Peptide Targets for Glioblastoma Against Novel Cytomegalovirus Antigens
Phase: PHASE1
Conditions: Glioblastoma, Glioblastoma Multiforme
Sponsor: Eric Thompson, M.D.

 
PERFORMANCE: 
Peptide Targets for Glioblastoma Against Novel Cytomegalovirus Antigens 
 
Investigational Review Board Number Pro00034208  
Food and Drug Administration Investigational New Drug Permit 15,846 
 
 
Duke University Medical Center 
Gary E. Archer, Ph.D. 
Annick Desjardins, M.D. 
Henry S. Friedman, M.D. 
James E. Herndon, II, Ph.D. 
Denise Jaggers, RN, B.S.N. 
Margaret Johnson, M.D. 
Roger E. McLendon, M.D. 
David M. Ashley, MBBS, FRACP, PhD (Principal Investigator)  
Sarah Woodring 
Elizabeth S. Miller 
Katy Peters, M.D., Ph.D. 
Kent Weinhold, Ph.D. 
Weihua Xie, M.S. 
John H. Sampson, M.D., Ph.D. 
Dina Randazzo, D.O. 
 

 
 
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Version 
Date 
Sections 
Revised 
Purpose 
Institutional 
Dates 
Federal 
Dates 
(1571 #) 
20111018 
 
Initial Protocol 
 
 
20120221 
Study 
Synopsis, 
Sections 10.1, 
16.1  
Initial Protocol IRB Modifications 
 
Updated pregnancy testing 
language 
20120227 
20131126 
(0) 
20130219 
Cover Page 
 
 
Study 
Synopsis, 
Sections 11.1, 
11.4.4 
 
Study 
Synopsis, 
Sections 7.1, 
11.1, 11.4.4 
 
Study 
Synopsis, 
Sections 7.2, 
8.1, 8.2, 11.1, 
11.4.4 
New IND Submission and Updated 
Cover Page 
 
 
Clarified PEP-CMV dose 
 
 
 
Clarified and Corrected GM-CSF 
dose 
 
 
 
Clarified day of vaccine 
administration in relation to 
temozolomide cycles 
 
 
 
20131211 
via email 
20130307 
Study 
Synopsis, 
Section 11.1 
 
Study 
Synopsis, 
Section 11.1 
 
Table 3 
 
 
Section 19.1 
 
 
 
Section 15.3 
Revised DLT definition per FDA 
recommendation 
 
 
Primary Safety Assessment 
changed to occur on day 28 of the 
1st adjuvant cycle of TMZ 
 
Table provided for Schedule of 
Events 
 
Inserted standard language for 
prophylaxis treatment for 
Pneumomcystis Carinii 
 
Clarified Primary Endpoint for 
Immune Monitoring and comparison 
between the 3 arms 
 
20131211 
via email 
20131220 
Section 11.5.1 
 
 
Clarified vaccine treatment will end 
if DLT occurs 
 
20140724 
(AMD0003) 
20131223 
via email 
and via 

 
 
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Sections 11.6, 
11.7, 11.8, 
11.9, 11.10 
 
 
Section 16.1 
Added these sections to cover 
treatment period, end of treatment, 
end of study, follow up and 
withdrawal of subjects  
 
Corrected name of contact to 
receive pathology slides 
FED EX 
(1) 
20140801 
Cover Page 
and Section    
11.5.4 
Changed the PI 
20140808 
(AMD0005) 
 
20160714 
Study 
Synopsis, 
Study Schema 
Section 7.2, 
Sections 8, 11, 
12, 13, 15, 16 
& 19 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Updated and clarified the 
hypotheses and objectives; 
removed the reference to following 
RTOG 0525 for eligibility and 
updated the inclusion/exclusion 
criteria to match BTC studies, 
removing supratentorial component, 
clarifying complete or partial 
resection, & increasing KPS to 70% 
and adding Curran Group Status; 
removed one of the 3 
randomization arms; added Td 
booster as a requirement & Td pre-
conditioning before vaccine #1; 
changed enrollment numbers & 
clarified time of enrollment; clarified 
primary objective; changed first 3 
vaccines to bi-weekly; clarified 
difference between 5-day & 21-day 
TMZ and delay between cycles 1 
and 2; added definition and 
information on pseudoprogression; 
removed radiographic response 
section & referred to BTC SOPs; 
changed from term “DLT” to 
“unacceptable toxicities” and 
updated definition; updated/clarified 
immune monitoring time points; 
updated randomization model; 
updated/clarified definitions of 
“evaluable subject”, “on study”, “off 
study”, and “end of study”; removed 
XRT/TMZ time restraint and 
referred to SOC as a guideline thus 
removing those sections and 
referring reader to Appendices; 
updated follow up requirements; 
updated Schedule of Events; 
changed from paper documentation 
to electronic medical record; 
20160714 
(AMD0014) 
20160714 
(4) 

 
 
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Cover Page 
clarified progressive disease 
requires removal from study; 
deleted data and specimen 
collection section & outdated 
recruitment section & enrollment 
tables; included an accrual pause 
(rather than halt) for safety 
evaluation & updated the statistics, 
updated name of contact for 
pathology slides; added DSMB/COI 
language; updated references and 
bibliography throughout. 
Added Dr. Fecci to cover page. 
20160929 
Cover Page 
 
 
Study 
Synopsis, 
Table of 
Contents, 
Sections 7, 8, 
9, 10, 11, 12, 
13,15, 18, and 
Appendices 
Removed Sharon Norman and 
added Denise Jaggers 
 
Updated study drug, limited total 
vaccines to 20, changed timing of 
consent to after XRT/TMZ, updated 
hypotheses/objectives, updated 
CDX-11- drug information, included 
information on ELEVATE trial, 
added in missing exclusion 
criterion, removed all sections on 
XRT/TMZ, clarified TMZ therapy 
information, clarified timing of 
evaluable subject assessment, 
clarified CBC and CMP draws and 
window of vaccine administration, 
included the description of the Td 
pre-conditioning mixture, removed 
withdrawal information related XRT, 
updated follow-up requirements, 
updated immune monitoring and 
removed immunologic testing not 
being done due to change in study 
drug components, changed study 
“coordinator” to study “team”, 
updated schedule of events for 
these changes and included the 
study suspension after first 6 
subjects, updated the statistical 
section for change in PEP-CMV 
components, and included DCI 
SOC description. Finally, removed 
the reference to radiation and TMZ 
therapy in protocol as this is done 
prior to enrollment, as well as 
referred the reader to the TMZ PI 
uploaded in eIRB rather than in the 
 
20160929 
(5) 

 
 
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appendices, – and with these 
removals, updated the Table of 
Contents and References. 
20170106 
Study 
Synopsis, 
Sections 11 
and 12 
Clarified that screening consent will 
only be done if CMV status is not 
already known. 
20170106 
(AMD0027) 
 
20170110 
Study 
Synopsis and 
Section 10.1 
Added to inclusion criteria that the 
tumor must have a supratentorial 
component. 
20170110 
(AMD0028) 
 
20170221 
Sections 10, 
11.1, 11.2.2,  
12, 13, 14.2.2 
Removed one immune monitoring 
time point and clarified the other 
immune monitoring time point 
(Sections 11.1, 12, 13).   Modified 
TMZ completion requirement.  
Modified criteria for adjuvant TMZ 
initiation (Section 11.1.)  Updated 
inclusion/exclusion criteria (Section 
10).  Added dose reduction option 
(Section 11.2.2).  Section 14.2.2 
added to allow for the use of EMLA 
cream at the injection site. 
 
 
20170726 
Study 
Synopsis, 
Sections 10.1, 
11.1, 11.4.2, 
11.4.3, 11.5.1, 
11.5.2, 12.5, 
13.6, 15.3, 
and Table 3 
Curran Group status removed and 
specifications to steroid use added 
to the inclusion criteria (Sections 
10.1 and 12.5), required as 
baseline measurement.  A Study 
Drug Safety Arm was added to 
Sections 11.1, 11.4.3, and 15.3.  
Pre-medication with Zofran and 
extended follow-up procedure was 
added (Section 11.4.2). A post-
vaccine monitoring plan was added 
(Section 11.5.1).  Added Allergy 
Testing (Section 11.5.2).  Removed 
MMSE from Table 3 and Section 
13.6.  Removed appendix including 
the safety oversight committee. 
Changed PI to David Ashley.  
Removed protocol footer.  
 
 
20180124 
Sections 
11.4.3, 15.3, 
Table 3, and 
Figure 13  
Added immune monitoring blood 
draw volumes and modified tube 
top color (Table 3 and Section 
11.4.3).  Clarified the immune 
monitoring schedule for draws after 
vaccine #2 (Section 11.4.3).  
Updated Figure 13.  Modified Table 
3 footnote to clarify that pre- and 
post-XRT screening activities do 
 
 

 
 
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not need to occur at the same visit.  
Updated Schedule B, Vaccine #2 
corrective action (Section 15.3) to 
be consistent with wording in 
previous section, Section 11.4.3.  
20180622 
(submitted 
to FDA 
only) 
Sections 1, 9, 
11.1, 11.4.2, 
11.4.3, 11.5.1, 
11.6, 12.1, 13, 
15.1, and 15.3 
With this amendment, we complete 
the enrollment to the study drug 
safety arm. This amendment is to 
change the vaccine administration 
procedure after completion of the 
Study Drug Safety Arm. 
In addition to an administrative 
review, the following changes have 
been made: 
Updated key personnel on cover 
page. Added pre-medication with 
Tylenol (Sections 11.1 and 11.4.2). 
Updated the number of enrolled 
patients (Sections 1, 11.1, 15.1). 
Updated vaccine administration 
(Sections 1 and 11.1). Updated 
immune monitoring blood draws 
(Sections 1, 11.1, 12.1, 13). 
Removed old TMZ requirements 
(Section 11.1). Clarified clinical 
endpoint evaluable patients 
(Section 11.1). Specified that the 
Study Drug Safety Arms 
procedures are “not applicable to 
patients enrolled on protocol 
version 6/12/18 or later” (Sections 
11.1, 11.4.3, 15.3). Updated follow-
up schedule (Section 11.5.1). 
Removed accrual suspension after 
12 patients (Section 11.6). 
Toxicities will be formally monitored 
without accrual suspension 
according to Section 15.1. 
Additional administrative edits were 
made. 
 
 
20180719 
(includes 
20180622 
mods + 
FDA 
mods) 
Sections 1, 
10.1, 11.1, 
11.5.3, 14.2.4, 
and Table 3 
This amendment is in response to 
FDA concerns from amendment 
protocol version 20180622.  
Specified physiologic doses of 
hydrocortisone therapy (Sections 1 
and 10.1).  Added potential risk of 
steroid-induced adrenal 
suppression, including justification 
for steroid supplementation with 
physiologic levels of hydrocortisone 
 
 

 
 
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(Section 14.2.4).  Added cortisol 
levels to screening and prior to 
each vaccine (Table 3).  Updated 
unacceptable toxicity language to 
include vaccine-related ≥ Grade 3 
events, ≥ Grade 3 toxicity of any 
duration, and Grade 2 urticaria as a 
criteria for early withdrawal 
(Sections 1, 11.1, and 11.5.3).  
Minor administrative changes in the 
Appendix. 
20181218 
Sections 1, 
7.1, 8.1, 8.2, 
11.1, 15.1, 
11.4.2, 11.5.1, 
11.5.4, 12.6, 
13, 17.2, and 
Table 3 
Updated the clinical research 
coordinator to Kristen Fisher. 
Removed antibody analysis, which 
is irrelevant without Component B 
(Sections 1, 7.1, 8.1, 8.2, removed 
previous Section 13.3).  Created a 
Group 1 that follows the original 
treatment plan and then a Group 2 
that does not have Component B 
and has modified TMZ schedules - 
removed all but the initial adjuvant 
TMZ cycles for unmethylated 
patients (Sections 11.1, 15.1, Table 
3); removed Component B and 
changed Component A 
administration to be half in the right 
groin and half in the left groin for 
Group 2 (Sections 11.1 and 11.4.2). 
Made post-vaccine monitoring 
schedule consist with the rest of the 
protocol (Section 11.4.2). Removed 
FDA reporting of unrelated SAEs 
(Section 11.5.4). Due to the minimal 
toxicity seen in the 6 patient study 
drug safety cohort, the vital 
monitoring has been changed to 
every 30 (± 5) minutes (Sections 
11.4.2, 11.5.1, 12.6, Table 3). 
Added MGMT methylation 
screening to Table 3. Added a 3-
day interval to the 2nd and 3rd 
vaccine (Sections 1, 11.1, 11.4.2). 
Re-worded MRI schedule due to 
removal of TMZ cycles for 
unmethylated patients (Section 1). 
Updated immune monitoring 
schedule for Group 2 due to 
removal of Component B (Sections 
1, 7.2, 13, Table 3).  Clarified 
 
 

 
 
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Component A and Component B 
administration volumes (Sections 
11.4.2 and 11.4.4). 
20190312 
Sections 1, 
7.1, 7.2, 10.2, 
11.1, 11.4.1, 
11.4.2, 13, 
13.4, 15.1, 
15.2, 15.3, 
15.4.1, 15.4.2, 
15.4.3, 15.4.4, 
and Table 3 
Updated investigators.  Removed 
Component B for all active and 
future patients (Sections 1, 7.1, 7.2, 
11.1, 11.4.2, 15.1, 15.2, 15.3, 
15.4.1, 15.4.2, 15.4.4). Delete 
exclusion criterion for known allergy 
to ingredients of Component B 
(Sections 1, 10.2).  Change 
immune monitoring schedule due to 
removal of Component B (Sections 
1, 13, 15.4.3).  Change details 
regarding MGMT promoter 
methylation testing (Section 11.1, 
Table 3).  Remove incorrect 
language related to randomization 
(Section 11.4.1).  Fix and update 
footnote numbering in Table 3).  
Update Polyfunctional Analysis 
section for current practices 
(Section 13.4). 
 
 
20190730 
Sections 11.1, 
11.4.2, 11.5.1, 
12.1, 12.6, 
and Table 2 
Description of recent adverse 
events (Section 11.5.1).  
Revised/extended post-vaccine 
monitoring times after vaccines with 
associated updates to vital signs 
taken during the monitoring times 
(Sections 11.4.2, 12.1 [Table 2], 
and 12.6).  Addition of optional oral 
prednisone for patients with post-
vaccine reaction(s) (Sections 11.1 
and 11.4.2). 
 
 
20200117 
Sections 11.1, 
11.4.2, 11.5.1, 
11.5.3, 
11.10.1, 12.6, 
14.1, and 
14.2.1 
Add new supportive measures pre-
vaccine and in the event of a post-
vaccine reaction (Sections 11.1, 
11.4.2, and 11.5.1).  Qualify 
potential prednisone premedication 
as oral (Sections 11.5.1 and 12.6). 
Add history of vaccine reactions in 
study at interim analysis (Sections 
11.5.1 and 14.2.1).  Summarize 
results of recent immune analyses 
(Sections 11.5.1, 14.1, and 14.2.1).  
Add CRS Management Plan 
(Section 11.5.1).  Revise 
unacceptable toxicity definition 
(Section 11.5.3).  Reword criteria 
 
 

 
 
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for early withdrawal pertaining to 
corticosteroids (Sections 11.1 and 
11.10.1). Clarify enrollment goals 
for consenting, randomizing, and 
evaluable (Sections 1, 11.1, 15.1, 
and 15.2). 
20200826 
Sections 1, 5, 
11.1, 11.4.2, 
11.5.1, 11.5.2, 
11.5.4, 11.5.5, 
and 16.4 
Updated investigators.  Definition of 
unacceptable toxicity revised per 
FDA request under IND (Sections 
1, 11.5.4).  New abbreviation added 
(Section 5). Language added 
regarding how indeterminate 
MGMT methylation will be handled 
(Section 11.1).  Updates to safety 
precautions (Sections 11.1, 11.4.2).  
Revised CRS Management Plan 
per FDA request under IND 
(Section 11.5.1). New section 
added regarding use of reduced 
dose of bevacizumab for 
inflammatory response (Section 
11.5.2). CTCAE version clarified as 
4.03 (Sections 11.5.4, 11.5.5, 16.4) 
 
 
 
 
 

 
 
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1 STUDY SYNOPSIS 
Title 
PERFORMANCE Trial 
Study Drug: 
PEP-CMV is a vaccine originally comprised of two components (referred 
to as Component A and Component B).  Component A is a pp65 synthetic 
long peptide (SLP).  Component B consists of a neutralizing antibody 
epitope from human CMV glycoprotein B (gB) conjugated to KLH.  
Component B was removed from the vaccine as of 20190130 (change 
reflected in 20181218 version of the protocol). Component A is 
administered as a stable water:oil emulsion in Montanide ISA 51 
(Incomplete Freund’s Adjuvant). The previously included Component B 
was administered in aqueous solution with 150 µg of GM-CSF.   
Rationale: 
 
Radiation (RT) and temozolomide (TMZ) have efficacy in GBM, but are not 
curative. Adjuvant therapies are desperately needed.  CMV antigens have 
been 
identified 
in 
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. 
Therapeutic TMZ induces a profound lymphopenia that may enhance anti-
tumor vaccination responses when given during the homeostatic T-cell 
proliferation that occurs in response to lymphodepletion.  Other peptide 
vaccines given to patients with GBM during recovery from TMZ-induced 
lymphopenia have produced potent tumor-specific immune responses. Our 
Td pre-conditioning platform in the context of pp65 RNA-pulsed autologous 
dendritic cell (DC) vaccination also elicited superior anti-tumor responses 
compared to controls receiving DC vaccines without Td pre-conditioning. 
In our clinical trial (Pro00003877 ATTAC), patients with newly-diagnosed 
GBM who were administered the Td skin pre-conditioning before DC 
vaccination revealed significantly longer PFS and OS compared to the 
control cohort receiving unpulsed DCs. In evaluating the relationship 
between DC migration and clinical responses, we observed a modest 
positive association between levels of DC migration and survival.  
Primary Objectives: 
1. To assess the safety of PEP-CMV vaccination in combination with 
adjuvant TMZ. 
2. To determine the TMZ regimen that produces the highest number of 
T cells that specifically secrete IFNƴ by ELISPOT in response to 
component A of PEP-CMV.  
Secondary 
Objective: 
1. To determine if tumors are CMV antigen negative by 
immunohistochemical analysis and microarray analysis at the time of 
disease progression/recurrence. 
Exploratory 
Objectives: 
1. To quantitate the immune response to PEP-CMV vaccine by pp65 
ELISPOT. 
2. To determine the quality of the CMV pp65 immune response by 
polyfunctional flow cytometry  
3. To evaluate if Treg levels remain the same or decrease after 
vaccination. 
4. Identify if any HLA haplotypes respond better to the vaccine.  
5. To characterize immunologic cell infiltrate in tumors at the time of 
disease progression/recurrence.  
6. To describe PFS and OS within the two treatment groups. 
7. To estimate radiographic response rate to PEP-CMV in the subset of 
patients with residual disease. 
Inclusion Criteria: 
1. Age ≥ 18 years. 
2. Histopathologically proven newly-diagnosed primary glioblastoma 
with complete or partial surgical resection. Biopsy not acceptable. 
3. Patients must be CMV seropositive. 

 
 
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4. The tumor must be supratentorial. 
5. Karnofsky performance status of ≥ 70. 
6.  Stable or decreasing steroid dose (≤ 4 mg/day) at time of post-XRT 
adjuvant TMZ initiation.  If patients are decreasing steroid use, once 
they are at 2 mg/day, they may be supplemented with physiologic 
replacement hydrocortisone therapy (20-30 mg/day in divided doses), 
at the discretion of the treating oncologist.   
7. Hematology 
• 
ANC ≥ 1500 cells/µL 
• 
Platelet count ≥ 100,000 cells/µL 
• 
Hemoglobin ≥ 9.0 g/dl  
8. Chemistry 
• 
ALT/AST ≤ 3.0 times the upper limit of normal 
• 
Total bilirubin ≤ 1.5 x the upper limit of normal (Exception: 
Patient has known Gilbert’s Syndrome or patient has 
suspected Gilbert’s Syndrome, for which additional lab 
testing of direct and/or indirect bilirubin supports this 
diagnosis.  In these instances, a total bilirubin of ≤ 3.0 x 
ULN is acceptable.) 
Exclusion Criteria: 
1. Radiographic or cytologic evidence of leptomeningeal or multifocal 
disease at any time prior to randomization. 
2. Prior conventional antitumor therapy, other than steroids, RT or TMZ 
therapy given for glioblastoma. 
3. Pregnant or need to breast feed during the study period. 
4. Not adhering to pregnancy prevention recommendations. 
5. Active infection requiring intravenous antibiotics or an unexplained 
febrile (> 101.5o F) illness. 
6. Immunosuppressive disease or human immunodeficiency virus 
infection. 
7. Patients with unstable or severe intercurrent medical conditions such 
as severe heart or lung disease. 
8. Allergic or unable to tolerate TMZ for any reason. Any patient that 
successfully completed at least 5 weeks of Temodar during standard 
of care XRT/TMZ and whose blood counts meet the eligibility 
requirements (inclusion #7) within 5 weeks post XRT/TMZ is eligible.   
9. Patients with previous inguinal lymph node dissection, radiosurgery, 
brachytherapy, or radiolabeled monoclonal antibodies. 
10. Prior allogeneic solid organ transplant. 
11. Currently receiving or ever received immunosuppressive therapy for 
an autoimmune disorder or an organ transplant. 
Study Design: 
If their CMV serostatus is unknown, patients will be asked to consent to 
CMV screening either prior to or following XRT/TMZ.  Patients are enrolled 
to the study following XRT/TMZ and prior to initiation of post-XRT cycles 
of adjuvant TMZ, provided they meet all eligibility criteria.  After signing 
main consent, patients will undergo intramuscular (I.M.) Tetanus-
diphtheria booster vaccination with 0.5 mL of Td (tetanus, diphtheria 
toxoid, adsorbed). After meeting all eligibility criteria, patients will be 
randomized to one of two arms:  
 
• 
Arm 1 will receive  standard TMZ (150-200 mg/m2/day on days 1-5 of 
each 28-day cycle) with vaccination on Day 23 (-1 day, +2 days) of 
each TMZ cycle 
• 
Arm 2 will receive dose-intensified TMZ (75-100 mg/m2/day on days 1-
21 of each 28-day cycle) with vaccination on day 23 (±1 day) of each 
TMZ cycle.  

 
 
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Beginning with protocol version 20181218, patients who are MGMT 
unmethylated will only receive one adjuvant cycle of the TMZ regimen 
according to their assigned randomized arm. Patients who are MGMT 
methylated or whose methylation status is inconclusive will continue with 
up to 12 cycles of TMZ. All patients enrolled prior to approval of protocol 
version 20181218 will receive 6-12 cycles of TMZ, independent of their 
MGMT methylation status.  Patients who are dependent on steroid 
supplements above immune suppressive levels (4 mg) at time of 
vaccination or who are unable to tolerate TMZ will be withdrawn from the 
study and replaced before vaccination.    
 
Up to 70 patients may be consented on the main informed consent to meet 
the goal of 26 evaluable patients (13 per arm) for the primary outcomes.  
 
For both arms, the patients must be screened at Duke within 3 ± 1 weeks 
after the completion of standard of care radiation.  For both arms, the initial 
cycle of adjuvant TMZ will begin as soon as possible following 
randomization. For Arm 1, the adjuvant TMZ cycle(s) will be given at a 
standard targeted dose of 150-200 mg/m2/d for 5 days. For Arm 2, the 
adjuvant TMZ cycle(s) will be at the dose-intensified dose of 75-100 
mg/m2/d for 21 days.  If a patient enrolls after approval of protocol version 
20181218 and has an MGMT unmethylated tumor, they will discontinue 
TMZ after the 1st cycle.   
 
All patients will have blood drawn for immune monitoring and then receive 
a tetanus pre-conditioning injection intradermally (i.d.) in the RIGHT groin 
(as described below) on day 22 (+1 day) of cycle 1 of adjuvant TMZ. On 
the following day, the patients will receive study vaccine. The vaccine is 
administered as follows: 500 µg of PEP-CMV Component A mixed with 
Montanide ISA-51 administered i.d. with half on the right groin and half on 
the left groin. Vaccines #2 and #3 will be given at 2 week (+ 3 days) 
intervals; the MRI visit with immune monitoring will take place within 2 
weeks (+3 days) of vaccine #3.  Prior to the 20181218 amendment, 
vaccination consisted of two components: Component A and Component 
B. However, after that amendment, vaccination for all newly enrolled 
patients consisted of Component A only. 
 
For patients enrolled before approval of protocol version 20181218 and for 
patients enrolled after approval of protocol version 20181218 who have an 
MGMT methylated tumor or MGMT methylation status in inconclusive, 
TMZ cycle 2 will begin after that visit, but no sooner than 14 days post 
vaccine 3.  This will result in at least ~35-day delay before starting TMZ 
cycle 2.  MGMT unmethylated patients enrolled after approval of protocol 
version 20181218 will not receive subsequent cycles of TMZ, but will 
continue to receive vaccines approximately every 4 (+2) weeks. 
 
An unacceptable toxicity will be defined as any ≥ Grade 3 toxicity possibly, 
probably, or definitely related to the PEP-CMV vaccine with the following 
exceptions. A portion of the Grade 3 vaccine reactions noted in prior 
patient experiences with the PEP-CMV vaccine, described in Sections 
11.5.1 and 14.2.1, will not be considered unacceptable if they are indicative 
of immune response.  That is, toxicities such as Grade 3 flu-like symptoms, 
fever, and chills/rigors will not be considered unacceptable toxicities if the 
duration is less than 72 hours. Any ≥ Grade 3 organ toxicity (cardiac, renal, 
hepatic), including CRS-related toxicities such as hypotension and 

 
 
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tachycardia, of any duration will be considered an unacceptable toxicity.  
Although not considered an unacceptable toxicity, any patient with ≥ Grade 
2 urticaria will not receive further vaccines, will be withdrawn from the 
study, and will be replaced if less than 3 vaccines have been administered 
without unacceptable toxicity.  The prevalence of unacceptable toxicities 
occurring during the initial 3 bi-weekly vaccinations or the vaccinations 
administered concurrently with temozolomide will be continuously 
monitored.  If more than 25% of accrued patients experience unacceptable 
toxicities, then accrual will be suspended and reported toxicity will be 
carefully reviewed to determine if modifications to the protocol treatment 
should occur.  Peptide vaccinations employing Montanide ISA-51 as 
adjuvants have generally been well-tolerated in human patients in 
numerous phase I-III trials.  
 
Treatment Plan:  Patients will be vaccinated in conjunction with 
subsequent 28-day cycles for a total of 6 to 12 adjuvant TMZ cycles after 
RT if patient is continuing on TMZ (i.e., for patient’s who’s tumor is 
methylated or the status is inconclusive). Cycles are every 4 (+ 2) weeks, 
adjusting for slight delays on startup or scheduling of each 28-day cycle.  
For patients whose tumor is methylated or the status is inconclusive, the 
total number of TMZ cycles given will be at the discretion of the treating 
oncologist.  During the 28-day cycles, vaccinations will occur on day 23 (-
1 day, + 2 days) of each cycle for each arm: (Arm 1) standard TMZ (200 
mg/m2/day on days 1-5 of each 28-day cycle), or (Arm 2) dose-intensified 
TMZ (100 mg/m2/day on days 1-21 of each 28-day cycle).   
 
All vaccines will be given i.d. approximately 10 cm below the inguinal 
ligament bilaterally. Component A will be administered half in the RIGHT 
groin and half in the LEFT groin at a maximum of 0.2 mL per intradermal 
injection. A target of six cycles with maximum of twelve cycles of TMZ may 
be given to patients enrolled before approval of protocol version 20181218 
and to methylated patients enrolled after approval of protocol version 
20181218, at the discretion of the treating neuro-oncologist.  Unmethylated 
patients enrolled after approval of protocol version 20181218 will receive 
only one cycle of TMZ.  After the completion of a patient’s last TMZ cycle, 
vaccines will continue every 4-6 weeks for a maximum number of 20 
vaccines (unless tumor progression occurs). 
 
Patients will be imaged with contrast-enhanced MRI within 2 weeks (+3 
days) after vaccine 3 and then approximately every 8 weeks [every 2 
cycles thereafter (i.e., end of cycles 3, 5, 7, 9, 11)]. RANO criteria will be 
used for assessment of pseudo-progression, and patients demonstrating 
definitive progression will be removed from study. Any patient removed 
prior to immune monitoring post vaccine 3 will be replaced for immunologic 
endpoints. Clinical endpoint comparisons will be made amongst patients 
successfully randomized to adjuvant TMZ treatment arms and receiving at 
least one vaccine.  
 
Blood for immune monitoring will be obtained: 
• 
Prior to Td pre-conditioning 
• 
During vaccines 1 and 2 (just prior to Component A, 1 and 2 hours 
after Component A 
• 
Prior to vaccine 3 
• 
After vaccine 3 (within 2 weeks of vaccine 3 [+ 3 days]) 
• 
Prior to vaccines 4 and 6 

 
 
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• 
At tumor progression (if feasible)  
 
As part of standard care for these patients, upon tumor progression, 
participants may undergo stereotactic biopsy or resection.  As this is not a 
research procedure, consent will be obtained separately. Patients that 
have this procedure done here at Duke University Health System may be 
approached to participate in the Duke Brain Tumor Center Biorepository 
study (Pro00007434).  Tissue obtained from patients who consented to the 
Duke Brain Tumor Center Biorepository will be used to assess 
immunologic cell infiltration, antigen expression, and biomarkers for 
immunologic response. 
 
Study Drug Safety Arm: 
Following the severe adverse event that occurred on 4/5/17, a small study 
drug safety arm of 6 patients was included in this study.  These 6 patients 
will not contribute to the overall statistical evaluation of the study, but 
instead, will be used to determine how the study drug administration or the 
study drug itself should be modified to reduce the hypersensitivity reactions 
in patients. 
 
Protocol version 6/12/18 updates the protocol based on the information 
gathered from the completed Study Drug Safety Arm.  All patients enrolled 
on the study after completion of the Study Drug Safety Arm toxicity follow-
up (~6/6/18) will NOT follow the Study Drug Safety Arm modifications.   
Statistical Analysis 
Of primary interest in this study is a comparison of the 2 arms with respect 
to peak immune response (ELISPOT) in the immune monitoring blood that 
will be collected as described in the treatment plan. The ACT II study 
conducted at Duke provides antibody titer data that can be used to 
estimate an appropriate sample size for comparison of antibody 
response.  ELISPOT data is not available.  The ACT II dataset provides 
peak antibody titer to EGFRvIII data for 5 patients in Arm A (treated with 5 
day TMZ) and 10 patients in Arm B (treated with 21 day TMZ).  Given that 
the distribution of peak titers in the two arms is not normally distributed, 
resampling methods were used to estimate the power of a comparison 
using a one-tailed Wilcoxon rank sum test.  Each of 10000 simulations for 
a fixed sample size per arm involved random sampling with replacement 
the peak antibody data separately within arm A and B, and then computing 
the Wilcoxon rank sum test.  The percentage of tests that was statistically 
significant estimated the power of the statistical test.  With 2 primary 
endpoints, power calculations assumed a type I error rate of 0.025.  With 
13 evaluable patients per arm having immune monitoring done prior to 
vaccine 4, there is 81% power to detect a difference similar to that 
observed in ACT II.   
 
With the goal of 26 evaluable patients (13 per arm) for the primary 
outcomes, a maximum of 70 patients may be consented on the main 
informed consent to meet this goal.  With the 20190312 protocol 
amendment, the primary analysis dataset includes only patients who 
receive Component A alone (See Section 15.2).  Hence, after this 
amendment, the study targets the accrual of 26 patients (13 per arm) who 
receive only Component A and are evaluable for the assessment of 
immunologic response. 
 
Following the safety events of vaccine reactions that were initially reported 
on 20170407, the study was amended to investigate, in a separate 

 
 
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unrandomized drug safety cohort of 6 patients, the etiology of these 
events.  Within this cohort, patients were consented to receive study 
vaccine with standard of care (SOC) 5-day TMZ at 150-200 mg/m2/d for 
28-day cycles with various sequences of vaccine components A and B. 
Details of this safety assessment plan are provided in Section 11.4.3.  
Upon completion of this unrandomized portion of the study, the 
randomized study was allowed to reinitiate with changes to vaccine 
administration procedures based on observations made in the safety 
cohort (protocol v. 20180719). Subsequently, Component B was removed 
from the vaccine for future patients due to problems with availability and 
supply (protocol v.20181218). On 20190130, Component B was removed 
from the vaccine for all patients (current and future) due to two new safety 
events observed and reported in January 2019. 

 
 
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2 TABLE OF CONTENTS 
1 
STUDY SYNOPSIS ............................................................................................... 10 
2 
TABLE OF CONTENTS ........................................................................................ 16 
3 
LIST OF FIGURES ................................................................................................ 17 
4 
LIST OF TABLES .................................................................................................. 18 
5 
LIST OF POSSIBLE ABBREVIATIONS ................................................................ 19 
6 
DEFINITION OF TERMS ....................................................................................... 21 
7 
ABSTRACT AND STUDY SCHEMA ..................................................................... 22 
 
Abstract ..........................................................................................................22 
 
Study Schema ................................................................................................24 
8 
HYPOTHESES AND OBJECTIVES ...................................................................... 26 
 
Hypotheses ....................................................................................................26 
 
Objectives ......................................................................................................27 
9 
BACKGROUND AND RATIONALE ...................................................................... 28 
 
Disease and Current Therapy ........................................................................28 
 
Rationale for Immunotherapy .........................................................................28 
 
CMV in GBM ..................................................................................................29 
 
Polyfunctional T-cell Responses and Anti-Viral Immunity ...............................31 
 
Temozolomide, Lymphopenia, and Homeostatic Proliferation ........................33 
 
Tetanus Diphtheria Toxoid (Td) ......................................................................36 
 
Phase I/II Trials in GBM Targeting CMV .........................................................37 
 
Peptide Vaccines for Cancer ..........................................................................38 
 
Tumor-Specific Immunotherapy in Patients with GBM ....................................39 
10 
STUDY POPULATION .......................................................................................... 40 
 
Inclusion Criteria ............................................................................................40 
 
Exclusion Criteria ...........................................................................................41 
11 
INVESTIGATIONAL PLAN.................................................................................... 42 
 
Overview ........................................................................................................42 
 
Temozolomide Therapy .................................................................................45 
 
Pseudoprogression ........................................................................................46 
 
Treatment Procedures ...................................................................................46 
 
Safety, Toxicity, and Adverse Events .............................................................51 
 
Treatment Period ...........................................................................................55 
 
End of Treatment ...........................................................................................55 
 
Definition of Evaluable Subjects, On Study, and End of Study .......................56 
 
Follow-up Period ............................................................................................56 
 Early Withdrawal of Subject(s) .......................................................................56 
12 
DESCRIPTION OF STUDY EVALUATIONS ......................................................... 58 
 
Schedule of Events ........................................................................................58 
 
Medical History ..............................................................................................59 
 
Physical and Neurological Examination ..........................................................59 
 
Karnofsky Performance Status Score.............................................................60 
 
Curran Group Status ......................................................................................60 
 
Vital Signs ......................................................................................................60 
 
Clinical Laboratory Assessments ...................................................................60 
 
Measurement of Radiographic Response ......................................................60 
13 
IMMUNOLOGICAL RESPONSE AND SAFETY EVALUATIONS ......................... 61 
 
Serum Sample and PBMC Collection, Processing and Storage .....................61 
 
Enzyme-Linked Immunospot (ELISPOT) Assay .............................................61 

 
 
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TReg Levels ...................................................................................................62 
 
Polyfunctional Analysis ..................................................................................62 
 
Tetramer Analysis ..........................................................................................63 
14 
SAFETY MONITORING ........................................................................................ 63 
 
Potential Benefits ...........................................................................................63 
 
Potential Risks ...............................................................................................63 
 
Data and Safety Monitoring Plan ....................................................................67 
15 
STATISTICAL CONSIDERATIONS ...................................................................... 68 
 
Study Design and Conduct .............................................................................68 
 
Sample Size Justification for Immunologic Response Outcome .....................69 
 
Toxicity Monitoring .........................................................................................70 
 
Analytic Methods ............................................................................................71 
16 
DATA MANAGEMENT AND PROCESSING ......................................................... 73 
 
Study Documentation .....................................................................................73 
 
Data Management..........................................................................................73 
 
Data Management Procedures and Data Verification .....................................74 
 
Coding ...........................................................................................................74 
 
Study Closure ................................................................................................74 
17 
ADMINISTRATIVE AND ETHICAL CONCERNS .................................................. 74 
 
Regulatory and Ethical Compliance ...............................................................74 
 
DUHS Institutional Review Board and DCI Cancer Protocol Committee .........74 
 
Informed Consent ..........................................................................................75 
 
Privacy, Confidentiality, and Data Storage .....................................................75 
 
Data and Safety Monitoring ............................................................................76 
 
Protocol Amendments ....................................................................................76 
 
Records Retention .........................................................................................76 
 
Conflict of Interest ..........................................................................................76 
 
Registration Procedure ..................................................................................77 
18 
REFERENCES ...................................................................................................... 78 
19 
APPENDICES ....................................................................................................... 87 
 
Appendix A - Pneumocystis Carinii Management ...........................................87 
 
Appendix B – TMZ Package Insert .................................................................88 
 
Appendix C – DSMBplus Charter ...................................................................89 
 
Appendix D – SOPs and FORMs ...................................................................90 
 
3 LIST OF FIGURES 
Figure 1. Study Schema for Patients Enrolled Prior to Approval of Protocol v. 20181217
 ......................................................................................................................................24 
Figure 2. Study Schema for Patients Enrolled After Approval of Protocol v. 20181217 ..25 
Figure 3: Western Blot Detection of CMV IE1, pp65, and gB Proteins in Freshly 
Resected GBM Samples. ..............................................................................................29 
Figure 4:  IHC and ISH on GBM specimen ....................................................................30 
Figure 5: Polychromatic Flow Cytometry to Analyze Polyfunctional CD4+ and CD8+ T 
cells: ..............................................................................................................................32 
Figure 6: Lymphocyte Counts of Patients with Newly-Diagnosed GBM Receiving 
Standard Dose Adjuvant TMZ (200 mg/m2/5d) and Dose-Intensified TMZ (100 
mg/m2/21d). ..................................................................................................................33 
Figure 7: Antibody Titers in Patients During Standard-Dose TMZ (200 mg/m2/5d) and 
Dose-Intensified TMZ (100 mg/m2/21d) are Shown. ......................................................34 

 
 
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Figure 8: DTH Responses .............................................................................................35 
Figure 9: Progression Free Survival (PFS) Percentage .................................................38 
Figure 10: Increase in Frequency of pp65-Specific T cells in Patients with GBM 
Receiving DC Vaccination and Standard-Dose TMZ. ....................................................40 
Figure 11: DTH Responses to pp65 RNA Pulsed DC Vaccines. ....................................40 
Figure 12: Study Drug Safety Arm Study Schema for Vaccine #1. .................................48 
Figure 13: Study Drug Safety Arm Study Schema for Vaccine #2 if the Patient 
Experiences No Hypersensitivity Reaction to Vaccine #1. .............................................48 
4 LIST OF TABLES 
Table 1. Temozolomide Dose Delay, Reduction, or Discontinuation ..............................45 
Table 2. CRS Management Plan based upon the ASTCT Consensus Grading .............52 
Table 3: Schedule of Events ..........................................................................................58 
 

 
 
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5 LIST OF POSSIBLE ABBREVIATIONS 
Ab 
Antibody 
ABC 
Automated Blood Count 
ACD 
Acid Citrate Dextrose 
ACLS 
Advanced Cardiac Life Support 
ACTH 
Adrenocorticotropic Hormone 
APAAP 
Alkaline Phosphatase Antialkaline Phosphatase Complex 
ALT 
Autologous Lymphocyte Transfer 
AST 
Aspartate Aminotransferase 
ASTCT 
American Society for Transplantation and Cellular Therapy 
AT 
Ambient Temperature 
β-HCG 
Beta-Human Chorionic Gonadotropin 
BTSC 
Brain Tumor Stem Cells 
Ca++ 
Calcium 
cDNA 
Complimentary Deoxyribonucleic Acid 
CFA 
Complete Freund’s Adjuvant 
CFC 
Cytokine Flow Cytometry 
CLIA 
Clinical Laboratory Improvement Act 
CMP 
Comprehensive Metabolic Panel 
CMV 
Cytomegalovirus 
CNC 
Clinical Neurologic Change 
CNS 
Central Nervous System 
Con-A 
Concanavalin A 
CPC 
Cancer Protocol Committee 
CRS 
Cytokine Release Syndrome 
CT 
Computed Tomography 
CTL 
Cytotoxic T-Lymphocyte 
DC 
Dendritic Cell 
DI 
Dose Intensified 
DLT 
Dose Limiting Toxicity 
DTH 
Delayed-type Hypersensitivity 
EAE 
Experimental Autoimmune Encephalomyelitis 
EBRT 
External Beam Radiation Therapy 
ELISA 
Enzyme-Linked ImmunoSorbent Assay 
ELISPOT 
Enzyme-linked Immunospot 
EGFR 
Epidermal Growth Factor Receptor 
EGFRvIII-KLH 
EGFRvIII conjugated to Keyhole Limpet Hemocyanin 
FACS 
Fluorescence Activated Cell Sorting 
FDA 
Federal Drug Administration 
FEV 
Forced Expiratory Volume 
gB 
Glycoprotein B 
GBM 
Glioblastoma 
GM-CSF 
Granulocyte Macrophage Colony Stimulating Factor 
HIV 
Human Immunodeficiency Virus 
HLA 
Human Leukocyte Antigen 
I.D. 
Intradermal 
IFN-γ 
Interferon-gamma 

 
 
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IHC 
Immunohistochemistry 
IL-4 
Interleukin-4 
IL-12 
Interleukin-12 
IL-13 
Interleukin-13 
IRB 
Institutional Review Board 
KLH 
Keyhole Limpet Hemocyanin 
KPS 
Karnofsky Performance Status 
MAb 
Monoclonal Antibody 
MG 
Malignant Glioma 
MHC 
Major Histocompatibility Complex 
MRI 
Magnetic Resonance Imaging 
mRNA 
Messenger Ribonucleic Acid 
MTD 
Maximally Tolerated Dose 
NA 
Non-adherent 
NCI CTC 
National Cancer Institute Common Toxicity Criteria 
NIH 
National Institutes of Health 
NK 
Natural Killer 
OS 
Overall Survival 
PBMC 
Peripheral Blood Mononuclear Cells 
PD 
Progressive Disease 
PHA 
Phytohemagglutinin 
PFS 
Progression Free Survival 
PI 
Principle Investigator 
PT 
Prothrombin Time 
PTT 
Partial Thromboblastin Time 
RANO 
Revised Assessment in Neuro-Oncology 
RNA 
Ribonucleic Acid 
RT 
Radiation Therapy 
RT-PCR 
Reverse Transcriptase Polymerase Chain Reaction 
SAE 
Severe Adverse Event 
SLPs 
Synthetic Long Peptides 
SOC 
Standard of Care or Safety Oversight Committee 
SPORE 
Specialized Program of Research Excellence 
TCR 
T-cell Receptor 
TGF-β 
Transforming Growth Factor-β 
TMZ 
Temozolomide 
TNF-α 
Tumor Necrosis Factor-α 
TRegs 
Regulatory T-cells 
TTP 
Time to Progression 
TTRNA 
Total Tumor mRNA 
WHO 
World Health Organization 
XRT 
External Beam Radiation Therapy 
XRT/TMZ 
Temozolomide During Concomitant Radiation Therapy 
 

 
 
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6 DEFINITION OF TERMS 
Term 
Definition 
Date of diagnosis 
Earliest confirmation by radiographic or 
histologic examination 
Date of randomization 
Date TMZ regimen is assigned. 
Date of progression 
Earliest date of surgical confirmation of 
disease progression. Where surgery is 
not feasible, earliest date of radiographic 
confirmation of progression. 
Enrolled patient 
Patient who signs the informed consent 
form and meets all eligibility criteria. 

 
 
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7 ABSTRACT AND STUDY SCHEMA 
 Abstract 
Despite aggressive, computer-guided surgery, high-dose focused radiation, and toxic, 
multimechanistic chemotherapy, 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[1].  The inherent biologic 
specificity of immunotherapy, however, offers the prospect of targeting neoplastic cells 
more precisely. 
The recent discovery that GBMs, but not surrounding normal brain tissue, serve as a 
refuge for CMV reactivation  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 CNS, and antitumor immunotherapy targeting viral proteins in human CNS 
tumors associated with Epstein-Barr virus (EBV), another Herpesvirus, have been 
curative[2-7].  Similarly, vaccinations directed against the highly immunogenic antigens of 
human papilloma virus have been shown to reduce the incidence of cervical intraepithelial 
neoplasia in a prospective, randomized, double-blind trial[8]. 
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 who are MGMT methylated, and TMZ is now 
frequently given to these patients during and after RT.  Therapeutic TMZ, however, also 
induces a profound lymphopenia.  ALT after TMZ therapy may provide a source of T-cells 
that remain receptive to vaccination in this context while potentially protecting the patient 
from opportunistic infections as well.  In addition, the homeostatic proliferation of T-cells 
that is triggered in response to lymphopenia may even potentiate anti-tumor vaccine or 
adoptive T-cell responses [9]. 
Our recent clinical trial with DC-based vaccination targeting the immunodominant CMV 
antigen pp65 has produced immunologic responses in patients with GBM undergoing 
standard of care TMZ along with encouraging PFS (15.4 months, CI95: 10.0, 27.8).  In 
order to advance vaccines targeting CMV in GBM, we have developed a CMV-specific 
peptide (PEP-CMV). PEP-CMV is a vaccine comprised of two components (referred to as 
Component A and Component B).  Component A, pp65 synthetic long peptide (SLP) is a 
single peptide from human CMV matrix protein pp65SLP.  Component B consists of a 
neutralizing antibody epitope from human CMV glycoprotein B conjugated to KLH (gB-
KLH).  Component A will be administered as a stable water:oil emulsion in Montanide ISA 
51 (Incomplete Freund’s Adjuvant). The gB-KLH conjugate (referred to as Component B) 
will be administered in aqueous solution with 150 µg of GM-CSF. Due to insufficient 
manufacturing of Component B followed by safety events in a subset of patients who 
received Component B, no patients will receive Component B as of 20190130. 
Our preliminary data demonstrating the capacity to increase DC migration to VDLNs 
via Td pre-conditioning of the vaccine site offer potential therapeutic interventions whereby 
we can enhance the immunologic responses to ultimately overcome the inherent 
challenges in faithfully eradicating established tumors.  Our Td pre-conditioning platform 
in the context of DC vaccination also elicited superior anti-tumor responses compared to 
controls receiving DC vaccines without Td pre-conditioning. In our clinical trial 
(Pro00003877 ATTAC), patients with newly-diagnosed GBM who were administered the 

 
 
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Td skin pre-conditioning before DC vaccination revealed significantly longer PFS and OS 
compared to the control cohort receiving unpulsed DCs. In evaluating the relationship 
between DC migration and clinical responses, we observed a positive association 
between levels of DC migration and survival.  In addition, based on our preliminary data 
that Td vaccine site pre-conditioning enhances the immunogenicity of PEP-CMV in HLA-
A2 transgenic mice, all patients will also receive a Td vaccine site pre-conditioning in the 
RIGHT groin and the following day receive the PEP-CMV vaccine.  
In this protocol, the goal is to determine the safety of PEP-CMV vaccine with adjuvant 
TMZ, and what TMZ regimen produces the highest number of T cells that specifically 
secrete IFNγ by ELISPOT in response to PEP-CMV component A. 

 
 
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 Study Schema  
 
1Figure 1. Study Schema for Patients Enrolled Prior to Approval of Protocol v. 20181217 
  
 
 
                                               
1 Originally, patients received both Component A and Component B and methylation status did not inform 
TMZ schedule.  Please note that a decision was made to eliminate Component B for all patients on study for 
safety reasons (January 2019) and this immediate change was approved via a protocol deviation. 

 
 
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2Figure 2. Study Schema for Patients Enrolled After Approval of Protocol v. 20181217 
 
 
 
                                               
2 With approval of Protocol v.20181217, newly enrolled patients receive Component A alone and methylation 
status informs subsequent TMZ schedule.  Please note that Component B was initially eliminated in protocol 
v.20181217 for future patients due to issues with supply.  The decision mentioned above in Footnote 1 and 
the aforementioned protocol deviation eliminated Component B for all current patients, in addition to future 
patients.   

 
 
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8 HYPOTHESES AND OBJECTIVES 
 Hypotheses 
• 
Our primary hypothesis is that: 
 
o Treatment of adult patients with newly diagnosed GBM using PEP-
CMV during recovery from TMZ-induced lymphopenia in patients that 
are seropositive for CMV will be safe. 
 
o Treatment of adult patients with newly diagnosed GBM using PEP-
CMV during recovery from TMZ-induced lymphopenia in patients that 
are seropositive for CMV will induce cellular immune responses 
through IFNƴ secretion of total Component A specific T cells using 
ELISPOT.  
 
• 
Secondary hypotheses include: 
 
o Tumors are CMV antigen negative by immunohistochemical analysis 
and 
microarray 
analysis 
at 
the 
time 
of 
disease 
progression/recurrence. 
 
• 
Exploratory hypotheses include: 
 
o The immune response to the PEP-CMV vaccine is detectable by pp65 
ELISPOT. 
 
o The quality of immune responses determined by polyfunctional flow 
cytometry is multifunctional or monofunctional using the pp65 peptide 
in PEP-CMV.  
 
o Tregs will decrease after vaccination with PEP-CMV in combination 
with TMZ. 
 
o Different HLA haplotypes respond better to PEP-CMV vaccine in 
combination with TMZ. 
 
o 
At the time of disease recurrence, immunologic tumor cell infiltrate 
will be evident. 
 
o    That PFS and OS within the 2 arms will provide information on the 
effects between the 2 TMZ regimens.  
 
Treatment of adult patients with newly diagnosed GBM using PEP-CMV during 
recovery from TMZ-induced lymphopenia in patients that are seropositive for 
CMV will induce objective radiographic responses in this patient population in the 
subset of patients with residual disease. 
 

 
 
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 Objectives 
8.2.1 Primary Objective 
• 
To assess the safety of PEP-CMV vaccination in combination with 
adjuvant TMZ. 
• 
To determine the TMZ regimen that produces the highest number of T 
cells that specifically secrete IFNƴ by ELISPOT in response to PEP-
CMV component A. 
8.2.2 Secondary Objectives 
• 
To 
determine 
if 
tumors 
are 
CMV 
antigen 
negative 
by 
immunohistochemical analysis and microarray analysis at the time of 
disease progression/recurrence. 
8.2.3 Exploratory Objectives 
• 
To quantitate the immune response to the PEP-CMV vaccine by pp65 
ELISPOT. 
• 
To determine the quality of the immune response by polyfunctional flow 
cytometry using the pp65 peptide in PEP-CMV. 
• 
To evaluate if Treg levels remain the same or decrease after 
vaccination. 
• 
Identify if any HLA haplotypes respond better to the vaccine.  
• 
To characterize immunologic cell infiltrate in tumors at the time of 
disease progression/recurrence.  
• 
To describe PFS and OS within the 2 arms. 
• 
To estimate radiographic response rate to PEP-CMV in the subset of 
patients with residual disease. 
 
 

 
CONFIDENTIAL 
  
9 BACKGROUND AND RATIONALE 
 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 [10], 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[11-16]. 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. 
Moreover, the non-specific nature of conventional therapy for brain tumors often results in 
incapacitating damage to surrounding normal brain and systemic tissues[1, 17]. 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. 
 Rationale for Immunotherapy 
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 precise antitumor therapy. Recently, TMZ, a myelosuppressive alkylating 
chemotherapy, has shown a benefit in patients with GBM who are MGMT methylated, but 
median survival for GBM is still less than 15 months[14]. Moreover, these conventional 
therapies lack specificity and result in incapacitating damage to surrounding normal brain 
and systemic tissues[1]. Immunotherapy may provide an opportunity to eliminate altered 
neoplastic cells without adding additional toxicity to multi-modality therapy, but the 
lymphopenia induced by cycle(s) of adjuvant TMZ, now the standard-of-care in patients 
with GBM, would be predicted to curtail the induction of productive antitumor immune 
responses. However, our vaccines targeting EGFRvIII, when given to patients with GBM 
during recovery from TMZ-induced lymphopenia, have produced strong immunologic 
responses that eliminate tumor cells expressing the targeted antigen and are accompanied 
by complete radiographic responses in all patients with residual disease and patient 
survival that significantly exceeds that of historical controls[18-21]. We believe, and our 
preliminary data in mice and humans strongly support, that an environment uniquely 
susceptible to the induction of potent immune responses is created within the host during 
recovery from TMZ-induced lymphopenia. This hypothesis is supported by basic 
investigations into lymphocyte homeostasis, which have shown that, during periods of 
lymphopenia, recovering lymphocytes have a significantly lowered threshold for activation, 
but they remain dependent on limiting amounts of homeostatic cytokines and antigen-
specific stimulation for survival and proliferation.  As a result, lymphocytes that encounter 
their cognate antigen during homeostatic proliferation, perhaps in the form of a vaccine, 
are given a competitive advantage. Although we have been able to successfully target 

 
 
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EGFRvIII in this context, only 25-30% of patients with GBM express this mutation and the 
outgrowth of recurrent tumors that no longer express EGFRvIII highlights the need to 
effectively target more ubiquitously expressed antigens in GBMs.  
 CMV in GBM 
The recent discovery and confirmation by five independent laboratories[20, 22-26], 
including our own, that cytomegalovirus (CMV) propagates within a high proportion of 
GBMs (>90% of examined GBMs) without infecting surrounding normal brain provides an 
unparalleled opportunity to subvert the highly immunogenic antigens from CMV as tumor-
specific targets.  Cobbs et al., first reported the associated of CMV with malignant 
gliomas[22]. We recently reported the first confirmatory report of the expression of CMV 
antigens in greater than 90% of GBM specimens without expression in normal brain[23]. 
To date, five independent laboratories from major academic medical centers (University of 
Alabama, Duke University, UCLA, MD Anderson Cancer Center, and the Karolinska 
Institute in Stockholm, Sweden) have confirmed this association.   We and others have 
demonstrated the presence of CMV early and late antigens in GBM tumors by 
immunohistochemisty, in situ hybridization, real-time PCR, viral DNA sequencing, and 
electron microscopy in published materials[22, 23, 25, 26].  In additional data, we have 
confirmed that the detection of CMV proteins by monoclonal antibodies in IHC is specific 
for CMV antigens using Western blot analysis of freshly resected GBM specimens (Figure 
3).  
 
Figure 3: Western Blot Detection of CMV IE1, pp65, and gB Proteins in Freshly Resected 
GBM Samples.  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 the 
abundance of CMV proteins in laboratory strain infected lysates 1/40th the amount of lysate relative 
to GBM sample was loaded onto gels for Western blot detection.  Detection of IE1 has been 
observed in 14/22 (63.6%) analyzed GBM samples and pp65 detection was positive in 19/22 
(86.3%) analyzed tumors.  gB envelope protein was detected in 16/16 (100%) tumors chosen 
based on positivity for pp65.  Normal brain lysates derived from autopsy specimens were negative 
in 5/5 cases (median age 64) not shown (0%), p<0.0001. 
 
CMV is an endemic β-Herpesvirus that does not usually cause significant clinical 
disease in adults.  Herpesviruses have previously been implicated in a number of human 
malignancies including lymphoma, nasopharyngeal cancer, cervical cancer, and Kaposi’s 
sarcoma[20, 27, 28]. Expression of proteins unique to CMV has been reported in a large 
proportion of GBMs[22].  Near universal detection of the CMV immunodominant proteins 
pp65, glycoprotein B (UL55), and immediate early gene 1 protein (IE1) in GBM has been 
 
 
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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confirmed by us and others by immunohistochemistry (IHC), in situ hybridization (ISH), and 
polymerase chain reactions (PCR)[22-25] (Figure 4).   
Presence of the virus in these samples was confirmed by electron microscopic 
detection of intact virions[22].  CMV antigens were not detected in surrounding normal  
 
 
Figure 4:  IHC and ISH on GBM Specimen  IHC on GBM specimen: a.  Isotype control antibody; 
b.  anti-smooth muscle actin c.  anti-CMV IE1 antibody (40x); d.  anti-CMV IE1 antibody (200x); e. 
anti-CMV pp65 antibody (40x); f.  anti-CMV pp65 antibody (100x).  ISH on GBM specimens:  Two 
GBM specimens were examined GBM1: g. negative control DNA probe; i. positive control DNA 
probe (Alu sequence); k. CMV genomic DNA probe; GBM2: h. negative DNA probe; j. positive 
control DNA probe (Alu sequence); l. CMV genomic DNA probe.  (Figure from Mitchell et al., 
Neuro-Oncology, 2008, 10(1):10-18). 
 
brain samples, meningiomas, or brains affected by ischemia, Alzheimer’s disease, 
paraneoplastic encephalitis, or Cryptococcal cerebritis.  Our analysis of viral strains by PCR 
amplification and DNA sequencing revealed that several clinical isolates of CMV were 
associated with these tumors, consistent with a low level systemic viral reactivation in 
patients with GBM[23].  While the biologic and clinical significance of the association of 
CMV with malignant gliomas is still under investigation, a preponderance of published data 
and preliminary data presented in this application verifies the expression of CMV 
intracellular and cell surface antigens in primary gliomas. One of the advantages of 
immunotherapeutic targeting of viral antigens in cancer, is that the immune system does 
not distinguish whether the target plays a critical role in maintaining the malignant 
phenotype of the cell, but rather triggers a host of mechanisms to kill the cell expressing 
the antigen, regardless of whether the antigen has oncogenic relevance.  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 viral-antigen targeted immunotherapy[2-7, 20, 29, 30].  
Recent publications in the New England Journal of Medicine highlighted the effectiveness 
of synthetic peptide vaccines targeting HPV antigens in the eradication of vulvar intrapethial 
neoplasia in vaccinated women[20, 31].  These examples, and others, demonstrate the 
perhaps unique capacity of the immune system to eradicate tumors associated with strong 
viral antigens. 
 
                            IHC on GBM 
                             ISH on GBM 
a.
c.
d.
e.
f.
b.
g.
i.
j.
k.
l.
h.

 
 
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CMV has also been reported to be associated with a number of other malignancies 
including colorectal cancer and prostate cancer[32-39], and while this data awaits further 
confirmation, it suggests that an effective therapeutic CMV vaccine may have utility in 
disease settings outside of treatment of malignant brain tumors.  
 
The immunodominant CMV proteins are well-conserved and induce a robust immune 
response that naturally eliminates cells expressing CMV proteins and confines the virus to 
a latent state.  As a result, the immunologic responses to CMV have been well-
characterized.  In addition to our own work, DCs pulsed with CMV antigens have been 
shown to be potent inducers of CMV-specific immune responses in several studies[40-44].  
There has also been a vast amount of experience with both the safety and efficacy of 
immunotherapy targeting CMV[45]. Vaccination against CMV has safely reduced the risk 
of viral infection and transmission to fetuses in animal models[46-48] and in clinical 
trials[49-54].  The induction of potent CMV-specific T-cell immunity has been used to safely 
and successfully protect against CMV reactivation  and to treat acute CMV infections[55, 
56] in myelodepleted bone marrow transplant patients.  In addition, T-cell mediated 
immunotherapy has proven highly effective in the treatment of CMV-associated disease 
within the CNS[57] and in the treatment of acute CMV infections[55, 56].   
 Polyfunctional T-cell Responses and Anti-Viral Immunity 
Protective immunity against acute and chronic viral infections requires effective T cell 
responses. While CD4 and CD8 T cell responses against viral infections that are rapidly 
cleared are characterized by polyfunctional effector functions including cytotoxicity 
(expression of CD107a), and the secretion of IFNγ, IL-2, and TNFα chronic infections often 
are typified by reactive T cells that are mono- or dual-functional, largely restricted to the 
secretion of IFNγ and TNFα, and unable to express and secrete IL-2[58-61]. Recent studies 
in HIV+ patients have demonstrated that the salient feature of T cell responses in patients 
who are long term non-progressors (LTNPRs) versus those who progress to AIDS, is the 
maintenance of polyfunctional HIV-specific T cell responses in LTNPRs[62-67]. Recent 
studies have demonstrated that polyfunctional T cell responses correlate with protection 
against CMV infection as well. Our preliminary data demonstrate that patients with GBM 
exhibit deficits in baseline CMV-specific polyfunctional immune responses in vivo but these 
deficits can be reversed in vitro by immunogenic stimulation with CMV antigens presented 
by autologous DCs. These findings may explain why CMV-infected tumor cells may persist 
in hosts with GBM and also suggests that proper vaccination in vivo could restore 
polyfunctional immune responses and mediate clearance of CMV-associated malignant 
gliomas (Figure 5). 

 
 
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Figure 5: Polychromatic Flow Cytometry to Analyze Polyfunctional CD4+ and CD8+ T cells:  
T cells isolated from a normal donor were stimulated for 6h in vitro with CMV pp65 peptide mix.  T 
cells were analyzed using 11-color polychromatic flow cytometry.  CD4 and CD8 T cells were 
characterized as 1] Naive (N), 2] Central Memory (CM) and 3] Effector Memory (EM) based on the 
expression of CD45RO and CD27.  Terminal Effector (TE) cells and Effector (E) cells were 
identified based on CD45RO and CD57 expression.  The activation state of each T cell sub-
population was analyzed by measuring 1] CD107, 2] IFN-γ, 3] IL-2 and 4] TNF-α production.  
Polyfunctional responses were defined as indicated in the figure. 
 
 
Ungated
Singlets
CD3+ Exclusion-
Scatter
Basic Gates (3)
CD4+CD8-
CD8+CD4-
CD4+CD8+
88.3
FSC-H
FSC-W
SSC-A
FSC-A
99.3
Exclusion 
CD3 AmCyan
41.4
0.79
CD4 PerCP-Cy5.5
CD8 Alexa700
57.8
36.3
Maturational
Gates
(15: 5/basic gate)
CD57 FITC
CD27 APC-Alexa750
CD45RO ECD
Naive Central
Memory
Effector
Memory
Terminal
Effector Effector
43
54.1
2.58
0.33
56.4
28.6
8.46
6.55
0.12
1.07
55.9
42.9
5.67
13.2
24.2
56.9
22
62.5
11.7
3.98
3.98
22.9
51.7
21.5
CD57 FITC
CD27 APC-Alexa750
CD45RO ECD
CD57 FITC
CD27 APC-Alexa750
CD45RO ECD
N
CM
EM
TE
E
N
CM
EM
TE
E
N
CM
EM
TE
E
Naive Central
Memory
Effector
Memory
Terminal
Effector Effector
Naive Central
Memory
Effector
Memory
Terminal
Effector Effector
2.59
1.14
0.31
4.19
CD107 PE-Cy5
IFN−γ PE-Cy
IL-2  PE
TNF-α A700
Cytokine
Gates
(60: 4/maturational gate)
CD8+
Central
Memory
Boolean Gates
[288: 16/gate x (3 basic + 15 maturational)]
7+g+2+T+
7+g+2+T-
7+g+2-T+
7+g-2+T+
7-g+2+T+
7+g+2-T-
7+g-2+T-
7+g-2-T+
7-g+2+T-
7-g+2-T+
7-g-2+T+
7+g-2-T-
7-g+2-T-
7-g-2+T-
7-g-2-T+
7-g-2-T-
Polyfunctional (1: ++++ ) and (4:+++)
Polyfunctional (4: +++)
Bifunctional (6: ++)
Monofunctional (4: +)
Nonfunctional (1: ----)
Key:
7 = CD107
g = IFN-γ
2 = IL-2
T = TNF-α

 
 
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 Temozolomide, Lymphopenia, and Homeostatic Proliferation 
TMZ is an alkylating chemotherapeutic that has recently been shown to prolong survival 
in patients with GBM[14].  As a result, it has become part of the standard regimen used to 
treat these patients.  TMZ induces a transient lymphopenia, and although 
counterintuitive, we and others have shown that following periods of lymphopenia, immune 
responses can be markedly enhanced[9, 68-71].  Probably as a result of a surge in 
cytokines (IL-7, IL-15) in response to lymphopenia, lymphocytes undergoing homeostatic 
proliferation enjoy a reduced activation threshold[72, 73], differentiate directly into effector 
memory T-cells capable of rapid and intense response to antigen[74], display increased 
expression of anti-apoptotic molecules, and are less sensitive to immunosuppressive NK 
cell-mediated lysis[74].  Still, lymphocytes must encounter their cognate antigen and 
compete for limiting amounts of these homeostatic cytokines to proliferate under these 
conditions[73]. Thus, B- or T-cells specific for antigens that predominate during this 
recovery period, like those provided in the form of a vaccine, have a competitive 
advantage and become disproportionately over-represented in the recovering 
lymphocyte population both in murine models [9, 75] and in humans [70]. These skewed 
homeostatic responses have been shown to enhance antitumor immunity [9, 75, 76] but 
can also increase the risk of autoimmunity.   
 
 
 
Figure 6: Lymphocyte Counts of Patients with Newly-Diagnosed GBM Receiving Standard 
Dose Adjuvant TMZ (200 mg/m2/5d) and Dose-Intensified TMZ (100 mg/m2/21d).  Standard 
dose TMZ induces Grade 2 lymphopenia in 100% of patients (<800 cells/uL; blue line) and 
transient Grade 3 lymphopenia in 40% of patients. Sustained Grade 3 lymphopenia (<500 cells/uL; 
red line) was induced by dose-intensified TMZ cycle #3 in all patients. 
 
Leveraging this principle, Rosenberg and colleagues have produced dramatic clinical 
responses [71, 77-80], along with some autoimmune toxicity, in patients with advanced 
malignant melanoma [68, 69].  After lymphodepletion, T-cells can expand dramatically and 
tumor-specific T-cells can constitute up to 90% of the host’s T-cell repertoire and can 
be maintained for months [68, 81], and in these studies clinical regressions correlate 
with the frequency of tumor-specific T-cells achieved in the peripheral blood and 
persistence of these cells in vivo [31, 77-80, 82-84].  
 
 
Vaccine 1
Vaccine 2
Vaccine 3
Pre-TMZ
TMZ Cycle-1
TMZ Cycle-2
TMZ Cycle-3
TMZ Cycle-4
TMZ Cycle-5
TMZ Cycle-6
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
Standard Dose TMZ (200mg/m2/5d) (N=5)
Dose-Intesified TMZ (100mg/m2/21d) (N=8)
Grade 3 (<500 cells/µL)
Grade 2 (<800 cells/µL)
Absolute Lymphocyte Count (Day 21)
(cells/µL blood)

 
 
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Figure 7: Antibody Titers in Patients During Standard-Dose TMZ (200 mg/m2/5d) and Dose-
Intensified TMZ (100 mg/m2/21d) are Shown. All patients receiving the 21 day dose regimen 
developed EGFRvIII-specific antibodies with titers detectable at greater than 1:100,000 dilution.  
Humoral and cellular immune responses have been dramatically better in patients receiving the 
higher TMZ dose. 
 
TMZ produces a survival benefit in patients with GBM who are MGMT methylated and 
has become a routine part of the therapy for these patients. Due to the known 
myelosuppressive and immunosuppressive effects of TMZ and its potential negative 
implications for immunotherapy targeting GBMs, we evaluated the impact of TMZ 
treatment, in the context of our EGFRvIII peptide vaccine trial (now rindopepimut, Celldex 
Therapeutics), on the lymphocyte compartment and immunologic responses of patients 
with newly-diagnosed GBM undergoing two different dose regimens of adjuvant TMZ [85]. 
After concurrent TMZ with EBRT, patients received TMZ for either 5 days (200 mg/m2) or 
21 days (100 mg/m2) of each 28 day cycle. 100% of patients receiving the standard 5 day 
schedule exhibited Grade 2 lymphopenia (<800 cells/uL) (Figure 6) with nadirs occurring 
14-21 days after the first dose (n=5). Grade 3 lymphopenia was observed in only 1 patient 
with this regimen. Sustained Grade 3 lymphopenia (<500 cells/uL) was induced in all 
patients receiving the 21 day regimen, however, by the fifth cycle of TMZ (Figure 6). 
Despite the profound lymphopenia, we have been able to induce and maintain potent 
EGFRvIII-specific immune responses (Figure 7 and Figure 8) in patients with GBM 
receiving serial cycles of TMZ [86]. 
 
Vaccine 3
Vaccine 4
Vaccine 5
Vaccine 6
Vaccine 7
Vaccine 8
10,000
100,000
1,000,000
200 mg/m2/5d
100 mg/m2/21d
1 / Titer
P=0.0093, Two-way 
ANOVA, repeated 
measures, For 
Interaction
ACTIVATE: 7/15
ACT II: 15/15
(P = 0.0022, 
Fisher’s exact)

 
 
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Figure 8: DTH Responses to EGFRvIII vaccination (Left) in patients receiving lymphodepletive 
TMZ (100 mg/m2/d x 21 days) cycles (black) were increased when compared with standard dosing 
(200 mg/m2/d x 5 days) with continued vaccination.  DTH responses to the recall antigen Candida 
(Right) in patients receiving lymphodepletive TMZ (100 mg/m2/d x 21 days) cycles (black) were 
decreased when compared with standard dosing (200 mg/m2/d x 5 days) with continued cycles of 
TMZ. 
 
Vaccine DTH Response
Pre Vaccine
Dose 1 TMZ
Dose 3 TMZ
Dose 5 TMZ
0
20,000
40,000
60,000
80,000
100,000
Standard  200mg/m2/5d
Undectable
Undectable
Dose-Intensified 100mg/m 2/21d
mm squared
Candida DTH Response
Pre Vaccine
Dose 1 TMZ
Dose 3 TMZ
Dose 5 TMZ
0
2,000
4,000
6,000
8,000
Standard  200mg/m2/5d
Undetectable
Dose-Intensified 100mg/m 2/21d
mm squared
 
 

 
 
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 Tetanus Diphtheria Toxoid (Td) 
The current use of Td toxoid is for active immunization in children and adults against 
infection with the bacteria Clostridium tetani and Corynebacterium diphtheria. Tetanus 
infection is manifested primarily by neuromuscular dysfunction caused by a potent exotoxin 
released by C. tetani. Diphtheria is an acute toxin-mediated infectious disease caused by 
toxigenic strains of C. diphtheriae. Protection against disease is due to the development of 
neutralizing antibodies to the diphtheria toxin. Td toxoids adsorbed are readily available as 
several approved administrations [i.e., Daptacel (DTaP), Infanrix (DTap), Tenivac (Td 
adult), Boostrix (Tdap)] [87, 88]. Protection against disease is due to the development of 
neutralizing antibodies to the tetanus toxin. A serum tetanus antitoxin level of at least 0.01 
IU/mL, measured by neutralization assays, is considered the minimum protective level. A 
level ≥0.1 IU/mL by ELISA has been considered as protective[89]. A serum diphtheria 
antitoxin level of 0.01 IU/mL, measured by neutralization assays, is the lowest level giving 
some degree of protection; a level of 0.1 IU/mL by ELISA is regarded as protective. 
Diphtheria antitoxin levels ≥1.0 IU/mL by ELISA have been associated with long-term 
protection. 
 
Following deep s.c./i.m. administration of the tetanus toxoid vaccine, toxoid molecules 
are taken up at the vaccination site by immature DCs, which are professional antigen-
presenting cells. Within these cells, they are processed through the endosomal pathway 
(involving the phagolysosome) where they are bound to MHC type II molecules on the 
surface of DCs. The MHC II:toxoid complex then migrates to the cell surface. While this 
process is happening within the cell, the now activated mature DC at the vaccine site 
migrates along lymph channels to the draining lymph node where they encounter naive 
TH2 cells, each with their own unique TCR. Identifying and then binding of the MHC 
II:toxoid to the specific TH2 receptor then activates the naive T cell, causing it to proliferate. 
Simultaneously, toxoid molecules not taken up by DCs pass along lymph channels to the 
same draining lymph nodes where they come into contact with B cells, each with their own 
unique B-cell receptor (BCR). Binding to the B cell through the specific immunoglobulin 
receptor that recognizes tetanus toxoid results in the internalization of toxoid, processing 
through the endosomal pathway and presentation on the cell surface as an MHC II:toxoid 
complex, similarly to DCs undergoing the same process [90].  
 
These two processes occur in the same part of the lymph node with the result that the 
B cell with the MHC II:toxoid complex on its surface now comes into contact with the 
activated TH2 whose receptors are specific for this complex. The process, termed linked 
recognition, results in the TH2 activating the B cell to become a plasma cell with the 
production initially of IgM, with a later switch to IgG antibodies produced. Additionally, a 
subset of these B cells becomes memory cells [90]. 
 
The novelty of using Td toxoid vaccination lies in the ability of this potent recall antigen 
to enhance antitumor responses as part of a cancer vaccination protocol. Td toxoid induces 
an inflammatory milieu within the intradermal vaccine site, thereby promoting the migration 
of injected tumor-specific DCs. Additionally, in the context of vaccinating the host with 
tumor-derived peptides, conditioning the vaccine site with Td toxoid has demonstrated 
enhanced immunogenicity with these peptides.  
 
Our data from the ATTAC clinical trial demonstrating the capacity to enhance DC 
migration to VDLNs via Td pre-conditioning of the vaccine site offer potential therapeutic 
interventions whereby we can enhance the immunologic responses to ultimately overcome 

 
 
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the inherent challenges in faithfully eradicating established tumors [91]. In a completed 
randomized clinical trial, we found that migration of injected DCs to VDLNs following 
vaccine site pre-conditioning with Td toxoid was significantly increased compared to 
controls and that the efficiency of DC migration was strongly associated with clinical 
outcomes of patients with newly-diagnosed GBM, the most fatal type of malignant brain 
tumors. To address this observation, we took our Td pre-conditioning platform back into 
the preclinical setting using transgenic mouse models and were able to corroborate the 
effects of Td pre-conditioning on increasing the lymph node homing of intradermally 
administered DCs. Moreover, Td administration at a single vaccine site increases the 
migration of a bilateral DC vaccine to both inguinal lymph nodes. Regardless of the side of 
the Td intradermal skin prep, DC migration to bilateral inguinal VDLNs was equally 
increased, supporting a systemic response to recruit peripherally administered DCs. 
  
Our Td pre-conditioning platform in the context of DC vaccination also elicited superior 
anti-tumor responses compared to controls receiving DC vaccines without Td pre-
conditioning. In our clinical trial, patients with newly-diagnosed GBM who were 
administered the Td skin prep before DC vaccination revealed significantly longer 
progression-free and overall survival rates compared to the control cohort. In evaluating 
the relationship between DC migration and clinical responses, we observed a modest 
positive correlation between levels of DC migration and survival. In our preclinical model, 
Td pre-conditioning prior to vaccination with tumor antigen-specific DCs dramatically 
suppressed the growth of established and highly aggressive B16-F10/OVA tumors. The 
use of Td with a DC vaccine increased antitumor responses in an antigen-specific manner, 
as non-specific DC vaccines were not potentiated with Td pre-conditioning. Furthermore, 
in a challenge setting, where mice are administered the treatment platform prior to 
challenge with tumor inoculation, Td pre-conditioning at the vaccine site induced a 
significant survival benefit compared to controls. 
 
Because of these findings, we have opened a trial enrolling patients to a randomized 
dendritic cell immunotherapy trial where the two groups receive vaccine site 
preconditioning with tetanus versus conditioning with non-antigen loaded DC, 
(Pro000054740 ELEVATE). This trial is powered to determine if greater migration to the 
draining lymph node correlates with overall survival. To obtain further confirmation that pre-
conditioning the vaccine site with Td toxoid increases DC migration to VDLNs and improves 
overall survival. Due to these promising results, we have included Td pre-conditioning 
following Td booster in all of our immunotherapy trials including this one. 
 Phase I/II Trials in GBM Targeting CMV 
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).  
This trial has enrolled 9 patients with newly diagnosed GBM who underwent gross total 
resection (>95%) followed by standard external beam radiation (XRT) (60 Gy) and 
concurrent TMZ (75 mg/m2/d) for six weeks followed by monthly 5 day TMZ (150-200 
mg/m2/d) for six cycles.  Leukapheresis harvested post-surgical resection and prior to 
initiation of XRT/TMZ was used to generate pp65 RNA electroporated autologous DCs.  
Following the first cycle of TMZ (100 mg/m2/21 days) 2 x 107 DCs i.d. mixed with 150 µg of 
GM-CSF were administered every two weeks for the first three doses and monthly 
thereafter on day 22 of each cycle.  Patients were monitored by MRI (every two months) 
for tumor progression and blood collected monthly for immunologic monitoring.   Patients 
exhibit a median PFS of 29.2 months (Figure 9) and OS that has not reached median at 40 

 
 
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months of follow-up. This is favorable compared to historical controls (p = 0.0002). 
Immunologic responses are shown in (Figure 10 and Figure 11). 
 
Our preliminary data indicate that the recovery phase from TMZ therapy provides an 
immunologic environment favoring enhanced immune responses to vaccination.  This 
approach has been successful in targeting EGFRvIII positive tumors [91].   
 
Figure 9: Progression Free Survival (PFS) Percentage Promising PFS in patients with GBM 
receiving DC vaccines plus GM-CSF targeting CMV pp65. 9 patients with GBM were treated with 
DC vaccines targeting CMV pp65 on the ATTAC GM trial (FDA IND-BB 12839; Duke IRB 3108). 
Progression free survival (PFS) is favorable compared to historical controls (ATTAC GM PFS 29.2 
months vs Historical controls 6.7 months P=0.0002). 
 Peptide Vaccines for Cancer 
Peptide vaccines encoding minimal CD8+ cytotoxic T cell (CTL) epitopes have been 
demonstrated in many contexts to induce protective immunologic responses in 
experimental animals and mediate regressions of established tumors [92, 93]. However, 
with few several notable exceptions[31, 94-97], vaccine-based cancer immunotherapy in 
humans is in need of significant improvement in immunogenicity and clinical outcomes [71].   
 
The addition of CD4+ T cell helper epitopes to peptide vaccine formulations is widely 
believed to be an advantageous strategy for enhancing anti-tumor immunity [98].  This 
presumption is supported by the observation that CD4+ T cell directed therapy can mediate 
effective anti-tumor immunity in humans and experimental animals [99-101]. CD4+ T helper 
cells deliver assistance for CD8+ effector cells by fully activating dendritic cells through the 
CD40-CD40L signaling pathway [102]. However, vaccination with T helper epitope 
encoding peptides has yielded mixed results in human clinical trials as a method to 
enhance anti-tumor cytotoxic T cell responses in vivo [100, 103-105] and thus warrants 
systematic evaluation.  
 
Recent studies have highlighted the use of longer peptides (15-30 amino acids in 
length) instead of minimal CTL epitopes (typically 8-10 amino acids) for induction of a more 
robust, long lived CD8+ cytotoxic T cell responses in experimental animals [31, 106]. The 
expanded length of the peptide requires it be processed by antigen-presenting cells. 
Minimal CTL epitopes do not require processing by antigen-presenting cells and thus are 
free to bind directly to T-cell receptors. This can induce tolerance due to a lack of co-
0
20
40
60
0
10
20
30
40
50
60
70
80
90
100
ATTAC GM
Matched Controls
 (n=17)
Months
Progression Free Survival
(PFS) Percentage

 
 
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stimulatory molecules that are presented to T-cells during antigen presentation by dendritic 
cells, however this area of peptide formulation requires further evaluation [107].      
 
We believe the use of serial vaccination during recovery from chemotherapy-induced 
lymphopenia, as described above, is a novel platform for enhancing the efficacy of tumor-
specific vaccines and we have observed prolonged and potent immunologic responses in 
TMZ treated mice using minimal epitope vaccines.   
 
Peptide vaccination in humans has exhibited an excellent safety profile with virtually no 
dose limiting toxicities [31, 104]. Most toxicities occur when peptide vaccines are used in 
conjunction with immunostimulatory cytokines such as IL-2, which are expected results of 
IL-2 therapy [108]. This feature of peptide vaccinations allows it to be used in combinatorial 
immunotherapeutic strategies and/or as salvage therapies in resistant disease [109, 110]. 
 Tumor-Specific Immunotherapy in Patients with GBM 
Our studies in adults with malignant primary brain tumors have shown that tumor-
specific antigens in the form of RNA or proteins are capable of inducing T-cell proliferation, 
antibody induction, cytokine secretion, and specific lysis of tumor cells. We have completed 
a Phase II multi-institutional tumor-specific immunotherapy study in adults. In this study, 
patients with newly-diagnosed with GBM are vaccinated monthly after receiving standard 
radiation and TMZ therapy and are followed for clinical and radiographic responses. This 
study showed that a vaccination approach targeting the tumor-specific mutation of the 
epidermal growth factor receptor, EGFRvIII, was capable of universally eliminating the 
targeted cell population such that all patients with recurrent tumors no longer expressed 
the targeted antigen. These studies have also produced remarkably consistent and 
impressive median survival of >126 weeks which is significantly better than TMZ-treated 
historical controls matched for treating institution, known prognostic factors, and eligibility 
criteria (P<0.001). Although patients with residual disease were excluded from our Phase 
II studies, a small number of patients with residual disease were treated on our initial Phase 
I studies. All of these patients had nearly complete radiographic responses. Remarkable 
radiographic responses have also been seen in our recent studies using mRNA-loaded 
DCs in patients with GBM as well. 
 
Using human Cytomegalovirus antigens as a model platform, we have demonstrated 
the capacity for RNA-loaded DCs to induce antigen-specific CD8+ and CD4+ T cell 
responses in vitro including lytic reaction against antigen expressing targets, proliferative 
responses (Figure 10), and cytokine-secretion in response to antigenic restimulation 
(Figure 11). 
RNA DCs 

 
 
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Figure 10: Increase in Frequency of pp65-Specific T cells in Patients with GBM Receiving 
DC Vaccination and Standard-Dose TMZ.  Pre and Post vaccine PBMC from patients with GBM 
receiving cycles of TMZ (150-200 mg/m2 x 5 days) and autologous pp65 RNA-pulsed DC vaccines 
were stained with CD8-FITC and CD3-APC in conjunction with PE conjugated CMVpp65 tetramers 
(Beckman Coulter, HLA-A*0201, HLA-A*2402, HLA-A*0101, HLA-B*0702, HLA-B*0801, HLA-
B*3501) and analyzed by FACS. Dot plots above show percent tetramer positive of total 
CD3+CD8+ lymphocytes. 
 
 
Figure 11: DTH Responses to pp65 RNA Pulsed DC Vaccines.  To assess for patients’ 
baseline and progressive status of cellular immunity, routine skin tests were performed before the 
first immunization, at the time of post-immunization leukapheresis. For skin testing, 1 x 106 pp65 
RNA transfected mature DCs and 1 x 106 untransfected mature DCs were injected intradermally to 
assess for the development of  a DTH reaction.  In addition, in order to assess the 
immunocompetence of the patients they received DTH skin test to the common recall antigens 
Trichophyton and Candida. 
10 STUDY POPULATION  
 Inclusion Criteria 
1. Age ≥ 18 years. 
2. Histopathologically proven newly-diagnosed primary glioblastoma with complete or 
partial surgical resection. Biopsy not acceptable. 
3. Patients must be CMV seropositive. 
4. The tumor must be supratentorial. 
0.32%
1.86%
0.05%
0.22%
1.76%
2.85%
3.66%
6.94%
Pre Vaccine
Post Vaccine
Patient 1
HLA-A*0101 
Patient 1
HLA-B*0702
Patient 2
HLA-A*0101
Patient 3
HLA-B*3501
0.32%
1.86%
0.05%
0.22%
1.76%
2.85%
3.66%
6.94%
Pre Vaccine
Post Vaccine
Patient 1
HLA-A*0101 
Patient 1
HLA-B*0702
Patient 2
HLA-A*0101
Patient 3
HLA-B*3501

 
 
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5. Karnofsky performance status of ≥ 70. 
6. Stable or decreasing steroid dose (≤ 4 mg/day) at time of post-XRT adjuvant TMZ 
initiation.  If patients are decreasing steroid use, once they are at 2 mg/day, they 
may be supplemented with physiologic hydrocortisone therapy (20-30 mg/day in 
divided doses), at the discretion of the treating oncologist.   
7. Hematology 
• 
ANC ≥ 1500 cells/µL 
• 
Platelet count ≥ 100,000 cells/µL 
• 
Hemoglobin ≥ 9.0 g/dl  
8. Chemistry 
• 
ALT/AST ≤ 3.0 times the upper limit of normal 
• 
Total bilirubin ≤ 1.5 x the upper limit of normal (Exception: Patient has 
known Gilbert’s Syndrome or patient has suspected Gilbert’s Syndrome, 
for which additional lab testing of direct and/or indirect bilirubin supports 
this diagnosis.  In these instances, a total bilirubin of ≤ 3.0 x ULN is 
acceptable.)   
 Exclusion Criteria 
1. Radiographic or cytologic evidence of leptomeningeal or multifocal disease at any 
time prior to study entry. 
2. Prior conventional antitumor therapy, other than steroids, RT or TMZ therapy given 
for glioblastoma. 
3. Pregnant or need to breast feed during the study period. 
4. Not adhering to pregnancy prevention recommendations. 
5. Active infection requiring intravenous antibiotics or an unexplained febrile (> 101.5o 
F) illness. 
6. Immunosuppressive disease or human immunodeficiency virus infection. 
7. Patients with unstable or severe intercurrent medical conditions such as severe 
heart or lung disease. 
8. Allergic or unable to tolerate TMZ for any reason. Any patient that successfully 
completed at least 5 weeks of Temodar during standard of care XRT/TMZ and 
whose blood counts meet the eligibility requirements (inclusion #7) within 4 weeks 
post XRT/TMZ is eligible.   
9. Patients with previous inguinal lymph node dissection, radiosurgery, brachytherapy, 
or radiolabeled monoclonal antibodies. 
10. Prior allogeneic solid organ transplant. 
11. Currently receiving or ever received immunosuppressive therapy for an 
autoimmune disorder or an organ transplant. 
 

 
 
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11  INVESTIGATIONAL PLAN 
 Overview 
If their CMV serostatus is unknown, patients will consent to CMV screening either prior 
to or following XRT/TMZ.  Patients are enrolled to the study following XRT/TMZ and prior 
to initiation of post-XRT cycles of TMZ provided they meet all other eligibility criteria (if CMV 
status is not already known, a screening consent will be obtained for CMV screen only to 
confirm eligibility).  After signing main consent, patients receive a Tetanus-diphtheria 
booster vaccination with 0.5 mL of Td (tetanus, diphtheria toxoid, adsorbed) 
intramuscularly. After meeting all eligibility criteria, patients are randomized to one of two 
arms: 
• 
Arm 1) will receive standard TMZ (150-200 mg/m2/day on days 1-5 of each 28-day 
cycle).  
• 
Arm 2 will receive dose-intensified TMZ (75-100 mg/m2/day on days 1-21 of each 28-
day cycle).  
 
New patients who enroll on study after protocol version 20181218 is approved and who 
are MGMT unmethylated will only receive one adjuvant cycle of their assigned TMZ 
regimen. Patients who enroll on study before protocol version 20181218 is approved or 
enroll on study after protocol version 20181218 is approved and either are MGMT-
methylated or their methylation status is inconclusive will continue with up to 12 cycles of 
TMZ. 
 
MGMT gene promoter methylation status is obtained at Duke using validated testing 
from LabCorp by PCR and/or from Caris by pyrosequencing. If MGMT gene promoter 
methylation status has already been conducted outside of Duke, the results will be used, 
as long as the testing was performed by a validated method/test.  If the results of testing 
by LabCorp, Caris, or other external, validated test is indeterminate or if the results of more 
than one type of testing are in disagreement, the results will be considered methylated and 
the patient will be treated per protocol as methylated based on their randomization.  That 
is, Arm 1 will receive standard adjuvant dosing of temozolomide for the first 5 days of each 
cycle for 6 to 12 cycles.  Arm 2 will receive dose-intensified temozolomide for 21 days of 
each cycle for 6 to 12 cycles. 
 
Patients who are dependent on steroid supplements above immune suppressive levels 
(4 mg daily) at time of vaccination or who are unable to tolerate TMZ will be withdrawn from 
the study before vaccination therapy (see Section 10.2).   In order to meet the goal of 
obtaining 26 evaluable patients who received Component A only (13 patients per arm) for 
the primary outcomes, up to 70 patients may be consented on the main informed consent.  
 
For both study arms, the initial Duke visit will be within 3 ± 1 weeks after completion of 
standard of care radiation. For Arm 1, the initial cycle of TMZ will begin as soon as possible 
after randomization at a standard targeted dose of 150-200mg/m2/d for 5 days.  For Arm 2, 
the initial cycle of TMZ will begin, as soon as possible after randomization at the dose-
intensified dose of 75-100 mg/m2/d for 21 days.  All patients will have immune monitoring 
blood drawn and then receive a tetanus pre-conditioning injection (Td 1 flocculation unit, 
Lf, in 0.4 mLs of saline) in the RIGHT groin (as described below) on day 22 (+1 day). 
Additional safety measures were implemented in this study during the latter half of 2019 
and the beginning of 2020, resulting from safety events on study.  Patients will receive their 
1st vaccine in the Oncology Treatment Center (OTC) and will receive a normal saline bolus 

 
 
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by IV prior to vaccine administration.  The IV saline lock will remain in place during the post-
vaccine monitoring period for the purpose of quick administration of additional fluids and/or 
medication should either be necessary.  Supportive medications for post-vaccine reactions 
may include the following by IV: normal saline bolus, Benadryl, Solu-medrol.  On Day 23, 
all patients will be pre-medicated with Zofran® and Tylenol®.  If a patient experiences any 
post-vaccine reactions (please refer to Sections 11.5.1 and 14.2.1 for a description of 
previous reactions), the patient also may be pre-medicated with oral prednisone at one or 
more subsequent vaccine visits, at the discretion of the patient and their provider.  Patients 
on study who have experienced a significant post-vaccine reaction with previous vaccines 
(i.e., ≥ Grade 2 toxicity thought to be possibly, probably, or definitely related to study 
vaccine), but who are not removed from study, will receive a normal saline bolus by IV prior 
to vaccine administration for all subsequent vaccines and the IV saline lock will remain in 
place as a precautionary measure, in case supportive medications are needed.  Patients 
on study who have never experienced a post-vaccine reaction or only experienced a Grade 
1 toxicity with earlier vaccines will not be required to have an IV saline lock placed prior to 
administration of subsequent vaccines, although the treating provider may opt to proceed 
with this precautionary measure. If a patient enrolls after approval of protocol version 
20181218 and has an MGMT unmethylated tumor, they will only receive a single cycle of 
TMZ. 
 
Previously, patients received the vaccine as follows: 500 µg of PEP-CMV Component A 
mixed with Montanide ISA-51 intradermally administered in the RIGHT groin and 2 hours 
later, 500 µg of PEP-CMV Component B mixed in 150 µg of GM-CSF intradermally 
administered in the LEFT groin.  With revisions made in protocol v. 20181218, patients will 
receive the vaccine as follows: 500 µg of PEP-CMV Component A mixed with Montanide 
ISA-51 administered intradermally with half in the RIGHT groin and half in the LEFT groin. 
Vaccines #2 and #3 will be given at 2 week (+3 days) intervals, the MRI visit with immune 
monitoring will take place within 2 weeks (+3 days) of vaccine #3.  For patients who enrolled 
before approval of protocol version 20181218 for those who are MGMT methylated or with 
inconclusive MGMT methylation status, TMZ cycle 2 will begin after the aforementioned 
visit, but no sooner than 14 days post vaccine 3.  This will result in an at least an ~35-day 
delay before starting TMZ cycle 2 MGMT unmethylated patients enrolled after approval of 
protocol version 20181218 will not receive subsequent cycles of TMZ, but will continue to 
receive vaccines approximately every 4 (+2) weeks. 
 
Patients who receive more than 1 cycle of TMZ will be vaccinated in conjunction with 
each subsequent 28-day TMZ cycle for a total of 6 to 12 cycles of TMZ after RT followed 
by further vaccination up to 20 vaccines.  Patients who receive only 1 cycle of TMZ will be 
vaccinated on a 28-day cycle with up to 20 vaccines. All cycles, with or without TMZ, may 
be given every 4 (+ 2) weeks, in order to adjust for slight delays on startup of each 28-day 
cycle, and the total number of cycles are given at the discretion of the treating oncologist.  
During vaccine (+/- TMZ) cycles, vaccinations will occur on day 23 (-1 day, + 2 days) of 
each cycle for each arm: (Arm 1) standard TMZ (150-200 mg/m2/day on days 1-5 of each 
28-day cycle), or (Arm 2) dose-intensified TMZ (75-100 mg/m2/day on days 1-21 of each 
28-day cycle).  All vaccines will be given i.d. approximately 10 cm below the inguinal 
ligament bilaterally.  A target of six cycles with a maximum of twelve cycles of TMZ may be 
given to patients enrolled prior to approval of protocol version 20181218 and those with 
MGMT/inconclusive methylation status if enrolled after approval of protocol version 
20181218, at the discretion of the treating neuro-oncologist.  After the completion of a 
patient’s TMZ cycles, vaccines will continue to be administered every 4 (+ 2) weeks for a 
maximum of 20 vaccines (unless tumor progression occurs). 

 
 
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Patients will be imaged with contrast-enhanced MRI after vaccine 3 (at cycle 2 initiation 
visit) and then approximately every 8 weeks [every 2 cycles thereafter (i.e., end of cycles 
3, 5, 7, 9, 11)].  RANO criteria [111] will be used for assessment of pseudoprogression and 
patients demonstrating definitive progression will be removed from study. Any patient 
removed prior to immune monitoring post vaccine 3 will be replaced for immunologic 
endpoints. Clinical endpoint comparisons will be made amongst patients randomized to 
adjuvant TMZ treatment arms who have received at least one vaccine.  
 
Blood for immune monitoring will be obtained:  
• 
Prior to Td pre-conditioning 
• 
During vaccines 1 and 2 (just prior to Component A, 1 and 2 hours after 
Component A) 
• 
Prior to vaccine 3 
• 
After vaccine 3 (within 2 weeks of vaccine 3 [+ 3 days]) 
• 
Prior to vaccines 4 and 6 
• 
At tumor progression (if feasible)   
 
Patients seen by the Duke neuro-oncology team between these vaccine visits may have 
blood drawn for immune monitoring at the discretion of the study team. 
 
As part of standard care for these patients, upon tumor progression, participants may 
undergo stereotactic biopsy or resection.  As this is not a research procedure, consent will 
be obtained separately. Patients who have this procedure done here in the Duke University 
Health System may be approached to participate in the Duke Brain Tumor Center 
Biorepository study (Pro00007434).  Tissue obtained from patients who consented to the 
Duke Brain Tumor Center Biorepository will be used to assess immunologic cell infiltration, 
antigen expression, and biomarkers for immunologic response. 
 
Study Drug Safety Arm (added 5/16/17 following safety events that occurred on 
4/5/17, not applicable for patients enrolled on protocol version 6/12/18 or later) 
 
Definition of Study Drug Safety Arm: 
The purpose of this 6 patient safety arm is to investigate which component or 
combination of components of the PEP-CMV vaccine (PEP-CMV is administered as two 
components: Component A and Component B) results in a hypersensitivity reaction, similar 
to that seen in the first 3 patients treated with the vaccine.  Serious Adverse Events for the 
first 3 patients receiving study drug vaccine were submitted to FDA and the IRB on 4/10/17.  
These patients experienced rapid onset (within 2 hours of vaccine) of a combination of the 
following symptoms: nausea, vomiting, diarrhea, chills, fever, myalgias, dyspnea, 
generalized muscle weakness, back pain, and cough.  We believe that the combination of 
the vaccine components administered at the same time resulted in this intense 
hypersensitivity reaction and by administering the components with a delay, we may 
alleviate the severity of these reactions.  Additionally, by administering the components 
separately, we can determine if it’s an individual component or a combination of the 
components that results in the reaction.  After we determine which component or 
combination of components is related to the hypersensitivity reaction, we will put the study 
on a temporary hold and investigate how we can modify the study drug or administration 
procedure in order to reduce the occurrence of these reactions.   
 

 
 
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Rationale of Study Drug Safety Arm: 
Our previous experience with peptide vaccines is from the RESIST study (Pro00054746) 
which has given vaccines to 15 patients without observing any systemic hypersensitivity 
reaction.  This vaccine has tetanus preconditioning and a single IDH-targeting peptide that 
is co-administered with GM-CSF and Montanide ISA 5.1.  Our current study differs from 
the RESIST study by using different peptides, a pp65 synthetic long peptide and human 
CMV glycoprotein B (gB), that are meant to target the CMV antigens inthe tumor cells.  
Additionally, Component B of the vaccine has the gB conjugated to KLH, a component that 
is not present in the RESIST peptide.  Furthermore, Component B has a mixture of KLH 
and GM-CSF.  Given that GM-CSF and Montanide administered together in the RESIST 
study has not led to systemic hypersensitivity reactions, it is likely that KLH alone or the 
combination of two or more of Montanide, GM-CSF, KLH, and/or the tetanus 
preconditioning results in the reaction in these patients.   
  Temozolomide Therapy 
11.2.1 Temozolomide Therapy: After Radiation 
With the exception to the delay between cycles 1 and 2, TMZ will be administered after 
standard external beam RT as part of this protocol every 4 (+ 2) weeks according to 
assignment arm (5-day or 21-day) at the discretion of the treating oncologist using standard 
guidelines as outlined in the TMZ Package Insert (uploaded in electronic IRB). 
11.2.2 
Temozolomide Dose Reduction 
All subjects randomized to the standard dose of TMZ will start 5-day TMZ at a dose of 150-
200 mg/m2/day, at the discretion of the treating physician.  All subjects randomized to dose-
intensified TMZ will start 21-day TMZ at a dose of 75-100 mg/m2/day, at the discretion of 
the treating physician.  During 5-day or 21-day TMZ treatment (28 day cycles), dose 
adjustments to TMZ, if needed, may be conducted as outlined below:   
 
Table 1. Temozolomide Dose Delay, Reduction, or Discontinuation 
Toxicity  
Delay TMZ Dose a 
Reduce TMZ by 25%   
Discontinue TMZ  
Absolute Neutrophil 
Count  
>0.5 and <1.0 x 109/L 
>0.5 and <1.0 x 109/L 
<0.5 x 109/L  
Platelet Count  
>10 and <100 x 109/L 
>10 and <100 x 109/L 
<10 x 109/L  
CTC Non-hematological 
Toxicity (except for 
alopecia, nausea, 
vomiting)  
CTC Grade 3 
CTC Grade 3  
CTC Grade 4 b  
 
a: If dose is delayed, treatment with TMZ can resume when the 
following conditions are met: absolute neutrophil count >1.0 x 109/L; 
platelet count >100 x 109/L; CTC non-hematological toxicity resolved to 
baseline (except for alopecia, nausea, vomiting). 
b: TMZ is to be discontinued if a 25% dose reduction is required more 
than 1 time or if the same Grade 3 non-hematological toxicity (except 
for alopecia, nausea, vomiting) recurs after dose reduction or delay.   

 
 
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  Pseudoprogression 
There is now a well-documented phenomenon of pseudoprogression in patients with 
GBM characterized by MRI changes during radiation with temozolomide and up to 2-3 
months beyond.  These changes resemble tumor growth but actually are due to treatment-
related changes.  Based on current treatment guidelines [112], patients with radiographic 
changes consistent with pseudoprogression that are neurologically and clinically stable are 
recommended to continue standard temozolomide and be reassessed by MRI.  Such 
patients will undergo randomization and receive vaccine as described in the protocol after 
consultation with the treating neuro-oncologist. 
  Treatment Procedures 
11.4.1 Patient Number Assignment and Randomization 
Patients will be assigned a study ID number serially once they have signed the CMV 
consent form or main study consent form if the CMV consent form is not required, this 
number will stay with them as they are checked for study eligibility. This number should be 
referenced on all patient-specific study-related material. A permuted-block randomization 
scheme developed by the study biostatistician will be used to assign patients to treatment 
arms. The randomization module for the Duke electronic database system (Title 21 CFR 
Part 11 Compliant) will be used for this purpose.   
11.4.2 PEP-CMV Vaccination 
As described above, patients will be randomized to one of two arms (1:1) to receive: (1) 
standard TMZ (150-200 mg/m2/day on days 1-5 of each 28-day cycle), or (2) dose-
intensified TMZ (75-100 mg/m2/day on days 1-21 of each 28-day cycle).  
 
During the first cycle of adjuvant TMZ, all patients will receive a tetanus pre-conditioning 
injection in the RIGHT groin on Day 22 (+1 day) and the following day receive the vaccine.  
Previously, patients received vaccine prepared as follows: 500 µg of PEP-CMV Component 
A mixed with Montanide ISA-51 i.d. administered in the RIGHT groin and 2 hours later, 500 
µg of PEP-CMV Component B mixed in 150 µg of GM-CSF i.d. administered in the LEFT 
groin. With revisions made in protocol v. 20181218, all newly enrolled patients will have the 
vaccine prepared as follows: 500 µg of PEP-CMV Component A mixed with Montanide 
ISA-51 administered i.d. with half in the RIGHT groin and half in the LEFT groin.   With the 
20190318 amendment, Component B will be removed from the vaccination regimen for all 
patients previously receiving Component B as part of the regimen. The subsequent 2 
vaccines will be administered every 2 weeks (+ 3 days) for a total of 3 vaccines. This will 
result in a ~35-day delay in the second cycle of TMZ.  MGMT unmethylated patients, after 
protocol version 20181218 is approved, will not receive subsequent cycles of TMZ, but will 
continue to receive vaccines approximately every 4 (+2) weeks. 
 
Patients will be vaccinated in conjunction with subsequent TMZ cycles every 4 (+2) 
weeks for a total of 6 to 12 cycles of TMZ after RT. Additional vaccines may be administered  
up to 20 vaccines, at the discretion of the treating neuro-oncologist. All vaccines will be 
given i.d. approximately 10 cm below the inguinal ligament bilaterally.  For MGMT 
unmethylated patients after protocol version 20181218 is approved, only one cycle of TMZ 
will be provided; however, vaccines will continue every 4 (+ 2) weeks after the single TMZ 
cycle for a maximum of 20 (unless tumor progression occurs). 
 

 
 
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PEP-CMV is administered as Component A alone (500 µL per side). As described 
above, vaccines will be delivered intradermally.  Details of the procedure will be recorded 
on the appropriate eCRF.  Patients will receive their 1st vaccine in the OTC with an IV bolus 
of normal saline prior to vaccination, and the IV saline lock will remain in place in case quick 
administration of fluids or medications is needed for a reaction. Patients will be pre-
medicated with Zofran® and Tylenol® and optionally with oral prednisone at the discretion 
of the patient and his/her provider if patient has previously experience post-vaccine 
reaction(s).  Patients will be monitored in the clinic for approximately 4 hours post-
immunization for the development of any adverse reactions after vaccines #1 and #2 for a 
total of 8 sets of vital signs (i.e., every 30 minutes).  Supportive medications for post-
vaccine reactions may include the following by IV: normal saline bolus, Benadryl, Solu-
medrol.  If no reaction(s) occur during vaccines #1 and #2, patients will be monitored for 2 
hours for a total of 4 sets of vital signs (i.e., every 30 minutes) after vaccine #3, as long as 
the reason for no reactions is not pre-medication with oral prednisone.  If no reaction(s) 
occur after vaccine #3, patients will be monitored for 30 minutes with one set of vital signs 
for subsequent vaccines, as long as the reason for no reactions is not pre-medication with 
oral prednisone.  If a patient experiences a post-vaccine reaction at any point, the 
monitoring period will revert to 4 hours until such time as he/she have 2 consecutive 
vaccines with no reaction.  The PI or sub-investigator may extend monitoring periods at 
any given visit if they feel it is clinically indicated for the patient’s safety.  Patients who have 
experienced a significant post-vaccine reaction at any previous vaccine (i.e., ≥ Grade 2 
toxicity considered possibly, probably, or definitely related to the study vaccine), but who 
are not removed from study, will receive a bolus of normal saline by IV before study vaccine 
and the saline lock will remain in place as a precautionary measure. Patients who have 
never experienced a post-vaccine reaction or only experienced a Grade 1 toxicity with 
earlier vaccines will not be required to have an IV saline lock placed prior to administration 
of  subsequent vaccines, although the treating provider may opt to proceed with this 
precautionary measure.  The immunization procedures will be supervised by a nurse or 
physician that has completed Basic Life Support (BLS) course.  A cardiac resuscitation cart 
will be available in the vicinity when performing these immunizations in case of severe 
allergic reactions.  

 
 
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11.4.3 
Treatment Plan for Study Drug Safety Arm (not applicable for 
patients enrolled on protocol version 6/12/18 or later) 
 
 
 
Figure 12: Study Drug Safety Arm Study Schema for Vaccine #1. 
 
 
 
Figure 13: Study Drug Safety Arm Study Schema for Vaccine #2 if the Patient Experiences 
No Hypersensitivity Reaction to Vaccine #1. 
 

 
 
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Following the safety events that were initially reported on 4/10/17, 6 additional patients 
were consented to receive study vaccine with standard of care (SOC) 5-day TMZ at 150-
200 mg/m2/d for 28-day cycles.  These patients were not randomized according to the 
original study design to either 21 day or 5-day Temodar; they received only SOC 5 day 
Temodar cycles.  Of these 6 patients, 3 patients were assigned to Schedule A and 3 
patients were assigned to Schedule B.  They were assigned to Schedule A or B such that 
every other patient that receives vaccine is on a different schedule, i.e., patient 1 received 
Schedule A, patient 2 received Schedule B, patient 3 received Schedule A, etc.  There was 
a planned interval of at least 48 hours between each individual patient receiving study 
vaccine.  
 
Vaccine #1 was administered according to the schematic shown in Figure 12.  Every 
patient received a tetanus preconditioning injection in the RIGHT groin on Day 22 (+1 day) 
of the TMZ cycle, and the following day received a single component of the vaccine.  
Component A consisted of 500 µg of PEP-CMV mixed with Montanide ISA-51 i.d. 
administered in the RIGHT groin and Component B consists of 500 µg of PEP-CMV, gB 
conjugated to KLH, mixed in 150 µg of GM-CSF i.d. in the LEFT groin.  Patients assigned 
to Schedule A were infused with only Component A at the first vaccine.  Patients assigned 
to Schedule B were infused with only Component B at the first vaccine.  Blood for immune 
monitoring (~ 20 mL3) was drawn just prior to component administration, 1 hour, and 2 
hours after component administration.  If no hypersensitivity reaction was observed after 
the administration of only a single Component, the patient received both components at 
the subsequent vaccine administration, according to Figure 13.  If a hypersensitivity 
reaction occurred in 2 out of 3 Schedule A patients after receiving only Component A, 
further administration of Component A would be stopped and the study team would 
evaluate potential modifications for Component A.  If a hypersensitivity reaction occurred 
in 2 out of 3 Schedule B patients after receiving only Component B, but no hypersensitivity 
reactions had been experienced by Schedule A patients receiving only Component A, 
further administration of Component B to Schedule B patients would be stopped and all 
remaining vaccinations would be done with Component A alone.  If 2 out of 3 patients on 
both Schedule A and B experienced hypersensitivity reactions after Vaccine #1, the study 
team would evaluate potential modifications to both components and no patient would 
proceed to Vaccine #2.     
 
Those patients who received Vaccine #1 with no hypersensitivity reaction proceeded 
to Vaccine #2, administered according to Figure 13.  Patients assigned to Schedule A had 
Component A administered in the RIGHT groin and were observed for 2 hours (up to 4 
hours), followed by administration of Component B in the LEFT groin.  Patients had to be 
asymptomatic for at least 2 hours before proceeding to administration of Component B.  
Blood draws occurred every hour, beginning at just prior to Component A administration 
and ending at the 2 hour observation time point following Component B administration.  
Blood (~ 20 mL3) was drawn to monitor immune activity through analysis of, but not limited 
to, TNFα, GM-CSF, IFNα, IFNγ, IL-6, and IL-1.  Patients assigned to Schedule B had 
Component B administered in the LEFT groin and were observed for 2 hours (up to 4 
hours), followed by administration of Component A administered in the RIGHT groin.  Blood 
draws occurred every hour, beginning at just prior to Component B administration and 
ending at the 2-hour observation time point following Component A administration.  These 
6 patients would continue to receive study vaccine according to their assigned schedule, if 
                                               
3 One 10 mL EDTA tube, most frequently lavender top for plasma collection, such as BD-36643.  One 10 mL 
serum tube, most frequently red top for serum collection, such as BD-VT6430. 

 
 
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no hypersensitivity reactions occurred, for as long as they remained on study.  If patients 
in Schedule A experienced a hypersensitivity reaction after Vaccine #2, they would 
continue all further injections according to Vaccine #1, Component A only.  If patients in 
Schedule B experienced a hypersensitivity reaction after Vaccine #2, but there was no 
hypersensitivity reactions to Schedule A patients with Component A alone, they would 
continue all further injections with Component A only.  After completing Vaccine #2, the 
immune monitoring blood draws and MRIs followed the schedule described in Table 3 and 
did not include the additional immune monitoring blood draws that were drawn in Vaccine 
#1 and Vaccine #2 (Figure 12 and Figure 13).  The timing of the first 3 vaccines could result 
in a potential ~35-day delay in the second cycle of TMZ. 
 
Patients were vaccinated in conjunction with subsequent TMZ cycles every 4 (+2) 
weeks for a total of 6 to 12 cycles of TMZ after RT at the discretion of the treating neuro-
oncologist. All vaccines were given i.d. approximately 10 cm below the inguinal ligament 
bilaterally (Component A in the RIGHT groin and Component B in the LEFT).  Vaccines 
continued 4 (+ 2) weeks after TMZ cycles for a maximum of 20 (unless tumor progression 
occurred). 
 
PEP-CMV was administered as two components, Component A in a total volume of 1 
mL and Component B in a total volume of 800 µL, as described above, that was delivered 
intradermally.  Patients were pre-medicated with Zofran® and were monitored in the clinic 
for 2 hours post-immunization for the development of any adverse effects. Vital signs were 
obtained every 15 minutes until discharge from the clinic.  Additional follow-up with the 
patients was done by phone at 24 and 48 hours to inquire about additional 
symptoms/adverse events experienced in the 48 hours following vaccine.  Vaccine 
administration was supervised by a nurse or physician that has completed Basic Life 
Support (BLS) course.  A cardiac resuscitation cart was available in the vicinity when 
performing these immunizations in case of severe allergic reactions. 
 
11.4.4 Drug Accountability  
The investigator and pharmacist are responsible for correct storage of vaccine. The 
vaccine made available for this clinical trial must be used in accordance with the protocol 
and must only be handled by the investigator or appropriately designated individuals. 
Documentation of receipt and disposition of the vaccine will be managed in accordance 
with study operational procedures. At completion of the trial, or during routine monitoring 
visits, the PI or his designee will perform a full drug accountability check. 
 
The master record must include: 
• 
Date of receipt of vaccine shipment 
• 
All batch numbers 
• 
Quantities received 
The dispensing record must include: 
• 
Quantities dispensed 
• 
Identification of the person to whom vaccine was administered 
• 
Date of each dispensing 
These master and dispensing records are separate from records kept for individual trial 
patients.  

 
 
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  Safety, Toxicity, and Adverse Events 
11.5.1 Post-Vaccine Management Plan 
Following a safety event on 4/5/17 in which a patient was hospitalized for post-vaccine 
reactions including nausea, vomiting, fever, chills, myalgias, dyspnea, and diarrhea, A post-
vaccine management plan was initiated to increase patient safety. A detailed treatment 
plan of the first 6 patients scheduled to receive vaccine following this safety event is 
described in Section 11.4.3.  For these 6 patients and subsequent patients, subjects were 
observed for approximately 2 hours following the first 3 vaccine administrations and follow-
up phone calls occurred at 24 and 48 hours (after the first 3 vaccines only).  For all 
subsequent vaccines, Vaccine #4+, subjects were observed for approximately 30 minutes 
following vaccination.  Monitoring included vitals approximately every 30 minutes from the 
previous vitals measurement. 
 
Additional safety events were observed in two patients treated with their first vaccine in 
June 2019.  Following discharge from our clinic after protocol-specified monitoring for 2 
hours, both patients experienced reactions. One patient experienced grade 1 fever, chills, 
and sinus tachycardia. The 2nd patient experienced grade 1 chills and nausea, grade 2 
dehydration, and grade 3 flu-like symptoms. Both patients received the protocol-specified 
pre-medications (Zofran®, Tylenol®) at their Vaccine #1 visits. At their Vaccine #2 visits, 
both patients were also pre-medicated with oral prednisone and did not experience similar 
reactions.  In response to these safety events, an extended post-vaccine monitoring period 
is being implemented in which patients will be observed in clinic for approximately 4 hours 
following Vaccines #1 and #2 with regular vital signs monitoring every 30 minutes and will 
be asked to remain in the area overnight to lessen the chance they are travelling should 
they experience a reaction.  Patients will continue to receive follow-up phone calls at 24 
and 48 hours (after the first 3 vaccines only). 
 
During an interim review of safety on November 27, 2019, the following Grade 3 
unacceptable toxicities (as defined in protocol v. 20190730) were noted in 4 out of 15 
patients:  
Patient ID 1125 
hypotension, lactic acidosis, flu-like symptoms 
Patient ID 1131 
hypotension 
Patient ID 1149 
flu-like symptoms, hypotension, lactic acidosis 
Patient ID 1163 
hypotension 
 
Further safety measures were implemented in the latter half of 2019 and the beginning 
of 2020, resulting from safety events on study.  Patients will receive their 1st vaccine in the 
Oncology Treatment Center (OTC) and will receive a normal saline bolus by IV prior to 
vaccine administration.  The IV saline lock will remain in place during the post-vaccine 
monitoring period for the purpose of quick administration of additional fluids and/or 
medication should either be necessary.  Supportive medications for post-vaccine reactions 
may include the following by IV: normal saline bolus, Benadryl, Solu-medrol.   
 
To date (January 10, 2020), we have analyzed immune responses in 16 patients who 
received vaccine 1 and were batch assayed.  We believe that the observed toxicities are 
related to cytokine release syndrome (CRS).  The immune responses across 4 degrees of 
reactions (ranging from no reactions to Grade III reactions) demonstrate a significant 
difference between levels of cytokines (G-CSF, GM-CSF, IFN-γ, IL-10, IL-2, IL-8, MIP1-α, 
and TNF-α) two hours after vaccine, as compared to those levels pre-vaccination, with IL-

 
 
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6 levels also nearing significance.  For these cytokines, subsequent pairwise comparisons 
included comparisons involving those experiencing Grade III adverse events after Tukey 
adjustment. These data suggest that the adverse event reactions following PEP-CMV 
vaccination are indeed immune-related.   
 
The cumulative data indicate that PEP-CMV vaccination is inducing the activation of T 
cells specific for the target antigen pp65. Furthermore, in patients with AEs, the pp65-
specific T cell activation may be inducing a temporary elevation of pro-inflammatory 
cytokines.  We believe that the vaccine reactions we have been observing in 
PERFORMANCE are likely indicative of vaccine potency.  Patients who have demonstrable 
Grade III reactions also have measurable cellular responses to the PEP-CMV vaccines, 
which may be indicative of clinical responses.   
 
CRS is associated with elevated circulating levels of cytokines including IL-6 and IFNγ. 
Commonly referred to as an infusion reaction, it results from the release of cytokines from 
cells targeted by the antibody, as well as immune effector cells recruited to the area. When 
cytokines are released into the circulation, systemic symptoms such as fever, nausea, 
chills, hypotension, tachycardia, asthenia, headache, rash, scratchy throat, and dyspnea 
can result. In most patients, the symptoms are mild to moderate in severity and are 
managed easily. However, some patients may experience severe, life-threatening 
reactions that result from massive release of cytokines. Massive cytokine release is an 
oncologic emergency, and special precautions must be taken to prevent life-threatening 
complications.  A CRS Management Plan based upon the ASTCT Consensus Grading 
[113] is provided in Table 2 below for the mitigation of symptoms of CRS. Please note that 
ASTCT Consensus Grading guidelines refer to the CTCAE v.5 description of constitutional 
symptoms for its CRS term. 
 
Table 2. CRS Management Plan based upon the ASTCT Consensus Grading 
Grade of 
Toxicity 
ASTCT Definition  
Management Plan 
1 
Fever ≥ 38°C, with or without 
constitutional symptoms4, not 
attributable to another cause 
Symptomatic management only 
2 
Fever ≥ 38°C with hypotension 
not requiring vasopressors 
and/or hypoxia requiring the use 
of oxygen delivered by low-flow 
nasal cannula (≤6 L/minute) or 
blow-by 
 
Hypotension:  Clearly establish a baseline 
blood pressure; if hypotension develops, 
give fluids. 
Other reactions: Patients with grade 2 
toxicity will be individually tailored, 
depending on the patient age and medical 
co-morbidity, and therefore clinical 
judgement will be crucial prior to use of 
immunosuppression for grade 2 toxicity.  
Patients with grade 2 toxicity will be 
monitored in the OTC with very close 
cardiac monitoring. 
                                               
4 The associated constitutional symptoms may be reported, but do not affect CRS grade per ASTCT 
Consensus Grading Guidelines.  

 
 
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3 
Fever ≥ 38°C with hypotension 
requiring 1 vasopressor with or 
without vasopressin and/or 
hypoxia requiring high-flow 
nasal cannula (>6 L/minute), 
facemask, 
nonrebreather mask, or venturi 
mask not attributable to another 
cause 
If grade 3 toxicity develops where fluid 
resuscitation and 1 low dose vasopressor 
are not sufficient to reverse the 
hypotension, the patient will be transferred 
and monitored closely in the emergency 
department. All patients with grade 3 
toxicity will receive immunosuppressive 
agents including corticosteroids, such as 
2mg/kg Solu-medrol.  In severe cases, 
Tocilizumab, antihuman IL-6R mAb, will be 
administered.  This drug is stocked in the 
Duke pharmacy. 
4 
Fever ≥ 38°C with hypotension 
requiring multiple vasopressors 
(excluding vasopressin) and/or 
hypoxia requiring positive 
pressure (e.g., CPAP, bilevel 
positive airway pressure, 
intubation, mechanical 
ventilation) not attributable to 
another cause 
All patients with immediate, life-
threatening toxicity will be treated with full 
support, including mechanical ventilation, 
immunosuppressive agents, Tocilizumab 
to prevent inflammatory cascade, and 
close comprehensive organ monitoring. 
 
11.5.2 
Special Consideration 
Due to the impact of high dose steroids on the development of an optimal immune 
response, if a patient demonstrates neurologic or cerebral radiographic signs suggestive 
of a localized inflammatory reaction, secondary to the immune response triggered by 
PEP-CMV vaccination, that requires an increase in dexamethasone dose, every effort 
should be made to not increase the dose above 4 mg per day at any time. Instead, 
patients should be treated with bevacizumab at the reduced dose of 7.5 mg/kg IV 
approximately every 3 weeks.  If a patient requires planned treatment for their tumor with 
bevacizumab > 7.5 mg/kg approximately every 3 weeks, they will be considered off study 
and enter the follow-up phase. Neuroimaging (MRI) will be performed according to 
protocol schedule and, at that time, it will be assessed whether further treatment with 
bevacizumab is needed to control the cerebral inflammation. Bevacizumab will not be 
provided by the study. Every attempt should be made to reduce or discontinue 
dexamethasone, when clinically possible, so as not to mitigate immune response. 
 
If there are AEs or other circumstances prohibiting the use of bevacizumab, 
corticosteroids, surgery, or other interventions deemed more appropriate for the patient 
by the treating physician will be used, if needed, to treat any localized inflammatory 
reaction secondary to DC vaccination. 
 
 
11.5.3 
Allergy Testing 
Patients who experience a hypersensitivity reaction as described in Section 15.3 may 
be offered optional Allergy Testing.  To complete the optional Allergy Testing, patients must 
sign the Allergy Test Consent Form.  Allergy testing will only be done on patients who have 
experienced a hypersensitivity reaction following vaccine administration, in an attempt to 
uncover which specific element of Component A and/or Component B results in the 

 
 
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reaction.  Patients will receive intradermal injections of potential allergens to both the left 
and the right groin.  The allergens include components that are present in Component A 
and Component B of the PEP-CMV vaccine, as relevant.  Each intradermal injection will 
be administered 1 hour apart and blood draws (~20 mL) will be taken 1 hour prior to the 
first injection and 1 hour following the final injection.  Blood (~20 mL) will be drawn to 
monitor immune activity through analysis of TNFα, GM-CSF, IFNα, IFNγ, IL-6, and IL-1, as 
appropriate.  Patients will receive SLP and Montanide in the right groin and GM-CSF, gB, 
and KLH in the left groin.  If the patient has only received either Component A or 
Component B, they will only receive intradermal injections of the elements that were part 
of the vaccine that they received.  Patients will be monitored according to Allergy and 
Immunology standard of care practices in the clinic.    
11.5.4 Unacceptable Toxicities 
The original definition of unacceptable toxicities in this study was any vaccine-related 
or any non-neurologic ≥ Grade 3 toxicity of any duration not attributable to TMZ, 
bevacizumab, and/or disease progression; any Grade 4 toxicity, including neurologic 
events not due to progressive disease; or any life threatening-event not attributable to 
concomitant medication, co-morbid event, or disease progression.  The definition of 
unacceptable toxicity was revised with v.20200117 of the protocol and will be defined now 
as any life-threatening ≥ Grade 3 toxicity that is possibly, probably, or definitely related to 
the PEP-CMV vaccine with the following exceptions (updated in the v.20200504 protocol).  
A portion of the Grade 3 vaccine reactions noted in prior patient experiences with the PEP-
CMV vaccine, described in Sections 11.5.1 and 14.2.1, will not be considered unacceptable 
if they are indicative of immune response.  That is, toxicities such as Grade 3 flu-like 
symptoms, fever, and chills/rigors will not be considered unacceptable toxicities if the 
duration is less than 72 hours. Please see Section 11.5.1 for guidelines on management 
of immune response, including cytokine release syndrome (CRS).  Any ≥ Grade 3 organ 
toxicity (cardiac, renal, hepatic), including CRS-related toxicities such as hypotension and 
tachycardia, of any duration will be considered an unacceptable toxicity. Toxicities will be 
tracked on all subjects who received PEP-CMV vaccine using the NCI CTCAE (Version 
4.03) criteria. Patients who experience unacceptable toxicities will not continue to receive 
vaccine therapy. Although not considered an unacceptable toxicity, any patient with ≥ 
Grade 2 urticaria will not receive further vaccines, will be withdrawn from the study, and will 
be replaced if less than 3 vaccines have been administered without unacceptable toxicity. 
 
The prevalence of unacceptable toxicities occurring during the initial 3 bi-weekly 
vaccinations or the vaccinations administered concurrently with temozolomide will be 
continuously monitored.  If more than 25% of accrued patients experience unacceptable 
toxicities, then accrual will be suspended and reported toxicity will be carefully reviewed to 
determine if modifications to the protocol treatment should occur.  Peptide vaccinations 
employing Montanide ISA-51 as adjuvants have generally been well tolerated in human 
patients in numerous phase I-III trials.  
11.5.5 Adverse Event Reporting and Documentation 
An “Adverse Event” will be defined as any adverse change from the subject’s pre-
treatment baseline condition (which is based on the physical and neurologic assessment 
done at the SOC clinic visit prior to initiation of post-radiation chemotherapy and vaccine 
therapy), including any clinical or laboratory test abnormality that occurs during the course 
of research after vaccine treatment has started.  Adverse events will be categorized and 
graded in accordance with the NCI CTCAE (Version 4.03). 

 
 
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A “Serious Adverse Event” will be defined as an undesirable sign, symptom or medical 
condition which: 1) is fatal or life threatening; 2) requires inpatient hospitalization or a 
prolongation of existing hospitalization; 3) results in persistent or significant 
disability/incapacity; 4) constitutes a congenital anomaly or a birth defect and/or; 5) is 
medically significant such that it may jeopardize the subject, and may require medical or 
surgical intervention to prevent one of the outcomes listed above. 
 
A summary of all adverse events (not just those considered related to the DC vaccine) 
will be kept which will categorize the event by organ system, relationship to treatment, its 
grade of severity, and resolution.  The PI will periodically review the collective adverse 
events with the intention of identifying any trends or patterns in toxicity.  If any such trends 
are identified, depending on their severity and frequency, a protocol amendment will be 
considered. 
 
All adverse events which are serious and unexpected (as defined by 21CRF312.32[a]) 
should be reported immediately to Dr. David Ashley (Pager: 919-206-3433) or his designee 
(919-684-5301).  Fatal or life-threatening, unexpected adverse events that are related or 
possibly related to the research will be reported to the FDA by telephone, facsimile, or in 
writing as soon as possible, but no later than 7 calendar days after first knowledge by the 
sponsor followed by as complete a report as possible within 8 additional calendar days.  
Serious, unexpected adverse events that are related or possibly related to the research 
and are not fatal or life-threatening will be reported to the FDA by telephone, facsimile, or 
in writing as soon as possible, but no later than 15 calendar days after first knowledge by 
the sponsor. 
 
All adverse events that are considered serious, unanticipated, and related or possibly 
related to the research (as defined by 21CRF312.32[a]) will be reported to the Duke 
University Medical Center IRB using the appropriate SAE report form.  At the time of the 
annual progress report to the Duke University Medical Center IRB, a summary of the overall 
toxicity experience will be provided. 
  Treatment Period 
Patients that have consented onto this study will receive vaccines as indicated above. 
Once patients have received vaccine therapy, they will be considered as “treated” on this 
study. 
 
Treatment procedures for the Study Drug Safety Arm are described in Section 11.4.3.  
Further details of potential study drug modification and treatment period pausing criteria is 
described in Section 15.3.  
  End of Treatment 
One month after final vaccination or once all the blood for immune monitoring has been 
collected following progression (whichever comes first), the treatment phase of the study 
will be completed.  

 
 
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  Definition of Evaluable Subjects, On Study, and End of 
Study 
Subjects evaluable for the analysis of the primary endpoints will include all randomized 
patients who undergo immune monitoring post vaccine #3, prior to 2nd vaccine/TMZ cycle.  
Survival analyses will include all randomized patients evaluable for the primary outcome.  
 
Safety analyses will include all patients who receive vaccine treatment. 
 
Once the patient signs an ICF, that subject will be considered “on study”. Rationale for 
taking patient off protocol treatment (see Section 11.10.1 below) will be documented. 
  Follow-up Period 
Patients evaluable for the primary outcome will be followed for progression and survival 
and data recorded by the study team. 
  Early Withdrawal of Subject(s) 
11.10.1 Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time. The PI may also withdraw 
a subject from the study at any time based on his/her discretion. Reasons for withdrawal 
may include at the PI’s discretion may include, but are not limited to, the following: 
• 
Progressive disease as documented by MRI or physical examination at any 
time after the initiation of immunotherapy 
• 
Development of unacceptable toxicity 
• 
Pregnancy 
• 
Patients requiring an increase in corticosteroids, with the exception of nasal 
or inhaled steroid, such that at the time of first vaccination they require a dose 
above immune suppressive levels, will be removed from the study and 
replaced.  For the purposes of this study, immune suppressive dose will be 
defined as ≤4 mg of dexamethasone / day or equivalent. Once vaccinations 
have been initiated, if patients subsequently require increased steroids, they 
will still be permitted to remain on the study, but every effort will be made to 
minimize steroid requirements 
• 
Abnormal laboratory values 
• 
Upon request of the subject 
• 
If, in the investigator’s medical judgment, further participation would be 
injurious to the subject’s health or wellbeing 
• 
Development of intolerable symptoms 
• 
Protocol deviation 
• 
Administrative reasons, such as a major violation of the clinical trial protocol 
• 
Non-compliance of the subject 
• 
Clinical decline  
• 
Allergic or hypersensitivity reaction to Td vaccine 
• 
Allergic or hypersensitivity reaction to PEP-CMV 
• 
Failure to tolerate TMZ therapy for any reason 
• 
Development of any of the following co-morbidities: 
o Unstable 
angina 
and/or 
congestive 
heart 
failure 
requiring 
hospitalization. 

 
 
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o Transmural myocardial infarction within the last 6 months. 
o Hepatic insufficiency resulting in clinical jaundice and/or coagulation 
defects; note, however, that laboratory tests for liver function and 
coagulation parameters are not required for entry into this protocol. 
o Acquired Immune Deficiency Syndrome (AIDS) based upon current 
CDC definition; note, however, that HIV testing is not required for entry 
into this protocol. The need to exclude patients with AIDS from this 
protocol is necessary because the treatments involved in this protocol 
may be significantly immunosuppressive. 
o Major medical illnesses or psychiatric impairments that in the 
investigator's opinion will prevent administration or completion of 
protocol therapy. 
11.10.2 Follow-up Requirements for Early Withdrawal 
Subjects that are withdrawn by the PI prior to randomization will be considered eligibility 
failures and thus not followed for survival. 
 
Randomized subjects treated on this study that are withdrawn by the PI for any of the 
aforementioned reasons after the immune monitoring post-vaccine 3 will continue to be 
followed for progression and survival by the study team until death or are lost to follow up 
unless the subject voluntarily withdrew permission to follow for survival.  
 
Randomized subjects that withdraw before the immune monitoring post-vaccine 3 will 
be followed for survival by the study team unless permission to do so has been withdrawn.  
11.10.3 Replacement of Early Withdrawal(s) 
Subjects who voluntarily withdraw prematurely or who are withdrawn by the PI prior to 
immune monitoring post vaccine #3 will be replaced. However, the data from these patients 
will be included in all appropriate analyses.   
11.10.4 Off Study 
Subjects evaluable for the primary outcome are considered off study if lost to follow up 
or deceased. 
 
 

 
 
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12 DESCRIPTION OF STUDY EVALUATIONS 
 Schedule of Events 
Table 3: Schedule of Events 
 
Pre-
XRT/TMZ 
Screening5 
Post- 
XRT/TMZ 
screening6 
TMZ 
Cycle 17 
 
Td pre-
conditioning8 
Vaccines 
2 & 3 
TMZ 
Cycles  
2-129 
Vaccines 
4-2010 
Progression 
CMV consent, if 
needed 
X 
X 
 
 
 
 
 
 
Main study 
consent 
 
X 
 
 
 
 
 
 
Td booster 
 
X  
 
 
 
 
 
 
Medical History 
 
X 
 
 
 
 
 
 
History and 
Physical 
 
X  
 
X 
X 
 
X 
 
Neurological 
Exam 
 
X  
 
X 
X 
 
X 
 
KPS 
 
X 
 
X 
X 
 
X 
 
Curran 
 
X  
 
 
 
 
 
 
CMV screen 
X 
X 
 
 
 
 
 
 
MGMT 
methylation 
 
X 
 
 
 
 
 
 
CBC w/ 
differential11 
 
X 
X 
X 
X 
X 
X 
 
CMP11 
 
X  
X 
X 
X 
X 
X 
 
Beta HCG 
(quantitative) 
 
X  
 
 
 
 
 
 
Cortisol 
Levels11 
 
X 
X 
 
X 
 
X 
 
                                               
5 If patients have yet to undergo XRT/TMZ when they are presented with the study, they will undergo the pre-
XRT/TMZ screening.  If the patient is CMV positive, they will undergo standard of care XRT/TMZ and then 
return post XRT/TMZ for the next screening portion.  If patients have already been tested for CMV, they will 
not need to sign the CMV consent or have the CMV test repeated. 
6 If patients have already undergone XRT/TMZ when they are presented with the study, they will undergo both 
pre- and post-XRT/TMZ screening after completing XRT/TMZ, prior to Cycle 1 of adjuvant TMZ.  If patients 
have already been tested for CMV, they will not need to sign the CMV consent or have the CMV test repeated. 
7 Temodar cycle 1 should start 2-6 weeks post the end of XRT/TMZ and will last until 2 weeks post Vaccine 
#3.  Patients will only take either 5 days of Temodar on days 1-5 (Arm 1) or 21 days on days 1-21 (Arm 2) 
based on the randomization results. 
8 Td pre-conditioning occurs on day 22 (+1 day) and is given in the RIGHT groin for all patients.  Vaccine #1 
is given on day 23 (+2 days).   
9 Temodar cycles 2 – 12, if applicable, are 28-day cycles (Patients who enrolled prior to approval of protocol 
version 20181217 and patients who enrolled after approval of protocol version 20181218 who are MGMT-
methylated.  Cycles may be given every 4 (+2) weeks, in order to adjust for slight delays on startup of each 
28-day cycle, and the total number of cycles are given at the discretion of the treating oncologist.   
10 Vaccines #4 - #20 will be given every 4-6 weeks or on day 23 (-1 day/+2 days) of each TMZ cycle depending 
on MGMT methylation status. 
11 CBC w/diff and CMP to be drawn at post XRT/TMZ screening, days 14, 21, and 28 (+/- 2 days) of each TMZ 
cycle, and at the discretion of the treating oncologist. 
11 At post-XRT/TMZ screening for baseline and prior to each vaccine. 

 
 
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Pre-
XRT/TMZ 
Screening5 
Post- 
XRT/TMZ 
screening6 
TMZ 
Cycle 17 
 
Td pre-
conditioning8 
Vaccines 
2 & 3 
TMZ 
Cycles  
2-129 
Vaccines 
4-2010 
Progression 
Immune 
Monitoring 
 
 
X13 
X14 
X13 
X15 
X16 
X17 
Vaccine 
 
 
 
X 
X 
 
X 
 
Vitals18 
 
X 
 
X 
X 
 
X 
 
Randomization 
 
X 
 
 
 
 
 
 
MRI 
 
X 
 
 
 
X19 
X20 
 
Pathology 
testing21 
 
X 
 
 
 
 
 
X17 
Adverse 
Events22 
 
Ongoing 
Con Meds22 
 
Ongoing 
 Medical History 
The medical history is to include any current active diagnoses and any previously 
treated illnesses, including the approximate date of onset (at minimum indicate the year or 
approximate year) and date of resolution if applicable, and description of any prior 
surgeries. The body systems to be reviewed and any concurrent or past diagnoses 
documented include but are not limited to: eyes, ears, nose, and throat; respiratory; 
cardiovascular; gastrointestinal; musculoskeletal; neurological; endocrine/metabolic; 
hematologic/lymphatic; dermatological; genitourinary; psychiatric and allergies. In addition, 
the patient’s date of birth, gender, race, and ethnicity should be documented. If serum CMV 
testing was not done and/or results are not available, a screening consent will be obtained 
and CMV testing will be performed. 
  Physical and Neurological Examination 
Physical examinations are to be completed by the investigator or designated 
participating clinician. The minimum body systems to be examined for any abnormalities 
and symptoms include: general appearance; skin; lymphatic; head, eyes, ears, nose, 
throat; extremities; respiratory; cardiovascular; abdominal; musculoskeletal; neurological 
and genitourinary. 
 
General neurological assessments are to be completed by the investigator or 
designated participating clinician.  The assessment includes but it not limited to: a general 
                                               
13 During vaccine 1: just prior to Component A, 1 and 2 hours after Component A (~20 mL, 1 EDTA lavender 
top tube and 1 serum red top tube at each blood draw). Just prior to vaccine 2 (1 EDTA lavender top tube, 1 
serum red top tube, and 6 yellow ACD tubes). During vaccine 2: 1 and 2 hours after Component A (~20 mL, 1 
EDTA lavender top tube and 1 serum red top tube at each blood draw). Just prior to vaccine 3 (9 yellow ACD 
tubes). 
14 Prior to Td pre-conditioning (~90 mL, 9 yellow ACD tubes and 2 red serum tubes) 
15 At the clinic visit 2 weeks after vaccine 3 [+3 days] (~90 mL, 9 yellow ACD tubes and 2 red serum tubes) 
16 Prior to vaccines #4 and #6 (~90 mL, 9 yellow ACD tubes and 2 red serum tubes) 
17 Immune monitoring blood and pathology testing performed at progression, if possible 
18 Vital signs will be measured at all protocol-indicated visits before and approximately every 30 minutes after 
vaccination during the post-vaccine monitoring times described in Sections 11.4.2 and 12.6, until discharged 
from the clinic.  Measurements to be reported include temperature in Celsius or Fahrenheit, pulse (beats per 
minute), respiratory rate (breaths per minute) and blood pressure. 
19 MRI at prior to TMZ cycle 2 clinic visit (within 2 weeks +3 days after vaccine 3) 
20 MRI visits take place at the vaccine visit that coincides with the end of TMZ/vaccine cycles 1, 3, 5, 7, 9, 11 
and then after every other vaccine visit during the post-TMZ period. 
21 Pathology testing is performed per BTC standard of care practice and is obtained at the time of new patient 
evaluation 
22 Adverse events and con meds will be collected throughout the study starting with the post-XRT/TMZ 
screening visit. 

 
 
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mental status examination (orientation, memory, attention/concentration, and language), 
assessment of cranial nerves, motor, coordination and gait, reflexes, and sensory 
evaluation.   
  Karnofsky Performance Status Score 
Patients will all be graded according to the Karnofsky performance scale at enrollment 
and recorded in the electronic medical record. 
  Curran Group Status 
Patients will be assessed for baseline Curran Group Status of I-IV at Screening (see 
Appendices for Curran and Eligibility Forms). 
 Vital Signs 
Vital signs will be measured at all protocol-indicated visits before and approximately 
every 30 minutes after each vaccination and documented in the patient’s electronic medical 
record. Patients will be monitored for approximately 4 hours after vaccines 1 and 2 for a 
total of 8 sets of vital signs.   This monitoring time can be reduced to approximately 2 hours, 
for a total of 4 vital signs, after vaccine #3, if the patient experiences no reactions to the 
first 2 vaccines, as long as the reason for no reactions is not pre-medication with oral 
prednisone. If no reaction(s) occur after vaccine #3, patients will be monitored for 30 
minutes with one set of vital signs for subsequent vaccines, as long as the reason for no 
reactions is not pre-medication with oral prednisone.  If a patient experiences a post-
vaccine reaction at any point, the monitoring period will revert to 4 hours until such time as 
the patient has 2 consecutive vaccines with no reaction.  The PI or sub-investigator may 
extend monitoring periods at any given visit if they feel it is clinically indicated for the 
patient’s safety.  Each ~30 minute interval will be counted from the time of the previous 
vitals measurement.  It will not be considered a deviation if the vitals are obtained within 10 
minutes of the 30 minute interval.  Measurements to be reported include temperature in 
Celsius or Fahrenheit, pulse (beats per minute), respiratory rate (breaths per minute) and 
blood pressure. 
  Clinical Laboratory Assessments 
Whole blood samples will be collected and submitted to the institution’s local laboratory 
according to the institution’s standard procedures. Standard laboratory procedures and 
institutional guidelines will be followed to analyze and report findings for hematology, 
coagulation times and serum biochemistry.  
 
Normal ranges with the units of measure for all required parameters will be provided to 
the study sponsor by the local laboratory conducting the clinical laboratory analysis. All 
results may be reported in conventional units if this is the typical clinic report method for 
the local laboratory; otherwise, the International System of units may be used. 
  Measurement of Radiographic Response 
Patients with newly-diagnosed GBM will be imaged by MRI as per standard of care for 
eligibility and baseline measurements, to assess progression after vaccine 3 (at the TMZ 
cycle 2 initiation visit, if applicable) and then approximately every 8 weeks (or every other 
vaccine), coinciding with every 2 cycles of TMZ, if applicable (i.e., end of cycles 3, 5, 7, 9, 
11). Although the purpose of this study is not to detect tumor responses, any evidence of 

 
 
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tumor response will be determined according to the Duke PRTBTC SOP (see  Appendix D 
– SOPs and FORMs). RANO criteria [111] will be used for overall assessment of tumor 
response and pseudoprogression. Tumor progression will need to be documented 
histologically, unless there are clinical contraindications, to exclude inflammatory 
responses presenting as radiographic or clinical changes, which could indicate potentially 
toxic or therapeutic responses and not tumor progression. If tissue is obtained, it will be 
used to confirm tumor progression histologically and to assess immunologic cell infiltration 
and antigen escape using IHC. Upon progression, patients will be taken off study treatment 
and may be treated on other therapies as directed by the treating oncologist.  
13 IMMUNOLOGICAL RESPONSE AND SAFETY EVALUATIONS 
Immunological response will be evaluated from peripheral blood obtained prior to Td 
pre-conditioning, during vaccine #1 (just prior to Component A, 1 and 2 hours after 
Component A), during vaccine #2 (just prior to Component A, 1 and 2 hours after 
Component A), prior to vaccine #3, after vaccine #3 (at the clinic visit prior to starting the 
2nd cycle of TMZ), prior to vaccines #4 and #6, and at progression (if feasible). Patients 
seen by the Duke neuro-oncology team between these vaccine visits may have blood 
drawn for immune monitoring at the discretion of the study team.  The total amount of blood 
required for this purpose will be ~90 mL, blood draw amounts for the other time points 
mentioned above are described in Table 3.  A comparison of pre-therapy lymphocyte 
functions and antibody levels to those at intervals after each immunization will be made.  
These tests may provide evidence for the development of immune responses following 
PEP-CMV immunization and will play an important role in the design of future clinical trials.   
  Serum Sample and PBMC Collection, Processing and 
Storage 
For cellular immune assays, PBMC will be separated from blood at the clinical site 
within 8 h of blood draw, using a standard SOP, frozen to -70 ˚C at -1 ˚C/min (“Mr. Frosty” 
freezing container, Thermo Fisher Scientific, Rochester, NY) and stored in liquid nitrogen.   
For detection of antibodies, serum will be separated from whole blood in a red top tube 
using a standard SOP and aliquoted at 0.2 mL per tube and stored at -135°C. 
  Enzyme-Linked Immunospot (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-γ ELISPOT allows for the direct visualization of human γ -interferon cytokine release 
from a single cell, which has been widely reported to be an indicator of activation of an 
antigen-specific immune response. Testing will include, but not be limited to, interferon 
gamma detection. 
 
On the day of testing, PBMC will be thawed quickly, washed, resuspended in R-10 
medium and cell counts and viability measured by Guava Counter (Guava Technologies, 
Inc., Hayward, CA).  Cells will then be rested overnight at 37˚C, 5% CO2, washed, and cell 
counts and viability measured by Guava Counter.  PBMCs (250,000/well) will be stimulated 
overnight with synthetic peptide pp65 in Component A of the PEP-CMV vaccine. Aliquots 
of the peptide 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-

 
 
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γ  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-γ MAb for 1 h at 
room temperature, washed with PBS, incubated with avidin-peroxidase complex for 1 h 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. If a response above background is detected to the peptide, responses will be 
measured by polyfunctional T cell assay to the pp65 peptide and to the gB peptide 
depending on availability of cells. 
 TReg Levels 
TRegs levels will be determined from PBMCs collected from blood obtained prior to 
starting post-RT TMZ therapy[114], then prior to Td pre-conditioning, at the clinic visit prior 
to 2nd cycle of TMZ, if applicable, (~2 weeks after vaccine #3), prior to vaccines 4, 6, and 
at progression, as described by us previously[114], using combinations of titrated 
antibodies against CD4 (RPA-T4), CD8 (RPA-T8), CD127 (IL-2r), and CD25 (BD 
Biosciences, San Jose, California). Following incubation, fixation/permeabilization buffer 
(eBioscience) is added to each sample. Cells are then washed in 1X permeabilization buffer 
(eBioscience), pelleted, and stained with foxp3-APC (e Bioscience, clone PCH101) for 30 
minutes in the dark at 4oC in the presence of Permeabilization Buffer (eBioscience). 
Samples are washed and analyzed on a FACSCaliber LSRII Fortessa flow cytometer (BD 
Biosciences). Data analysis will be performed using BD FloJo software. CD25+foxp3+ will 
be gated from CD4+ lymphocytes to enumerate TReg lymphocytes.   
  Polyfunctional Analysis 
Using our tiered approach to immune monitoring, if a CMV pp65 specific immune 
response is detectable by ELISpot assay, a polyfunctional analysis of 10-12 markers of T-
cell phenotype and function will be performed.  PBMCs will be stained for the surface 
markers CD3 (to identify it as a T cell), CD4 (to define as specific helper cell), CD8 (to 
define antigen specific cytotoxic T cells). The maturation state and the activation status of 
samples will be detected by polychromatic flow cytometry using optimized panels which 
will include CCR7, CD45RA, HLA-DR, and CD69. Levels of naïve (TN – CD45RA+CCR7+), 
central memory (TCM – CD45RA-CCR7+), effector memory (TEM – CD45RA-CCR7-), or 
terminally differentiated effector memory (TEMRA – CD45RA+CCR7+). To determine T-
cell reactivity to the vaccine we will include antibodies specific against the intracellular 
cytokines IFN-γ, TNF-a, IL-2 and CCL3, CD107a along with a vital-dye reagent. Briefly, 
cryopreserved PBMC samples will be thawed and rested overnight at 370C/5% CO2 in 
RPMI media containing 10% fetal calf serum. Cells are adjusted to 2x106/well with or 
without stimulation with a mixture of the  peptides to Component A in the vaccine in the 
presence of Brefeldin A (5 μg/ml Sigma-Aldrich, St. Louis, MO) monensin (1µg/ml; 
Golgistop, BD Biosciences, San Diego, CA), and antibodies against CCL3 and CD107a for 
5-6 hr at 370C and 5% CO2. Following stimulation, cells will be treated with EDTA for 15 
minutes at ambient temperature (AT, 18-22ºC). The cells will be washed, and stained with 
MAbs specific for CD4, CD8, CCR7, CD45RA, and HLA-DR. and a vital-dye reagent 
(LIVE/DEAD) for 20 minutes at AT. After two washes, 1x BD FACS Lysing solution (BD 
Biosciences, San Jose, CA) will be added and samples will be incubated for 10 minutes at 
AT.  After one wash, 1x BD FACS Permeabilizing Solution 2 (BD Biosciences, San Jose, 
CA) will be added and samples incubated for 10 minutes at AT. After one wash, cells will 

 
 
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be stained with CD3, CD69, IFN-γ, TNF-α, and IL-2 for 30 minutes on ice, washed, and 
fixed in PBS containing 1% formaldehyde (Sigma-Aldrich, St. Louis, MO).  In all 
experiments, a negative control (cells alone), and a positive control (SEB, 10 μg/ml, Sigma-
Aldrich) will be included. The samples are acquired on a custom LSRII polychromatic flow 
cytometer (BD Immunocytometry System, San Jose, CA) equipped for detection of 17 
fluorescent parameters.  We are planning to collect a minimum of 500,000 total 
lymphocytes from each sample, because we expect the frequency of responding cells to 
be between 0.05 and 1.0%.  This number of events is required based on calculations 
performed by Dr. Holden Maecker (BD Bioscience, personal communication) to detect a 
statistically significant number of positive events that can be used for the analysis of the 
data and the characterization of the different populations. 
  Tetramer Analysis 
CMV pp65- conjugated tetramers will be available (Beckman Coulter) that cover 
immunodominant CD8+ T-cell epitopes from HLA haplotypes A*0201, A*0101, A*2402, 
B*0702, B*3501, and B*0801.  PBMC from patients with GBM will be labeled with CD8-
FITC (BD Bioscience) and CD3-APC (BD Bioscience) in conjunction with the appropriate 
PE-conjugated CMVpp65 or HIV gag tetramers for 30 minutes in dark.  Cells will be 
incubated with FACS Lyse (BD Bioscience) for 30 minutes in the dark, washed, and 
analyzed on a BD FACS Calibur.  A minimum of 30,000 CD3+CD8+ events will be collected 
and sample probe will be rinsed between samples to avoid carry-over.   
14 SAFETY MONITORING 
  Potential Benefits 
Based on experience with immunization in our previous vaccine trials, immunotherapy 
may be of benefit to patients with MGs.  Of course, because individuals respond differently 
to therapy, no one can know in advance if it will be beneficial in an individual case.  The 
potential benefits may include reduction and/or remission of the patient’s brain cancer.  
Because this procedure is experimental, it cannot be guaranteed that patients will receive 
any benefit as a result of participating in this research study.  The information collected in 
this research may help scientists better understand the mechanisms involved in the 
immune system’s ability to fight cancer.  If such an understanding comes from this 
research, then it may benefit society by furthering the development of improved treatment 
methods for human malignant brain tumors in the future. 
 
Most recently, we examined immune data on sixteen study participants who had received 
vaccine 1 and had been batch assayed. These data indicate that PEP-CMV vaccination is 
inducing the activation of T cells specific for the target antigen pp65.  Likely, this is a result 
of vaccine potency against CMV.  
  Potential Risks 
14.2.1 Allergic Reactions to PEP-CMV Immunization 
Injection of the PEP-CMV vaccine 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 locally may include pain, redness and 
swelling at the injection site.  Grade 3 vaccine reactions have been experienced by patients 

 
 
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who have received the PEP-CMV vaccine in this study and include hypotension, flu-like 
symptoms, and lactic acidosis.  Patients also have experienced Grade 1 and 2 vaccine 
reactions, such as fever, chills, headache, sinus tachycardia, nausea, and dehydration. Our 
recent analysis of patients with AEs on this study indicates that pp65-specific T cell 
activation may be inducing a temporary elevation of pro-inflammatory cytokines.  We 
believe that the vaccine reactions we have been observing in PERFORMANCE are likely 
indicative of vaccine potency.  Patients who have demonstrable Grade III reactions also 
have measurable cellular responses to the PEP-CMV vaccines, which may be indicative of 
clinical responses.   
14.2.2 
Injection Site  
EMLA® cream, or equivalent topical analgesic, can be offered to subjects who 
experience local pain at the injection site. 
14.2.3 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 PEP-CMV injections. Patients 
will be monitored throughout the course of the study.  
14.2.4 
Steroid Induced Adrenal Suppression 
It is widely recognized that many brain tumor patients will suffer from steroid induced 
adrenal suppression. This is often related to chronic systemic therapy with dexamethasone 
on a daily or twice daily regimen.  
 
It is well supported that the best predictor of adrenal suppression is the patient's current 
glucocorticoid dosage. Patients who receive doses of dexamethasone of more than 2 mg 
per day for periods of more than three weeks and those who appear to have cushingoid 
features are highly likely to have adrenal suppression. 
 
As patients suffering from adrenal suppression are at risk of acute adrenal crisis in 
association with an acute medical event such as allergic reactions, it is strongly 
recommended that patients on this study receive physiologic replacement hydrocortisone 
therapy (20-30 mg/day in divided doses) if a wean of dexamethasone is considered 
indicated. 
14.2.5 Infection 
The PEP-CMV injections may include the risk of infection due to potential contamination 
of the peptides in the laboratory.  This may result in localized redness, swelling, or 
induration at the injection site.  In the most extreme situation, this may lead to systemic 
bacterial/fungal sepsis and possibly death.  The probability of this risk is relatively low, 
given the small injection volume (1 mL divided between >2 intradermal locations) and the 

 
 
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fact that the peptides will be strictly tested for sterility prior to each injection.  The risk of 
infection due to potential contamination of the peptides in the laboratory will be minimized 
by biosafety quality assurance and testing.  All cell cultures will be handled under sterile 
conditions in a core tissue culture facility dedicated to the processing of human cells.  Prior 
to injection into patients, peptides must pass sterility tests in thiglycolate broth, tryptic soy 
blood agar, and inhibitory Sabouraud agar.  Following injections, patients will be monitored 
throughout the course of the study for any signs and symptoms of infection. There have 
been no infections to date in the recent VICTORI (IRB #3108-05-9R4) or ACTIVATE (IRB 
#5421-05-1R1) clinical trials testing similar approaches in a similar patient population.  If 
an active infection is suspected, patients will be cultured and treated with appropriate 
antibiotics.  
14.2.6 Delayed Autoimmune Diseases 
It is possible that delayed autoimmune disease(s) may develop as a result of injection 
with PEP-CMV.  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 peptide vaccination in glioma patients has not demonstrated evidence of 
autoimmunity in treated patients.  Furthermore, the doses of TMZ used in this study for 
induction of lymphopenia are standard doses administered to patients with GBM.  It, 
therefore, is unknown what the risk of delayed autoimmune disease is for this study.  
14.2.7 Phlebotomy 
Drawing blood or inserting an intravenous catheter into an arm vein may result in 
bruising or swelling in the area of the insertion, bleeding at the site of the needle puncture, 
light headedness, fainting and very rarely, local infection, which may be severe. These risks 
are reduced by the fact that the blood will be drawn by a qualified physician, nurse or 
phlebotomist (a professional trained to draw blood). 
14.2.8 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 be excluded from the study. Patients will also be checked to make sure that 
they do not bring any metal objects into the MRI facility.  Dental fillings are less affected by 
the magnetic fields generated and are therefore permitted.  It will be asked that patients let 
the physicians conducting this study know of any metal in their bodies other than dental 
fillings.   
14.2.9 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.  

 
 
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Patients with mild allergies (i.e., rash only) will be pretreated with Tylenol and Benadryl 
prior to injection of the contrast agent. 
14.2.10 Temozolomide 
TMZ has been well tolerated by both adults and children with the most common toxicity 
being mild myelosuppression.  Other, less likely, potential toxicities include nausea and 
vomiting, constipation, headache, alopecia, rash, burning sensation of skin, esophagitis, 
pain, 
diarrhea, 
lethargy, 
hepatotoxicity, 
anorexia, 
fatigue 
and 
hyperglycemia.  
Hypersensitivity reactions have not yet been noted with TMZ.  As in the case with many 
anti-cancer drugs, TMZ may be carcinogenic.  Rats given TMZ have developed breast 
cancer.  The significance of this finding for human is not presently known. TMZ therapy will 
be followed but given as standard of care. If toxicities occur, the Principle investigator and 
primary physician will titrate therapy based on standard clinical guidelines as outlined in 
the TMZ package insert (please see TMZ package insert uploaded in the electronic IRB). 
14.2.11 Allergic Reactions to KLH 
Injection of KLH (applies only to patients who received Component B) 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.  
14.2.12 GM-CSF  
Injection of GM-CSF (applies only to patients who received Component B) 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. 
14.2.13 Montanide ISA 51 
Montanide ISA 51 adjuvant is well-tolerated. Local reactions may include granuloma, 
local pain, tenderness and erythema.  Montanide ISA 51 may also cause flu-like 
symptoms, nausea and vomiting.  
14.2.14 Td Risks 
Injection of Td toxoid may produce inflammation, edema, induration, erythema, or 
pruritus at the injection site. Intradermal administration may cause occasional pain and 
discomfort up to three days after the vaccine is given. 
 
14.2.15 Unknown Risks 
The overall risk classification of this research is unknown.  Clinical trials using peptide-
based immunizations on brain tumor patients only recently published in the literature. From 
our experience with 40 patients in ongoing and previous trials, we have not seen any 
toxicities or serious unexpected adverse events.  
 
14.2.16 Confidentiality 
Participation in research investigations may result in a loss of confidentiality.  However, 
all data from preoperative and postoperative evaluations will be coded to protect the 

 
 
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patient’s identity.  The coding, and the results of these studies will be available only to the 
individuals involved with the study, the clinical staff administering the study, representatives 
of the National Institutes of Health, and representatives of the U.S. Food and Drug 
Administration.  Any publications resulting from this study will not use patient identifying 
data. 
 
14.2.17 Treatment Alternatives & Financial Reimbursement 
Alternative treatments for newly-diagnosed malignant brain tumors include additional 
surgery, radiation, and/or chemotherapy.  If the patient chooses not to participate in this 
trial, they certainly may seek alternative treatment.  If the patient fails treatment through 
this trial, these alternatives may still be available to the patient.  There will be no financial 
reimbursement to patients for study participation. 
 
  Data and Safety Monitoring Plan 
The DCI Monitoring Team will conduct monitoring visits to ensure subject safety and to 
ensure that the protocol is conducted, recorded, and reported in accordance with the 
protocol, standard operative procedures, good clinical practice, and applicable regulatory 
requirements.  As specified in the DCI Data and Safety Monitoring Plan, the DCI Monitoring 
Team will conduct routine monitoring after the third subject is enrolled, followed by annual 
monitoring of 1-3 subjects until the study is closed to enrollment and subjects are no longer 
receiving study interventions that are more than minimal risk. 
 
The Safety Oversight Committee (SOC) will perform annual reviews on findings from 
the DCI Monitoring Team visit and additional safety and toxicity data submitted by the 
Principal Investigator. 
 
Additional monitoring may be prompted by findings from monitoring visits, unexpected 
frequency of serious and/or unexpected toxicities, or other concerns and may be initiated 
upon request of DUHS and DCI leadership, the CPC, the Safety Oversight Committee 
(SOC), the sponsor, the Principal Investigator, or the IRB.  All study documents must be 
made available upon request to the DCI Monitoring Team and other authorized regulatory 
authorities, including but not limited to the National Institute of Health, National Cancer 
Institute, and the FDA.  Every reasonable effort will be made to maintain confidentiality 
during study monitoring. 
 
This clinical research study will also be monitored internally by the PI.  In terms of 
internal review the PI will continuously monitor and tabulate adverse events.  Appropriate 
reporting to the Duke University Medical Center IRB will be made.  If an unexpected 
frequency of grade III or IV events occur, depending on their nature, action appropriate to 
the nature and frequency of these adverse events will be taken.  This may require a protocol 
amendment, dose de-escalation, or potentially closure of the study.  The PI of this study 
will also continuously monitor the conduct, data, and safety of this study to ensure that: 
 Interim analyses occur as scheduled; 
 Stopping rules for toxicity and/or response are met; 
 Risk/benefit ratio is not altered to the detriment of the subjects; 
 Appropriate internal monitoring of adverse events and outcomes is done; 
 Over-accrual does not occur; 
 Under-accrual is addressed with appropriate amendments or actions; 
 Data are being appropriately collected in a reasonably timely manner. 

 
 
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15 STATISTICAL CONSIDERATIONS 
  Study Design and Conduct 
A total of 70 patients may be consented on the main informed consent, in order to 
randomize a sufficient number of patients with newly diagnosed GBM who completed 
standard of care radiotherapy with temozolomide into one of two treatment arms.  Patients 
will receive 3 bi-weekly (every other week) vaccinations during the first cycle of either: (Arm 
1) 5-day standard of care temozolomide at 150-200 mg/m2/day, or (Arm 2) 21-day 
temozolomide at 75-100 mg/m2/day post chemo-radiation (please refer to study overview 
for timing of vaccine administration).  We will target the treatment of 26 patients (13 per 
arm) who are evaluable for the primary immune endpoint. All patients will continue to 
receive PEP-CMV vaccine as described in the Treatment Plan at 4 (+2) week intervals for 
up to 20 vaccines, these vaccine administrations will coincide with TMZ cycles for those 
who are receiving TMZ.  
 
With the 20190312 protocol amendment, the study’s primary analysis has focused on 
patients who receive Component A only.  Hence, we target the treatment of 26 patients (13 
per arm) who received only Component A and are evaluable for the immune endpoints. 
 
Once 6 subjects have been accrued to each arm of this study and the last patient has 
received the third vaccination and has been followed for approximately 2 weeks, toxicities 
up to that point will be formally monitored. Further accrual will be dependent upon the 
prevalence of unacceptable toxicity within each arm.  See Section 15.4 for further details 
of monitoring guidelines. 
 
Following the safety events of vaccine reactions that were initially reported on 
20170407, the study was amended to investigate, in a separate unrandomized drug safety 
cohort of 6 patients, the etiology of these events.  Within this cohort, patients were 
consented to receive study vaccine with standard of care (SOC) 5-day TMZ at 150-200 
mg/m2/d for 28-day cycles with various sequences of vaccine components A and B. Details 
of this safety assessment plan are provided in Section 11.4.3.  Upon completion of this 
unrandomized portion of the study, the randomized study was allowed to reinitiate with 
changes to vaccine administration procedures based on observations made in the safety 
cohort (protocol v. 20180719). Subsequently, Component B was removed from the vaccine 
for future patients due to problems with availability and supply (protocol v.20181218). On 
20190130, Component B was removed from the vaccine for all patients (current and future) 
due to two new safety events observed and reported in January 2019. 
 
As of 20190312, 13 patients have been enrolled on the study: 
 
Periods of Study Conduct 
Patient Enrollment Description as of 20190312 
Initial – Randomized 
3 patients randomized and treated with 1-2 vaccinations that 
included Component A and B as originally planned before 
study suspension: 
• 
2 patients assigned to standard TMZ on days 1-5 
• 
1 patient assigned to dose-intensified TMZ on days 1-21 
Safety Cohort  
(See Section 11.4.3) 
6 patients treated with standard TMZ with different 
sequences of Component A and B 
Resumed – Randomized 
Once randomization resumed, the 1st 4 patients randomized: 

 
 
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• 
2 patients assigned to standard TMZ with vaccine that 
included Component B; Component B was dropped for 
these 2 patients due to safety events (one patient 
received only one vaccine containing Component B, and 
the other patient received 3 vaccines containing 
Component B).  
• 
2 patients assigned to dose-intensified TMZ – 
Component B was dropped before these patients started 
treatment; hence, only Component A was part of 
vaccine. 
 
For all subsequent patients enrolled, the vaccine includes 
Component A only.  
 
As of protocol version 20190312, the primary analysis of this study will focus on only 
those patients who receive Component A alone.  
 
As of 20200110, 15 patients have received only Component A of which 8 are currently 
evaluable for the immune response primary endpoint.  
 Sample Size Justification for Immunologic Response 
Outcome 
Of primary interest is a comparison of the 2 arms with respect to the peak immune 
response (ELISPOT) in the immune monitoring blood that will be collected as described in 
the treatment plan. A patient will be considered evaluable and included in these analyses 
if the patient had their immune monitoring blood work drawn prior to initiation of post-RT 
TMZ cycle 2, if applicable.  
 
Given that humoral responses are known to be surrogates for cellular immune 
responses (ELISPOT), we will use antibody titer data from the ACT II study, a study 
conducted at Duke, to estimate an appropriate sample size for pairwise comparison of peak 
immune response (ELISPOT). The ACT II dataset provides peak antibody titer to EGFRvIII 
data for 5 patients in arm A and 10 patients in arm B.  Given that the distribution of peak 
titers in the two arms is not normally distributed, resampling methods were used to estimate 
the power of a comparison using a one-tailed Wilcoxon rank sum test.  The median peak 
titer (and range) in arm A and B were 51200 (2560 – 204800) and 385000 (20480, 
20480000) respectively.  Each of 10000 simulations for a fixed sample size per arm 
involved randomly sampling with replacement the peak antibody data separately within arm 
A and B, and then computing the Wilcoxon rank sum test.  The percentage of tests that 
was statistically significant estimated the power of the statistical test.  With 2 primary 
endpoints, power calculations assumed a type I error rate of 0.025.  With 13 evaluable 
patients in each arm, there is 81% to detect a difference similar to that observed in ACT II.  
In order that there are 26 evaluable patients, we anticipate a need to consent up to 70 
patients on the main informed consent form. 
 
With the 20190312 protocol amendment, the study has focused on patients who 
receive Component A only.  Hence, we target the treatment of 26 patients who have 
received only Component A and are evaluable for the assessment of immunologic 
response. As of 20200110, 15 patients have received only Component A of which 8 are 
evaluable for immune response. 

 
 
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  Toxicity Monitoring 
After the 20190312 protocol amendment, only the monitoring described below in the 
section entitled “Overall Adverse Event Monitoring” is applicable. 
 
Initial Study Monitoring for Adverse Events:  Once 6 subjects have been accrued to 
each arm of this study and the last patient has received the third vaccination and has been 
followed for approximately 2 weeks, toxicities up to that point will be formally monitored. A 
patient who does not complete this follow-up period without experiencing an unacceptable 
toxicity will be replaced for this initial monitoring of adverse events.  If 1 or less of the 6 
subjects within each arm experience an unacceptable toxicity, accrual will continue.  
Otherwise, if 2 or more of the 6 subjects within an arm experience unacceptable toxicity, 
the study will be carefully reviewed to determine if modifications to the protocol or treatment 
plan need to occur. 
 
Study Drug Safety Arm Monitoring for Hypersensitivity Reactions (not applicable for 
patients enrolled on protocol version 6/12/18 or later): For the study drug safety arm 
(Section 11.4.3), unacceptable toxicity is defined as any hypersensitivity reaction within 6 
hours after vaccine reaction that requires intervention to prevent life threatening symptoms, 
according to the PI and the study team.  The PI will review the reaction to determine if it is 
unacceptable. 
Corrective Action for Hypersensitivity Reactions in Study Drug Safety Arm (not 
applicable for patients enrolled on protocol version 6/12/18 or later):  If 2 out of 3 Schedule 
A patients experience hypersensitivity reactions after Component A injection (Vaccine #1), 
the study will be put on hold in order to evaluate potential modifications to Component A to 
increase patient safety.  
 
If 2 out of 3 Schedule A patients experiences hypersensitivity reactions after 
Component B injection (Vaccine #2), we will discontinue the use of Component B and 
continue all further vaccinations with Component A alone.  
 
If 2 out of 3 Schedule B patients experience hypersensitivity reactions after Component 
B injection (Vaccine #1), but Schedule A patients have not experienced hypersensitivity 
reactions after Vaccine #1, we will discontinue the use of Component B in Schedule B 
patients and continue all further vaccinations with Component A alone. 
 
If 2 out of 3 Schedule B patients experience hypersensitivity reactions after Component 
A injection during Vaccine #2, but Schedule A patients have not experienced 
hypersensitivity reactions after Vaccine #1, Schedule B patients will discontinue the use of 
Component B and continue all further vaccinations with Component A alone.    
 
If 2 out of 3 patients from both Schedule A and B experience hypersensitivity reactions 
after Vaccine #1, the study will be put on hold and the study team will work on modifying 
the study drug to decrease the hypersensitivity reaction.   
 
Overall Adverse Event Monitoring:  The prevalence of unacceptable toxicities occurring 
during the initial 3 bi-weekly vaccinations administered concurrently with temozolomide will 
be monitored.  If more than 25% of accrued patients experience unacceptable toxicities, 
then accrual will be suspended and reported toxicity will be carefully reviewed to determine 

 
 
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if accrual should be terminated or modifications to the protocol treatment should occur.  
This review will consider that patients will have different lengths of follow-up and duration 
of vaccination treatment.   
 
As described in Section 11.5.3, the definition of unacceptable toxicity has been modified 
with v.20200117 of the protocol, and now excludes vaccine reactions indicative of immune 
response. Therefore, as of the time of this amendment, no patient had experienced an 
unacceptable toxicity under the new definition. 
  Analytic Methods 
15.4.1 Primary Analyses 
Safety:  To assess the safety of PEP-CMV vaccination in combination with adjuvant 
TMZ, the percentage of patients with unacceptable toxicity will be estimated within each 
arm.  All patients who received any PEP-CMV vaccine will be included in these safety 
analyses.   
 
As described in Section 15.1, the vaccination treatment regimen has been modified 
over time due to safety issues.  Hence, toxicity will be summarized for each period of study 
conduct as follows: 
 
• 
Initial randomization period where patients were assigned to one of two TMZ regimens 
with both Component A and B.  The 2 patients assigned to standard TMZ after 
randomization resumed who received both Component A and B will also be included in 
this summary. 
• 
Safety Cohort Period in which all patients received standard TMZ with vaccine 
Component A and B 
• 
Randomization period in which patients received at least one cycle of Component B 
prior to discontinuation 
• 
Randomization period in which patients are assigned to one of two TMZ regimens and 
receive only vaccine Component A 
 
Immunologic Response:  The primary analysis will focus on patients who have follow-
up immunologic monitoring after the 3rd vaccination and before initiation of the second 
TMZ/vaccine cycle.  Such a patient is considered “evaluable” for the immunologic response 
primary analyses.   
 
The Wilcoxon rank sum test will compare treatment groups with regard to the median 
peak number of T cells that secrete IFNγ by ELISPOT in response to component A of PEP-
CMV.  Analyses will include only those patients who have an assessment of immune 
response after receiving 3 vaccinations. 
 
Though the original vaccination included Component A and B, the primary immunologic 
analysis will focus on only those patients who received Component A alone.  Additional 
exploratory analyses may include data from the few “evaluable” patients who received both 
vaccine Components A and B. 
15.4.2 Secondary Analyses 
CMV Status at Recurrence: Among patients who provide pathology at the time of tumor 
recurrence, a chi-square test will be used to compare treatment arms with respect to the 

 
 
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percentage of patients observed to be positive for antigen escape outgrowth at tumor 
recurrence.  A paired t-test will be used to compare within each treatment arm the mean 
intensity of CMV antigen expression for primary and recurrent tumors.  Alternative 
nonparametric methods may be considered if assumptions for parametric analytic methods 
are not satisfied. Though the original vaccination included Component A and B, the primary 
analysis of CMV status at recurrence will focus on only those patients who received 
Component A alone.  However, additional exploratory analyses may include data from the 
few patients who received both vaccine Components A and B for whom we have info about 
CMV status at recurrence. 
15.4.3 Exploratory Immunologic Analyses 
The following immunologic analyses will focus on patients who are evaluable for the 
immunologic monitoring primary analysis, i.e., patients who had immunologic monitoring 
conducted at baseline and prior to the second cycle of TMZ.  These analyses will focus 
primarily upon patients who received only Component A.  However, additional analyses 
may be conducted besides those described below. 
 
Quality of Immune Response:  The quality of the immune response will be assessed 
by polyfunctional flow cytometry.  The analysis used here for polyfunctionality will be one 
that has been successful at distinguishing HIV progressors and non-progressors based on 
T-cell phenotype [115, 116]. All data will be background subtracted. For each measure, a 
lower threshold corresponding to two standard deviations above background is set to 0 
based on a Poisson model essentially allowing T-cells to be designated as positive or 
negative for a certain phenotypic marker. The number of positive phenotypic markers post-
vaccination will be calculated for each patient.  The Kruskal-Wallis test will be used to 
compare treatment arms with respect to the number of phenotypic markers observed post-
vaccination 3, as well as at vaccination 6.  Additional exploratory analyses will be conducted 
using a Fisher’s exact test that will compare treatment groups with respect to the proportion 
of patients with 5 positive markers, and 4 or more positive markers.  Analyses similar to 
those described for the primary immunologic analyses will be used to examine the 
percentage of tetramer positive cells. 
 
Analysis of covariance will be used to compare treatment groups with respect to the 
change between baseline and post-vaccination time-points in mean proliferative response 
and cytokine production, with baseline levels included in the model as a covariate.  If 
assumptions of normality are not satisfied, the Kruskal-Wallis test will be used to compare 
treatment arms.  Analyses will focus on the change between baseline and post-vaccination 
3 assessments, as well as the change between baseline and vaccine 6. 
 
Effect on Tregs:  The mean levels of Tregs at baseline and at each follow-up 
assessment will be estimated within each treatment group.  A mixed model that accounts 
for the correlation of repeated measurements within a patient will be used to compare 
treatment groups with respect to changes in Treg levels over time. 
 
Effect of HLA haplotypes: Initially the distribution of HLA haplotypes (A, B, C, DR) will 
be described.  Depending up the distribution of these haplotypes, the effect of HLA 
haplotype on outcome may be explored using the proportional hazards model (for OS and 
PFS) or linear regression (for some immune response outcomes).  Analyses will adjust for 
treatment group.  If possible, analyses will appropriately adjust for confounding variables.  
 

 
 
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Immunologic Cell Infiltrate:  Within each treatment arm, changes in levels of infiltrating 
lymphocytes detected in primary and recurrent tumor specimens will be assessed using a 
paired t-test or its nonparametric analogue among patients who provide pathology at the 
time of tumor recurrence.  When appropriate, a two-sample t-test will compare arms with 
respect to the change. 
15.4.4 Other Exploratory Analyses 
Progression-Free Survival and Overall Survival:  The Kaplan-Meier estimator will be 
used to describe the progression-free survival (PFS) and overall survival (OS) experience 
of patients in each treatment group.  These analyses will focus on those patients who have 
received at least 3 vaccines.  PFS is defined as the time between the third vaccine and first 
documentation of death or disease progression/recurrence.  Patients remaining alive 
without disease progression will have PFS censored at their last follow-up. OS is defined 
as time between the third vaccine and death.  It should be noted that these analyses will 
focus on only those patients who received Component A alone as we have with other 
analyses. Additional analyses will describe OS and PFS from randomization.  
 
Radiographic Response:  Among the subset of patients with residual disease, the 
proportion of patients with radiographic response will be estimated with 95% confidence 
interval. 
16 DATA MANAGEMENT AND PROCESSING 
  Study Documentation 
Study documentation includes but is not limited to source documents, case report 
forms, monitoring logs, appointment schedules, study team correspondence with sponsors 
or regulatory bodies/committees, and regulatory documents that can be found in the DCI-
mandated “Regulatory Binder”, which includes but is not limited to signed protocol and 
amendments, approved and signed informed consent forms, FDA Form 1572, CAP and 
CLIA laboratory certifications, and clinical supplies receipts and distribution records. 
 
Source documents are original records that contain source data, which is all information 
in original records of clinical findings, observations, or other activities in a clinical trial 
necessary for the reconstruction and evaluation of the trial. Source documents include but 
are not limited to hospital records, clinical and office charts, laboratory notes, memoranda, 
subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified after verification as being accurate 
copies, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject 
files, and records kept at the pharmacy, at the laboratories and at medico-technical 
departments involved in the clinical trial. When possible, the original record should be 
retained as the source document. However, a photocopy is acceptable provided that it is a 
clear, legible, and an exact duplication of the original document. 
  Data Management 
The subject’s medical records will be the primary source document for the study. 
Source documents include all information in original records and certified copies of original 
records of clinical findings, observations, or other activities in a clinical investigation used 
for reconstructing and evaluating the investigation. Source documentations may also 
include paper eligibility checklists, data flowsheets, patient reported outcomes and other 

 
 
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paper documents. The PI, the study coordinator, the data management team, and the 
clinical trials manager are permitted to make entries, changes, or corrections in the source 
documents or database per the study delegation of authority log. 
 
Errors on the source documents will be crossed out with a single line, and this line will 
not obscure the original entry. Changes or corrections will be dated, signed, initialed, and 
explained (if necessary). Database changes will be tracked via electronic trail 
automatically. 
  Data Management Procedures and Data Verification 
The DCI IT Shared Resource has developed Title 21 CFR Part 11 compliant databases 
for cancer clinical trials. DCI IT has extensive expertise in database quality assurance, data 
standards, and use of caBIG tools to support cancer researchers.  
 
Data queries will be generated automatically by the eCRF system. These data queries 
signify the presence of data inconsistencies. The study and data management team will 
cross-reference the data to verify accuracy. Missing or implausible data will be highlighted 
for the PI requiring appropriate responses (i.e., confirmation of data, correction of data, 
completion or confirmation that data is not available, etc.). 
 
The database will be reviewed and discussed prior to database closure, and will be 
closed only after resolution of all remaining queries. 
  Coding 
All medical terms will be coded using CTCAE (version 4.03). 
  Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following 
activities: 
 
• 
Data clarification and/or resolution 
• 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
• 
Review of site study records for completeness 
• 
Shipment of all remaining laboratory samples to the designated laboratories. 
17 ADMINISTRATIVE AND ETHICAL CONCERNS 
  Regulatory and Ethical Compliance 
This protocol was designed and will be conducted and reported in accordance with the 
ICH Harmonized Tripartite Guidelines for Good Clinical Practice, the Declaration of 
Helsinki, and applicable federal, state, and local regulations. 
  DUHS Institutional Review Board and DCI Cancer Protocol 
Committee 
The protocol, informed consent form, advertising material, and additional protocol-
related documents must be submitted to the DUHS IRB and DCI CPC for review. The study 
may be initiated only after the Principal Investigator has received written and dated 
approval from the CPC and IRB. 

 
 
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The Principal Investigator must submit and obtain approval from the IRB for all 
subsequent protocol amendments and changes to the informed consent form. The CPC 
should be informed about any protocol amendments that potentially affect research design 
or data analysis (i.e., amendments affecting subject population, inclusion/exclusion criteria, 
agent administration, statistical analysis, etc.). 
 
The Principal Investigator must obtain protocol re-approval from the IRB within 1 year 
of the most recent IRB approval. The Principal Investigator must also obtain protocol re-
approval from the CPC within 1 year of the most recent IRB approval, for as long as the 
protocol remains open to subject enrollment. 
  Informed Consent 
The informed consent form must be written in a manner that is understandable to the 
subject population. Prior to its use, the informed consent form must be approved by the 
IRB. 
 
The Principal Investigator or authorized key personnel will discuss with the potential 
subject the purpose of the research, methods, potential risks and benefits, subject 
concerns, and other study-related matters. This discussion will occur in a location that 
ensures subject privacy and in a manner that minimizes the possibility of coercion. 
Appropriate accommodations will be made available for potential subjects who cannot read 
or understand English or are visually impaired. Potential subjects will have the opportunity 
to contact the Principal investigator or authorized key personnel with questions, and will be 
given as much time as needed to make an informed decision about participation in the 
study. 
 
Before conducting any study-specific procedures, the Principal Investigator must obtain 
written informed consent from the subject. The original informed consent form will be stored 
with the subject’s study records, and a copy of the informed consent form will be provided 
to the subject. The Principal Investigator is responsible for asking the subject whether the 
subject wishes to notify his/her primary care physician about participation in the study. If 
the subject agrees to such notification, the Principal Investigator will inform the subject’s 
primary care physician about the subject’s participation in the clinical study. 
  Privacy, Confidentiality, and Data Storage 
The Principal Investigator will ensure that subject privacy and confidentiality of the 
subject’s data will be maintained. RDSPs will be approved by the appropriate institutional 
Site Based Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to 
subjects during the course of the study. Prospective participants will be consented in an 
exam room where it is just the research staff, the patient and his family, if desired. For all 
future visits, interactions with research staff (study doctor and study coordinators) regarding 
research activities will take place in a private exam room. All research related interactions 
with the participant will be conducted by qualified research staff who are directly involved 
in the conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets 
under lock and key accessible only by the research staff. Subjects will be identified only by 

 
 
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a unique study number and subject initials. Electronic records of subject data will be 
maintained using an electronic database, which is housed by the DCI. Access to electronic 
databases will be limited to the Principal Investigator, key personnel, statisticians, and the 
PRTBTC data manager. Data stored on portable memory devices will be de-identified. The 
security and viability of the IT infrastructure will be managed by the DCI and/or Duke 
Medicine.  
 
Upon completion of the study, research records will be archived and handled per DUHS 
HRPP policy.  
 
Subject names or identifiers will not be used in reports, presentations at scientific 
meetings, or publications in scientific journals. 
  Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and 
Safety Monitoring Plan. The DCI Safety Oversight Committee (SOC) is responsible for 
annual data and safety monitoring of DUHS sponsor-investigator phase I and II, therapeutic 
interventional studies that do not have an independent DSMB. The primary focus of the 
SOC is review of safety data, toxicities and new information that may affect subject safety 
or efficacy. Annual safety reviews includes but may not be limited to review of safety data, 
enrollment status, stopping rules if applicable, accrual, toxicities, reference literature, and 
interim analyses as provided by the sponsor-investigator. For a more detailed description 
of the DSMP for this protocol, refer to Section 16.    
  Protocol Amendments 
All protocol amendments must be initiated by the Principal Investigator and approved 
by the IRB prior to implementation. IRB approval is not required for protocol changes that 
occur to protect the safety of a subject from an immediate hazard. However, the Principal 
Investigator must inform the IRB and all other applicable regulatory agencies of such action 
immediately. 
 
Though not yet required, the CPC should be informed about any protocol amendments 
that potentially affect research design or data analysis (i.e., amendments affecting subject 
population, inclusion/exclusion criteria, agent administration, etc.). 
  Records Retention 
The Principal Investigator will maintain study-related records for the longer of a period 
of: 
- 
at least two years after the date on which a New Drug Application is approved by 
the FDA 
- 
at least two years after formal withdrawal of the IND associated with this protocol 
- 
at least six years after study completion (Duke policy). 
  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, 

 
 
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speaking honoraria, advisory board appointments, publicly-traded or privately-held 
equities, stock options, intellectual property, and gifts. 
 
The Duke University School of Medicine’s RIO reviews and manages research-related 
conflicts of interest. The Principal Investigator and Sub-Investigators must report conflicts 
of interest annually and within 10 days of a change in status, and when applicable, must 
have a documented management plan that is developed in conjunction with the Duke RIO 
and approved by the IRB/IEC. 
 
Due to potential for COI in relation to proprietary interest in the PEP-CMV vaccine, a 
Data Safety and Monitoring Board (DSMBplus) will be established.  Please see Appendix 
C – DSMBplus Charter for detail on the Duke PRTBTC DSMBplus Charter. 
  Registration Procedure 
After patients have been enrolled, subject registration will be entered into the Duke 
electronic research system and the subject’s visits associated in the Duke Epic Maestro 
Care system with this protocol, which is entered after Duke IRB approval. 

 
CONFIDENTIAL 
 
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19 APPENDICES 
  Appendix A - Pneumocystis Carinii Management 
Pneumocystis Carinii Prophylaxis  
Both corticosteroid therapy and continuous temozolomide therapy induce 
lymphocytopenia. Patients receiving any of these drugs or both concomitantly are at an 
increased risk for opportunistic infections. Therefore, a prophylaxis against P. carinii 
pneumonia is recommended for all patients receiving temozolomide during radiotherapy: 
trimethoprim-sulfamethoxazole (Bactrim forte®, Bactrim DS®) 1 tablet 3 times per week or 
monthly pentamidine inhalations (300 mg via aerosol monthly) or dapsone 100 mg p.o. 
each day (except in patients with G6-PD deficiency). Prophylaxis is recommended to 
continue for the duration of radiotherapy, regardless of the lymphocyte count and will be 
up to the discretion of the treating neuro-oncology team. After completion of the 
chemoradiation, patients with a lymphocyte count < 500/mm2 should have CD4 
quantification. If the CD4 is < 200, then prophylaxis is recommended to continue and the 
CD4 should be quantified on a monthly basis. If the lymphocyte count is ≥ 500 or the CD4 
is > 200, then prophylaxis can be stopped. 
 
 
 

 
 
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  Appendix B – TMZ Package Insert 
Uploaded separately in electronic IRB. 

 
 
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 Appendix C – DSMBplus Charter  
Uploaded separately in electronic IRB. 
 
 

 
 
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  Appendix D – SOPs and FORMs 
Uploaded separately in electronic IRB.