Pilot Trial of Sipuleucel-T, With or Without pTVG-HP DNA Booster Vaccine, in Patients With Castrate-Resistant, Metastatic Prostate Cancer
Phase: PHASE2
Conditions: Prostate Cancer
Sponsor: University of Wisconsin, Madison

9/3/15 version 
 
 
Pilot Trial of Sipuleucel-T, with or without pTVG-HP DNA Booster Vaccine, in Patients 
with Castrate-Resistant, Metastatic Prostate Cancer  
 
CO11816 
 
Investigational Agent: 
 
 
BB IND 12109 - pTVG-HP DNA encoding human prostatic acid phosphatase  
 
 
STUDY SITE INFORMATION 
 
Study Sites: 
University of Wisconsin Carbone Cancer Center (UWCCC) 
 
1111 Highland Avenue 
 
Madison, WI 53705 
 
Study Principal Investigator: Douglas G. McNeel, M.D., Ph.D. 
 
UWCCC 
 
7007 Wisconsin Institutes for Medical Research 
 
1111 Highland Ave.  
 
Madison, WI 53705 
 
Tel:  (608) 263-4198 
 
Fax: (608) 265-0614 
 
dm3@medicine.wisc.edu 
 
 
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 
 
 
 
 
 
 
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 

 
 9-3/2015Version 
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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 
 
 
Start Date: 
 
 
 
February 11, 2013 
 
Projected End of Accrual Date: 
December 31, 2015 
 
Projected End of Study Date:  
December 31, 2016 
 

 
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TABLE OF CONTENTS 
 
SYNOPSIS:..................................................................................................................................... 4 
 
1. 
Introduction ....................................................................................................................... 6 
2. 
Background and Rationale ................................................................................................. 7 
3. 
Objectives ........................................................................................................................ 12 
4. 
Product Information ......................................................................................................... 13 
 
A. 
Sipuleucel-T .......................................................................................................... 13 
 
B. 
Plasmid DNA vaccine ........................................................................................... 14 
5. 
Patient Selection .............................................................................................................. 16 
 
A. 
Inclusion Criteria .................................................................................................. 16 
 
B. 
Exclusion Criteria ................................................................................................. 17 
6. 
Experimental Design ....................................................................................................... 18 
 
A. 
Study Arm Assignment and Toxicity Assessment................................................ 18 
 
B. 
Endpoints .............................................................................................................. 19 
7. 
Measurement of Effect .................................................................................................... 20 
8. 
Definition and Management of Limiting Toxicities and Adverse Events ....................... 26 
9. 
Plan of Treatment ............................................................................................................ 26 
10. 
Response Monitoring ....................................................................................................... 31 
 
A. 
Immunological Monitoring ................................................................................... 31 
 
B. 
Circulating Tumor Cell (CTC) Enumeration ........................................................ 34 
 
C. 
Serum PSA Doubling Time Response .................................................................. 35 
11. 
Reporting Adverse Events ............................................................................................... 35 
12. 
Statistical Considerations ................................................................................................ 36 
 
A. 
Overview ............................................................................................................... 36 
 
B. 
Objectives ............................................................................................................. 36 
 
C. 
Definitions/Endpoints ........................................................................................... 37 
 
D. 
Study Design ......................................................................................................... 38 
 
E. 
Randomization and Stratification ......................................................................... 39 
 
F. 
Sample Size Estimation and Accrual .................................................................... 39 
 
G. 
Statistical Analysis Plan ........................................................................................ 40 
13. 
Administrative Considerations ........................................................................................ 44 
14. 
Data and Safety Monitoring Plan .................................................................................... 44 
15. 
Potential Risks and Benefits, and Procedures to Minimize Risk .................................... 49 
16. 
Study Data Management and Procedural Issues .............................................................. 52 
17. 
References: ...................................................................................................................... 56 
 
APPENDIX A ............................................................................................................................... 61 
APPENDIX B ………………………………………………………………………………… 62

 
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SYNOPSIS: 
 
 Primary Objectives: 
 
1. To evaluate whether immunization with a DNA vaccine encoding prostatic acid 
phosphatase (PAP, pTVG-HP) can augment PAP-specific effector and memory T cells 
following treatment with sipuleucel-T.  
 
Secondary Objectives: 
 
1. To determine 12-month radiographic progression-free survival  
2. To determine median time-to-radiographic progression as a function of baseline time 
point (pretreatment or 3-months post treatment) 
3. To evaluate effects of treatment on changes in serum PSA level and changes in PSA 
doubling time  
 
Laboratory and Exploratory Objectives: 
 
1. To determine overall survival 
2. To evaluate effects of treatment on number of circulating tumor cells as determined by 
PSA ELISPOT 
3. To evaluate PAP-specific antibody and T-cell immune responses following treatment 
with sipuleucel-T and DNA vaccine 
4. To determine whether PAP-specific immune response is associated with prolonged (1 
year) radiographic progression-free survival 
5. To determine whether baseline immune responses (Th1 type or regulatory type) predict 
for immune responses elicited/augmented following treatment with sipuleucel-T +/- DNA 
vaccine 
6. To determine whether treatment elicits immunologic antigen spread to other prostate 
associated antigens 
 
 
Study Scheme: 

 
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Study Calendar: 
 
 
 
Vaccine / Treatment Visit: 
 
 
Pre-Screen 
(within 4 weeks of Screen) 
Screen/Randomization 
(within  2 weeks of day 1) 
Day 1 – sipuleucel-T collection 
#1 
Day 4 – sipuleucel-T   infusion 
#1 
Day 15 – sipuleucel-T collection 
#2 
Day 18 – sipuleucel-T infusion 
#2 
Day 29 – sipuleucel-T collection 
#3 
Day 32 – sipuleucel-T infusion 
#3 
Day 43 – sipuleucel-T follow-up 
/ DNA imm #1a  
Day 57 – DNA imm #2a 
Day 71 – DNA imm #3a 
3-Month  Followup 
(Day 85) – DNA imm #4a 
6-Month Follow up 
(Week 24) – DNA imm #5a 
9-Month Follow-up 
(Week 36) – DNA imm #6a 
12-Month (Week 48) 
Follow-up and/or  
End of study visit
History & Physical Exam 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam, ECOG PS 
 
X 
 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Vital signs 
 
X 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Toxicity assessment b 
 
X 
 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Injection site inspection 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
Lab Tests 
CBC with differential and 
platelets c 
 
X 
 
 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Creatinine, alk phos, SGOT, 
total bilirubin, amylase, LDH 
 
X 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
X 
Serology for HIV Ab/Ag 
combo, Hepatitis B Surface Ag, 
Hepatitis C AB 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Serum testosterone, ALT 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
PSAd and PAP 
 
X 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
X 
Procedures 
CT abdomen/pelvis e 
X 
 
 
 
 
 
 
 
 
 
 
Xf 
X 
X 
Xg 
Bone scan e 
X 
 
 
 
 
 
 
 
 
 
 
Xf 
X 
X 
Xg 
Leukapheresis 
 
Xh 
X 
 
X 
 
X 
 
 
 
 
 
Xh,i 
 
 
Sipuleucel-T infusion 
 
 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
Tetanus immunization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
DNA immunization –ARM 2 
PATIENTS ONLY a 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
 
Blood for CTC analysis 
 
X 
 
 
 
 
 
 
X 
 
 
X 
X 
X 
X 
Blood for immune studies j 
 
X  
 
 
 
 
 
 
X 
 
 
X 
X 
X 
Xk 
Note: Grey regions indicate those tests or procedures or visits only applicable to subjects treated on Arm 2. Acceptable delays in 
study treatments or procedures may result in corresponding delays of subsequent visits. Annual long term follow up will occur 
for up to five years. See Section 9 for more details. 
a ARM 2 Only: Biweekly DNA immunization (#1-3) can be +/- 3 days; quarterly DNA immunization (#4-6) can be +/- 7 days from expected date 
determined from prior visit. 
b Study coordinator or research nurse review of systems, including ECOG PS 
c CBC with differential and platelets to be performed within 72 hours prior to leukapheresis (for Sipuleucel-T treatment) unless an event occurs 
that would require it be drawn sooner for safety considerations, or if required sooner per institutional requirements or MD discretion. A CBC 
result within 7 days prior to leukapheresis for research is ideal. 
d PSA measurements must be performed at the 3-month intervals defined, but can be more frequent if clinically indicated. 
e Radiographic studies (CT scans and bone scans) can be performed +/- 7 days of the study visit 
f Staging studies (CT and bone scan): scan progression between Screening and Month 3 will not be used to define radiographic progression for 
the purposes of this study and will not necessitate subjects to come off study. Patients should come off study at this time point only due to 
symptomatic progression requiring other treatment for prostate cancer. 
g Radiographic studies not required at off-study time point if subject already met time-to-progression endpoint and/or studies already performed 
within 1 month; scans otherwise performed every 12 weeks 
h Leukapheresis collected (+/- 7 days) for research, not preparation of sipuleucel-T product 
i If leukapheresis is not able to be obtained at Month 6 visit, then up to 150 ml may be obtained via peripheral blood draw as an alternative. 
j Blood for immune studies is to be collected per calendar (+/-7 days). This should still be collected for subjects that come off study early, 
however the quarterly interval time points can correspond to the patient’s standard of care visits.  
k Blood for immune studies is not necessarily collected at “End of Study” visit; it’s collected at quarterly intervals for up to one year. 
 

 
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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 have died as a result of prostate cancer in 2010 [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, abiraterone, an oral androgen synthesis inhibitor, was 
similarly approved in the setting of docetaxel-refractory, castrate-resistant metastatic prostate 
cancer following a prospectively randomized, blinded, placebo-controlled trial demonstrating 
nearly a 4-month median improvement in overall survival [8].  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 [9].  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 [10-13].  Consequently, 
evaluating active immunotherapies in combination with other immunotherapy or traditional 
agents, or earlier in the course of disease, are rational directions to further improve on these 
therapies to attempt to further improve on the survival benefit observed [14].   
 
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 [15].  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 [16].  Data from independent labs 
has demonstrated that, in a rat model, vaccine strategies targeting PAP can result in PAP-specific 

 
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CD8+ T-cells, the presumed population mediating tumor cell destruction, and anti-tumor 
responses [17-20].  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 
[21].  In rodent studies, we have similarly demonstrated that PAP can be immunologically 
targeted using genetic vaccines, and a plasmid DNA vaccine in particular [18, 19].   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 [22].  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 [23].  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 boosting immune responses from other priming immunizations [23].  
These findings have justified further evaluation of this vaccine in a randomized phase II clinical 
trial, a study currently underway, and also specifically suggest that the pTVG-HP might be used 
in combination with sipuleucel-T to augment PAP-specific T-cell responses primed by that 
immunization strategy, as both vaccine approaches target the same antigen.  This serves as the 
primary 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 [15, 24].  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 [25].  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 [26, 27].  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 [28].  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 [23, 28]. 
 
B. 
Sipuleucel-T:  The first anti-tumor vaccine FDA approved for the treatment of cancer 
 

 
 9-3/2015Version 
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We have previously reviewed the preclinical and early clinical development of sipuleucel-T 
elsewhere [14].  Sipuleucel-T has been evaluated in three Phase III clinical trials in patients with 
metastatic, castrate-resistant prostate cancer.  In the first phase III trial (D9901), Small and 
colleagues enrolled 127 men with asymptomatic disease, randomized 2:1 to receive three 
biweekly infusions of sipuleucel-T versus placebo.  Patients on the sipuleucel-T arm received as 
many autologous cells, cultured in vitro with PA2024 (PAP-GM-CSF fusion protein), as could 
be prepared from a standard leukapheresis product (with a median of 3.65x109 nucleated cells 
per infusion [29]).  The patients receiving placebo underwent a leukapheresis, and cells were re-
infused without being pulsed with PA2024.  In this study, the primary endpoint was TTP (either 
radiographic or development of pain).  In this study, treatment with sipuleucel-T was found to be 
well tolerated.  The most common side effects associated with sipuleucel-T infusion were Grade 
1 or 2 rigors and pyrexia (54.9% and 37.8% of patients, respectively), with a lower frequency of 
patients having Grade 1 or 2 tremors (26.8%) [29].  T-cell proliferative responses were detected 
to the PA2024 fusion protein [29].  However, while serum PSA decreases of greater than 25% 
were detected in 6.8% of patients receiving sipuleucel-T (with no decreases in the control 
patients), the primary endpoint of TTP did not reach statistical significance: patients receiving 
sipuleucel-T had a median TTP of 11.7 weeks (95% confidence interval (CI): 9.1-16.6) 
compared with 10.0 weeks for patients receiving placebo (95% CI: 8.7-13.1) [29].   
 
While the D9901 trial was ongoing, a second companion phase III trial was also underway, 
accruing patients with asymptomatic metastatic, castrate-resistant disease (D9902A).  This trial 
was being conducted as a confirmatory study with the same population, study design, and 
randomization scheme.  While this trial was still accruing patients, the results from the D9901 
trial showing a lack of difference in TTP were unblinded, and due to lack of significance in the 
primary endpoint of TTP, the D9902A trial was abandoned.   
 
A subset analysis of D9901 found that patients with a Gleason score ≤7 treated with sipuleucel-T 
had a significant increase in TTP compared with those receiving placebo.  This led to another 
Phase III trial (D9902B), which was designed similarly to the D9901 trial with 127 patients and a 
primary endpoint of TTP, but with the plan to only accrue asymptomatic metastatic, castrate-
resistant patients with a Gleason score of 7 or less.   However, as this trial was beginning to 
accrue, a later analysis of the D9901 trial revealed a significant difference in overall survival, 
with patients receiving sipuleucel-T having a three-year overall survival of 25.9 months, 
compared with 21.4 months of patients receiving placebo (hazard ratio (HR) 1.43, p=0.01) [30].  
Additionally, as described above, it was observed that nearly half of progression events occurred 
within the first 12 weeks of the study, possibly before an optimal immunologic effect could have 
occurred, and therefore possibly accounting for a lack of difference in TTP [29, 30].  
Furthermore, an integrated analysis of the D9901 and D9902A trials suggested that patients 
receiving sipuleucel-T had a median survival of 23.2 months (95% CI: 19.0-31.0) versus 18.9 
months (95% CI: 13.5-25.3) in the placebo control group (HR: 1.5, p=0.011) [30].  Due to these 
findings, the D9902B protocol was amended, changing the primary endpoint from TTP to overall 
survival, and increasing the planned patient enrollment from 127 to 512 to sufficiently power this 
study to detect a difference in overall survival.  This trial (IMPaCT, Immunotherapy for Prostate 
Adenocarcinoma Treatment) confirmed that treatment with sipuleucel-T provided a significant 
increase in overall survival: patients receiving an infusion of PA2024-pulsed cells had a median 
survival of 25.8 months compared to 21.7 months in the placebo group, consistent with the 
previous trials [7].  This survival difference reflected a significant difference in the risk of death 

 
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in patients treated with sipuleucel-T compared to the placebo group (hazard ratio (HR), 0.78.  
95% CI: 0.61-0.96, p=0.03) [7].  This survival benefit was also present regardless of prior or 
subsequent treatment with docetaxel and prednisone.  Similar to previous trials, no difference in 
time to progression was observed (14.6 weeks in the sipuleucel-T group versus 14.4 weeks in the 
placebo group, p=0.63), contradicting trends observed in the earlier phase studies [7].  With the 
significant survival benefit detected in the IMPACT phase III trial, FDA approved sipuleucel-T 
in April 2010 for the treatment of asymptomatic or minimally symptomatic metastatic, castrate-
resistant prostate cancer.   
 
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 [31-33], 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 [19, 34].  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 [35] and CD8+ T-cells specific for the 
targeted antigen [31, 36-39].  In animal models, the use of an intradermal route of vaccine 
administration, in particular, tends to promote this Th1/CTL-biased immune response [35, 40, 
41].  It should also be acknowledged that the clinical efficacy of a DNA vaccine encoding 
tyrosinase for the treatment of canine melanoma, delivered with GM-CSF as a vaccine adjuvant, 
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.  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.   
 
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 [18, 19].  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 [42].  
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 

 
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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, similar to the 
approach used in the canine melanoma vaccine trials, with each immunization.  13 additional 
subjects were treated in an expanded cohort at the 1500 µg DNA dose.  Safety evaluation 
included monthly blood tests (CBC, creatinine, urinalysis, anti-nuclear antibody tests, liver 
function tests, and serum amylase) and physical examination and review of systems.  Subjects 
were also monitored for one hour after each vaccination for any immediate allergic reactions.  
No serious adverse events were observed, and no significant laboratory anomalies were observed 
apart from a single grade 3 amylase that was determined to not be treatment-related.  No other 
events > grade 2 were observed in any of the 22 subjects treated.  Common events observed were 
grade 1/2 fevers, chills, and local site reactions lasting typically less than 24 hours.  2/22 subjects 
experienced grade 2 back pain occurring ~ 45 minutes after treatment and lasting < 10 minutes.  
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.  For this 
evaluation autologous dendritic cells were prepared by culture of flask-adherent monocytes for 
six days in serum-free medium supplemented with GM-CSF and IL-4.  CD8+ T cells, purified 
from PBMC by negative magnetic bead selection, were cultured with autologous dendritic cells 
and protein antigens for 96 hours, without in vitro restimulation, and ELISPOT was used as a 
quantitative direct assessment of effector T-cell response.  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 [22].  In 
addition, several subjects, including the subject with the greatest magnitude ELISPOT response, 
were also found to have PAP-specific memory T-cell recall responses as identified by 
proliferating CD4+ and CD8+ T cells in a 96-hour proliferation assay following antigen-
stimulation [22].   Finally, 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 [23].  In addition, one 
patient went on to receive booster immunizations with 100 µg pTVG-HP at monthly intervals.  
As shown in Figure 1, booster immunizations were able to augment PAP-specific CD4+ and 
CD8+ proliferative T cells, as well as PAP epitope-specific CD8+ T cells (Figure 2) months after 
the initial immunization course.  Taken together, these results demonstrate that pTVG-HP can 
elicit PAP-specific T-cell immune responses, CD8+ T-cell responses in particular, and these 
responses could be elicited at even the lowest dose (100 µg) of plasmid DNA vaccine tested.    
 
Figure 1:  PAP-specific cellular immune responses can be boosted months after initial immunization.  Peripheral blood mononuclear 
cells were obtained from a patient 18 months after the initial immunization series (pre boosters), and after 2, 4, or 6 monthly booster 
immunizations with 100 µg pTVG-HP.  Antigen-specific T cells were identified by antigen-specific T-cell proliferation by BrdU 
incorporation and flow cytometric analysis (BrdU proliferation kit, BD Biosciences).  Shown is the proliferation index relative to a 
media only control of CD4+ T cells (panel A) and CD8+ T cells (panel B).  Panel C shows patient’s serum PSA levels prior to and after 
treatment, indicating stable disease.   

 
 9-3/2015Version 
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Given the ability of a DNA vaccine to elicit PAP-specific T cells in some patients with prostate 
cancer, the observation that immune responses elicited tended to be after multiple 
immunizations, and the further observation that in at least one individual these immune 
responses could be boosted with subsequent reimmunization [23], 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.  While this trial is 
still underway and results are preliminary, to date we have identified that many patients 
developed PAP-specific immune responses after as few as 3-6 immunizations (Figure 3, subject 
#2), and some individuals (Figure 3, subject #1), no immune responses were detectable after 
even 24 biweekly immunizations.  These findings suggest that more potent immunization 
strategies might be necessary to elicit PAP-specific immunity in some individuals that could be 
further boosted with a DNA vaccine, and hence a rationale for the current trial.  In addition, 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 – this trial is currently underway.   
 
E. 
Summary 
 
The current protocol will examine the use of a plasmid DNA vaccine encoding PAP to induce 
and/or augment T-cell immune responses to PAP in patients with castrate-resistant, metastatic 
prostate cancer following treatment with sipuleucel-T.  Based on published results from trials 
conducted with sipuleucel-T, PA2024 and PAP antibody and proliferative T cell responses have 
been observed, suggesting a possible CD4+ T-cell-biased immune response.  In 25 individuals 
who have received multiple immunizations with the pTVG-HP DNA vaccine, no PAP-specific 
antibody responses have been observed, and multiple individuals have had detectable Th1-biased 
PAP-specific CD4+ and CD8+ responses.  These results suggest that different types of immune 
responses to the same antigen might be elicited depending on the type of immunization, and 
hence a combination able to elicit a broad immune response might have greater anti-tumor effect.  
Given that a Th1/CD8+/CTL response would be predicted to be the most effective immune 
response elicited capable of lysing PAP-expressing prostate cancer cells, the identification of 
these specific subsets will be the primary endpoint of this pilot trial.  The trial will be conducted 
in patients with minimally symptomatic, castrate-resistant, metastatic prostate cancer, as this is 
the approved indication for treatment with sipuleucel-T.  In addition, given previous observations 
from clinical trials using sipuleucel-T, and multiple other anti-tumor vaccine trials, suggesting 
that clinical effects (unlike with standard cytotoxic therapies) might occur months after treatment 
Figure 2:  PAP-derived epitope-specific cellular 
immune responses can be detected long-term after 
initial immunization.  Peripheral blood mononuclear 
cells were obtained from an HLA-A2-expressing 
patient at multiple intervals following an initial 6 
biweekly immunization series and 6 monthly booster 
immunizations 18 months later (red bars indicate 
timing of these immunizations).  Cells were stimulated 
in vitro for 1 week with peptides and assayed for the 
frequency 
of 
epitope-specific 
T 
cells 
among 
CD3+CD8+ cells by HLA-A2-specific PAP peptide-
specific pentamers (ProImmune, Inc.).   

 
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Figure 3:  Patients with the same stage of prostate cancer respond differently to treatment with a DNA vaccine.  Patients 
with castrate-resistant, non-metastatic prostate cancer were immunized at two-week intervals with a DNA vaccine 
encoding PAP 24 times (subject 1, panels A and B) or 6 times, followed by immunization at 3-month intervals (subject 2, 
panels C and D), as indicated by the red asterisks.  Immune response was assessed using freshly obtained PBMC, without 
in vitro stimulation by T-cell proliferation using PKH26 dye dilution, using as stimulator antigens PAP (blue), PSA (pink, 
negative control), and tetanus toxoid (green), and assessed by flow cytometry after 7 days in antigen-specific culture 
(panel B).  Precursor frequency of antigen-specific CD8+ T cells was estimated using ModFit software.  In addition, 
immune response was assessed by antigen-specific IFNγ (panel A and C) or granzyme B (panel D) release by ELISPOT 
after 48- or 24-hour antigen-specific culture without in vitro stimulation.   
concurrent with the generation of immune responses, exploratory clinical endpoints will be to 
determine whether treatment affects progression-free survival using a delayed (12-week) time 
point as the baseline for subsequent radiographic comparisons.  Additional clinical endpoints 
will be to examine the effects of treatment on circulating tumor cell number, PSA kinetics, and 
whether favorable delays in disease progression are associated with the detection of persistent 
immune responses to PAP or other “off-target” prostate cancer antigens after treatment, or 
negatively associated with unfavorable tolerant-type immune responses existing pre-treatment.  
If successful, this trial could provide rational clinical endpoints and immune biomarkers that 
could be used in the design of future studies using sipuleucel-T in combination with other agents, 
and/or a larger, confirmatory study in combination with booster DNA vaccines.   
 
 
 
3. 
Objectives 
 
A. 
Primary Objectives 
 
1. To evaluate whether immunization with a DNA vaccine encoding prostatic acid 
phosphatase (PAP, pTVG-HP) can augment PAP-specific effector and memory T cells 
following treatment with sipuleucel-T.  
 

 
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B. 
Secondary Objectives: 
 
1. To determine 12-month radiographic progression-free survival  
 
2. To determine median time-to-radiographic progression as a function of baseline time 
point (pretreatment or 3-months post treatment) 
 
3. To evaluate effects of treatment on changes in serum PSA level and changes in PSA 
doubling time  
 
C. 
Laboratory and Exploratory Objectives: 
 
1. To determine overall survival  
 
2. To evaluate effects of treatment on number of circulating tumor cells as determined by 
PSA ELISPOT 
 
3. To evaluate PAP-specific antibody and T-cell immune responses following treatment 
with sipuleucel-T and DNA vaccine 
 
4. To determine whether PAP-specific immune response is associated with prolonged (1 
year) radiographic progression-free survival  
 
5. To determine whether baseline immune responses (Th1 type or regulatory type) predict 
for immune responses elicited/augmented following treatment with sipuleucel-T +/- DNA 
vaccine 
 
6. To determine whether treatment elicits immunologic antigen spread to other prostate 
associated antigens 
 
 
4. 
Product Information 
 
A. 
Sipuleucel-T  
 
PA2024 
 
PA2024 is a recombinant fusion protein consisting of human PAP and GM-CSF.  GM-CSF acts 
as a growth factor and differentiation factor for antigen-presenting cells, including dendritic 
cells; the purpose of the fusion protein is to direct the PAP antigen to antigen-presenting cells in 
vitro, and promote antigen uptake and processing of the PAP antigen.   
 
 
Preparation of sipuleucel-T 
 
Patients will undergo a standard 1.5-2.0 blood volume leukapheresis to harvest peripheral blood 
mononuclear cells.  This procedure will be performed either at the UWHC Infusion Center or a 
contract provider authorized by Dendreon Corporation.  Immediately after collection, the product 

 
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will be transported by air carrier to a Dendreon-approved national cell processing facility.  There, 
the cell product will be enriched for antigen-presenting cells by proprietary buoyant density 
centrifugation techniques, and cultured in vitro in the presence of PA2024 for 36-44 hours.  After 
the culture period, the cells will be washed and resuspended in Lactated Ringer’s Injection, USP.  
The sipuleucel-T cell product will then be shipped back to the UWHC for infusion at the UWHC 
Infusion Center.  During shipment, sterility testing and cell count data are gathered for final 
product release for re-infusion.  These procedures will all be performed as part of the standard-
of-care for the FDA-approved sipuleucel-T product and re-infusion.   
 
B. 
Plasmid DNA vaccine 
 
 
pTVG-HP (pTVG4 vector containing cDNA for human PAP) 
 
The sequence of the pTVG-HP plasmid has been confirmed by standard DNA sequencing to 
confirm its identity.  A bacterial strain was transformed with this plasmid and transferred to the 
National Gene Vector Laboratories (NGVL) manufacturing facility (Center for Biomedicine and 
Genetics, City of Hope/Beckman Research Institute, Duarte, CA) from which a master cell bank 
was prepared.  This master cell bank was shipped in 2008 to the Waisman Clinical 
Biomanufacturing Facility (WCBF) at the University of Wisconsin.  Bacteria from this bank was 
grown and amplified in culture under kanamycin selection, and two separate GMP-grade lots of 
plasmid DNA have been prepared (one at the Beckman Research Institute for the initial phase I 
trial, and one lot at the WCBF).  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 were 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 future lot release.  The vaccine will be supplied in single-use vials containing 0.6 
mL 0.2 mg/mL pTVG-HP in phosphate-buffered saline.  Vials containing phosphate-buffered 
saline only will be used for preparing the placebo/GM-CSF.   Vials will be stored at –80°C until 
the day of use.   
 
GM-CSF 
 
GM-CSF (Leukine®, Sargramostim), will be obtained from commercial vendor (Genzyme, 
Cambridge, MA) 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 [43, 44].  The use of GM-CSF is 
associated with little toxicity [45-47].  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 

 
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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 [48].  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 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 [47]. 

 
 9-3/2015Version 
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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. 
 
 
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 standard of care androgen deprivation treatment 
before trial entry (surgical castration versus GnRH analogue or antagonist 
treatment), and subjects receiving GnRH analogue or antagonist must continue 
this treatment throughout the time on this study. 
b. Patients may have been treated previously with a nonsteroidal antiandrogen, with 
evidence of disease progression (defined below) subsequently.  Subjects must be 
off use of anti-androgen for at least 4 weeks (for flutamide) or 6 weeks (for 
bicalutamide or nilutamide) prior to registration.   
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. Castration levels of testosterone (< 50 ng/dL) within 2 weeks of registration 
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 [49] or RECIST 1.1 during or after completing last therapy: 
a. PSA:  At least two consecutive rises in serum PSA, obtained at a minimum of 1-
week intervals, and each 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 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). 

 
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6. Must have > 3 serum PSA values obtained over at least a 12 week  period of time prior to 
registration, including the day of screening, to calculate a PSA doubling time 
(http://www.mskcc.org/applications/nomograms/prostate/PsaDoublingTime.aspx).  Note: 
PSA’s are not required to be obtained at the same laboratory. Use all PSA values that 
have been done in last 6 months to calculate PSA doubling time.  
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 14 
days of first peripheral blood collection for Sipuleucel-T. 
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. Negative serology tests for HIV Ab/Ag Combo and for active Hepatitis B and Hepatitis 
C, within 14 days of first peripheral blood collection for Sipuleucel-T. 
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 
12. 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 prostate cancer histology 
2. Patients may not be receiving other investigational agents or be receiving concurrent 
anticancer therapy other than androgen deprivation 
3. Symptomatic metastatic disease, as defined by the need for opioid analgesics for the 
treatment of pain attributed to a prostate cancer metastatic lesion; patients receiving 
opioids must receive approval from the PI for eligibility 
4. Patients may not have been treated with prior sipuleucel-T  
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 1 mg prednisone daily); 
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 
• Any other hormonal agent or supplement being used with the intent of cancer 
treatment 

 
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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 (e.g. docetaxel, mitoxantrone, cabazitaxel) within 6 months 
of registration is prohibited 
9. Patients with a history of life-threatening autoimmune disease 
10. Patients who have undergone splenectomy 
11. Patients must not have other active malignancies other than non-melanoma skin cancers 
or carcinoma in situ of the bladder.  Subjects with a history of other cancers who have 
been adequately treated and have been recurrence-free for > 3 years are eligible.   
12. Patients with known brain metastases 
13. Any antibiotic therapy or evidence of infection within 1 week of registration 
14. Any other medical intervention or condition, which, in the opinion of the PI could 
compromise patient safety or adherence with the study requirements 
15. Patients cannot have concurrent enrollment on other phase I, II, or III investigational 
treatment studies. 
 
 
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 booster immunizations with a DNA vaccine encoding PAP 
with rhGM-CSF adjuvant following treatment with sipuleucel-T.  Study arms will be defined as 
follows: 
 
Arm 1: 
sipuleucel-T – administered intravenously (i.v.) per standard of care biweekly, 
 
weeks 0, 2, and 4 
 
Arm 2: 
sipuleucel-T – administered intravenously (i.v.) per standard of care biweekly, 
 
weeks 0, 2, and 4 
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally 
(i.d.)  biweekly 4 times, then every 3 months to complete a 1-year 
treatment period (i.e. weeks 6 (day 43), 8 (day 57), 10 (day 71), 12 (month 
3), then months 6 and 9) 
 
A. 
Study Arm Assignment and Toxicity Assessment 
 
Patients with asymptomatic-to-minimally-symptomatic, castrate-resistant, metastatic prostate 
cancer, with evidence of progressive disease, and a calculable PSA doubling time with at least 3 
serum PSA values collected over a minimum of 3 months, will be invited to participate.  These 
are subjects who would be eligible to receive sipuleucel-T as routine standard of clinical care.  
Subjects will be randomly assigned to a treatment group, stratified by pre-treatment estimated 
survival (< 18 months, > 18 months), using the Halabi nomogram [50], and using 
laboratory/clinical data collected at screening for this determination.  This stratification was 
chosen given the identification in a previous clinical trial that patients with more indolent disease 
characteristics appeared to have a greater benefit from vaccine treatment in terms of overall 

 
 9-3/2015Version 
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survival, and the specific use of an 18-month cutoff is currently being used in prospective 
evaluation of other vaccine trials [51].  Given the absence of significant adverse events observed 
in previous clinical trials with either sipuleucel-T or pTVG-HP, no adverse events > grade 2 are 
anticipated.  However, all study patients will be evaluated for evidence of toxicity, and further 
accrual to either treatment arm will be discontinued if the toxicity rate is deemed to be excessive.  
A toxicity rate of 20% for Grade 3 events (or Grade > 1 autoimmune events) or 10% 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.     
 
Evidence that the toxicity rate is excessive will be considered sufficient if 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., 20% for Grade 3 events and 10% 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 (or Grade >1 autoimmune events) are present in at least 3 
out of 5, 4 out of 9 (or less), 5 out of 13 (or less), or 6 out of 15 (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 2 out of 5, 3 out of 11 (or less), or 4 out of 15 
(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 by the UW DSMC (data and safety monitoring committee) to consider safety and dose 
modifications 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 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 other therapies for 
prostate cancer are warranted. Patients should be discouraged from discontinuing protocol 
treatment for PSA rise only.  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. The 6 
month visit, in particular, is important to collect for the purpose of endpoint analysis.  Subjects 
who have not come off study earlier for these reasons will come off study at the end of the one-
year treatment period. 
 
The primary endpoint of the trial will be an immunological endpoint – the detection of 
augmented numbers of PAP-specific effector (IFNγ-secreting and/or granzyme B-secreting) and 
memory (proliferating) T-cell immune response after immunization, using blood obtained at 6 
months for the primary analysis compared with pre-treatment.  Immune response will be 
determined as a static time point at 6 months, and “durable” immune responses will also be 
determined by evaluation at 3, 9, and 12 months after study entry.   
 

 
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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 will be defined using both RECIST 1.1 and PCWG2 published criteria [49],  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.  Time to progression will be determined from registration for all subjects.  
1-year progression-free survival will be calculated for each treatment arm, as well as the median 
progression-free survival.   
 
PSA doubling times will be calculated from all available values obtained within 3-6 months from 
the same clinical laboratory (when feasible) prior to study enrollment, up to and including the 
baseline value, and used to determine a pre-treatment PSA doubling time.  Similarly, a post-
treatment doubling time will be calculated from all PSA values obtained beginning with the PSA 
value at day 1 and continuing until the end-of study (or 1-year) value.   
 
 
7. 
Measurement of Effect  
 
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. 
 
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 at day 85 (3 months).  Objective 
progression at Month 3 from the pre-treatment scans will not be used to alter treatment unless the 

 
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patient has symptomatic disease requiring other therapy and thereby needs to discontinue 
treatment.   
 
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. 
 
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 

 
 9-3/2015Version 
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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.  
 
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, 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 at 
least 6 weeks later demonstrating > 2 new lesions.  This is to eliminate the 
possibility of flair responses seen on bone scans. 
 
3. Symptomatic Deterioration  

 
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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. 
 
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 
 

 
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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  
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 

 
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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 immunization prolongs 
the time to progression using a delayed baseline evaluation after the treatment has been 
given time to elicit an 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 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. 
 

 
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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) 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 immunization.  For purposes of this study, a Grade 2 autoimmune toxicity 
will be considered equivalent to a Grade 3 toxicity.  If a patient develops Grade 3 toxicity with 
an attribution of at least possibly related to vaccine, the vaccination schedule will be held until 
the toxicity resolves to Grade 2 or less (Grade 1 for autoimmune toxicity).  There will be no dose 
reductions due to adverse events.  If a patient develops a second Grade 3 event (or Grade 2 
autoimmune toxicity) that is believed at least possibly due to treatment, or any Grade 4 event, no 
further vaccinations will be given and the patient will be removed from study.  If the adverse 
event occurs during the initial 12 weeks of treatment, a treatment delay of up to one week will be 
permitted, with the expectation that the event resolve to < grade 1 for retreatment, and that 
subsequent immunizations will follow at 2-week intervals.  A delay > 1 week will result in a 
skipped dose.  If the adverse event occurs during the 3-month booster phase, vaccination will be 
held in the event of toxicity attributed to vaccination.  If the toxicity observed is believed to be a 
result of the immune response generated, a regimen of corticosteroids may be administered, as 
clinically indicated, and after discussion with the study and local PIs.  The following dose 
schedule could be used: 
 
 
Day 1:  Intravenous Solu-Medrol at 1 mg/kg IV q12 hr 
 
Day 2:  Intravenous Solu-Medrol at 1 mg/kg IV x 1 
 
Day 3-4:  Prednisone at 30 mg p.o. bid 
 
Day 5-6:  Prednisone at 15 mg p.o. bid 
 
Day 7-8:  Prednisone at 10 mg p.o. bid 
 
Day 9-10:  Prednisone at 10 mg p.o. qd 
 
Day 11-12:  Prednisone at 5 mg p.o. qd 
 
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. 
 
 
9. 
Plan of Treatment 
 
The following section describes the schedule for prescreening, sipuleucel-T collections and 
infusions, vaccine inoculations, and clinical and laboratory evaluations.  

 
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When a chemistry panel is indicated the following tests are performed:  creatinine, 
SGOT, total bilirubin, alkaline phosphatase, amylase, and LDH.  Whenever a CBC is indicated, 
this shall include differential and platelet count.  
 
Sipuleucel-T infusions should be 14 days apart, but may be up to 21 days apart to 
accommodate scheduling difficulties. The study calendar will then be delayed accordingly by the 
same amount of time. For a delay greater than a week, the nature of this delay will be discussed 
with the medical monitor or PI to determine the appropriate course of action. For subjects on 
Arm 2, biweekly DNA vaccination (Immunization #1-3) can be +/- 3 days and quarterly 
vaccinations (Immunizations #4-6) can be +/- 7 days from the expected date (determined from 
prior visit).  
 
All subjects should have blood drawn for immune monitoring per study calendar (+/- 7 
days) unless they come off study early. Subjects that come off study early should still have all of 
the immune draws done, but the blood draws can correspond with standard of care visits (i.e. 
once off study, they don’t need to come to clinic just for the immune draw). This plan is 
summarized in the Study Schema (pg. 4), Study Calendar (pg. 5), and Blood Draws for Research 
table (Appendix B) 
 
A. 
Prescreen (performed within 4 weeks of Day 1) 
 
 
1. 
Confirm potential eligibility by history, pathology, diagnosis, and serial serum 
PSA levels; no exclusions by history 
2. 
CT scan of abdomen and pelvis, bone scan 
3. 
Sign consent form 
 
B. 
Screening Evaluation (performed within 2 weeks of Day 1; can coincide with prescreen 
evaluation) 
 
1. 
Physical examination, including vital signs, symptoms assessment, and ECOG 
performance score 
2. 
Evaluation of blood cell counts (CBC with differential and platelets), chemistry 
panel,  serum prostate specific antigen (PSA), serum PAP, serum testosterone, 
serology tests for HIV1, HIV2, Hepatitis B, and Hepatitis C. Tests ordered are the 
following: serology for  HIV Ab/Ag Combo, Hepatitis B Surface Ag, and 
Hepatitis C AB 
3. 
Determination of predicted survival (< 18 months, > 18 months) for stratification 
purposes using laboratory values and clinical data (ECOG performance score, 
presence or absence of visceral metastases, Gleason score, screening PSA, 
screening LDH, screening alkaline phosphatase, and screening hemoglobin) and 
the Halabi nomogram [50]. 
4. 
Randomization – treatment arm assignment  
5. 
Baseline research leukapheresis (50-100 mL approximate total volume, for 
immunological monitoring, not for preparation of sipuleucel-T) – to take place 2 
weeks (+/- 7 days) prior to day 1.   
6. 
Tetanus immunization (to take place after baseline leukapheresis and prior to day 
1) 

 
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7. 
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 
 
C. 
Peripheral Blood Collection for Sipuleucel-T – Day 1 
 
1. 
Standard 1.5-2.0 liter peripheral blood cell leukapheresis – conducted at 
Dendreon-authorized center for sipuleucel-T collection. 
 
D. 
Sipuleucel-T Infusion #1 – Day 4  
 
1. 
Reinfusion of sipuleucel-T product – conducted at UWHC – standard-of-care 
protocol 
2. 
Monitor patient for 30 minutes after completing infusion.  Monitoring should 
include vital signs (including temperature, heart rate and blood pressure) and brief 
symptoms assessment by nursing staff prior to infusion and 30 minutes after 
infusion complete.   
 
E. 
Peripheral Blood Collection for Sipuleucel-T – Day 15 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score 
(these can occur up to 72 hours prior to leukapheresis) 
2. 
Blood draw for CBC with differential and platelets (can occur up to 72 hours prior 
to leukapheresis) 
3. 
Standard 1.5-2.0 peripheral blood cell leukapheresis – conducted at Dendreon-
authorized center for sipuleucel-T collection. 
 
F. 
Sipuleucel-T Infusion #2 – Day 18 
 
1. 
Reinfusion of sipuleucel-T product – conducted at UWHC – standard-of-care 
protocol 
2. 
Monitor patient for 30 minutes after completing infusion.  Monitoring should 
include vital signs (including temperature, heart rate and blood pressure) and brief 
symptoms assessment by nursing staff prior to infusion and 30 minutes after 
infusion complete.   
 
G. 
Peripheral Blood Collection for Sipuleucel-T – Day 29 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score (can 
occur up to 72 hours prior to leukapheresis) 
2. 
Blood draw for CBC with differential and platelets (can occur up to 72 hours prior 
to leukapheresis) 
3. 
Standard 1.5-2.0 peripheral blood cell leukapheresis – conducted at Dendreon-
authorized center for sipuleucel-T collection. 
 
H. 
Sipuleucel-T Infusion #3 – Day 32 
 

 
 9-3/2015Version 
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1. 
Reinfusion of sipuleucel-T product – conducted at UWHC – standard-of-care 
protocol 
2. 
Monitor patient for 30 minutes after completing infusion.  Monitoring should 
include vital signs (including temperature, heart rate and blood pressure) and brief 
symptoms assessment by nursing staff prior to infusion and 30 minutes after 
infusion complete.   
 
I. 
Sipuleucel-T Follow-Up Visit Day 43 (+/- 3 days) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score  
2. 
Blood draw for CBC with 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 
 
 
ARM 2 SUBJECTS ONLY:  DNA Immunization #1: 
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 and injection site inspection.   
 
J. 
ARM 2 SUBJECTS ONLY: DNA Immunization #2 – Day 57 (+/- 3 days)  
 
1. 
Immunization/GM-CSF and post-treatment monitoring as described above in 
section 9.I.4-5 
 
K. 
ARM 2 SUBJECTS ONLY:  DNA Immunization #3 – Day 71 (+/- 3 days) 
 
1. 
Immunization/GM-CSF and post-treatment monitoring as described above in 
section 9.I.4-5 
 
L. 
3-Month Follow-up – Day 85 (+/- 7 days) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score. 
2. 
Blood draw for CBC with differential and platelets, 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) 
ARM 2 SUBJECTS ONLY: DNA Immunization #4:  
5. 
Immunization/GM-CSF and post-treatment monitoring as described above in 
section 9.I.4-5 
 

 
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M. 
6-month Follow-up – Week 24 (+/- 7 days) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score 
2. 
Blood draw for CBC with differential and platelets, chemistry panel, serum PSA 
and PAP (can occur up to 7 days prior to leukapheresis) 
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).  If leukapheresis is 
not able to be obtained, then up to 150 ml of peripheral blood (green-top 
heparinized tubes) may be obtained via peripheral blood draw for the T-cell 
response evaluation. 
ARM 2 SUBJECTS ONLY: DNA Immunization #5 
6. 
Immunization/GM-CSF and post-treatment monitoring as described above in 
section 9.I.4-5 
 
N. 
9-Month Follow-up – Week 36 (+/- 7 days) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score. 
2. 
Blood draw for CBC with differential and platelets, 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) 
ARM 2 SUBJECTS ONLY: DNA Immunization #6 
5. 
Immunization/GM-CSF and post-treatment monitoring as described above in 
section 9.I.4-5 
 
O. 
OFF STUDY VISIT AND/OR 12-Month Follow-up – (week 48 +/- 7 days) 
 
1. 
Physical examination, symptoms assessment, and ECOG performance score. 
2. 
Blood draw for CBC with differential and platelets, 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) 
 
P. 
Long-term Follow-Up 
 

 
 9-3/2015Version 
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1. 
Subjects will be contacted by telephone (if not already being seen in clinic) 
annually (from registration) for up to 5 years 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. 
Immunological Monitoring 
 
Blood will be collected by either peripheral blood draw (up to 150 mL) or leukapheresis (50-100 
mL) pre-immunization, after the initial three sipuleucel-T treatments, and at 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 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.  
Some assays may assess immune responses to the PA2024 (PAP-GM-CSF) fusion antigen or to 
GM-CSF. 
 
A.1. 
Quantitative assessment of PAP-specific CD8+ T-cell effector immunity 
 

 
 9-3/2015Version 
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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 < 1:10,000 cells) and also permits simultaneous analysis of 
cryopreserved batched specimens [23].  IFNγ and granzyme B will be preferred analytes 
evaluated, as these are specifically associated with inflammatory/tissue-destructive (Th1-type, 
cytolytic) immune responses (see Figure 3).  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), 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 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.  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.   
 
A.2. 
Assessment of PAP-specific memory T-cell immunity 
 
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 (see Figure 3).  Specifically, PBMC will be labeled in vitro with 
PKH26 dye (Sigma, St. Louis, MO) according to 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 1 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 

 
 9-3/2015Version 
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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.   
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.  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.   
 
A.3. 
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 will be evaluated by ELISA 
using an indirect method similar to that described previously [26].  Specifically, Immulon-4 
ELISA plates (Dynex Technologies Inc.) will be coated with 2 µg/ml purified PAP protein 
(Research Diagnostics, Inc.) 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 [26]. 
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). 
 
A.4. 
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.  Antigen–driven swelling will be determined as previously 
described [52].  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 

 
 9-3/2015Version 
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swelling will be defined as the antigen-specific swelling subtracted for 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), or 1 µg of mouse anti-human CTLA-4 
monoclonal Ab (clone AS32, Ab Solutions, Mountain View, CA) 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 [53].  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.   
 
A.5. 
Assessment of antigen-spread to other prostate-associated antigens 
 
High-throughput immunoblot (HTI):  A secondary 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.  Exploratory studies will 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 [54-56].  IgG specific for 126 antigens, including 29 cancer-
testis antigens [57, 58] and 97 prostate antigens frequently immunologically recognized [59-62] 
will be identified by screening a high-density phage array expressing these individual antigens, 
as we have previously reported [54, 56, 57].  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 if 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.  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.   
 
B. 
Circulating Tumor Cell (CTC) Enumeration 
 
CTC will be enumerated at the same time points as for immune evaluation (pre-treatment, after 6 
weeks, and at quarterly intervals for up to one year) using an ELISPOT methodology as 
described above, however using capture and detection antibodies specific for PSA [63].  This 
methodology has been used to count PSA-secreting cells directly from peripheral blood with a 
frequency < 1 per million PBMC.  Given the rarity of these cells, they will not be normally 
distributed among individual wells of an ELISPOT plate.  Hence, PBMC will be plated at 

 
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200,000 cells/well of a 96-well plate, and all spots will be counted (i.e. screening for events 
among ~20 x 106 total PBMC).  Multiple replicates to determine standard deviation are not 
feasible.  Other methods of CTC enumeration, such as by flow cytometry, may also be used. 
 
C. 
Serum PSA Doubling Time Response 
 
See Section 12 for further details.  Serum PSA doubling-time response will be considered a 
secondary endpoint.  The pre-treatment serum PSA doubling time will be calculated from all 
serum PSA values available for the three-month period prior to study treatment, not on other 
treatments for prostate cancer, up to and including the PSA value obtained at screening, using the 
Memorial 
Sloan-Kettering 
Cancer 
Center 
nomogram 
(http://www.mskcc.org/applications/nomograms/prostate/PsaDoublingTime.aspx).  The post-
treatment serum PSA doubling time will be calculated using the same algorithm and using all 
serum PSA values available during the time patients are on study beginning with the PSA value 
obtained at day 1.   
REPORTING AND RESPONSE DEFINITION:  PSA doubling times for the pre-treatment and 
post-treatment periods will be defined as above.  An increase in the PSA doubling time to at least 
double the baseline value will be defined as a PSA doubling time “response” [64]. 
 
 
11. 
Reporting Adverse Events 
 
Please see section 14 below, which contains in detail the policy and procedures for patient safety 
monitoring and adverse event reporting. 
 
A. 
Definition 
 
An adverse event is defined as any unfavorable and unintended sign (including abnormal 
laboratory finding unless they are grade 1 and deemed clinically insignificant), symptom or 
disease temporally associated with a medical treatment or procedure, regardless of whether it is 
considered related to the treatment or procedure. Note that for this study grade 1 laboratory 
values deemed clinically insignificant will not be reported as adverse events.  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.  
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 that the event is believed to be at least 
possibly related to study treatment.  Serious adverse events are any events occurring that result in 
any of the following outcome: 
 
1. 
Subject death 
2. 
Life-threatening adverse event 
3. 
In patient hospitalization or prolongation of existing hospitalization 
4. 
Persistent or significant disability/incapacity 
5. 
Congenital anomaly or birth defect 
 

 
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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 
 
4 
Life-threatening or disabling adverse event 
 
5 
Fatal adverse event 
 
B. 
Procedure for Reporting Adverse Events 
 
Regulations defining the responsibilities for reporting adverse events are defined by the National 
Cancer Institute Cancer Therapy Evaluation Program (NCI CTEP, http://ctep.info.nih.gov).  
Please see the details of adverse event reporting in Section 14.C. 
 
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 treatment-related will be repeated at appropriate intervals 
until the course is determined or a return to normal values occurs.  Information will be recorded 
as noted above.   
 
 
12. 
Statistical Considerations 
 
A. 
Overview 
 
This a pilot trial to evaluate the immunological and clinical effect of booster immunizations with 
a DNA vaccine encoding PAP with rhGM-CSF adjuvant following treatment with sipuleucel-T. 
The rationale for conducting this trial is summarized in Section 2 (E). 
 
B. 
Objectives 
 
The primary objective of this study is to evaluate whether immunization with a DNA vaccine 
encoding prostatic acid phosphatase (PAP, pTVG-HP) can augment PAP-specific effector and 
memory T cells following treatment with sipuleucel-T.  
 
The secondary objectives are: 
1. To determine 12-month progression-free survival  
2. To determine median time-to-progression as a function of baseline time point 
(pretreatment or 3-months post treatment) 
3. To evaluate effects of treatment on changes in serum PSA level and changes in PSA 
doubling time  
 
The laboratory and clinical exploratory objectives are: 
1. To determine overall survival 

 
 9-3/2015Version 
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2. To evaluate effects of treatment on number of circulating tumor cells as determined by 
PSA ELISPOT 
3. To evaluate PAP-specific antibody and T-cell immune responses following treatment 
with sipuleucel-T and DNA vaccine 
4. To determine whether PAP-specific immune response is associated with prolonged (1 
year) progression-free survival 
5. To determine whether baseline immune responses (Th1 type or regulatory type) predict 
for immune responses elicited/augmented following treatment with sipuleucel-T +/- DNA 
vaccine 
6. To determine whether treatment elicits immunologic antigen spread to other prostate 
associated antigens 
 
C. 
Definitions/Endpoints 
 
C.1. 
Primary Immunological Endpoint: Immune Response 
 
An immune response will be defined based on the detection of augmented numbers of PAP-
specific effector (IFNγ-secreting and/or granzyme B-secreting) and/or memory (proliferating) T-
cell immune response after immunization, using blood obtained at 6 months for the primary 
analysis compared with pre-treatment.  Specifically, a positive antigen-specific response 
resulting from immunization will be defined as a PAP-specific response by two or more of these 
three tests detectable at the 6-month post-treatment time point that is significantly (p<0.05, two-
sided using a two-sample t-test) higher than to media only, at least 3-fold higher than the mean 
baseline value, and with a frequency > 10 per 106 PBMC, based on 8-well replicate assays (for 
ELISPOT-based assays) or triplicate assays (for proliferation analysis).  Otherwise, a patient will 
be declared as an immune non-responder.  
  
Immune response will be determined as a static time point at 6 months, and “durable” immune 
responses will also be determined by the presence of a PAP-specific immune response by the 
criteria above detectable at 3, 9, or 12 months after study entry.   
 
C.2. 
Secondary Clinical and Immunological Endpoints 
 
C.2.1. Time to 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. 
 

 
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The rationale for defining time to progression as a function of baseline time points (date of 
randomization and 3-month disease assessment) is described in Section 6 (B). 
 
C.2.2. Twelve-month Progression Free Survival Rate 
 
The 12-month progression free survival rate will be defined as the proportion of patients 
progression free at the 12 month (post-randomization) disease assessment time point. 
 
C.2.3. Overall 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. 
 
C.2.4. PSA Response 
 
PSA complete response will be defined a decrease in PSA to <0.2 ng/mL.  The decrease must be 
confirmed with PSA measurement a minimum of four weeks later (confirmed PSA CR).  PSA 
partial response will be defined as reduction of at least 50% in baseline PSA.  
 
C.2.5. PSA Doubling Time 
 
PSA doubling time (PSADT) will be defined as the time it takes for the PSA value to double.  
PSADT will be calculated for each patient pre-treatment and post-treatment.  The pre-treatment 
PSADT will be calculated from all available values obtained within 3-6 months prior to study 
enrollment, up to and including the baseline value. The post-treatment PSADT will be calculated 
from all PSA values obtained beginning with the PSA value at day 1 and continuing until the 
end-of study value.  The pre-treatment and post-treatment PSADT will be calculated for each 
patient as the log 2 divided by the slope of the log PSA line, based on the linear regression model 
of log (PSA value) on time. 
 
C.2.6. Evaluable for Immune Response 
 
All patients who complete sipuleucel-T treatment at weeks 0, 2, and 4, and who have an immune 
response assessment at month 6 will be evaluable for immune response.  
 
C.2.7. Analysis Populations 
 
The ITT population consists of all patients who are randomized.  The per-protocol population is 
defined as all patients who have been treated according to protocol, i.e., all inclusion/exclusion 
criteria are satisfied, and adequate treatment compliance of sipuleucel-T treatment and vaccine 
(pTVG-HP) is achieved.  
  
D. 
Study Design 
 
The study is designed as a randomized, two-arm, open-label, single institution pilot trial.  Study 
arms will be defined as follows: 
 

 
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Arm 1: 
sipuleucel-T – administered intravenously (i.v.) per standard of care biweekly, 
weeks 0, 2, and 4. 
 
Arm 2: 
sipuleucel-T – administered intravenously (i.v.) per standard of care biweekly,  
weeks 0, 2, and 4  
pTVG-HP (100 µg) with rhGM-CSF (208 µg) administered intradermally (i.d.) 
biweekly 4 times, then every 3 months to complete a 1-year treatment period (i.e. 
weeks 6, 8, 10, 12, then months 6, 9) 
 
Eligible subjects will be randomly assigned in 1:1 fashion to either Arm 1 or Arm 2.  
 
E. 
Randomization and Stratification 
 
Eligible patients will be randomized to the two study arms with an allocation ratio of 1:1. The 
randomization will be based on permutated blocks of size 2-4, stratified by estimated survival (< 
18 months, > 18 months) using the Halabi nomogram  [50]. 
 
F. 
Sample Size Estimation and Accrual 
 
The endpoint of this pilot trial will be an immunological endpoint, i.e., the immune response rate 
at 6 months.  Based on published results from a randomized trial using sipuleucel-T only in this 
population, in which proliferative T-cell responses specific for PAP were detected in 15 of 55 
(27.3%) patients after immunization [7], and results from a phase I trial with this DNA vaccine 
only in which PAP-specific cytolytic T-cell responses were amplified after immunization in 7 
out 12 (58%) evaluable patients [23], it is anticipated that the immune response rate in the 
sipuleucel-T alone arm (Arm 1) will be 25% and that the addition of the pTVG-HP vaccine (Arm 
2) will boost the response rate to at least 75%.  An increase of 50% in the immune response rate 
will be considered as a clinically important difference.  The following table shows the required 
sample sizes (per arm) to detect various differences in the immune response rates between the 
two arms with 80% power at the one-sided 5% significance level.  These calculations are based 
on the stratified Mantel-Haenszel test with exact p-values. 
 
Table 12.1 Required sample sizes (per arm) to detect various differences in the immune response 
rates between the two arms with 80% power at the two-sided 5% significance level 
 
 
 
Immune Response Rate: Arm 2 
(sipuleucel-T + pTVG-HP vaccine arm) 
Immune Response  
Rate Arm 1  
(sipuleucel-T alone 
arm) 
70% 
75% 
80% 
85% 
15% 
12 
10 
9 
9 
20% 
15 
12 
10 
9 
25% 
18 
14 
12 
10 
30% 
23 
18 
15 
12 
 

 
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A sample size of 14 patients per arm is proposed for this trial.  This sample size is sufficient to 
detect an anticipated difference in the immune response rate of at least 50% (i.e., ≥75% in Arm 2 
vs. 25% in Arm 1) with adequate power (≥80%) at the one-sided 5% significance level.  The 
following table shows the attainable power levels to detect various differences in the immune 
response rates between arms at the one-sided 5% significance level, assuming a sample size of 
28 evaluable (for immune response) patients. 
 
Table 12.2: Attainable power levels to detect various differences in the immune response rates 
between the two arms at the one-sided 5% significance level, assuming a sample size of 14 
evaluable patients per arm 
 
 
Immune Response Rate: Arm 2 
(sipuleucel-T + pTVG-HP vaccine arm) 
Immune Response  
Rate Arm 1 
(sipuleucel-T alone 
arm) 
70% 
75% 
80% 
85% 
15% 
85% 
91% 
96% 
98% 
20% 
76% 
85% 
91% 
96% 
25% 
65% 
80% 
84% 
91% 
30% 
53% 
65% 
76% 
85% 
 
A difference in the immune response rate of 55% between the two arms will be detected with at 
least 84% power.  Analogously, a difference in the efficacy response rate of only 40% between 
the two arms will be detected with 53% power at the one-sided 5% significance level.  In order 
to account for unevaluable patients, 30 eligible patients (15 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 30 patients will be accrued within 18 months.  Each 
patient will be followed for at least 12 months.  
 
G. 
Statistical Analysis Plan 
 
G.1. 
General 
 
This section outlines the statistical analysis strategy and procedures for the study.  If after the 
study has begun changes are made to primary and/or key secondary objectives, or the statistical 
methods related to those objectives, then the protocol will be amended. 
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 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 9.2 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 

 
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multiple comparisons.  Except for the primary immunological endpoint, where the comparison 
between arms will be conducted at the one-sided 0.05 significance level, all other comparisons 
will be made using a two-sided test at a significance level of 0.05 without adjustment for 
multiplicity. 
 
G.2. 
Baseline Comparability 
 
All measurements (variables) collected at baseline will be summarized and compared between 
study arms.  These include demographic variables, lab parameters (CBC and platelets, creatinine, 
alk phos, SGOT, total bilirubin, amylase, LDH, serum testosterone and PSA and PAP) and 
medical history information.  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.  
 
G.3. 
Analysis of the Primary Immunological Endpoint:  Immune Response Rate 
 
Immune responses, obtained at 6 months, will be summarized in tabular format.  Ninety-five 
percent confidence intervals for the proportions of patients with an immune response will be 
constructed for each study arm.  The comparison of immune response rates between study arms 
will be performed using the Cochran-Mantel-Haenszel test, stratified by estimated survival (< 18 
months, > 18 months) using the Halabi nomogram [50].  Due to the small sample size, exact p-
values will be computed using Zelen’s exact test for equal odds ratios for stratified 2 X 2 tables.  
 
As a secondary analysis, “durable” immune responses will be determined by the presence of 
immune responses at one or more of the 3, 9, and 12 month time points evaluated after 
randomization.  Immune responses determined at the 3, 9 and 12 month time points will be 
summarized in tabular format and compared between study arms using the Cochran-Mantel-
Haenszel test, stratified by estimated survival (< 18 months, > 18 months) using the Halabi 
nomogram [50].  Furthermore, a generalized linear mixed effects model for binomial data with a 
logit link function will be employed to evaluate the longitudinal effects of immune responses 
measured at the 3-month intervals.  
 
G.4. 
Analysis of Secondary and Exploratory Clinical and Immunological Endpoints 
 
G.4.1. Analysis of 12-month Progression-Free Survival Rate 
 
The 12-month progression-free survival rate will be estimated for each arm using the Kaplan-
Meier method.  The 12-month progression-free survival rate, along with the corresponding two-
sided 95% confidence intervals, will be reported for each arm.  In order to evaluate the 12-month 
progression-free rate 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.  
 
G.4.2. Analysis of Progression-Free Survival  
 

 
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Progression-free survival will be estimated for each arm using the Kaplan-Meier method.  The 
median progression-free survival, along with the corresponding two-sided 95% confidence 
intervals, will be reported for each arm.  The comparison of progression-free survival between 
study arms will be performed using the stratified (by estimated survival at baseline, < 18 months 
vs. > 18 months) log-rank test.  In order to evaluate progression-free rate 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.  
 
G.4.3. Analysis of Overall Survival  
 
Overall survival will be estimated for each arm using the Kaplan-Meier method.  The median 
overall survival, along with the corresponding two-sided 95% confidence intervals, will be 
reported for each arm.  The comparison of overall survival between study arms will be 
performed using the stratified log-rank test. 
 
G.4.4. Analysis of Changes in PSA Values, PSA Kinetics and PSA Doubling 
Time 
 
PSA values will be summarized using standard descriptive statistics for each defined assessment 
time point (baseline, 3, 6, 9, and 12 months) in terms of number of observations, means, standard 
deviations, medians and ranges, stratified by study arm.  Percentage changes from baseline to the 
3, 6, 9, and 12 months assessment will be calculated.  Changes within each arm will be evaluated 
using a paired t-test or nonparametric Wilcoxon Signed Rank test, if the percentage changes are 
not normally distributed. Analysis of variance, which will include study arm and baseline 
predicted survival (< 18 months vs. ≥ 18 months) will be used to compare percentage changes in 
PSA values between study arms.  If the percentage changes are not normally distributed and if a 
normality-improving data transformation is unavailable, then the stratified, nonparametric 
Wilcoxon rank-sum tests will replace the planned ANOVA.  Changes in PSA levels will be 
presented graphically using waterfall plots.  
 
PSA response rates (CR / PR) will be summarized in tabular format and compared between study 
arms using the Cochran-Mantel-Haenszel test, stratified by baseline estimated survival (< 18 
months vs. ≥ 18 months). 
 
PSA doubling time for each patient is estimated as: PSADT = log(2)/b, where b is the OLS 
estimator of slope parameter of the linear regression model log(PSA) = α +β*month + ε.  This 
method may result in negative PSA doubling time estimates.  For analysis purposes, negative 
PSA doubling time estimates will be replaced by fixed positive PSA doubling time values (e.g., 
by the largest observed positive PSA doubling time value).  Serum PSA doubling time will be 
summarized for each study arm by standard descriptive statistics in terms of means, standard 
deviations, medians and ranges.  Percentage changes between pre-treatment and post-treatment 
PSA doubling times will be computed.  Changes between pre- and post-treatment serum PSA 
doubling time within each arm will be assessed using a paired t-test and/or non-parametric 
Wilcoxon Signed Rank test.  A two-sample t-test or non-parametric Wilcoxon Rank Sum test 
will be used to compare the changes in PSA doubling time from the pre- to the post-treatment 
assessments between study arms.  

 
 9-3/2015Version 
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G.5. 
Correlative Analyses 
 
G.5.1. Analysis for evaluating effects of treatment on the number of circulating 
tumor cells as determined by PSA ELISPOT 
 
The number of circulating tumor cells per million PBMC will be summarized in terms of 
medians and ranges, stratified by study arm and assessment time point.  The nonparametric 
Wilcoxon Signed Rank test will be used to evaluate the effects of treatment on the number of 
circulating tumor cells within each study arm.  The comparison of changes in the number of 
circulating tumor cells between study arms will be performed using the nonparametric, stratified, 
Wilcoxon Rank Sum test.  Results will be presented graphically over time, and trends (increase, 
decrease, stable) will be assessed over time.   
 
G.5.2. Analysis for evaluating PAP-specific antibody and T-cell immune 
responses following treatment with sipuleucel-T and DNA vaccine 
 
PAP-specific antibody and T-cell immune responses, evaluated by multiple different measures 
described in Section 10.A, following treatment with sipuleucel-T and DNA vaccine will be 
summarized in tabular format. 
 
G.5.3. Analysis for evaluating whether PAP-specific immune response is 
associated with prolonged (1 year) progression-free survival 
 
Logistic regression analysis will be conducted to evaluate whether PAP-specific immune 
response is associated with prolonged (1-year) progression-free survival. The dependent 
variables will be 12-month progression free survival while treatment arm and PAP-specific 
immune responses (no vs. yes) will be included as covariates.  The results will be reported in 
terms of odds ratios and the corresponding 95% confidence intervals.  
 
G.5.4. Analysis for evaluating whether baseline immune responses (Th1 type or 
regulatory type) predict for immune responses elicited/augmented 
following treatment with sipuleucel-T +/- DNA vaccine 
 
Logistic regression analysis will be conducted to evaluate whether baseline immune responses 
(Th1-type or regulatory type) predict for immune responses elicited/augmented following 
treatment with sipuleucel-T +/- DNA vaccine.  6-month and durable immune response at 3,6 or 
12 months will be included as dependent variables in this analysis while treatment arm and 
baseline PAP-specific immune responses (no vs. yes) will be included as covariates.  Odds ratios 
and the corresponding 95% confidence intervals will be reported.  
 
G.5.5. Analysis for evaluating whether treatment 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.  In addition, logistic regression 
analysis will be conducted to evaluate whether antigen spread is associated with the generation 

 
 9-3/2015Version 
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of PAP-specific immune responses elicited as a result of treatment.  The dependent variables will 
be the detection of IgG responses to > 1 “off-target” antigen at 6 months (and not present pre-
treatment) while treatment arm and 6-month PAP-specific immune responses (no vs. yes) will be 
included as covariates.  The results will be reported in terms of odds ratios and the corresponding 
95% confidence intervals.  
 
 
13. 
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.  Lymphocytes collected will be stored in liquid nitrogen, and sera will be 
aliquoted and stored at –80°C in the research laboratory of the PI for immune analysis.  
 
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 local 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.   
 
 
14. 
Data and Safety Monitoring Plan 
 
A. 
Oversight and Monitoring Plan 
The UWCCC Data and Safety Monitoring Committee (DSMC) is responsible for 
monitoring data quality and subject safety for all UWCCC clinical studies.  A summary 
of DSMC activities follows: 
• 
Review of all clinical trials conducted at the UWCCC for data integrity and safety 
• 
Review of all serious adverse events requiring expedited reporting as defined in the 
protocol 
• 
Review of reports generated by the UWCCC data quality control review process  
• 
Submit recommendations for corrective action to the CRC 

 
 9-3/2015Version 
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• 
Notify the  Local Principal Investigator of the DSMC’s recommendation to the CRC 
• 
Work in conjunction with the Health Sciences IRB in the review of protocol deviations, 
violations and unanticipated problems reported by the UWCCC DOWGs.  
• 
The committee ensures that notification is provided to all external sites participating in 
multiple-institutional clinical trials coordinated by the UWCCC of serious adverse events 
requiring expedited reporting. 
 
Phase II Trials 
 
Data related to these trials are discussed at regularly scheduled Disease Oriented Working Group 
meetings where the result of each subject’s treatment is discussed and the discussion is 
documented in the minutes. The discussion will include for each treatment arm/dose level, the 
number of subjects, significant toxicities as described in the protocol, dose adjustments, and 
responses observed.  Protocol Summary Reports are required for submission to the Data and 
Safety Monitoring Committee for review. 
 
B. 
Review and Oversight Requirements 
 
B.1. 
Serious Adverse Event  – Reported Within 24 Hours : 
Serious Adverse Events requiring reporting within 24 hours (as described in 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.  A 24 hr. initial “SAE Details” Report, 
generated in the UWCCC database, must be attached to the email along with any pertinent 
information available at the time of initial reporting. The DSMC Chair will review the 
information and determine if immediate action is required. Within 10 working days, all 
subsequent SAE documentation must be submitted electronically along with a 24 hour follow-up 
“SAE Details” Report and a completed UWCCC SAE Routing Form to 
saenotify@uwcarbone.wisc.edu. All information is entered and tracked in the UWCCC database. 
If the SAE occurs on a multiple-institutional clinical trial coordinated by the UWCCC, the 
Outreach Coordinator will ensure that all participating sites are notified of the event and resulting 
action within one working day of the determination. 
See Section D for detailed instructions on SAE reporting. 
 
B.2. 
Serious Adverse Event  – Reported within 10 Days 
Serious Adverse Events requiring reporting within 10 working days (as described Table D-2l) 
will also be sent to the UWCCC DSMC Chair via email to saenotify@uwcarbone.wisc.edu. A 10 
day “SAE Details” report, generated in the UWCCC database must be attached to the email 
along with pertinent information regarding the SAE and the UWCCC SAE Routing Form. The 
Committee Chair will review the information and determine if further action is required.  This 
information is entered and tracked in the UWCCC database. 
If the SAE occurs on a multiple-institutional clinical trial coordinated by the UWCCC, the 
Outreach Coordinator will ensure that all participating sites are notified of the event and resulting 
action within one working day of the determination. 
 

 
 9-3/2015Version 
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See Section D for detailed instructions on SAE reporting. 
 
C. 
Study Progress Review 
Protocol Summary Reports (PSR) are required to be submitted to the DSMC commensurate with 
the Phase of the study. The PSR provides a cumulative report of serious adverse events, as well 
as any protocol violations, deviations or unanticipated problems, toxicities and responses that 
have occurred on the protocol in the timeframe specified. PSRs are reviewed at each DSMC 
meeting. 
 
Protocol Summary Reports enable DSMC committee members to assess whether significant 
benefits or risks are occurring that would warrant study closure. This information is also 
provided by Disease Oriented Working Group meeting minutes, internal audit and/or response 
review reports.  In addition, the DSMC requires the DOWG or Local Principal Investigator to 
submit external DSMB reports or any other significant 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.  The DSMC ensures that the Local  PI reports any 
temporary or permanent suspension of a clinical trial to the sponsor (e.g., NCI Program Director, 
Industry Supporter(s), Cooperative Group Study Chair, etc.) and other appropriate agencies. 
 
D. 
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.   
 
D.1. 
 SAE Requiring 24 Hour Reporting Occurs at UWCCC: 
 
a.    
To the FDA and NIH OBA: 
        
      
Report the SAE to the FDA using the FDA Med Watch form available online. 
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 reporting form available online. 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 must be reported to 
Dendreon within 24 hours of being made aware of the SAE. Notification can be 

 
 9-3/2015Version 
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made via phone or telefacsimile using an SAE Report Form to be provided by 
Dendreon. 
  
Dendreon Corporation  
Attn:  
Safety Manager 
Facsimile:  
(206) 829-1647 
Phone:  
(206) 219-7189 
After Hours: (206) 274-6774 
  
Significant new information regarding an ongoing SAE and the resolution must 
be sent to Dendreon within 3 business days of awareness of the new information 
to Dendreon on the SAE Report Form. 
 
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 
 
D.2. 
SAE Requiring 10 Day Reporting Occurs at UWCCC:  
 
a.    
To the FDA and NIH OBA  
        
Report the SAE to the FDA using the FDA Med Watch form available online. 
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 reporting form available online. 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 must be reported to 

 
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Dendreon within 24 hours of being made aware of the SAE. Notification can be 
made via phone or telefacsimile using an SAE Report Form to be provided by 
Dendreon. 
  
Dendreon Corporation  
Attn:  
Safety Manager 
Facsimile:  
(206) 829-1647 
Phone:  
(206) 219-7189 
After Hours: (206) 274-6774 
  
Significant new information regarding an ongoing SAE and the resolution must 
be sent to Dendreon within 3 business days of awareness of the new information 
to Dendreon on the SAE Report Form. 
 
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 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Expedited Reporting Table D-2 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE:  Investigators MUST immediately report to the study PI 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). 
 
 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization  
≥ 24 hrs 
10 Calendar Days 
24-Hour 5 
Calendar Days 
Not resulting in 
Hospitalization  
≥ 24 hrs 
Not required 
10 Calendar Days 
1Serious adverse events that occur more than 30 days after the last administration of investigational 
agent/intervention 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 4, and Grade 5 AEs 
Expedited 10 calendar day reports for: 
• Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• Grade 3 adverse events 
 
 
NOTE:  FOR THIS STUDY, GRADE 2 AUTOIMMUNE REACTIONS WILL BE 
TREATED AS GRADE 3 EVENTS FOR REPORTING PURPOSES. 
 
 
15. 
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 

 
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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 monitored for 60 minutes following each 
treatment with evaluation of vital signs and examination of the skin site of treatment.  Subjects 
will also be asked to keep a record of unusual site or other reactions for two days after 
immunization.   
 
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 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 a previous phase I 
clinical trial with this DNA vaccine.  However, 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.  While no autoimmune 
treatment-limiting adverse events have been observed to date, it is conceivable that by using a 
prime-boost strategy with different immunization approaches targeting the same PAP antigen 
could lead to more autoimmune events than either 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 [65].  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 [47].  
Uncommon and rare side effects, which have been less frequently observed with daily 

 
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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. 
 
A.3. 
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.4. 
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.5. 
From tetanus immunization 
 
A tetanus booster vaccine will be delivered prior to receiving sipuleucel-T 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.6. 
From sipuleucel-T 
 
Sipuleucel-T has been given to over 1000 patients with castrate-resistant metastatic prostate 
cancer.  In the phase III IMPACT trial that led to its approval, the primary adverse events 
experienced by patients receiving sipuleucel-T more commonly than placebo included fever, 
chills, nausea, emesis, headache, dizziness, pain, myalgias, hypertension, sweating, and 
influenza-like symptoms, all typically of limited (1-2 day) duration.  Given that sipuleucel-T will 

 
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be given in the same population of subjects, and prior to the other experimental treatment, we do 
not expect other unusual adverse events from this treatment.   
 
B. 
Potential Benefits 
 
No benefits are guaranteed.  It is hoped that individual patients treated with the DNA vaccine 
following sipuleucel-T will derive a clinical response as suggested by a delay in time to disease 
progression.  In addition, sipuleucel-T is the standard of care for patient with this stage of 
disease, based on a demonstrated improvement in overall survival.  Hence it is hoped that further 
booster immunization will provide additional benefit that could be demonstrated by an 
improvement in 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.  Thus, it is hoped that future patients will benefit 
from research participation in the current study. 
 
 
16. 
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 (Jane Straus RN, Dottie Horvath RN, Mulusew Yayehyirad, RN 
or Mary Jane Staab RN) 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.  

 
 9-3/2015Version 
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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 On-line Clinical Oncology Research Environment (ONCORE). 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 ONCORE.  The system will prompt the user to the forms that are required based 
upon the patient’s enrollment and treatment dates. 
 
The Local 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.   
 
 
Long-term follow-up form 
 
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 maintained in individual laboratory research 
charts for each subject.  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 Drs. McNeel, Liu, Kyriakopoulos, Bruce, Lang and Wilding, 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, Dr. 
Jens Eickhoff, 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, 

 
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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 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.  This research database will only be available to the study investigators, 
not other laboratory personnel, to maintain confidentiality.  Subject sera will be maintained 
indefinitely in the laboratory of the study principal investigator, stored in aliquots at –20°C to –
80°C.  Peripheral blood mononuclear cells will be stored indefinitely in the laboratory of the 
study principal investigator, stored in aliquots in liquid nitrogen. 
 
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, repeated 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 
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. 
 

 
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F.  
Roles and Responsibilities of Study Personnel at UWCCC 
 
Study Principal Investigator: 
 
Dr. Douglas McNeel MD PhD, Associate 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 several other clinical trials, and 
has had formalized training in the ethics and conduct of clinical trials and human subjects 
protection.  He will be overall responsible for the conduct of the trial and its analysis, but will not 
participate in patient accrual, treatment of patients on study, or interpretation of clinical data.  Dr. 
McNeel’s involvement will center on the scientific aspects of the project, including overall trial 
design and analysis of samples from the trial.  Dr. McNeel will participate as author on any 
manuscripts derived from this research. 
 
UWCCC Local 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 be the UWCCC local 
principal investigator for this study.  As UWCCC local PI he will be responsible for activities 
involving direct patient care and interaction, including recruitment, consent, clinical care and 
adverse event reporting. The UWCCC local PI will also assume the role of principal investigator 
on the protocol and UW HS-IRB submission, working with the UW HS-IRB as the responsible 
investigator to obtain and maintain UW HS-IRB approval for the study.  The UWCCC local PI 
will also be responsible for reviewing significant adverse events that occur at UWCCC and will 
oversee the care provided by the UWCCC co-investigators on this study. 

 
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17. 
References: 
 
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Oefelein M. G., Smith N. D., Grayhack J. T., Schaeffer A. J., and McVary K. T. (1997) 
"Long-term results of radical retropubic prostatectomy in men with high grade carcinoma 
of the prostate." J Urol 158:1460-1465. 
3. 
Crawford E. D., Eisenberger M. A., McLeod D. G., Spaulding J. T., Benson R., Dorr F. 
A., Blumenstein B. A., Davis M. A., and Goodman P. J. (1989) "A controlled trial of 
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Petrylak D. P., Tangen C., Hussain M., Lara P., and al. e. (2004) "SWOG 99-16:  
Randomized phase III trial of docetaxel (D)/estramustine (E) versus mitoxantrone 
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de Bono J. S., Oudard S., Ozguroglu M., Hansen S., Machiels J. P., Kocak I., Gravis G., 
Bodrogi I., Mackenzie M. J., Shen L., Roessner M., Gupta S., and Sartor A. O. (2010) 
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Redfern C. H., Ferrari A. C., Dreicer R., Sims R. B., Xu Y., Frohlich M. W., and 
Schellhammer P. F. (2010) "Sipuleucel-T immunotherapy for castration-resistant prostate 
cancer." N Engl J Med 363:411-422. 
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de Bono J. S., Logothetis C. J., Molina A., Fizazi K., North S., Chu L., Chi K. N., Jones 
R. J., Goodman O. B., Jr., Saad F., Staffurth J. N., Mainwaring P., Harland S., Flaig T. 
W., Hutson T. E., Cheng T., Patterson H., Hainsworth J. D., Ryan C. J., Sternberg C. N., 
Ellard S. L., Flechon A., Saleh M., Scholz M., Efstathiou E., Zivi A., Bianchini D., Loriot 
Y., Chieffo N., Kheoh T., Haqq C. M., and Scher H. I. (2011) "Abiraterone and increased 
survival in metastatic prostate cancer." N Engl J Med 364:1995-2005. 
9. 
Kirk T. N. and Moyad M. A. (2006) "National survey of advanced prostate cancer 
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McNeel D. G. (2007) "Prostate cancer immunotherapy." Curr Opin Urol 17:175-181. 
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McNeel D. G. (2007) "Cellular immunotherapies for prostate cancer." Biomed 
Pharmacother 61:315-322. 
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Ward J. E. and McNeel D. G. (2007) "GVAX: an allogeneic, whole-cell, GM-CSF-
secreting cellular immunotherapy for the treatment of prostate cancer." Expert Opin Biol 
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Alam S. and McNeel D. G. (2010) "DNA vaccines for the treatment of prostate cancer." 
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14. 
Olson B. M. and McNeel D. G. (2011) "Sipuleucel-T:  immunotherapy for advanced 
prostate cancer." Open Acc. J. Urol. 3:49-60. 

 
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Gutman A. B. and Gutman E. B. (1938) "An acid phosphatase in the serum of patients 
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APPENDIX A 
 
NCI Common Terminology Criteria – Version 4 
 
http://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/ctcaev4.pdf 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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APPENDIX B 
 
 
Blood Draws for Research 
 
 
Screen 
Day 43 
Month 3 
Month 6 
Month 9 
Month 12 
 
Research 
Leukapheresis 
 
OR 
 
Alternative* 
(150mL – peripheral 
blood) 
 
50-
100mL 
 
 
 
 
N/a 
 
 
 
 
 
50-
100mL 
 
-----or---- 
 
 
150mL* 
 
 
Sera for Antibody 
20mL 
10mL 
10mL 
10mL 
10mL 
10mL 
Blood for CTC and/or 
T-cell response 
20 mL 
100mL 
100mL 
20mL 
100mL 
100mL 
Approximate Total 
90-
190mL 
110mL 
110mL 
80-
180mL 
110mL 
110mL 
* Peripheral blood draw may be done as an alternative to leukapheresis only at Month 6. Patients 
should not go on study if leukapheresis cannot be done at screening. 
Volumes in the table are approximate and are to be used for determining how many and which 
tubes are to be drawn per the protocol. Blood sera for antibody testing is to be collected 
in red top tubes and blood for CTC analysis/T-Cell response is to be collected in green 
top heparinized tubes.