Pilot Trial of pTVG-HP DNA Vaccine and Pembrolizumab in Patients With Castration-Resistant, Metastatic Prostate Cancer
Phase: PHASE1
Conditions: Hormone-Resistant Prostate Cancer, Metastatic Malignant Neoplasm in the Bone, Metastatic Malignant Neoplasm in the Soft Tissues, Metastatic Prostate Carcinoma, Prostate Adenocarcinoma, Recurrent Prostate Carcinoma, Stage IV Prostate Cancer
Sponsor: University of Wisconsin, Madison

Vaccine Therapy and Pembrolizumab in Treating Patients with Hormone-Resistant, 
Metastatic Prostate Cancer
NCT02499835
PI: Glenn Liu, MD
04-06-2021

04/06/2021 version 
 
 
Pilot trial of pTVG-HP DNA Vaccine and Pembrolizumab in Patients with Castration-
Resistant, Metastatic Prostate Cancer  
 
UW15014 
 
INVESTIGATIONAL AGENT: 
 
 
 
 
Referencing:  BB IND 12109 - pTVG-HP DNA encoding human prostatic acid  
 
 
 
 
phosphatase  
 
 
 
 
 
STUDY SPONSOR / PROTOCOL PRINCIPAL INVESTIGATOR:  
Douglas G. McNeel, MD PhD 
 
 
STUDY SUPPORTER: 
Madison Vaccines, Inc., 505 S. Rosa Road, Madison, WI  
Prostate Cancer Foundation (MSN 175454) 
 
 
STUDY SITE INFORMATION 
 
Study Sites: 
 
University of Wisconsin Carbone Cancer Center (UWCCC) 
 
 
 
1111 Highland Avenue 
 
 
 
Madison, WI 53705 
 
Local Principal Investigator:  
 
 
 
Glenn Liu, M.D. 
 
 
 
UWCCC 
7051 Wisconsin Institutes for Medical Research 
1111 Highland Ave.  
Madison, WI 53705 
Tel:  (608) 265-8689 
Fax:  (608) 265-5146 
 
 
 
gxl@medicine.wisc.edu 
 
Other Investigators: Christos Kyriakopoulos, M.D. – Clinical Investigator  
 
 
 
Joshua Lang, M.D. M.S. – Clinical Investigator 
 
 
 
Hamid Emamekhoo, M.D. – Clinical Investigator 
 
 
 
Robert Jeraj, Ph.D. – Physicist, molecular imaging 
 
 
 
Scott Perlman, M.D. – Nuclear Medicine 
 
 
 
Humberto, Rosas M.D. – Interventional Radiology 
 
 
 
 
 
 

 
 
Version 04/06/2021 
2 
 
 
Study Coordinator: 
Mary Jane Staab, R.N. B.S.N. 
 
 
 
UWCCC 
 
 
 
600 Highland Ave. K6/5 CSC 
 
 
 
Madison, WI  53792 
 
 
 
Tel:  (608) 263-7107 
 
 
 
mjs@medicine.wisc.edu 
 
 
Study Biostatistician: Jens C. Eickhoff, Ph.D. 
 
 
 
UWCCC 
 
 
 
600 Highland Ave.  K4/438 CSC 
 
 
 
Madison, WI  53792 
 
 
 
Tel:  (608) 265-6380 
 
 
 
eickhoff@biostat.wisc.edu 
 
 
 
Projected Start Date:  
 
June 1, 2015 
 
Projected End of Accrual Date: 
September 1, 2018 
 
Projected End of Study Date:  
September 1, 2019 
 

 
 
Version 04/06/2021 
3 
 
TABLE OF CONTENTS 
 
SYNOPSIS:..................................................................................................................................... 4 
 
1. 
Introduction ..................................................................................................................... 10 
 
2. 
Background and Rationale............................................................................................... 12 
 
3. 
Objectives ........................................................................................................................ 19 
 
4. 
Product Information ......................................................................................................... 20 
 
5. 
Patient Selection .............................................................................................................. 23 
 
A. 
Inclusion Criteria .................................................................................................. 23 
 
B. 
Exclusion Criteria ................................................................................................. 24 
 
6. 
Experimental Design ....................................................................................................... 26 
 
7. 
Measurement of Effect (PCWG2 Recommendations) .................................................... 28 
 
8. 
Definition and Management of Limiting Toxicities and Adverse Events ....................... 34 
 
9. 
Plan of Treatment ............................................................................................................ 37 
 
10. 
Response Monitoring ....................................................................................................... 51 
 
A. 
Safety .................................................................................................................... 51 
 
B. 
Immunological Monitoring ................................................................................... 51 
 
C. 
Circulating Tumor Cell (CTC) Evaluation ........................................................... 56 
 
D. 
Histopathology Evaluation .................................................................................... 56 
 
E. 
FLT PET/CT Molecular Imaging ......................................................................... 57 
 
F.  
Na PET/CT Molecular Imaging……………………………………………….... 59 
 
11. 
Statistical Considerations ................................................................................................ 60 
 
12. 
Administrative Considerations ........................................................................................ 67 
 
13. 
Data and Safety Monitoring Plan .................................................................................... 68 
 
14. 
Potential Risks and Benefits, and Procedures to Minimize Risk .................................... 75 
 
15. 
Study Data Management and Procedural Issues .............................................................. 79 
 
16. 
Roles and Responsibilities of Study Personnel at UWCCC ............................................ 82 
 
17. 
References: ...................................................................................................................... 83 

 
 
Version 04/06/2021 
4 
 
SYNOPSIS: 
 
Primary Objectives: 
 
1. To evaluate the safety of pembrolizumab in combination with pTVG-HP in patients with 
castration-resistant, metastatic prostate cancer 
2. To determine the 6-month progression-free survival and median time to radiographic 
progression in patients with castration-resistant metastatic prostate cancer treated with 
pembrolizumab in combination with pTVG-HP 
3. To evaluate the anti-tumor response rates (objective response rate and PSA response rate, 
using PCWG2 criteria) in patients with castration-resistant metastatic prostate cancer 
treated with pembrolizumab in combination with pTVG-HP 
 
Secondary Objectives: 
 
1. To determine whether either treatment sequence, or PAP-specific immune response, is 
associated with prolonged (6-month) radiographic progression-free survival 
2. To evaluate effects of schedule (concurrent versus delayed administration of 
pembrolizumab) on the magnitude of PAP-specific T-cell responses, PD-1 expression on 
circulating T cells, and PD-L1 expression on circulating epithelial cells (CEC) and on 
tumor biopsies 
3. To determine the median time to radiographic progression using concurrent administration 
schedules 
 
Exploratory Objectives: 
 
1. To evaluate effects of treatment on the number of circulating tumor cells  
2. To evaluate PAP-specific antibody responses following treatment with pembrolizumab and 
pTVG-HP DNA vaccine 
3. To determine whether treatment with either sequence elicits immunologic antigen spread 
to other prostate associated antigens 
4. To determine whether pre-existing or vaccine-induced PD-L1 expression on CEC or tumor 
biopsies is predictive of objective clinical response 
5. To determine whether treatment elicits expression of other regulatory molecules on tumor-
specific T cells (e.g. TIM3, BTLA, and LAG3) or tumor cells (e.g. HVEM, phosphatidyl 
serine, PD-L2)   
6. To determine whether PD-1-regulated antigen-specific T-cells identified by trans vivo 
DTH testing can identify patients who develop objective clinical responses with PD-1 
blockade therapy in combination with pTVG-HP 
7. To determine whether changes in lymph nodes and soft tissue tumor lesions are observed 
by FLT PET/CT after treatment with vaccine with or without pembrolizumab  
8. To determine if PD-1 inhibitor therapy in combination with pTVG-HP will change number 
and activity (SUV) in osteoblastic metastases as measured by NaF PET/CT. 
 

 
 
Version 04/06/2021 
5 
 
Study Scheme: 
 
 
 
 
 
 
 
 
 
Extended treatment Arm 3: 
 
 
 
 
 
Extended treatment Arm 4: 
 
 
 

 
 
Version 04/06/2021 
6 
 
Plan of Treatment:   ARM 1 (Dark grey indicates those days and procedures specific to Arm 1) 
 
 
 
Vaccine / 
Treatment 
Visit: 
 
 
 
SCREENING 
Day 1  
Day 15 (+/- 3 days)  
Day 22 (7 +/- 3 days 
after day 15)
Day 29 (14 +/3 days  
after day 15)
Day 43 (14 +/- 3 days 
after day 29)
Day 57  (14 +/- 3 days 
after day 43)
Day 64 (7 +/- 3 days 
after day 57)
Day 71 (14 +/- 3 days 
after day 57)
Day 85 (14 +/- 3 days 
after day 71) – 3 month 
Da 106 (21 +/- 3 days 
after day 85) 
Da 127 (21 +/- 3 days 
after day 106)
Day 148 (21 +/- 3 days 
after day 127)
Day 169 (21 +/- 3 days 
after day 148) – 6 m 
9-month F/U (84 +/- 10 
days after day 169 
OF F STUDY  
12-month F/U – (84 +/- 
10 days after 9moth 
Within 6 
weeks of day 1 
Within 4 
weeks of day 1 
History and Physical Exam 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam, 
ECOG PS 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Tox assessmenta 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 Lab Tests 
CBC 
X 
 
X 
 
X 
 
X 
 
X 
 
Xg 
X 
X 
X 
X 
X 
X 
Chemistry 
panelf  
X 
 
X 
 
X 
 
X 
 
X 
 
Xg 
X 
X 
X 
X 
X 
X 
PT/INR 
 
Xk 
 
 
 
 
 
 
 
 
Xk 
 
 
 
 
 
 
Serum 
testosterone 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PSA and PAP 
X 
 
X 
 
X 
 
X 
 
X 
 
Xg 
X 
X 
X 
X 
X 
X 
Procedures 
CT chest/ 
abdomen/pelvis 
X 
 
 
 
 
 
 
 
 
 
Xgj 
 
 
 
Xhj 
Xhj 
Xij 
Bone scanl 
X 
 
 
 
 
 
 
 
 
 
Xg 
 
 
 
Xh 
Xh 
Xi 
Leukapheresis 
 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
Xbh 
 
 
FLT PET scanm 
 
Xc 
 
 
 
 
 
 
 
 
Xg 
 
 
 
 
 
 
Tissue biopsym 
 
Xd 
 
 
 
 
 
 
 
 
Xg 
 
 
 
 
 
 
Tetanus 
immunization 
 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for 
immune 
analyses 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
Blood for CTC 
analysis 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
 
X 
X 
X 
Treatments 
DNA 
immunization 
 
 
X 
X 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
Pembrolizumab 
infusion  
 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
a Study coordinator or research nurse review of systems   
 
 
b For patients unable to undergo leukapheresis, a 200-mL blood draw (green top tubes) may be performed instead 
c FLT PET scan – within 4 weeks (28 days) of day 1 – to provide baseline study for comparison prior to treatment 
d Biopsy within 2 weeks prior to day 1 
e Tetanus immunization to take place any time after leukapheresis and prior to day 1  
f Where chemistry labs indicated, the following serum tests are to be performed: Chem 7 (sodium, potassium, bicarbonate, BUN, 
creatinine, glucose), ALT, AST, bilirubin, alkaline phosphatase, amylase, TSH (thyroid stimulating hormone), and LDH 
g Procedures can be +/- 7 days of this visit, scans to be done prior to biopsy (and study treatment for Arm 2) 
h Can be +/- 7 days of this visit 
i Can be +/- 7 days of this visit, and not required if this is off-study visit and previous scans already confirmed disease progression 
j CT of chest at this time point only as clinically indicated  
k PT/INR only required for those subjects who will be undergoing a biopsy.  May be performed up to 30 days prior to biopsy, 
required same day if subject is on coumadin 
l If NaF PET/CT is available as standard of care procedure, this may be substituted for bone scan (bone scintigraphy), however all 
pre-treatment and post-treatment tests must use the same modality (NaF PET/CT or bone scintigraphy) 
m See section 9 for whether or not biopsy and FLT PET is required 

 
 
Version 04/06/2021 
7 
 
Plan of Treatment:   ARM 2 
 
 
 
Vaccine / 
Treatment 
Visit: 
 
 
 
SCREENING 
Day 1  
Day 15 (+/- 3 days) 
Day 29 (14 +/3 days  
after day 15)
Day 43 (14 +/- 3 days 
after day 29)
Day 57  (14 +/- 3 days 
after day 43)
Day 71 (14 +/- 3 days 
after day 57)
Day 85 (14 +/- 3 days 
after day 71) – 3 month 
Da 106 (21 +/- 3 days 
after day 85)
Da 127 (21 +/- 3 days 
after day 106) 
Day 148 (21 +/- 3 days 
after day 127)
Day 169 (21 +/- 3 days 
after day 148) – 6 m 
9-month F/U (84 +/- 10 
days after day 169 
OF F STUDY  
12-month F/U – (84 +/- 
10 days after 9moth 
F/U) 
Within 6 
weeks of day 1 
Within 4 
weeks of day 1 
History and Physical Exam 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam, 
ECOG PS 
 
X 
X 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
Tox assessmenta 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 Lab Tests 
CBC 
X 
 
X 
 
 
X 
 
 
Xg 
X 
X 
X 
X 
X 
X 
Chemistry 
panelf  
X 
 
X 
 
 
X 
 
 
Xg 
X 
X 
X 
X 
X 
X 
PT/INR 
 
Xk 
 
 
 
 
 
 
Xk 
 
 
 
 
 
 
Serum 
testosterone 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PSA and PAP 
X 
 
X 
 
 
X 
 
 
Xg 
X 
X 
X 
X 
X 
X 
Procedures 
CT chest/ 
abdomen/pelvis 
X 
 
 
 
 
 
 
 
Xgj 
 
 
 
Xhj 
Xhj 
Xij 
Bone scanl 
X 
 
 
 
 
 
 
 
Xg 
 
 
 
Xh 
Xh 
Xi 
Leukapheresis 
 
Xb 
 
 
 
 
 
 
 
 
 
 
Xbh 
 
 
FLT PET scanm 
 
Xc 
 
 
 
 
 
 
Xg 
 
 
 
 
 
 
Tissue biopsym 
 
Xd 
 
 
 
 
 
 
Xg 
 
 
 
 
 
 
Tetanus 
immunization 
 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for 
immune 
analyses 
 
X 
 
 
 
X 
 
 
Xg 
 
 
 
X 
X 
X 
Blood for CTC 
analysis 
 
X 
 
 
 
X 
 
 
Xg 
 
 
 
X 
X 
X 
Treatments 
DNA 
immunization 
 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Pembrolizumab 
infusion 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
 
 
a Study coordinator or research nurse review of systems   
 
 
b For patients unable to undergo leukapheresis, a 200-mL blood draw (green top tubes) may be performed instead 
c FLT PET scan – within 4 weeks (28 days) of day 1 – to provide baseline study for comparison prior to treatment 
 
d Biopsy within 2 weeks prior to day 1 
e Tetanus immunization to take place any time after leukapheresis and prior to day 1  
f Where chemistry labs indicated, the following serum tests are to be performed: Chem 7 (sodium, potassium, bicarbonate, BUN, 
creatinine, glucose), ALT, AST, bilirubin, alkaline phosphatase, amylase, TSH (thyroid stimulating hormone), and LDH 
g Procedures can be +/- 7 days of this visit; scans to be done prior to biopsy (and study treatment for Arm 2) 
h Can be +/- 7 days of this visit 
i Can be +/- 7 days of this visit, and not required if this is off-study visit and previous scans already confirmed disease progression 
j CT of chest at this time point only as clinically indicated 
k PT/INR only required for those subjects who will be undergoing a biopsy. May be performed up to 30 days prior to biopsy, 
required same day if subject is on coumadinl If NaF PET/CT is available as standard of care procedure, this may be substituted for 
bone scan (bone scintigraphy), however all pre-treatment and post-treatment tests must use the same modality (NaF PET/CT or 
bone scintigraphy) 
m See section 9 for whether or not biopsy and FLT PET is required. 

 
 
Version 04/06/2021 
8 
 
Plan of Treatment:   ARM 3 – EXTENDED TREATMENT COHORT 
 
 
 
Vaccine / 
Treatment Visit: 
 
 
 
SCREENING 
Day 1  
Week 3 (21 +/- 3 
days from Day 1)
Week 6 (21 +/- 3 
days from week 3)
Week 9 (21 +/- 3 
days from week 6)
Week 12 (21 +/- 3 
days from week 9)
Week 15 (21 +/- 3 
days from week 12)
Week 18 (21 +/- 3 
days from week 15) 
Week 21 (21 +/- 3 
days from week 18)
Week 24 (21 +/- 3 
days from week 21)
Week 27 (21 +/- 3 
days from week 24)
Week 30 (21 +/- 3 
days from week 27) 
Week 33 (21 +/- 3 
days from week 30) 
Week 36 (21 +/- 3 
days from week 33) 
Week 39 (21 +/- 3 
days from week 36) 
Week 42 (21 +/- 3 
days from week 39) 
Week 45 (21 +/- 3 
days from week 42) 
END OF STUDY  
Week 48 (21 +/- 3 
days after week 45) 
28-Day Follow Up  
(28 +/- 7 days after 
off-treatment visit) 
Within 6 wks 
of day 1 
Within 4 wks 
of day 1 
 
History and Physical Exam 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam, 
ECOG PS 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Tox assessmenta 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Lab Tests 
CBC 
X 
 
X 
Xi Xi Xi Xi Xi 
Xi 
Xi Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
X 
Chemistry panelf  
X 
 
X 
Xi Xi Xi Xi Xi 
Xi 
Xi Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
X 
PT/INR 
 
Xk 
 
 
 
 
Xk 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum 
testosterone 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PSA and PAP 
X 
 
X 
Xi Xi Xi Xi Xi 
Xi 
Xi Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
Xi 
X 
 
Procedures 
CT chest/ 
abdomen/pelvis 
X 
 
 
 
 
 
Xgj 
 
 
 
Xhj 
 
 
 
Xhj 
 
 
 
Xhj 
 
Bone scan 
X 
 
 
 
 
 
Xg 
 
 
 
Xh 
 
 
 
Xh 
 
 
 
Xh 
 
Leukapheresis 
 
Xb 
 
 
 
 
 
 
 
 
Xbh 
 
 
 
 
 
 
 
 
 
FLT PET scanm 
 
Xc 
 
 
 
 
Xg 
 
 
 
 
 
 
 
 
 
 
 
 
 
NaF PET/CT 
scanm 
 
X 
 
 
Xg 
 
Xg 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tissue biopsym 
 
Xd 
 
 
 
 
Xg 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tetanus 
immunization 
 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for 
immune analyses 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
(X)n 
Blood for CTC 
analysis 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
(X)n 
 
Treatments 
DNA 
immunization 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Pembrolizumab 
infusion 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
a Study coordinator or research nurse review of systems   
 
 
b For patients unable to undergo leukapheresis, a 200-mL blood draw (green top tubes) may be performed instead 
c FLT PET scan – within 4 weeks (28 days) of day 1 – to provide baseline study for comparison prior to treatment 
 
d Biopsy within 2 weeks prior to day 1 
e Tetanus immunization to take place any time after leukapheresis and prior to day 1  
f Where chemistry labs indicated, the following serum tests are to be performed: Chem 7 (sodium, potassium, bicarbonate, BUN, 
creatinine, glucose), ALT, AST, bilirubin, alkaline phosphatase, amylase, TSH (thyroid stimulating hormone), and LDH 
g Procedures can be +/- 7 days of this visit; scans to be done prior to biopsy  
h Can be +/- 7 days of this visit 
i Blood draws can be up to 3 days prior to this visit 
j CT of chest at this time point only as clinically indicated 
k PT/INR only required for those subjects who will be undergoing a biopsy.  May be performed up to 30 days prior to biopsy, 
required same day if subject is on coumadin 
m See section 9 for whether or not biopsy, FLT, or NaF PET is required. 
n Blood collected for immune analysis only if not collected within previous 60 days 
 
 

 
 
Version 04/06/2021 
9 
 
Plan of Treatment:   ARM 4 – EXTENDED TREATMENT COHORT  
 
 
 
 
 
 
 
Vaccine / Treatment Visit 
Screening 
Day 1 
Wk 2 (14 +/- 3 days from Day 1) 
Wk 4 (14 +/- 3 days from Wk 2) 
Wk 6 (14 +/- 3 days from Wk 4) 
Wk 8 (14 +/- 3 days from Wk 6) 
Wk 10 (14 +/- 3 days from Wk 8) 
Wk 12 (14 +/- 3 days from Wk 10) 
Wk 14 (14 +/- 3 days from Wk 12) 
Wk 16 (14 +/- 3 days from Wk 14) 
Wk 18 (14 +/- 3 days from Wk 16) 
Wk 20 (14 +/- 3 days from Wk 18) 
Wk 22 (14 +/- 3 days from Wk 20) 
Wk 24 (14 +/- 3 days from Wk 22) 
Wk 26 (14 +/- 3 days from Wk 24) 
Wk 28 (14 +/- 3 days from Wk 26) 
Wk 30 (14 +/- 3 days from Wk 28) 
Wk 32 (14 +/- 3 days from Wk  30) 
Wk 34 (14 +/- 3 days from Wk 32) 
Wk 36 (14 +/- 3 days from Wk 34) 
Wk 38 (14 +/- 3 days from Wk 36) 
Wk 40 (14 +/- 3 days from Wk 38) 
Wk 42 (14 +/- 3 days from Wk 40) 
Wk 44 (14 +/- 3 days from Wk 42) 
Wk 46 (14 +/- 3 days from Wk 44) 
END OF TREATMENT  Wk 48 
(14 +/- 3 days from Wk 46)
28-Day Follow Up (28 +/- 7 days 
after off-treatment visit) 
Within 6 wks of day 1 
Within 4 wks of day 1 
History and Physical Exam 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam, ECOG PS 
 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Tox assessmenta 
 
X 
X X X X X X X X X X X X X X X X X X X X X X X X 
X 
X 
Lab Tests 
CBC 
X 
 
X 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
Xf 
Chemistry paneld  
X 
 
X 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
Xf 
Serum testosterone 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PSA and PAP 
X 
 
X 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
 
Xf 
Xf 
Procedures 
CT chest/ abdomen/pelvis 
X 
 
 
 
 
 
 
 Xeg  
 
 
 
 Xeg  
 
 
 
 Xeg  
 
 
 
 
Xeg 
 
Bone scan 
X 
 
 
 
 
 
 
 
Xe 
 
 
 
 
 
Xe 
 
 
 
 
 
Xe 
 
 
 
 
 
Xe 
 
NaF PET/CT scani 
 
X 
 
 
 
Xe 
 
 
Xe 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Leukapheresisb 
 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 Xbe  
 
 
 
 
 
 
 
 
 
 
 
 
Tetanus immunizationc 
 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for immune analyses 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
(X)h 
Blood for CTC analysis 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
(X)h 
Treatments 
DNA immunization 
 
 
X X X X X X X X X X X X X X X X X X X X X X X X 
 
 
Pembrolizumab infusion 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
a Study coordinator or research nurse review of systems   
 
 
b For patients unable to undergo leukapheresis, a 200-mL blood draw (green top tubes) may be performed instead 
c Tetanus immunization to take place any time after leukapheresis and prior to day 1  
d Where chemistry labs indicated, the following serum tests are to be performed: Chem 7 (sodium, potassium, bicarbonate, BUN, 
creatinine, glucose), ALT, AST, bilirubin, alkaline phosphatase, amylase, TSH (thyroid stimulating hormone), and LDH 
e Can be +/- 7 days of this visit 
f Blood draws can be up to 3 days prior to this visit 
g CT of chest at this time point only as clinically indicated 
h Blood collected for immune analysis only if not collected within previous 60 days 
i There will be up to 10 subjects total between Arms 3 and 4 who will undergo NaF PET/CT scan.   
 
 
 

 
 
Version 04/06/2021 
10 
 
1. 
Introduction 
 
Prostate cancer is the most common tumor among men, and the second leading cause of male 
cancer-related death in the United States [1].  Despite advances in screening and early detection, 
over 30,000 U.S. men are estimated to die as a result of prostate cancer in 2014 [1].  Treatment 
with surgery and radiation remain effective for presumed organ-confined disease, however 
approximately one third of these patients will have progressive or metastatic disease at 10 years 
[2].  Prostate cancer, once it becomes metastatic, is not curable and is generally initially treated 
with androgen deprivation, and androgen deprivation remains the cornerstone on which other 
therapies are added [3].  Unfortunately, within typically 2-3 years the disease becomes refractory 
to androgen deprivation, and castrate-resistant prostate cancer is the lethal phenotype of the 
disease.  Within the last several years, several therapies have been approved by FDA based on 
their ability to prolong overall survival in this population of patients.  Specifically, docetaxel was 
approved in 2004 after two large randomized trials showed a 2-3 month median improvement in 
overall survival compared with mitoxantrone [4, 5].  Cabazitaxel was approved in 2010 for patients 
with disease refractory to docetaxel after an international trial demonstrated a similar 2-3 month 
median improvement in overall survival compared with mitoxantrone [6].  Also in 2010, 
sipuleucel-T (Provenge®, Dendreon Corporation) was approved for patients with minimally 
symptomatic castrate-resistant, metastatic prostate cancer, based on the results of a prospectively 
randomized, blinded, phase III placebo-controlled clinical trial, and supporting data from previous 
phase III clinical trials, demonstrating a median 4-month improvement in overall survival [7].  
Finally, in 2011 and 2013, abiraterone and enzalutamide, agents targeting androgen synthesis or 
signaling, were similarly approved in the setting of docetaxel-refractory, castrate-resistant 
metastatic prostate cancer following prospectively randomized, blinded, placebo-controlled trial 
demonstrating a 2-3-month median improvement in overall survival [8, 9].  These recent advances 
have clearly improved the situation for patients with advanced prostate cancer, however have 
presented new challenges in terms of the optimal sequence and approach to the management of 
castrate-resistant disease.   
 
Despite the impact of chemotherapies for advanced prostate cancer, many patients and treating 
physicians believe that the small overall survival benefit provided by chemotherapy may not justify 
its use in all patients, in part due to potential side effects [10].  Vaccine-based strategies, also 
known as active immunotherapies, are particularly appealing as potentially safer treatments, and 
the trials that led to the approval of sipuleucel-T showed markedly fewer adverse events than are 
typically seen with chemotherapy agents [7].  Many vaccines for prostate cancer are in clinical 
development, all of which have demonstrated similar safety profiles, and some of which have 
demonstrated anti-tumor activity, as we have recently reviewed [11, 12, 13, 14].  There has also 
been interest in developing antigen non-specific immune-activating therapies, such as through T-
cell checkpoint blockade.  Inhibitors of PD/PD-L1 or CTLA-4, for example, have demonstrated 
remarkable efficacy alone or in combination for metastatic melanoma [15, 16, 17], and PD/PD-L 
blockade inhibitors have demonstrated activity as single agents for several solid tumor types [18, 
19].  Targeting PD-1, in particular, should be a universal therapy, as it targets the T-cell 
compartment rather than the tumor directly.  However, in separate phase I trials with PD-1 
blocking antibodies, there has been no objective response observed to patients (n=25) with 
metastatic prostate cancer [18, 19].  The difference in response to certain tumors, with higher 
frequencies of responses observed in patients with renal cell cancer, melanoma and non-small cell 
lung cancer compared to prostate cancer, for example, suggest that differences are likely due to 

 
 
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differences in the T-cells of responding and non-responding patients.  In addition, early phase 
clinical trials using PD-1 or PD-L1 have identified that the expression of at least one of the ligands 
for PD-1 (PD-L1) on the target tumor cell by biopsy is associated with clinical response to therapy 
[18].  This is expected, given that tissue-infiltrating T cells can induce the expression of PD-L1 
via the expression of IFNγ.  Notwithstanding, some tumors that are known to express PD-L1, such 
as prostate cancer [20], have demonstrated little response to treatment with PD-1 blockade as 
single-agent therapies in early phase trials [18, 19].   
  
In preliminary studies (further described below), we have recently identified in a murine model 
using a vaccine targeting a tumor antigen that DNA immunization leads to the generation of CD8+ 
T cells with cytolytic activity, and that these cells express PD-1. Moreover, immunization leads to 
a compensatory response in the tumor to increase expression of PD-L1.  In further studies, efforts 
to increase the number and avidity of CD8+ T cells with cytolytic activity by means of changes 
introduced into the DNA vaccine led to an unanticipated inferior anti-tumor immune response due 
to this PD-1/PD-L1 upregulation.  Blockade of this pathway using antibodies blocking either PD-
1 or PD-L1 restored the anti-tumor activity of the vaccine encoding the tumor-specific target 
antigen in this murine model, an effect not observed with anti-PD-1 treatment alone, as 
monotherapy had no demonstrable effect.  Importantly, immunization with the vaccine encoding 
the native (unmodified) antigen in combination with an antibody blocking PD-1 similarly 
demonstrated an improved anti-tumor response compared to vaccination alone.  As a result, 
evaluating active immunotherapies in combination with PD-1 checkpoint inhibitors are a rational 
direction to further improve on the efficacy of these therapies and may have durable, objective 
anti-tumor responses in patients with metastatic cancer [21]. 
 
Prostatic acid phosphatase (PAP) is a model antigen for vaccine-based treatment strategies 
targeting prostate cancer.  PAP is a well-defined protein whose expression is essentially restricted 
to normal and malignant prostate tissue [22].  It is also one of only a few known prostate-specific 
proteins for which there is a rodent homologue, thereby providing an animal model for evaluating 
vaccine strategies and assessing toxicity [23].  Data from independent labs has demonstrated that, 
in a rat model, vaccine strategies targeting PAP can result in PAP-specific CD8+ T-cells, the 
presumed population mediating tumor cell destruction, and anti-tumor responses [24, 25, 26, 27].  
PAP is the target antigen of the autologous antigen-presenting cell sipuleucel-T vaccine in which 
autologous peripheral blood mononuclear cells are loaded ex vivo with a PAP-GM-CSF fusion 
protein.  A separate phase I clinical trial evaluated dendritic cells loaded with a murine homologue 
of PAP, and demonstrated immunogenicity of this approach [28].  In rodent studies, we have 
similarly demonstrated that PAP can be immunologically targeted using genetic vaccines, and a 
plasmid DNA vaccine in particular [25, 26].   We have previously reported the results of a phase 
I/II trial conducted in patients with early, PSA-recurrent (clinical stage D0) prostate cancer using 
this same DNA vaccine (pTVG-HP).  No significant adverse events were observed in 22 subjects 
treated over a 12-week period of time.  Moreover, several patients developed evidence of PAP-
specific CD4+ and CD8+ T-cells, and several patients experienced a prolongation in the PSA 
doubling time, demonstrating immunological efficacy and suggesting a possible anti-tumor effect 
[29].  The presence of long-term IFNγ-secreting immune responses to PAP, detectable at multiple 
times months after immunization, were associated with increases in PSA doubling time, suggesting 
this might serve as a rational biomarker for efficacy [30].  Moreover, it was found that immune 
responses could be augmented months later with repeated immunizations, suggesting that DNA 
vaccines might provide a simple means of eliciting tumor-specific CD8+ T cells [31].  These 

 
 
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findings have justified further evaluation of this vaccine in a randomized phase II clinical trial, a 
study currently underway (NCT01341652).  Finally, further preliminary studies have suggested 
that patients previously treated with pTVG-HP have circulating PAP-specific CD8+ T cells with 
PD-1 expression and circulating EpCam+ circulating epithelial cells (CEC) with PD-L1 
expression, analogous to our findings in murine models.  These finding suggest that the pTVG-HP 
vaccine might specifically be used to elicit CD8+ T cells specific for prostate tumors, the efficacy 
of which could be augmented with concurrent treatment with PD-1 blockade.  Collectively, these 
findings form the rationale for the current trial.   
 
 
2. 
Background and Rationale 
 
A. 
PAP is a tumor antigen in prostate cancer and PAP-specific CD8+ CTL can lyse prostate 
cancer cells 
 
PAP was first identified in 1938 and was initially used as a serum marker for the detection of 
prostate cancer [22, 32].  PAP expression in normal and malignant prostate cells is well-
documented, and is still used in immunohistochemical staining to establish a prostate origin of 
metastatic carcinoma [33].  The ubiquitous expression of PAP in prostate tissue makes it an 
appealing antigen as a potential “universal” target for immune-directed therapies of prostate 
cancer, unlike specific oncogenes that may or may not be expressed by a particular tumor.  
Moreover, it has been demonstrated that some patients with prostate cancer have preexisting 
antibody and T-cell responses to PAP, suggesting that tolerance to this “self” protein can be 
circumvented in vivo [34, 35].  In particular, the demonstration of Th1-like immune responses 
specific for PAP suggest that an immune environment permissive of an anti-tumor response can 
exist in patients even without immunization [36].  Moreover, we have previously demonstrated 
that CD8+ T cells specific for PAP, with cytolytic activity for prostate cancer cells, can exist in 
patients with prostate cancer, and can be augmented with vaccination [30, 36]. 
 
B. 
PD-1 blockade 
 
A major mechanism by which tumors can avoid immune detection is by expression of PD-L1 or 
PD-L2, ligands for a receptor on T cells (PD-1), activation of which can decrease T-cell function 
and lead to immune tolerance.  There is currently great enthusiasm to specifically develop PD/PD-
L blockade inhibitors given the relative paucity of adverse events observed with these agents in 
clinical trials, and long-term disease response observed in some instances in early phase clinical 
trials.    Targeting PD-1, in particular, should be a universal therapy, as it targets the T-cell 
compartment rather than the tumor directly.  However, clinical trial experience to date suggests 
that patients with some solid tumor types (notably renal cell cancer, melanoma and non-small cell 
lung cancer) experience more benefit than patients with other histologies, including prostate cancer 
[18, 19].  This disparity suggests that differences are due to differences in the T-cells of responding 
and non-responding patients.  In particular, higher frequencies of tumor-infiltrating lymphocytes 
(TIL) are typically observed in patients with renal cell cancer and melanoma than prostate cancer 
[37].  In addition, early phase clinical trials using PD-1 or PD-L1 have identified that the 
expression of at least one of the ligands for PD-1 (PD-L1) on the target tumor cell by biopsy is 
associated with clinical response to therapy [18].  This is expected, given that tissue-infiltrating T 
cells can induce the expression of PD-L1 via the expression of IFNγ, and ligand binding of PD-1 

 
 
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leads to decrease in T-cell effector function.  It has been demonstrated that prostate cancers can 
express PD-L1, and can have infiltrating PD-1-expressing T cells [20].  Taken together, these 
results suggest that the efficacy of anti-tumor immunotherapy could be increased for prostate 
cancer by combining agents able to increase the number of tumor-specific T cells, such as through 
vaccination, and by PD-1/PD-ligand blockade.   
 
Given the anti-tumor responses observed in early phase clinical trials with antibodies targeting 
either PD-1 or PD-L1, several pharmaceutical companies have been developing related agents.  At 
the time of this writing, only one agent has yet been approved as a therapy, pembrolizumab 
(Keytruda®, Merck).  Specifically, pembrolizumab was approved in September 2014 for the 
treatment of ipilimumab-refractory advanced melanoma as a “breakthrough” therapy on the basis 
of an open-label, international, multicenter expansion cohort of a phase I trial of patients with 
advanced (metastatic) melanoma whose disease had progressed following treatment with 
ipilimumab [38].  In that trial, 173 patients received pembrolizumab at one of two doses (2 mg/kg 
or 10 mg/kg) at 3-week intervals until disease progression or intolerable toxicity.  An overall 
response rate of 26% was observed, irrespective of dose [38].  Grade 3 fatigue was the only drug-
related grade 3 or 4 adverse event reported in more than one patient.  Given these findings, 
pembrolizumab is currently FDA approved for the treatment of patients with ipilimumab-
refractory melanoma, dosed at 2 mg/kg intravenously every 3 weeks until disease progression or 
intolerable adverse effects.  Of note, however, earlier phase clinical trials have suggested that 
treatment can lead to prolonged responses even after discontinuing treatment [15].    
  
C. 
DNA vaccines can elicit antigen-specific Th1/CTL immune responses 
 
Over the last decade, there has been considerable interest in the development of plasmid DNA-
based vaccines, a strategy that offers several distinct advantages over other methods of antigen 
delivery.  DNA can be rapidly and inexpensively purified, and there are no problems with 
solubility as there frequently are with peptides and recombinant protein vaccines.  In addition, 
because the DNA in nucleic acid vaccines has been demonstrated to be taken up and expressed by 
host tissues and presented by host antigen-presenting cells (APC) directly [39, 40, 41], antigen 
presentation occurs through naturally processed epitopes, and does not require autologous cell 
processing.  DNA vaccines can therefore be theoretically employed in an HLA-independent 
fashion unlike peptide-based vaccines that are necessarily HLA-restricted.  This strategy is ideal 
in the HLA-diverse human situation.  In many ways, this method of immunization is similar to the 
use of viral immunization vectors, however without the additional foreign antigens introduced with 
a viral vector and consequently less of a risk of an overwhelming immune response to the vector 
itself [26, 42].  Of importance in tumor immunization models, several groups have demonstrated 
that immunization with plasmid DNA encoding a target antigen is a potent means of eliciting Th1-
biased immune responses [43] and CD8+ T-cells specific for the targeted antigen [39, 44, 45, 46, 
47].  In animal models, the use of an intradermal route of vaccine administration, in particular, 
tends to promote this Th1/CTL-biased immune response [43, 48, 49].  It should also be 
acknowledged that the clinical efficacy of a DNA vaccine encoding tyrosinase for the treatment of 
canine melanoma was approved by the USDA in early 2010 based on the results of clinical studies 
demonstrating an improved survival of companion dogs with oral melanoma [50, 51].  In fact, this 
represents the first vaccine approved in the U.S. for the treatment of existing cancer.  Thus, this 
approach bears further investigation in human clinical trials.   
 

 
 
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D. 
DNA vaccine encoding PAP (pTVG-HP) can elicit antigen-specific CD4+ and CD8+ T 
cells in patients with prostate cancer 
 
We have previously reported that DNA vaccines encoding either the human or rat homologue of 
PAP can elicit PAP-specific CD4+ and CD8+ T cells in rats, suggesting a feasible means of 
eliciting PAP-specific anti-tumor immune responses in patients [25, 26].  We have subsequently 
reported the results from a phase I/II clinical trial in which subjects with non-castrate, non-
metastatic prostate cancer were immunized six times at two-week intervals with this same DNA 
vaccine.  The primary objectives of that trial were to evaluate the safety and immunological 
efficacy of the pTVG-HP DNA vaccine in patients with clinical stage D0 prostate cancer [52].  
This was a dose-escalation study, with an expanded cohort of subjects treated at the maximum 
tolerated dose.  In the dose-escalation portion, nine subjects were treated in three dose cohorts of 
100 µg, 500 µg, or 1500 µg DNA, administered intradermally every two weeks for six total 
immunizations.  200 µg GM-CSF was co-administered as a vaccine adjuvant with each 
immunization.  13 additional subjects were treated in an expanded cohort at the 1500 µg DNA 
dose.  No serious adverse events were observed, and no significant laboratory anomalies were 
observed.  Common events observed were grade 1/2 fevers, chills, and local site reactions lasting 
typically less than 24 hours.  The primary immunological endpoint of this trial was the induction 
of PAP-specific IFNγ-secreting effector CD8+ T cells detectable two weeks following the final 
immunization.  As previously reported, three patients had a significant increase in the number of 
PAP-specific IFNγ-secreting CD8+ T cells after immunization compared with pre-immunization, 
one patient from each dose cohort [29].  Several individuals experienced a prolongation in PSA 
doubling time over one year following treatment compared with pre-treatment.  Overall the median 
PSA doubling time was 6.5 months in the four months pre-treatment and 8.5 months in the 4-
month on-treatment period (p = 0.033).  Long-term PAP-specific IFNγ-secreting T-cell responses 
were observed in several patients up to one year after immunization; the presence of this persistent 
immunity was associated with favorable changes in PSA doubling time [30].   
 
A second pilot trial was initiated evaluating different schedules of immunization in patients with 
castrate-resistant, non-radiographically metastatic prostate cancer.  This trial was designed to 
answer the question of whether six immunizations were insufficient in some individuals to develop 
an immune response, and whether ongoing repetitive immunization might be necessary.  This trial 
demonstrated that PAP-specific immune responses developed in some individuals after as few as 
3-6 immunizations, and some individuals developed no detectable immune responses after even 
24 biweekly immunizations [31].  Favorable changes in PSA doubling time were again observed, 
and tended to be greatest in patients with evidence of long-term immunity [31].  The findings from 
both of the trials above have provided the rationale for a randomized phase II clinical trial 
evaluating this same DNA vaccine to determine whether vaccination prolongs time to disease 
progression, a multicenter trial that is currently underway (NCT01341652).   
 
E. 
DNA vaccine with PD pathway blockade elicits anti-tumor response in murine model 
 
Given our findings in human trials that some patients did not develop evidence of immune 
response, and even those that did still showed evidence of disease progression, we have sought to 
evaluate methods to increase the efficacy of DNA vaccines and evaluate potential mechanisms of 
tumor escape.  For these efforts we have focused on vaccine strategies targeting SSX2, a 
“neoantigen,” and one for which we have identified that there are only two HLA-A2-restricted 

 
 
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epitopes, one dominant (p103-111) and one subdominant (p41-49), providing a convenient 
preclinical model [53].  Using the HHD-II strain of mice, which expresses human HLA-A2 and 
HLA-DR1 and is knocked out for murine MHC class I and II [54], we have demonstrated that 
immunization with a DNA plasmid encoding SSX2 elicits robust CD8+ T cells that can be 
identified by tetramer staining and IFNγ ELISPOT [36, 53, 55].  We have also generated a 
syngeneic sarcoma cell line in this HHD-II strain, and transfected this to express SSX2 
(Sarc/SSX2).  Using this system, we have explored whether the CD8+ effector T-cell response can 
be augmented using plasmid DNA vaccines modified to encode stronger MHC class I binding 
epitopes.  Using peptides with point mutations demonstrated to increase HLA-A2 binding affinity, 
we demonstrated that direct immunization could elicit CD8+ T cells with cross-reactive 
recognition for the native epitope, and that these cells could lyse peptide-loaded target cells, and 
human prostate tumor cells expressing native SSX2 and HLA-A2 [56].  Having identified altered 
peptide ligands (APL) for each epitope that elicited a higher frequency of epitope-specific T cells, 
we next encoded these APL within the coding sequence of an SSX2 plasmid DNA vaccine.  HHD-
II mice, without tumors, were immunized with plasmids encoding one or two modified epitopes, 
modified at single amino acids.  We have reported that immunization with a plasmid encoding 
both modified epitopes elicited a greater frequency of T cells specific for both epitopes compared 
with the native vaccine (pTVG-SSX2) [56].  Immunization with a DNA vaccine with point 
mutations ablating HLA-A2 binding elicited no peptide-specific T cells.  The plasmid DNA with 
two HLA-A2-optimized point mutations (pTVG-SSX2opt) was then evaluated for anti-tumor 
efficacy in studies in which Sarc/SSX2 tumors were implanted prior to immunization.  As shown 
in Figure 1, animals receiving the native SSX2 gene by DNA immunization had a marked decrease 
in tumor growth rate, but complete tumor eradication was not observed.  Unexpectedly, and 
replicated in several additional studies, tumor-bearing mice receiving the pTVG-SSX2opt DNA 
vaccine had greater tumor growth compared with the native pTVG-SSX2 vaccine.  To evaluate 
possible means of immune avoidance, we evaluated tumor cells for expression of PD-L1.  
Sarc/SSX2 tumor cells treated with IFNγ in vitro upregulated PD-L1 as detected by flow cytometry 
(not shown).  Moreover, as shown in Figure 2, Sarc/SSX2 tumors from mice immunized with 
either pTVG-SSX2 or pTVG-SSX2opt, but not vector control, upregulated PD-L1 expression, 
suggesting that PD-L1 expression was induced by the antigen-specific T cells.  This was further 
demonstrated by culturing splenocytes from mice immunized with pTGV-SSX2opt with tumor cells 
in vitro – PD-L1 expression was increased compared with treatment of tumor cells with 
splenocytes from control-immunized mice (data not shown).  Next, as shown in Figure 3, antigen-
specific CD8+ T cells detected by tetramer staining were highly PD-1 expressing in mice 
immunized with pTVG-SSX2opt compared with mice immunized with the native pTVG-SSX2 
vector.  Finally, as shown in Figure 4, combining vaccine treatment with an antibody blocking PD-
1 (clone G4, courtesy of Dr. Leiping Chen) was found to elicit a greater anti-tumor response than 
treatment with either vaccine or anti-PD1 alone.  Similar results were demonstrated in separate 
studies with the native vaccine alone in combination with anti-PD-1 treatment (not shown), and 
tumor eradication was observed in 5/10 animals treated with either vaccine combination (not 
shown).  Taken together, these findings demonstrate that PD-L1 expression by tumors, induced by 
antigen-specific CD8+ T cells, is a mechanism of tumor immune escape, and this can be abrogated 
by PD-1 blockade.  
 
Given these findings, we next asked whether other immune regulatory markers were increased 
following immunization.  Splenocytes from animals treated with a DNA vaccine encoding native 
or optimized SSX2 were collected and antigen-specific CD3+CD8+ T cells were assessed for 

 
 
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expression of PD-1, TIM-3, LAG3, BTLA, CD160, or CD244.  As shown in Figure 5, in addition 
to PD-1, expression of TIM-3 and LAG3 were also increased on T cells following immunization 
with the modified construct.  Expression of CD244 was higher on tetramer+ cells from animals 
immunized with the native vaccine than with the control or modified construct.  These findings 
demonstrate that combining T-cell regulatory receptor blockade with anti-PD-1 with a T-cell 
activating therapy (DNA vaccine) is a rational clinical direction, and this is the primary endpoint 
of this trial.  Moreover, our murine studies suggest that other regulatory receptors might also be 
affected by immunization, and hence the evaluation of this will be an exploratory endpoint of this 
trial.   
 
In further preclinical studies to evaluate the possible role of PD-1 in patients who previously 
received a DNA vaccine encoding PAP, we used a delayed-type hypersensitivity (DTH) model 
(developed by UW investigator Dr. William Burlingham, PhD) to evaluate mechanisms of immune 
regulation, similar to studies we have previously reported [57].  While a small number, we have 
found in 2 of 2 patients evaluated prior to and after DNA vaccination that PD-1 blockade, like 
CTLA-4 blockade, could restore effector function (as measured by DTH) of PAP-specific T cells 
elicited with vaccination (Figure 6).  We also found in a patient previously treated with pTVG-
HP, who then subsequently received sipuleucel-T for metastatic disease, that PD-L1 expression 
could be directly detected on  circulating CD45-EpCAM+ cells (Figure 7).  These studies form the 
basis for further exploratory studies proposed, to determine whether the trans vivo DTH method 
used can identify patients with antigen-specific PD-1-mediated regulation as either a predictive 
biomarker or measure of response, and whether similarly the presence of PD-L1 can be detected 
on circulating epithelial (tumor) cells as a predictive biomarker or measure of response, similar to 
what has been proposed for other studies using PD-1 blocking antibodies [18].   
 
 
 
 
 
 
 
 
 
Figure 1:  Modifications to HLA-A2-specific epitopes encoded 
by DNA vaccine do not elicit greater anti-tumor response.  
Groups of 6 HHD-II mice per group were implanted 
subcutaneously with 105 Sarc/SSX2 tumor cells and then  
immunized weekly with 100 µg plasmid DNA encoding SSX2 
(pTVG-SSX2, red), or encoding SSX2 containing both 
“optimized” epitopes (pTVG-SSX2opt, green), or vector control 
(pTVG4, blue).   Tumor growth was followed over time.  As 
shown, animals immunized with DNA encoding both modified 
epitopes had continued tumor growth compared with animals 
immunized with the native DNA vaccine.   
Figure 2:  Immunization of SSX2+ tumor-bearing 
mice with SSX2-encoding DNA vaccines elicits PD-
L1 expression in tumors.  Tumors obtained from mice 
immunized as in Figure 4 were evaluated by flow 
cytometry (panel A) or IHC (panel B) for PD-L1 
expression.  As shown, animals immunized with 
either DNA vaccine encoding SSX2 (native or both 
modified epitopes) had increased PD-L1 expression 
on tumors relative to controls.   

 
 
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PD-1 Expression on Splenocytes
MFI
Tetramer +
CD8
Tetramer +
CD8
0
1000
2000
3000
4000
5000
P<0.0001
SSX-2                  Optimized
Figure 3:  Immunization of mice with optimized SSX2-encoding DNA vaccine 
elicits higher PD-1 expression on antigen-specific CD8+ T cells.  Splenocytes from 
animals immunized with pTVG-SSX2 (SSX-2) or pTVG-SSX2opt (Optimized) were 
stained for p103 and p41 (HLA-A2) tetramers and PD-1. Shown is the mean 
fluorescence 
intensity 
(MFI) 
of 
PD-1 
expression 
on 
antigen-specific 
CD3+CD8+tetramer+ (tetramer+) cells compared with CD3+CD8+tetramer- (CD8) 
popula-tions.  As shown, animals immunized with the optimized vaccine had 
increased PD-1 expression on antigen-specific CD8+ T cells.   
Figure 4:  Immunization of mice with optimized SSX2-encoding DNA 
vaccine with anti-PD-1 elicits greater anti-tumor response.  Groups of 4-
6 HLA-A2 transgenic mice were implanted subcutaneously with 2x104 
Sarc/SSX2 tumor cells and then  immunized weekly with 100 µg 
plasmid DNA encoding SSX2 (pTVG-SSX2), or encoding SSX2 
containing both “optimized” epitopes (pTVG-SSX2opt), or vector control 
(pTVG4), as in Figure 4, and with or without 100 µg anti-mouse PD-1 
(clone G4) administered 24 hours after each immunization.   Tumor 
growth was followed over time.  As shown, the anti-tumor efficacy of 
the “optimized” vaccine was improved with anti-PD1, and no anti-tumor 
effect was seen with anti-PD1 treatment alone.   
Figure 5:  Immunization of mice with APL-modified SSX2-encoding DNA vaccine elicits PD-1 and TIM-3 expressing tetramer+ CD8+ 
T cells.  Groups of 4-6 HLA-A2 transgenic mice were implanted subcutaneously with 2x104 Sarc/SSX2 tumor cells and then immunized 
weekly with 100 µg plasmid DNA encoding SSX2 (pTVG-SSX2), or encoding SSX2 containing both APL-modified epitopes (pTVG-
SSX2opt), or vector control (pTVG4), as in Figure 4.  Splenocytes recovered from animals were stained for tetramer positive events 
among CD8+ T cells and costained for the markers indicated.  Top panels represent p41-specific tetramer positive events, bottom panels 
represent p103-specific tetramer positive events.  Of note, tetramer positive events are detectable in mice with Sarc/SSX2 tumors even 
with control immunization (pTVG4); these events not detectable in non-tumor bearing mice (not shown).   

 
 
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F. 
Summary 
 
The current protocol will examine the use of a plasmid DNA vaccine encoding PAP to induce 
and/or augment therapeutic T-cells specific for the prostate tumor antigen PAP in patients with 
castrate-resistant, metastatic prostate cancer with concurrent or subsequent treatment with a PD 
pathway inhibitor, pembrolizumab.  The primary endpoint, objective response, is based on 
observations from previous early phase clinical trials demonstrating no objective disease response 
in patients treated with PD-1 blockade alone, and our preliminary studies demonstrating that in a 
similar murine model of tumor cells that express PD-L1 that there was no evidence of tumor 
growth delay or eradication unless PD pathway blockade was combined with tumor antigen-
specific vaccination.  This trial will explore whether there is a preferred sequence of treatment, 
and will in secondary and exploratory endpoints evaluate whether sequential or concurrent 
treatment affects the character and magnitude of the immune response.  Further studies will explore 
whether the identification of antigen-specific PD-1-regulated T-cells is predictive for response to 
combined treatment.  An additional exploratory endpoint will be to identify whether changes in 
FLT PET scans, particularly in systemic lymph nodes or lymph nodes in the area of immunization, 
might serve as a molecular imaging tool to identify immunological activation following 
immunization with or without pembrolizumab. The trial will be conducted in patients with 
castrate-resistant, metastatic prostate cancer, as this is the lethal form of prostate cancer and one 
for which there is a high need for therapies with a greater safety profile and greater magnitude of 
benefit.  Moreover, based on published studies, the median time to disease progression in this stage 
Figure 6:  Patients treated with pTVG-HP develop T cell 
responses to PAP, the function of which is regulated by 
PD-1.  PBMC obtained from 2 patients pre (blue) or 2 
weeks post (red) a 12-week treatment with pTVG-HP 
were placed in the footpads of SCID mice with the 1 µg 
of the antigens listed (TT/D = tetanus toxoid, positive 
control, PSA = negative control), with or without 1 µg of 
anti-CTLA-4 or anti-PD1.  DTH responses were read at 
24 hours.  As shown, both patients had responses to PAP 
after immunization that were “uncovered” by either 
CTLA-4 or PD-1 blockade.  
Figure 7:  PD-L1 can be detected directly on circulating epithelial 
cells (presumed circulating tumor cells) by flow cytometry. PBMC 
were obtained from a patient who had previously received pTVG-HP, 
and then subsequently received standard sipuleucel-T for the 
treatment of metastatic prostate cancer.   Cells were stained for CD45, 
EpCAM, and PD-L1.  The gating shows live (DAPI-) CD45-EpCAM+ 
cells (a population not detectable in non-cancer population – not 
shown)for their PD-L1 expression.   

 
 
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of disease is on the order of 3 months, and few PSA responses have been observed following 
vaccination alone in published studies in this stage of disease [7, 58], simplifying the identification 
of potential benefit, and potential magnitude of such benefit, for planning future confirmatory 
clinical trials.   
 
In May 2017 this trial will be modified to further explore the concurrent administration of these 
two agents.  This is based on emerging data from this trial that suggests that this treatment has 
evidence of anti-tumor efficacy, but is limited by being only delivered over a 3-month period of 
time.  As such, after completion of the two-arm trial with at least 12 evaluable subjects per study 
arm, those arms will be closed to further accrual, with an extended treatment cohort (Arm 3) that 
will be opened to accrual as described below.   
 
In early 2018 this trial will be modified again to further explore a schedule of concurrent 
administration of the two agents, but with less frequent pembrolizumab dosing. This is based on 
emerging immunological biomarker data from the trial that the vaccine schedule at every-two-
week intervals may be preferred, and with the goal of minimizing the frequency of adverse events 
related to pembrolizumab observed in Arm 3. In January 2018, 11 patients had been accrued to 
Arm 3 and had received at least one treatment with DNA and pembrolizumab. At that point, 2 of 
11 patients treated in Arm 3 had no evidence of PSA decline from baseline, which was lower than 
expected from what had been observed in Arm 1 (in which 8 of 13 patients had some PSA decline 
from baseline, p=0.04). In addition, while not a clinical parameter, the frequency of immune 
response to the PAP target antigen was observed in the laboratory to be overall lower than what 
had been observed in either Arm 1 or Arm 2.  Given these findings, and given that the primary 
change from Arm 1 to Arm 3 was in changing the vaccine schedule from every 2 weeks to every 
3 weeks, it was decided to add an additional treatment arm that would keep the vaccine schedule 
at 2-week intervals, but continuing combined treatment beyond 12 weeks.  The schedule for 
pembrolizumab was increased to every 4 weeks for patient convenience and with the goal of 
reducing toxicity, since adverse events > grade 2 experienced to date were all attributed to 
pembrolizumab. This extended treatment cohort (Arm 4) will open to accrual after completion of 
enrollment to Arm 3. 
 
 
3. 
Objectives 
 
A. 
Primary Objectives 
 
1. To evaluate the safety of pembrolizumab in combination with pTVG-HP in patients with 
castration-resistant, metastatic prostate cancer 
 
2. To determine the 6-month progression-free survival and median time to radiographic 
progression in patients with castration-resistant metastatic prostate cancer treated with 
pembrolizumab in combination with pTVG-HP 
 
3. To evaluate the anti-tumor response rates (objective response rate and PSA response rate, 
using PCWG2 criteria) in patients with castration-resistant metastatic prostate cancer 
treated with pembrolizumab in combination with pTVG-HP  
 

 
 
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B. 
Secondary Objectives: 
 
1. To determine whether either treatment sequence, or PAP-specific immune response, is 
associated with prolonged (6-month) radiographic progression-free survival 
 
2. To evaluate effects of schedule (concurrent versus delayed administration of 
pembrolizumab) on the magnitude of PAP-specific T-cell responses, PD-1 expression on 
circulating T cells, and PD-L1 expression on circulating epithelial cells (CEC) and on 
tumor biopsies 
 
3. To determine the median time to radiographic progression using concurrent administration 
schedules  
 
C. 
Laboratory and Exploratory Objectives: 
 
1. To evaluate effects of treatment on number of circulating tumor cells  
 
2. To evaluate PAP-specific antibody responses following treatment with pembrolizumab and 
pTVG-HP DNA vaccine 
 
3. To determine whether either treatment sequence elicits immunologic antigen spread to 
other prostate-associated antigens 
 
4. To determine whether pre-existing or vaccine-induced PD-L1 expression on CEC or tumor 
biopsies is predictive of objective clinical response 
 
5. To determine whether treatment elicits expression of other regulatory molecules on tumor-
specific T cells (e.g. TIM3, BTLA, and LAG3) or tumor cells (e.g. HVEM, phosphatidyl 
serine, PD-L2)   
 
6. To determine whether PD-1-regulated antigen-specific T cells identified by trans vivo DTH 
testing can identify patients who develop objective clinical responses with PD-1 blockade 
therapy in combination with pTVG-HP 
 
7. To determine whether changes in lymph nodes and soft tissue tumor lesions are observed 
by FLT PET/CT after treatment with vaccine with or without pembrolizumab 
 
8. To determine if PD-1 inhibitor therapy in combination with pTVG-HP will change number 
and activity (SUV) in osteoblastic metastases as measured by NaF PET/CT. 
 
 
4. 
Product Information 
 
A. 
Anti-PD-1 monoclonal antibody (Pembrolizumab, Keytruda®)  
 
Pembrolizumab (Keytruda®, Merck) is a human programmed death receptor-1 (PD-1)-blocking 
antibody indicated for the treatment of patients with unresectable or metastatic melanoma and 

 
 
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disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF 
inhibitor.  While this agent is commercially available and will be purchased for use, its use is 
investigational under this protocol.  The same dose and schedule will be used in this protocol as 
for the approved melanoma indication.  Specifically, it will be administered at 2 mg/kg as an 
intravenous infusion over 30 minutes every 3 weeks (up to four maximum doses).  Actual current 
body weight will be used to calculate the dose for each treatment. Pembrolizumab will be 
administered as an intravenous infusion over 30 minutes using an IV line containing a sterile, non-
pyrogenic, low-protein binding 0.2 µm to 5 µm in-line or add-on filter, and according to its 
package insert.   
 
NOTE:  For the expansion cohort (Arm 3), this will similarly use a dose of 2 mg/kg 
pembrolizumab, but with 200 mg maximum dose, administered as an intravenous infusion over 30 
minutes every 3 weeks for up to a maximum of 16 doses.  For the second expansion cohort (Arm 
4), this will similarly use a dose of 2 mg/kg pembrolizumab, but with 200 mg maximum dose, and 
administered as an intravenous infusion over 30 minutes every 4 weeks for up to a maximum of 
12 doses.   
 
The most common adverse reactions (reported in > 20% of patients) included fatigue, cough, 
nausea, pruritis, rash, decreased appetite, constipation, arthralgia, and diarrhea.  Immune-mediated 
adverse reactions have also been observed, including immune-mediated pneumonitis, colitis, 
hepatitis, hypophysitis, nephritis, hyperthyroidism and hypothyroidism.  The management of 
suspected adverse reactions is discussed in Section 8 below.  
 
B. 
Plasmid DNA vaccine 
 
 
pTVG-HP (pTVG4 vector containing cDNA for human PAP) 
 
pTVG-HP is a plasmid DNA, produced in E. coli, that encodes the cDNA for human prostatic acid 
phosphatase (PAP).  DNA from the master cell bank bacterial strain has been confirmed by 
standard DNA sequencing to confirm its identity.  The biological activity of each lot has been 
tested in rodent studies demonstrating that T-cell immune responses specific for PAP can be 
elicited in vivo following immunization.  Lots are tested for appearance, plasmid homogeneity, 
DNA identity by restriction endonuclease evaluation, protein contamination, RNA contamination, 
genomic DNA contamination, sterility, endotoxin, and pH, and criteria for each of these have been 
established for lot release.  The vaccine will be supplied in single-use vials containing at least 0.6 
mL 0.2 mg/mL pTVG-HP in phosphate-buffered saline.  Vials will be stored at <–60°C until the 
day of use.   
 
GM-CSF 
 
GM-CSF (Leukine®, Sargramostim), will be obtained from commercial vendor (such as 
Genzyme, Cambridge, MA, or other commercial vendor) and used as a vaccine adjuvant and will 
be provided without charge to subjects participating in this trial. GM-CSF, as described above, is 
a growth factor that supports the survival, clonal expansion and differentiation of hematopoietic 
progenitor cells including dendritic antigen presenting cells.  In preliminary animal experiments 
and human clinical trials GM-CSF has been shown to be safe and serve as an effective adjuvant 
for the induction of antibody and T-cell responses to the immunized antigen [59, 60].  The use of 

 
 
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GM-CSF is associated with little toxicity [61, 62, 63].  GM-CSF is a sterile, white, preservative-
free, lyophilized powder supplied in 250 µg-dose vials.   
 
Recombinant human GM-CSF (rhGM-CSF), when administered intravenously or subcutaneously 
is generally well tolerated at doses ranging from 50 to 500 µg/m2/day.  Severe toxic manifestations 
are extremely rare in patients treated with rhGM-CSF.  Diarrhea, asthenia, rash and malaise were 
the only events observed in more than 5% of the rhGM-CSF group compared with the placebo 
group in phase III controlled studies of patients undergoing autologous bone marrow 
transplantation.  In uncontrolled phase I/II studies for various indications, the adverse effects 
reported most frequently were fever, chills, nausea, vomiting, asthenia, headache and pain in the 
bones, chest, abdomen, joints, or muscles.  Most of these systemic events reported with rhGM-
CSF administration were mild to moderate in severity and rapidly reversed by the administration 
of analgesics or antipyretics.  Other events reported infrequently were dyspnea, edema, local 
injection site reactions, and rash.  Thrombosis and cardiac arrhythmia have also been reported, and 
there have been infrequent reports of tissue sloughing, leukemic progression, congestive heart 
failure, hepatomegaly, and intracranial bleeding; and isolated reports of Guillain-Barré syndrome 
and increased histiocytes.  Neutropenia, although usually a pre-existing condition in the patients 
receiving rhGM-CSF, has been reported in association with administration.  There was an 
increased frequency of severe thrombocytopenia in patients receiving concurrent chemotherapy 
and radiotherapy with GM-CSF.  Eosinophilia and other blood abnormalities may occur. 
 
There have been rare reports of 1) sequestration of granulocytes in the lungs with respiratory 
symptoms; 2) a syndrome characterized by respiratory distress, hypoxia, flushing, orthostatic 
hypotension, and partial loss of consciousness; 3) peripheral edema, pericardial or pleural 
effusions, and capillary leak of fluid; and 4) serious allergic or anaphylactic reactions. 
 
Administration of rhGM-CSF may aggravate fluid retention in patients with pre-existent edema, 
capillary leak syndrome, or pleural or pericardial effusions.  In some patients with pre-existing 
renal or hepatic dysfunction, elevation of the serum creatinine or bilirubin and hepatic enzymes 
has occurred during the administration of rhGM-CSF.  Dose reduction or interruption of rhGM-
CSF administration has resulted in a decrease to pretreatment values.  Occasional transient and 
reversible supraventricular arrhythmia has been reported in uncontrolled studies, particularly in 
patients with a previous history of cardiac arrhythmia. 
 
Stimulation of marrow precursors with rhGM-CSF may result in a rapid rise in white blood cell 
count.  Dosing should be stopped if the ANC exceeds 20,000/cm3.  rhGM-CSF may stimulate the 
growth of myeloid malignancies; therefore, caution must be exercised in its use in these 
malignancies or myelodysplastic syndromes. 
 
Because GM-CSF will only be administered at most once every two weeks on the current protocol, 
most of these side effects are not anticipated.  In previous vaccine studies using rhGM-CSF as an 
adjuvant in an identical fashion, local skin reactions consisting of erythema and induration have 
occurred in many patients.  These resolved without need for treatment over a 2-3 week period for 
some, and over 2-3 days for most.  Transient leukopenia (lasting only a couple of hours) has been 
observed in some patients, and resolved without treatment and without clinical sequelae [64].  In 
previous clinical trials with this DNA vaccine and GM-CSF, transient dyspnea and chest pain have 
been observed, occurring within 45 minutes of treatment and resolving within 10 minutes without 

 
 
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treatment; these reactions have been attributed to the GM-CSF.  One allergic reaction with tongue 
swelling and lip edema has been observed in one patient following 11 immunizations, resolving 
following treatment with antihistamines, and without sequelae.  Furthermore, the generation of a 
transient immune response to rhGM-CSF following its use as a vaccine adjuvant has been 
observed, however no clinical sequelae were noted, and the significance of this observation is not 
clear at present [63]. 
 
DNA vaccine preparation and administration 
 
Vials will be thawed, and the plasmid DNA itself will be used to reconstitute the GM-CSF, on the 
day of administration.  Specifically: 
 
For each of the DNA immunizations:  0.6 mL of 0.2 mg/mL pTVG-HP will be withdrawn and 
used to reconstitute 250 µg GM-CSF.  0.25 mL will then be drawn into each of two tuberculin 
syringes.  This effectively provides a 100-µg dose of DNA and 208 µg GM-CSF. 
 
The vaccine will then be administered intradermally, on the lateral aspect of the arm (left arm, 
preferentially) in two adjacent sites, with the total syringe contents (0.25 to 0.3 mL) administered 
at each site.   
 
 
5. 
Patient Selection 
 
A. 
Inclusion Criteria 
  
1. Age ≥ 18 years. 
2. Histologically confirmed diagnosis of prostate cancer (adenocarcinoma of the prostate) 
3. Metastatic disease as evidenced by the presence of soft tissue and/or bone metastases on 
imaging studies (CT of abdomen/pelvis, bone scintigraphy) 
4. Castrate-resistant disease, defined as follows: 
a. All patients must have received (and be receiving) standard of care androgen 
deprivation treatment (surgical castration versus GnRH analogue or antagonist 
treatment); subjects receiving GnRH analogue or antagonist must continue this 
treatment throughout the time on this study. 
b. Patients may or may not have been treated previously with a nonsteroidal 
antiandrogen.  For patients previously treated with an antiandrogen, they must be 
off use of anti-androgen for at least 4 weeks (for flutamide) or 6 weeks (for 
bicalutamide or nilutamide) prior to registration.  Moreover, subjects who 
demonstrate an anti-androgen withdrawal response, defined as a > 25% decline in 
PSA within 4-6 week of stopping a nonsteroidal antiandrogen, are not eligible until 
the PSA rises above the nadir observed after antiandrogen withdrawal. 
c. Patients must have a castrate serum level of testosterone (< 50 ng/dL) within 6 
weeks of day 1 
5. Progressive disease while receiving androgen deprivation therapy defined by any one of 
the following as per the Prostate Cancer Clinical Trials Working Group 2 (PCWG2) bone 
scan criteria [65] or RECIST 1.1 during or after completing last therapy: 

 
 
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a. PSA:  At least two consecutive rises in serum PSA, obtained at a minimum of 1-
week intervals, with the final value > 2.0 ng/mL.  
b. Measurable disease:  > 50% increase in the sum of the cross products of all 
measurable lesions or the development of new measurable lesions.  The short axis 
of a target lymph node must be at least 15mm by spiral CT to be considered a target 
lesion.   
c. Non-measurable (bone) disease:  The appearance of two or more new areas of 
uptake on bone scan (or NaF PET/CT) consistent with metastatic disease compared 
to previous imaging during castration therapy.  The increased uptake of pre-existing 
lesions on bone scan will not be taken to constitute progression, and ambiguous 
results must be confirmed by other imaging modalities (e.g. X-ray, CT or MRI). 
6. Prior treatment with abiraterone or enzalutamide is permitted, but patients must have been 
off prior corticosteroid treatment for at least 28 days.   
7. Life expectancy of at least 6 months 
8. Patients must have an ECOG performance status of 0, 1, or 2. 
9. Adequate hematologic, renal and liver function as evidenced by the following within 6 
weeks of day 1: 
WBC  
> 2000 / mm3 
ANC  
> 1000 / mm3  
HgB 
 
> 9.0 gm/dL 
Platelets 
> 100,000 / mm3 
Creatinine 
< 2.0 mg/dL 
AST, ALT 
< 2.5 x institutional upper limit of normal  
10. No known history of HIV 1 and 2, HTLV-1, or active Hepatitis B or Hepatitis C 
11. Patients must be at least 4 weeks from any prior treatments and have recovered (to < Grade 
2) from acute toxicity attributed to this prior treatment, unless considered chronic 
12. Patients must be willing and able (in the opinion of the treating physician) to undergo two 
research biopsies for the investigational component of this trial. See section 6.A for more 
details. 
13. Patients must be willing to undergo two leukapheresis procedures for the investigational 
component of this trial 
14. Patients must be willing to undergo FLT PET/CT or NaF PET/CT scans for the 
investigational component of this trial and have no known allergies to FLT or NaF. See 
section 9 for more details. 
15. For those patients who are sexually active, they must be willing to use barrier contraceptive 
methods during the period of treatment on this trial (and for four weeks after the last DNA 
immunization treatment for patients in Arm 1) 
16. Patients must be informed of the experimental nature of the study and its potential risks, 
and must sign an IRB-approved written informed consent form indicating such an 
understanding 
 
B. 
Exclusion Criteria 
 
1. Small cell or other variant (non-adenocarcinoma) prostate cancer histology, unless there is 
evidence that the tumor expresses PAP 
2. Patients may not be receiving other investigational agents or be receiving concurrent 
anticancer therapy other than standard androgen deprivation therapy 

 
 
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3. Concurrent bisphosphonate therapy is not excluded, however patients should not start 
bisphosphonate therapy while on this study; those patients already receiving 
bisphosphonate therapy should continue at the same dosing and schedule as prior to study 
entry 
4. Rapidly progressive symptomatic metastatic disease, as defined by the need for increased 
opioid analgesics within one month of registration for the treatment of pain attributed to a 
prostate cancer metastatic lesion; patients receiving opioids must receive approval from the 
PI for eligibility 
5. Treatment with any of the following medications within 28 days of registration, or while 
on study, is prohibited: 
• Systemic corticosteroids (at doses over the equivalent of 5 mg prednisone daily) – not 
permitted within 1 month of registration; inhaled, intranasal or topical corticosteroids 
are acceptable 
• PC-SPES  
• Saw Palmetto 
• Megestrol 
• Ketoconazole 
• 5-α-reductase inhibitors – patients already taking 5-α-reductase inhibitors prior to 28 
days prior to registration may stay on these agents throughout the course of therapy, 
but these should not be started while patients are on study  
• Diethyl stilbesterol 
• Abiraterone 
• Enzalutamide 
• Radium 223 (Xofigo®) 
• Any other hormonal agent or supplement being used with the intent of cancer treatment 
6. External beam radiation therapy within 4 weeks of registration is prohibited, or anticipated 
need for radiation therapy (e.g. imminent pathological fracture or spinal cord compression) 
within 3 months of registration. 
7. Major surgery within 4 weeks of registration is prohibited 
8. Prior cytotoxic chemotherapy (for example, but not limited to, docetaxel, mitoxantrone, 
cabazitaxel) within 6 months of registration is prohibited 
9. Patients with a history of life-threatening autoimmune disease 
10. Patients with a history of allergic reactions to GM-CSF or the tetanus vaccine 
11. Patients who have undergone splenectomy 
12. Patients must not have other active malignancies other than non-melanoma skin cancers or 
superficial bladder cancer.  Subjects with a history of other cancers who have been 
adequately treated and have been recurrence-free for > 3 years are eligible.   
13. Patients with known brain metastases 
14. Any antibiotic therapy or evidence of infection within 1 week of registration 
15. Any other medical intervention or condition, which, in the opinion of the PI or treating 
physician, could compromise patient safety or adherence with the study requirements 
(including biopsies or leukapheresis procedures) over the primary 3-6 month treatment 
period. 
16. Patients cannot have concurrent enrollment on other phase I, II, or III investigational 
treatment studies. 

 
 
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17. NOTE:  There is no exclusion for prior immune-based therapy.  This includes patients 
previously treated on Arms 1 or 2 who are otherwise eligible for treatment on Arm 3 or 4.   
 
 
6. 
Experimental Design 
 
This will be a 1:1 randomized, open-label, single institution pilot trial designed to evaluate the 
immunological and clinical effect of a DNA vaccine encoding PAP with rhGM-CSF adjuvant 
given with (concurrently or sequentially) pembrolizumab.  Study arms will be defined as follows: 
 
Arm 1: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.) 
 
biweekly 6 times 
Pembrolizumab 2 mg/kg, administered intravenously every 3 weeks 4 times,  
 
beginning on day 1 after the first pTVG-HP vaccination  
 
Arm 2: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.) 
 
biweekly 6 times 
 
Pembrolizumab 2 mg/kg, administered intravenously every 3 weeks 4 times, the 
first dose administered two weeks after the last pTVG-HP vaccination 
 
Extended treatment Arm 3: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.)  
 
every 3 weeks, for a maximum of 16 doses 
Pembrolizumab 2 mg/kg, with a maximum dose of 200 mg, administered 
 
intravenously  every 3 weeks, for a maximum of 16 doses, beginning on 
 
day 1 after the first pTVG-HP vaccination  
 
Extended treatment Arm 4: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.)  
 
every 2 weeks, for a maximum of 24 doses 
Pembrolizumab 2 mg/kg, with a maximum dose of 200 mg, administered 
 
intravenously  every 4 weeks, for a maximum of 12 doses, beginning on 
 
day 1 after the first pTVG-HP vaccination  
 
 
A. 
Study Arm Assignment and Toxicity Assessment 
 
Patients with castrate-resistant, metastatic prostate cancer, with evidence of progressive disease by 
scans (but not by pain symptoms) or rising PSA, who may or may not have been previously treated 
with other therapies (including chemotherapy, abiraterone, or enzalutamide), will be invited to 
participate.  Subjects will be randomly assigned to a treatment group for the first two study arms.  
Once at least 12 evaluable subjects have been accrued to Arms 1 and 2, and once Arm 3 is open to 
accrual, Arms 1 and 2 will be closed and all further accrual will be to Arm 3.  Given the absence 
of significant adverse events observed in previous clinical trials with pTVG-HP, no adverse events 
> grade 2 are anticipated related to that agent.  While not common, previous immune-associated 
toxicities have been observed with pembrolizumab, including immune-mediated pneumonitis, 
colitis, hepatitis, hypophysitis, nephritis, hyperthyroidism, and hypothyroidism.  Hence these may 

 
 
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be anticipated events.  In 89 patients treated with pembrolizumab at 2 mg/kg every 3 weeks in a 
study of patients with unresectable or metastatic melanoma, < 20% of patients experienced any 
adverse reaction > grade 2; the most common adverse reaction was fatigue (7% of patients).  Three 
grade 4 laboratory abnormality events (hyperglycemia, increased ALT, and anemia) were reported.   
In a larger trial of 411 patients with advanced melanoma, 9% of patients discontinued treatment 
due to adverse reactions.  However, patients in the approval trial received a median of 9 doses, 
more doses than will be used in the current trial.  In the current trial patients will be evaluated for 
evidence of toxicity, and further accrual to a specific treatment arm will be discontinued if the 
toxicity rate is deemed to be excessive for that arm.  Given the more limited exposure to this agent 
(4 doses total), a toxicity rate of 35% for Grade 3 events or 20% Grade 4 events given an attribution 
of at least possibly related to study treatment will be considered excessive.  Grade 3 limited site 
reactions (lasting < 48 hours) and fevers/chills (lasting < 48 hours) will be excluded from this 
assessment, as expected possible adverse events of limited duration from vaccination.     
 
Evidence that the toxicity rate is excessive will be considered sufficient if, within any arm, the 
lower limits of the 90% one-sided confidence intervals for the estimates of the true toxicity rates 
exceed the toxicity limits defined above (i.e., 35% for Grade 3 events and 20% for Grade 4 events), 
after the first 5 patients have been accrued.  Operationally, this will occur if any of the following 
events occurs within a study arm:  Grade ≥ 3 events are present in at least 4 out the first of 5, 5 out 
of the first 7 (or less), 6 out of the first 10 (or less), or 7 out of the first 12 (or less) or 8 out the first 
14 (or less) or 9 out of the first 16 (or less) of the subjects accrued (within a study arm).  
Furthermore, accrual will be temporarily suspended if any of the following events occur:  Grade 4 
events are present in at least 3 out of the first 5, 4 out of 9 (or less), 5 out of the first 13 (or less) or 
6 out of 16 (or less) of the subjects accrued (within a study arm).   
 
If one of these thresholds is reached, accrual will be temporarily suspended and the study will be 
reviewed for safety, dose modification and safety modification, and the study will be reviewed for 
safety by the UW DSMC (data and safety monitoring committee) with respect to treatment arm.  
Once the study is suspended, it would only be reopened if modifications could be made, and 
approved by the DSMC and the UW-IRB, to assure that patient safety is reestablished.  If not, the 
study would be closed.   
 
B. 
Endpoints 
 
Patients will come off study at the time of radiographic (CT and/or bone scintigraphy) disease 
progression, with progression being defined only AFTER the first 3-month staging evaluation, at 
the time of undue toxicity (as defined below), or at the discretion of the patient and treating 
physician that continuing treatment is not in the best interest of the patient and/or that other 
standard therapies for prostate cancer are warranted due to symptomatic progression.  Patients 
should be discouraged from discontinuing protocol treatment for PSA rise only. NaF and FLT 
PET/CT scans will be investigational and will not be used for making individual subject treatment 
decisions with respect to progression necessitating continuing or discontinuing protocol treatment.  
However, findings on NaF or FLT PET/CT that might pose medical risk to individual subjects will 
be communicated to the treating physician such that dedicated clinical imaging/intervention could 
be pursued, if necessary.  Such findings on NaF or FLT PET/CT, by themselves, will not be used 
to define disease progression necessarily requiring patients to come off trial.  Patients who, in the 
discretion of the treating physician, appear to be benefiting (e.g. declining serum PSA and/or 

 
 
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improvement in symptoms despite new radiographic lesions), may continue in protocol 
treatment/observation after discussion with the PI and study sponsor.  Patients will continue to 
have blood drawn for immune studies, as per study calendar, up to one year after screening, even 
if off study prior to that time. Patients who undergo an initial research biopsy and/or NaF/FLT 
PET scan should also undergo the subsequent scans, as long as they are willing, even if they are 
off study prior to Month 3. In particular, evaluations performed at 6-month time point should be 
performed even if off-study prior to that time, if feasible.  All subjects who have not come off 
study earlier for these reasons will come off study at the end of one year. 
 
The primary endpoints of the trial will be clinical endpoints (safety, 6 month PFS, and objective 
response rates).  All subjects will undergo radiographic imaging (CT of abdomen and pelvis and 
bone scan) prior to treatment, and at 3-month intervals (or as clinically indicated).  Radiographic 
disease progression and response will be defined using PCWG2 published criteria [65], and as 
detailed in Section 7.  Based on the prior trials leading to the approval of sipuleucel-T, and because 
immune therapies are expected to require up to several months before radiographic responses or 
stabilization might be observed, the radiographic studies obtained at 3 months will not be used to 
define progression, rather will be used as the baseline for future comparison.  That is, while 
baseline studies will be obtained prior to study registration, and radiographic progression may be 
expected in 50% of patients at 3 months, this will not be used to define progression necessitating 
patients come off study, rather disease progression occurring after this initial 3-month period. 
These criteria, in addition to being more standard for contemporary clinical trials in advanced 
prostate cancer, were also specifically designed to capture late radiographic objective responses 
that have been observed with immune-based therapies, including pembrolizumab and other T-cell 
checkpoint inhibitors [80]. Time to progression will be determined from registration for all 
subjects.  6-month progression-free survival will be calculated for each treatment arm, as well as 
the median progression-free survival.  
 
The primary endpoint for the extended treatment Arms 3 and 4 will be time to radiographic 
progression. 
 
 
7. 
Measurement of Effect (PCWG2 Recommendations) 
 
A. 
Malignant Disease Evaluation 
 
To assess objective response, it is necessary to estimate the overall tumor burden at baseline (for 
purposes of comparison, “baseline” imaging will be that obtained at 3-month time point) to which 
subsequent measurements will be compared. Measurable disease is defined by the presence of at 
least one measurable lesion. 
 
All measurements should be recorded in metric notation by use of a ruler or calipers. The same 
method of assessment and the same technique should be used to characterize each identified lesion 
at baseline and during follow-up. All pre-treatment evaluations should be performed as closely as 
possible to the beginning of treatment and not more than four weeks before registration. 
 
The term evaluable in reference to measurability will not be used because it does not provide 
additional meaning or accuracy. 

 
 
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At baseline, tumor lesions will be characterized as either measurable or non-measurable. 
 
NOTE:  While progression will be documented from pre-treatment staging studies in all 
individuals, the intent of these measurements in this trial will be to determine whether “baseline” 
evaluation for immune-based treatments is preferable at a later time point after the therapy has 
been given the opportunity to exert an anti-tumor effect.  Hence, the “baseline” evaluation for 
determining progression will be the staging studies performed ~ day 85 (3 months).  Objective 
progression at that time point (Month 3, day 85) from the pre-treatment scans will not be used to 
alter treatment unless the patient has symptomatic disease requiring other therapy and thereby 
needs to discontinue treatment.  For all of these measurements, PCWG2 criteria will be used to 
identify “measurable” lesions at each of the baseline (pre-treatment and 3 month time points), and 
RECIST 1.1 criteria will be used to monitor for change over time (consistent with PCWG2 
criteria).  
 
1. Measurable  
Lesions that can be accurately measured in at least one dimension (longest diameter 
to be recorded) as ≥ 20 mm (2.0 cm) with conventional techniques or as > 10 mm (1.0 
cm) with spiral CT scan.  For a lymph node to be considered pathologically enlarged 
and measurable, it must be > 15mm in short axis when assessed by spiral CT.  
 
If the measurable disease is restricted to a solitary lesion, its neoplastic nature should 
be confirmed by cytology/histology. 
 
Tumor lesions that are situated in a previously irradiated area are not considered 
measurable. 
 
2. Non-Measurable  
All other lesions, including small lesions [longest diameter < 20 mm (2.0 cm) with 
conventional techniques or < 10 mm (1.0 cm) with spiral CT scan], lymph nodes < 
15mm in short axis, and truly non-measurable lesions. 
 
Lesions considered to be truly non-measurable include the following: bone lesions, 
leptomeningeal 
disease, 
ascites, 
pleural/pericardial 
effusion, 
lymphangitis 
cutis/pulmonis, abdominal masses that are not confirmed and followed by imaging 
techniques, and cystic lesions. 
 
B. 
Definitions of Response 
 
1. Target Lesions  
All measurable lesions, up to a maximum of two lesions per organ and five lesions in 
total, representative of all involved organs. Target lesions should be selected on the 
basis of their size (those with the largest diameters) and their suitability for accurate 
repeated measurements. 
 

 
 
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The sum of the diameters (long axis for non-nodal, short axis for nodal) of all target 
lesions will be calculated at baseline and reported as the baseline sum diameter. The 
sum diameter will be used to characterize the objective tumor response.  
 
a. Complete Response (CR)  
The disappearance of all target lesions. To be assigned a status of complete response, 
changes in tumor measurements must be confirmed by repeat assessments performed 
no less than four weeks after the criteria for response are first met.  PSA must also 
be < 0.2 ng/mL.  All disease must be assessed using the same technique as at baseline.  
Lymph nodes that shrink to less than 1.0 cm are considered normal. 
 
b. Partial Response (PR)  
At least a 30% decrease in the sum of the diameters of target lesions (long axis for 
non-nodal lesions, short axis for nodal lesions), taking as reference the baseline sum 
diameter. To be assigned a status of partial response, changes in tumor measurements 
must be confirmed by repeat assessments performed no less than four weeks after the 
criteria for response are first met.  There must be no new lesions.  All target measurable 
lesions must be assessed using the same techniques as at baseline. 
 
c. Progressive Disease (PD)   
At least a 20% increase in the sum of the diameters of target lesions (long axis for 
non-nodal lesions, short axis for nodal lesions), and a 0.5 cm absolute minimum 
increase, taking as reference the smallest sum diameter recorded since the Month 3 
baseline measurements, or the appearance of one or more new lesion(s).  
 
d. Stable Disease (SD)  
Change in tumor size is less than that required to identify a partial response or 
progressive disease. To be assigned a status of stable disease, measurements must have 
met the stable disease criteria at least once after study entry at a minimum interval of 
12 weeks.  
 
2. Nontarget Lesions  
All other lesions or sites of disease that do not meet the criteria for target lesions, 
including those on bone scintigraphy. Measurements of these lesions are not required, 
but the presence or absence of each should be noted throughout the study. 
 
a. Complete Response (CR)  
The disappearance of all nontarget lesions and undetectable PSA tumor marker levels. 
To be assigned a status of complete response, changes in tumor measurements must 
be confirmed by repeat assessments performed no less than four weeks after the 
criteria for response are first met. 
 
b. Incomplete Response/Stable Disease (SD)  
The persistence of one or more nontarget lesion(s) and/or the persistence of detectable 
serum PSA tumor marker levels.  To be assigned a status of stable disease, 
measurements must have met the stable disease criteria at least once after study entry 
at a minimum interval of 12 weeks.  

 
 
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c. Progressive Disease (PD)  
The appearance of one or more new lesion(s) and/or unequivocal progression of 
existing nontarget lesions.   
 
NOTE:  For lesions only detectable by bone scan (or NaF PET/CT), the appearance 
of > 2 new lesions, with symptoms, will constitute disease progression.  Without 
symptoms, and if no other evidence of disease progression (no progressive disease by 
PSA or measurable disease criteria), progression must be documented with repeat 
bone scintigraphy (or NaF PET/CT) at least 6 weeks later demonstrating > 2 new 
lesions.  This is to eliminate the possibility of flair responses seen on bone imaging 
scans. 
 
3. Symptomatic Deterioration  
Patients with a global deterioration of health status requiring discontinuation of 
treatment without objective evidence of disease progression at that time should be 
classified as having symptomatic deterioration. 
 
4. PSA Progression 
While PSA values will be collected, and PSA kinetics (PSA responses and 
maximal changes in PSA by waterfall plots) monitored as secondary endpoints, 
PSA rise will not be used to determine objective disease progression.   Similar to 
the radiographic studies above, PSA progression or response will be defined 
using both pre-treatment (study entry) and 3-month values as baseline values.   
 
a. PSA Complete Response:  Decrease in PSA to <0.2 ng/mL and confirmed with 
PSA measurement a minimum of four weeks later (confirmed PSA CR).  There 
must be no evidence of radiographic progression. 
 
b. PSA Partial Response: Greater than or equal to 50% reduction in baseline PSA. 
There must be no evidence of radiographic progression. 
 
c. Best Response: This is calculated from the sequence of objective statuses.  The 
date of response will be defined as the first date at which PSA declined from 
baseline or normalized.   
 
i. Confirmed PSA PR: Two or more objective statuses of PSA PR or better 
a minimum of four weeks apart documented before progression. Best 
response for objective disease must be stable/no response or better.  
 
ii. Unconfirmed PSA PR: One objective status of PSA PR documented before 
progression, but not qualifying for confirmed PSA PR. Best response for 
objective disease must be Stable/no response or better. 
 
iii. No PSA Response: Objective PSA status does not qualify as a PSA PR or 
unconfirmed PSA PR. 
 

 
 
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iv. Inadequate Assessment, response unknown: When best response for 
objective disease is inadequate or unknown or when PSA has been 
inadequately assessed, then PSA response will be coded likewise. 
 
d. PSA Progression: Although not considered progression per the primary objective 
of this study, time to PSA progression will be used for secondary analyses.  PSA 
progression will be defined as a 50% increase in PSA over the nadir PSA, and > 2 
ng/mL above the nadir, confirmed by a second value 3 or more weeks later (i.e. 
confirmed rising trend). If no on-study reduction has occurred, nadir would be the 
baseline value (pre-treatment or month 3 time point for analyses).   
 
C. 
Evaluation of Patient’s Best Overall Response 
 
The best overall response is the best response recorded from baseline until disease 
progression/recurrence, taking as reference for progressive disease the smallest measurements 
recorded after baseline. The table below provides overall responses for all possible combinations 
of tumor responses in target and nontarget lesions, with or without new lesions. 
 
To be assigned a status of complete or partial response, changes in tumor measurements must be 
confirmed by repeat assessments performed no less than four weeks after the criteria for response 
are first met. 
 
To be assigned a status of stable disease, measurements must have met the stable disease criteria 
at least once after study entry at a minimum interval of 12 weeks. 
 
Overall Response for all Possible Combinations of Tumor Response 
Target 
Lesions 
Nontarget 
Lesions 
New 
Lesions 
Overall 
Response 
CR 
CR 
No 
CR 
CR 
Incomplete 
response/SD 
No 
PR 
PR 
Non-PD 
No 
PR 
SD 
Non-PD 
No 
SD 
PD 
Any 
Yes or No 
PD 
Any 
PD 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease 
 
1. First Documentation of Response  
The time between initiation of therapy and first documentation of PR or CR. 
 
2. Confirmation of Response  
To be assigned a status of complete or partial response, changes in tumor 
measurements must be confirmed by repeat assessments performed no less than four 
weeks after the criteria for response are first met. 
 
3. Duration of Response  

 
 
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Duration of overall response – the period measured from the time that measurement 
criteria are met for complete or partial response (whichever status is recorded first) 
until the first date that recurrent or progressive disease is objectively documented, 
taking as reference the smallest measurements recorded since treatment started. 
 
a. Duration of Overall Complete Response  
The period measured from the time measurement criteria are met for complete 
response until the first date that recurrent disease is objectively documented. 
 
b. Duration of Stable Disease  
A measurement from baseline (3 month) until the criteria for disease progression is 
met, taking as reference the smallest measurements recorded since baseline. To be 
assigned a status of stable disease, measurements must have met the stable disease 
criteria at least once after study entry at a minimum interval of 12 weeks.  
 
4. Survival  
Overall survival will be defined as the time interval from randomization to death from 
any cause or to the last follow-up in censored patients. 
 
5. Time to Objective Disease Progression  
Time to progression will be defined as a function of baseline time points in two ways:  
(1) 
Time to progression from randomization: Time elapsed from randomization 
until disease progression or death.  If a patient doesn’t experience a disease 
progression or death event before the end of the follow-up period, then the 
observation for this patient will be censored. 
 
(2) 
Time to progression from 3 months post-randomization:  Time elapsed from 
the 3 months disease progression assessment until disease progression or death. If 
a patient doesn’t experience a disease progression or death event before the end of 
the follow-up period, then the observation for this patient will be censored. 
 
The intent of defining time to progression as a function of baseline time points (date of 
randomization and 3-month disease assessment) is to determine if treatment prolongs the 
time to progression using a delayed baseline evaluation after the treatment has been given 
time to elicit an immunologically-mediated anti-tumor response.   
 
6. Methods of Measurement  
Imaging based evaluation is preferred to evaluation by clinical examination. The same 
imaging modality must be used throughout the study to measure disease. 
 
a. 
CT and MRI  
CT and magnetic resonance imaging (MRI) are the best currently available and 
most reproducible methods for measuring target lesions. Conventional CT and MRI 
should be performed with contiguous cuts of 10 mm or less in slice thickness. Spiral 
CT should be performed by use of a 5 mm contiguous reconstruction algorithm. 
This specification applies to tumors of the chest, abdomen, and pelvis, while head 

 
 
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and neck tumors and those of the extremities require specific procedures.  
Ultrasound should not be used for measurement methods.   
 
b. 
Tumor Markers  
 
 
The PSA tumor marker alone will not be used to assess response.  PSA values will 
be collected for separate reporting of PSA kinetics, as described above.   In addition, 
PSA must decline to < 0.2 ng/mL for a patient to be considered in complete clinical 
response when all tumor lesions have disappeared. 
 
c. 
Clinical Examination  
 
Clinically detected lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes). For skin lesions, 
documentation by color photography, including a ruler to estimate size of the 
lesion, is recommended.  Photographs should be retained at the institution. 
 
 
8. 
Definition and Management of Limiting Toxicities and Adverse Events 
 
A treatment-limiting toxicity will be defined as any Grade 3 or greater toxicity (using the NCI 
Common Terminology Criteria version 4, Appendix C), or a specific Grade 2 event as described 
below that affects agent dosing, with an attribution of at least possibly related to the study or 
treatment procedures, and occurring between the pre-study visit and within one month of the final 
study vaccine treatment or within 90 days of the last treatment with pembrolizumab (whichever is 
later).  In general, if a patient develops Grade 3 toxicity with an attribution of at least possibly 
related to treatment (vaccine and/or pembrolizumab), the treatment schedule will be held until the 
toxicity resolves to Grade 1 or less.  There will be no dose reductions due to adverse events.  If a 
patient develops a second Grade 3 event both believed at least possibly due to treatment, or any 
Grade 4 event, no further treatments will be given.  The patient will remain on study, however, 
with collection of radiographic and laboratory data as per protocol.  The rationale for this is that 
patients could continue to benefit in terms of anti-tumor response and this could in fact be related 
to the development of other autoimmune toxicities.  If the adverse event occurs during the initial 
12 weeks (Arm 1) or 24 weeks (Arm 2) of treatment, a treatment delay of up to one week will be 
permitted, with the expectation that the event resolve to < grade 2 for retreatment, and that 
subsequent treatment will resume at the original study intervals.  A delay > 1 week will result in a 
skipped dose/treatment.  While rare overall, autoimmune events observed following 
pembrolizumab treatment of patients with melanoma typically resolved and did not necessarily 
lead to discontinuation of treatment.    
 
For patients specifically treated in Arms 3 or 4, the following management rules will apply:  If a 
patient develops Grade 3 toxicity with an attribution of at least possibly related to pembrolizumab 
and unlikely related (or unrelated) to pTVG-HP vaccine, the pembrolizumab treatment will be held 
until the toxicity resolves to Grade 1 or less, with the vaccine schedule continuing as per protocol.  
If a patient develops a second Grade 3 event (or any Grade 4 event) believed to be at least possibly 
due to pembrolizumab, but unlikely related (or unrelated) to pTVG-HP vaccine, no further 
pembrolizumab treatments will be given.  The patient will continue with pTVG-HP administration 
only as per protocol.  However, if a patient develops a Grade 3 toxicity with an attribution of at 
least possibly related to pTVG-HP vaccine, both pTVG-HP and pembrolizumab treatment will be 

 
 
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held until the toxicity resolves to Grade 1 or less.  If a patient develops a second Grade 3 event (or 
any Grade 4 event) believed at least possibly related to pTVG-HP vaccine, no further treatments 
will be given.  The patient will remain on study, however, with collection of radiographic and 
laboratory data as per protocol.  The rationale for these modifications is that patients could continue 
to benefit in terms of anti-tumor response and this could in fact be related to the development of 
other autoimmune toxicities, and patients who have toxicity from pembrolizumab could still 
potentially benefit from continued pTVG-HP vaccination.   
 
Specific symptoms and potential adverse events that have been identified in previous trials 
with pembrolizumab are described below.  Management for these specific conditions will be 
conducted as described, consistent with what has been previously recommended in the FDA 
prescribing information “package insert”: 
 
Infusion-related reactions: 
 
Treatment with pembrolizumab will be permanently discontinued for grade 3 or 4 infusion-related 
reaction.   
 
Constitutional symptoms: 
 
Mild, low-grade fevers and chills, and local skin site reactions, are expected events following 
immunization and/or treatment with GM-CSF.  Grade 1 events will not be treated with anti-
inflammatory agents unless they persist 48 hours after an immunization.  Constitutional 
inflammatory events > grade 1, or persisting beyond 48 hours after immunization, will be managed 
with ibuprofen, if needed, up to 600 mg three times daily. 
 
Fatigue was reported in 47% (and 7% grade 3 fatigue) of 89 patients with melanoma who received 
pembrolizumab in the trial leading to its approval.  As this is a potentially expected event, and not 
life-threatening, treatment will not be held for fatigue grade 3 or lower.  
 
Hematologic: 
 
Anemia was reported in 14% (and 5% grade 3 anemia) of 89 patients with melanoma who received 
pembrolizumab in the trial leading to its approval.  Anemia is also common in patients with 
advanced prostate cancer.  In the absence of findings to suggest this is an immune-mediated event, 
treatment will not be held for anemia grade 3 or lower.  
 
Pulmonary adverse events: 
 
Pneumonitis occurred in 12 (2.9%) of 411 melanoma patients receiving pembrolizumab, including 
8 (1.9%) with grade 3 and 1 (0.2%) with grade 4 events.  The median time to development of 
symptoms was 4.9 months.  Patients will be monitored for signs and symptoms of pneumonitis, 
with radiographic imaging (chest X-ray or CT) and specialist referral as clinically indicated.  
Specifically, a CT of the chest will be performed at baseline as a basis for comparison, and then 
subsequently as clinically indicated.  Pembrolizumab will be held for grade 2 pneumonitis, and 
permanently discontinued for grade 3 or higher events.  Symptomatic patients will be treated with 
high-dose systemic corticosteroids (> 40 mg prednisone or equivalent per day) for at least one 

 
 
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week followed by taper, or as clinically indicated.  If retreatment is possible, treatment with 
pembrolizumab will continue to be held until the toxicity resolves to < grade 2. 
 
Immune-mediated colitis adverse events: 
 
Colitis (including microscopic colitis) occurred in 4 (1%) of 411 melanoma patients receiving 
pembrolizumab, including 1 patient with grade 2 and 2 patients with grade 3 events.  The median 
time to development of symptoms was 6.5 months.  Patients will be monitored for signs and 
symptoms of colitis (abdominal pain +/- diarrhea), with radiographic imaging and/or colonoscopy 
as clinically indicated.  Pembrolizumab will be held for grade 2 or grade 3 colitis, and permanently 
discontinued for grade 4 events.  Symptomatic patients will be treated with high-dose systemic 
corticosteroids (> 40 mg prednisone or equivalent per day) for one week followed by taper, or as 
clinically indicated.  If retreatment is possible, treatment with pembrolizumab will continue to be 
held until the toxicity resolves to < grade 2. 
 
Immune-mediated hepatitis adverse events: 
 
Hepatitis (including liver-associated pain and/or elevation of serum liver function tests) occurred 
in 2 (0.5%) of 411 melanoma patients receiving pembrolizumab, including 1 patient with a grade 
4 event.  The time to development of symptoms was 22 days.  Patients will be monitored by 
laboratory tests for changes in liver function.  Pembrolizumab will be held for grade 2 or higher 
liver function test abnormalities, and permanently discontinued for grade 3 or 4 events.  Patients 
with grade 2 or higher events will be treated with high-dose systemic corticosteroids (> 40 mg 
prednisone or equivalent per day) for at least one week followed by taper, or as clinically indicated.  
If retreatment is possible, treatment with pembrolizumab will continue to be held until the toxicity 
resolves to < grade 2. 
 
Immune-mediated hypophysitis adverse events: 
 
Hypophysitis occurred in 2 (0.5%) of 411 melanoma patients receiving pembrolizumab, including 
1 patient with grade 2 and 1 patient with grade 4 events.  The median time to development of 
symptoms was 1.5 months.  Patients will be monitored for signs and symptoms of hypophysitis, 
which could include headache, visual disturbances, polyuria, and polydipsia; lab tests will be 
performed as clinically indicated.  Pembrolizumab will be held for grade 2 or higher hypophysitis, 
and permanently discontinued for grade 4 events.  Patients with grade 2 or higher events will be 
treated with high-dose systemic corticosteroids (> 40 mg prednisone or equivalent per day) for at 
least one week followed by taper, or as clinically indicated.  If retreatment is possible, treatment 
with pembrolizumab will continue to be held until the toxicity resolves to < grade 2. 
 
Immune-mediated nephritis adverse events: 
 
Nephritis occurred in 3 (0.7%) of 411 melanoma patients receiving pembrolizumab, including 1 
patient each with grade 2, 3, or 4 events.  Two of these patients developed acute interstitial 
nephritis.  The time to development of symptoms was 11 months, and 5 months after the last dose 
in at least one patient.  Patients will be monitored for changes in renal function, with other lab tests 
as clinically indicated.  Pembrolizumab will be held for grade 2 or higher nephritis, and 
permanently discontinued for grade 3 or higher events.  Patients with grade 2 or higher events will 

 
 
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be treated with high-dose systemic corticosteroids (> 40 mg prednisone or equivalent per day) for 
at least one week followed by taper, or as clinically indicated.  If retreatment is possible, treatment 
with pembrolizumab will continue to be held until the toxicity resolves to < grade 2. 
 
Immune-mediated thyroid function adverse events: 
 
Hyperthyroidism occurred in 5 (1.2%) of 411 melanoma patients receiving pembrolizumab, 
including 2 patients with grade 2 events and 1 patient with a grade 3 event.  The median time to 
development of symptoms was 1.5 months.  Patients will be monitored for serum changes in 
thyroid function, and subsequently with other lab tests as clinically indicated.  Pembrolizumab will 
be held for grade 3 or higher hyperthyroidism, and permanently discontinued for grade 4 events.  
Patients with grade 3 or higher events will be treated with high-dose systemic corticosteroids (> 
40 mg prednisone or equivalent per day) for at least one week followed by taper, or as clinically 
indicated.  If retreatment is possible, treatment with pembrolizumab will continue to be held until 
the toxicity resolves to < grade 2. 
 
Hypothyroidism occurred in 34 (8.3%) of 411 melanoma patients receiving pembrolizumab, 
including 1 patient with a grade 3 event.  The median time to development of hypothyroidism was 
3.5 months.  Patients will be monitored for serum changes in thyroid function, and subsequently 
with other lab tests if clinically indicated.  Isolated hypothyroidism will be managed with thyroid 
hormone replacement therapy without treatment interruption and without corticosteroids.   
 
Other immune-mediated adverse events: 
 
Other clinically significant, immune-mediated adverse reactions have been observed in <1% of 
patients treated with pembrolizumab, including exfoliative dermatitis, uveitis, arthritis, myositis, 
pancreatitis, hemolytic anemia, partial seizures, adrenal insufficiency, myasthenic syndrome, optic 
neuritis, and rhabdomyolysis.  For suspected immune-mediated adverse events apart from those 
previously listed, efforts should be made to confirm the etiology and exclude other causes.  Based 
on the severity of symptoms, and as directed by the treating physician, pembrolizumab should be 
either discontinued or held until improvement in symptoms to < grade 2.  Corticosteroids may be 
administered if clinically indicated, again as directed by the treating physician. 
 
 
9. 
Plan of Treatment 
 
The following section describes the schedule for prescreening, treatment, and clinical and 
laboratory evaluations.  When a chemistry panel is indicated the following tests are performed:  
Chem 7 (including sodium, potassium, bicarbonate, BUN, creatinine, and glucose), ALT, AST, 
bilirubin, alkaline phosphatase, amylase, thyroid stimulating hormone (TSH) and LDH.  Whenever 
a CBC is indicated, this will include differential and platelet count.  This plan is summarized in 
the Schema. 
 
Because one biomarker endpoint is to evaluate metastatic tissue prior to and after treatment, 
patients with disease amenable to biopsy will initially be required to undergo one pretreatment 
biopsy and one post-treatment biopsy roughly three months after the start of treatment. It is our 
intent to have at least 6 patients per study arm with evaluable pre-treatment and post-treatment 

 
 
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biopsies.  If this is reached, subsequent patients with viable lesions amenable to biopsy may not 
be required to undergo these procedures. It is expected that some patients may have a baseline 
biopsy and not be able to undergo a repeat biopsy, and some patients may not have viable disease 
to biopsy, and these instances will not affect study participation.  Similarly, up to 6 subjects per 
arm will be required to complete the FLT PET scan at baseline and again at month 3 (Week 12).  
If there are subjects who complete the first but not the second scan, more than 6 subjects in an arm 
may, after approval from the Study PI, undergo the FLT PET scans in order to have at least 6 
subjects who complete both scans. A maximum of 10 evaluable subjects between Arms 3 and 4 
will be required to undergo the NaF PET scans. Selected subjects in Arm #3 will undergo either 
the FLT PET or the NaF PET based on the area of their disease (soft tissue vs bone).   Subjects in 
Arm 4 will not receive additional biopsies but may undergo NaF PET.   
 
FIRST COHORT:  STUDY ARMS 1 and 2: 
 
A. 
Prescreen / Eligibility (performed within 6 weeks of Day 1) 
 
 
1. 
Confirm potential eligibility by history, pathology, diagnosis; no exclusions by 
history 
2. 
CT scan of abdomen and pelvis, bone scan 
3. 
Sign consent form 
4. 
Evaluation of CBC, chemistry panel, serum prostate specific antigen (PSA), serum 
PAP, and serum testosterone. 
 
B. 
Pre-Treatment Evaluations (within 4 weeks of Day 1; can coincide with prescreen 
evaluation) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score 
2. 
Randomization – treatment arm assignment  
3. 
Baseline research leukapheresis (50-100 mL approximate total volume, for 
immunological monitoring) – to take place within 4 weeks prior to day 1.  
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc 
blood draw may be substituted (twenty 10-mL green-top heparinized tubes). 
4. 
Tetanus immunization (to take place after baseline leukapheresis and prior to day 
1) 
5. 
20 mL peripheral blood (red-top tubes) for sera to evaluate baseline antibody 
responses, and 20 mL peripheral blood (green-top tubes) for CTC baseline 
evaluation 
6. 
FLT PET/CT scan (to occur within 4 weeks prior to day 1). 
7. 
Tissue biopsy – metastatic site – to take place within 2 weeks prior to day 1 
 
C. 
Immunization #1 (Day 1) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score  
2. 
Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP 
3. 
Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid 
area of the lateral arm (preferentially left arm) with 0.25 mL administered at each 
of two adjacent sites. 

 
 
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39 
 
4. 
Following vaccinations, the subjects will be observed for 60 minutes for 
unanticipated adverse events.     
 
D. 
ARM 1 SUBJECTS ONLY:  Anti-PD-1 #1 (Day 1, 1-3 hours after day 1 immunization)  
 
1. 
Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
2. 
Subjects will be observed for additional 30 minutes for unanticipated adverse 
events.  Vital signs (temperature, heart rate and blood pressure) will be assessed 
and recorded by the staff prior to, and at the end of, the treatment period.   
 
E. 
Immunization #2 - Day 15 (+/- 3 days) 
 
1. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
2. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
 
F. 
ARM 1 SUBJECTS ONLY:  Anti-PD-1 #2 - Day 22 (7 +/- 3 days after day 15 visit)  
 
1. Physical examination, symptoms assessment, and ECOG performance score  
2. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP 
3. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
4. Subjects will be observed for additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.   
 
G. 
Immunization #3 - Day 29 (14 +/- 3 days after day 15 visit) 
 
1. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
2. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
 
H. 
Immunization #4 - Day 43 (14 +/- 3 days after day 29 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score  
2. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP  
3. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis  
4. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
5. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
 

 
 
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40 
 
I. 
ARM 1 SUBJECTS ONLY:  Anti-PD-1 #3 (Day 43, 1-3 hours after day 43 immunization)  
 
1. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
2. Subjects will be observed for additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.   
 
J. 
Immunization #5 - Day 57 (14 +/- 3 days after day 43 visit) 
 
1. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
2. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.   
 
K. 
ARM 1 SUBJECTS ONLY:  Anti-PD-1 #4 – Day 64 (7 +/- 3 days after day 57 visit)  
 
1. Physical examination, symptoms assessment, and ECOG performance score  
2. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP  
3. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
4. Subjects will be observed for an additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.   
 
L. 
Immunization #6 - Day 71 (14 +/- 3 days after day 57 visit) 
 
1. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
2. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
 
M. 
3-Month Follow-up - Day 85 (14 +/- 3 days after day 71 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed +/- 7 
days of this visit) 
3. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
4. CT scan of abdomen and pelvis, bone scintigraphy (can be performed +/- 7 days of this 
visit, but prior to pembrolizumab treatment for patients in Arm 2) 
5. FLT PET/CT scan (can be performed +/- 7 days of this visit, but prior to 
pembrolizumab treatment for patients in Arm 2) 
6. Tissue biopsy of same metastatic lesion as in pre-treatment period (can be performed 
+/- 7 days of this visit, but prior to pembrolizumab treatment for patients in Arm 2) 
 

 
 
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41 
 
ARM 2 SUBJECTS ONLY – Anti-PD-1 #1: 
 
7. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
8. Subjects will be observed for an additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.  
 
N. 
DAY 106 – (21 +/- 3 days after day 85 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP  
 
ARM 2 SUBJECTS ONLY – Anti-PD-1 #2: 
 
3. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
4. Subjects will be observed for an additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.  
 
O. 
DAY 127 – (21 +/- 3 days after day 106 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP  
 
ARM 2 SUBJECTS ONLY – Anti-PD-1 #3: 
 
3. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
4. Subjects will be observed for an additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.  
 
P. 
DAY 148 – (21 +/- 3 days after day 127 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP  
 
ARM 2 SUBJECTS ONLY – Anti-PD-1 #4: 
 
3. Pembrolizumab 2 mg/kg administered intravenously over 30 minutes 
4. Subjects will be observed for an additional 30 minutes for unanticipated adverse events.  
Vital signs (temperature, heart rate and blood pressure) will be assessed and recorded 
by the staff prior to, and at the end of, the treatment period.  
 
Q. 
6-month Follow-up – Day 169 (21 days +/- 3 days after day 148 visit) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score  
2. 
Blood draw for CBC, chemistry panel, serum PSA and PAP 

 
 
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42 
 
3. 
CT scan of abdomen/pelvis and bone scintigraphy (can be performed +/- 7 days of 
this visit) 
4. 
10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 
20 ml peripheral blood (green-top heparinized tubes) for CTC analysis 
5. 
Leukapheresis (~1.0 blood volume, 100 mL collection) for T-cell response 
evaluation (can be performed +/- 7 days of this study visit) 
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc 
blood draw may be substituted (twenty 10-mL green-top heparinized tubes). 
 
R. 
9-Month Follow-up (84 days +/- 10 days following 6-month study visit) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score. 
2. 
Blood draw for CBC, chemistry panel, serum PSA and PAP  
3. 
10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 
100 ml peripheral blood (green-top heparinized tubes) for T-cell response 
evaluation and CTC analysis 
4. 
CT scan of abdomen and pelvis, bone scintigraphy (can be performed +/- 7 days of 
this visit) 
 
S. 
12-Month Follow-up (84 days +/- 10 days following 9-month study visit)  
OFF-STUDY Visit 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score. 
2. 
Blood draw for CBC, chemistry panel, serum PSA and PAP  
3. 
10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 
100 ml peripheral blood (green-top heparinized tubes) for T-cell response 
evaluation and CTC analysis 
4. 
CT scan of abdomen and pelvis, bone scintigraphy (can be performed +/- 7 days of 
this visit, and does not need to be repeated for off-study visit if progression of 
disease already confirmed within 1 month) 
 
T. 
Long-term Follow-Up 
 
1. 
Subjects will be contacted by telephone (if not already being seen in clinic) annually 
for 2 years following the last DNA vaccination to collect clinical information to 
identify any potential long-term risks.  This information has been requested by FDA 
for all gene delivery trials to assess potential long-term risks.  The specific 
information to be collected annually will include:   
 Date of contact 
 Current medications 
 Hospitalizations (dates and reasons for hospital admission) 
 Stage of prostate cancer, treatments for prostate cancer, and recent 
serum PSA level 
 New cancer diagnoses 
 New autoimmune disorders 
 New hematologic or neurologic disorders 
 Other new medical diagnoses 

 
 
Version 04/06/2021 
43 
 
 Date of death if patient deceased 
 
EXTENDED TREATMENT COHORT – ARM 3: 
 
U. 
Prescreen / Eligibility (performed within 6 weeks of Day 1) 
 
 
1. Confirm potential eligibility by history, pathology, diagnosis; no exclusions by history 
2. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scan 
3. Sign consent form 
4. Evaluation of CBC, chemistry panel, serum prostate specific antigen (PSA), serum 
PAP, and serum testosterone. 
 
V. 
Pre-Treatment Evaluations (within 4 weeks of Day 1; can coincide with prescreen 
evaluation) 
 
1. Physical examination, symptoms assessment, and ECOG performance score 
2. Baseline research leukapheresis (50-100 mL approximate total volume, for 
immunological monitoring) – to take place within 4 weeks prior to day 1.  
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc blood 
draw may be substituted (twenty 10-mL green-top heparinized tubes). 
3. Tetanus immunization (to take place after baseline leukapheresis and prior to day 1) 
4. 20 mL peripheral blood (red-top tubes) for sera to evaluate baseline antibody responses, 
and 20 mL peripheral blood (green-top tubes) for CTC baseline evaluation 
5. FLT PET/CT scan – to occur within 4 weeks prior to day 1 (up to  6 evaluable patients 
per cohort) 
6. NaF PET/CT scan – to occur within 4 weeks prior to day 1 (max of 10 evaluable 
patients) 
7. Blood for PT/INR for patients undergoing biopsy (may be 30 days up to day of biopsy 
for patients not taking Coumadin) 
8. Tissue biopsy – metastatic site – to take place within 2 weeks prior to day 1 (minimum 
of 6 patients per cohort) 
 
W. 
Immunization #1 (Day 1) 
 
1. Physical examination, symptoms assessment, and ECOG performance score  
2. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
3. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
4. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
5. Subjects will be observed for additional 30 minutes for unanticipated adverse events.     

 
 
Version 04/06/2021 
44 
 
 
X. 
Immunization #2 - Week 3 (21 +/- 3 days after Day 1 visit) 
Immunization #4 - Week 9 (21 +/- 3 days after Week 6 visit) 
Immunization #6 - Week 15 (21 +/- 3 days after Week 12 visit) 
Immunization #8 - Week 21 (21 +/- 3 days after Week 18 visit) 
Immunization #10 - Week 27 (21 +/- 3 days after Week 24 visit) 
Immunization #12 - Week 33 (21 +/- 3 days after Week 30 visit) 
Immunization #14 - Week 39 (21 +/- 3 days after Week 36 visit) 
Immunization #16 - Week 45 (21 +/- 3 days after Week 42 visit) 
 
1. Symptoms assessment  
2. Blood draw for CBC, differential and platelets, and chemistry panel ( can be performed 
+/- 3 days of this visit). 
3. Blood draw for PSA and PAP (can be performed +/- 3 days of this visit) 
4. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
5. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
6. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
7. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
 
Y. 
Immunization #3 – Week 6 (21 +/- 3 days after Week 3 visit) 
 
8. Physical examination, symptoms assessment, and ECOG performance score  
9. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP  
10. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
11. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
12. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
13. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
14. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
15. NaF PET/CT scan –+/- 7 days from this visit (only if baseline NaF PET/CT obtained) 
 
 
Z. 
Immunization #5 – Week 12 (21 +/- 3 days after Week 9 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 

 
 
Version 04/06/2021 
45 
 
3. Blood for PT/INR for patients undergoing biopsy (may be 30 days up to day of biopsy 
for patients not taking Coumadin)  
4. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
5. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
6. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
7. Pembrolizumab 2 mg/kg, maximum dose 200 mg, administered intravenously over 30 
minutes 
8. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
9. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
10. FLT PET/CT scan for those patients with baseline scan (can be performed +/- 7 days 
of this visit) 
11. NaF PET/CT scan –+/- 7 days from this visit (only if baseline NaF PET/CT obtained) 
 
12. Tissue biopsy of same metastatic lesion as in pre-treatment period (can be performed 
+/- 7 days of this visit) 
 
AA. 
Immunization #7 – Week 18 (21 +/- 3 days after Week 15 visit) 
Immunization #11 – Week 30 (21 +/- 3 days after Week 27 visit) 
Immunization #15 – Week 42 (21 +/- 3 days after Week 39 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
4. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
5. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
6. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
BB. 
Immunization #9 – Week 24 (21 +/- 3 days after Week 21 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. Leukapheresis (~1.0 blood volume, 100 mL collection) for T-cell response evaluation 
(can be performed +/- 7 days of this study visit) 
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc 
blood draw may be substituted (twenty 10-mL green-top heparinized tubes). 

 
 
Version 04/06/2021 
46 
 
4. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 20 
ml peripheral blood (green-top heparinized tubes) for CTC analysis 
5. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
6. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
7. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
8. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
9. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
 
CC. 
Immunization #13 – Week 36 (21 +/- 3 days after Week 33 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
4. Following vaccinations, the subjects will be observed for 60 minutes for unanticipated 
adverse events.     
5. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
6. Subjects will be observed for additional 30 minutes for unanticipated adverse events.  
7. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
 
DD. 
Week 48 Visit (End of Study) (21 +/- 3 days after Week 45 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
4. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
 
EE. 
28-Day Follow Up Visit (28 +/- 7 days after Off-study date, if prior to week 48) 
 
Patients will come off study at the time of disease progression, undue toxicity, or at the 
discretion of the patient and/or treating physician that it is not in the interest of the patient 
to continue treatment.  A separate follow-up visit is not required for patients completing 
the week 48 evaluation, however for patients coming off study prior to week 48, 28-day 
follow up visit will include the following: 
 

 
 
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47 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP  
3. IF NOT ALREADY COLLECTED WITHIN PREVIOUS 60 DAYS:  10 ml peripheral 
blood (red-top tubes) for sera to evaluate antibody responses, and 100 ml peripheral 
blood (green-top heparinized tubes) for T-cell response evaluation and CTC 
analysis 
 
FF. 
Long-term Follow-Up 
 
1. Subjects will be contacted by telephone (if not already being seen in clinic) annually 
for 2 years following the last DNA vaccination to collect clinical information to identify 
any potential long-term risks.  This information has been requested by FDA for all gene 
delivery trials to assess potential long-term risks.  The specific information to be 
collected annually will include:   
 Date of contact 
 Current medications 
 Hospitalizations (dates and reasons for hospital admission) 
 Stage of prostate cancer, treatments for prostate cancer, and recent 
serum PSA level 
 New cancer diagnoses 
 New autoimmune disorders 
 New hematologic or neurologic disorders 
 Other new medical diagnoses 
 Date of death if patient deceased 
 
 
EXTENDED TREATMENT COHORT – ARM 4: 
 
GG. 
Prescreen / Eligibility (performed within 6 weeks of Day 1) 
 
 
1. Confirm potential eligibility by history, pathology, diagnosis; no exclusions by history 
2. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scan 
3. Sign consent form 
4. Evaluation of CBC, chemistry panel, serum prostate specific antigen (PSA), serum 
PAP, and serum testosterone. 
 
HH. 
Pre-Treatment Evaluations (within 4 weeks of Day 1; can coincide with prescreen 
evaluation) 
 
1. Physical examination, symptoms assessment, and ECOG performance score 
2. Baseline research leukapheresis (50-100 mL approximate total volume, for 
immunological monitoring) – to take place within 4 weeks prior to day 1.  
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc blood 
draw may be substituted (twenty 10-mL green-top heparinized tubes). 
3. Tetanus immunization (to take place after baseline leukapheresis and prior to day 1) 
4. 20 mL peripheral blood (red-top tubes) for sera to evaluate baseline antibody responses, 
and 20 mL peripheral blood (green-top tubes) for CTC baseline evaluation 

 
 
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48 
 
5. NaF PET/CT scan – to occur within 4 weeks prior to day 1 (max of 10 evaluable 
patients) 
 
 
II. 
Immunization #1 (Day 1) 
 
1. Physical examination, symptoms assessment, and ECOG performance score  
2. Blood draw for CBC, differential and platelets, chemistry panel, PSA and PAP 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
6. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.     
7. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
8. Subjects will be observed for additional 30 minutes for unanticipated adverse events.     
 
JJ. 
Immunization #2 - Week 2 (14 +/- 3 days after Day 1 visit) 
Immunization #6 - Week 10 (14 +/- 3 days after Week 8 visit) 
Immunization #8 - Week 14 (14 +/- 3 days after Week 12 visit) 
Immunization #10 - Week 18 (14 +/- 3 days after Week 16 visit) 
Immunization #12 - Week 22 (14 +/- 3 days after Week 20 visit) 
Immunization #14 - Week 26 (14 +/- 3 days after Week 24 visit) 
Immunization #16 - Week 30 (14 +/- 3 days after Week 28 visit) 
Immunization #18 - Week 34 (14 +/- 3 days after Week 32 visit) 
Immunization #20 - Week 38 (14 +/- 3 days after Week 36 visit) 
Immunization #22 - Week 42 (14 +/- 3 days after Week 40 visit) 
Immunization #24 - Week 46 (14 +/- 3 days after Week 44 visit) 
 
1. Symptoms assessment  
2. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
3. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.      
 
KK. 
Immunization #4 – Week 6 (14 +/- 3 days after Week 4 visit) 
 
1. Symptoms assessment  
2. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
4. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.     
5. NaF PET/CT scan –+/- 7 days from this visit (only if baseline NaF PET/CT obtained) 

 
 
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49 
 
 
 
 
 
LL. 
Immunization #3 - Week 4 (14 +/- 3 days after Week 2 visit) 
Immunization #5 - Week 8 (14 +/- 3 days after Week 6 visit) 
Immunization #9 - Week 16 (14 +/- 3 days after Week 14 visit) 
Immunization #11 - Week 20 (14 +/- 3 days after Week 18 visit) 
Immunization #15 - Week 28 (14 +/- 3 days after Week 26 visit) 
Immunization #17 - Week 32 (14 +/- 3 days after Week 30 visit) 
Immunization #21 - Week 40 (14 +/- 3 days after Week 38 visit) 
Immunization #23 - Week 44 (14 +/- 3 days after Week 42 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
4. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.     
5. Pembrolizumab 2 mg/kg, maximum dose 200 mg, administered intravenously over 30 
minutes 
6. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
 
MM. Immunization #7 - Week 12 (14 +/- 3 days after Week 10 visit) 
Immunization #19 - Week 36 (14 +/- 3 days after Week 34 visit) 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
4. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
5. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
6. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.     
7. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
 
8. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
 

 
 
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50 
 
9. AT WEEK 12 ONLY (NOT week 36): NaF PET/CT scan –+/- 7 days from this visit 
(only if baseline NaF PET/CT obtained) 
 
 
NN. 
Immunization #13 – Week 24 (14 +/- 3 days after Week 22 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. Leukapheresis (~1.0 blood volume, 100 mL collection) for T-cell response evaluation 
(can be performed +/- 7 days of this study visit) 
NOTE:  For patients unable to undergo leukapheresis at this time point, a 200-cc 
blood draw may be substituted (twenty 10-mL green-top heparinized tubes). 
4. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses 
5. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
6. Subjects will receive pTVG-HP DNA immunizations intradermally on the deltoid area 
of the lateral arm (preferentially left arm) with 0.25 mL administered at each of two 
adjacent sites. 
7. Following vaccinations, the subject will be observed for 60 minutes for unanticipated 
adverse events.     
8. Pembrolizumab 2 mg/kg, maximum 200 mg dose, administered intravenously over 30 
minutes 
9. Subjects will be observed for additional 30 minutes for unanticipated adverse events.   
 
OO. 
Week 48 Visit (End of Treatment) (14 +/- 3 days after Week 46 visit): 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP (can be performed up to 3 
days prior to this visit) 
3. 10 ml peripheral blood (red-top tubes) for sera to evaluate antibody responses, and 100 
ml peripheral blood (green-top heparinized tubes) for T-cell response evaluation and 
CTC analysis (can be performed +/- 7 days of this visit) 
4. CT scan of abdomen and pelvis (CT of chest as clinically indicated), bone scintigraphy 
(can be performed +/- 7 days of this visit) 
 
PP. 
28-Day Follow Up Visit (28 +/- 7 days after Off-study date, if prior to week 48) 
 
Patients will come off study at the time of disease progression, undue toxicity, or at the 
discretion of the patient and/or treating physician that it is not in the interest of the patient 
to continue treatment.  A separate follow-up visit is not required for patients completing 
the week 48 evaluation, however for patients coming off study prior to week 48, 28-day 
follow up visit will include the following: 
 
1. Physical examination, symptoms assessment, and ECOG performance score. 
2. Blood draw for CBC, chemistry panel, serum PSA and PAP  
3. IF NOT ALREADY COLLECTED WITHIN PREVIOUS 60 DAYS:  10 ml peripheral 
blood (red-top tubes) for sera to evaluate antibody responses, and 100 ml peripheral 

 
 
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51 
 
blood (green-top heparinized tubes) for T-cell response evaluation and CTC 
analysis 
4. Patients to remain in telephone contact to report new symptoms up to 90 days after 
treatment with last dose of pembrolizumab 
 
QQ. 
Long-term Follow-Up 
 
1. Subjects will be contacted by telephone (if not already being seen in clinic) annually 
for 2 years following the last DNA vaccination to collect clinical information to identify 
any potential long-term risks.  This information has been requested by FDA for all gene 
delivery trials to assess potential long-term risks.  The specific information to be 
collected annually will include:   
 Date of contact 
 Current medications 
 Hospitalizations (dates and reasons for hospital admission) 
 Stage of prostate cancer, treatments for prostate cancer, and recent 
serum PSA level 
 New cancer diagnoses 
 New autoimmune disorders 
 New hematologic or neurologic disorders 
 Other new medical diagnoses 
 Date of death if patient deceased 
 
 
10. 
Response Monitoring 
 
A. 
Safety 
 
All subjects will be assessed at regular intervals, as per the study calendar, for evidence of adverse 
events by exam, symptoms, and clinical laboratory findings.  For these assessments, we will use 
the 
NCI 
common 
terminology 
criteria, 
version 
4 
(http://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcaev4.pdf). 
 
B. 
Immunological Monitoring 
 
Blood will be collected by either peripheral blood draw (up to 120 mL), leukapheresis (50-100 
mL), or leukapheresis replacement draw (200 mL) pre-immunization, at weeks 6, 12 and 24 of 
treatment, and at months 9 and 12 (quarterly intervals up to one year), for immunological 
monitoring.  From the heparinized blood, peripheral blood mononuclear cells (PBMC) will be 
prepared by density centrifugation over Ficoll-Paque using standard techniques.  PBMC will be 
used directly for analysis, and residual material cryopreserved in liquid nitrogen using 90% 
autologous serum collected at the time of blood draw, or 90% fetal calf serum, and 10% DMSO.  
Sera will be prepared from the red-top tubes and stored in aliquots at –80°C for antibody analyses.  
IFNγ and granzyme B ELISPOT analysis, PAP-specific T-cell proliferation, flow cytometric 
assays of antigen-specific cytokine secretion, and ELISA tests for antigen-specific antibodies, will 
be the primary methods of analysis.  The primary antigens tested will be PAP (experimental), PSA 
(negative control), and tetanus toxoid (positive control).  The primary immune analysis will be 

 
 
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conducted at the 6-month time point, and compared with the pre-treatment time point, and for 
patients to be evaluable for immune response (primary endpoint), blood (PBMC and serum) from 
this time point must be available for analysis.  However, immune monitoring will be conducted at 
the other time points indicated in secondary analyses to evaluate kinetic measures of immunity, 
and evaluate whether durable immune responses of particular phenotypes are elicited and/or 
maintained.  Assays may be conducted at the time of sample collection (fresh) and/or batched and 
performed at one time from multiple cryopreserved samples collected at different time points.  
Other methods of effector and regulatory T-cell response to PAP and other human tissue antigens 
may be used.   
 
B.1. 
Quantitative assessment of PAP-specific CD8+ T-cell effector immunity 
 
PAP-specific IFNγ- and granzyme B-secreting T-cell precursor frequency quantification by 
ELISPOT: ELISPOT will be used as the preferred methodology, as it permits analysis of low-
frequency events (LOD ~ 1:100,000 cells) and also permits simultaneous analysis of cryopreserved 
batched specimens [30].  IFNγ and granzyme B will be preferred analytes evaluated, as these are 
specifically associated with inflammatory/tissue-destructive (Th1-type, cytolytic) immune 
responses.  Specifically, cryopreserved PBMC from subjects at the various time points will be 
thawed, rested, and then transferred to 96-well nitrocellulose microtiter (ELISPOT) plates 
previously coated with monoclonal capture antibodies specific for IFNγ or granzyme B.  105 cells 
per well will be cultured in the presence of media (RPMI 1640 supplemented with L-glutamine, 
penicillin/streptomycin, ß-mercaptoethanol and 10% human AB serum) only (no antigen), 2 µg/ml 
PAP protein, 2 µg/ml PSA protein (negative control), 2 µg/ml of peptide libraries specific for PAP 
or control, 250 ng/ml tetanus toxoid, or 2.5 µg/ml PHA (positive mitogenic control) for 24-48 
hours.  Plates will then be washed with PBS containing 0.05% Tween-20 and incubated for 2.5 
hours at room temperature with 50 µl/well PBS containing 5 µg/ml biotinylated detection 
antibodies for either IFNγ or granzyme B.  After incubation, wells will be washed with PBS, and 
further incubated with 100 µl/well streptavidin-labeled alkaline phosphatase (BioRad, Hercules, 
CA) and then developed with 100 µl/well BCIP/NBT colorimetric substrate (BioRad).  The 
colorimetric reaction will be stopped by rinsing the plates under cool tap water, and wells will be 
allowed to dry completely before spots are enumerated with an ELISPOT automatic plate reader.   
 
REPORTING AND RESPONSE DEFINITION:  Results will be presented as previously reported 
as the mean (+/- standard deviation) number of spot-forming-units (sfu) per 106 cells (frequency), 
calculated by subtracting the mean number of spots obtained from the no antigen control wells 
from the mean number obtained in the experimental wells, normalized to 106 starting PBMC, from 
8-well replicate assays [31].  Comparison of experimental wells with control, no antigen, wells 
will be performed using a two-sample t-test, with p < 0.05 (two-sided) defined as a significant 
antigen-specific T-cell response.  A significant antigen-specific response resulting from 
immunization will then be defined as a PAP-specific response detectable at the 6-month post-
treatment time point (or other post-treatment time point evaluated) that is significantly higher than 
to media only (as above), at least 3-fold higher than the mean baseline value, and with a frequency 
> 10 per 106 PBMC.   
 
B.2. 
Assessment of PAP-specific memory T-cell immunity 
 

 
 
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Ability of T-cell lines to proliferate in response to antigenic stimulation:  T-cell proliferation in 
response to antigen stimulation as a measure of memory T-cell responses will be assessed by a 
PKH26 dye dilution assay as previously reported [31].  Specifically, PBMC will be labeled in vitro 
with PKH26 dye (Sigma, St. Louis, MO) according to the manufacturer’s recommendation.  T-
cell cultures with and without antigens will be established in replicates using 2x105 peripheral 
blood mononuclear cells (PBMC)/well, plated in 96-well round bottom microtiter plates (Corning, 
Cambridge, MA), in media consisting of RPMI 1640 (Gibco) and supplemented with L-glutamine, 
penicillin/streptomycin, ß-mercaptoethanol and 10% human AB serum (ICN Flow, Costa Mesa, 
CA).  Antigens may include 2 µg/ml of a pool of peptide spanning the amino acid sequence of 
PAP, 2 µg/ml PAP protein (Research Diagnostics Inc., Flanders, NJ), 2 µg/ml PSA (Research 
Diagnostics Inc.), 250 ng/ml tetanus toxoid, and 2.5 µg/ml phytohemaglutinin (PHA).  After 6-7 
days of culture at 37oC/5% CO2, cell surface markers will be stained to characterize the T-cell 
phenotype and memory phenotype of proliferating cells (CD45RO, CCR7).  Flow cytometery will 
be used to identify and enumerate CD3+CD4+ and CD3+CD8+ T cells co-staining for PKH26.  
The frequency of antigen-specific CD3+CD4+ and CD3+CD8+ T cells will be determined by 
determining the precursor frequency of PKH26+ events among CD4+ or CD8+ events (estimated 
from the number of cells and number of cell divisions by dye dilution using ModFit software, 
Verity Software House, Topsham, ME), and subtracting the mean precursor frequency of 
proliferating cells under media-only conditions.  Other cell surface markers may be assessed, other 
antigens evaluated, and/or other methods of antigen-specific T-cell proliferation.   
 
REPORTING AND RESPONSE DEFINITION:  Data will be reported as a mean and standard 
deviation of antigen-specific (PAP-specific) proliferative precursors per 106 PBMC using triplicate 
assessments for each antigen-stimulation condition, as previously reported [31].  Comparison of 
experimental wells with control, no antigen, wells will be performed using a two-tailed Student’s 
t test, with p < 0.05 defined as a significant antigen-specific proliferative T-cell response.  A 
significant antigen-specific response resulting from immunization will then be defined as a PAP-
specific response detectable at the 6-month post-treatment time point (or other post-treatment time 
point evaluated) that is significantly higher than to media only (as above), at least 3-fold higher 
than the mean baseline value, and with a frequency > 100 per 106 CD4+ or CD8+ T cells.   
 
B.3. 
Assessment of PAP-specific T cells by intracellular cytokine staining 
 
PAP-specific T-cell cytokine expression by intracellular cytokine staining:  As another method of 
evaluating AR LBD-specific T cell responses, intracellular cytokine staining will be used.  
Specifically, fresh or cryopreserved PBMC from subjects at the various time points will be rapidly 
thawed, cultured in 96-well microtiter plates in the presence of test antigens (PAP (protein or pool 
of overlapping 15-mer peptides), PSA, tetanus, PHA, or media only) for 4-24 hours, and then will 
be analyzed for intracellular cytokine expression.  Specifically, stimulated cells will be treated 
with monensin for 4-8 hours at 37oC/5%CO2.  Cells will then be washed with PBS/3%FCS, 
followed by staining for surface molecules (including CD3, CD4, and CD8 to identify T cell 
subsets, as well as CCR7 and CD45RO to identify memory populations, and potentially other cell 
surface markers).  Cells will then be fixed, permeabilized, and then stained for intracellular 
expression of a panel of cytokines (including, but not limited to, IFNγ, TNFα, IL-2, IL-17, and IL-
10), or control IgG labeled with the appropriate fluorochromes.  Cells will be analyzed on a flow 
cytometer, and results will be determined by identifying the frequency of T cells (either total T 
cells and/or T cell subsets, including CD4+ and CD8+ T cells, as well as various memory 

 
 
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populations or regulatory populations) expressing each individual cytokine, or cells expressing 
multiple cytokines.   
 
REPORTING AND RESPONSE DEFINITION:  Data will be reported as a mean and standard 
deviation of antigen-specific (PAP-specific) CD4+ or CD8+ T cells expressing one or more 
cytokines per 106 PBMC using triplicate assessments for each antigen-stimulation condition, as 
previously reported [31].  Comparison of experimental wells with control, no antigen, wells will 
be performed using a two-tailed Student’s t test, with p < 0.05 defined as a significant antigen-
specific T-cell response.  A significant antigen-specific response resulting from immunization will 
then be defined as a PAP-specific response detectable at the 6-month post-treatment time point (or 
other post-treatment time point evaluated) that is significantly higher than to media only (as 
above), at least 3-fold higher than the mean baseline value, and with a frequency > 100 per 106 
CD4+ or CD8+ T cells. 
  
B.4. 
Assessment of antigen-specific antibody immunity 
 
Enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies responses to PAP:  
The presence of a coexisting humoral immune response to PAP (or other antigens) will be 
evaluated by ELISA using an indirect method similar to that described previously [34].  
Specifically, Immulon-4 ELISA plates (Dynex Technologies Inc.) will be coated with 2 µg/ml 
purified PAP protein (Research Diagnostics, Inc., or other antigens or commercial sources) in 0.1 
M NaHCO3/Na2CO3 buffer (pH 9.6) overnight at 4oC.  After blocking with PBS/1% BSA for 1 
hour at room temperature, wells will be washed with PBS + 0.05% Tween-20 (PBS-Tween) and 
then incubated for 1 hour with human sera diluted 1:25, 1:50, 1:100 and 1:200.  After washing, 
plates will then be sequentially incubated with a peroxidase-conjugated anti-human IgG detection 
antibody (Amersham), followed by peroxidase enzyme TMB substrate (Kierkegaard and Perry 
Laboratories).  The color reaction will be stopped with 1N H2SO4 and the optical density measured 
at 450 nm.  Antibody titers for PAP-specific IgG antibodies will be determined as previously 
described [34]. 
 
REPORTING AND RESPONSE DEFINITION:  These are not strictly quantitative assays.  IgG 
response will be reported graphically demonstrating sera dilution curves, and by titer – defined as 
the highest sera dilution at which IgG responses are detectable above the mean + 3 standard 
deviations of the negative control.  A positive IgG response resulting from immunization will be 
defined as an antigen-specific (anti-PAP) IgG titer at least 4-fold higher than the baseline titer 
detectable at the 6-month post-treatment time point (or other post-treatment time point evaluated). 
 
B.5. 
Assessment of antigen-specific regulatory immune responses 
 
Trans-vivo delayed-type hypersensitivity (tvDTH) evaluation:  7.5-10 x 106 PBMC obtained from 
patients prior to and after immunization will be co-injected into the footpads of 6-to-8-week old 
SCID mice with 1 µg of PAP protein (Fitzgerald Industries, Acton, MA), or tetanus toxoid (TT/D; 
Aventis Pasteur, Bridgewater, NJ) as a recall antigen, versus phosphate-buffered saline (PBS) 
alone as a negative control.  Other antigens may be evaluated as well.  Antigen–driven swelling 
will be determined as previously described [66].  DTH reactivity after 24 hours will be shown as 
the change in footpad thickness in multiples of 10-4 inches, measured using a dial thickness gauge 
(Mitutoyo, Japan), and net swelling will be defined as the antigen-specific swelling subtracted for 

 
 
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the contribution obtained with PBMC plus PBS.  To determine the effect of neutralizing 
antibodies, PBMC will be mixed with 1 µg of PAP antigen and 25 µg of either control IgG or 
rabbit anti-human TGF-β, goat anti-human IL-10 (R&D Systems, Minneapolis, MN), 1 µg of 
mouse anti-human CTLA-4 monoclonal Ab (clone AS32, Ab Solutions, Mountain View, CA), or 
1 µg of mouse anti-human PD-1 monoclonal Ab (or potentially other antibodies to other regulatory 
molecules), and injected into the footpads of SCID mice as above.  The extent of bystander 
suppression, defining an antigen-specific regulatory response, will be measured as inhibition of 
recall antigen (tetanus) response in the presence of PAP antigen (or prostate-specific antigen (PSA) 
as a negative control) and calculated as previously described [67].  Given the nature of the testing 
and the requirement for SCID mice, data will be shown derived from single measurements, but 
with key experiments repeated 2-3 times for confirmation.   
 
REPORTING AND RESPONSE DEFINITION:  These are not strictly quantitative assays.  An 
antigen-specific (PAP-specific) regulatory response for these purposes will be defined as an 
inhibition of a recall antigen (tetanus toxoid) DTH response by > 50% when performed in the 
presence of PAP; i.e. a DTH response in the presence of PAP+TT that is < 50% of that obtained 
with TT only.  The identification of PD-1 (or CTLA-4) regulated responses will be defined as the 
identification of an increase (>15 x 10-4 inches) in a PAP-specific response in the presence of anti-
PD-1 (or anti-CTLA-4) versus IgG control, as in Figure 6.   
 
B.6. 
Assessment of antigen-spread to other prostate-associated antigens 
 
High-throughput immunoblot (HTI):  An exploratory objective of the study will be to determine 
if patients treated develop “off-target” prostate cancer antigen-specific immune responses as 
evidence of antigen spread.  Future studies may evaluate T-cell responses to non-targeted antigens.  
However, the primary evaluation will be to evaluate IgG responses to a panel of prostate-associated 
antigens as we have previously reported in patients treated with vaccines or other immune-
modulating agents [68, 69, 70].  IgG specific for 126 antigens, including 29 cancer-testis antigens 
[71, 72] and 97 prostate antigens frequently immunologically recognized [73, 74, 75, 76] will be 
identified by screening a high-density phage array expressing these individual antigens, as we have 
previously reported [68, 70, 71].  An alternative to this approach may include the evaluation of 
responses to panels of protein antigens fixed to support membranes provided by commercial 
vendors.  The primary analysis will be conducted using sera obtained at 6 months, and compared 
with IgG responses identified at baseline, to determine whether IgG specific for individual antigens 
are elicited over time.  Other time points will be assessed to determine the durability and kinetics 
of immune response development, and confirmatory ELISA studies will be performed where 
feasible.   
 
REPORTING AND RESPONSE DEFINITION:  A positive IgG response will be defined as an 
immunoreactive spot to a defined antigen, scored by at least 3 of 4 independent reviewers, and 
detectable to at least 2 of 3 replicates per immunoblot membrane as previously described [68, 77], 
or by statistically defined criteria using commercial antigen sources.  An IgG response resulting 
from immunization will be defined as an immunoreactive antigen identified at the 6-month (or 
other post-treatment time for subsequent analyses) time point that was not identified at the pre-
treatment time point.   
 

 
 
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C. 
Circulating Tumor Cell (CTC) Evaluation 
 
CTC will be enumerated and characterized at the same time points as for immune evaluation (pre-
treatment, after 6 weeks, and at quarterly intervals for up to one year) using flow cytometry.  
Specifically, as in Figure 7, PBMC obtained at these time points will be stained with fluorochrome-
labeled antibodies specific for, and including at least, CD45, EpCAM, PD-1, CTLA-4, and DAPI.  
CTC will be defined as CD45-EpCAM+DAPI- cells, and the percentage of these events among all 
live cellular events will be determined at the different time points.  The % of CTC expressing PD-
1 or CTLA-4 will also be determined.  Results will be reported in tabular format from the different 
time points, and general trends will be assessed.  These results will not be strictly quantitative, as 
multiple replicates to determine standard deviation are not feasible.  Other methods of CTC capture 
and enumeration may also be used.  Further investigational studies may be conducted with CTC 
collected by these methods.   
 
D. 
Histopathology Evaluation 
 
Tissue biopsies will be obtained from metastatic lesions (the same lesion per patient) prior to 
treatment and at week 12.  The purpose of these studies is to determine whether immunization 
affects PD-L1 expression in the tumor (likely by eliciting tumor antigen-specific T cells secreting 
IFNγ), whether concurrent treatment with anti-PD-1 mAb leads to an increase in infiltration of 
CD8+ T cells, and whether treatment increases expression of other T-cell regulatory ligands on T 
cells (PD-1, CTLA-4, TIM-3, BTLA, LAG-3) or tumors (e.g. HVEM, phosphatidyl serine, PD-
L2).  Consequently, biopsy specimens obtained pre-treatment and after 12 weeks will be stained 
with antibodies specific for CD3, CD4, CD8, FoxP3, PD-1, CTLA-4, TIM3, BTLA, LAG-3, PD-
L1, PD-L2, phosphatidyl serine, HVEM and potentially other markers.  Staining and quantification 
will be reviewed by a pathologist blinded to the treatment groups to determine CD8+ T cells per 
field, CD4+FoxP3+ (Treg):CD8+ T cell ratio, PD-L1 expression, and whether these or the 
expression of CD8+ T cells expressing one regulatory receptors (or tumor cells expressing one or 
more regulatory ligands) change from pre-treatment to the 12 week time point.   
 
Logistically, patients will be scheduled for a CT-guided biopsy of a bone or soft tissue abnormality 
consistent with prostate cancer metastasis.  This site of biopsy will be determined by the 
interventional radiology staff in conjunction with the local PI after review of available scans (bone 
scans, CT scans, MRI), with preference given to lesions meeting the following factors:  (1) size of 
lesion (preference towards the largest lesion, given the need for biopsy and re-biopsy after 12 
weeks); (2) safety of biopsy (with obvious preference to lesions with the least risk to the patient); 
and (3) for bone lesions with the greatest intensity activity noted on bone scintigraphy.  
Coordination and acquisition of biopsy material will be performed by the UWCCC TSB 
(Translational Science Biocore).  Standard laboratory tests to assess bleeding and coagulation will 
also be completed prior to the procedure.  Conscious sedation will be used as necessary (typically 
by using intravenous fentanyl and midazolam) and with appropriate nursing support.  In order to 
obtain adequate tumor sample, and as long as it is deemed safe, and based on accessibility, target 
lesion size, specimen integrity, specimen appearance, and intraprocedural bleeding, an attempt will 
be made to obtain 2-6 biopsy samples.  Biopsies will be performed using sterile technique and 
lidocaine local anesthetic.   For bone lesions, at least an 11-13 gauge bone-cutting needle, with or 
without a co-axial trocar, will be used.  2-6 biopsy samples will be obtained of the selected lesion, 
with multiple samples obtained by: 1) sequential advancement of needle passes through a fixed 

 
 
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trocar, such that each needle pass progresses more deeply into the bone lesion; or 2) re-insertion 
and re-direction of the needle and/or trocar through another region of the bone lesion.  At the 
completion of the procedure, CT imaging will be repeated to assess for immediate complications.  
The patient will be observed for 2-4 hours before discharge.   
 
Samples will be transported to the UWCCC TRIP (Translational Research Initiatives in Pathology) 
lab for formalin fixation, paraffin embedding, sectioning, H&E staining, and ultimately for IHC 
analysis as described above.  Other types of gene expression profiling may be used as well to 
evaluate gene expression changes, and changes in T cell subtypes infiltrating tumors prior to and 
after treatment.  
 
 
E. 
FLT PET/CT Imaging 
 
3’-Deoxy-3’-[18F]Fluorothymidine (FLT) positron emission tomography (PET)/computed 
tomography (CT) uses a thymidine analogue that accumulates in proliferating tissue.  Proliferating 
tissue could include malignant lesions or immune cells.  Following vaccination we have observed 
a proliferation of CD4+ and CD8+ T cells specific for PAP, at least as assessed in vitro following 
reencounter with the PAP antigen [29, 31].  In a study of patients with melanoma treated 
intranodally with a dendritic cell vaccine, FLT PET/CT could detect uptake in treated lymph nodes 
compared to control lymph nodes following vaccination, uptake that persisted for up to 3 weeks 
[78].  Consequently, FLT PET/CT may be useful as a molecular imaging tool to detect immune 
activation after immunization, at the same time as detecting decreases in proliferating tumor 
masses as a result of treatment.  We hypothesize that FLT PET/CT will be able to detect increased 
uptake in left axillary draining lymph nodes (draining site of intradermal immunization) at 12 
weeks compared with pre-treatment studies, and that a decrease in uptake will be observed in soft 
tissue metastatic deposits.  FLT PET/CT will be performed at baseline (within 4 weeks of starting 
treatment) and at week 12, thus enabling comparative evaluation with other imaging obtained at 
that time and biopsy results. 
 
FLT PET/CT IMAGING PROCEDURES:  To ensure uniformity as well as optimal data 
interpretation, analyses will be conducted under the supervision of Dr. Robert Jeraj, PhD (co-
investigator) at the University of Wisconsin Image Analysis Core (IMAC) facility, with all 
radiographic analysis by Dr. Jeraj and designees blinded to study treatment arm.  
 
• 
At the beginning of each imaging session, a CT scan will be obtained on the combined 
PET/CT scanner. The CT scan is an integral part of the PET/CT acquisition and serves for 
attenuation correction calculations and better PET/CT image co-registration. It will enable better 
delineation of the patient anatomy, which will be important when comparing different scans. An 
FLT-PET scan will follow. The patients will be injected up to 15 mCi (555 MBq) of 18F-FLT.   
 
• 
Dynamic imaging (15 cm field of view) will be performed first to obtain arterial input 
function and post injection kinetics of the tracer. The field of view will be positioned so that either 
a tumor mass or axillary draining nodes will be in the field of view. If applicable, the region that 
will allow at least a partial heart view will be selected. The dynamic scan will be performed for 30 
minutes. The arterial blood decay curve of 18F-FLT will be estimated from the time-dependent 

 
 
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activity in the left ventricular blood pool. The arterio-venous difference will be quantitatively 
assayed from the time-dependent activity in the right cardiac chambers. 
 
• 
After the initial dynamic imaging of the heart region, a static whole body scan will be 
initiated. The whole body scan will be initiated at approximately 60 minutes after the injection. To 
minimize errors of the relative change assessment, the subsequent whole body scans will be 
initiated within 5 minutes of the initiation time of the baseline scan. The whole body scan will be 
performed with up to 10 minutes/scanning position, lasting up to 60 minutes. The patient will be 
scanned “head first”. Together with the dynamic imaging, the total body scanning time will be up 
to 90 minutes (30 minutes dynamic plus up to 60 minutes static whole body scan).  
 
• 
All the scans will be acquired in a 3D mode to increase spatial resolution of the imaging 
data.  
 
 
FLT-PET/CT image reconstruction: 
 
• 
For both the dynamic and whole body scans, two PET image reconstructions will be used 
– optimal qualitative and optimal quantitative reconstructions. The optimal quantitative 
reconstruction will be used for quantitative evaluation; the optimal qualitative reconstruction will 
be used for clinical read of the FLT PET/CT scans.  
 
 
FLT-PET/CT image analysis: 
 
• 
All the PET image data will be coregistered to the baseline (pretreatment) image data to 
enable comparison of the spatially dependent changes of the investigated tumor and metastasis 
parameters during the therapy. The scans will be coregistered based on the CT data. Each of the 
metastases will be coregistered locally, segmented and treated individually. Up to five metastases 
will be used in this analysis. Both the CT data, as well as the corresponding FLT PET data, will 
be analyzed for spatial correlation. The correlation coefficients, as well as joint histograms, will 
be used to evaluate similarity of the distributions.  
 
• 
The CT data will be analyzed to establish anatomical changes in tumor size. Evaluation 
will be based on the change of the volume of the metastatic mass, as segmented from a CT scan: 
ΔV=Vfollow-up-Vbaseline. 
 
• 
The dynamic FLT-PET/CT imaging data will be used to perform kinetic analysis of the 
imaging data, thus allowing increase correlation to the biological parameters (cell proliferation 
index).  
 
• 
The static total body FLT-PET/CT imaging data will be used to identify tumor masses. 
Each of the metastasis will be treated individually. Standardized uptake values (SUV) will be used 
to assess the FLT PET uptake. SUVmax, SUVmean and SUVtotal will be recorded and analyzed. 
In addition, the spatial distribution of the change will be determined, in case of heterogeneous 
tumor uptake distribution.  

 
 
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F. 
NaF PET/CT Imaging 
 
One of the best PET imaging agents for detection of bone metastasis is 18F-Sodium fluoride (NaF). 
NaF uptake is characterized by high and rapid bone uptake accompanied by very rapid blood 
clearance, which results in a high bone-to-background ratio in a short time. The retention and 
clearance mechanisms of NaF are well understood (16). NaF retention is a 2-phase process (17). 
NaF is rapidly taken up by the bone, which leads to rapid clearance from the plasma. It is cleared 
from plasma in a biexponential manner -- the first phase has a half-life of 0.4 h, and the second 
phase has a half-life of 2.6 h (18). Retention of NaF in the bone is almost perfect -- essentially all 
the NaF that is delivered to bone by the blood is retained in the bone (19), illustrated by the fact 
that one hour after administration of NaF, only about 10% of the injected dose remains in the blood 
(20). In the first phase, the 18F2 ion exchanges for an OH2 ion on the surface of the hydroxyapatite 
matrix of bone. In the second phase, the 18F2 ion migrates into the crystalline matrix of bone, where 
it is retained until the bone is remodeled; therefore its retention directly reflects active bone 
metabolism. As a result, its accumulation is proportional to both blood flow and osteoblastic 
(metabolic) activity; thus, it represents an excellent PET tracer for imaging of bone metastasis (21, 
22). NaF PET has been validated clinically as a diagnostic imaging modality. In this context, NaF 
PET has been shown to be superior to both FDG PET and 99mTc-MDP SPECT in detection of bone 
metastases (16, 25). NaF is a FDA approved imaging tracer for use with PET/CT. We hypothesize 
that NaF PET/CT will be able to detect early changes in number of bone metastases, as well as 
changes in osteoblastic activity that may reflect inflammatory effects. Three NaF PET/CT will be 
obtained in 6 - 10 patients with known osteoblastic metastases (baseline, at week 6 (+/- 1 wk), and 
again at week 12 (+/- 1 wk)).  
 
Sodium Fluoride F 18 (NaF) Injection 
 
NaF is commercially available.  Ordering, Dosimetry, Quality Assurance, Quality Control, and 
Storage will be per usual protocols. 
 
“Sodium Fluoride F 18 Injection is provided as a ready-to-use, isotonic, sterile, pyrogen-free, 
clear and colorless solution. Each mL of the solution contains 10–400 mCi fluoride 18F at the end 
of synthesis (EOS) reference time in 0.9% aqueous sodium chloride. Fluoride 18F ions decay by 
positron emission with a half-life of 109.7 minutes. The drug product complies with the United 
States Pharmacopeia (USP) monograph for Sodium Fluoride F-18 Injection, and is manufactured 
following procedures that conform to the radiopharmaceuticals for PET compounding standards 
(USP <823>)” (Investigator’s Brochure. Edition 1, July 2008.  p. 10). Following the standard 
dose, patients will be injected with 5 mCi of NaF.   
 
For full details regarding Physical, Chemical, and Pharmaceutical Properties and Formulation; 
Nonclinical Studies; and Previous Human Experience, please refer to the commercial package 
insert for Sodium Fluoride F-18 injection. 
 
Imaging Procedures: 
 
To ensure uniformity, the established UW Nuclear Medicine image acquisition and reconstruction 
procedures for NaF PET/CT will be utilized. Please refer to the UW Nuclear Medicine Standard 
Operating Procedure for image acquisition and other image-related details. 

 
 
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Image Analysis: 
 
Image analysis will be performed in Nuclear Medicine and led by Dr. Perlman (co-investigator). 
Changes in number of lesions, SUVtotal/SUVmax/SUVmean will be calculated for individual 
lesions using established PERCIST criteria [81]. 
 
 
11. 
Statistical Considerations 
 
A. 
Overview 
 
This is a pilot trial to evaluate the immunological and clinical effect of combining a DNA vaccine 
encoding PAP (and using a rhGM-CSF adjuvant) with a monoclonal antibody (pembrolizumab) 
specific for PD-1.  The rationale for conducting this trial is summarized in Section 2 (E). 
 
B. 
Objectives 
 
The primary objectives are to: 
 
1. To evaluate the safety of pembrolizumab in combination with pTVG-HP in patients with 
castration-resistant, metastatic prostate cancer 
2. To determine the 6-month progression-free survival and median time to radiographic 
progression in patients with castration-resistant metastatic prostate cancer treated with 
pembrolizumab in combination with pTVG-HP 
3. To evaluate the anti-tumor response rates (objective response rate and PSA response rate, 
using PCWG2 criteria) in patients with castration-resistant metastatic prostate cancer 
treated with pembrolizumab in combination with pTVG-HP 
 
The secondary objectives are to: 
 
1. To determine whether either treatment sequence, or PAP-specific immune response, is 
associated with prolonged (6-month) radiographic progression-free survival 
2. To evaluate effects of schedule (concurrent versus delayed administration of 
pembrolizumab) on the magnitude of PAP-specific T-cell responses, PD-1 expression on 
circulating T cells, and PD-L1 expression on circulating epithelial cells (CEC) and on 
tumor biopsies 
3. To determine the median time to radiographic progression using concurrent administration 
schedules 
 
The exploratory objectives are to: 
 
1. To evaluate effects of treatment on the number of circulating tumor cells  
2. To evaluate PAP-specific antibody responses following treatment with pembrolizumab and 
pTVG-HP DNA vaccine 
3. To determine whether treatment with either sequence elicits immunologic antigen spread 
to other prostate associated antigens 

 
 
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4. To determine whether pre-existing or vaccine-induced PD-L1 expression on CEC or tumor 
biopsies is predictive of objective clinical response 
5. To determine whether treatment elicits expression of other regulatory molecules on tumor-
specific T cells (e.g. TIM3, BTLA, and LAG3) or tumor cells (e.g. HVEM, phosphatidyl 
serine, PD-L2)   
6. To determine whether PD-1-regulated antigen-specific T-cells identified by trans vivo 
DTH testing can identify patients who develop objective clinical responses with PD-1 
blockade therapy in combination with pTVG-HP. 
7. To determine whether changes in lymph nodes and soft tissue lesions are observed by FLT 
PET/CT after treatment with vaccine with or without pembrolizumab 
8. To determine if PD-1 inhibitor therapy in combination with pTVG-HP will change number 
and activity (SUV) in osteoblastic metastases as measured by NaF PET/CT. 
 
C. 
Study Design 
 
The study is designed as a randomized, two-arm, open-label, single institution pilot trial.  Study 
arms will be defined as follows: 
 
Arm 1: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.) 
biweekly 6 times beginning at day 1 pembrolizumab 2 mg/kg, administered 
intravenously every 3 weeks 4 times, beginning at day 1  
 
Arm 2: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.) 
biweekly 6 times beginning at day 1 pembrolizumab 2 mg/kg, administered 
intravenously biweekly 6 times, the first dose administered two weeks after the last 
pTVG-HP vaccination 
 
Eligible subjects will be randomly assigned in 1:1 fashion to either Arm 1 or Arm 2.  
 
During accrual to these 2 study arms, clinical observations suggested it could be advantageous to 
further investigate the combined treatment arm (Arm 2), but over a longer period of time.  
Specifically, objective responses and PSA declines were observed during the 3 months of 
treatment, but progression occurred after discontinuing therapy.  Hence a third, non-randomized 
arm was proposed after 12 evaluable subjects were accrued to Arms 1 and 2 to further investigate 
concurrent treatment, and specifically timed with vaccine administration.  
  
Extended treatment Arm 3: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.)  
 
every 3 weeks, for a maximum of 16 doses 
Pembrolizumab 2 mg/kg, with a maximum dose of 200 mg, administered 
 
intravenously  every 3 weeks, for a maximum of 16 doses, beginning on 
 
day 1 after the first pTVG-HP vaccination  
 
During accrual to Arm 3, laboratory observations suggested it may be preferable to use vaccine at 
every 2-week intervals rather than every 3-week intervals.  In order to evaluate this schedule with 
concurrent pembrolizumab, but ideally reduce the potential toxicity from pembrolizumab by 

 
 
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decreasing the frequency of administration, a separate extended treatment arm (Arm 4) is proposed 
after completion of accrual to Arm 3.   
 
Extended treatment Arm 4: 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.)  
 
every 2 weeks, for a maximum of 24 doses 
Pembrolizumab 2 mg/kg, with a maximum dose of 200 mg, administered 
 
intravenously  every 4 weeks, for a maximum of 12 doses, beginning on 
 
day 1 after the first pTVG-HP vaccination,  
 
D. 
Randomization and Stratification 
 
Eligible patients will be randomized to the first two study arms with an allocation ratio of 1:1. The 
randomization will be based on permutated blocks of size 2-4.  Treatment arm assignment will be 
made by biostatics/investigational pharmacy after a patient has been registered.   
 
E. 
Sample Size and Power Calculation 
 
The expected 6-months progression-free survival rate in Arm 2 (pembrolizumab is administered 
sequentially after pTVG-HP) is 25%.  It is hypothesized that the treatment of pTVG-HP, when 
administered concurrently with pembrolizumab (Arm 1), will result in a substantial increase in the 
6-month progression-free survival rate. Specifically, it is anticipated that the 6-month progression-
free survival rate in Arm 1 will be at least 65-75%. The following table shows the required sample 
sizes for detecting various differences in the 6-month progression-free survival rates between the 
two study arms, assuming 80% power and a one-sided 0.10 significance level. Since this is a pilot 
study, a less stringent significance level of 0.10 rather the traditional 0.05 level will be used for 
the primary efficacy evaluation. 
 
 
 
6-month progression free survival rate Arm 1 
6-month progression free  
survival  rate Arm 2 
0.65 
0.70 
0.75 
0.80 
0.20 
15 
11 
10 
9 
0.25 
17 
15 
11 
10 
  
 
A sample size of 15 patients per arm is proposed. This sample size will provide 80% power to 
detect an anticipated absolute difference of at least 45% (e.g., 25% vs. 70% or 20% vs. 65%) in 
the 6-months progression free survival rates between the two arms. These calculations are based 
on Fisher’s exact test.  Furthermore, the proposed sample size of 15 patients per arm is also 
sufficient to provide accuracy in estimating toxicity rates.  Specifically, the toxicity rates (i.e., of 
grade ≥ 2, grade ≥ 3, grade 4 toxicities) for each arm will be estimated with a standard error of less 
than 15% and the 95% confidence interval of the toxicity rates will be no wider than 45%. The 
probability of observing at least one event of a serious adverse event within a study arm is >95% 
if the adverse event rate is 20%. Analogously, the probability of observing at least one event of 
serious adverse event is at least 79% if the adverse event rate is only 10%.  
 

 
 
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In order to account for unevaluable (for efficacy evaluation) patients, 32 eligible patients (16 per 
arm) will be accrued and randomized.  Based on past experience with this patient population 
treated at our institution, we anticipate that the proposed total number of 32 patients will be accrued 
within 18 months.   
 
 
A total sample size of 20 evaluable patients is proposed for the extended treatment Arm 3 to 
evaluate the median time to radiographic progression. The proposed concurrent administration 
schedule will be considered as ineffective if it results in a median time to radiographic progression 
of 3 months or less. Hence, the null hypothesis that the median time to radiographic progression 
is at most 3 months will be tested against the alternative hypothesis that the median is greater than 
3 months. A median time to radiographic progression of 6 months or more, on the other hand, will 
be considered as promising to warrant further clinical evaluation of the proposed concurrent 
administration schedule. With the proposed sample size of 20 evaluable patients for the extended 
treatment Arm 3, a median time to radiographic progression of 6 months will be detected with 
90% power at the one-sided 0.05 significance level, assuming an accrual period of 12 months with 
a uniform accrual pattern and a minimum follow-up period of 12 months.  Furthermore, a total 
sample size of 20 evaluable patients is also proposed for the extended treatment Arm 4, to evaluate 
the median time to radiographic progression for this study arm.  The null hypothesis that the 
median time to radiographic progression is at most 3 months will be tested against the alternative 
hypothesis that the median is greater than 3 months. With the proposed sample size of 20 evaluable 
patients for the extended treatment Arm 4, a median time to radiographic progression of 6 months 
will be detected with 90% power at the one-sided 0.05 significance level, assuming an accrual 
period of 12 months with a uniform accrual pattern and a minimum follow-up period of 12 months. 
Furthermore, the proposed sample size of 20 patients for both Arm 3 and Arm 4 is adequate to 
estimate toxicity rates within each arm with sufficient accuracy. Specifically, toxicity rates will be 
estimated with a standard error of less than 12% and the corresponding two-sided 95% confidence 
intervals will be no wider than 40%.  
 
NaF PET/CT will be obtained in 6 - 10 patients with known osteoblastic metastases at baseline, 
week 6 and week 12. This sample size will provide at least 80% power to detect moderate to large 
effect sizes ranging from 1.0-2.0 for the differences in SUV metrics between time points at the 
two-sided 0.05 significance level.   
 
 
 
F. 
Analysis 
 
F.1. 
General 
 
This section outlines the statistical analysis strategy and procedures for the study.  Prior to the 
analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will be written describing 
all analyses that will be performed. 
Summary tabulations will be presented that will display the number of observations, mean, 
standard deviation, median, range, minimum, and maximum for continuous variables and the 
number and percent per category for ordinal and categorical data.  All endpoints of this study will 

 
 
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be presented graphically where possible using boxplots and histograms for continuous variables 
and (stacked) bar charts for categorical variables.  Data analysis will be performed using SAS® 
(SAS Institute Inc., Cary, North Carolina) version 9.3 or greater.  
 
There are many comparisons made between the two trial arms for multiple measurements, both 
clinical and laboratory.  However, since this a pilot study, no adjustment will be made for multiple 
comparisons.  A two-sided test at a significance level of 0.05 will be used for all comparisons, 
unless noted otherwise.  
 
F.2. 
Analysis Populations 
 
The ITT population consists of all patients who are randomized.  The per-protocol population 
consists of all evaluable patients, i.e., patients who have been treated according to protocol (all 
inclusion/exclusion criteria are satisfied, and adequate treatment compliance (complete treatment 
to at least day 43). The safety population consists of all subjects who received at least one dose of 
pTVG HP alone or in combination with pembrolizumab. All primary, secondary and correlative 
outcomes will be analyzed for both the IIT and per-protocol population. The IIT population will 
be the primary analysis population. Safety will be evaluated using the safety population.   
 
F.3. 
Baseline Comparability 
 
All measurements (variables) collected at baseline will be summarized and compared between 
study arms.  These include demographic variables, ECOG PS, lab parameters (CBC, chemistry 
panel, serum testosterone, PSA and PAP) medical history information and CTCs.  Comparisons 
between study arms of baseline variables on a continuous scale will be performed using a two-
sample t-test and/or nonparametric Wilcoxon Rank Sum test.  Comparisons between study arms 
of baseline variables on a categorical scale will be performed using Fisher’s exact test.  
 
F.4. 
Analysis of Primary Endpoints 
 
F.4.1. Analysis of Toxicity Rates and Serum Chemistry Parameters 
 
Subjects will be evaluated at each visit by a review of systems based on the most recent version of 
the NCI common toxicity criteria.  Furthermore, serum chemistries, including renal function tests, 
blood counts, liver function tests, and serum amylase, will be evaluated at 6-12 week intervals. 
Toxicities will be summarized by type and severity in tabular format. Toxicity rates (grade 2, grade 
3, grade 4, grade ≥ 2, grade ≥ 3, etc.) will be calculated for each study arm and reported along the 
corresponding 95% confidence intervals. The 95% confidence intervals will be constructed using 
the Wilson score method. Fisher’s exact test will be used to compare toxicity rates between study 
arms. Serum chemistry and amylase parameters will be summarized using standard descriptive 
statistics. Changes from the baseline assessment will be evaluated using a paired t-test. 
 
F.4.2. 6-Month Progression Free Survival Rate and Median Time to Radiographic 
Progression 
 
The 6-month progression-free survival rate, along with the corresponding two-sided 95% 
confidence intervals, will be reported for each arm, and for the overall combined study. The Wilson 

 
 
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score method will be used to construct the confidence intervals. Fisher’s exact test will be used to 
compare the 6-month progression-free survival rates between study arms.  
 
Median time to radiographic progression will be estimated for each arm using the Kaplan-Meier 
method. The median time to radiographic progression will be reported for each of the four arms 
along with the corresponding 95% confidence intervals which will be constructed using the 
Brookmeyer-Crowley method. The log-rank test will be used to perform the comparison of time 
to radiographic progression between Arm 1 versus Arm 2. A one-sided 0.10 significance level will 
be used to conduct the comparison of the 6-month progression-free survival rate and time to 
radiographic progression between study arms.  Within Arm 3 and Arm 4, the null hypothesis that 
the median time to radiographic progression is at most 3 months will be tested against the 
alternative hypothesis that the median time to radiographic progression is greater than 3 months.  
 
In order to evaluate the 6-month progression-free rate and median time to radiographic progression 
as a function of baseline time point (pretreatment or 3-months post treatment, the analysis will be 
conducted using two different baseline values: (1) Date of randomization, and (2) 3-months disease 
assessment.  
 
F.4.3. Objective Response Rate and PSA Response Rate 
 
The objective response rate and PSA response rate will be calculated for each study arm and for 
both arms combined along with the corresponding 95% confidence intervals. The Wilson score 
method will be used to construct the confidence intervals. Fisher’s exact test will be used to 
compare the objective response and PSA response rates between study arms. Duration of response 
will be analyzed using the Kaplan-Meier method.  
 
F.5. 
Analysis of Secondary Endpoints 
 
F.5.1. PAP-Specific Immune Response 
 
The number and frequencies of PAP-specific immune responses will be summarized in tabular 
format for each study arm and both study arms combined. A log-linear model will be used to 
evaluate whether PAP-specific immune response predicts the 6-months progression-free survival 
rate. The interaction between treatment (pembrolizumab administered sequentially versus delayed) 
and PAP-specific immune response will be included in the model. Analogously, a Cox 
proportional hazard regression model will be used to examine the association between PAP-
specific immune response and the time to radiographic progression.  
 
F.5.2. PAP-Specific T-Cell Response, PD-1 Expression, PD-L1 Expression 
 
PD-1 and PD-L1 expression levels will be summarized in terms of means, standard deviations and 
ranges for each study arm separately and for both arms combined. The number and frequencies of 
PAP-specific T-cell responses will be summarized in tabular format. Fisher’s exact test will be 
used to compare the PAP-specific T-cell response rates between study arms. A linear regression 
model or a negative binomial regression model will be utilized to evaluate the effects of schedule 
(concurrent versus delayed administration of pembrolizumab) on PD-1 expression on the number 
of circulating T cells, and PD-L1 expression on the number of circulating epithelial cells. The 

 
 
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choice of the model will be dependent on the distribution of the outcome variables (number of 
circulating T-cells and number of circulating epithelial cells).  
 
F.6. 
Exploratory Analyses 
 
F.6.1. Evaluation of Treatment Effects on the Number of Circulating Tumor Cells 
 
The number of circulating tumor cells will be summarized in terms of means and standard 
deviations. Within each arm, and for both arms combined, changes in the number of circulating 
tumor cells from the pre-treatment (date of screening/randomization) to the post-treatment 
assessments (day 43, day 85, day 169, month 9, and off-study) assessments will be evaluated using 
a paired t-test. A two-sample t-test will be used to compare the number of circulating tumor cells 
at each time point, and the changes in the number of circulating tumor cells from the pre-treatment 
assessment, between study arms.  In order to evaluate longitudinal changes in the number of 
circulating tumor cells, linear mixed effects modeling with subject specific random effects will be 
conducted.  
 
F.6.2. Evaluating of Treatment Effects on PAP-specific Antibody Responses 
 
The number and frequency of PAP-specific antibody responses will be summarized in tabular 
format, stratified by study arm and for both arms combined. A generalized linear mixed effects 
model with subject specific random effects will be used to evaluate the treatment effect of  the 
proposed pTVG-HP DNA vaccine and pembrolizumab (sequentially versus concurrently) 
combination on  PAP-specific antibody response. 
 
F.6.3. Evaluation whether Treatment with either Sequence Elicits Immunologic 
Antigen Spread to other Prostate Associated Antigens 
 
The detection of antigen spread to other prostate associated antigens, and the identification of 
specific antigens recognized, will be analyzed descriptively.  Furthermore, linear regression 
analysis will be conducted to evaluate whether treatment with either sequence (pembrolizumab 
administered sequentially or concurrently) elicits immunologic antigen spread to other prostate 
associated antigens. 
 
F.6.4. Evaluation of Pre-Existing or Vaccine Induced PD-L1 Expression on CEC 
or Tumor Biopsies as a Predictor for Clinical Response 
 
Logistic regression analysis will be conducted to examine whether pre-existing or vaccine induced 
PD-L1 expression on CEC or tumor biopsies predict clinical response. This analysis will be 
conducted for both arms combined. The results will be summarized in terms of odds ratios along 
with the corresponding 95% confidence intervals.  
 
F.6.5. Evaluation whether Treatment with either Sequence Elicits other 
Regulatory Molecules on Tumor-Specific T cells or Tumor Cells 
 
Linear regression analysis will be conducted to determine whether treatment with either sequence 
elicits other regulatory molecules on tumor-specific T cells or tumor cells. 

 
 
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F.6.6. Evaluation whether PD-1-Regulated Antigen-specific T-cells Identified by 
trans vivo DTH Testing Can Identify Patients who Develop Objective 
Clinical Responses with PD-1 Blockade Therapy in Combination with 
pTVG-HP 
 
The number and frequencies of PD-1-regulated antigen specific T-cell responses will be 
summarized in tabular format.  Logistic regression analysis will be conducted to examine whether 
PD-1 regulated antigen-specific T-cells predict clinical response. The sensitivity, specificity, 
negative predictive value and positive predictive value of PD-1 regulated antigen specific T-cell 
responses for predicting clinical response will be calculated and reported along with the 
corresponding 95% confidence intervals.  
 
F.6.7. Evaluation of changes in FLT uptake by FLT PET/CT after Treatment with 
pTVG-HP with or without Pembrolizumab 
 
Standard PET parameters, including SUVtotal, SUVmax, SUVmean, and the number of lesions, in 
terms of soft tissue lesions and major lymph node groups, will be obtained.  Parameters will be 
summarized in terms of means, standard deviations and ranges. Within each arm and for both arms 
combined, changes in parameters will be evaluated using a paired t-test (for SUV) or 
nonparametric Wilcoxon signed rank test (number of lesions). Since the distribution of SUV values 
is generally skewed, a log-transformation will be utilized before conducting the comparisons. A 
two-sample t-test or nonparametric Wilcoxon rank sum test will be used to compare changes in 
parameters between study arms.  
 
F.6.8 Evaluation of changes in NaF uptake by NaF PET/CT after treatment with 
pTVG-HP with or without Pembrolizumab 
 
Standard PET parameters, including SUVtotal, SUVmax, SUVmean, and the number of lesions, in 
terms of osteoblastic lesions, will be obtained using established PERCIST criteria.  To ensure 
uniformity, the established UW Nuclear Medicine image acquisition and reconstruction 
procedures for NaF PET/CT will be utilized. Please refer to the UW Nuclear Medicine Standard 
Operating Procedure for image acquisition and other image-related details. Image analysis will be 
performed in Nuclear Medicine and led by Dr. Perlman (co-investigator).  
 
 
12. 
Administrative Considerations 
 
A. 
Specimen Handling 
 
Upon entry into the study, each subject will be assigned a unique identification number.  
All materials collected on that subject will be labeled with that number only, for reasons 
of confidentiality, and specimens may be listed with unique codes for individual subjects 
and obtained at the different time points.  Lymphocytes collected will be stored in liquid 
nitrogen, and sera will be aliquoted and stored at –80°C in the research laboratory of the 
protocol sponsor (Dr. McNeel) for immune analyses.  
 

 
 
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B. 
Institutional Review Board 
 
In accordance with federal regulations (21 CFR 312.66), an Institutional Review Board (IRB) that 
complies with the regulations in 21 CFR 56 must review and approve this protocol and the 
informed consent form prior to the initiation of the study at each investigator site.  In addition, the 
study cannot be instituted without FDA approval of the vaccine formulations.  Finally, the trial 
will be conducted with adherence to this protocol, following good clinical practice (GCP) 
guidelines, and in compliance with other applicable regulatory requirements.  Any modifications 
to the protocol must follow the procedure as outlined in Section 16 below. 
 
C. 
Consent 
 
The Principal Investigator, co-investigators, or their associates must explain verbally and in 
writing the nature, duration, and purpose of the study and possible consequences of the treatment.  
Patients must also be informed that they may withdraw from the study at any time and for any 
reason without jeopardizing their future treatment.  In accordance with Federal regulations (21 
CFR 312), all patients must sign the IRB-approved consent form.   
 
 
13. 
Data and Safety Monitoring Plan 
 
A. 
Definitions 
 
An adverse event is defined as any unfavorable and unintended sign (including abnormal 
laboratory finding), symptom or disease temporally associated with a medical treatment or 
procedure, regardless of whether it is considered related to the treatment or procedure.  Adverse 
events are categorized as definite, probable, possible, unlikely or unrelated in relation to the 
medical treatment or procedure performed.  A life-threatening event is defined as any adverse 
event that places the subject, in the view of the investigator, at immediate risk of death from the 
reaction.  Serious adverse events are any events occurring that result in any of the following 
outcomes: 
 
1. Subject death 
2. Life-threatening adverse event 
3. Inpatient hospitalization or prolongation of existing hospitalization 
4. Persistent or significant disability/incapacity 
5. Congenital anomaly or birth defect 
 
Adverse events are classified by organ system and graded by severity according to the current NIH 
Common Terminology Criteria, as described in Appendix A.  The defined grades use the following 
general guidelines: 
 
 
0 
No adverse event or within normal limits 
 
1 
Mild adverse event 
 
2 
Moderate adverse event 
 
3 
Severe adverse event 

 
 
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4 
Life-threatening or disabling adverse event 
 
5 
Fatal adverse event 
 
All adverse events will be recorded from time of informed consent through 30 days after the final 
dose of study drug, as well as any time after these 30 days for events that are believed to be at least 
possibly related to study treatment. 
 
Appropriate clinical, diagnostic, and laboratory measures to attempt to delineate the cause of the 
adverse reaction in question must be performed and the results reported.  All tests that reveal an 
abnormality considered to be drug- or vaccine-related will be repeated at appropriate intervals until 
the course is determined or a return to normal (or pre-treatment) values occurs.  Information will 
be recorded as noted above.   
 
 
B. 
Oversight and Monitoring Plan 
The UWCCC Data and Safety Monitoring Committee (DSMC) is responsible for the regular 
review and monitoring of all ongoing clinical research in the UWCCC.  A summary of DSMC 
activities are as follows: 
• 
Reviews all clinical trials conducted at the UWCCC for subject safety, protocol 
compliance, and data integrity. 
• 
Reviews all Serious Adverse Events (SAE) requiring expedited reporting, as defined 
in the protocol, for all clinical trials conducted at the UWCCC, and studies conducted 
at external sites for which UWCCC acts as an oversight body. 
• 
Reviews all reports generated through the UWCCC DSMS elements (Internal Audits, 
Quality Assurance Reviews, Response Reviews, Compliance Reviews, and Protocol 
Summary Reports) described in Section II of this document. 
• 
Notifies the Local Principal Investigator of DSMC decisions and, if applicable, any 
requirements for corrective action related to data or safety issues. 
• 
Notifies the CRC of DSMC decisions and any correspondence from the DSMC to the 
Local Principal Investigator. 
• 
Works in conjunction with the UW Health Sciences IRB in the review of relevant 
safety information as well as protocol deviations, non-compliance, and unanticipated 
problems reported by the UWCCC research staff.  
• 
Ensures that notification is of SAEs requiring expedited reporting is provided to 
external sites participating in multi-institutional clinical trials coordinated by the 
UWCCC. 
 
 
C. 
Monitoring and Reporting Guidelines 
Phase I/II and Phase II Trials 
 

 
 
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UWCCC quality assurance and monitoring activities are determined by study sponsorship and risk 
level of the protocol as determined by the UWCCC protocol review and monitoring committee 
(PRMC).  All protocols (including Intervention Trials, Non-Intervention Trials, Behavioral and 
Nutritional Studies, and trials conducted under a Training Grant) are evaluated by the PRMC at 
the time of committee review.  UWCCC monitoring requirements for trials without an acceptable 
external DSMB are as follows: 
a) Intensive Monitoring 
Protocols subject to intensive monitoring generally include UW Institutional Phase I 
and Institutional Trials of any phase involving recombinant DNA/gene transfer.  These 
protocols undergo continuous review of data and subject safety at weekly Phase 
I/Disease Oriented Working Group (DOWG) meetings where the results of each 
subject’s treatment are discussed and the discussion is documented in the DOWG 
meeting minutes. The discussion includes the number of subjects enrolled, significant 
toxicities, dose adjustments, and responses observed. Protocol Summary Reports are 
submitted on a quarterly basis by the study team for review by the DSMC. 
 
 
D. 
Review and Oversight Requirements 
 
Serious Adverse Event – Reported Within 24 Hours  
Serious Adverse Events requiring reporting within 24 hours (as described Table D-2) must also be 
reported to the Data and Safety Monitoring Committee (DSMC) Chair via an email to 
saenotify@uwcarbone.wisc.edu within one business day.  The OnCore SAE Details Report must 
be submitted along with other report materials as appropriate (NCI AdEERS form or FDA 
Medwatch Form #3500 and/or any other documentation available at that time of initial reporting).  
The DSMC Chair will review the information and determine if immediate action is required.  
Within 10 working days, all available subsequent SAE documentation must be submitted 
electronically 
along 
with 
a 
24 
hour 
follow-up 
SAE 
Details 
Report 
to 
saenotify@uwcarbone.wisc.edu. All information is entered and tracked in the UWCCC database. 
The Protocol Principal Investigator notifies all investigators involved with the study at the 
UWCCC, the IRB, the sponsor, and the funding agency and provides documentation of these 
notifications to the DSMC. 
If the SAE occurs on a clinical trial in which the UW PI serves as the sponsor-investigator, the PI 
reviews the event to determine whether the SAE requires reporting to the FDA and other 
participating investigators.   
For a multiple-institutional clinical trial the Protocol Principal Investigator (Investigator sponsor) 
is responsible for ensuring SAEs are reported to the FDA as well as to all participating investigators 
See Section E.1 for detailed instructions on SAE reporting. 
 
Serious Adverse Event  – Reported within 10 Days 
Serious Adverse Events requiring reporting within 10 days (as described in the protocol) must also 
be reported to the Data and Safety Monitoring Committee (DSMC) Chair via an email to 
saenotify@uwcarbone.wisc.edu.  The OnCore SAE Details Report must be submitted along with 

 
 
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other report materials as appropriate (NCI AdEERS form or FDA Medwatch Form #3500 and/or 
any other documentation available at that time of initial reporting). The DSMC Chair will review 
the information and determine if further action is required. All information is entered and tracked 
in the UWCCC database. 
The Protocol Principal Investigator notifies all investigators involved with the study at the 
UWCCC, the IRB, the sponsor, and the funding agency and provides documentation of these 
notifications to the DSMC. 
If the SAE occurs on a clinical trial in which the UW PI serves as the sponsor-investigator, the PI 
reviews the event to determine whether the SAE requires reporting to the FDA and other 
participating investigators.   
For a multiple-institutional clinical trial the Protocol PI (Investigator sponsor) is responsible for 
ensuring SAEs are reported to the FDA as well as to all participating investigators. 
 
See Section E.2 for detailed instructions on SAE reporting. 
 
Sponsor-Investigator Responsibilities for SAE Review 
 
In the event the UWCCC Principal Investigator is acting as the Sponsor-Investigator (i.e., the PI 
holds the IND), the PI assumes responsibilities of the study sponsor in accordance with FDA 21 
CFR 312.32.  In this capacity, the UWCCC PI reviews all reports of serious adverse events 
occurring on the study at the UWCCC and participating external sites and makes a determination 
of 1) suspectedness (i.e., whether there is a reasonable possibility that the drug caused the AE); 
and 2) unexpectedness (the event is not listed in the Investigator’s Brochure or is not listed at the 
specificity or severity that has been observed) in the context of this study.  SAE with suspected 
causality to study drug and deemed unexpected are reported as IND Safety Reports by the UWCCC 
PI to the FDA, all participating investigators on the study, and the external global sponsor (if 
applicable) within 15 calendar days. All fatal or life-threatening SAE that are unexpected and have 
suspected causality to the study drug will be reported by the UWCCC PI to the FDA, all 
participating investigators on the study, and the external global sponsor (if applicable) within 7 
calendar days. 
 
 
Study Progress Review 
Protocol Summary Reports (PSR) are required to be submitted to the DSMC in the timeframe 
determined by the risk level of the study (quarterly; semi-annually; or annually). The PSR provides 
a cumulative report of SAEs, as well as instances of non-compliance, protocol deviations, and 
unanticipated problems, toxicities and responses that have occurred on the protocol in the 
timeframe specified.  PSRs for those protocols scheduled for review are reviewed at each DSMC 
meeting. Cumulative reports will also be due annually to FDA (pertaining to this IND), and to the 
NIH OBA (pertaining to this OBA reference number). 
 
Protocol Summary Reports enable DSMC committee members to assess whether significant 
benefits or risks are occurring that would warrant study suspension or closure. This information is 
evaluated by the DSMC in conjunction with other reports of quality assurance activities (e.g., 
reports from Internal Audits, Quality Assurance Reviews, etc.) occurring since the prior review of 
the protocol by the DSMC.  Additionally, the DSMC requires the study team to submit external 

 
 
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DSMB or DSMC reports, external monitoring findings for industry-sponsored studies, and any 
other pertinent study-related information.  
 
In the event that there is significant risk warranting study suspension or closure, the DSMC will 
notify the Local PI of the DSMC findings and ensure the appropriate action is taken for the protocol 
(e.g., suspension or closure).  The DSMC ensures that the PI reports any temporary or permanent 
suspension of a clinical trial to the sponsor (e.g., NCI Program Director, Industry Sponsor Medical 
Monitor, Cooperative Group Study Chair, etc.) and other appropriate agencies. DSMC findings 
and requirements for follow-up action are submitted to the CRC.   
 
E. 
Expedited Reporting of Adverse Events 
 
Depending on the nature, severity, and attribution of the serious adverse event an SAE report will 
be phoned in, submitted in writing, or both according to Table D-2 below.  All serious adverse 
events must also be reported to the UWCCC Data and Safety Monitoring Committee Chair.  All 
serious adverse events must also be reported to the UW IRB (if applicable), and any 
sponsor/funding agency not already included in the list. 
 
Determine the reporting time line for the SAE in question by using the following table D-2.   
 
E.1. 
SAE Requiring 24 Hour Reporting Occurs at UWCCC: 
 
a.    
To the FDA and NIH OBA: 
 
Report the SAE to the FDA using the current FDA Med Watch form (cuttently 
available 
at      
http://www.fda.gov/downloads/Safety/MedWatch/HowToReport/DownloadForm
s/UCM082728.pdf) or another comparable form such as the National Institute of 
Health Office of Biotechnology Activities form listed below.     
        
 
Print the completed Med Watch form and fax it to FDA at (800) 332-0178.     
 
Report the SAE to the National Institute of Health Office of Biotechnology 
Activities 
using 
their 
current 
reporting 
form 
(currently 
located 
at 
http://oba.od.nih.gov/oba/rac/Adverse_Event_Template.pdf).   Completed reports 
may be sent either e-mail or facsimile to the addresses listed on the form. 
        
 
 
b.   
To the Industry Supporter: 
 
All SAEs that occur from the signing of the study specific consent through the 
duration of the post-therapy adverse event collection period (30 days after the last 
treatment administration) must be reported to Madison Vaccines Inc. within 24 
hours of being made aware of the SAE. Notification can be made via phone or 
telefacsimile a copy of the FDA Med Watch form to: 
  
Madison Vaccines Inc.  
Attn:  
Safety Manager 
Phone:  
(608) 467-5269 

 
 
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Fax:  
To be provided  
  
Significant new information regarding an ongoing SAE and the resolution must be 
sent to Madison Vaccines Inc. within 3 business days of awareness of the new 
information via the same reporting mechanism.  
 
c.   
To the IRB: 
 
       
 
Consult the UW-IRB website for reporting guidelines.   
      
d. 
To the UWCCC: 
 
Reference the SAE SOP (Standard Operating Procedure) and the SAE Reporting 
Workflow for DOWGs on the UWCCC website (http://www.uwccc.wisc.edu) for 
specific instructions on how and what to report to the UWCCC for 24-hour initial 
and follow-up reports.  A follow-up report is required to be submitted within 10 
days of the initial 24-hour report.   
 
For this protocol, the following entities are required to be notified: 
 
 
1. 
saenotify@uwcarbone.wisc.edu 
2.  
Any other appropriate parties listed on the SAE Routing Form (for follow-
up reports only) 
3. 
UW Institutional Biosafety Officer (via First Report form  
(http://www2.fpm.wisc.edu/biosafety/emergency_prep.htm)) within one 
working day of the event 
 
E.2. 
SAE Requiring 10-Day Reporting Occurs at UWCCC:  
 
a.    
To the FDA and NIH OBA  
        
Report the SAE using the current FDA Med Watch form (currently available at      
http://www.fda.gov/downloads/Safety/MedWatch/HowToReport/DownloadForm
s/UCM082728.pdf) or another comparable form such as the National Institute of 
Health Office of Biotechnology Activities reporting form listed below. 
        
 
 
Print the completed Med Watch form and fax it to (800) 332-0178.      
        
 
 
Report the SAE to the National Institute of Health Office of Biotechnology 
Activities 
using 
their 
current 
reporting 
form 
(currently 
located 
at 
http://oba.od.nih.gov/oba/rac/Adverse_Event_Template.pdf).  Completed reports 
may be sent either e-mail or facsimile to the addresses listed on the form. 
        
 
 
b.   
To the Industry Supporter: 
All SAEs that occur from the signing of the study specific consent through the 
duration of the post-therapy adverse event collection period (30 days after the last 
treatment administration) must be reported to Madison Vaccines Inc. within 24 

 
 
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hours of being made aware of the SAE. Notification can be made via phone or 
telefacsimile a copy of the FDA Med Watch form to: 
  
Madison Vaccines Inc.  
Attn:  
Safety Manager 
Phone:  
(608) 467-5269 
Fax:  
To be provided  
  
Significant new information regarding an ongoing SAE and the resolution must be 
sent to Madison Vaccines Inc. within 3 business days of awareness of the new 
information via the same reporting mechanism. 
 
c.   
To the IRB: 
 
       
 
Consult the UW-IRB website for reporting guidelines.   
 
d.  
To the UWCCC: 
 
Reference the SAE SOP and the SAE Reporting Workflow for DOWGs on the 
UWCCC website (http://www.uwccc.wisc.edu) for specific instructions on how 
and what to report to the UWCCC for 10-day reports.   
 
For this protocol, the following entities are required to be notified: 
1. 
saenotify@uwcarbone.wisc.edu 
2. 
Any appropriate parties listed on SAE Routing Form 
3.  
UW Institutional Biosafety Officer (via First Report form  
(http://www2.fpm.wisc.edu/biosafety/emergency_prep.htm) within one 
working day of the event 
 
Expedited Reporting Table D-2 
FDA Reporting Requirements for Serious Adverse Events (21 CRF Part 312) 
NOTE: Investigators MUST immediately report to the PI  and any other parties outlined in the protocol ANY 
Serious Adverse Events, whether or not they are considered related to the investigational 
agent(s)/intervention (21 CFR 312.64). 
An adverse event is considered serious if it results in ANY of the following outcomes: 
1) 
Death. 
2) 
A life-threatening adverse event. 
3) 
An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization 
for ≥ 24 hours. 
4) 
A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
5) 
A congenital anomaly/birth defect. 
6) 
Important Medical Events (IME) that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they may 
jeopardize the patient or subject and may require medical or surgical intervention to prevent one of 
the outcomes listed in this definition (FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
ALL SERIOUS adverse events that meet the above criteria* MUST be immediately reported to the UWCCC 
within the timeframes detailed in the table below: 

 
 
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Hospitalization 
Grade 1 and Grade 2 Timeframes 
Grade 3-5 
Timeframes 
Resulting 
in 
hospitalization  ≥ 
24 hrs 
10 Calendar Days 
24 Hour; 5 Calendar Days 
Not resulting in 
Hospitalization ≥ 
24 hrs 
Not required 
Expedited AE reporting timelines are defined as: 
• 
24-Hour; 5 Calendar Days – The AE must initially be reported within 24 hours of learning of the 
AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour report. 
• 
10 Calendar Days – A complete expedited report on the AE must be submitted within 10 calendar 
days of learning of the AE 
1 Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention (or 90 days for pembrolizumab) and have an attribution of possible, probable, or 
definite require reporting as follows: 
 
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
• 
All Grade 3, 4, and 5 AEs 
Expedited 10 calendar day reports for: 
• 
Grade 2 AEs resulting in hospitalization or prolongation of hospitalization 
2 For studies using PET or SPECT IND agents, the AE reporting period is limited to 10 radioactive half-
lives, rounded UP to the nearest whole day, after the agent/intervention was last administered.  Footnote 
“1” above applies after this reporting period. 
 
 
 
14. 
Potential Risks and Benefits, and Procedures to Minimize Risk 
 
A. 
Potential Risks 
 
A.1. 
From immunization with DNA plasmid encoding PAP 
 
Two potential toxicities might be predicted to occur from DNA-based vaccines.  The first would 
be immediate toxicity due to the vaccination itself, and a second would be due to immunological 
consequences of the vaccination targeting other unrelated tissues.  An intradermal route of 
administration will be used, and is preferred given the presence of Langerhans’ antigen-presenting 
cells in the dermis.  Intradermal administrations, however, carry a risk of immediate allergic 
reactions.  For that reason, subjects will be remain for 60 minutes following each treatment to 
evaluate the skin site of treatment and for any immediate allergic events.  Subjects will also be 
asked to keep a record of unusual site or other reactions for two days after immunization.  To date, 
in studies using pTVG-HP only without anti-PD-1, allergic reactions have been rare (grade 3 
angioedema observed in 1 of over 50 treated subjects, <5% individuals). 
 
With respect to eliciting unwanted immunological reactions, PAP, a protein whose expression is 
essentially restricted to the prostate, does share homology with other tissue phosphatases, notably 
lysosomal acid phosphatase (LAP), most prevalent in pancreatic tissue.  No such toxicity has been 
reported in clinical trials targeting PAP by means of a dendritic cell vaccine, and the generation of 

 
 
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PAP-specific CTL in rat models similarly did not elicit detectable evidence of autoimmune disease 
in non-prostate tissues.  Moreover, this was not seen in previous clinical trials with this DNA 
vaccine with over 50 subjects.  This could theoretically be potentiated, however, with the anti-PD-
1 therapy.  Consequently, in order to further evaluate this potential toxicity in humans, subjects 
will be evaluated at each visit by a review of systems based on the NCI common toxicity criteria.  
In addition, subjects will be examined and serum chemistries, including renal function tests, blood 
counts, liver function tests, and serum amylase, will be evaluated at 6-12 week intervals, as 
outlined above.  The serum amylase will be used as a serum marker to monitor for evidence of 
subclinical pancreatic inflammation.  Again, while no autoimmune treatment-limiting adverse 
events have been observed to date in other trials, it is conceivable that by using a checkpoint 
inhibitor such as anti-PD-1 with an immunization approach targeting the PAP antigen that more 
autoimmune events could be observed than with either agent alone.  For this reason, these blood 
tests will be continued to be monitored in this trial.   
 
While there has been a suggestion that the plasmid DNA could insert into the host chromosomal 
DNA, this has not been documented in any other study, and several laboratory investigations have 
suggested the possibility of this occurring is less than the spontaneous mutation rate, and therefore 
not a real risk [79].  Even should this occur, there is little reason to suspect that this would put 
subjects at any increased risk, and in this patient population with treated prostate cancer, there is 
little risk of transfer to offspring.  There are no known risks to blood donation during or after 
immunization, however subjects will be requested to not donate blood from study entry until 
completion of all study procedures. 
 
The DNA itself is not hazardous (in that it encodes no viral proteins, is not radioactive, and is not 
itself a carcinogen) and does not pose additional risk to subjects or study personnel.  However, 
standard precautions to reduce the risk of needle sticks to study personnel will be performed. 
 
A.2. 
From intradermal treatment with rhGM-CSF 
 
GM-CSF, administered intradermally with the vaccine, is being used as a vaccine adjuvant, and 
has demonstrated safety from multiple other vaccine trials in humans.  Common side effects 
include erythema and induration at the site of immunization (lasting for several days), and a 
transient decrease in peripheral white blood cell counts (lasting for several hours), mild flu-like 
symptoms (lasting for several hours).  It has also been observed and reported that T-cell and 
antibody responses can occur to GM-CSF, but without any known clinical sequelae [63].  
Uncommon and rare side effects, which have been less frequently observed with daily 
administration of higher doses of GM-CSF, include: vomiting; diarrhea; fatigue; weakness; 
headache; decreased appetite; feeling of faintness; facial flushing; pain in the bones, muscles, 
chest, abdomen, or joints; blood clots or unusual bleeding symptoms; rapid or irregular heartbeat 
or other heart problems; kidney and liver dysfunction; fluid accumulation or worsening of pre-
existing fluid accumulation in arms and legs, in the lungs, and around the heart that may result in 
breathing problems and heart failure; allergic reactions; sloughing of skin; liver enlargement; 
Guillain-Barré syndrome; hypotension; loss of consciousness; dyspnea.  In a previous phase I trial 
with this DNA vaccine and GM-CSF, two patients experienced chest and back pain, attributed to 
the GM-CSF, which lasted less than 10 minutes and occurred within one hour of receiving GM-
CSF.  For this reason, all subjects will be monitored for one hour after receiving each DNA 
immunization treatment. 

 
 
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A.3. 
From treatment with pembrolizumab 
 
Pembrolizumab (Keytruda®, Merck) is a human programmed death receptor-1 (PD-1)-blocking 
antibody.  Given that PD-1 is expressed on T cells, and that ligation of PD-1 by one of its ligands 
causes inactivation of T-cell function, blocking PD-1 by means of an antibody such as 
pembroliziumab can release the function of T cells that may have reactivity to normal cells.  In 
fact, the goal of this therapy is to “unleash” T cells, and cytolytic CD8+ T cells in particular, that 
recognize tumor cells.  In this trial, the goal is to elicit a specific population of CD8+ T cells that 
recognize prostate tumors, and allow them to lyse prostate tumor by not permitting expression of 
the ligand PD-L1 on prostate tumors to inhibit their function.  But because PD-1 blockade is itself 
not specific, the release of T cells with specificity to other normal tissues can result, leading to 
autoimmune adverse events.  To date, while autoimmune adverse events have been relatively 
uncommon (occurring in <10% of patients with melanoma treated), multiple different immune 
adverse events have been observed.  These have included immune-mediated pneumonitis, colitis, 
hepatitis, hypophysitis, nephritis, hyperthyroidism and hypothyroidism.  Other clinically 
significant, immune-mediated adverse reactions have been observed in <1% of patients treated 
with pembrolizumab, including exfoliative dermatitis, uveitis, arthritis, myositis, pancreatitis, 
hemolytic anemia, partial seizures, adrenal insufficiency, myasthenic syndrome, optic neuritis, and 
rhabdomyolysis.  Most of these events resolved following discontinuation of pembrolizumab, or 
in some cases following treatment with systemic corticosteroids.  But because of these rare, but 
potentially serious immune adverse events, patients will be followed closely for unusual new 
symptoms.   
 
In addition to these immune-mediated adverse events, other common adverse reactions (reported 
in > 20% of patients) included fatigue, cough, nausea, pruritis, rash, decreased appetite, 
constipation, arthralgia, and diarrhea.   
 
A.4. 
From blood tests 
 
Drawing blood may cause temporary discomfort and bruising at the site of venipuncture.  Skin 
infections, while possible, are extremely rare as a result of blood draws 
 
A.5. 
From leukapheresis 
 
Adverse events from leukapheresis are uncommon.  Bruising at the site of venipuncture for venous 
access is possible, as is a rare risk of infection related to this venipuncture.  Anticoagulants (ACD) 
are used during cell collection, but these have not been associated with subsequent bleeding 
problems.  There is a rare risk for citrate toxicity as a result of the citrate anticoagulation used 
during cell collection.  This can cause hypocalcaemia and muscle spasms.  There is no associated 
increased risk of infection from removing white blood cells, however a mild anemia can result for 
1-2 days, and for this reason a hematocrit must be measured within 1 week of each leukapheresis; 
an hematocrit < 30% and/or platelet count <50,000 will be used as a contraindication for 
leukapheresis.   
 
A.6. 
From tetanus immunization 
 

 
 
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A tetanus booster vaccine (commercially available vaccine delivered as an intramuscular injection) 
will be delivered prior to receiving  the DNA vaccine as an immunological positive control for the 
laboratory analysis.  While this is a standard, well tolerated immunization, rare potential side 
effects of this vaccine could include: difficulty in breathing or swallowing; hives; swelling of the 
eyes, face or inside of the nose; confusion; convulsions; headaches; sleepiness; lymphadenopathy; 
and vomiting.  Other mild adverse effects not requiring medical attention include: chills; fever; 
mild irritability or tiredness; skin rash; or pain, tenderness, redness, itching or swelling at the site 
of injection. 
 
A.7. 
From tissue biopsies 
 
Standard laboratory tests will be done to assess bleeding risk before the procedure is done.  The 
sample will be taken from an area of reduced risk of complications.    Possible side effects of a 
biopsy include bleeding, infection, bruising, pain or discomfort at the biopsy site and possible side 
effects from the local anesthetic (pain or bruising at the site where anesthetic is given).  The main 
discomfort associated with this test is pain when the bone or tissue is being withdrawn. In order to 
make the procedure more comfortable, subjects will get a local anesthetic to numb the area. A mild 
sedative may also be given. While sedated, subjects will be able to respond to commands.  It is 
recommended that subjects be observed for 2-4 hours after the biopsy. 
 
CT scans for biopsies:  At the time of biopsy, a CT scan may be done to determine where the 
biopsy will be done.  After the biopsy, a CT scan may be done over the biopsy site to look for any 
immediate complications.  These scans will be performed in addition to the scans as part of normal 
cancer care.  The scans involve exposure to radiation in the form of x-rays.  The level of radiation 
used is kept to a minimum to prevent damage to body cells.  Under some rare circumstances of 
prolonged, high-dose exposure, x-rays can cause adverse health effects, such as skin reddening 
(erythema), skin tissue injury, hair loss, and cataracts.  The amount of radiation from the additional 
scans in this study is not known to be associated with any serious health risks. The exposure related 
to this CT scan equals approximately one year of standard environmental background radiation. 
 
A.8. 
From FLT PET/CT and NaF PET/CT 
 
The FLT PET/CT and NaF PET/CT scans will expose subjects to a small amount of radiation, 
approximately equal to one bone scan.. The amount of radiation subjects will receive from this 
study is within the limits of the federal government’s rules and regulations and is thought to be 
safe.  Risks generally associated with insertion of a catheter (for the administration of the tracer) 
include pain, bruising, lightheadedness, and on rare occasions, infection.   
 
Reproductive risks of the FLT PET/CT and NaF PET/CT scans: The effects of the FLT and NaF 
tracers on the developing fetus are unknown.  Subjects should discuss the use of reliable methods 
of birth control like condoms and spermicidal foam or sexual abstinence with their study doctor(s).  
Procreative sex should be avoided prior to study entry and for the duration of study participation.  
If a subject’s partner is of child-bearing potential, the subject must be surgically sterile or use an 
acceptable method of birth control.  If a subject’s partner becomes pregnant while the subject is 
participating in this study, she should inform her treating physician immediately. 
 
A.9. 
Confidentiality 
 

 
 
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The research staff will make their best effort to keep samples and data confidential at all times.  
However, absolute confidentiality cannot be guaranteed.  There is a slight risk of breach of 
confidentiality which could be embarrassing or stigmatizing. 
 
 
B. 
Potential Benefits 
 
No benefits are guaranteed.  It is hoped that individual patients treated with the DNA vaccine either 
together with, or in sequence with, pembrolizumab will derive a clinical response as suggested by 
an objective disease response, decline in serum PSA, and/or a prolonged time to disease 
progression.  While no direct benefit is guaranteed, the results from this trial will guide the 
direction of future vaccine trials targeting PAP or potentially other antigens for the treatment of 
prostate cancer, and could suggest means by which anti-PD-1 therapies could be used in prostate 
cancer, as these agents have demonstrated substantial benefit for patients with other types of 
malignancies other than prostate cancer.  Thus, it is hoped that study participants and future 
patients will benefit from research participation in the current study. 
 
 
15. 
Study Data Management and Procedural Issues 
 
A. 
Study Enrollment Procedures – Recruitment and Informed Consent Process: 
 
Potentially eligible subjects at the UWCCC site will be patients regularly followed or referred to 
the University of Wisconsin Hospital and Clinics, and seen in the Medical Oncology, Urology, or 
Radiation Oncology outpatient clinics at the University of Wisconsin.  No specific advertisement 
or recruiting tools will be used.  Subjects will be identified by their primary radiation, surgical, or 
medical oncologist, and informed about this study, alternatives to this study, and the possible risks 
and benefits.  Potentially eligible subjects will be informed that their decision to participate or not 
participate will in no way affect their ongoing medical care.  Subjects who are interested at that 
point in obtaining more information will then be introduced to one of the GU research nurses who 
will then present the study (review rationale, describe time commitment, discuss again possible 
risks and benefits, and answer procedural questions) to the subject and provide them with a consent 
form.  In order to allow research subjects time to review the consent form thoroughly with their 
family and referring or primary physician, the subject will be instructed to take the consent form 
home without signing.  If the patient wishes to proceed with enrollment, they are then asked to 
contact the GU research office (608) 263-7107.  To avoid the possibility of unintended coercion, 
incarcerated subjects and subjects unable to provide their own informed consent will not be 
considered eligible.  In addition, the research staff will call the patient to further discuss the study 
and its requirements.  Any questions the subject has will be addressed by the research staff or 
investigators, and the time commitment and alternatives to treatment will again be reviewed.  If 
the subject agrees to participate in the study, they will be instructed to return to clinic to meet with 
the research staff and to sign and date the consent form. Our research staff will sign and date the 
consent form as the person obtaining consent.  No screening procedures done solely for purposes 
of the study will be obtained prior to the subject signing the consent form. 
 
Assignment to treatment arm will be based on the randomization list which will be generated by 
the study biostatistician.  

 
 
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B. 
Data Collection Procedures 
 
Electronic case report forms (e-CRFs) will be submitted to the UWCCC GU Oncology Office via 
UWCCC’s internal database. Completion of the e-CRFs will be done in accordance with the 
instructions provided by the UWCCC GU office in a study-specific data capture plan.  The e-CRFs 
are found in the study specific calendar that has been created in the database.  The system will 
prompt the user to the forms that are required based upon the patient’s enrollment and treatment 
dates. 
 
The Principal Investigator will be responsible for assuring that all the required data is entered onto 
the e-CRFs accurately and within 2 weeks of the date in which the previous cycle was completed.   
 
All clinical safety and clinical response data will be collected by the study coordinators on 
electronic case report forms as noted in section 16.B.  
 
Research data evaluating immune responses will be maintained in individual laboratory research 
charts.  All laboratory analysis conducted at the UWCCC will be maintained in research charts in 
the laboratory of Dr. McNeel and stored a minimum of 7 years.   
 
Adverse events, clinical responses, and issues related to disease progression are reviewed each 
week by the Genitourinary Malignancy clinical research Disease Oriented Working Group 
(DOWG), attended by GU medical oncologists, GU malignancy research nurses, data 
coordinators, and the GU malignancy program manager.  Minutes from these meetings are 
recorded, and bi-annual reports from this group are submitted to and reviewed by the UWCCC 
Data Safety and Monitoring Committee.  All clinical safety, immunological response, and clinical 
response data will be analyzed by the study statistician, with the University of Wisconsin 
Department of Biostatistics and Medical Informatics. 
 
All subject research charts will be maintained at individual treatment sites for a minimum of seven 
years after study completion.  All clinical information maintained in the UWCCC clinical trials 
database will be stored indefinitely.  All research samples and data derived from these specimens 
will be maintained indefinitely in the laboratory of Dr. McNeel.  All information with patient 
identifiers or medical history information will be kept in locked cabinets or secured databases 
available only to the study personnel to maintain patient confidentiality.   
 
C. 
Description of Procedures to Maintain Confidentiality of Research Specimens 
 
All specimens obtained for the immunological evaluation of this trial (blood specimens) will be 
delivered to the laboratory of Dr. McNeel.  Specimens received by Dr. McNeel’s lab will only be 
handled by laboratory personnel who have undergone HIPAA training and annual UWHC-
mandated blood-borne pathogen safety training.  Receipt of specimens will be entered into a 
database that will provide a unique code for each specimen.  All stored specimens (sera, peripheral 
blood mononuclear cells) will be labeled with this unique code and the date of preparation.  Thus, 
all patient identifiers will be removed from the final stored samples, and any data generated will 
contain only the unique code as identifier.  A database will be maintained to link individual coded 
specimens with an individual subject (name, hospital medical record number, date/time point) in 

 
 
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order to be able to compare information from samples obtained at different time points from a 
specific individual, and ultimately for transfer of research data to the clinical trials database, if 
necessary.  This research database will only be available to the study investigators, not other 
laboratory personnel, to maintain confidentiality.  Subject sera will be maintained in Dr. McNeel’s 
laboratory, stored in aliquots at –20°C to –80°C.  Peripheral blood mononuclear cells will be stored 
in Dr. McNeel’s laboratory, stored in aliquots in liquid nitrogen. If warranted, samples may be sent 
to an external institution or a contract research organization for the purpose of collaboration and 
/or analysis.  
 
Subjects will have the option to consent to future research using both their samples and data which 
may be used independently. Samples remaining after the primary analysis has been completed and 
available for future research will be “banked” and stored as they were for this study unless they 
are selected to be sent for research elsewhere, which may be within or outside of the University of 
Wisconsin. Subjects wishing to withdraw their banked samples will be required to provide written 
notice to the principal investigator, otherwise their samples may be used indefinitely for cancer 
research. All research samples and data derived from these specimens will be maintained 
indefinitely in the laboratory of Dr. McNeel.  All information with patient identifiers or medical 
history information will be kept in locked cabinets or secured databases available only to the study 
personnel to maintain patient confidentiality.  
 
D. 
Modifications of Protocol and Deviations from Protocol 
 
Any changes to the preceding protocol after approval by the University of Wisconsin IRB must be 
submitted as an amendment to this IRB with a description of specific changes.  All changes must 
be approved by the IRB before implementation.  If these changes were related to possible adverse 
events that could potentially affect subject safety, the consent form will also be modified, and 
submitted with the protocol to the IRB.  After approval, all enrolled subjects will be asked to 
review and sign the new consent form to proceed with treatment.  If these changes were related to 
a severe adverse event, or could affect the scientific integrity of the study, the events would also 
be reviewed internally by the DSMC, as described above, and the study could be placed on hold 
pending a protocol and consent form amendment, as described above, or study closure.  In addition, 
any information that might affect the immediate safety of currently enrolled subjects will be 
communicated with them directly as well. 
  
Deviations from the study protocol, for reasons other than patient safety, are not permitted.  Any 
possible deviations, intended or not, will be documented in the subjects’ research chart.  Deviations 
that are unlikely to affect subject safety, such as missing a specific study lab draw, will be 
documented in the research chart and discussed with the study PI.  Deviations that could potentially 
affect subject safety, such as missing safety labs, will be documented, performed as soon as 
possible, and reviewed at the weekly GU clinical research (DOWG) meetings, with prompt 
reporting as soon as any deviation is identified to the IRB if there are concerns for patient safety 
or scientific integrity of the study.     
 
E. 
Withdrawal from Study Protocol 
 
Subjects are informed during the consenting process, and in the consent form, that their 
participation is voluntary and they may withdraw consent at any time and for any reason.  If 

 
 
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subjects wish to withdraw from the study, they will be asked, but not required, to perform the off-
study procedures/blood draws.  They will be again informed that their decision to participate or 
not participate at any time will not affect their routine medical care or any other benefits to which 
they were otherwise entitled.  In addition, subjects will be removed from study participation if 
there is a concern for patient safety, as described above, if they are unable to comply with study 
procedures, or if the study is terminated by local or national regulatory agencies. 
 
 
16. 
Roles and Responsibilities of Specific Study Personnel at UWCCC 
 
Protocol sponsor: 
 
Dr. Douglas McNeel MD PhD, Professor of Medicine, is a genitourinary medical oncologist, with 
a clinical research and laboratory interest in immune-based therapies for prostate cancer.  He has 
served as the principal investigator for multiple other clinical trials, and has had formalized training 
in the ethics and conduct of clinical trials and human subjects protection.  He is overall responsible 
for the design of the trial, interpretation of the protocol as study sponsor, and its laboratory 
analysis, and holds the physician IND for the pTVG-HP DNA vaccine.  Because of his ownership 
interest in the pTVG-HP vaccine that has been licensed by the WARF (Wisconsin Alumni 
Research Foundation, intellectual property manager for the University of Wisconsin) to a company 
that is seeking to commercialize this vaccine (Madison Vaccines, Inc), however, he will not be 
involved in any direct patient care aspects of this trial (recruitment, treatment of subjects on trial, 
review of imaging studies, or review of clinical laboratories as they pertain to the treatment of 
individual subjects).   
 
 
UW Principal Investigator: 
 
Dr. Glenn Liu MD, Associate Professor of Medicine, is a genitourinary medical oncologist with a 
primary interest in genitourinary cancer clinical trials research. He will serve as principal 
investigator and in this capacity will be both a clinical co-investigator for this study, involved in 
the recruitment, treatment and review of individual treated subjects, and will also oversee all 
UW/IRB aspects related to direct patient care issues. 
 
 

 
 
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