove 
red cells and debri. Tumor cells were harvested and lysates prepared in the presence of proteinase 
inhibitors and Western blot analysis performed using IE1-specific MAb (810, Chemicon) and pp65 
MAb (sc 71229, Santa Cruz). CMV- and VSV-infected fibroblast lysates were used as positive and 
negative control samples respectively. Due to abundance of CMV proteins in laboratory strain virus 
infected lysates, 1/40 the amount of lysate was loaded for positive control lanes. Detection of IE1 
has been observed in 14/22 (63.6%) analyzed GBM samples and pp65 detection was positive in 
19/22 (86.3%) tumors analyzed by WB.  gB envelope protein was detected in 16/16 (100%) tumors 
chosen based on positivity for pp65 (not shown).  Normal brain lysates derived from autopsy 
specimens were negative in 5/5 cases (median age 64) not shown (0%), p<0.0001. 
CMV
(CMV + 
Control)
VZV
(CMV –
Control)
pp65 65KD
IE1 68KD
GBM
S200
GBM
S346
GBM
S418
GBM
S922
GBM
S536
GBM
S609
GBM
S666
GBM
S865
GBM
S878
GBM
S968
Beta-Actin 43KD


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papilloma virus-related cervical intraepithelial neoplasia in a prospective, randomized, double-
blind trial(Koutsky, Ault et al. 2002). 
 
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(Kondo and 
Mocarski 1995; Prosch, Docke et al. 1999).  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(Sissons, Sinclair et al. 1991; Larsson, Soderberg-
Naucler et al. 1998; Reeves, MacAry et al. 2005).   
 
Vaccination specifically against CMV(Gonczol, Ianacone et al. 1989; Plotkin, Higgins et al. 1994; 
Britt, Fay et al. 1995; BenMohamed, Krishnan et al. 2002; Pepperl-Klindworth, Frankenberg et al. 
2002) has effectively reduced the risk of viral infection and transmission to fetuses in animal 
models(Minamishima 1977; Morello, Ye et al. 2002; Cicin-Sain, Brune et al. 2003) and in clinical 
trials(Sachs, Simmons et al. 1984; Gonczol, Ianacone et al. 1989; Plotkin, Higgins et al. 1994; 
Adler, Shaw et al. 1995; Adler, Hempfling et al. 1998; Drulak, Malinoski et al. 2000). Human 
clinical trials have also demonstrated some benefit of administering neutralizing antibodies in the 
treatment of human CMV infection(Ohizumi, Suzuki et al. 1994; Falagas, Snydman et al. 1997; 
Hammond 1999; Tzakis 2001), 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(Steinman 2001), and DCs pulsed in vitro with CMV antigens 
have been shown to be potent inducers of CMV-specific CTL responses in several studies(Cho, 
Han et al. 2001; Kleihauer, Grigoleit et al. 2001; Szmania, Galloway et al. 2001; Peggs, Verfuerth 
et al. 2002; Raftery, Schwab et al. 2002), in addition to our own work which is outlined below. 
 
6.6 Targeting CMV in Patients with Malignant Gliomas 
6.6.1 DCs Electroporated with pp65-LAMP mRNA 
In preparation for the clinical trial outlined in this protocol, we wanted to demonstrate that DCs 
generated from patients with GBM, when loaded with CMV pp65-LAMP mRNA, could be used 
to generate antigen-specific autologous T-cells.  These in vitro assays showed that after 15 days of 
co-culture with pp65 RNA-loaded DCs, the number of CD8+/pp65/A2 tetramer+ cells increased 
from <1% at baseline to 42.8% of all lymphocytes after 15 days while the absolute number of 
positive cells increased from 1.5x105 to 1.17x107 (78-fold expansion).  Further experiments 
demonstrated the ability to expand CD8+/HLA-A2 tetramer+ T cells from patients with GBM by 
2.51 + 0.83 logs (n=5, range 1.73-3.74) with a purity of 54.0 + 24.9% (n=5, range 35.9-88.4). 
 
To establish that T-cells stimulated with pp65-LAMP mRNA transfected mature DCs reacted in 
an antigen specific manner against CMV pp65, we assayed for specific IFN- release by ELISPOT.  
DCs that had been previously been transfected with pp65-LAMP mRNA, matured and frozen, 


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were thawed, washed, counted and used to stimulate 
autologous T-cells.  At the end of 14 days, the effector T-
cells were incubated with target DCs transfected with 
CMV pp65-LAMP mRNA, CEA LAMP mRNA, pp65 
peptide mix (138 peptides, 15 mers with 11 amino acid 
overlaps), HIV pol peptide mix, or untransfected DCs at 
different effector to target ratios in nitrocellulose wells 
coated with anti-IFN- MAb.  Effector T-cells generated 
by in vitro stimulation with CMV pp65-LAMP transfected 
DCs secreted IFN- only in response to pp65 transfected 
targets, (Fig. 4).  Cytokine flow cytometry (CFC) also 
demonstrated specific response to CMV pp65 peptide after 
a clinical scale expansion of cells from a patient with 
GBM.  Briefly, cells were incubated for 6 hours with 
mixed overlapping CMV pp65 peptides (138 peptides, 15 
mers with 11 amino acid overlaps) and brefeldin A 
(10ug/mL) before fixation and permeabilization.  Samples 
were labeled with CD4 (or CD8)-FITC, CD69-PE, CD3-
PE/Cy5.5, and IFN--APC (along with appropriate isotype controls).  Flow cytometry analysis of 
pre and post expansion indicated that after expansion CD8+/CD69+/CD3+/ IFN-+ increased from 
3.28% to 52.47% while CD4+/CD69+/CD3+/ IFN-+ increased from 0.43% to 78.08%.  It is also 
worth noting that in this particular patient sample there was greater percent of CD8 T-cells 
secreting IFN- (57.84%) than were CMV-pp65 tetramer positive (21.9%).  Finally, to verify that 
these effector cells were capable of killing malignant astrocytes as well, HLA-A-2+ U251MG cells 
were 51Cr-labelled and either pulsed with an HLA-A2 specific pp65 peptide (NLVPMVATV) 
(PEP-CMV) or the control HIV p1777-85 
peptide 
(SLYNTVATL) 
(PEP-HIV) 
(Anaspec), or infected with CMV.  As 
expected, CMV infection significantly 
reduced MHC class I expression on 
U251MG 
target 
cells 
with 
mean 
fluorescent intensity of HLA-A2 staining 
decreasing from 827.5 in uninfected cells 
to 593.9 10 days after infection.  Still 
effector cells were able to specifically kill 
the malignant astrocytes infected with 
CMV as well as peptide-pulsed MG targets 
(Fig. 5).  These responses were dependent 
on CD8+ T-cells and HLA-A2.  Another 
CLT assay demonstrated that T-cells 
stimulated by DCs transfected with CMV 
pp65-LAMP RNA and cultured for 14 days 
show high specific response against CMV 
pp65 peptide pulsed target cells.  Specific 
killing was demonstrated by utilizing the 
2:1
5:1
40:1 
0
100
200
300
400
500
600
pp65 LAMP Targets
CEA LAMP Targets
CMV Peptide Targets
HIV Peptide Targets
Naive Targets
Effector:Target Ratio
Spots per Well
Fig. 4: T-cells stimulated by CMV pp65-LAMP 
electroporated DCs secrete IFN-γ specifically.  DCs 
were mixed with T-cells in a 1:10 ratio and incubated in 
Aim V media supplemented with 2% HABS.  IL-2 
(10U/mL) was added on day 3 and the cells were grown 
for 14 days.  After an 18 hour incubation the wells were 
washed and the nitrocellulose was developed and the 
spots counted. 
 
Fig. 
5: 
T-cells 
stimulated 
by 
CMV 
pp65-LAMP 
electroporation generate specific CTL response against CMV-
pp65 pulsed target cells.  Briefly, target T2 cells are concurrently 
labeled with a combination of fluorescent membrane dye and 
either CMV-pp65 or HIV p17 peptides.  Labeled targets were then 
co-cultured for 1 hour with effector cells in media containing a 
fluorogenic caspase substrate.  In analysis, targets are 
differentiated by the fluorescent membrane dye while killing is 
defined by cells containing the fluorescently cleaved substrate.  
Figure above represents effectors IVS-7 and IVS-8 with CMV-
pp65 or HIV-p17 peptide pulsed T2 cells. 
CTL  Assay
0.0
20.0
40.0
60.0
80.0
100.0
0
5
10
15
20
25
30
Effector:Target
% Target cell Killing
IVS-7:T2p17
IVS-7:T2pp65
IVS-8:T2p17
IVS-8:T2pp65


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highly 
sensitive) 
CyToxiLux® 
Plus 
assay 
kit 
(OncoImmunin, Inc., Gaithersburg, MD)(Liu, Chahroudi 
et al. 2002)(not shown). 
 
6.6.2 Recognition of Human 
CMV antigens by TILs 
We have recently gathered evidence that recognition of 
human CMV antigens may occur at a significant level in 
patients with GBM even without vaccination.  PBLs and 
tumor-infiltrating lymphocytes (TILs) were isolated from 
patients and briefly expanded in a non-specific fashion in 
vitro with CD3/CD28 beads (Dynal) and IL-2. 
Tetramer analysis of an HLA-A.2+ TIL sample revealed a 
frequency of 10.26% pp65/A2 tetramer+ cells among the 
TILs isolated (Fig. 6).  PBLs from the same patient, 
cultured in an identical fashion, had a starting frequency of 
0.99% pp65/A2 tetramer+ cells, but this number instead 
actually declined during the same culture period to 0.46%.  In an ELISPOT assay, these same 
TILs, when exposed to autologous tumor, HLA-A2 matched irrelevant tumor, or T2 cells loaded 
with HLA-A2 restricted CMV peptide epitope, also demonstrated CMV-specific secretion of IFN-
 (Fig. 7).  Incidentally, this patient’s tumor stained very strongly for CMV antigens in our IHC 
assay. 
 
TIL recognition of pp65 in an HLA-A2-negative patient was also confirmed using cytokine 
florescent cytometry (CFC) after stimulation with CMV pp65 or HIV p17 peptide mix (BD) that 
stimulates both CD4+ and CD8+ T-cells.  This patient’s TILs consisted almost entirely of CD8+ 
T-cells and 41.12% of the T-cells became CD69+/IFN-+ after exposure to CMV pp65 peptide 
mix, indicating a strong reaction to CMV-specific 
peptides (Fig. 8). HIV peptide stimulation did not 
stimulate cytokine production above background 
(0.87%). 
0
50
100
150
200
250
Autologous
Tumor
A431
Tumor
T2
CMV pp65
T2
HIV
p17
Spots per Well
Fig. 8: ELISPOT showing that TILs from an HLA-
A2+ patient with GBM recognize CMV.  T-cells 
specifically secrete IFN- in response to autologous 
tumor and PEP-CMV-pulsed T2 cells, but not an 
irrelevant HLA-A2 tumor (A431) or PEP-HIV-pulsed 
T2 cells. (P=0.019, ANOVA) 
 
Fig. 6: T-cells stimulated by CMV pp65-
LAMP electroporated DCs lysis CMV-
specific targets CTL assay showing lysis of 
U251MG cells infected with CMV (open 
circles) or pulsed with HLA-A2 CMV pp65 
peptide (black diamonds). or HIV p17 peptide 
(grey squares).(P=0.002, t-test) 
0
10
20
30
40
50
60
70
0
5
10
Effector:Target
% Specific Lysis
 
 
Fig. 
7: 
Tetramer 
analysis 
showing 
that 
a 
disproportionate percentage of TILs from a HLA-A2+ 
patient with GBM are pp65-specific. HIV pol/A2 
tetramer was used as negative control. (P<0.0001, 
binomial proportions) 
0.46% 
PBLs 
10.26%
TILs 


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6.7 ATTAC and ATTAC-GM Clinical Trials 
A Phase I/II clinical trial of autologous pp65 RNA loaded DCs was initiated at our center (ATTAC 
Protocol- FDA-IND-BB-12839; Duke IRB Protocol 8108; PI: Duane A. Mitchell).  Twenty-four 
patients with newly-diagnosed GBM who underwent gross total resection (>95%) followed by 
standard external beam radiation (60Gy) and concurrent TMZ (75 mg/m2/day) for 6 weeks 
followed by adjuvant monthly TMZ were enrolled into two cohorts.  Cohort 1, referred to as the 
ATTAC trial (n=13 patients) received standard-dose TMZ (150-200 mg/m2 x 5 days per cycle) 
and were randomized to receive different inflammatory skin preparations (unpulsed mature DCs 
or Td booster) prior to intradermal vaccination with pp65 RNA-loaded DCs.  At the vaccine 4, 
patients received Indium-111 labeled DCs and migration to VDLNs was monitored by SPECT/CT 
imaging.  
Cohort 2 (n=11 patients), referred to the ATTAC-GM trial, received dose-intensified TMZ and 
pp65 RNA-pulsed DCs mixed with GM-CSF 
(150ug).  SPECT/CT imaging of DCs was also 
conducted at the fourth vaccine to evaluate the 
impact 
of 
GM-CSF 
on 
DC 
migration.  
Leukapheresis harvested post-surgical resection 
and prior to initiation of TMZ was used to generate 
DCs and pp65 RNA electroporated autologous DCs 
(2x107 DCs i.d.) were administered every two 
weeks for first three doses after first TMZ cycle and 
monthly thereafter on day 22-24 of each cycle.  
Patients in both trials were monitored by MRI 
(every two months) for tumor progression and 
blood 
collected 
monthly 
for 
immunologic 
monitoring.  Treatment was well tolerated with no 
vaccine related adverse events on cohort 1 
(ATTAC).  A single patient on cohort 2 (ATTAC 
GM) experienced a Grade 3 hypersensitivity 
reaction that was attributed to GM-CSF.  This 
patient received additional DC vaccines without the 
inclusion of GM-CSF without incident.  
Expansion of CMV-specific T cells was observed 
by tetramer responses in 4 of 13 patients on cohort 
1 after the third vaccine. Subsequent immune 
 
Fig. 9: CFC showing that TILs from an HLA-A2 - patient with GBM recognize CMV.  TILs were 
activated (CD69+) and produced IFN- in response to CMV peptide mix, but not in response to HIV 
peptide mix. (P<0.0001, binomial proportions) 
0.87% 
41.12% 
HIV peptide mix 
CMV peptide mix 
  
Fig. 10: Overall survival in patients with GBM 
receiving pp65 RNA-pulsed DC vaccines + GM-CSF 
during dose-intensifed TMZ. Dose-intensified TMZ 
has been shown to convey no treatment benefit in 
patients with GBM in recent phase III trial (RTOG 0525) 
compared to standard-dose TMZ.  Overall survival of 
patients (n=11) with newly-diagnosed GBM receiving 
pp65 RNA-pulsed DCs + GM-CSF (150ug) during dose-
intensified TMZ exceeds 40 months.   EORTC and 
RPA/Curran nomogram predicted survival curves for 
patients enrolled are depicted.  Patient survival observed 
significantly exceeds predicted survival and historical 
data for patients with GBM.  


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monitoring was conducted using ELISPOT analysis which detected expansion of CMV-specific T 
cells in 10 of 11 subjects enrolled on cohort 2.   
 
 
Median overall survival for all patients enrolled on cohort 1 is 20.6 months with patients 
randomized to Td skin prep exhibiting greater DC migration to VDLNs and superior clinical 
outcomes (P<0.01).  There was a strong correlation between successful migration of DCs to 
VDLNs (measured at vaccine 4) and clinical outcomes in cohort 1 and patients randomized to 
receive Td + DC vaccine had prolonged survival (>36.6 months) compared to patients randomized 
to undpulsed DC preparation (18.5 months).   Cohort 2 (ATTAC-GM) has a median PFS and OS 
that exceeds 40 months (Fig. 10).   
 
6.8 T Cell Homeostatic Proliferation 
Homeostatic proliferation is a mechanism by which the body maintains normal physiologic levels 
of circulating lymphocytes.  Within the normal host, homeostatic proliferation involves the 
relatively slow replication of naïve and memory T-cells along with the generation of new 
lymphocytes from the bone marrow to maintain normal numbers of T-cells (and other 
lymphocytes). 
 
Under conditions that induce profound lymphopenia, however, such as during non-myeloablative 
chemotherapy, naïve and memory T-cells enter a rapid proliferative state in order to replenish the 
drastically diminished lymphocyte numbers.  During physiologic hematopoietic recovery, new T-
cells must be regenerated from the bone marrow or by division of the few remaining T-cells in the 
peripheral lymphoid organs or circulation and is a prolonged process that may take weeks to 
months to recover normal T-cell counts.  Following a period of lymphopenia, there is a 
spontaneous repopulation of the host’s T-cells to re-establish the T-cell repertoire(Cho, Rao et al. 
2000). Tanchot et al.(Tanchot, Rosado et al. 1997) propose that T-cells remaining after 
lymphodepletion compete for a finite amount of cytokine and antigenic stimulation.  Adoptively 
transferred lymphocytes when transplanted into lymphopenic hosts, also undergo rapid 
proliferation and differentiation into effector and memory cells and can provide enhanced 
protection against tumor outgrowth in experimental animals(Wrzesinski and Restifo 2005).  
Furthermore, populations of tumor-specific lymphocytes when transferred into lymphopenic 
melanoma patients have been shown to be capable of eradicating disseminated malignant disease 
and maintaining high levels of tumor-specific memory T-cells in the circulation of treated 
patients(Dudley, Wunderlich et al. 2002; Dudley, Wunderlich et al. 2005; Hughes, Yu et al. 2005; 
Zhou, Dudley et al. 2005).  These studies indicate that homeostatic proliferation may be leveraged 
to augment anti-tumor immunity by using T-cells educated to be reactive against tumor either 
through ex vivo expansion or possibly DC vaccination in vivo. 
 
Recently it has also been demonstrated that depletion of the host’s native lymphocyte pool with 
subsequent vaccination during endogenous homeostatic T-cell reconstitution enhanced the 
protection of mice bearing established extracranial tumors(Asavaroengchai, Kotera et al. 2002).  
Similarly, adoptive reconstitution with tumor-reactive T-cells after intentional non-myeloablative 
lymphodepletion allowed for a marked preferential expansion and maintenance of tumor-specific 
transferred T-cells resulting in dramatic clinical responses, along with some autoimmune toxicity, 
in patients with advanced malignant melanoma(Dudley, Wunderlich et al. 2002).  Later studies 
have also indicated that ex vivo manipulation of the adoptively transferred cells, for example by 


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CONFIDENTIAL 
depletion of Tregs, further enhances the reconstitution of the host with tumor antigen-specific T-
cells(Antony, Piccirillo et al. 2005).  These approaches take advantage of still undefined 
homeostatic forces that provide an environment conducive to T-cell proliferation during recovery 
from lymphopenia.  We propose to use tetanus prior to DC vaccination in patients with treatment-
induced lymphopenia in order to determine whether this addition increases total uptake at 48 hours 
compared to those patients that received PBMCs with or without tetanus. 
 
Our previous studies have demonstrated the safety of DC vaccination targeting the EGFRvIII 
mutation in patients with MGs, and we expect the targeting of CMV antigens to be well tolerated.  
There is considerable clinical experience with T-cell based immunotherapy targeting CMV in bone 
marrow and organ transplant patients and these studies have demonstrated the safety and efficacy 
of immunotherapy against CMV, even in CNS-involved disease.  Lymphopenia will be induced in 
our patient population using standard doses of TMZ, an alkylating agent used routinely in the 
treatment of MGs at our institution and abroad as outlined below.  We, therefore, expect that DC 
+ GM-CSF pretreated with or without Td should be safe and highly efficacious in patients with 
MGs. 
 
Lymphodepletion may be advantageous in patients with GBMs for several reasons.  First, 
lymphodepletion will eliminate the defective T-cells that are well-described in patients with 
GBM(Brooks, Netsky et al. 1972).  Second, lymphodepletion will eliminate Tregs, an inhibitory 
subset shown by us to be significantly upregulated in patients with GBMs (Fig. 11) and, in fact 
TMZ may preferentially deplete these suppressive cells(Su, Sohn et al. 2004).  By transferring 
tumor-specific T-cells into such an environment, especially in the context of tumor-specific 
vaccination, the tumor-specific T-cells may have a selective advantage.  It has also been shown in 
lymphopenic hosts, that proliferating T-cells differentiate directly into memory T-cells capable of 
rapid and intense response to antigen re-
exposure(Tanchot, Rosado et al. 1997).  Grossman 
and Paul propose that T-cells respond to 
lymphopenia by tuning their activation threshold 
down to a level at which T-cells are driven to 
proliferate, even in the absence of foreign 
antigens(Grossman and Paul 2000).  Also in line 
with our thinking, others(Dudley, Wunderlich et 
al. 2002; Hu, Poehlein et al. 2002) propose that, if 
tumor 
antigens 
are 
present 
during 
this 
repopulation, the population of T-cell could be 
skewed to create a tumor antigen-targeted 
response.  Homeostatic proliferation can then be 
augmented with autologous transfer of antigen-
specific activated T-cells to skew the reconstituted 
pool resulting in an even more homogeneous T-
cell repertoire that preferentially targets tumor 
antigen. In fact, in other studies, it has been shown 
that transferred T-cells can expand dramatically in 
the host to the point where they constitute up to 
 
Fig. 11: Comparison of Treg fraction in peripheral 
blood from GBM patients (n=20) with fraction in 
healthy volunteers (n=10) and patients with benign 
intracranial tumors (meningioma n=5; acoustic 
neuroma n=1). Data are expressed as the % of CD4+ 
cells that are CD25+CD45RO+. Horizontal hash bars 
indicate mean levels for each group (volunteers = 6.07% 
± 0.39%, MG=15.94% ± 2.20%, benign=5.94% ± 
0.89%).  Mean fraction in patients with GBM is 
significantly elevated over that in both volunteers 
(P=0.004) and patients with benign intracranial tumors 
treated with equivalent doses of peri-operative steroids 
(P=0.023). 
GBM
Volunteers
Benign Tumors
0
10
20
30
40
CD4s: %CD25+CD45RO+


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CONFIDENTIAL 
90% of the host’s T-cell repertoire and can be maintained for months following transfer(Dudley, 
Wunderlich et al. 2002). 
 
6.9 Treatment-Induced Lymphopenia from TMZ 
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(Stupp, Dietrich et al. 2002; Hegi, Diserens et al. 2005; Stupp, Mason et al. 2005).  
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 or adoptive reconstitution with CMV-specific T-cells, 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(Su, Sohn et al. 2004).  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 whole body irradiation (WBI), as a positive control, as methods for 
induction of treatment-induced lymphopenia in order to determine the ability of TMZ-induced 
lymphodepletion to enhance active 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.  In this model system, we found that ALT 
coupled with DC vaccination (DC + ALT) is a potent mechanism for inducing antigen-specific T-
cell expansion after TMZ treatment.  TMZ was found to be an effective agent for inducing 
homeostatic proliferation of transferred CD4+ and CD8+ T-cells and for enhancing DC or peptide 
vaccinations with or without ALT (Fig. 12). 


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 STUDY RATIONALE 
 
We have demonstrated in murine models that DCs loaded with tumor-specific antigens in the form 
of peptides or RNA can induce potent and specific humoral and cell-mediated immune responses 
that are effective against murine intracerebral (i.c.) tumors, including a syngeneic murine 
astrocytoma, without inducing autoimmunity(Heimberger, Crotty et al. 2000; Heimberger, Archer 
et al. 2002; Heimberger 2003; Mitchell, Cui et al. 2011).  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 
 
 
Fig. 12: 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. 
No Vaccine
100
101
102
103
104
CD4 APC
R4
0.74%
CD8 APC
100
101
102
103
104
CD4 APC
R4
2.11%
DC OVA RNA
100
101
102
103
104
CD8 APC
R4
13.92%
OVA peptide/CFA
100
101
102
103
104
CD8 APC
R4
79.86%
OVA peptide/CFA
100
101
102
103
104
CD8 APC
R4
0.79%
No Vaccine
100
101
102
103
104
CD8 APC
R4
29.80%
DC OVA RNA
CD8 APC
Untreated
Temozolomide


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response may be important in the biology of these tumors. We have recently demonstrated that 
fusion of the immunodominant antigen CMVpp65 to full-length LAMP targeting protein enhances 
activation and expansion of CMV-specific T cells in vitro.  TMZ has recently shown efficacy in 
some patients with newly-diagnosed GBM and is now frequently given to these patients during 
initial RT.  Therapeutic TMZ induces a profound lymphopenia, however, that may inhibit anti-
tumor vaccination.  With these observations in mind, we are now proposing to determine the 
immunologic and clinical effects of CMV-specific vaccination after therapeutic TMZ-induced 
lymphodepletion with GM-CSF and with Td skin preparations in patients with newly-diagnosed 
GBM. 
 
Td is a routinely used vaccine in the normal human population that we have shown in pilot studies 
may function as a potent adjuvant to enhance DC trafficking to VDLNs. Our previous studies have 
shown that successful DC migration to VDLNs may be a requisite for clinical activity of RNA-
pulsed DCs and administration of Td prior to vaccination may improve the effectiveness of DC 
vaccines in patients with GBM.  
 
Adjuvants frequently used with vaccination include Freund’s incomplete adjuvant, bacilli 
Calmette-Guerin, QS-21, and diphtheria toxoid.  Supplemental cytokines have been used as well 
for the adjuvant immunological effects(Rosenberg, Yang et al. 1998). GM-CSF has been 
commonly used, as it is commercially available and well tolerated.  GM-CSF is capable of 
stimulating macrophage function, inducing proliferation and maturation of dendritic cells, and is 
able to enhance T-lymphocyte stimulatory function.  Intradermal administration of GM-CSF 
enhances the immunization efficacy at the site of administration in a dose dependent fashion at an 
optimal dose of 125 g(Kremer, Stevens et al. 2000).  Significant anti-tumor immunity has been 
demonstrated in preclinical murine studies in which irradiated, stably transfected tumor cell lines 
secreting GM-CSF have protected against subsequent tumor challenge, especially against 
intracerebral tumors(Sampson, Ashley et al. 1997; Mach, Gillessen et al. 2000).  Furthermore, the 
potency of GM-CSF has been demonstrated in a Phase I clinical trial in melanoma patients 
vaccinated with irradiated autologous melanoma cells engineered to secrete GM-CSF(Soiffer, 
Lynch et al. 1998).  The immunization sites were intensely infiltrated with T lymphocytes, 
dendritic cells, macrophages, and eosinophils in 100% of evaluable patients.  Extensive tumor 
destruction was seen in 11 of 16 patients.  Both cytotoxic T cells and antibody responses were 
associated with this tumor destruction. Hence, GM-CSF has an extensive track record both as a 
growth factor and an adjuvant, is commercially available and has an acceptable toxicity profile. 
The experience on the ACTIVATE study supports the use of GM-CSF and sets the precedent for 
this study. 
 
 SUBJECT ELIGIBILITY 
 
8.1 Inclusion Criteria 
Enrollment and randomization must occur prior to subjects undergoing standard of care chemo-
radiation.  
 
All the following inclusion criteria need to be met in order to randomize the subject: 
1. Age ≥ 18 years. 


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2. Confirmed diagnosis of de novo Glioblastoma (WHO Grade IV glioma) by histopathology 
or molecular studies. 
(Secondary GBM not eligible). 
3. The tumor must have a supratentorial component. 
4. Patient must have undergone surgical resection of tumor with less than 3cm x 3cm (9cm2) 
residual enhancing tumor as product of longest perpendicular planes by MRI. 
5. Patients must have recovered from the effects of surgery, postoperative infection, and other 
complications. 
6. A diagnostic contrast-enhanced MRI or CT scan (if MRI is not available) of the brain must 
be performed preoperatively and postoperatively. Post-op MRI must be performed  within 
28 days prior to study enrollment. If no post op MRI/CT within 28 days of enrollment is 
available, a new brain MRI (or CT) must be obtained in order to determine residual burden 
and eligibility prior to randomization.  
Patients unable to undergo MR imaging because of non-compatible devices can be 
enrolled, provided pre- and post-operative contrast-enhanced CT scans are obtained and 
are of sufficient quality. 
7. Karnofsky Performance Status (KPS) ≥ 70.  
8. Signed informed consent.  If the patient’s mental status precludes his/her giving informed 
consent, written informed consent may be given by the legally authorized representative. 
9. For females of childbearing potential, negative serum pregnancy test at enrollment (test 
will be repeated within 72 hours prior to starting TMZ). 
10. Women of childbearing potential (WOCBP) must be willing to use acceptable 
contraceptive method to avoid pregnancy throughout the study and for at least 24 weeks 
after the last dose of study drug.    
Refer to Appendix G for definition of WOCBP and guidance on acceptable contraceptive 
methods. 
11. Males with female partners of childbearing potential must agree to practice adequate 
contraceptive methods throughout the study and should avoid conceiving children for 24 
weeks following the last dose of study drug. 
Refer to Appendix G for guidance on acceptable contraceptive methods. 
 
To be assessed prior to initiation of adjuvant TMZ: 
1. Patients must have completed standard RT at the discretion of the treating Radiation 
Oncologist and concomitant TMZ therapy at a targeted dose of 75mg/m2/d for < 49 days 
without significant toxicity that persisted over 4 weeks.  Significant toxicity is defined as 
one or more of the following: 
a. ANC < 0.5 x 109/L (Grade 4)  
b. Platelet count < 10 x 109/L (Grade 4)  
c. Grade 3 or 4 non-hematologic toxicity (except alopecia, nausea and vomiting unless 
the patient has failed maximal antiemetic therapy, and fatigue). 
2. History & physical with neurologic examination within 14 days prior to initiation of 
adjuvant TMZ. 
3. For patients receiving steroids, daily dose must be < 4 mg.   
4. CBC with differential obtained within 14 days prior to initiation of TMZ with adequate 
bone marrow function as defined below: 
a. Absolute neutrophil count (ANC) ≥ 1500 cells/mm3. 


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CONFIDENTIAL 
b. Platelet count ≥ 100,000 cells/mm3. 
c. Hemoglobin ≥ 10 g/dl. (The use of transfusion or other intervention to achieve Hgb 
≥ 10 g/dl is acceptable.) 
5. Adequate renal function within 14 days prior to initiation of adjuvant TMZ as defined 
below: 
a. BUN ≤ 25 mg/dl 
b. Creatinine ≤ 1.7 mg/dl 
6. Adequate hepatic function within 14 days prior to initiation of adjuvant TMZ as defined 
below: 
a. Bilirubin ≤ 2.0 mg/dl 
b. ALT ≤ 5 times institutional upper limits of normal for age 
c. AST ≤ 5 times institutional upper limits of normal for age 
 
8.2 Exclusion Criteria 
All the following exclusion criteria need to be verified in order to randomize the subject:   
 
1. Prior invasive malignancy (except for non-melanomatous skin cancer) unless disease free 
for ≥ 3 years. (For example, carcinoma in situ of the breast, oral cavity, and cervix are all 
permissible.)  
2. Metastases detected below the tentorium or beyond the cranial vault and leptomeningeal 
involvement.  
3. Recurrent or multifocal malignant gliomas. 
4. HIV, Hepatitis B, or Hepatitis C seropositive. 
5. Known active infection or immunosuppressive disease. 
6. Prior chemotherapy or radiosensitizers (including Gliadel wafers) for cancers of the head 
and neck region, other than TMZ prescribed during radiation for GBM (Prior 
chemotherapy for a different cancer is allowable). 
7. Prior radiotherapy to the head or neck (except for T1 glottic cancer and that prescribed for 
GBM ≤60Gy), resulting in overlap of radiation fields.  Radiosurgery is not permitted. 
8. Severe, active co-morbidity, defined as follows: 
a. Unstable angina and/or congestive heart failure requiring hospitalization. 
b. Transmural myocardial infarction within the last 6 months. 
c. Acute bacterial or fungal infection requiring intravenous antibiotics at initiation of 
XRT/TMZ. 
d. Chronic Obstructive Pulmonary Disease exacerbation or other respiratory illness 
requiring hospitalization or precluding study therapy at initiation of XRT/TMZ. 
e. Hepatic insufficiency resulting in clinical jaundice and/or coagulation defects. 
f. Acquired Immune Deficiency Syndrome (AIDS) based upon current CDC 
definition. The need to exclude patients with AIDS from this protocol is necessary 
because the treatments involved in this protocol may be significantly 
immunosuppressive. 
g. Patients with autoimmune disease requiring medical management with 
immunosuppressants. 
h. Major medical illnesses or psychiatric impairments that, in the investigator's 
opinion, will prevent administration or completion of protocol therapy. 


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i. Active connective tissue disorders such as lupus or scleroderma that, in the 
investigator’s opinion, place the patient at high risk for radiation toxicity. 
9. Pregnancy or women of childbearing potential and men who are sexually active and who 
are unwilling or unable to use an acceptable method of contraception for the entire study; 
this exclusion is necessary because the treatment involved in this study may be significantly 
teratogenic.  
10. Pregnant or lactating women, due to possible adverse effects on the developing fetus or 
infant. 
11. Prior allergic reaction to TMZ, GM-CSF, or Td. 
12. Prior history of brachial neuritis or Guillain-Barré syndrome. 
13. Patients treated on any other therapeutic clinical protocols within 30 days prior to study 
entry. 
 
To be assessed prior to initiation of TMZ: 
1. Patients did not start RT and TMZ within 7 weeks of surgery. 
2. Progression of disease as defined by modified RANO criteria. 
3. More than 45 days after completion of RT and TMZ. 
 
 INVESTIGATIONAL PLAN 
 
9.1 Overview 
The eligibility criteria for this trial will be similar to those used in the RTOG 0525 study (Aldape 
2009).  Prior to the initiation of TMZ and external beam RT, patients will sign consent and undergo 
leukapheresis for PBMCs in accordance with SOP-UFBTIP-131.  
Up to 200 newly diagnosed WHO Grade IV glioma patients will be enrolled in the study. Eligible 
patients will be randomized, undergo leukapheresis and receive “standard-of-care” radiotherapy 
concurrent with TMZ under the assumption that approximately 120 patients will be vaccinated 
according to treatment arm in this placebo controlled, single blinded, randomized Phase II study.  
 
Prior to leukapheresis, patients will be randomized to receive one of three treatment regimens with 
dose-intensified TMZ treatment at 100 mg/m2/day for 21 days with an allocation ratio of 1:1:1 into 
one of three arms to receive: 
 
1)  pp65-shLAMP mRNA DCs with GM-CSF 150 µg (Td skin prep) 
2)  pp65-flLAMP mRNA DCs with GM-CSF 150 µg (Td skin prep) 
3)  Unpulsed PBMCs (saline skin prep) 
 
Randomization will be stratified by; 
- 
Age 69 years old and younger, who will receive dose-intensified adjuvant temozolomide; 
or 70 years old and older who will receive standard dose adjuvant temozolomide.  
- 
Recursive Partitioning Analysis (RPA) class (III, IV, or V). 
To determine RPA class, Performance Status (PS) will be determined from KPS conversion 
using the ECOG comparison scale for patients ≤ 50 years; a Mini-Mental State 
Examination will be performed at enrollment for patients ≥ 50 years.  
- 
CMV seronegative or seropositive 
 


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Within 7 weeks of surgery, patients will undergo standard RT with concurrent TMZ at a target 
dose of 75 mg/m2/day.   
 
Patients will receive the initial cycle of TMZ 4 (+/- 1) weeks after completing RT. Study drugs 
will be given intradermally at day 22-24 after the first TMZ cycle and divided equally between 
both inguinal regions in accordance with SOP-UFBTIP-128. All patients will receive Td booster 
(5 Lf) with vaccine #1 regardless of booster history.  Vaccine #2 and #3 will occur at 2 week 
intervals.  
 
All patients will undergo repeat leukapheresis 2 to 4 weeks after vaccine #3 for PBMCs to generate 
additional DC vaccines and for immunologic monitoring with specific assessment of baseline 
antigen-specific cellular and humoral immune responses.  TMZ cycle 2 will occur 24 hours to 1 
week after repeat leukapheresis. Patients may undergo additional leukapheresis, if needed, to 
generate up to 10 DC vaccines as outlined in section 9.6., Removal of PBMCs by Leukapheresis. 
 
Patients will then be vaccinated monthly in conjunction with subsequent TMZ cycles every 5(+/- 
1) weeks for a total of 6 to 12 cycles after RT as per guidelines defined in recent phase III clinical 
trial evaluating TMZ regimens in patients with GBM (RTOG 0525).  Vaccines will be given on 
day 22-24 of each TMZ cycle.  Patients will continue with monthly vaccinations until a total of 10 
vaccines have been administered or until tumor progression (whichever comes first).   
 
All patients will undergo vaccine site pretreatment with Td (1 Lf) or saline (depending on 
randomization arm) 6-24 hours prior to Vaccine #3, #6, and #9.  At vaccine #3, patients at the UF 
site will undergo MRI/MRS imaging of inguinal vaccine-site draining lymph nodes before vaccine 
site pretreatment, 48 hrs post vaccination, and 5 to 7 days post vaccination (optional). Peripheral 
blood and urine samples will be collected at these time points for soluble biomarker analysis. 
 
Patients who have been enrolled in the study and become ineligible to receive study treatment for 
reasons other than progression may be allowed to receive vaccines that have been generated for 
them at the discretion of the PI as outlined in section 9.8 DC or PBMC Vaccination.   
 
Blood for immune monitoring will be collected during initial leukapheresis, prior to vaccines 1-3, 
during MRI/MRS imaging time points surrounding vaccine #3, during the second leukapheresis, 
prior to vaccine 4, 7, 10, and at progression if feasible.  Peripheral blood will be processed in 
accordance with SOP-UFBTIP-126.   Urine samples will be collected at MRI/MRS imaging time 
points surrounding vaccine #3 and transferred to the UFBTIP laboratory for nMR analysis.  
 
Up to twelve cycles of TMZ may be given if the patient demonstrates continued improvement or 
stability on radiologic scan, decreased corticosteroid requirement, improvement in performance 
status, or improvement in neurologic function. 
 
Patients will be imaged bimonthly during TMZ cycles and every 3 months after the 12th TMZ 
cycle (or 1 year post surgery if TMZ discontinued prior to 12 cycles) without receiving any other 
prescribed antitumor therapy unless progression occurs. 
 


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The primary measure of response will be by serial measures of the product of the two largest 
cross-sectional diameters.  The modified RANO criteria (Ellingson, Wen, Cloughesy 2017) will 
be used for assessment of pseudoprogression, progression or response.  
 
Tumor progression should 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. As this is not a research procedure, consent for biopsy will be obtained separately. If 
tissue is obtained, it will be used to confirm tumor progression histologically and evaluate cellular 
infiltration and CMV antigen expression at the tumor site.  
If histopathology confirms treatment effect vs tumor progression, the patient may resume study 
treatment at the discretion of the PI in consultation with the treating physician, if the PI believes 
that it is safe to continue therapy and that the patient may benefit from the ongoing treatment 
regimen as described in section 11.11 Confirmation of Tumor Progression by Histopathology. 
 
Patients will be followed until death due to any cause. MRI and clinical evaluation for assessment 
of disease progression will be conducted bi-monthly for the first 12 months and then every 3 
months thereafter. 
 
9.2 Registration Procedures 
To register a patient to this study, patient demographic data will be entered into the UFHCC 
OnCore database by the participating site coordinator after subject consent has been obtained and 
documented.  The Oncore database will assign a study ID to the patient after eligibility is 
confirmed and an “On Study” Date is entered in Oncore. Patients who signed consent but are 
deemed not eligible to participate, will be assigned a failed-screening ID by the Coordinating 
Center Project Coordinator.   
 
For patients enrolled at an outside participating site who will receive study treatment at the 
University of Florida, the ATTAC Registration Form will be submitted to the Protocol 
Coordinating Center along with applicable supporting documentation.  Once the patient has been 
entered in the UFHCC database and the registration form has been received, the Protocol 
Coordinating Center will confirm eligibility and randomize eligible patients. The patient’s 
assigned study ID will be forwarded to the participating site coordinator by email. 
 
9.3   Randomization Procedures 
Patients will be randomized by the designated site personnel where study treatment will be 
administered in accordance with SOP-UFBTIP-140.01. 
 
9.4 Radiation Therapy 
Standard external beam RT will be administered concomitantly with TMZ (Appendix A) 
according to standard guidelines which were adopted from the RTOG 0525 protocol and per the 
recommendation of the treating Radiation Oncologist.  Institutional practices for administration of 
external beam RT for patients with GBM may be followed. 
 
Patients enrolled in the study will be encouraged to receive Radiation Therapy (and concomitant 
chemotherapy) at their participating study site. However, to limit travel and finance-related burden 


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associated with daily travel to participating study site, patients should be allowed to receive 
standard chemo-radiation with their local treating oncologist.  
 
9.5 Temozolomide Therapy 
TMZ will be administered concomitantly with standard external beam RT.  
 
Management of chemotherapy will be done by the patient’s treating neuro-oncologist in 
accordance with institutional practices. 
 
Post RT, patients will receive Adjuvant cycles of doses-intensified TMZ. Dose intensified TMZ 
will be administered orally once per day for 21 consecutive days (days 1-21) of a 35 (+/- 7) days 
cycle at 100 mg/m2/day.  Cycle #1 will last up to 12 weeks to allow time to administer 3 bi-weekly 
vaccines and repeat leukapheresis.  Based on TMZ-associated adverse events during the previous 
cycles, dose reduction of dose-intense TMZ may be done at the discretion of the site treating neuro-
oncologist.  Patients who experienced significant hematologic toxicity will not receive dose-
intense temozolomide. Patients who experienced hematologic toxicity grade ≥ 3 due to 
Temozolomide during their chemo-radiation treatment will receive standard dose Temozolomide 
at 150-200 mg/m2/day for 5 consecutive days of a 35 (+/7) days cycle. For these patients, one dose 
reduction to 100 mg/m2/day for 5 days will be allowed based on AE on previous cycle. 
 
Appendix B, C and D are provided as guidelines for dose modifications and chemo management.  
 
Patients age 70 and above, will receive standard dose Temozolomide at 150-200 mg/m2/day for 5 
consecutive days of a 35 (+/7) days cycle. For these subjects, one dose reduction to 100 mg/m2/day 
for 5 days will be allowed based on AE on previous cycle. 
 
In the event that chemo adjuvant cycle is delayed past the timeframe provided in Appendix C and 
D, the treatment with Temozolomide may resume when toxicity has resolved (or returned within 
treatment limits) if patient has not experienced tumor progression at the discretion of the PI in 
consultation with the treating physician, if the PI believes that it is safe to continue therapy and 
that the patient may benefit from additional chemo cycles. Study vaccines will be given at day 22-
24 of each cycle when chemo is restarted. 
 
9.6 Removal of PBMCs by Leukapheresis 
Prior to the initiation of TMZ and external beam RT and again 2 to 4 weeks after vaccine #3, 
subjects will undergo leukapheresis in accordance with SOP-UFBTIP-131. Intravenous access 
will be obtained by placing two large gauge catheters into the antecubital veins bilaterally or by 
using a dialysis-type central venous catheter that will allow for large volume apheresis. In addition 
to research protocol consent, additional surgical/procedural consent will be obtained for placement 
of the central venous catheter by the surgeon/proceduralist. Placement of this special catheter may 
require use of fluoroscopy. The procedure will be conducted using the COBE®Spectra™ Apheresis 
System (or equivalent).  A sample of each leukapheresis product will be used for immunologic 
monitoring. 
 
Subjects may undergo additional leukapheresis, if needed, to generate up to 10 DC vaccines. 
Additional leukapheresis procedures may be performed at anytime at the discretion of the PI to 


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limit treatment interruption/delay as much as possible. When feasible, additional leukapheresis 
should be scheduled no earlier than 1 week following DC vaccination and prior to the following 
TMZ cycle. After the leukapheresis, TMZ cycle may occur as scheduled.  
 
9.6.1 PBMC Generation, Storage, and Testing 
PBMCs will be extracted from leukapheresis by density centrifugation in the UF cGMP facility.  
The cells are washed and processed according to BR-UFBTIP-1001 and BR-UFBTIP-1001-C 
for PBMC to be used as a clinical product. The PBMCs are then stored until needed in liquid 
nitrogen. After freezing and prior to release of PBMC as a clinical product, an aliquot of 
cryopreserved cells will be thawed and sent for QA/QC release testing according to SOP-
UFBTIP-122. Testing will include sterility, aerobic and anaerobic bacterial cultures, fungal 
cultures, mycoplasma and endotoxin testing.  
 
9.6.2 DC Generation, Storage, and Testing 
Dendritic Cells will be manufactured according to BR-UFBTIP-1002 or BR-UFBTIP-1002-C 
from either fresh or frozen PBMC. Immature DCs will be generated from adherent cells of the 
PBMCs under the culture condition that contains GM-CSF and IL-4 for 7 days.  At the end of the 
7 day incubation, the cells are then harvested and electroporated with pp65-shLAMP mRNA or 
pp65-flLAMP mRNA per randomization assignment. The DCs are placed in a flask with AIM V 
media containing HABS + GM-CSF + IL-4+ TNF-α + IL-6 + IL-1β and incubated at 37OC in 5% 
CO2 for 18-24 hours for maturation.  The antigen loaded mature DCs are harvested, washed, and 
then stored until needed in a liquid nitrogen freezer.  After freezing and prior to release, an aliquot 
of cells will be thawed and sent for QA/QC release testing according to SOP-UFBTIP-
122. Testing will include sterility, aerobic and anaerobic bacterial cultures, fungal cultures, and 
endotoxin testing.   Production of PBMC product will be in accordance with BR-UFBTIP-1001-
C. 
 
9.6.3 Preparation of DCs or PBMCs for Vaccination 
For each vaccination, DCs or PBMCs will be rapidly thawed at 37oC, washed with PBS and 
counted. The cell concentration will be adjusted to 2x107 cells per 400µL of preservative free 
saline containing 150 µg of GM-CSF for DC vaccines and saline only for PBMC vaccines and 
loaded into a 1 mL tuberculin syringe with an appropriately sized needle in accordance with SOP-
UFBTIP-1124. 
 
9.7 Tetanus and Diphtheria Toxoid Booster and site pre-
treatment 
At time of first vaccination, all patients, regardless of tetanus vaccination history, will receive a 
Td booster (5 Lf) intramuscularly into the deltoid muscle.   
 
Subjects will undergo vaccine site pretreatment with a one-fifth dose of Td (1 Lf) (treatment arms 
1 and 2) or saline (arm 3) intradermally 6-24 hours prior to Vaccine #3, #6, and #9, alternating site 
for each pretreatment 
 


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9.8 DC or PBMC Vaccination 
On day 22-24 of the first TMZ cycle, all patient groups will receive their first intradermal 
immunization in accordance with SOP-UFBTIP-128 (per randomization assignment).  The 2 
subsequent immunizations will be given every 2 (+/- 1) weeks for a total of three doses.  Each 
immunization will be divided equally between both inguinal regions with a total volume of 200 
µL per side.  Vaccines target dose is 2 x 10 ^7 cells. If less cells (DCs or PBMCs) are available at 
time of vaccination, injection may proceed with available cells and patients will be informed of 
the lower dose.  Patients will be monitored for thirty minutes to one hour post-immunization for 
the development of any adverse effects.  The immunization procedures will be supervised by a 
nurse or physician that has completed an Advanced Cardiac Life Support (ACLS) course.  A 
cardiac resuscitation cart will be available in the immediate vicinity when performing these 
immunizations in case of severe allergic reactions.   
 
After vaccine #3, patients will undergo a repeat leukapheresis for PBMCs and immunologic 
monitoring.  
 
Subsequently, patients will be immunized on day 22-24 of every cycle of TMZ. 
 
Monthly vaccinations will continue until a total of 10 vaccines have been administered or until 
tumor progression (whichever comes first).  Missed vaccines during cycles 1-8 may be given with 
subsequent cycles of Temozolomide (or monthly for patients off Temozolomide) if there is no 
tumor progression until patients receive a total of 10 vaccines.   
 
Patients who experienced toxicity during temozolomide adjuvant cycles for whom it is deemed 
not safe to resume chemo cycles may continue to receive monthly study vaccines at the discretion 
of the PI in consultation with the treating physician, if the PI believes that it is safe to continue 
therapy and that patients may benefit from the study treatment. Vaccination with investigational 
product may be given until the patient receives a total of 10 vaccines or until progression, 
whichever comes first.   
 
Patients who can no longer receive Temozolomide and start other anti -cancer therapies before 
experiencing disease progression will not receive additional study vaccines. 
 
Patients who have been enrolled in the study but are no longer eligible to receive study treatment 
for reasons other than progression are allowed to receive vaccines that have been generated for 
them at the discretion of the PI. They will be replaced for purposes of study endpoint if they do 
not meet criteria of an evaluable subject (received standard XRT/TMZ and received at least one 
DC vaccine).  Such subjects would include: subjects who do not complete standard radiation 
treatment with concomitant TMZ, have prolonged interruption of treatment beyond allowable 
study guidelines. These patients will be included in the intent to treat analysis but excluded from 
the evaluable subjects analysis if disqualified prior to receiving first vaccine. Such subjects may 
receive QA/QC qualified DC vaccines according to study schedule with or without TMZ at 
discretion of the PI and in consultation with the treating neuro-oncologist  (i.e. subjects removed 
for intolerance of TMZ treatment may discontinue or reduce TMZ dose as clinical management 
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Given absence of curative treatment for GBM, release of vaccines to patients who have consented 
to the study but are unable to meet study requirements (most often chemotherapy intolerance) is 
deemed in best interest of the enrolled subjects and best utilization of cGMP manufactured 
autologous products. While these patients may be excluded from primary endpoint analysis of 
evaluable subjects, all toxicity monitoring and DLT stopping rules will apply to any subjects 
receiving DC vaccination. 
 
9.9 MRI/MRS Imaging of DC Migration 
All patients will undergo vaccine site pre-treatment with Td (1Lf) (Martin-Fontecha, Sebastiani et 
al. 2003) or saline 6-24 hours prior to Vaccine #3, #6, and #9. 
 
At vaccine #3, patients at the University of Florida site will undergo MRI/MRS imaging before 
vaccine site pretreatment, 48hrs post vaccination, and 5 to 7 days post vaccination (optional) to 
evaluate impact of DC vaccination and unpulsed PBMC vaccination at inguinal draining lymph 
nodes.  MRI/MRS imaging 5 to 7 days post vaccination may be performed when feasible for 
patients. Regions of interest will be analyzed in a blinded fashion by qualified radiologists within 
the Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) facility and UF Health 
Department of Radiology.  Peripheral blood and urine will be collected at these time points for 
soluble biomarker analysis. These correlative studies are designed to determine whether changes 
in VDLNs can be assessed by MRI/MRS and correlation between changes and clinical outcome. 
 
 STUDY REQUIREMENTS 
 
10.1 Enrollment 
• Signed informed consent. 
• History, physical & neurological exam 
• MMSE for subject ≥ 50 years. 
• Histopathology report confirming GBM (WHO Grade IV Glioma) and molecular 
diagnostics. 
If molecular testing were not performed at time of diagnosis, promoter 
hypermethylation of the O6-methylguanine-methyl-transferase (MGMT) gene, and 
the mutation status of the IDH1 and IDH2 genes will be obtained for study data 
analysis. 
• Preoperative MRI (or CT). 
• Postoperative MRI (or CT) within 28 days of study enrollment. If the postoperative 
MRI is older than 28 days, a new MRI will be obtained prior to patient randomization. 
The new MRI will serve as baseline MRI. 
• Karnofsky Performance Status. 
• CBC with differential and CD4+/CD8+ T cell ratio. 
• Infectious disease testing to include: HIV, Hepatitis B, Hepatitis C and CMV. 
• For females of child-bearing potential, β-HCG serum pregnancy test. 
• Pathology slides (if available) consisting of 1 hematoxylin and eosin (H&E) and 19 
unstained slides, from all prior resections, prepared on Fischer Plus glass or Histostix 
coated slides.   


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• Confirm eligibility. 
 
10.2 Prior to Leukapheresis (Vaccine Cell Generation) 
• Perform randomization prior to leukapheresis #1. 
• CBC with differential and CD4+/CD8+ T cell ratio. 
• Comprehensive metabolic panel 
• For females of child-bearing potential, β-HCG serum pregnancy test. 
• Infectious disease testing in accordance with institutional practices.  
• Blood for immune monitoring. 
 
• Dialysis type central venous catheter for patients with poor venous access. 
• Oral Tums, 2 tablets, BID for 2 days prior to the leukapheresis procedure (if standard-
of-care) to prevent the development of hypocalcemia from the citrate. 
• Document adverse events. 
• Document concomitant medications. 
 
10.3 Chemoradiation 
• For females of child-bearing potential, β-HCG serum pregnancy test within 72 hours 
prior to starting TMZ. 
• Administer TMZ continuously from day 1 of RT to the last day of RT for a maximum 
of 49 days. 
• Standard RT at the discretion of the treating Radiation Oncologist  
• Radiation Therapy summary to include start and end dates, number of fractions, any 
interruptions, and total dose delivered. 
 
10.4 Adjuvant TMZ Cycles 
• History, physical & neurological exam 
• Confirm eligibility prior to TMZ cycle 1. 
• Administer Td booster with vaccine 1. 
• Administer TMZ. 
• Administer study vaccines.  
• Perform vaccine site pretreatment with Td (1Lf) or saline intradermal prior to Vaccine 
#3, #6, and #9.   
• Inguinal MRI/MRS imaging with Vaccine 3 (University of Florida site only). For 
women of childbearing potential a urine pregnancy test will be obtained prior to 
imaging. 
• Blood for immune monitoring: 
o Leukapheresis 1 
o Prior to vaccine 1 
o Prior to vaccine 2 
o Prior to vaccine 3 
o At MRI/MRS imaging time points (UF site only) 
o Leukapheresis 2 
o Prior to vaccine 4 
o Prior to vaccine 7 
o Prior to vaccine 10 


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o At progression 
• Urine for immune monitoring 
o Prior to vaccine 1 
o At MRI/MRS imaging time points (UF site only) 
 
Patients seen by the neuro-oncology team between these vaccine visits may have blood 
drawn for immune monitoring at the discretion of the study team.  
For immune monitoring, up to 80 mLs of blood will be collected in 10 yellow top tubes 
and processed in accordance with SOP-UFBTIP-126. For humoral response, 
approximately 3.5 mLs will be collected in a gold top tube.   
• CBC with differential and CD4+/CD8+ T cell ratio at each vaccine visit and, if 
clinically indicated, at routine clinic visits.  
• MRI (or CT) at the following time points: 
o bimonthly during TMZ cycles and every 3 months after the 12th TMZ cycle (or 
1 year post surgery if TMZ discontinued prior to 12 cycles) 
o Response and progression will be assessed following Modified RANO Criteria 
as described in Section 11.Response Evaluation. Initial assessment of disease 
status will be done by the treating neuro-oncologist/study investigators, upon 
review of surveillance brain MRI during routine clinic visit followed by study 
vaccine administration or chemo cycle initiation. Clinical assessment of disease 
status will be followed by formal measurements of enhancing lesions for 
modified RANO criteria. 
• Review and document adverse events at each visit. 
• Review and document concomitant medications at each visit. 
10.5 At Progression 
• MRI (or CT) documenting progression. 
• Blood for immunologic monitoring if feasible. 
• Pathology slides (if available) consisting of 1 hematoxylin and eosin (H&E) and 19 
unstained slides, prepared on Fischer Plus glass or Histostix coated slides.   
 
10.6 Concomitant Medication 
The following medications will be recorded with start date, stop date, and indication: 
▪ Steroids  
▪ Anticonvulsants  
▪ Bevacizumab 
▪ Any medication used in the treatment of a study drug related AE/SAE 
▪ Any medication deemed to possibly be interactive with study drug by PI or 
designee.  
Concomitant medication will be recorded at the time of vaccine #1 and continue until 30 
days after the last dose of study drug and at leukapheresis time point. 
 
Use of bevacizumab: 


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Bevacizumab is not a study drug on this trial. However, for patients who develop radiation necrosis 
symptoms, a short trial of bevacizumab can be considered at the discretion of the PI in consultation 
with the treating physician, if the PI believes that the patient may benefit from the treatment.  
Dose and frequency of bevacizumab treatment will be at the discretion of the investigator in 
keeping with standard of practice (bevacizumab 5-10 mg/kg every 2-3 weeks) for up to 3 months 
treatment. 
 
NOTE: Bevacizumab is to be used for participants who develop symptomatic radiation necrosis, 
and is not intended to be used to treat the underlying tumor.  
 


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10.7 Schedule of Study Assessments 
 
 
 
 
                           
 
                                  Visits 
 
 
 
 
 
 
        Study Requirements 
Pre-Treatment 
Treatment Period 
Follow-up 
Enrollment 
Randomization
Leukapheresis #1 
Chemo-radiation ¹⁹ 
Adjuvant TMZ Cycle 1 
Vaccine #1 
Vaccine #2 
Vaccine #3 
Leukapheresis  #223 
Adjuvant TMZ Cycle 2 
Vaccine #4 
Adjuvant TMZ Cycle 3 
Vaccine #5 
Adjuvant TMZ Cycle 4 
Vaccine #6 
Adjuvant TMZ Cycle 5 
Vaccine #7 
Adjuvant TMZ Cycle 6 
Vaccine #8 
Adjuvant TMZ Cycle 7 
Vaccine #9 
Adjuvant TMZ Cycle 8 
Vaccine #10 
Up to 12 Cycles of TMZ 
 
 
 
 
 
 
Progression 
 
Follow-up until 
tumor progression 
or death 
4 wks after RT 
completion +/-1
Day 22-24 of Cycle 1 
2 weeks after  
Vaccine #1 +/-1
2 weeks after  
Vaccine #2 +/-1
 2 to 4 weeks after  
vaccine #3  
1 day to 1 week after 
2nd Leukapheresis
Day 22-24 of Cycle 2 
5 weeks after  
Cycle #2 +/-1
Day 22-24 of Cycle 3 
5 weeks after 
Cycle #3 +/-1
Day 22-24 of Cycle 4 
5 weeks after Cycle 
#4 +/-1
Day 22-24 of Cycle 5 
5 weeks after 
Cycle #5 +/-1
Day 22-24 of Cycle 6 
5 weeks after 
Cycle #6 +/-1
Day 22-24 of Cycle 7 
5 weeks after 
Cycle #7 +/-1
Day 22-24 of Cycle 8 
Every 5 weeks   +/-1 
Inclusion and Exclusion criteria 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Brain MRI or CT scan 
X¹ 
 
 
 
˂-------------------------------------------------------------------X⁸------------------------------------------------------------------˃ 
X8 
Karnofsky Performance Status 
X 
 
 
 
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Serum pregnancy  test 
X 
 
X 
X2 
 
 
 
X2 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor Tissue 
X3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X3 
Randomization 
 
X12 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC w/ differential 
X 
 
X 
 
 
X9 
X9 
X9 
 
 
X9 
 
X9 
 
X9 
 
X9 
 
X9 
 
X9 
 
X9 
 
 
Immunophenotyping CD4+/CD8+ T cell ratio 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Peripheral blood for immune monitoring 
 
 
X14 
 
 
X14 
X14 
X14 
X14 
 
X14 
 
 
 
 
 
X14 
 
 
 
 
 
X14 
 
X14 
Comprehensive metabolic panel 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Infectious disease testing 
X22 
 
X4 
 
 
 
 
 
X4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Oral Tums 
 
 
X5 
 
 
 
 
 
X5 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Leukapheresis 
 
 
X 
 
 
 
 
 
X16 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Temozolomide 
 
 
 
X6 
X10 
 
 
 
 
X10 
 
X10 
 
X10 
 
X10 
 
X10 
 
X10 
 
X10 
 
X10 
 
External Beam RT 
 
 
 
X7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
History, Physical and Neurological Exam 
X21 
 
 
 
X11 
 
 
 
 
X11 
 
X11 
 
X11 
 
X11 
 
X11 
 
X11 
 
X11 
 
X11 
 
Td Booster 
 
 
 
 
 
X15 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blinded vaccine site pre-treatment 
 
 
 
 
 
 
 
X17 
 
 
 
 
 
 
X17 
 
 
 
 
 
X17 
 
 
 
 
Blinded study vaccine 
 
 
 
 
 
X13 
X13 
X13 
 
 
X13 
 
X13 
 
X13 
 
X13 
 
X13 
 
X13 
 
X13 
 
 
MRI/MRS Inguinal imaging (UF site only) 
 
 
 
 
 
 
 
X18 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine Immune monitoring (UF site only) 
 
 
 
 
 
 X18 
 
X18 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medications 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Adverse event assessment 
 
 
X20 
 
 
X20 
X20 
X20 
X20 
X20 
X20 
X20 
X20 X20 
X20 
X20 
X20 
X20 
X20 
X20 
X20 
X20 
X20 
 
 
Footnotes on next page


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Footnotes for Schedule of Study Assessments: 
1 
A diagnostic contrast-enhanced MRI or CT scan of the brain must be performed preoperatively and 
postoperatively.If no post op MRI/CT within 28 days of enrollment is available, a new brain MRI (or 
CT) must be obtained in order to determine residual burden and eligibility prior to randomization. 
2 
Negative serum pregnancy test within 72 hours prior to starting TMZ and negative urine pregnancy test 
prior to Inguinal MRI/MRS (UF site only). 
3 
1 H&E and 19 unstained slides from original surgery and at progression, if applicable. 
4 
Pre-leukapheresis testing in accordance with institutional practices.  
5 
Oral Tums, 2 tablets, for 2 days prior to leukapheresis (if standard-of-care). 
6 
Administer TMZ continuously from day 1 of RT to the last day of RT at a daily oral dose of 75 mg/m2 
for a maximum of 49 days. 
7 
Standard RT at the discretion of the treating Radiation Oncologist   
8 
Bimonthly during TMZ cycles and every 2 to 3months after the 12th TMZ cycle (or 1 year post surgery 
if TMZ discontinued prior to 12 cycles). Measurements for all imaging will be recorded.  Prior to 
vaccination, assessment for tumor progression can be made by the treating investigator, but all CT/MRI 
measurements for study endpoints must be made by designated study personnel. 
9  
 CBC with differential and CD4+/CD8+ T cell ratio at each vaccine visit and, if clinically indicated, at 
routine clinic visits. 
10 
Up to 12 cycles of dose-intensified TMZ may be given.  Cycle #1 should be started 4 (+/- 1) weeks after 
completing chemoradiation.  Administer once per day for 21 consecutive days (days 1-21) of a 35 (+/- 
7) day cycle. The starting dose for the first cycle will be 75 mg/m2/day, with a single dose escalation to 
100 mg/m2/day in subsequent cycles if no adverse events are noted. 
11 
Within 14 days prior to initiation of TMZ and prior to subsequent TMZ cycles. 
12 
Randomization will occur prior to leukapheresis. 
13 
Administer vaccine #1 intradermally at day 22-24 after the first TMZ cycle.  Vaccines #2 and #3 will 
occur at 2 (+/- 1) week intervals.  Vaccines #4-10 will be given on day 22-24 of each TMZ cycle. 
Continue until a total of 10 vaccines have been administered or until tumor progression (whichever 
comes first).  Each vaccination will be equally divided between both inguinal regions.   Monitor for 30-
60 minutes post-immunization for any adverse effects.   
14 
Up to 10 yellow top tubes (ACD-A) and 1 gold top tube (SST) during initial leukapheresis, prior to 
vaccines 1-3, during second leukapheresis, prior to vaccine 4, 7, 10, at MRI/MRS imaging time points 
(UF site only) and at progression if feasible.  Peripheral blood will be processed in accordance with 
SOP-UFBTIP-126.  
15 
All patients will receive IM Td booster (5 Lf) with vaccine #1. 
16 
2 to 4 weeks after vaccine #3, repeat leukapheresis for DC or PBMC generation.  
17 
All patients will undergo vaccine site pretreatment with Td (1 Lf) or saline 6-24 hours prior to Vaccine 
#3, #6, and #9.   
18 
Patients at University of Florida site will undergo MRI/MRS imaging of inguinal draining lymph nodes 
before vaccine #3 site pretreatment, 48hrs post vaccination, and 5 to 7 days post vaccination (optional). 
Urine samples will be collected prior to vaccine #1 and at MRI/MRS imaging time points. 
19  
Within 7 weeks of surgery. 
20    
AE collection will begin at the time of administration of vaccine #1 and continue until 30 days after 
the last dose of study drug.  Any adverse events associated with leukapheresis will also be recorded. 
21   
Including MMSE for patients age ≥ 50 years. 
22  
Results from infectious disease markers performed within 30 days of enrollment can be used to satisfy 
criteria. 
23  
Additional leukapheresis may be performed if needed to generate additional vaccines.


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 RESPONSE EVALUATION 
 
The modified RANO provides clinical guidelines for continuing therapy beyond suspected 
radiographic progression if the treating physician believes there may be a therapeutic benefit and 
provides criteria for defining progression and early drug failure while also allowing for the 
possibility of pseudoprogression (PsP) and pseudoresponse (PsR). An update to the RANO 
criteria proposes strategies to establish a general framework for response assessment in neuro-
oncology that is agnostic to the mechanism of action of the particular therapy (anti-angiogenic, 
immunotherapy...) to address the challenges associated with interpretation of radiographic 
changes in patients with glioblastoma in clinical trials. The modified RANO proposes to use the 
post-radiation time point as the baseline for response evaluation and to consider only 
objectively defined, measurable enhancing disease in the definition of response and progression 
(i.e. exclusion of qualitative assessed T2/FLAIR changes) 
 
11.1 General Methodology for Determining Tumor Measurements 
Newly diagnosed GBM patients will initially undergo a pre-entry MRI scan for initial diagnosis 
prior to entry in the study and prior to therapy. The post-operative scan will determine residual 
enhancing disease which may be used as a covariate in survival analyses. Patients will then start 
on standard therapy, Temozolomide with concurrent radiation therapy (RT). The Post-RT 
scan will be required and used as the baseline scan for which response will be determined. 
Following the first cycles of adjuvant therapy, patients will receive additional required MRI 
scans. 
 
All radiographic responses will be based on MRI scans of the brain obtained with and without 
contrast. Tumor size will be based on the product of the maximal diameters in 2 different planes. 
Response will be assessed as a percentage change in tumor size from baseline. Tumor 
measurements in the follow-up scans will be compared to those of the post-RT scan to document 
response.  
 
Definition of Measurable Disease, Non-Measurable Disease, and Target Lesions 
Measurable disease should be defined as contrast enhancing lesions with a minimum size of both 
perpendicular measurements greater than or equal to 10mm (if the largest diameter is greater than 
10mm but the perpendicular diameter is less than 10mm, this would constitute non-measurable 
disease).  Up to a total of five target measurable lesions should be defined and ranked from largest 
to smallest. 
 
Algorithm for identifying measurable and target lesions: 
 


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11.2 Complete Response (CR) 
Requires all of the following: 
1. Disappearance of all enhancing measurable and non-measurable disease sustained for at 
least 4 weeks. The first scan exhibiting disappearance of all enhancing measurable and 
non-measurable disease is considered “preliminary CR”. If the second scan exhibits 
measurable enhancing disease with respect to the “preliminary CR” scan, then the 
response is not sustained, noted as pseudoresponse, PsR, and is now considered 
“preliminary PD” (note: confirmed PD requires at least two sequential increases in tumor 
volume). If the second scan continues to exhibit disappearance of enhancing disease or 
emergence of non-measurable disease (less than 10mm bidimensional product), it is 
considered a durable CR and the patient should continue on therapy until confirmed PD 
is observed. 
2. Patients must be off corticosteroids (or on physiologic replacement doses only). 
3. Stable or improved clinical assessments. 
Note: Patients with non-measurable disease only at baseline cannot have CR; the best response 
possible is stable disease (SD). 
 
 
 
 


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11.3 Partial Response (PR) 
 Requires all of the following: 
1. ≥50% decrease in sum of products of perpendicular diameters of all measurable 
enhancing lesions compared with baseline, sustained for at least 4 weeks. The first scan 
exhibiting ≥50% decrease in sum of products of perpendicular diameters of all 
measurable enhancing lesions compared with baseline is considered “preliminary PR”. If 
the second scan exhibits PD with respect to the “preliminary PR” scan, then the response 
is not sustained, noted as pseudoresponse, PsR, and is now considered “preliminary PD” 
(note confirmed PD requires at least two sequential increases in tumor volume). If the 
second scan exhibits SD, PR, or CR, it is considered a durable PR and the patient should 
continue on therapy until confirmed PD is observed. 
2. Steroid dose should be the same or lower compared with baseline scan. 
3. Stable or improved clinical assessments. 
Note: Patients with non-measurable disease only at baseline cannot have PR; the best response possible is stable 
disease (SD). 
 
11.4 Stable Disease (SD) 
 Requires all of the following: 
1. Does not qualify for complete response, partial response, or progressive disease. Note 
this also applies to patients that demonstrate PsR when the confirmation scan does not 
show PD or PsP when the confirmation scan does not show PR/CR. 
In the event that corticosteroid dose was increased (for new symptoms/signs) without confirmation 
of disease progression on neuroimaging, and subsequent follow-up imaging shows that the steroid 
increase was required because of disease progression, the last scan considered to show stable 
disease will be the scan obtained when the corticosteroid dose was equivalent to the baseline dose. 
 
11.5 Progressive Disease 
 Defined by any of the following: 
1. At least two sequential scans separated by at ≥4 weeks both exhibiting ≥25% increase in 
sum of products of perpendicular diameters of enhancing lesions. The first scan 
exhibiting ≥25% increase in sum of products of perpendicular diameters of enhancing 
lesions should be compared to the smallest tumor measurement obtained either at 
baseline (if no decrease) or best response (on stable or increasing steroid dose) and is 
noted as “preliminary PD.” If the second scan at least 4 weeks later exhibits a subsequent 
≥25% increase in sum of products of perpendicular diameters of enhancing lesions 
relative to the “preliminary PD” scan, it is considered “confirmed PD” and the patient 
should discontinue therapy. If the second scan at least 4 weeks later exhibits SD or 
PR/CR, this scan showing “preliminary PD” is noted as “pseudoprogression”, PsP, and 
the patient should continue on therapy until a second increase in tumor size relative to the 
PsP scan is observed. Note that any new measurable (>10mm x 10mm) enhancing 
lesions should not be immediately considered PD, but instead should be added to the sum 


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of bidimensional products or total volume representing the entire enhancing tumor 
burden. 
2. In the case where the baseline or best response demonstrates no measurable enhancing 
disease (visible or not visible), then any new measurable (>10mm x 10mm) enhancing 
lesions are considered PD after confirmed by a subsequent scan ≥4 weeks exhibiting 
≥25% increase in sum of products of perpendicular diameters of enhancing lesions 
relative to the scan first illustrating new measurable disease. The first scan exhibiting new 
measurable disease is noted as “preliminary PD.” If the second scan at least 4 weeks later 
exhibits a subsequent ≥25% increase in sum of products of perpendicular diameters of 
enhancing lesions relative to the “preliminary PD” scan it is considered “confirmed PD” 
and the patient should discontinue therapy. If the second scan at least 4 weeks later 
exhibits SD, CR, PR, or becomes non-measurable, this scan showing “preliminary PD” is 
noted as “pseudoprogression”, PsP, and the patient should continue on therapy until a 
second increase in tumor size relative to the “preliminary PD”, or PsP, scan is observed. 
Note that any new measurable(>10mm x 10mm) enhancing lesions on the subsequent 
scan following the preliminary PD scan should not be immediately considered confirmed 
PD, but instead should be added to the sum of bidimensional products or total volume 
representing the entire enhancing tumor burden. 
3. Clear clinical deterioration not attributable to other causes apart from tumor (e.g. 
seizures, medication adverse effects, therapy complications, stroke, infection) or 
attributable to changes in steroid dose. 
4. Failure to return for evaluation as a result of death or deteriorating condition. 
11.6 Symptomatic Deterioration & Reporting Clinical Status 
Patients with global deterioration of health status requiring discontinuation of treatment without 
objective evidence of disease progression at that time, and not either related to study treatment or 
other medical conditions, should be reported as PD due to “symptomatic deterioration.” Every 
effort should be made to document the objective progression even after discontinuation of 
treatment due to symptomatic deterioration.  
Neurological exam data should be provided to the study designated neuro-radiologists as “stable, 
improved, declined” in case report forms. Clinical status should be recorded as “declined” if the 
neurological exam is worse, otherwise the clinical status should be set to “improved” or “stable”. 
In the event that necessary clinical data is not available, clinical status should be recorded as “not 
available” and that particular time point can only be reviewed for PD (otherwise “non-evaluable”). 
Neurological data must be within ±7 days of the time-point response date, otherwise the data is 
considered “not available”. 
 
11.7 Steroid Use and Dose 
Steroid use should be derived from the concomitant medications on the case report forms and 
recorded as “Yes”, “No”, or “not available”. A value of “No” should be assigned if, at the time-
point, the subject is not on steroids or on physiologic replacement doses only (<2 mg 
dexamethasone or equivalent per day). 
Average steroid dose no greater than 2 mg change from baseline should be abstracted to “stable”. 
If outside this range the steroid dose should be abstracted to “increased” or “decreased” 
accordingly. Steroid data should be within ±5 days of the time-point response date, otherwise the 
data is considered “not available”. 


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11.8 Overall Objective Status 
The overall objective status for an evaluation should be determined by combining the patient’s 
radiographic response on target lesions, new disease, neurological status, and steroid dose/usage 
as defined in the table below for patients with measurable (>10mm x 10mm) disease. Note that 
patients with possible PsP or pseudoresponse should be given the Objective Status of “Preliminary 
Progression” or “Preliminary Response”, respectively. Once PsP, pseudoresponse, or true 
progression/response are confirmed, the Objective Status can be changed accordingly. 
 
 


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Guidelines for determining comprehensive objective status 
Target lesions 
(current scan) 
Target lesions (previous 
scan) 
New sites of 
measurable diseasea 
Neurological 
status 
Steroid 
usage 
Steroid dose 
Overall objective status 
CR 
Not Evaluated 
No 
Stable/Better 
No 
N/A 
Preliminary CR 
PR 
Not Evaluated 
No 
Stable/Better 
Any 
Stable/Decreasing Preliminary PR 
PD 
Not Evaluated 
Yes or No 
Stable/Better 
Any 
Stable/Increasing 
Preliminary PD 
PD 
Preliminary or Confirmed 
PR/CR 
No 
Stable/Better 
Any 
Stable/Increasing 
Preliminary PD 
SD 
Preliminary or Confirmed 
CR/PR or SD/NE 
No 
Stable/Better 
Any 
N/A 
SD 
PR 
Preliminary PR 
Yes or No 
Stable/Better 
Any 
Stable/Decreasing Confirmed PR 
SD 
Preliminary PR 
Yes or No 
Stable/Better 
Any 
Stable/Decreasing SD (Preliminary PR 
→Confirmed PR) 
SD 
Preliminary CR 
Yes or No 
Stable/Better 
Any 
Stable/Decreasing SD (Preliminary CR 
→Confirmed CR) 
CR 
Preliminary CR 
No 
Stable/Better 
No 
N/A 
Confirmed CR 
SD 
Preliminary PD 
No 
Stable/Better 
Any 
Stable/Decreasing SD 
(Confirmed PsP) 
CR/PR/SD 
PD/NE 
CR/PR/SD/PD/NE 
Yes or No 
Worse 
Any 
Stable/Increasing 
Confirmed PD 
PD 
Preliminary PD 
Yes or No 
Any 
Yes 
Stable/Increasing 
Confirmed PD 
aNote that new sites of measurable disease are added to the sum of bidimensional products or total lesion volume, or constitutes preliminary PD in the case of 
no measurable disease at baseline or best response 


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11.9 Detailed Modified Radiographic Response Assessment 
 
The diagram below indicates the modififed RANO treatment algorithm for the assessment of 
modified radiographic response in newly diagnosed glioblastoma: 
 
 
 
Preliminary Radiographic Progression 
If the lesion size has increased ≥25% sum of bidirectional product between MRI Scan 1 and N, 
these patients should be categorized as “preliminary radiographic progression”. If the 
investigator believes the patient can safely continue on therapy, then they should continue to 
treat and acquire a follow-up confirmatory scan [MRI(N + 1)] at the next scan interval (8 
weeks ± 4 weeks from MRI Scan (N) or no less than 4 weeks minimum duration between 
preliminary PD and confirmed PD scans) to verify tumor growth and progression. For patients 
with gross-total resection (GTR) and no measurable enhancing disease, preliminary radiographic 
progression is defined as a transition from no measurable disease to non-measureable (but 
present) disease(<10mm x 10mm) or measurable disease (>10mm x 10mm). If the investigator 
feels it is safe to keep the patient on, a confirmatory scan at MRI(N + 1) should be obtained to 
verify tumor progression. 
Confirmed Progression 
If the patient has an increase ≥25% sum of bidirectional product between MRI Scan N and N + 1, 
this is “Confirmed Progression”, the patient should stop therapy and the date of radiographic 
progression is the date of suspected progression, MRI(N). If the patient has SD/PR/CR on 
MRI(N + 1) with respect to MRI(N), PsP is confirmed and the patient should continue on 
therapy. Patients will then continue on therapy and receive additional follow-up MRI scans 
[MRI(M)]. If the lesion size has increased ≥25% sum of bidirectional product on MRI(M) 
relative to the smaller of Nadir or MRI(N + 1), then the patient has “Confirmed Progression”, the 
patient should stop therapy and the date of radiographic progression is the new date, MRI(M). 
For patients with no measurable disease at the Post-RT baseline, “Confirmed Progression” will 
be defined as a transition from non-measurable (but present) disease (<10mm x <10mm) on 
MRI(N) to measurable disease (>10mm x 10mm) on MRI(N + 1). For patients with confirmed 
PsP and no measurable disease at Nadir, “Confirmed Progression” should be defined as a 
transition from no measurable disease to measurable disease (>10mm x 10mm). In all cases, 
patients with confirmed progression should stop therapy. 


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Preliminary Radiographic Response  
If a measurable lesion has decreased ≥50% sum of bidirectional product between MRI(1) and 
MRI(N), these patients should be categorized as “preliminary radiographic responders” and will 
be monitored for an additional time point and/or treatment cycle. After an additional cycle of 
therapy (8 weeks ± 4 weeks from MRI(N)), patients will receive a confirmatory MRI(N + 1). 
Confirmed Radiographic Response 
If the lesion(s) have increased ≥25% sum of bidirectional product between MRI Scan N and 
N + 1, (indicating radiographic progression from MRI(N)), this is considered an “unsustained 
radiographic response” or “pseudoresponse”. These patients should be categorized as 
“preliminary radiographic progression”. If the investigator believes the patient can safely 
continue on therapy, then they should continue to treat and acquire a follow-up confirmatory 
scan [MRI(M)] at the next scan interval (8 weeks ± 4 weeks from MRI Scan (N+1) or no less 
than 4 weeks minimum duration between preliminary PD and confirmed PD scans) to verify 
tumor growth and progression. If the patient has an increase ≥25% sum of bidirectional product 
between MRI Scan N+1 and M, this is “Confirmed Progression”, the patient should stop therapy 
and the date of radiographic progression is the date of suspected progression, MRI(N+1). If the 
patient has SD/PR/CR on MRI(M) with respect to MRI(N+1), PsP is confirmed and the patient 
should continue on therapy. Patients will then continue on therapy and receive additional follow-
up MRI scans [MRI(M)]. 
Alternatively, if the lesion has not increased from MRI(N), this is considered a “durable 
radiographic response,” the patient will continue on therapy, and the date 
of preliminary radiographic progression is the time point of an increase ≥25% sum of 
bidirectional product (from Nadir) during the remainder of the study. The investigator can then 
decide whether to continue safely on therapy until progression has been confirmed and at the 
subsequent time point stop therapy if they feel the patient cannot safely continue therapy. 
Stable Disease 
If the lesion size has not increased or decreased beyond the set thresholds between Scan 1 and N, 
the patient is considered “stable.” Such patients will continue on therapy, and the date 
of preliminary progression is the time point of an increase ≥25% sum of bidirectional product 
(from Nadir) during the remainder of the study. Upon preliminary progression the investigator 
can choose to either continue therapy and confirm progression or discontinue therapy. For cases 
with significant neurologic decline at the time of imaging progression as determined from 
MRI(N), a confirmatory scan at time point MRI(N + 1) may not be possible or necessary. For 
these cases, it is appropriate to define MRI(N) as the progression time point. 
 
11.10 Clinical Judgement 
Treatment effects, necrosis, vasogenic edema and true tumor progression may appear similar on 
post contrast brain MRI. There is a continued effort to develop new tools and method to 
characterize these different outcomes. However, differentiating treatment effects from true 
progression in patients with high-grade glioma remains an ongoing challenge. 
Consequently, we propose to provide some allowance for clinical judgement when deciding 
whether to discontinue DC vaccination when there is a reasonable doubt regarding disease 
progression versus treatment effects. 


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In the event of radiographic changes that meet modified RANO criteria for disease progression 
in clinically stable or improved patients, vaccination treatment may be continued at the discretion 
of the PI in consultation with the treating physician, if the PI believes that it is safe to continue 
therapy and that patients may benefit from the ongoing treatment regimen.  Subsequent 
evaluation by radiographic imaging, clinical evaluation, and/or surgery/biopsy that declares true 
tumor progression will result in establishing the date of tumor progression at the time when the 
patient first met modified RANO criteria for documented tumor progression.  Therefore, it will 
not be deemed a protocol deviation for patients to have received subsequent DC vaccines during 
the interval of assessment for resolving tumor progression.  Given the primary endpoint of 
overall survival for this phase 2 study and above described difficulties in radiographic 
assessment in this patient population, an allowance for clinical judgement on treatment 
discontinuation is warranted.  
 
11.11 Confirmation of Tumor Progression by Histopathology 
Due to the challenge of recognizing treatment effect from tumor progression, histological 
confirmation of tumor progression should be obtained when clinically indicated to avoid premature 
termination 
of 
effective 
treatment 
or 
continuing 
ineffective 
treatment.  
Tissue obtained by biopsy or resection will be used to confirm tumor progression histologically. 
If tumor progression is confirmed, ongoing treatment will be stopped and best of available care at 
the 
time 
of 
confirmed 
progression 
will 
be 
discussed 
with 
the 
patient.  
If obtained in sufficient quantity, available tissue will be used to evaluate cellular infiltration and 
CMV antigen expression at the tumor site.  
In the event that histopathology reveals inflammatory response, indicating toxic or therapeutic 
responses and not tumor progression, the patient may resume ongoing treatment at the discretion 
of the PI in consultation with the treating physician after the patient has recovered from the effect 
of biopsy/surgery. 
 
 IMMUNOLOGICAL RESPONSE EVALUATIONS 
 
Immunological response evaluations for baseline values will be conducted on PBMCs collected at 
the time of the initial leukapheresis sample.  Immunologic response evaluations will also be 
performed from peripheral blood obtained prior to select vaccinations.  The primary comparison 
for immunologic endpoints will be baseline response (pre-leukapheresis or P1) compared to 
response post vaccine #3 (post-leukapheresis or P2).  Our previous studies demonstrated a 
significant increase in immunologic responses in vaccinated patients that peaked after the third 
vaccine in most subjects.  A comparison of pre-therapy lymphocyte function to additional vaccine 
time points may be conducted on an exploratory basis. Arms 1 and 2 (pp65-shLAMP RNA-pulsed 
DCs and pp65-flLAMP RNA-pulsed DCs) will be analyzed separately for immunologic endpoint 
comparisons.  Studies examining DNA vaccines using full-length LAMP1 fusion antigens 
compared to truncated versions of the LAMP protein have demonstrated that increased antigen 
expression, secretion of LAMP fusion proteins, enhanced antibody titers, increased cytokine 
release by CD4+ and CD8+ T cells, improved central memory T cell differentiation, and enhanced 


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polyfunctional T cell responses, all were dependent on full-length LAMP1 fusion 
antigens(Godinho, Matassoli et al. 2014).  Truncated LAMP fusions that did not contain the 
luminal domain of LAMP were much less efficient in this wide spectrum of immunologic analyses.  
As a primary biologic analysis and secondary endpoint of this study, we will compare the 
immunologic responses in patients vaccinated with CMV pp65-flLAMP RNA-pulsed DCs (Arm 
1) to patients vaccinated with CMV pp65-shLAMP RNA-pulsed DCs (Arm 2) across this spectrum 
of responses.   
 
12.1 ELISPOT Assay 
The ELISPOT assay, our primary assay to detect cellular immune responses is a sensitive detection 
assay for evaluation of antigen-specific cytokine producing T-cells.  The IFN- ELISPOT allows 
for the direct visualization of human  -interferon cytokine release from a single cell, which has 
been widely reported to be an indicator of activation of an antigen-specific immune response.  
 
On the day of testing, PBMCs will be thawed quickly, washed, resuspended in R-10 medium and 
cell counts and viability measured PBMCs (250,000/well) will be stimulated overnight with 
synthetic peptides consisting of a peptide cocktail derived from human CMV proteins.  Single-use 
aliquots of the peptide pools will be resuspended to a final concentration of 1 μg/mL for each 
peptide.  Each assay will include PBMC cultured with no peptide or PHA (2.5 μg/mL, 0.25 ug/mL) 
and positive and negative control PBMC for each antigen if available.  PBMC are added to 
duplicate wells of 96-well ELISPOT assay plates coated with mouse IgG1 anti-human IFN-  
monoclonal antibody (MAb) will be incubated overnight at 37°C, 5% CO2, washed with 
PBS/Tween 20, incubated with biotinylated mouse IgG1 anti-human IFN-  MAb 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 min at room 
temperature and spot development stopped by distilled water rinse.  Plates will be dried and 
shipped to Zellnet Consulting (New York, NY) for spot enumeration by automated analysis with 
a Zeiss KS ELISPOT system.  Results will be expressed as the mean spot-forming cells (SFC)/106 
PBMC after subtraction of counts from cells cultured with no peptide.  
 
12.2 Polyfunctional T cell Responses, T cell Phenotype, and Other 
Type of Immune Cell Phenotype  
The laboratory of UFBTIP will conduct a flow cytometric analysis that aims to quickly detect the 
activation of T cells through CD69 and exhaustion via PD-1. A 13-color assay will be used to 
monitor the polyfunctional immune response of patients' CD4 and CD8 cells. The analysis will 
also include an examination of the phenotypic distribution of T cells that respond to the assay, 
including naive, effector, central memory, effector memory, and terminal effector cells. 
Additionally, the same panel will be used to analyze regulatory T cells (identified as 
CD3+CD4+CD25+FoxP3+), natural killer cells (CD56+), natural killer T cells (CD56+CD3+), 
invariant natural killer T cells (CD56+CD3+TCR-va24+), and cell viability (measured through the 
Live/Dead Fixable Blue Viability Assay) (as seen in Figure 13). 
 
 
 


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Immediately preceding select vaccinations peripheral blood will be drawn into vacutainer tubes 
containing ACD.  PBMC will be separated by density gradient centrifugation on ficoll-Hypaque 
and then cryopreserved until the day of immune monitoring operation.. 
 
    
12.3 Multiplex Cytokine Quantification 
The UFBTIP laboratory will perform a Multiplex cytokine analysis to detect multiple cytokines 
secreted by lymphocytes. This analysis will use a multiplex kit on the Meso Scale Discovery 
platform to simultaneously examine Type 0,1,2, and 3 cytokines (IFN-g, IL-1b, IL-2, IL-4, IL-6, 
IL-8, IL-10, IL12p70, IL-13, and TNF-a) secreted by T cells. Prior to select vaccinations, 
peripheral blood will be drawn into vacutainer tubes containing silicone and micronized silica 
particales (SST) to isolate patient serum. This serum will be cryopreserved until the day of the 
Multiplex cytokine assay.  
  
12.1 Single Cell Gene Expression and Immune Profiling For 
Monitoring Changes in Glioblastoma Patients with Sustained 
Radiographic Response to CMV pp64-LAMP RNA-Pulsed 
Dendritic Cell-Based Vaccines. 
The application of single cell sequencing as a novel immune monitoring platform can be used to 
identify the molecular mechanisms of immune response to dendritic cell- based vaccines, trace the 
cell types and states involved, and uncover novel biomarkers for immunotherapy.  We will perform 
 
         
 
 
Fig. 13: Polyfunctional Lymphocytes Gating Strategy. Lymphocytes can be expanded and phenotyped to identify 
T cells and NK cells. Within the T cells population, CD4 and CD8 T cells are further identified and their phenotypic 
distribution, along with regulatory T cells, is determined. These findings demonstrate that CMV pp65-LAMP RNA-
pulsed DCs can expand polyfunctional T cells in patients with GBM. 


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single-cell RNA Seq analysis of longitudinal peripheral blood mononuclear cells (PBMCs) in 
selected patients. 
 
Cell preparation: Frozen cell vials with PBMCs collected at different time points following DC 
vaccination of selected ATTAC patients will rapidly be thawed in a 37°C water bath. Thawed 
PBMCs will washed and filtered with a cell drainer. Cells will be collected by centrifugation at 
and subsequently counted with hemocytometer. For Single cell RNA-seq, single cell suspension 
will be loaded onto Chromium Controller according to the manufactures specifications and TCR-
seq libraries will be prepared via PCR amplification with primers specific to the TCR  and for 5’ 
gene expression library construction. Pre-processing of scRNA-seq data will be performed using 
specific software for identification of cell types. 
 
 SAFETY MONITORING AND REPORTING 
 
13.1 Dose-limiting Toxicity 
DLTs will be evaluated according to NCI CTCAE (Version 5.0) criteria.   
 
A DLT will be defined as a possibly, probably or definitely drug-related: 
- 
Grade IV toxicity (with the exception of grade IV cerebral edema) or any non-neurologic 
toxicity ≥ Grade III toxicity of any duration.  
- 
Grade III neurologic toxicity if not improved to Grade ≤ II within 7 days of clinical 
management will be declared a DLT. 
Neurotoxicity that improves to Grade II within 7 days will be allowed an additional 21-day 
management period to resolve to a Grade I or return to patient baseline. If the event does 
not improve to a Grade I or return to patient’s baseline within 21 days of management, it 
will then be declared a DLT 
 
Immune-related neuro toxicities are a recognized adverse event associated with immunotherapy. 
Symptoms may present throughout the course of treatment and involve the peripheral and central 
nervous system, and usually respond well to steroids, if started early. Early recognition of 
neurological toxicity is critical for the initiation of clinical management or surgical intervention to 
prevent or potentially reverse neurological sequelae. If neurological toxicity improved to ≤ Grade 
I within 28 days of clinical management, patient may resume treatment. 
 
Non-neurologic Grade III and any Grade IV toxicity or any life-threatening event not 
attributable to a concomitant medication, co-morbid event, or disease progression, even if 
reversible, will be considered a DLT.  If biopsy cannot be obtained, any new radiographic or 
clinical changes not attributable to tumor progression that produce a non-neurologic Grade III or 
any Grade IV or life-threatening toxicity will be considered at DLT.  Life-threatening events, as 
described above, even if considered a Grade III toxicity, will still be considered a DLT even if 
reversible and in such cases no further vaccinations will be given. 
 
If a neurologic Grade III NCI CTC 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 ≤ I or until the KPS score returns to within 10 points of 


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baseline.  Grade III neurologic toxicities will be declared irreversible if they cannot be reversed 
within 4 weeks of clinical management.  If the event has not improved to a Grade ≤ II within 7 
days of clinical management (then to ≤ Grade I or baseline within 21 days), a DLT will be declared 
for that patient and no further vaccinations or study-related procedures will be performed. If the 
event is reversed, but Grade III NCI CTC neurologic toxicity is again seen with subsequent 
vaccinations, all further vaccinations and study-related procedures will be withheld and a DLT 
will be declared.  Medical therapy or surgical intervention, may be used to reverse any toxicity if 
necessary. 
 
In the case of hypersensitivity reactions, treatment measures deemed medically appropriate will 
be initiated and the PI notified of the event. Although not considered a DLT, any patient with ≥ 
Grade II urticaria will not receive further vaccines and will be withdrawn from the study.     
 
Cerebral edema toxicity exception: 
Cerebral edema normally presents in glioblastoma patients as part of the disease process and can 
be exacerbated by standard of care chemotherapy and radiation. Furthermore, an effective anti-
tumor immune response may involve inflammatory response and edema in infiltrative tumor cells.  
 
Therefore, we propose that worsening cerebral edema (noted by MRI) that is not associated with 
changes in neurological symptoms will not constitute a DLT, but worsening cerebral edema in 
association with Grade 3 or greater other neurologic toxicities will be managed with above outlined 
criteria (improvement to < Grade 2 within 7 days and to Grade 1 or baseline within 21 days or will 
be declared a DLT).  Radiographic improvement of cerebral edema will be noted, but since NCI 
CTCAE v5.0 guidelines do not allow for an improvement in cerebral edema Grade < 3, the CTCAE 
grading criteria for cerebral edema will be exempted from DLT assessment. 
Grade III or greater toxicities associated with DC-based immunotherapy have been rare.  Adoptive 
T cell therapy using tumor-infiltrating lymphocytes and high-dose IL-2 in patients with melanoma 
and treatment with anti-CLTA-4 monoclonal antibody blockade have been the immunotherapy 
regimens most often associated with treatment related toxicities.  To take the most conservative 
approach to assessing possible toxicities associated with this treatment, we will be vigilant for any 
similar toxicity associated with this therapy, in addition to autoimmune toxicity specific to the 
CNS. Possible immune-mediated disorders that have been observed in patients who have received 
immunotherapy in early phase trials have involved the skin (vitiligo and cutaneous 
leukocytoclastic vasculitis), the thyroid gland (autoimmune thyroiditis), the liver (autoimmune 
hepatitis) and the pituitary (hypophysitis). Abnormal lab results, which may be immune-mediated, 
include elevations of serum lipase and amylase and liver function tests. If a patient has an AE that 
is thought to be possibly related to autoimmune antibodies (eg, thyroiditis, hepatitis, 
thrombocytopenia) the PI will send a blood sample for appropriate autoimmune antibody testing. 
If specific autoantibodies are present, the serum sample taken for storage at baseline will be tested 
for the presence of those autoantibodies. Further immunotherapy treatment in that patient will be 
held until the etiology of the event is established. 
 
 
13.2 Stopping Rules 
 


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To continuously monitor vaccine-related toxicity, we will use a one-sided Pocock-type sequential 
boundary (Ivanova et al. 2005) to repeatedly test whether a maximum acceptable DLT rate 
T=14.3% (1 patient in 7) has been exceeded. The sequential boundary will consist of the minimum 
cumulative number of patients experiencing DLTs per concurrent number of patients enrolled that 
would result in a rejection of the null hypothesis T≤14.3% and halting of the trial. The sequential 
boundary will be adjusted so that the overall probability of stopping the trial across the accrual of 
all 80 patients in the two treatment arms of the trial (i.e. the overall Type 1 error rate) is 0.05 when 
T=14.3%. If the trial runs to completion without being halted for excessive toxicity, insight into 
the magnitude of the true DLT rate can be gained by noting that the sequential boundary 
established for T=14.3% has a >80% overall chance of stopping the trial if in fact T is actually 
greater than 27.1%. The sequential boundary for T=14.3% and stopping probability 0.05 is 
displayed in the following table: 
 
N    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19 20 
PB   -  -  3  4  4  4  4  5  5  5  6  6  6  6  7  7  7  7  8  8 
 
N   21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 
PB   8  8  9  9  9  9  9 10 10 10 10 11 11 11 11 11 12 12 12 12 
 
N   41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 
PB  12 13 13 13 13 13 14 14 14 14 15 15 15 15 15 16 16 16 16 16 
 
N   61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 
PB  16 17 17 17 17 17 18 18 18 18 18 19 19 19 19 19 20 20 20 20 
 
N = Number of patients enrolled 
PB = Pocock boundary, the minimum cumulative number of patients  
     experiencing vaccine-related toxicity that should stop the trial  
     when the concurrent number of patients enrolled in the trial is N 
 
The safety monitoring rule described for vaccine-related toxicity was derived using methods 
described by Ivanova et al. (Ivanova A, BF Qaqish, and MJ Schell, 2005. Continuous toxicity 
monitoring in phase II trials in oncology. Biometrics 61: 540-545). Pocock boundaries were 
calculated using an online calculator maintained by one of the authors at the UNC Lineberger 
Comprehensive Cancer Center website:  
(http://cancer.unc.edu/biostatistics/program/ivanova/ContinuosMonitoringForToxicity.aspx). 
 
 
13.3 Adverse Events 
An AE is any untoward medical occurrence associated with use of a drug, whether or not 
considered drug related. 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 study drug, whether or not related to use of the study drug. 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. Elective and semi-elective surgery in asymptomatic patients should not be 
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An adverse drug reaction is any adverse event caused by a drug.  A suspected adverse reaction 
means any adverse event for which there is a reasonable possibility that the drug caused the adverse 
event. For the purposes of IND safety reporting, "reasonable possibility" means there is evidence 
to suggest a causal relationship between the drug and the adverse event. 
 
AE collection will begin at the time of administration of vaccine #1 and continue until 30 days 
after the last dose of study drug.  Grade 2 suspected adverse reactions and all Grade 3 or greater 
AEs must be recorded in the subject’s research record and entered into the OnCore eCRF within 
72 hours of PI discovery/notification of the event.  Any Grade 3 or greater adverse events 
associated with leukapheresis will also be recorded.  AEs must also be reported to the Coordinating 
Center as described in 13.3.   
 
AEs will be assessed according to the CTCAE version 5.0 (term and grade) and documented.  If 
CTCAE term does not exist, the PI should select the most appropriate CTC category and indicate 
“other”.  If CTCAE grading does not exist for an AE, the severity of the AE will be graded as (1) 
mild, (2) moderate, (3) severe, (4) life-threatening, or (5) fatal. 
 
Attribution of AEs will be assigned by the PI as follows: 
▪ Definite:  The AE is clearly related to the study drug 
▪ Probable:  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 
 
An adverse event is considered a Serious Adverse Event if it results in any of the following 
outcomes: 1) fatal or life threatening; 2) inpatient hospitalization or a prolongation of existing 
hospitalization; 3) a significant, persistent or permanent change, impairment, damage or disruption 
in the patient's body function/structure, physical activities and/or quality of life; 4) 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. 
 
An unexpected adverse event or unexpected suspected adverse reaction is an adverse event or 
suspected adverse reaction that is not listed in the investigator brochure or is not listed at the 
specificity or severity that has been observed or is not consistent with the risk information 
described in the general investigational plan or elsewhere in the IND application (if there is no 
investigator brochure). 
 
A summary of all Grade 3 or greater adverse events (not just those considered related to the 
vaccine) will be maintained.  Events will be categorized by organ system, relationship to treatment, 
grade or severity, and outcome.  The Protocol Chair will periodically review the adverse event 
summary with the intention of identifying any trends or patterns in toxicity.  If any such trends are 
identified, depending on their severity, frequency, and timing with respect to treatment, the 
Protocol Chair may request that the events be evaluated by a convened data safety monitoring 
board. 
 


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13.4 Adverse Event and Serious Adverse Event (SAE) Reporting 
All adverse events grade 2 or greater where there is evidence to suggest a causal relationship 
(possible, probable or definite) between the AE and investigational drug or study participation and 
all serious adverse events (regardless of causality) occurring at participating sites during the AE 
reporting periods require expedited reporting to the Coordinating Center.  
 
Adverse events meeting the definition of an SAE and any grade 2 or greater AEs where there is a 
possible, probable or definite relationship between the AE and investigational drug must be 
reported to the Coordinating Center within 24 hours of the investigator’s discovery.      Initial 
notification may be via e-mail.  A formal written initial report using UFBTIP Serious Adverse 
(SAE) Report must be sent to the Coordinating Center per the reporting timeframes outlined 
above.  SAEs and AE requirings expedited reporting must be entered into the OnCore eCRF as 
soon as possible but no later than 72 hours after discovery.    
   
All other AEs grade 3 or greater will be documented in the OnCore database for Protocol Chair 
review during the investigation. 
 
The Protocol Chair will promptly review expedited investigator reports and communicate the AE 
assessment to the reporting investigator and IND sponsors, as applicable.  If the AE meets FDA 
reporting requirements as described in 13.3.2, site PIs and IND sponsors will be notified and the 
IND sponsors will be responsible for reporting to FDA under the INDs regardless of which site 
initially reported the AE.   
 
If a death occurs within 30 days of DC vaccine administration, that is not attributable to progressive 
disease or other obvious non-study related cause (i.e. motor vehicle accident), enrollment of new 
subjects will be suspended until review by the Protocol Chair and IND Sponsor has been completed 
and the FDA notified.  If attribution of death is determined by the Protocol Chair, IND Sponsor or 
FDA to be possibly, probably or definitely related to trial drug, continued treatment of all enrolled 
subjects with trial drug will be suspended until the review is completed and recommendations for 
study continuation have been issued and approved by FDA.  Any death attributed to trial drug, 
regardless of the timeline with respect to last treatment, will result in suspension of enrollment and 
suspension of continued treatment for enrolled subjects until review by FDA is complete, and 
recommendations for study continuation issued and approved.  The IRB and DSMB will be 
notified of subject deaths per local policy.  Deaths or life-threatening adverse events will be 
reported to FDA by the IND sponsors as soon as possible but no later than 7 calendar days after 
the sponsor's initial receipt of the information (21 CFR 312.32).  
  
 
Grade 
Relation to IP 
Report to 
Coordinating 
Center 
Report to IRB and 
DSMB 
Report to FDA 
AE Grade 3 
No 
Enter data in 
OnCore 
Report per local 
policy 
No 
AE ≥ Grade 2  
Possible, 
probable or 
definite 
Report within 
24 hours via 
email  
Report per local 
policy 
IND sponsor will 
report to FDA if 


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meets definition 
below 
SAE 
All 
Report within 
24 hours via 
email 
Report per local 
policy 
IND Sponsor will 
report to FDA within 
15 calendar days if 
meets FDA 
requirement 
Fatal or life 
threatening 
All 
Report within 
24 hours by 
phone and via 
email 
Report per local 
policy 
IND Sponsor will 
report to FDA within 
7 calendar days if 
meets FDA 
requirement 
 
13.4.1 Adverse Event Reporting to IRB 
Adverse events and SAEs will be reported to the IRB of record per local IRB policy.    
 
13.4.2 Adverse Event Reporting to the DSMB 
Adverse events and SAEs will be reported to the DSMB of record per local policy.  The UFHCC 
DISC will be notified of serious adverse events within 5 business days of Coordinating Center 
notification. 
  
13.4.3 Adverse Event Reporting to the FDA 
Safety reporting will be in accordance with FDA requirements.  It is the responsibility of the IND 
sponsor to report to the FDA in accordance with 21 CFR 312.32 as follows: 
 
• Report any unexpected fatal or life-threatening suspected adverse reactions associated with use 
of the drug by telephone or fax as soon as possible but no later than 7 calendar days after 
initial receipt of the information.   
• Report any serious and unexpected suspected adverse reactions if there is evidence to suggest 
a causal relationship between the drug and the adverse event, as well as results from animal 
studies that suggest significant clinical risk within 15 calendar days after receipt of this 
information.   
 
A summary of Grade II (related) and Grade III toxicities, all IND safety reports and subject deaths 
during participation in the investigation will be submitted by the sponsor with the IND annual 
report. 
 
13.4.4 Adverse Event Reporting to Sites 
The Coordinating Center will notify site PIs of any adverse events associated with the use of the 
investigational vaccine that is both serious and unexpected within 15 calendar days of the 
notification of the event.  These reports should be submitted to the IRB of record in accordance 
with local policy.  Any changes to the protocol and/or consent will be initiated by the Coordinating 
Center and distributed to participating sites for review by the IRB.   
 


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13.5 Potential Risks 
13.5.1 Allergic Reactions to DC Immunization 
Injection of antigen presenting cells may result in an allergic reaction, which could include redness 
and swelling at the injection site, itching, hives, low blood pressure, difficulty breathing, or in the 
most extreme circumstances, death.  In addition, if the immune system becomes overly activated, 
potential discomforts may include pain, redness and swelling at the injection site.   
 
13.5.2 Cerebral Edema 
Cerebral edema may be secondary to the disease process itself, the surgical procedure, necrosis 
from previous radiation, or inflammation due to immune infiltration of the brain or destruction of 
tumor cells.  Symptoms may include, but are not limited to, severe headache, confusion, lethargy, 
unresponsiveness, coma, or focal neurological deficits.  Patients will be monitored throughout the 
course of the study and those patients with any signs or symptoms of cerebral edema may need 
their steroid doses increased, treatment with an osmotic diuretic, or surgical decompression.  
Edema that fails to respond to aggressive therapy may lead to permanent neurological impairment.  
The probability of this risk can be predicted to some degree based upon tumor size, location, pre-
operative neurological impairment, and post-operative course prior to DC injections. Patients will 
be monitored throughout the course of the study.  
 
13.5.3 Infection 
The DC or PBMC injections may include the risk of infection due to potential contamination in 
the laboratory.  This may result in localized redness, swelling, or induration at the injection site.  
In the most extreme situation, this may lead to systemic bacterial/fungal sepsis and possibly death.  
The probability of this risk is relatively low, given the small injection volume and the fact that the 
DCs and PBMCs will be strictly tested for sterility.  The risk of infection due to potential 
contamination of the DCs and PBMCs in the laboratory will be minimized by biosafety quality 
assurance and testing.  All cell cultures will be handled in a core tissue culture facility dedicated 
to the processing of human cells.  Following injections, patients will be monitored throughout the 
course of the study for any signs and symptoms of infection. If an active infection is suspected, 
patients will be cultured and treated with appropriate antibiotics.  
 
13.5.4 Delayed Autoimmune Diseases 
It is possible that delayed autoimmune disease(s) may develop as a result of injection with DCs or 
PBMCs.  This means that the immune system may be stimulated to attack natural tissue in the 
body.  Animal studies have reported the development of autoimmunity in the context of 
vaccination and recovery from lymphopenia.  However, our current experience with DC and 
PBMC vaccination in glioma patients has not demonstrated evidence of autoimmunity in treated 
patients.  It therefore, is unknown what the risk of delayed autoimmune disease is for this study.  
 
13.5.5 Phlebotomy 
Drawing blood or inserting an intravenous catheter into an arm vein may result in bruising or 
swelling in the area of the insertion, bleeding at the site of the needle puncture, light headedness, 
fainting and very rarely, local infection, which may be severe.  
 


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13.5.6 Leukapheresis 
As with any donation of blood, a variety of minor reactions may occur with leukapheresis, which 
include fainting, dizziness, or nausea.  Uncommon but serious complications may also result, 
which include bleeding, infection, an adverse reaction to the anticoagulant or replacement fluids, 
hypocalcemia, hypotension, shock, convulsions, air emboli, heart failure, or the inability to 
transfuse blood back into the patient.  These risks are reduced by the fact that the procedure will 
be performed by qualified staff at a specialized clinical hemapheresis unit.  Patients will be 
carefully monitored throughout the procedure by trained nursing and medical staff.  Calcium 
gluconate (2 mg PO) may be given to minimize the risks of hypocalcemia, fluid supplementation 
may be given to minimize hypotension, and blood will be routinely screened for infectious disease 
in accordance with institutional practices to minimize the risk of transmitting infection. 
 
13.5.7 Central Venous Catheter 
Some patient may require placement of a dialysis-type central venous catheter for the 
leukapheresis procedure. Separate operative/procedural consent will be obtained from the 
surgeon/proceduralist placing the central line. The surgeon/proceduralist may use fluoroscopy to 
place the central venous catheter and ensure that it is in a satisfactory position. Fluoroscopy 
involves low dose radiation and is low risk.  Risk of central line placement include (but not limited 
to): bruising, bleeding, injury to the vessel which may be permanent, possible injury to adjacent 
structures/organs, pneumothorax, infection and postoperative pain/discomfort. Removal of the 
central line is low risk but does include possible thrombosis or stenosis of the vessel. This may 
prevent future use of this vessel for additional leukapheresis.  
 
13.5.8 MRI 
The risks and/or discomforts associated with the performance of MRI include the anxiety produced 
from being in a tight, enclosed space (claustrophobia).  In addition, the machine operates using a 
large and powerful magnet.  The magnetism of the machine attracts certain metals: therefore, 
people with these metals in their bodies (specifically pacemakers, infusion pumps, metal aneurysm 
clips, metal prostheses, joints, rods or plates) will imaged by CT scan. Patients will also be checked 
to make sure that they do not bring any metal objects into the MRI facility.  Dental fillings are less 
affected by the magnetic fields generated and are therefore permitted.  It will be asked that patients 
let the physicians conducting this study know of any metal in their bodies other than dental fillings.   
 
13.5.9 GM-CSF 
Injection of GM-CSF may increase the risk of infection, lower platelets, or cause fluid retention.  
GM-CSF also may result in an allergic reaction, which could include redness and swelling at the 
injection site, itching, hives, flushing, syncope, low blood pressure, difficulty breathing, or in the 
most extreme circumstances, death.  In addition, if the immune system becomes overly activated, 
potential discomforts may include pain, redness and swelling at the injection site. 
 
13.5.10 Td 
The following adverse events have been identified during post-approval use of MassBiologics’ 
Td.  Because these reactions are reported voluntarily from a population of uncertain size, it is not 
always possible to reliably estimate their frequencies or to establish a causal relationship to 
vaccination.  The following adverse events were included because of seriousness or frequency of 
reporting: injection site reactions, including pain, tenderness, erythema, induration, pruritis, 


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swelling and warmth; peripheral edema, pyrexia, malaise; dizziness, headache, convulsions, 
myalgia, musculoskeletal stiffness or pain, arthralgia; rash; nausea; and cellulitis. 
 
13.5.11 Allergic Reactions to Contrast Agents 
During the MRI, patients will be given a contrast agent.  The agent is given routinely to obtain 
enhanced MRI scans of the brain.  The agent is administered through the vein and requires the 
placement of an IV catheter.  The catheter placement is similar to drawing blood except that the 
catheter remains in the vein during the time the agent is actively delivered.  The risks of a blood 
draw and insertion of a catheter are similar.  There have been a few, rare cases of allergies to the 
agent used in MRI contrast enhanced scans. Patients with any known severe allergies to contrast 
agents will be excluded from the study.  Patients with mild allergies (i.e., rash only) will be 
pretreated with Tylenol and Benadryl prior to injection of the contrast agent. 
 
13.5.12 Temozolomide 
TMZ has been well tolerated by both adults and children with the most common toxicity being 
mild myelosuppression.  Other, less likely, potential toxicities include nausea and vomiting, 
constipation, headache, alopecia, rash, burning sensation of skin, esophagitis, pain, diarrhea, 
lethargy, hepatotoxicity, anorexia, fatigue and hyperglycemia.  Hypersensitivity reactions have not 
yet been noted with TMZ.  As in the case with many anti-cancer drugs, TMZ may be carcinogenic.  
Rats given TMZ have developed breast cancer.  The significance of this finding for human is not 
presently known. TMZ therapy will be followed but given as standard of care. If toxicities occur, 
the Principal Investigator and primary physician will titrate therapy based on standard clinical 
guidelines. 
 
13.5.13 Confidentiality 
Participation in research may result in a loss of privacy if confidentiality is breached.  Only 
designated members of the study team will have access to individually identifiable private 
information.  Data collected from clinical records or patients will be recorded in OnCORE that 
uses Gatorlink authentication.  Clinical records containing PHI will be maintained as source 
documentation and stored in secure, limited access, locked offices.  Records will be made 
available to individuals involved with the study, the clinical staff administering the study, and 
regulatory representatives such as the IRB, FDA, OHRP and funding agency. Any publications 
resulting from this study will not include patient identifying data. 
 
 QUALITY CONTROL AND QUALITY ASSURANCE 
 
14.1 Data and Safety Monitoring Plan 
14.1.1 Data Monitoring Plan 
Adverse events meeting the definition of an SAE and any grade 2 or greater AEs where there is a 
possible, probable or definite relationship between the AE and investigational drug must be 
reported to the Coordinating Center within 24 hours of the investigator’s discovery.  These will be 
assessed by the Protocol Chair and reported to the IRB, DSMB, FDA and other participating 
investigators as outlined in the protocol. 
 


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The UFHCC SRMC conducts initial review of IITs and determines the level of risk which 
corresponds with DISC review requirements.  The UFHCC DISC will conduct monitoring and 
audit activities as outlined in the DISC Charter.   The results of such activities will be provided to 
the PI, IND sponsor and IRB. 
 
The UF study team will conduct annual QA self-assessments that include review of regulatory 
documentation (e.g., IRB documentation, FDA IND records, IND Sponsor-Investigator 
responsibilities), recruitment procedures, informed consent process/form documentation, subject 
selection, AE and SAE reporting, CRF and source documents. 
 
The Coordinating Center will periodically review data entry and source documents uploaded into 
OnCore or other electronic source, and notify sites of missing, incomplete or inconsistent data as 
applicable. 
 
Orlando Health Cancer Institute site data will be monitored by the coordinating center lead 
coordinator and regulatory manager by reviewing source documents uploaded in the study 
database (UF OnCore). 
 
Duke will manufacture their own IP under their own IND and will perform site-specific monitoring 
that will be reported to the Coordinating Center/Protocol Chair as described in the protocol. 
 
14.1.2 Data Safety Monitoring Board 
Oversight and monitoring of study related activities such as the safety of research participants, 
appropriateness of the study, and integrity of the data, will be provided by the University of Florida 
Health Cancer Center DSMB, now called the Data Integrity and Safety Committee (UFHCC 
DISC), in accordance with established policies, procedures and timeframes.  DISC reports and 
correspondence will be provided to participating investigators for review and submission to local 
oversight committees as required.. All reports generated by Duke pertaining to study conduct, will 
be sent to the University of Florida PI and Protocol Chair.  DSMB reports should be submitted to 
the IRB per local policy. 
 
14.2 Audits 
An IRB or other local oversight group may conduct audits to evaluate compliance with the protocol 
and the principles of GCP. The PI will provide the auditor(s) with direct access to all relevant 
documents and to allocate his/her time and the time of the study team to the auditor(s) in order to 
discuss findings and any relevant issues. 
 
Audits are designed to protect the rights and well-being of human research subjects. 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. 
 


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Audits examine research studies/clinical trials methodology, processes and systems to assess 
whether the research is conducted according to the protocol approved by the 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. 
 
Duke University and the University of Florida have an auditing mechanism in place and each 
institution will be responsible for auditing their respective data.  Additionally, Duke University 
will send copies of all audit findings to the University of Florida who, based on these findings, can 
request additional documentation or conduct an on-site visit. 
 
14.3 Data Management and Processing 
14.3.1 Study Documentation 
Study documentation includes but is not limited to source documents, case report forms, 
screening/enrollment, delegation of authority and monitoring logs (as applicable), appointment 
schedules, correspondence with sponsors or regulatory bodies/committees, and regulatory 
documents that can be found in the “Regulatory Binder” (electronic or hard copy), which includes 
but is not limited to signed protocol, amendments, and investigator’s brochure (as applicable),  
approved and signed informed consent forms, IRB submission documents, approvals and 
correspondence, CVs for investigators and sub-investigators, FDA Form 1572, financial disclosure 
forms, laboratory certifications and reference ranges, 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 Site Activation 
A site is considered activated when the site has received official written documentation from the 
Protocol Coordinating Center that the site has been approved to begin enrollment.  At a minimum, 
each participating clinical site must have the following documentation on file at UF prior to study 
activation: 
• IRB approval of the study in the form of a letter or other official document from the 
participating institution’s IRB of record.  This documentation must show the version 
of the protocol approved by the IRB. 
• IRB approval of an informed consent form. The consent must include a statement that 
data will be shared with UF, including the UFHCC, the DSMB (if applicable), and the 


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UF study team. 
• Protocol signature page signed and dated by the investigator at each participating site. 
 
The applicable IND sponsor is responsible for obtaining and maintaining signed and dated FDA 
Form 1572, and the CVs, applicable licenses and FDFs of all investigators listed on the 1572. 
 
The Coordinating Center is responsible for disseminating study updates, protocol and/or 
investigator brochure amendments, reportable adverse events, and informed consent modifications 
to all participating sites.  Protocol/consent modifications and IB updates will be forwarded 
electronically to participating sites within 4 weeks of UF IRB approval.  Activated participating 
sites are expected to submit protocol/consent/IB modifications to their local IRBs within 4 weeks 
of receipt unless otherwise noted.  Documentation of IRB approval should be provided to the 
Coordinating Center within 2 weeks of receipt of approval.  The Coordinating Center should 
review all revised consent forms prior to submission to the IRB. 
 
Documentation of participating sites’ IRB approval of annual continuing reviews, protocol 
amendments or revisions, SAE reports, and any protocol violations/deviations/exceptions must be 
on file at UF.  
 
The investigator or a designee from each institution must participate in conference calls, as 
scheduled, to update and provide information regarding the progress of the trial. 
 
14.3.3 Case Report Forms 
Electronic case report forms (eCRFs) will be utilized for this trial.  Only the PI and key study 
personnel listed on the study delegation log are permitted to make entries, changes, or corrections 
in the case report forms.  Source documents that support data entry should be available for 
monitoring or audit.  The Coordinating Center will periodically review data entry and notify sites 
of missing, incomplete or inconsistent data. 
 
14.3.4 Data Management Procedures and Data Verification 
Users of the electronic CRF will have access based on their delegated specific roles on the study.  
Completeness of entered data will be checked and cross-referenced to verify accuracy. Missing or 
implausible data will be highlighted for the PI requiring appropriate responses (i.e. confirmation 
of data, correction of data, completion or confirmation that data is not available, etc.). 
 
The database will be reviewed and discussed prior to database closure, and will be closed only 
after resolution of all remaining queries. An audit trail will be kept of all subsequent changes to 
the data. 
 
 STATISTICAL METHODS AND DATA ANALYSIS 
 
15.1 Study Design Overview 
The primary goal of this randomized phase II study is to determine whether the addition of pp65-
LAMP mRNA DC vaccination in adult patients with newly-diagnosed GBM receiving dose-
intensified TMZ will be a treatment worthy of investigation in a large phase III study.  A secondary 


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objective will be examination of the immunologic responses induced by full-length vs short LAMP 
sequences.  
 
Although this randomized phase II study is comparative, it will not be definitive as the assessment 
will be conducted with a large false-positive rate.  We have included a concurrently followed 
control arm in the trial design in lieu of a historical control group to ensure that appropriate 
comparisons can be made without concern that the patients receiving standard treatment differ in 
prognostic factors or standards of care (Rubinstein, 2009; Ratain and Sargent, 2009; Rubinstein et 
al., 2005). 
 
We will randomize eligible  patients, after enrollment, to one of 3 study arms in which they will 
receive 21-day TMZ concurrently with one of the following treatments: 
 
Arm # 
Vaccine 
Skin Preparation 
1 
pp65-shLAMP mRNA DCs with GM-CSF  
Tetanus 
2 
pp65-flLAMP mRNA DCs with GM-CSF 
Tetanus 
3 
PBMCs  
Saline 
 
Randomization will be stratified by: 
- 
Age 69 years old and younger; or 70 years old and older  
- 
RPA Class (III, IV, or V) 
- 
CMV seronegative or seropositive 
 
We will use a permuted block randomization scheme with a 1:1:1 study arm allocation ratio.and a 
block size of 6 within each stratum.   
 
 
Adapted RPA Stages For Malignant Gliomas (Survival With Concurrent Temozolomide 
and Radiation Therapy vs Radiation Therapy alone) 
Stage 
Characteristics 
Median OS 
2-year OS 
p-value vs. 
control 
III Age <50, PS 0 
21 vs. 15 mo 
43% vs. 
20% 
p<0.0001 
IV Age <50, PS 1-2 
Age >=50, Surgery, MMSE >=27 
16 vs. 13 mo 
28% vs. 
11% 
p<0.01 
V 
Age >=50 and either Biopsy only or 
MMSE <27 
10 vs. 9 mo 
17% vs. 6% 
p=0.05 
 
Approximately 200 patients will be enrolled in the study, randomized and receive “standard-of-
care” radiotherapy concurrent with TMZ under the assumption that approximately 120 patients 
will be vaccinated according to treatment arm. 
 
A Data Sharing Agreement has been established with Immunomic Therapeutics, Inc. and the 
University of Florida.  A Limited Data Set will be sent to evaluate immunogenicity and clinical 
efficacy of the vaccine products used within this clinical trial (for patients treated at the UF site 


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only) in which Immunomic Therapeutics, Inc. has scientific and potential commercial interest. 
Data will be provided within 6 months of study completion.  Applicable patient identifiers will be 
removed before sending the limited data set.  A separate data sharing agreement may be established 
by Immunomic Therapeutics, Inc. with Duke University. 
  
15.2 Sample Size Justification for Vaccine Comparison 
The primary focus of this trial will be to assess the worthiness of pp65-LAMP mRNA DC vaccine 
for further investigation by comparing overall survival (OS) between the active treatment group 
(Arms 1 and 2 combined) and the control group (Arm 3).  Because this test for efficacy will be 
made within the framework of a phase II trial, we have constrained sample size requirements to 
yield adequate power to detect a clinically relevant improvement in OS at the expense of the false 
positive rate (α-level or Type I error rate).  Adopting guidelines proposed by Ratain and Sargent 
(2009) and Rubinstein et al (2005), our proposed sample size of 40 + 40 = 80 patients in the 
treatment group and 40 patients in the control group will allow detection of a clinically relevant 
improvement in the median OS time of the treatment group with 90% power at a one-sided α-level 
of 0.2. 
 
In determining the effect size that can be detected by our proposed sample size, we considered 
using as a control reference level the median OS estimate of 14.6 months (95% CI: 13.2 to 16.8) 
reported by Stupp et al (2005) for patients receiving radiotherapy plus TMZ (RT+TMZ) for newly-
diagnosed GBM (40% of patients in the Stupp trial were grossly resected).  In the Stupp trial, 
randomization occurred prior to any post-surgical treatment, and the timing of randomization in 
our study will be similar. However, in our study, patients will not begin to experience potential 
differential effects due to treatment until after their first vaccination, which will occur 
approximately 15 weeks after randomization. To determine an appropriate control reference level 
for median OS for an index day 15 weeks after randomization, we carefully transferred the 
coordinates of multiple points along the Stupp RT+TMZ OS curve into a statistical software 
package. We determined that median OS relative to a starting time 15 weeks after the Stupp index 
day would be 13.8 months.  We also observed that RT+TMZ OS times appeared to approximately 
follow a Weibull distribution with a shape parameter = 1.20, indicating a slightly accelerating 
baseline hazard function.  Upon examining in a similar manner the RT+TMZ OS curve reported 
in the paper by Gilbert et al (2013), we also observed approximately Weibull-distributed OS times 
with shape parameter = 1.16. 
 
We therefore used 13.8 months as the median OS reference level for our control group and 
assumed Weibull-distributed OS times with a common shape parameter value for both the 
treatment and control groups.  This is equivalent to assuming Weibull-distributed OS times with 
proportional hazards between the treatment and control groups. 
 
Using methods adapted from Wu and Xiong (2014), we calculated improvement in median OS (or 
equivalently, the largest treatment vs. control hazard ratio less than 1) that could be detected by a 
log rank test with 90% power at 1-sided α=0.20, for selected Weibull shape parameter values 
ranging from 1.00 (equivalent to exponential survival with constant hazard) to 1.25 (accelerating 
hazard).  We assumed 120 patients would be randomized and proceed to vaccination at a uniform 
rate over a 3-year accrual period. These patients would then be followed after the end of accrual 
for one additional year.  As summarized in the following table, 77 deaths (expected by follow-up 


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times ranging from 45.1 to 48.9 months, depending on the degree of hazard acceleration assumed) 
will allow a treatment vs. control hazard ratio of 0.6 to be detected with sufficient power.  
Equivalently, improvement in median OS ranging from 7.0 to 9.2 months (again, depending on 
the degree of hazard acceleration assumed) can be detected with sufficient power. 
 
Improvement in median overall survival (Surv50) that can be detected at 90% power and a 
one-sided alpha-level of 0.20, for possible Weibull shape parameters characterizing the 
degree of hazard acceleration: 
                                                                
 
 
 
 
 
 
 
 
 
 
Detectable effect 
                                                                 at 90% power, 
                                                  Expected #   1-tail alpha=0.20 
        Weibull                                   of months    ----------------- 
TRT:     Shape    Baseline hazard                 to observe    Surv50   Hazard 
CTL N  parameter  function trend                  77 deaths    (months)   ratio 
-------------------------------------------------------------------------------- 
80:40    1.00     Constant (exponential survival)    48.9        + 9.2     0.60 
80:40    1.16     Accelerating (Gilbert et al)       46.3        + 7.6     0.60 
80:40    1.20     Accelerating (Stupp et al)         45.7        + 7.3     0.60 
80:40    1.25     Accelerating                       45.1        + 7.0     0.60 
                                                               
 
 
 
 
 
 
 
 
 
 
 
15.3 Analytic Methods  
15.3.1 Primary Objective and Secondary Clinical Objectives 
We will use Kaplan-Meier survival curves and the log rank test to characterize and compare PFS 
and OS in patients who received pp65-LAMP mRNA DC vaccine (Arms 1 and 2) and in control 
patients (Arm 3).  PFS is defined as the time between first vaccination and first documentation of 
either disease progression/recurrence, or death without prior progression/recurrence.  Patients 
remaining alive without disease progression will have PFS censored at last follow-up.  OS will be 
defined as the time between first vaccination and death, and will be censored at the last follow-up 
if death has not occurred.  We will also perform a stratified analysis incorporating the three 
stratification variables (age, RPA class, and CMV sero-status) using Cox proportional hazards 
regression. The index day for all of our primary comparisons will be the time of the first 
vaccination. Because patients who fail to proceed to the first vaccination because of GBM 
recurrence or for other reasons cannot experience differential effects due to treatment arm 
membership, we will not perform an intent-to-treat analysis that includes these patients. We will 
perform an intent-to-treat analysis of OS and PFS that includes patients who begin the vaccination 
sequence but who do not receive at least three vaccinations because of recurrence or other reasons. 
OS and PFS times for adequately vaccinated patients who are lost to follow-up will be considered 
right-censored at the time of loss and will be included in all of our survival comparisons. 
 
All subjects who receive at least one dose of DC vaccine will be considered evaluable for purposes 
of safety and toxicity. 
 
All subjects who complete chemoRT and receive dose-intense adjuvant TMZ and at least 3 DC 
vaccines will be considered evaluable for purposes of the primary and secondary endpoints.  
 


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The impact of pp65-LAMP mRNA DC vaccine on PFS and OS in patients with gross total 
resection will also be explored using the log rank test and Kaplan-Meier curves, although our trial 
design may not necessarily achieve sufficient power to detect a clinically meaningful effect within 
this patient subgroup. 
 
15.3.2 Secondary Biological Objectives  
We will estimate within-patient changes in immune response (ELISPOT, cytokines array, flow 
cytometric analysis and NK cell activity) from baseline to vaccine #3 and compare these changes 
between treatment and control groups using the Wilcoxon rank sum test. To assess the effect of 
these changes in immune response on OS and PFS subsequent to vaccine #3, we will conduct a 
landmark analysis within the framework of Cox proportional hazards models of post-baseline OS 
and PFS. These models will incorporate Indicators for treatment arm, change in immune response 
parameters from baseline to vaccine #3, interaction between treatment arm and immune response 
changes, and potential confounders.   
 
 REGULATORY REQUIREMENTS AND ETHICAL 
CONSIDERATIONS 
 
The study will be conducted in accordance with the protocol, FDA Regulations 21 CFR parts 50, 
54, 56 and 312, HHS Regulations 45 CFR part 46, the International Conference on 
Harmonization guidelines for Good Clinical Practice (ICH E6), and applicable institutional, local 
and state requirements. All personnel involved in the conduct of this study have completed 
human subjects protection training in accordance with local institutional requirements. 
 
16.1 Institutional Review Board 
Each site will obtain Institutional Review Board (IRB) approval of the protocol and protocol 
related documents including consent forms, prior to initiating the study.  The study may be initiated 
only after the Principal Investigator has received written and dated approval from the IRB and 
notification of activation from the Coordinating Center. Amendments to the protocol must 
originate with the Protocol Chair and be approved by the IRB prior to implementation.  The FDA 
will be notified as required under 21 CFR 312.30. 
 
The Principal Investigator must submit and obtain approval from the IRB for all subsequent 
protocol amendments and changes to the informed consent form in accordance with 21 CFR 56 
and local policy prior to implementation except where necessary to eliminate apparent immediate 
hazard to the subject.  If a change is made to eliminate an apparent immediate hazard, the PI must 
notify the IRB in accordance with local policy, the Protocol Chair and the sponsor within 24 hours.  
The IND sponsor will notify the FDA of such change in accordance with 21 CFR 312.30.   Each 
investigator will follow the requirements of their local IRB for periodic reporting of study progress, 
reporting of serious adverse events, unanticipated problems, and protocol deviations or violations, 
safety monitoring reports and study completion.  Protocol exceptions must have approval from the 
Protocol Chair, FDA (as applicable through the sponsor) and IRB prior to implementation. 
 
The Principal Investigator must obtain protocol re-approval as required by the IRB, but not less 
than once per year.   
 


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16.2 Informed Consent 
Informed consent will be obtained and documented in accordance with 21 CFR 50 and the 
requirements of the IRB.  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.  For potential subjects whose primary language is not English, investigators 
should follow the requirements and determinations of the reviewing IRB and any applicable 
institutional procedures. 
 
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 or a legally authorized representative. The original informed 
consent form will be maintained 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. 
 
16.3 Privacy, Confidentiality, and Data Storage 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s data 
will be maintained.   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. After agreeing to participate, some patient 
identifiers will be collected, used and/or recorded, including, but not limited to name, date of 
birth, telephone number, address, dates from health records and a unique patient number. This 
information may be kept in patient binders as well as recorded in the respective eCRF. Images 
collected will be as de-identified as possible. Only approved study personnel and those included 
in the HIPAA Authorization will have access to identified, partially identified, and/or coded data. 
Data and specimens will be collected, stored and transported with consideration for subject 
privacy and confidentiality. Electronic records of subject data will be maintained in a password-
protected computer.  Access to electronic databases will be limited to the Principal Investigator 
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Upon completion of the study, research records will be archived and handled per local policy.  
 
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or 
publications in scientific journals. 
 
16.4 Protocol Amendments 
All protocol amendments (including consent form changes) must be initiated by the Protocol Chair 
and approved by the IRB prior to implementation.  The FDA will be notified as required under 21 
CFR 312.30.  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, the 
Protocol Chair and the sponsor as described in 16.1. 
 
16.5 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; or 
▪ in compliance with local record retention policies. 
 
16.6 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.  Investigators and 
key personnel must provide financial disclosures prior to trial participation and as interests change 
and complete any applicable institutional conflict of interest documentation per local policy. 


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 APPENDICES 
 
18.1 Appendix A: Radiation Therapy 
The total targeted RT dose will be at the discretion of the treating Radiation Oncologist.  Optimally, 
one treatment of 1.8-2.0 Gy/fraction will be given daily 5 days per week for a total targeted dose 
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 will 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 will be delayed or interrupted if the platelet count is < 20,000. Radiation will 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 
5.0. 
 
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. 
 
 
 


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18.2 Appendix B: Temozolomide Therapy during Radiation 
TMZ should be administered continuously from day 1 of radiotherapy to the last day of radiation 
at a daily oral dose of 75 mg/m2 for a maximum of 49 days. The drug will be administered orally 
1 hour before each session of radiotherapy during weekdays (Monday through Friday).  During 
weekends without radiotherapy (Saturday and Sunday), the drug will be taken in the morning.   
The dose will be determined using the body surface area (BSA) calculated at the beginning of the 
concomitant treatment. The BSA will be calculated from the height obtained at the pretreatment 
visit and the weight obtained at the visit immediately before the first day of treatment. Capsules of 
TMZ are available in 5, 20, 100, 140, 180 and 250 mg. The daily dose will be rounded to the 
nearest 5 mg.  Patients will be instructed to swallow the capsules whole, in rapid succession, 
without chewing them. If vomiting occurs during the course of treatment, no re-dosing of the 
patient is allowed before the next scheduled dose. The capsules should be taken on an empty 
stomach, therefore a minimum of 2 hours after a meal and with no food ingestion for 1 hour after 
TMZ administration.  Antiemetic prophylaxis is usually not required for the continuous daily 
dosing schedule. However, prophylaxis with a 5-HT3 antagonist is recommended prior to 
administration of the first few TMZ doses and should be administered orally 30 to 60 minutes 
before TMZ treatment. 
 
No dose reduction should be made, but delay or discontinuation of TMZ administration should be 
decided weekly according to hematologic and non-hematologic AEs.  If one or more of the 
following are observed:  
• ANC < 1.0 x 109/ L  
• Platelet count < 75 x 109/L  
• Grade 3 non-hematologic AE (except for alopecia, nausea and vomiting if controlled by 
maximal antiemetic therapy, and fatigue if controlled by pharmacologic or non-
pharmacologic therapy) 
then treatment with concomitant TMZ will be withheld until all of the following conditions are 
met: 
• ANC ≥ 1.0 x 109/L  
• Platelet count ≥ 75 x 109/L  
• Grade ≤ 1 non-hematologic AE(except for alopecia, nausea and vomiting if controlled by 
maximal antiemetic therapy, and fatigue if controlled by pharmacologic or non-
pharmacologic therapy) 
 
In case of hematologic AE as defined above, a CBC should be performed at least twice weekly. In 
case of non-hematologic AE, the patient should be assessed at least weekly with relevant 
laboratory test(s). As soon as all of the above conditions are met, the administration of TMZ will 
resume at the same dose as used initially.  
If one or more of the following are observed:  
• ANC < 0.5 x 109/L (Grade 4)  
• Platelet count < 10 x 109/L (Grade 4)  
• Grade 3 or 4 non-hematologic AE (except for alopecia, nausea and vomiting if controlled 
by maximal antiemetic therapy, and fatigue if controlled by pharmacologic or non-
pharmacologic therapy) 
then treatment with concomitant TMZ should be stopped. 
 


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If the duration of radiotherapy exceeds 7 weeks, then concomitant treatment with TMZ should be 
stopped after 49 days of TMZ treatment. If the administration of TMZ has to be interrupted, the 
radiotherapy will proceed normally. Missed doses of TMZ will not be made up at the end of 
radiotherapy. The total number of days and total dose of TMZ will be recorded. 
 
Table 1:  Summary of TMZ Delay or Discontinuation during Concomitant Radiation 
Therapy AE 
 
 
Value 
Grade 
Action 
ANC  
≥ 0.5 and < 1.0 x 109/L  
2, 3 
Delay TMZ until:  
• ANC > 1.0 x 109/L  
• Plt > 75 x 109/L  
• Non-hem AE ≤ 1  
Platelet count  
≥ 10 and < 75 x 109/L  
2, 3 
Non-hematologic (except for 
alopecia, nausea/vomiting if 
controlled by maximal 
antiemetic therapy)  
NA  
3 
ANC  
< 0.5 x 109/L  
4 
Stop concomitant TMZ  
Platelet count  
< 10 x 109/L  
4 
Non-hematologic (except for 
alopecia, nausea/vomiting if 
controlled by maximal 
antiemetic therapy)  
NA  
4 
 
 
 


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18.3 Appendix C: Temozolomide Therapy after Radiation 
TMZ will be administered orally once per day for 21 consecutive days (days 1-21) of a 35 (+/- 7) 
day cycle. The starting dose for the first cycle will be 75 mg/m2/day, with a single dose escalation 
to 100 mg/m2/day in subsequent cycles if no adverse events > grade 2 are noted.  
 
Twelve cycles of TMZ may be given if the patient demonstrates continued improvement on MR 
scan, decreased corticosteroid requirement, improvement in performance status, or improvement 
in neurologic function. 
 
Dosing Modifications 
Dosing is based on adverse events (AEs) during the prior treatment cycle. If multiple AEs are seen, 
the dose administered should be based on the dose reduction required for the most severe grade of 
any single AE as shown in Table 2. 
 
Table 2:  Dosing Modification Schedule for TMZ per RTOG 0525 Regimen 
 
Dose Level 
Dose, mg/m2/day 
x 21 days 
Remarks  
-2 
35 
Reduction if prior AE  
-1 
50 
Reduction if prior AE  
0 
75 
Starting dose for cycle 1, increase to 100 mg/m2 for 
cycle 2 and beyond if no hematologic toxicity ≥ grade 2 
and non-hematologic toxicity > grade 2 
+1 
100 
Highest possible dose level (adjuvant) 
 
First cycle  
TMZ will be started at a dose of 75 mg/m2/day.  
 
Second cycle  
The dose of TMZ will be determined according to (1) non-hematologic AE during the preceding 
treatment cycle, as well as (2) the nadir (lowest/worst) ANC and platelet counts.  
 
Cycles 3-12 
Any dose reductions of TMZ will be determined according to (1) non-hematologic AE during the 
preceding cycle, as well as (2) the nadir (lowest/worst) ANC and platelet counts.  No dose 
escalation should be attempted. The same dose reductions as for the second cycle should be 
applied.  If the dose was reduced or delayed for adverse events, there will be no dose escalation.  
 
Delay  
On day 1 of each cycle (within the prior 72 hours), ANC ≥ 1 x 10/L, platelet count ≥ 100 x 10/L 
and all grade 3 or 4 non-hematologic AEs (except for alopecia, nausea, and vomiting if controlled 
by maximal antiemetic therapy) must have resolved (to grade ≤ 1).  
 
If AEs persists, treatment should be delayed by 1 week for up to 4 consecutive weeks. If, after 4 
weeks of delay, all AEs have still not resolved: then any further adjuvant treatment with TMZ 
should be stopped. 


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Dose escalation  
If, during the first cycle, all non-hematologic AEs observed were grade ≤ 2 (except for alopecia, 
nausea and vomiting if controlled by maximal antiemetic therapy) and with platelets ≥ 100 x 109/L 
and ANC ≥ 1 x109/L: then the TMZ dose should be escalated to dose level +1 and this dose should 
be used as the maintenance dose for subsequent cycles. If treatment after cycle 1 has to be delayed 
by any length of time because of ongoing hematologic AEs >2 and/or non-hematologic AEs of 
grade > 2 (except for alopecia, nausea and vomiting if controlled by maximal antiemetic therapy), 
then no escalation is possible.  If the dose was not escalated at cycle 2, then the dose should not be 
escalated in further cycles (3-12).  
 
Dose reductions  
If any non-hematologic AE observed was grade > 2 (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) and/or if platelets < 100 x 109/L and/or ANC < 1 x 
109/L, then the dose should be reduced by one dose level.  For patients who would require dose 
reductions to a dose level < 35 mg/m2/day (dose level -2), TMZ will be stopped.  Also, if any of 
the same non-hematologic grade 3 AE recurs (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) after reduction for that AE, then TMZ will be stopped.  
If any treatment-related non-hematologic AE observed was grade 4 (except for alopecia, nausea 
and vomiting if controlled by maximal antiemetic therapy) then adjuvant TMZ treatment should 
be stopped.  Please see Tables 3-6 for a summary of dose modification or discontinuation.  
 
The reason(s) for dose reduction and/or delay must be documented in the CRF. 


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Table 3: Summary of Dose Modification or Discontinuation During Post-Radiation 
Temozolomide Worst Non-Hematologic AE (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) During the Previous Cycles 
 
Grade  Dose Modification  
0-2  
No dose modifications for non-hematologic AEs. Dose escalations (only for cycle 
2) or reductions based on ANC and platelet counts are applicable.  
3  
Reduce by one dose level (except for alopecia, nausea and vomiting if controlled 
by maximal antiemetic therapy). Dose modifications (escalations or reductions) 
based on ANC and platelet counts are not applicable. No further escalation is 
possible. If the same non-hematologic grade 3 AE recurs (except for alopecia, 
nausea and vomiting if controlled by maximal antiemetic therapy) after reduction 
for that AE, then stop.  
4  
Stop (except for alopecia, nausea and vomiting if controlled by maximal antiemetic 
therapy). Dose modifications (escalations or reductions) based on ANC and 
platelet counts are not applicable.  
 
Table 4:  Worst Treatment-Related Hematologic AE during the Previous Cycles 
 
Worst AE 
 
Platelets 
≥100 x 109/L   
< 100 x 109/L   
 
ANC 
 
≥ 1.5 x 109/L   
Dose unchanged  
Reduce by 1 dose level 
≥1 & <1.5 x 109/L   
Dose unchanged  
Reduce by 1 dose level 
< 1 x 109/L   
Reduce by 1 dose level  Reduce by 1 dose level 
 
Note: A complete blood count will be performed prior to each vaccine and if clinically indicated 
on days 7, 14, 21 and 28 (+/- 72 hours) after the first daily dose of each adjuvant treatment cycle. 
 
Table 5:  Hematological AE on Day 1 of Each Cycle (within the prior 72 hours before Day 1 
 
AE  
Delay  
ANC< 1 x 109/L and/or 
Platelet count < 100 x 109/L  
Delay up to 4 weeks until all resolved. If 
unresolved after 4 weeks then stop. If 
resolved, dose delay/reductions based on 
hematologic and non-hematologic AEs are 
applicable.  
 
Table 6:  Non-Hematologic AE (except for alopecia, nausea and vomiting if controlled by 
maximal antiemetic therapy) on the day 1 of Each Cycle (within the prior 72 hours) 
 
Grade  
Delay  
2-3  
Delay up to 4 weeks until all resolved (to grade ≤ 1).  If 
unresolved after 4 weeks then stop. If resolved, dose 
delay/reductions based on hematologic and non-
hematologic AEs are applicable.  
4 
Stop (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy). 


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18.4 Appendix D: Alternative Temozolomide Therapy after 
Radiation 
 
If dose-intensified TMZ is delayed for more than 4 weeks or stopped due to any hematologic or 
non-hematologic AEs that were not grade 4, and less than 6 cycles of TMZ have been achieved, 
once the AEs have resolved to ≤ grade 1 but not more than 8 weeks after the previous dose-
intensified TMZ cycle, TMZ can be restarted at the standard Stupp protocol dosing schedule at 
150mg/m2 orally daily for 5 days on a 28+/-14 days cycle to achieve a maximal total of 12 cycles 
including the previous dose-intensified TMZ cycles.  No dose escalation will be allowed, but 1 
dose reduction to 100mg/m2 daily x 5 days on a 28 (+/-14) day cycle will be allowed. 
 
Table 7:  Dosing Modification Schedule for Maintenance TMZ per Stupp Regimen 
 
Dose Level 
Dose, mg/m2/day 
x 5 days 
Remarks  
-1 
100 
Reduction if prior AEs  
0 
150 
Starting dose for cycle 1 
 
Delay  
On day 1 of each cycle (within the prior 72 hours), ANC ≥ 1 x 10/L, platelet count ≥ 100 x 10/L 
and all grade 3 or 4 non-hematologic AEs (except for alopecia, nausea, and vomiting if controlled 
by maximal antiemetic therapy) must have resolved (to grade ≤ 1).  
 
If AEs persists, treatment should be delayed by 1 week for up to 4 consecutive weeks. If, after 4 
weeks of delay, all AEs have still not resolved: then any further adjuvant treatment with TMZ 
should be stopped. 
 
Dose escalation: No dose escalation is allowed  
 
Dose reductions  
If any non-hematologic AE observed was grade > 2 (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) and/or if platelets < 100 x 109/L and/or ANC < 1 x 
109/L, then the dose should be reduced by one dose level. For patients who would require dose 
reductions to a dose level < 100 mg/m2/day (dose level -1), TMZ will be stopped.  Also, if any of 
the same non-hematologic grade 3 AE recurs (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) after reduction for that AE, then TMZ will be stopped. 
If any treatment-related non-hematologic AE observed was grade 4 (except for alopecia, nausea 
and vomiting if controlled by maximal antiemetic therapy) then adjuvant TMZ treatment should 
be stopped.  Please see Tables 8-11 for a summary of dose modification or discontinuation.  
 
The reason(s) for dose reduction and/or delay must be documented in the CRF. 
 
 
Table 8: Summary of Dose Modification or Discontinuation during Post-Radiation 
alternative (Stupp) Temozolomide Worst Non-Hematologic AE (except for alopecia, nausea 
and vomiting if controlled by maximal antiemetic therapy) During the Previous Cycles 


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Grade  Dose Modification  
0-2 
No dose modifications for non-hematologic AEs. Dose reductions based on ANC 
and platelet counts are applicable.  
3 
Reduce by one dose level (except for alopecia, nausea and vomiting if controlled 
by maximal antiemetic therapy. Dose reduction based on ANC and platelet counts 
is not applicable. If the same non-hematologic grade 3 AE recurs (except for 
alopecia, nausea and vomiting if controlled by maximal antiemetic therapy) after 
reduction for that AE, then stop.  
4 
Stop (except for alopecia, nausea and vomiting if controlled by maximal antiemetic 
therapy. Dose reduction based on ANC and platelet counts is not applicable.  
 
Table 9:  Worst Treatment-Related Hematologic AE during the Previous Cycles 
 
Worst AE 
 
Platelets 
≥100 x 109/L   
< 100 x 109/L   
 
ANC 
 
≥ 1.5 x 109/L   
Dose unchanged  
Reduce by 1 dose level 
≥1 & <1.5 x 109/L   
Dose unchanged  
Reduce by 1 dose level 
< 1 x 109/L   
Reduce by 1 dose level  Reduce by 1 dose level 
 
 
Note: A complete blood count will be performed prior to each vaccine and if clinically indicated 
on days 7, 14, 21 and 28 (+/- 72 hours) after the first daily dose of each adjuvant treatment cycle. 
 
Table 10:  Hematological AE on Day 1 of Each Cycle (within the prior 72 hours before Day 1 
 
AE  
Delay  
ANC< 1 x 109/L and/or Platelet count < 
100 x 109/L  
Delay up to 4 weeks until all resolved. If 
unresolved after 4 weeks then stop. If 
resolved, dose delay/reductions based on 
hematologic and non-hematologic AEs are 
applicable.  
 
Table 11:  Non-Hematologic AE (except for alopecia, nausea and vomiting if controlled by 
maximal antiemetic therapy) On the day 1 of Each Cycle (within the prior 72 hours) 
 
Grade  
Delay  
2-3  
Delay up to 4 weeks until all resolved (to grade ≤ 1).  If 
unresolved after 4 weeks then stop. If resolved, dose 
delay/reductions based on hematologic and non-
hematologic AEs are applicable.  
4 
Stop (except for alopecia, nausea and vomiting if 
controlled by maximal antiemetic therapy) 
 
18.5 Appendix E: ECOG Performance Status to Karnofsky 
Performance Status Comparison Table 
 


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Performance Status (PS) will be determined from KPS conversion using the ECOG comparison 
scale for patients ≤ 50 years 
 
ECOG PERFORMANCE STATUS 
KARNOFSKY PERFORMANCE 
STATUS 
0—Fully active, able to carry on all 
pre-disease performance without 
restriction 
100—Normal, no complaints; no 
evidence of disease 
90—Able to carry on normal activity; 
minor signs or symptoms of disease 
1—Restricted in physically 
strenuous activity but ambulatory 
and able to carry out work of a light 
or sedentary nature, e.g., light house 
work, office work 
80—Normal activity with effort, 
some signs or symptoms of disease 
70—Cares for self but unable to carry 
on normal activity or to do active 
work 
2—Ambulatory and capable of all 
selfcare but unable to carry out any 
work activities; up and about more 
than 50% of waking hours 
60—Requires occasional assistance 
but is able to care for most of 
personal needs 
50—Requires considerable assistance 
and frequent medical care 
3—Capable of only limited selfcare; 
confined to bed or chair more than 
50% of waking hours 
40—Disabled; requires special care 
and assistance 
30—Severely disabled; 
hospitalization is indicated although 
death not imminent 
4—Completely disabled; cannot 
carry on any selfcare; totally 
confined to bed or chair 
20—Very ill; hospitalization and 
active supportive care necessary 
10—Moribund 
5—Dead 
0—Dead 
 
 
 


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18.6 Appendix G: GUIDANCE ON CONTRACEPTION 
 
Prior to study enrollment, women of childbearing potential must be advised of the importance of 
avoiding pregnancy during trial participation and the potential risk factors for an unintentional 
pregnancy.   
 
Women of childbearing potential (WOCBP) include any woman who has experienced menarche 
and who has not undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation, 
or bilateral oophorectomy) or who is not post-menopausal.  
Post-menopause is defined as: 
- Amenorrhea that has lasted for ≥ 12 consecutive months without another cause, or 
 
For the purposes of the proposed study, medically acceptable forms of contraceptive methods 
include the following: 
 
• Surgical sterilization at least 6 months before Study Drug administration 
• Implants 
• Levonorgestrel (LNG) and Copper T IUDs 
• Sexual abstinence 
• Injectable hormone depos 
• Oral contraceptive pill 
• Hormone patch 
• Vaginal ring 
• Condom 
 
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods) and 
withdrawal are NOT acceptable methods of contraception. 
 
http://www.cdc.gov/reproductivehealth/unintendedpregnancy/pdf/contraceptive_methods_508.p
df for a list of contraceptive methods and effectiveness. 
 

ICF_000.pdf: 
 
 
Study Title:  ATTAC-II: A Phase II Randomized, Blinded, and Placebo-Controlled Trial 
of CMV RNA Pulsed Dendritic Cells with Tetanus-Diphtheria Toxoid Vaccine in Patients 
with Newly-Diagnosed Glioblastoma 
 
NCT02465268 
 
Version Date of Consent Document: 10/21/2021 


IRB Project #:  201400697
Page 1 of 22
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INFORMED CONSENT FORM
to Participate in Research, and
AUTHORIZATION 
to Collect, Use, and Disclose Protected Health Information 
(PHI)
INTRODUCTION
If you are a legally authorized representative for the subject, as you read the 
information in this Consent Form, you should put yourself in their place to decide whether 
or not to allow us to collect research information about them and to allow them to take 
part in this study. If the subject you are representing becomes able to understand the 
information in this Consent Form while they are still participating in the study, he/she must 
then decide on their own if he/she wants to continue to take part in this research study. 
As you read the rest of this form, the word “you” refers to the subject.
Name of person seeking your consent:
Place of employment & position: 
Please read this form which describes the study in some detail. A member of the research 
team will describe this study to you and answer all of your questions.  Your participation is 
entirely voluntary.  If you choose to participate you can change your mind at any time and 
withdraw from the study.  You will not be penalized in any way or lose any benefits to which 
you would otherwise be entitled if you choose not to participate in this study or to withdraw.  
If you have questions about your rights as a research subject, please call the University of 
Florida Institutional Review Board (IRB) office at (352) 273-9600.   
GENERAL INFORMATION ABOUT THIS STUDY
1.
Name of Participant ("Study Subject")
___________________________________________________________________ 
Study ID:IRB201400697   Date Approved: 10/21/2021


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2.
What is the Title of this research study?
ATTAC-II: A Phase II Randomized, Blinded, and Placebo-Controlled Trial of CMV RNA-
Pulsed Dendritic Cells with Tetanus-Diphtheria Toxoid Vaccine in Patients with Newly-
Diagnosed Glioblastoma
3.  Who is paying for this research study? 
The sponsor of this study is the University of Florida Department of Neurosurgery.
4. Why is this research study being done? 
Agreeing to become involved in any research is always voluntary.  By signing this 
form, you are not waiving any of your legal rights.  If you decide not to participate in 
this research, you will not be penalized in any way and you will not lose any benefits 
to which you are entitled. If you have questions about your rights as a research 
subject, please call the University of Florida Institutional Review Board (IRB) office at 
(352) 273-9600.    
a) In general, what is the purpose of the research, how long will you be involved?  
The purpose of this research study is to determine if an investigational vaccine made 
from your own cells, called pp65 DC, is effective for the treatment of glioblastoma 
(GBM) brain tumor when given with stronger doses of routine chemotherapy. 
The use of a vaccine that activates your immune system is a type of immunotherapy.  
It is hoped that by giving the vaccine as a shot under the skin, the immune system 
will be activated to attack tumor cells in the brain while leaving normal cells alone.
The pp65 DC vaccine is investigational which means that it is not approved by the 
United States Food and Drug Administration (FDA) and is being tested in research 
studies.
You are being asked to be in this research study because you have been newly-
diagnosed with glioblastoma. 
Treatment with study drug will continue until 10 doses are given over the course 
of about 9 months.  
b) What is involved with your participation, and what are the procedures to be 
followed in the research? 
The following will be done to assess your eligibility and to conduct the study:
-
Blood test for eligibility and safety monitoring during the study participation.
-
Brain MRIs to evaluate your disease and groin MRIs (which is the study drug 
injection site) for research purposes.    
-
You will receive the following medications:
o
Chemotherapy: Temozolomide (TMZ) will be given as part of your normal 
care,
Study ID:IRB201400697   Date Approved: 10/21/2021


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o
Vaccination with Tetanus Diphtheria to boost your immune system, and
o
Study vaccine or placebo.
-
To make the study vaccine we will collect your white cells by performing a 
procedure called “Leukapheresis”. The procedure will take about 4-6 hours to 
complete and will require having venous access lines (similar to infusion lines) 
placed on each arm. The blood is drawn from one arm, the cells are filtered and 
the blood is returned through the other arm. If the veins in your arms are difficult 
to locate or to draw from, you will have a central line placed by physician in a 
large vein (e.g. in your neck or groin).  Placement of a central line may require a 
procedure (similar to an operation). The doctor performing the procedure may 
use special x-rays to assist with placement of the special IV. The central line will 
be removed after the cell collection is complete. Two or more leukapheresis 
procedures will be needed to produce 10 vaccines.
Once made, the study vaccine will be given with monthly cycles of TMZ.  In this 
study, you will take a higher dose of TMZ unless you are 70 years old or older.  You 
will receive three vaccines during the 1st cycle of chemo, then monthly vaccination 
will continue until you receive 10 vaccines. If your disease worsens before you 
receive the 10 vaccines, you will no longer receive the study drug and your treating 
neuro-oncologist will discuss the best treatment options available.
c) What are the likely risks or discomforts to you? 
Normal side effects and discomforts are expected due to the treatments and 
procedures. The risk related to the study drug and study procedures are listed 
below:
Study Drugs (pp65 DC Vaccine and PBMCs):  
-
Allergic reaction, swelling of the brain, infection and delayed autoimmune 
response.
Leukapheresis:
-
Light-headedness, fainting, vomiting, rapid breathing, chills, tingling, and 
nausea, blood clots.
-
You may need a special large IV placed in order to have leukapheresis. 
Placement of large IV in your arm or a central vein (like your neck or groin) 
include discomfort/pain, bruising, bleeding, and very rarely, injury to adjacent 
organs. The doctor placing the special IV will discuss these risks with you and 
you will sign a separate procedural informed consent form. 
The risks and discomforts associated with participation in this trial are described in 
more detail later in this document.
d) What are the likely benefits to you or to others from the research? 
The study vaccine may allow your immune system to better fight your tumor; 
however, it is possible that you will not benefit from taking part in this study.  
Study ID:IRB201400697   Date Approved: 10/21/2021


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Information gained from this study may help doctors advance the medical 
treatment for brain tumors. 
e) What are the appropriate alternative procedures or courses of treatment, if any, 
that might be helpful to you? 
If you do not want to be in this study, treatment may include surgery, radiation 
therapy with TMZ, and monthly cycles of TMZ, participation in another research 
study, if one is available and you qualify, or you can choose to have no treatment. 
Additional and more detailed information is provided within the remainder of this 
Informed Consent form, please read before deciding if you wish to participate in this 
study
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as 
required by US law.  This web site will not include information that can identify you.  At 
most, the web site will include a summary of the results. You can search this web site 
at any time.
WHAT CAN YOU EXPECT IF YOU PARTICIPATE IN THIS STUDY?
5. What will be done as part of your normal clinical care (even if you did not 
participate in this research study)?
If you chose not to participate in this research study, your normal clinical care would 
include the following 3 phases of treatment:  1) Surgery; 2) Radiation therapy with 
temozolomide; and 3) Monthly cycles of temozolomide.  
Surgery
You would have surgery to remove as much of the tumor as possible.  
Radiation therapy with temozolomide
After recovering from surgery, you would receive radiation therapy with temozolomide.  
One radiation treatment would be given daily, 5 days per week, for 6 total weeks.  
Temozolomide, a chemotherapy medication taken by mouth, would be taken once each 
day, including weekends, until the last radiation treatment. Throughout your treatment, 
you would be evaluated by your physician, have routine laboratory blood tests, and 
have a brain MRI (or CT scan if you cannot have an MRI) every 2-3 months to see if 
the tumor has returned or grown.  
An MRI is a type of scan which uses a magnet, radio waves, and computer equipment 
to produce images of structures in your body. It is a large machine with a hole in the 
middle that looks like a doughnut with a table going through it. During the MRI, the 
magnet makes tapping, thumping, or other loud noises. Earplugs or music may be 
provided to help muffle or block the noise.
Study ID:IRB201400697   Date Approved: 10/21/2021


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Monthly cycles of temozolomide
About 1 month after radiation therapy with temozolomide, you would start monthly 
temozolomide.  You would take temozolomide once each day for 5 straight days every 
28 days; this 28-day period is called a cycle.  Treatment with temozolomide would 
continue for 6-12 cycles, provided you are able to tolerate the temozolomide and your 
tumor has not returned or grown. Throughout your treatment, you would be regularly 
evaluated by your physician, have routine laboratory blood tests, and have a brain MRI 
(or CT scan if you cannot have an MRI) every 2-3 months to see if the tumor has 
returned or grown.  
6. What will be done only because you are in this research study? 
If you decide to participate in this study, some of the study procedures will be performed 
only at UF Health, as described below.  
Before you begin the study:
The study doctor or study staff will talk to you about the study to determine if you are 
willing to participate.  Study procedures cannot begin until you give your permission by 
signing this consent form. 
Enrollment Visit
If you agree to participate and after you sign the consent form, additional testing will be 
done to determine if you are eligible to enroll in the study. 
We will collect information on your past and current medical history to determine 
eligibility. The physician or the nurse practitioner will perform a physical and 
neurological exam. If you are 50 years of age or older, we will perform a brief exam to 
assess how your brain processes the information. 
Blood tests to check if you have ever been infected with any viruses that can be 
transmitted through the blood will also be done, including tests for HIV, Hepatitis B, 
Hepatitis C, and Cytomegalovirus (CMV).  
A positive HIV test means that your blood sample tested positive for HIV and that repeat 
testing will be performed to confirm (prove) this finding.  If your sample is positive for 
HIV, it means that you are a carrier of HIV and that you can pass the virus to others by 
intimate sexual contact, by sharing needles, and through donating blood and organs. 
A negative HIV test means that no antibody to HIV was found in your blood sample 
based on the result of the initial screening test, repeat screening tests, or a confirmatory 
test.
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There can be individuals who have HIV test results that are called “false positive” (for 
some reason, the test indicates that HIV antibodies are present in the blood when they 
are not).  There can also be false negative results which can have two possible 
meanings;  the person has been infected with HIV, but that person’s body has not yet 
made antibodies to the virus, or HIV antibody is present in the person’s blood, but for 
some reason the test failed to detect it.
A member of the study team will meet with you before and after the HIV test.  You may 
be asked to sign a separate consent form to have your blood tested for HIV.  Because 
we will test your blood for HIV (the virus that causes AIDS), Florida Law requires that 
we tell you that:

Your identity and HIV test result will be kept confidential to the extent provided 
by law.

Any physician who performs an HIV test that has a positive result or lab that 
processes a blood sample for HIV that has a positive result must report this 
information to County Health Department with information that will identify you.

If you want to get tested for HIV outside of this study, test sites that keep your 
results anonymous are available.  You can contact the County Health 
Department for information on those sites.
If you test positive for HIV, we will do the following:

Confirm the result by sending a sample of your blood for another HIV test.

If the second test result is positive, the positive test result and your identity will 
be reported to the County Health Department.  Florida law requires that we 
report this information.

Provide information on medical and support services available to you; and

Discuss information on the importance of notifying previous and current sexual 
partners that they may have been exposed to HIV and how to prevent HIV 
transmission.
If infected with HIV or Hepatitis, you will not be able to participate in this study.  Also, if 
you are a female and able to become pregnant, we will perform a pregnancy test.  If the 
results show that you are pregnant, you will not be able to participate in this study.  You 
will also be informed of any other findings that would exclude you from participating in 
this study. 
If you are not eligible, your study participation will end. You will not receive study drug. 
Your doctor will discuss appropriate standard treatments for you at that point.
Study Group Assignment:
If you are eligible to continue in the study and before leukapheresis to collect blood cells 
to make study drug,  you will be assigned into a treatment group, however you will not 
receive your first dose of study drug until after you complete standard treatment with 
radiation and temozolomide.  Study group assignment is done randomly (by chance) 
and is similar to the tossing of a coin.  Neither you nor your study doctor can decide 
which group you are assigned to.  In addition, your study group assignment is blinded; 
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meaning you will not know which study group you are in, or what treatment you are 
receiving.  This is done to prevent any bias so that the results of the study can be 
presented in a fair manner. 
You will be randomly assigned into one of three study drug groups:     
Group 1:
Experimental arm: pp65 DC vaccine (short) with GM-CSF and 
tetanus and diphtheria (Td)
Group 2:
Experimental arm: pp65 DC vaccine (long) with GM-CSF and tetanus 
and diphtheria (Td)
Group 3:
Placebo arm: Peripheral blood Mononuclear Cells (PBMCs) and 
saline skin preparation
 
You will have an equal (1 out of 3 or 33%) chance of  being assigned to one of the 
study drug groups above. 
pp65 DC short vaccine and pp65 DC long vaccine are both active study drugs. The 
difference between these 2 vaccines is the length of the pp65 protein.  We do not know 
if the response to the pp65 DC short vaccine and pp65 DC long vaccine will be 
different.  The responses to both vaccines will be measured during this research study 
and compared to each other. Peripheral blood mononuclear cells (PBMCs) that are 
collected by leukapheresis, will be used in this study as a placebo.  The placebo will 
look like and will be given in the same way as the pp65DC vaccine, but it will not contain 
the same cells.  The placebo will be used in this study to show what effect the pp65 
vaccine has compared to no vaccine at all.   If you are assigned to receive PBMCs, you 
will not receive the benefits of the pp65 DC vaccine, if there are any.  
Td is a vaccine that is routinely given to protect individuals from tetanus and diphtheria, 
infections caused by bacteria.  You will receive a Td booster before you get your first 
dose of study drug regardless of which group you are assigned.     In this study, Td skin 
preparation will be given to help the vaccine boost your immune system.  
The saline skin prep will be used in this study as a placebo.  The placebo will look like 
and will be given in the same way as the Td skin prep but it will not contain the same 
ingredients.  The placebo will be used in this study to show what effect the Td skin 
preparation has on boosting your immune system compared to no skin prep at all.   If 
you are assigned to receive the saline skin prep, you will not receive the benefits of the 
Td skin prep, if there are any.  
Leukapheresis:
After recovering from surgery and before radiation therapy with temozolomide, you will 
undergo a procedure called leukapheresis.  The purpose of leukapheresis is to collect 
large numbers of white blood cells from your blood and use them to make the study 
drugs.  All study participants will undergo all leukapheresis procedures at UF Health.  
Before the leukapheresis procedure, you will have a clinic visit at UF Health that will 
include a physical examination and blood tests.  Approximately 2 tablespoons, or 30cc, 
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of blood will be collected to check your blood counts, organ function, and electrolyte, or 
mineral, levels.  Blood tests for HIV, Hepatitis B, Hepatitis C, Syphilis, and 
Cytomegalovirus (CMV) will be repeated.  If you are a woman and able to have children, 
your blood will be tested to make sure you are not pregnant.  If you are infected with 
HIV or Hepatitis or you are pregnant, your study participation will end and you will not 
receive study drug.
Next, you will undergo leukapheresis.  In order to perform the procedure, a large needle 
(about the size of a sharpened pencil tip) will be placed in a vein in each arm.  After 
placing the needles, blood will be taken from one needle and continuously processed 
through a machine called a cell separator. This machine is designed to remove some 
of your white blood cells. The remainder of your blood will be returned to you through 
the needle in your other arm.  During the procedure, you will be asked to remain as still 
as possible.  The total time required to complete leukapheresis is approximately 4-6 
hours.  If the veins in your arms needed to perform the leukapheresis are difficult to 
locate or to draw from, you will have a separate procedure by physician to insert a 
temporary central line placed in a large vein either in the neck (the jugular vein) or in 
the groin (the femoral vein).  The doctor placing the line may use x-ray (called 
fluoroscopy) to assist with IV placement in the vein. This involves quick, low amounts 
of radiation. The doctor placing the line will explain the procedure and the risk and 
benefits associated with it and you will be asked to sign a separate consent form. The 
central line will be removed after the cells collection is complete. Pressure will be 
applied at the catheter insertion location for 5-10 minutes (maybe longer) and an 
occlusive bandage will be applied. 
The leukapheresis procedure will be repeated about 1 month after your third dose of 
study drug so that more white blood cells can be collected to make more study drug. If 
we were not able to make enough vaccines from the 2 leukapheresis procedures, you 
may undergo additional leukapheresis to complete the study.
Radiation and Temozolomide:
Following leukapheresis, you will undergo treatment with standard radiation and 
temozolomide as described in Section 6 above.  After you complete radiation therapy 
with temozolomide, the study doctor will determine if you are eligible to continue in the 
study based on how much temozolomide and radiation you received and blood test 
results.  If you are not eligible, your study participation will end and you will not receive 
study drug.  
If you are eligible to continue in the study, you will begin monthly cycles of 
temozolomide and scheduled study vaccines as described below. 
Temozolomide: 
About 4 weeks after you complete radiation therapy with temozolomide, you will begin 
monthly cycles of temozolomide.  In this study, you will take a higher dose of 
temozolomide once each day for 21 straight days every 35 days.  If you are 70 years 
or older, you will receive the standard dose of temozolomide for 5 straight days every 
35 days.  The number of days you receive temozolomide and the dose you receive may 
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be changed by your physician if you experience side effects or have problems with your 
laboratory blood test results.  
Throughout your treatment with temozolomide, you will have routine monitoring as 
described in Section 6 above.   
Study Drug Administration:
Study drug will be given in combination with your monthly temozolomide cycles.  The 
first dose will be given 1-3 days after completion of your first temozolomide cycle (day 
22-24).  The second dose will be given two weeks later and the third dose will be given 
two weeks after the second dose.  Starting with the fourth dose, study drug will be given 
on day 22-24 of each temozolomide cycle.  The fourth dose of study drug will be given 
after your second leukapheresis with cycle two of temozolomide.  All doses of study 
drug will be given at UF Health during study drug visits described below.  If you cannot 
or do not want to travel to the University of Florida for these visits, you will not be able 
to participate in this study. 
Treatment with study drug will continue until a total of 10 doses are given.  If the study 
doctor determines that your tumor has grown at any time during the study, or if you 
experience serious side effects, you will stop the treatments. You and your study doctor 
will decide what future treatment, if any, is right for you and you may receive alternate 
treatments at that time.
At the time of each vaccination, if fewer cells are available in the study drug than the 
target dose, you will still receive a vaccination with the available number of cells. It is 
not known if giving a lower number of cells in the study drug makes a difference in how 
well the vaccine works. Based on the information we have, there is not a clear 
relationship between number of dendritic cells in the study drug and effectiveness of 
the vaccine. We believe that it is reasonable to give each dose of vaccine with the 
number of dendritic cells that are available. Using a lower number of dendritic does not 
change the risks of giving the vaccine. You will always be informed if less than the 
target number of dendritic cells is being given with any vaccination.
The following procedures will be performed at study drug visits at UF Health:

You will receive a Td booster shot in the muscle of the upper arm before study 
drug dose #1 only. If you have received a Td booster shot in the past, you will 
still receive a Td booster shot before dose #1. 

You will receive the study drug as an injection just under the skin in both groin 
areas.  Each injection will only take a few minutes and you will be monitored for 
30 minutes to 1 hour after each injection to be certain you do not have a reaction.

You will receive the skin prep as an injection just under the skin in both groin 
areas 6-24 hours before doses #3, #6, and #9.  

Blood will be drawn to learn more about the way the immune system is working 
in your body and the effects of the treatment.  Blood (no more than 6 tablespoons 
or 90cc) will be collected at each of the following time points:

With the 1st leukapheresis

Before study drug doses #1, #2, and #3
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
48 hours and 5-7 days following study drug dose #3

With the 2nd leukapheresis

Before study drug doses #4, #7, and #10

Anytime there is suspicion that your tumor may have returned or grown.

A sample of your urine will be collected before you receive study drug dose #1 
and before the skin prep injection, 48 hours and 5-7 days following study drug 
dose # 3.  

We will ask you about any medications you are taking and if you are having any 
side effects. 
When you return to the clinic at UF Health before study drug dose #3, we will track 
where the study drug travels in the lymph nodes of your body by taking MRI (Magnetic 
Resonance Imaging)/MRS (Magnetic Resonance Spectroscopy) images of your groin. 
MRS is a type of scan similar to MRI which uses a magnet, radio waves, and computer 
equipment to produce images of the chemical content of tissue in your body. We will 
take these images and a sample of your urine before the skin prep injection, 48 hours 
and 5-7 days (optional) following study drug dose # 3. 
If you are a woman and able to have children, your urine will be tested before the 
MRI/MRS to make sure you are not pregnant.
Tests done only for research purposes will not be evaluated or used to diagnose or 
treat any of your medical problems. These tests may need to be repeated if required 
for your medical care in the future.
After completing treatment, we will collect information on how you are doing and if your 
tumor has returned or grown.  This information will be collected from your medical 
records or by contacting you by telephone every 2-3 months for as long as you allow us 
to do so.
It is possible that you become ineligible to receive the study drug during your 
participation in the study. For example if you cannot complete the full treatment of 
chemo-radiation, or if your chemo treatment is delayed for an extended time due to side 
effect or other unexpected events.  In such case, you may receive the vaccines that are 
available if the study physician and the principal investigator believe you could benefit 
from getting the vaccines and that it is safe for you. However, no additional vaccines 
will be generated. 
If the study physician decides you will benefit from starting another treatment, you will 
not receive the study vaccines. 
Once this research study is completed, any information that could identify you might be 
removed from any identifiable private information or identifiable biospecimens collected 
and that, after such removal, the information or biospecimens could be used for future 
research studies or distributed to another investigator for future research studies without 
additional informed consent from you or your legally authorized representative.
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If you have any questions now or at any time during the course, please contact one of 
the research team members listed in question 1 of the addendum.
7.
How long will you be in this research study?
Treatment with the study drug will continue until a total of 10 doses are given over the 
course of about 9 months.  After that, we would like to collect information on how you 
are doing for as long as you allow us to do so.
8.
How many people are expected to take part in this research study?
Approximately 200 people will take part in this study at the University of Florida, Orlando 
Health Cancer Institute and Duke University.  Up to 125 subjects may enroll at the 
University of Florida.
  
WHAT ARE THE RISKS AND BENEFITS OF THIS STUDY AND 
WHAT ARE YOUR OPTIONS?
9.
What are the possible discomforts and risks from taking part in this research 
study?
While in this study, you are at risk for possible side effects from the study drug and 
the study procedures. You should discuss these with the study doctor. The known 
side effects are listed below, but they will vary from person to person. There may also 
be other side effects that you experience that were not predicted. Many side effects 
go away shortly after the study drug is stopped, but in some cases, side effects may 
be serious, long lasting, and/or permanent and may even cause death. 
Side effects that may be associated with some of the procedures used in this study 
are as follows:
Study Drugs (pp65 DC Vaccine and PBMCs):
Allergic reaction: The study drug may cause an allergic reaction, which could include 
pain, redness or swelling at the injection site, itching, hives, low blood pressure, 
difficulty breathing, or in the most extreme circumstances, death.  
Cerebral edema or swelling of the brain: Cerebral edema may be caused by the 
tumor, surgery, radiation, immune cells in the brain, or the killing of tumor cells.  
Symptoms of cerebral edema may include severe headache, confusion, lack of 
energy, unconsciousness, coma, or loss of movement, sensation or function of 
certain areas of the body.  You will be monitored for signs of cerebral edema 
throughout the study and treated if necessary.  Cerebral edema that does not respond 
to treatment may lead to permanent damage.  
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Infection: If the study drug comes in contact with something unclean (contaminated) 
when it is being made, it could cause an infection. An infection may cause redness, 
swelling, and/or uncomfortableness at the injection site.  In extreme cases, this could 
lead to a severe infection of the blood (sepsis) and possibly death.  The risk of 
infection is very low because the study drugs are made in a special laboratory with 
strict rules and they are tested for germs.  You will be monitored for signs of infection 
throughout the study and treated if necessary.
Delayed autoimmune disease:  The study drug may activate the immune system to 
such a high degree that the immune system may start to attack normal brain tissue 
or other tissues in the body.  
Td Booster and Td Skin Prep:
Side effects that may be caused by Td include:

pain, tenderness, redness, hardness, itching, swelling, and/or warmth at the 
injection site

swelling of the arms and/or legs

fever and/or flu-like symptoms

dizziness, headaches, and/or convulsions (sudden, violent shaking of the 
muscles)

muscle, joint, and/or bone stiffness and/or pain

rash

nausea

cellulitis, an infection of the skin
Rarely, severe shoulder pain and reduced movement in the arm where the shot was 
given can happen.   Severe allergic reactions that cause redness or swelling at the 
injection site, itching, hives (raised red and sometimes itchy spots on the skin), low -
blood pressure, difficulty breathing, or death, are very rare. Rare cases of Guillain-
Barré syndrome (GBS) have been reported in people who receive Td.  GBS is a rare 
syndrome in which your body's immune system attacks your nervous system causing 
weakness and tingling in your extremities.  
Saline Skin Prep:
Side effects that may be caused by the saline skin prep include:

pain, tenderness, redness, hardness, itching, swelling, and/or warmth at the 
injection site

cellulitis, an infection of the skin
Temozolomide:
The most common side effects of temozolomide are loss of appetite, nausea, 
constipation and decrease in blood cell counts. A decrease in the number of white 
blood cells may increase your risk of infections. A reduction in the number of platelets 
may increase the risk of bleeding and a reduced number of red blood cells may 
increase fatigue or shortness of breath. Some people who have taken temozolomide 
also had the following side effects:
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
nausea and vomiting, especially on the first day of each cycle

back, abdominal and/or stomach pain, breast pain

diarrhea 

hair loss 

dry skin, skin redness, itching and/or rash

swelling of extremities (your arms, legs, fingers, or toes)

inflammation or swelling of the mouth, throat and/or sinuses

headache, confusion, loss of memory, dizziness, fatigue, fever, and/or 
weakness

anxiety, depression

joint and muscle pain

abnormal coordination when using your arms or legs (such as walking or 
feeding yourself) and/or abnormal feelings in your extremities

trouble sleeping or sleepiness

changes in your sense of taste

changes in your vision such as double or blurred vision

coughing or shortness of breath, respiratory tract infection

urinary incontinence/frequency, urinary tract infection

weight increase

seizures, hemiparesis (weakness on one side of the body)

adrenal hypercorticisim (elevated hormone levels)

allergic reactions, sometimes severe

hepatotoxicity (abnormal liver function tests); sometimes these may be severe, 
which is why liver function tests may be performed throughout temozolomide 
treatment cycles
Rarely, unusual (“opportunistic”) infections have occurred.  Rare cases of erythema 
multiforme (a skin condition that is similar to a bad rash) have been reported which 
got better after temozolomide was stopped and, in some cases, recurred upon 
restarting treatment with temozolomide.
Very rare side effects have included secondary cancers including leukemia and 
myelodysplastic syndrome (MDS).  MDS is a disorder of the bone marrow in which 
blood cells that do not function normally are produced.
Reproductive studies have not been done with people who are taking temozolomide. 
Immature sperm and testicular atrophy occurred in studies with rats and dogs, using 
doses of temozolomide 1/4 and 5/8 of the recommended human doses.  In animal 
studies, temozolomide caused death and multiple malformations in fetal rats and 
rabbits.
Leukapheresis:
Some side effects associated with the leukapheresis procedure may be similar to those 
experienced during blood donation and include light-headedness, fainting, vomiting, 
and rapid breathing.  Other side effects which may occur that are unique to the 
leukapheresis procedure include chills caused by cooling of the blood when it is 
contained in the special machine, and tingling and nausea caused by low blood calcium 
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levels due to the blood thinner (called citrate) used to maintain free flow of the blood 
through the machine.  These side effects can be controlled by altering the rate at which 
blood is withdrawn, by warm blankets, by altering the amount of blood thinner, by 
administration of calcium during the procedure, and by stopping the procedure.  
Rarely, the leukapheresis procedure may be associated with loss of blood, breakdown 
of the blood, clotting of the blood, allergic reactions, inadvertent infusion of air, and fluid 
overload or depletion.  These side effects can usually be controlled by stopping the 
procedure and providing appropriate medical care.  In addition, there is always the risk 
that very uncommon or previously unknown side effects may occur or that life-
threatening side effects may occur and death may result.
Central Venous Catheter Insertion for leukapheresis
If you require a special large intravenous (IV) line (called a “central venous catheter” 
or “catheter”) for the leukapheresis procedure, a temporary catheter will be placed in 
a vein either in the neck (the jugular vein) or in the groin (the femoral vein).  The 
doctor placing the catheter will describe the procedure and risks of the procedure and 
ask you to sign a separate consent form before the catheter placement.  Numbing 
medication (xylocaine) will be given by a small injection into the skin at the site of 
central venous catheter placement.  The catheter will be placed using ultrasound and 
live X-ray (fluoroscopy) for guidance.  The catheter will be held in place by stiches in 
the skin, which may be removed once the catheter is firmly in place.  
The risks of central catheter placement associated with this deeper venous catheter 
include: 

bruising around the catheter,

injury to the vein or other blood vessels, nerves and/or organs close to the 
area of catheter placement, 

collapse of the lung, bleeding or fluid in the chest that could require chest tube 
placement, 

bleeding under the skin,

infection,

allergic reaction to local numbing medication,

irregular heartbeat,

clotting of the catheter or the blood vessel or irritation/redness of the vein.

narrowing of the vein which may prevent future use of the vessel

removal of the central venous catheter may cause discomfort, small amount 
of bleeding and pressure will need to be held in the area for 5-10 minutes 
(maybe longer).
There is also a very rare possibility that you may have symptoms ranging from 
dizziness and fainting to allergic reactions such as anaphylaxis.
This research study may require a central venous catheter for the leukapheresis 
procedure.  The radiation you will receive from this procedure exposes a part of 
your body to a higher level of radiation than the rest of your body.  The typical 
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radiation exposure from this procedure is about 200 µSv (20 millirem) which is 
equal to about 26 days of natural background radiation exposure.
If you have a central venous catheter inserted, you may need conscious sedation, 
which is a combination of medicines to help you relax and to block pain during the 
procedure.  Conscious sedation lets you recover quickly and return to your everyday 
activities soon after your procedure. You will receive the medicine through an 
intravenous line (IV, in a vein).  You may feel pain from the initial needle stick when 
placing the IV. Possible bruising from where the needle went into the skin may occur, 
as well as rarely, an allergic reaction to the medication. Vomiting or inhaling food 
contents from the stomach, feeling confused, low blood pressure, drowsiness, 
allergic reaction, vein irritation are also risks of sedation. These risks are minimized if 
you do not eat for a minimum of 6-8 hours prior to any procedure.  You will sign a 
separate consent for this type of sedation.
MRI/MRS Imaging:
Magnetic resonance imaging (MRI) is a procedure that allows doctors to look inside 
the body by using a scanner that sends out a strong magnetic field and radio waves. 
This procedure is used routinely for medical care and is very safe for most people, but 
you will be monitored during the entire MRI scan in case any problems occur. The risks 
of MRI are: 
The MRI scanner contains a very strong magnet. Therefore, you may not be able to 
have the MRI if you have any type of metal implanted in your body, for example, any 
pacing device (such as a heart pacer), any metal in your eyes, or certain types of heart 
valves or brain aneurysm clips. Someone will ask you questions about this before you 
have the MRI.
There is not much room inside the MRI scanner. You may be uncomfortable if you do 
not like to be in close spaces ("claustrophobia"). During the procedure, you will be able 
to talk with the MRI staff through a speaker system, and, in the event of an emergency, 
you can tell them to stop the scan. If have claustrophobia, you may require medication 
to help you relax ("sedation"). If you do require medication to relax, you should not 
drive a car, take part in activities like riding a bike, or perform other similar tasks until 
the next morning because the medication(s) can affect your thinking for several hours 
and can slow down your reflexes.
The MRI scanner produces a loud hammering noise, which has produced hearing loss 
in a very small number of patients. You will be given earplugs to reduce this risk.
If you are a woman of childbearing potential, there may be unknown risks to the fetus. 
Therefore, before you can have the MRI, you must have a pregnancy test.
Reproductive Risks:
Because the treatment in this study might affect an unborn baby, you should not 
become pregnant while on this study.  Since this treatment will not be given to any 
patients who are pregnant, all women and adolescents of childbearing potential must 
take a pregnancy test prior to receiving any treatment on this study.  We encourage all 
women and adolescents enrolled on this study to use one of the effective birth control 
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methods during treatment and for six months after treatment is stopped.  These 
methods include total abstinence (no sexual intercourse), oral contraceptives ("the 
pill"), an intrauterine device (IUD), an etonogestrel implant (Implanon), or 
medroxyprogesterone acetate injections (Depo-Provera shots).  If one of these cannot 
be used, using contraceptive foam and a condom are recommended.  You must notify 
the doctor if you become pregnant during the course of this study. 
You should not nurse a baby while receiving treatment on this study or for six months 
after completing treatment.
We do not know whether using this treatment will affect sperm.  As a result, men and 
adolescents who have not had a vasectomy are advised to either (a) abstain from 
reproductive sexual intercourse or (b) use a condom and contraceptive foam during 
intercourse.  These precautions should be taken while on therapy and for at least one 
month after completing therapy.
HIV Testing:
Testing positive for human viruses (HIV) can be upsetting.  You will be provided with 
information on medical and support services that may be available to you.  If you test 
positive for HIV and that information were to become public, you could have problems 
getting insurance or a job.   Your identity and test results will be kept confidential to the 
extent permitted by Florida Law.  If test results are released in accordance with Florida 
Law, this disclosure will be accompanied by the following statement:  "This information 
has been disclosed to you from records whose confidentiality is protected by state law.  
State law prohibits you from making any further disclosure of such information without 
the specific written consent of the person to whom such information pertains, or as 
otherwise permitted by state law.  A general authorization for the release of medical or 
other information is not sufficient for this purpose."
Blood Draws: 
The risks of drawing blood from a vein include discomfort at the site of puncture; 
possible bruising and swelling around the puncture site; rarely an infection; and, 
uncommonly, faintness from the procedure.
Researchers will take appropriate steps to protect any information they collect about 
you.  However, there is a slight risk that information about you could be revealed 
inappropriately or accidentally.  Depending on the nature of the information, such a 
release of your information could upset or embarrass you, or possibly affect your 
insurability or employability.  Questions 17-21 in this form discuss what information 
about you will be collected, used, protected, and shared.
This study may include risks that are unknown at this time.
Personal Information: Taking part in this research may involve providing information 
that you consider confidential or private. Efforts such as coding research records, 
keeping research records secure and allowing only authorized people to have access 
to research records, will be made to keep your information safe. Researchers will take 
appropriate steps to protect any information they collect about you. However, there is 
a slight risk that information about you could be revealed inappropriately or 
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accidentally. Depending on the nature of the information, such a release could upset 
or embarrass you, or possibly affect your insurability or employability. Questions 13-
17 in this form discuss what information about you will be collected, used, protected, 
and shared.
Participation in more than one research study or project may further increase the risks 
to you. If you are already enrolled in another research study, please inform one of the 
research team members listed in question 1 of the addendum or the person reviewing 
this consent with you before enrolling in this or any other research study or project. 
Throughout the study, the researchers will notify you of new information that may 
become available and might affect your decision to remain in the study. If you wish to 
discuss the information above or any discomforts you may experience, please ask 
questions now or call one of the research team members listed in question 1 of the 
addendum form.
If you wish to discuss the information above, please ask questions by email or call the 
study coordinator listed in question 1 of the addendum.
10a. What are the potential benefits to you for taking part in this research study?
It is entirely possible that you will not benefit from taking part in this study.  However, it 
is hoped that the study vaccine may allow your immune system to better fight your 
tumor.
10b. How could others possibly benefit from this study?
Information gained from this study may help doctors advance the medical treatment   
for brain tumors. 
11. What other choices do you have if you do not want to be in this study? 
If you do not want to be in this study, treatment would include surgery, radiation 
therapy with temozolomide, and monthly cycles of temozolomide.  You may also 
choose to participate in another research study, if one is available and you qualify, or 
you can choose to have no treatment. 
Your participation in this study is voluntary and any decision to take part or not to 
participate in the study will in no way affect the quality of your care.
12a. Can you withdraw from this study?
You are free to withdraw your consent and to stop participating in this study at any 
time.  If you do withdraw your consent, you will not be penalized in any way and you 
will not lose any benefits to which you are entitled.  
If you decide to withdraw your consent to participate in this study for any reason, 
please contact one of the research team members listed in question 1 of the 
addendum.  
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If you have any questions regarding your rights as a research subject, please call the 
Institutional Review Board (IRB) office listed on the “Consent Addendum” attached.
12b.If you withdraw, can information about you still be used and/or collected?
If you withdraw from this study, no further information will be collected. Any 
information already collected during your participation may still be used.
12c. Can the Principal Investigator withdraw you from this study? 
You may be withdrawn from the study without your consent for the following reasons:

You are pregnant or become pregnant

You are infected with HIV or hepatitis

You did not receive enough temozolomide and/or radiation

We are unable to make or give the study drug

You do not follow the instructions given to you by the research staff

You experience a side effect and your physician feels it would be in your best 
interest to stop study treatment

The study is stopped
13. How will your health information be collected, used and shared?
If you agree to participate in this study, the Principal Investigator will create, collect, and 
use private information about you and your health. This information is called protected 
health information or PHI. In order to do this, the Principal Investigator needs your 
authorization  The following section describes what PHI will be collected, used and 
shared, how it will be collected, used, and shared, who will collect, use or share it, who 
will have access to it, how it will be secured, and what your rights are to revoke this 
authorization. 
Your protected health information may be collected, used, and shared with others to 
determine if you can participate in the study, and then as part of your participation in 
the study.  This information can be gathered from you or your past, current or future 
health records, from procedures such as physical examinations, x-rays, blood or urine 
tests or from other procedures or tests.  This information will be created by receiving 
study treatments or participating in study procedures, or from your study visits and 
telephone calls.  More specifically, the following information may be collected, used, 
and shared with others: 

Information that identifies or could be used to identify you such as name, date 
of birth, address, telephone numbers, medical record number and exact dates 
collected from your past, current or future health records

Complete past medical history

Records of physical and neurological exams

Laboratory, pathology, radiology, and other test results

HIV and other infectious disease test results
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
Records about medications and radiation therapy

Ability or potential ability to conceive a child

Records about side effects you may experience

Information on how you are feeling that is collected during telephone calls  
This information will be stored in locked filing cabinets or on computer servers with 
secure passwords, or encrypted electronic storage devices. 
Some of the information collected may be sent to a Immunomic Therapeutics, Inc., a 
healthcare company developing vaccine technology, located in Rockville Maryland.  If 
data is shared, it will be in the form of a limited data set, described below. 
Some of the information collected could be included in a "limited data set" to be used 
for other research purposes.  If so, the limited data set will only include information that 
does not directly identify you.  For example, the limited data set cannot include your 
name, address, telephone number, social security number, photographs, or other 
codes that link you to the information in the limited data set.  If limited data sets are 
created and used, agreements between the parties creating and receiving the limited 
data set are required in order to protect your identity and confidentiality and privacy.
14.  For what study-related purposes will your protected health information be 
collected, used, and shared with others?
Your PHI may be collected, used, and shared with others to make sure you can 
participate in the research, through your participation in the research, and to evaluate 
the results of the research study.  More specifically, your PHI may be collected, used, 
and shared with others for the following study-related purpose(s):
To find out if the investigational pp65 DC dendritic cell vaccine is effective for the 
treatment of glioblastoma when given with stronger doses of routine 
chemotherapy.
Once this information is collected, it becomes part of the research record for this study.  
15.  Who will be allowed to collect, use, and share your protected health 
information?
Only certain people have the legal right to collect, use and share your research 
records, and they will protect the privacy and security of these records to the extent 
the law allows. These people include:

the study Principal Investigator (listed in question 1 of the addendum) and 
research staff associated with this project.

other professionals at the institution where you are participating in this research 
study.

 the University of Florida Institutional Review Board which is responsible for the 
approval of this research study (IRB; an IRB is a group of people who are 
responsible for looking after the rights and welfare of people taking part in 
research)..  
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16.  Once collected or used, who may your protected health information be shared 
with?
Your PHI may be shared with:

the study sponsor (listed in Question 3 of this form).

Your treating physician and other professionals involved in your care.

The study’s Data and Safety Monitoring Board, who is responsible for monitoring 
the safety of the participants. 

National Institutes of Health, National Cancer Institute, who previously funded 
this study.

United States governmental agencies who are responsible for overseeing 
research, such as the Food and Drug Administration, the Department of Health 
and Human Services, and the Office of Human Research Protections
.

Government agencies who are responsible for overseeing public health 
concerns such as the Centers for Disease Control and federal, state and local 
health departments.

Your insurance company for the purposes of obtaining payment. 
Otherwise, your research records will not be released without your permission unless 
required by law or a court order. It is possible that once this information is shared with 
authorized persons, it could be shared by the persons or agencies who receive it and 
it would no longer be protected by the federal medical privacy law.  
17.  If you agree to take part in this research study, how long will your protected 
health information be used and shared with others?
Your PHI will be used and shared with others until the end of this study.
You are not required to sign this consent and authorization or allow researchers to 
collect, use and share your PHI. Your refusal to sign will not affect your treatment, 
payment, enrollment, or eligibility for any benefits outside this research study. 
However, you cannot participate in this research unless you allow the collection, use 
and sharing of your protected health information by signing this consent and 
authorization.
You have the right to review and copy your protected health information.  However, 
we can make this available only after the study is finished.
You can revoke your authorization at any time before, during, or after your 
participation in this study.  If you revoke it, no new information will be collected about 
you.  However, information that was already collected may still be used and shared 
with others if the researchers have relied on it to complete the research.  You can 
revoke your authorization by giving a written request with your signature on it to the 
Principal Investigator.
Since this research study is being conducted at several institutions, 
there is an Institution Specific “Addendum” to this consent form.  Please 
read this addendum prior to agreeing to participate in this research 
study.
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SIGNATURES
As an investigator or the investigator’s representative, I have explained to the participant 
the purpose, the procedures, the possible benefits, and the risks of this research study; 
the alternative to being in the study; and how the participant’s protected health information 
will be collected, used, and shared with others: 
       
Signature of Person Obtaining Consent and 
Authorization
Date
Consenting Adults.  You have been informed about this study’s purpose, procedures, 
possible benefits, and risks; the alternatives to being in the study; and how your protected 
health information will be collected, used and shared with others.  You have received a 
copy of this Form.  You have been given the opportunity to ask questions before you sign, 
and you have been told that you can ask other questions at any time.  
Adult Consenting for Self.  By signing this form, you voluntarily agree to participate in this 
study. You hereby authorize the collection, use and sharing of your protected health 
information as described above.  By signing this form, you are not waiving any of your legal 
rights. 
______________________________________________
_____________________
Signature of Adult Consenting & Authorizing for Self
Date
Parent/Adult Legally Representing the Subject.  By signing this form, you voluntarily 
give your permission for the person named below to participate in this study.  You hereby 
authorize the collection, use and sharing of protected health information for the person 
named below as described above.  You are not waiving any legal rights for yourself or the 
person you are legally representing.  After your signature, please print your name and your 
relationship to the subject.
______________________________________________
____________________
Consent & Authorization Signature 
Date
of Parent/Legal Representative
____________________________________
_____________________________
Print:  Name of Legal Representative 
Print:  Relationship to Participant:  
______________________________________________
Print:  Name of Subject  
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Participants Who Cannot Consent But Can Read and/or Understand about the Study.  
Although legally you cannot "consent" to be in this study, we need to know if you want to 
take part.  If you decide to take part in this study, and your parent or the person legally 
responsible for you gives permission, you both need to sign.  Your signing below means 
that you agree to take part (assent).  The signature of your parent/legal representative 
above means he or she gives permission (consent) for you to take part.
______________________________________________
_____________________
Assent Signature of Participant
Date
Study ID:IRB201400697   Date Approved: 10/21/2021