Conditions: Prostate Cancer Adenocarcinoma in Situ
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 29
Healthy volunteers: no

Brief summary:
This research is being done to see if an investigational prostate cancer vaccine, called GVAX, can safely be given together with a single intravenous injection of a drug called cyclophosphamide to men that will undergo surgery to remove their cancerous prostate glands who have also received standard hormonal therapy.

Detailed description:
Cancer immunotherapy refers broadly to approaches which attempt to treat cancer by activating immune responses directed against malignant tissue. Prostate GVAX is an allogeneic cell-based prostate cancer vaccine composed of two irradiated cell lines (PC3 and LNCaP) that have been genetically modified to secrete granulocyte-macrophage colony-stimulating factor (granulocytemacrophage-colony stimulating factor). The release of granulocytemacrophage-colony stimulating factor by these modified tumor cells serves to recruit dendritic cells which then present tumor antigens to T-cells, thus initiating antitumor immune responses.

However, abundant preclinical data show that, when used alone, cell-based immunotherapy is unable to break specific T-cell tolerance in tumor-bearing hosts. Studies in an autochthonous prostate cancer mouse model have shown that giving low-dose cyclophosphamide prior to a cell-based granulocytemacrophage-colony stimulating factor-secreting vaccine abrogates immune tolerance through augmentation of CD8+ T cell infiltration in the prostate, transient depletion of regulatory T cells (Tregs), and increased expression of dendritic cell maturation markers. Enhancement of antitumor immunity has also been observed in other preclinical models where cyclophosphamide was given in sequence with granulocytemacrophage-colony stimulating factor-secreting immunotherapy for the treatment of breast and pancreatic cancers. These preclinical data are supported by early-phase clinical trials combining GVAX with low-dose cyclophosphamide in pancreatic and breast cancers.

Furthermore, emerging evidence suggests that androgen deprivation therapy (ADT) itself has profound effects on the host immune system, resulting in thymic regeneration and enhancement of antitumor immunity. In addition, preclinical and clinical studies demonstrate that ADT augments prostate cancer-specific immune responses induced by immunotherapy, suggesting that ADT may act synergistically with immunotherapy. Based on data from mouse models as well as human clinical trials, it has been suggested that prostate cancer immunotherapy may be most effective when administered in the setting of an androgen-suppressed environment.

Building on these findings, investigators have designed a study to assess the use of ADT given alone or administered following immunization with low-dose cyclophosphamide and prostate GVAX, in patients undergoing radical prostatectomy. Investigators aim (1) to determine whether ADT is immunogenic in men with localized prostate cancer by evaluating T-cell infiltration in harvested prostate glands; (2) to determine whether administering ADT after low-dose cyclophosphamide and prostate GVAX augments immune infiltration into the prostate gland; and (3) to investigate whether this combinatorial immuno-hormonal approach is safe and feasible. Investigators hypothesize that the combination of ADT and cyclophosphamide/GVAX will produce significantly greater antitumor immune responses than would ADT used alone.

Arms:
arm 1: Degarelix will be administered as three 80 mg subcutaneous injections, for a total dose of 240 mg at 14 (±3) days prior to surgery. A telephone follow-up interview (or an in-person clinic visit) to evaluate for adverse events will occur 28 (±21) days after prostatectomy. Patients will then be followed by their urologists according to standard institutional practices, but will require prostate-specific antigen evaluations every 3 (±1) months during year 1 and every 6 (±2) months during years 2-3. arm 2: Cyclophosphamide will be given at a dose of 200 mg/m2 as a single intravenous infusion. 1 day later, prostate GVAX will be administered as five 0.8-mL intradermal injections of PC3 (2.5 × 108 cells) and five 0.5-mL intradermal injections of LNCaP (2.5 × 108 cells), for a total dose of 5 × 108 cells. On day 14, Degarelix will be administered as three 80 mg subcutaneous injections, for a total dose of 240 mg.

Interventions: intervention 1: degarelix acetate intervention 2: Cyclophosphamide intervention 3: GVAX
Intervention descriptions: intervention 1: Degarelix Acetate is a gonadotropin-releasing hormone (GnRH) receptor antagonist. It works by decreasing the amount of testosterone in the body,which the tumor needs to grow. intervention 2: Cyclophosphamide as a potent enhancer of immune responses to GVAX. cyclophosphamide is used as an immune suppressor in many autoimmune disorders. intervention 3: GVAX is granulocytemacrophage -colony stimulating factor -secreting allogeneic cell-based vaccine as immunotherapy for prostate cancer

Protocol document:
Prot_SAP_000.pdf: 
 
J1265: A neoadjuvant immunologic study of androgen deprivation therapy 
combined with a GM-CSF–secreting allogeneic prostate cancer vaccine and low-
dose cyclophosphamide in men with high-risk localized prostate cancer 
undergoing radical prostatectomy 
 
NCT01696877 
 
 
 Protocol Date: November 30, 2012 
 
 
Principal Investigator: 
Emmanuel S. Antonarakis, MD 
 


Androgen suppression with prostate GVAX and cyclophosphamide 
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A neoadjuvant immunologic study of androgen deprivation therapy 
combined with a GM-CSF–secreting allogeneic prostate cancer 
vaccine and low-dose cyclophosphamide in men with high-risk 
localized prostate cancer undergoing radical prostatectomy 
 
IRB #: NA_00073453 IND #: 9678 
Johns Hopkins University 
 
Principal Investigator 
Statistician 
Emmanuel S. Antonarakis, MD (Protocol Author) 
Marianna Zahurak, MS 
The Bunting-Blaustein Cancer Research Building 
1650 Orleans Street, CRB1 - Room 1M45 
Baltimore, MD 21231 
Phone: (443) 287-0553 
Fax: (410) 614-8397 
Johns Hopkins Oncology Biostatistics 
550 North Broadway, Suite 1103 
Baltimore, MD 21205 
Phone: (410) 955-4219 
Fax: (410) 955-0859 
Email: eantona1@jhmi.edu 
Email: zahurma@jhmi.edu 
Co-Principal Investigator 
Study Coordinator 
Edward M. Schaeffer, MD, PhD 
Haiyi Cao  
The Bunting-Blaustein Cancer Research Building 
1650 Orleans Street, CRB2 – Room 5M43 
Baltimore, MD 21231 
Phone: (410) 502-2733 
Fax: (410) 614-0549 
550 North Broadway 
Suite 827 
Baltimore, MD 21205 
Phone: (443) 287-6882 
Fax: (410) 614-7287 
Email: eschaef4@jhmi.edu 
Email: hcao7@jhmi.edu  
Co-Investigators 
Charles Drake, MD, PhD(cdrake@jhmi.edu) 
Michael Carducci, MD (carducci@jhmi.edu) 
Mario Eisenberger, MD (eisenma@jhmi.edu) 
Samuel Denmeade, MD (denmesa@jhmi.edu) 
Hans Hammers, MD, PhD (hhammer2@jhmi.edu) 
Jenny Kim, MD (jkim366@jhmi.edu) 
Janet Walczak, CRNP (walczja@jhmi.edu) 
Victoria Sinibaldi, CRNP (sinibvi@jhmi.edu) 
Misop Han, MD (mhan1@jhmi.edu) 
Alan Partin, MD, PhD (apartin2@jhmi.edu) 
Mohamad Allaf, MD (mallaf@jhmi.edu) 
Gary Rosner, PhD (grosner1@jhmi.edu) 
Donna Dowling, RN (ddowlin1@jhmi.edu) 
Monica Taylor, RN (mtaylo27@jhmi.edu) 
Caroline Pratz, CRNP (fidykca@jhmi.edu) 
Robin Gurganus, RN (rgurgan1@jhmi.edu) 
George Netto, MD (gnetto1@jhmi.edu) 
Jennifer Uram, PhD (juram1@jhmi.edu) 
Connie Collins, RN (ccolli23@jhmi.edu) 
Rana Sullivan, RN (tomalra@jhmi.edu) 
Avery Spitz, RN (aspitz2@jhmi.edu) 
Sara Solt (ssolt1@jhmi.edu) 
Anthony Wamwea (awamwea1@jhmi.edu) 
 
Protocol Date: November 30, 2012 
Protocol Type / Version # / Version Date: Protocol / Version 1.4 / November 30, 2012 


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CONFIDENTIALITY STATEMENT 
The information in this document is provided to you as an investigator, potential investigator, 
consultant, or contractor, for review by you, your staff, and the appropriate Institutional Review 
Board or Ethics Committee. By accepting this document, you agree that the information contained 
herein will not be disclosed to others without written authorization from the lead site/sponsor, 
except to the extent necessary to initiate the study or conduct study-related activities. 


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SYNOPSIS 
Title 
A neoadjuvant immunologic study of androgen suppression combined with a GM-CSF–
secreting allogeneic prostate cancer vaccine and low-dose cyclophosphamide in men 
with high-risk localized prostate cancer undergoing radical prostatectomy 
Institution 
Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD 
PI(s) 
Emmanuel S. Antonarakis, MD (PI); and Edward M. Schaeffer, MD, PhD (Co-PI) 
Sponsor 
This is an investigator-sponsored trial. Charles Drake, MD, PhD is the IND-holder. 
IRB # 
NA_00073453 
Investigational 
agents 
1) GM-CSF–secreting cell-based (PC3/LNCaP) prostate cancer immunotherapy 
2) Cyclophosphamide, intravenous 
3) Degarelix acetate, subcutaneous 
Phase 
Immunologic study (phase I/II) 
Indication 
Men with high-risk localized prostate cancer, prior to radical prostatectomy 
Target 
population 
Inclusion criteria: 
• 
histologically confirmed prostate adenocarcinoma 
• 
clinical stage T1c–T3b, N0, M0 
• 
Gleason sum 7-10 
• 
at least 2 positive cores 
• 
prior decision to undergo radical prostatectomy 
• 
adult male > 21 years of age 
• 
ECOG performance status 0-1, or Karnofsky score ≥ 70% 
• 
adequate kidney, liver, and bone marrow function 
• 
willingness to sign informed consent and adhere to study requirements 
Exclusion criteria: 
• 
presence of known lymph node involvement or distant metastases 
• 
prior radiation, hormones, biologics, or chemotherapy for prostate cancer 
• 
prior immunotherapy/vaccine therapy for prostate cancer 
• 
previous or concurrent use of cyclophosphamide 
• 
concomitant treatment with hormonal therapy or 5α-reductase inhibitors 
• 
use of experimental agents for prostate cancer within the past 3 months 
• 
known allergy to cyclophosphamide or G-CSF/GM-CSF 
• 
known sensitivity to materials of bovine origin 
• 
history of autoimmune disease requiring systemic immunosuppression 
• 
other concurrent malignancy 
• 
uncontrolled major infectious, cardiovascular, pulmonary, hematologic, or 
psychiatric illnesses that would make the patient a poor study candidate 
Start date/ 
Duration 
Initial patients are expected to be entered in October 2012. With an estimated 
enrollment rate of 4 patients per month, accrual is expected to last 8 months. 


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Sample size 
32 evaluable patients (i.e. those that undergo prostatectomy) 
Rationale 
Cancer immunotherapy refers broadly to approaches which attempt to treat cancer by 
activating immune responses directed against malignant tissue. Prostate GVAX is an 
allogeneic cell-based prostate cancer vaccine composed of two irradiated cell lines (PC3 
and LNCaP) that have been genetically modified to secrete granulocyte-macrophage 
colony-stimulating factor (GM-CSF). The release of GM-CSF by these modified tumor 
cells serves to recruit dendritic cells which then present tumor antigens to T-cells, thus 
initiating antitumor immune responses. 
However, abundant preclinical data show that, when used alone, cell-based 
immunotherapy is unable to break specific T-cell tolerance in tumor-bearing hosts. 
Studies in an autochthonous prostate cancer mouse model have shown that giving low-
dose cyclophosphamide prior to a cell-based GM-CSF–secreting vaccine abrogates 
immune tolerance through augmentation of CD8+ T cell infiltration in the prostate, 
transient depletion of regulatory T cells (Tregs), and increased expression of dendritic 
cell maturation markers. Enhancement of antitumor immunity has also been observed 
in other preclinical models where cyclophosphamide was given in sequence with GM-
CSF–secreting immunotherapy for the treatment of breast and pancreatic cancers. 
These preclinical data are supported by early-phase clinical trials combining GVAX with 
low-dose cyclophosphamide in pancreatic and breast cancers. 
Furthermore, emerging evidence suggests that androgen deprivation therapy (ADT) 
itself has profound effects on the host immune system, resulting in thymic regeneration 
and enhancement of antitumor immunity. In addition, preclinical and clinical studies 
demonstrate that ADT augments prostate cancer-specific immune responses induced by 
immunotherapy, suggesting that ADT may act synergistically with immunotherapy. 
Based on data from mouse models as well as human clinical trials, it has been suggested 
that prostate cancer immunotherapy may be most effective when administered in the 
setting of an androgen-suppressed environment. 
Building on these findings, we have designed a study to assess the use of ADT given 
alone or administered following immunization with low-dose cyclophosphamide and 
prostate GVAX, in patients undergoing radical prostatectomy. We aim (1) to determine 
whether ADT is immunogenic in men with localized prostate cancer by evaluating T-cell 
infiltration in harvested prostate glands; (2) to determine whether administering ADT 
after low-dose cyclophosphamide and prostate GVAX augments immune infiltration into 
the prostate gland; and (3) to investigate whether this combinatorial immuno-hormonal 
approach is safe and feasible. We hypothesize that the combination of ADT and 
cyclophosphamide/GVAX will produce significantly greater antitumor immune 
responses than would ADT used alone. 
Objectives 
Primary: 
• 
To quantify the extent of CD8+ T cell infiltration into the prostate from harvested 
prostate glands in men with high-risk localized prostate cancer undergoing radical 
prostatectomy 
• 
To evaluate the safety and tolerability of ADT following vaccination with  
cyclophosphamide and GVAX 4 weeks prior to radical prostatectomy 


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Secondary:
x
To evaluate the feasibility of administering ADT after immunization with low-dose 
cyclophosphamide and prostate GVAX, beginning 4 weeks prior to radical 
prostatectomy 
x
To quantify the extent of CD4+ T cell and Treg infiltration into the prostate, and to 
quantify the CD8+/Treg ratio as well as the CD4+/Treg ratio in prostate specimens 
x
To quantify tissue androgen concentrations (testosterone, dihydrotestosterone), 
and to quantify androgen receptor (AR) protein expression in prostate specimens 
x
To quantify markers of apoptosis (activated caspase 3) and proliferation (Ki-67) in 
prostate tumor specimens 
x
To evaluate the proportion of pathological complete responses (pCR) 
x
To evaluate the generation of novel antibodies to prostate-associated antigens in the 
serum of patients, after the initiation of protocol therapy 
x
To determine the PSA response rate and time-to-PSA-recurrence after radical 
prostatectomy 
Study design 
This is a single-center, randomized, 2-arm, open-label, prospective clinical trial 
evaluating the immunogenicity and safety of a single administration of LHRH antagonist 
(degarelix) used alone, or degarelix administered following vaccination with low-dose 
cyclophosphamide and GVAX, given 4 weeks prior to radical prostatectomy in men with 
localized prostate cancer. 
Patients will be recruited from the outpatient Urology clinic. Eligible patients will be 
randomized in a 1:1 ratio ȋ
ǣζ͹ͺ-10) to receive one of two 
treatment regimens (see Schema below), each beginning 28 (±3) days prior to radical 
prostatectomy. In Arm B, cyclophosphamide will first be given at a dose of 200 mg/m2 
as a single intravenous infusion. One day later, prostate GVAX will be administered as 
five 0.8-mL intradermal injections of PC3 (2.5 × 108 cells) and five 0.5-mL intradermal 
injections of LNCaP (1.6 × 108 cells), for a total dose of 4.1 × 108 cells.  On day 14, 
degarelix acetate will be administered as three 80-mg subcutaneous injections, for a 
total dose of 240 mg. Prostate glands will be harvested 14 (±3) days later, at the time of 
radical prostatectomy, and prostate tissue will be examined for the primary endpoint. In 
Arm A, an identical dose of degarelix acetate will be administered, 14 (±3) days prior to 
surgery.  A telephone follow-up evaluation (or an in-person visit) for adverse events will 
occur 28 (±21) days after surgery. Patients will then be followed by their urologists 
according to standard institutional practices, but will require PSA evaluations every 3 
(±1) months during year 1 and every 6 (±2) months during years 2-3. 
                                                          Treatment Schema: 
. 


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Baseline evaluations: 
• 
Informed consent 
• 
Medical history and physical examination 
• 
Review of medications 
• 
Vital signs, including height and weight 
• 
Performance status 
• 
Central pathologic review of prostate core biopsies 
• 
CT (If allergic to CT scan contrast, obtain MRI with contrast) and/or bone scan, if 
clinically indicated 
• 
Hematology, coagulation, and chemistry laboratories 
• 
Serum PSA measurement 
• 
Sera for immunoassays (Pre-treatment) 
On day 28 (±3 days): 
• 
Medical history and focused physical examination (including vital signs) 
• 
Review of medications 
• 
Performance status 
• 
Adverse events/toxicity evaluation 
• 
Hematology and chemistry laboratories, as clinically indicated 
• 
Serum PSA 
• 
Sera for immunoassays 
• 
Radical prostatectomy 
• 
Pathologic review of surgical specimen according to standard procedures 
• 
Evaluation of harvested prostate gland for CD8+ T cell infiltration 
Post-operative day 28 (±21 days): 
• 
Performance status (by telephone interview or clinic visit) 
• 
Adverse events/toxicity evaluation (by telephone interview or clinic visit) 
• 
PSA every 3 months (post-op year 1), then every 6 months (post-op years 2-3) 
Criteria for 
evaluation 
Primary endpoints:  
• 
Mean CD8+ T cell density in tumor tissue from harvested prostate glands of patients 
in each of the 2 treatment groups 
• 
Frequency, type, and severity of adverse events in each of the 2 treatment groups 
Secondary endpoints:  
• 
Mean Treg density in tumor tissue from harvested prostate glands of patients in each 
of the 2 treatment groups 
• 
Mean CD8+/Treg and CD4+/Treg ratios in tumor tissue from harvested prostate glands 
of patients in each of the 2 treatment groups 
• 
Mean CD4+ T cell density in tumor tissue from harvested prostate glands of patients 
in each of the 2 treatment groups 
• 
Mean levels of apoptotic markers (activated caspase 3) and proliferation markers 
(Ki-67) in prostate tumor specimens of patients in each of the 2 treatment groups 
• 
Mean tissue androgen concentrations (testosterone, dihydrotestosterone) and mean 
androgen receptor (AR) protein expression in prostate specimens 
• 
Proportion of pathological complete responses (pCR) in each treatment group 


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• 
Proportion of patients generating of novel antibodies to prostate-associated 
antigens after the initiation of protocol therapy in each treatment group 
• 
Proportion of patients achieving a PSA response rate (PSA <0.1 ng/mL) at 3 months 
after prostatectomy in each treatment group 
• 
Median time-to-PSA-recurrence (PSA ≥0.2 ng/mL) after radical prostatectomy in 
each of the 2 treatment groups 
Statistical 
methods 
Our primary hypothesis is that men receiving cyclophosphamide and GVAX followed by 
ADT (arm B) will have a 100% or greater CD8+ T cell infiltration of the prostate than 
will men receiving ADT alone (arm A). In the absence of any intervention prior to 
prostatectomy, we have previously determined that the mean staining percentage for 
CD8+ T cells in prostate tissue is 0.42% (standard deviation [SD] 0.36%). With 16 
patients per arm, and assuming an 86% coefficient of variation for the average CD8+ 
area/spot area ratios, a one-sided 0.05 α-level t-test of the logarithms of these ratios 
would have 82% power to detect a 2-fold (100%) increase of CD8+ T cells in the 
cyclophosphamide/GVAX + ADT arm compared to the ADT alone arm. Treating Gleason 
score as a block factor reduces the variance, which would be adequate to offset the loss 
of power due to the 1 degree of freedom associated with the blocking factor. 
The primary efficacy endpoint will be expressed as the mean CD8+ T cell staining 
percentage in prostate tumor tissues, reported separately for each treatment arm. High 
density (saturation) tissue microarrays (TMAs) using the highest-grade/largest tumor 
per patient and sampling it with 100-fold redundancy will be used to determine CD8+ T 
cell densities. Since the CD8+ T cell quantity is a ratio variable and the distribution is 
skewed, the log transformation will be used for this analysis. Log-transformed mean 
CD8+ T cell concentrations will be compared between treatment groups using a two-
way analysis of variance (ANOVA) with the stratification variable (Gleason score) 
treated as a block factor.  Boxplots and descriptive summaries by treatment arm will 
also be provided.  
In other analyses, continuous variables will be summarized by descriptive statistics 
using the mean, standard deviation, median, and range, and 95% confidence intervals 
will be reported where appropriate. Two-way ANOVA will be used to investigate 
comparisons between the two treatment arms. Proportions will be compared using a 
Mantel-Haenszel test. Kaplan-Meier analysis will be used to determine median time-to-
PSA-progression, and comparisons will be sought using the log-rank test, stratified by 
Gleason score. Statistical significance will be set at P <0.05. 
Safety analysis 
Frequency, types, and grades of adverse events in each treatment group will be 
measured using the NCI Common Toxicity Criteria version 4.0, and will be summarized 
using descriptive statistics. Formal safety assessments will be performed from the time 
of first administration of degarelix, cyclophosphamide or GVAX until the 28th 
postoperative day (±21 days). In addition, all patients that received GVAX will be 
enrolled on a separate long-term follow-up protocol and will be evaluated for late GVAX 
toxicities (e.g. autoimmune phenomena, second malignancies). These evaluations will 
occur annually for 3 years. 


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TABLE OF CONTENTS 
 
1. 
OBJECTIVES ........................................................................................................................................................... 10 
1.1 
Primary Objectives ................................................................................................................................................... 10 
1.2 
Secondary Objectives ............................................................................................................................................... 10 
2. 
BACKGROUND AND RATIONALE ...................................................................................................................... 10 
2.1 
Disease Background ................................................................................................................................................. 10 
2.2 
Treatment Background ........................................................................................................................................... 11 
2.3 
Rationale ...................................................................................................................................................................... 17 
3. 
PATIENT SELECTION........................................................................................................................................... 20 
3.1 
Target Population...................................................................................................................................................... 20 
3.2 
Inclusion Criteria ....................................................................................................................................................... 20 
3.3 
Exclusion Criteria ...................................................................................................................................................... 20 
3.4 
Inclusion of Minorities ............................................................................................................................................. 21 
3.5 
Prohibited Concomitant Medications ................................................................................................................. 21 
4. 
REGISTRATION AND ENROLLMENT PLAN .................................................................................................... 22 
4.1 
Registration Procedure ........................................................................................................................................... 22 
4.2 
Expected Enrollment ................................................................................................................................................ 22 
4.3 
Study Centers .............................................................................................................................................................. 22 
4.4 
Recruitment ................................................................................................................................................................ 23 
4.5 
Randomization ........................................................................................................................................................... 23 
5. 
STUDY PLAN .......................................................................................................................................................... 23 
5.1 
Overview and Schema.............................................................................................................................................. 23 
5.2 
Screening/Pretreatment Evaluation................................................................................................................... 26 
5.3 
Radical Prostatectomy ............................................................................................................................................. 26 
5.4 
Sera for Immunoassays ........................................................................................................................................... 27 
5.5 
Follow-Up Evaluation .............................................................................................................................................. 27 
5.6 
Duration of Therapy ................................................................................................................................................. 27 
6. 
THERAPEUTIC AGENTS ...................................................................................................................................... 28 
6.1 
Prostate GVAX Immunotherapy ........................................................................................................................... 28 
6.2 
Low-Dose Cyclophosphamide ............................................................................................................................... 32 
6.3 
Degarelix Acetate....................................................................................................................................................... 33 
6.4 
Concomitant Medications and Supportive Care.............................................................................................. 35 
6.5 
Drug Interactions/Cautions ................................................................................................................................... 36 
6.6 
Drug Dispensing Log ................................................................................................................................................ 36 
6.7 
Pregnancy .................................................................................................................................................................... 36 
7. 
ADVERSE EVENTS AND REPORTING REQUIREMENTS ............................................................................... 36 
7.1 
Recording and Grading ............................................................................................................................................ 37 
7.2 
Unexpected Adverse Events .................................................................................................................................. 38 
7.3 
Serious Adverse Events and Serious Adverse Drug Reactions .................................................................. 38 
7.4 
Perioperative Adverse Events............................................................................................................................... 40 
7.5 
Reporting Adverse Events ...................................................................................................................................... 40 
7.6 
Pregnancy .................................................................................................................................................................... 42 
8. 
OUTCOME ASSESSMENT ..................................................................................................................................... 42 
8.1 
Radical Prostatectomy Specimen......................................................................................................................... 42 
8.2 
Primary Endpoints .................................................................................................................................................... 43 
8.3 
Secondary Endpoints ............................................................................................................................................... 44 
9. 
REGULATORY AND REPORTING REQUIREMENTS ...................................................................................... 46 
9.1 
Regulatory Responsibilities ................................................................................................................................... 46 


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9.2 
Data Management ..................................................................................................................................................... 47 
9.3 
Study Monitoring and Quality Assurance ......................................................................................................... 47 
10. 
STATISTICAL CONSIDERATIONS...................................................................................................................... 48 
10.1 Study Design and Sample Size............................................................................................................................... 48 
10.2 Study Endpoints ......................................................................................................................................................... 49 
10.3 Analysis Populations ................................................................................................................................................ 52 
10.4 Safety Analysis............................................................................................................................................................ 52 
10.5 Statistical Procedures .............................................................................................................................................. 53 
11. 
PROTECTION OF HUMAN SUBJECTS ............................................................................................................... 53 
11.1 Ethical Considerations ............................................................................................................................................. 53 
11.2 Protocol Amendments ............................................................................................................................................. 54 
11.3 Written Informed Consent ..................................................................................................................................... 54 
11.4 Protection of Privacy ................................................................................................................................................ 54 
11.5 Study Termination or Modification ..................................................................................................................... 55 
12. 
REFERENCES ......................................................................................................................................................... 56 
APPENDIX A:  Performance Status Criteria ............................................................................................................. 59 
APPENDIX B:  Common Toxicity Criteria, Version 4.0 .......................................................................................... 62 
APPENDIX C:  Serious Adverse Event (SAE) Reporting Form.............................................................................. 63 
APPENDIX D:  Abbreviations and Acronyms ........................................................................................................... 65 
APPENDIX E:  Data and Safety Monitoring Plan (DSMP) ...................................................................................... 68 
LIST OF TABLES 
Table 1 
Study Calendars 
24 
Table 2 
Intensity of Adverse Event 
37 
Table 3 
Relationship of Adverse Event to Study Drug 
38 
LIST OF FIGURES 
Figure 1 
Clinical States of Prostate Cancer 
11 
Figure 2  
Cyclophosphamide Augments Antitumor Immunity Produced by GVAX 
16 
Figure 3  
Study Schema 
23 


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1. 
OBJECTIVES 
1.1 
Primary Objectives 
• 
To quantify the extent of CD8+ T cell infiltration into the prostate from harvested 
prostate glands in men with localized prostate cancer receiving neoadjuvant ADT 
alone (2 weeks prior to surgery), or cyclophosphamide and GVAX followed by ADT, 
(with CY/GVAX administered 4 weeks prior to prostatectomy, and ADT 
administered 2 weeks prior to prostatectomy). 
• 
To evaluate the safety and tolerability of ADT used alone (2 weeks prior to surgery), 
or cyclophosphamide and GVAX followed by ADT (with CY/GVAX administered 4 
weeks prior to prostatectomy, and ADT administered 2 weeks prior to 
prostatectomy). 
1.2 
Secondary Objectives 
• 
To evaluate the feasibility of administering ADT used alone (2 weeks prior to 
surgery), or cyclophosphamide and GVAX followed by ADT (with CY/GVAX 
administered 4 weeks prior to prostatectomy, and ADT administered 2 weeks prior 
to prostatectomy). 
• 
To quantify and compare the extent of CD4+ T cell and Treg infiltration into the 
prostate in prostate specimens of patients in each of the 2 treatment groups. 
• 
To quantify and compare CD8+/Treg and CD4+/Treg ratios in prostate specimens of 
patients in each of the 2 treatment groups. 
• 
To quantify and compare tissue androgen concentrations (testosterone, 
dihydrotestosterone), and to quantify androgen receptor (AR) protein expression in 
prostate specimens of patients in each of the 2 treatment groups. 
• 
To quantify markers of apoptosis (activated caspase 3) and markers of cell 
proliferation (Ki-67) in prostate tumor specimens of patients in each of the 2 
treatment groups. 
• 
To evaluate and compare the proportion of pathological complete responses (pCR) 
in prostate tumor specimens of patients in each of the 2 treatment groups. 
• 
To evaluate and compare the generation of novel antibody specificities to prostate-
associated antigens in the serum of patients in each group, after the receipt of 
protocol therapy. 
• 
To evaluate and compare PSA response rates and the time-to-PSA-recurrence after 
radical prostatectomy in each of the 2 treatment groups. 
2. 
BACKGROUND AND RATIONALE 
2.1 
Disease Background 
Prostate cancer is the second leading cause of cancer deaths in men. According to American Cancer 
Society estimates in 2008, as many as 186,320 American men will be diagnosed with prostate cancer, 
and nearly 28,660 will die of the disease (Jemal et al 2008). Yet, localized prostate cancer is often 
curable and even metastatic disease can respond to treatment. 


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The course of prostate cancer from diagnosis to death is best categorized as a series of clinical states 
(Figure 1). These clinical states involve the complex interplay of a network of signaling molecules 
that collectively promote net cell proliferation relative to cell death. Based on the extent of disease, 
hormonal status, and absence or presence of detectable metastases on imaging studies, the states 
are: localized disease, rising PSA after radiation therapy or surgery with no detectable metastases, 
and clinical metastases in the non-castrate or castrate settings. 
Treatment of localized prostate cancer usually consists of surgery or radiation therapy or both. For 
patients with high-risk or locally advanced disease, a combination of radiation therapy and hormonal 
therapy is often used. However, even after definitive local therapy, approximately 30-50% of patients 
will have a local or distant recurrence (Pound et al 1999, D’Amico et al 2000). The risk of recurrence 
depends on multiple factors including tumor stage, tumor Gleason score, and serum PSA at the time 
of diagnosis. Recently, PSA doubling time at recurrence has been shown to be a powerful predictor of 
distant metastases and survival (Antonarakis et al 2012; Freedland et al 2007). 
 
Figure 1 
Clinical States of Prostate Cancer 
 
Patients with metastatic prostate cancer have a median survival of 3-7 years, and most die of the 
disease. Treatment for metastatic disease involves surgical castration or hormonal manipulation 
using luteinizing hormone-releasing hormone (LHRH) agonists/antagonists, antiandrogens, or both. 
Although the majority of these patients initially respond to androgen deprivation therapy, all 
eventually progress to a state of castration-resistant prostate cancer (CRPC). Treatment options for 
these men are limited. Secondary hormonal manipulations, such as ketoconazole, are often used but 
these produce short-lived responses and do not improve survival (Small et al 2004). Moreover, these 
agents have unfavorable side effect profiles. Other acceptable approaches in these men include 
watchful waiting until the development of symptoms, or the initiation of cytotoxic chemotherapy. In 
this regard, docetaxel has been shown to improve overall survival in patients with CRPC, but only by 
a median of 2.9 months (Tannock et al 2004; Berthold et al 2008). However, because therapies for 
metastatic prostate cancer are palliative and not curative, there remains an urgent need to improve 
the outcome of primary therapies for prostate cancer. 
2.2 
Treatment Background 
2.2.1 
GM-CSF–secreting allogeneic cell-based vaccine as immunotherapy for prostate cancer 
Cancer immunotherapy refers broadly to approaches which attempt to treat or cure 
cancer by activating an immune response directed against malignant tissue (Pardoll 
2002). A number of different approaches fall broadly into this group, including dendritic 
cell vaccination, DNA vaccination, peptide vaccination, and others. Each of these 
methodologies has its own advantages and disadvantages. In particular, the use of 
granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjuvant has been 
widely evaluated in both murine models and in patients with cancer (Borrello and 


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Pardoll 2002). These studies suggest that GM-CSF based immunotherapy approaches 
function by recruiting and activating dendritic cells, now fairly well accepted as the most 
potent antigen presenting cells. Activated dendritic cells take up antigens from the 
tumor cell line or autologous tumor used in the biological formulation, and traffic to 
draining lymph nodes. In the lymphoid tissue, these activated dendritic cells present 
antigens derived from tumor peptides to T-cells, which then circulate to the sites of 
disease and (if successful) initiate a lytic immune response against the malignant tissue. 
After initial evaluation in murine models (Dranoff et al 1993), the first clinical 
applications of GM-CSF transduced tumor cells employed autologous tumor in the 
setting of metastatic renal cell carcinoma (Simons et al 1997). These studies 
demonstrated the relative safety of this approach, but also underscored the technical 
difficulty of using autologous tumor as a source of antigens for immunotherapy. 
Recently, two significant findings have impacted the use of GM-CSF immunotherapies. 
First, it has been shown that GM-CSF need not come directly from the immunizing tumor 
cells, and that irradiated bystander erythroleukemia cells transduced to produce GM-
CSF can provide identical response rates to directly transduced immunotherapy lines in 
murine models (Borrello et al 1999). Perhaps more significant, though, was the 
recognition that many common tumor antigens may indeed be shared between patients 
with the same tumor type, and that immortalized cell lines could thus be transduced 
with GM-CSF to serve as immunotherapies. The use of immortalized GM-CSF–secreting 
cell lines has been evaluated in patients with pancreatic cancer (Jaffee et al 2001), with 
preliminary results showing the safety of this immunotherapy, in addition to suggesting 
some degree of efficacy especially at higher immunotherapy doses. 
In prostate cancer, the initial immunotherapy approach also utilized irradiated 
autologous tumor cells transduced in vitro to secrete GM-CSF (Simons et al 1999; 
Dummer 2001). In a phase I trial, prostate tumor cells were harvested surgically and 
transduced with a replication-defective retrovirus. Doses of 10 million tumor cells were 
administered without significant adverse affects, and new delayed-type hypersensitivity 
(DTH) responses were documented in two out of eight treated patients. For phase II 
evaluation, an immunotherapy comprised of two common prostate cancer cell lines 
(LNCaP and PC3) has been developed (Higano et al 2008). LNCaP was originally isolated 
from a lymph node metastasis and expresses a number of prostate-restricted 
differentiation antigens including PSA and prostate-specific membrane antigen (PSMA) 
(Horoszewicz et al 1987; Israeli et al 1994). It also contains a mutant androgen receptor 
as well as the most common epigenetic abnormality in human prostate cancer: 
methylation of the glutathione-S-transferase gene (Lee et al 1994). The PC3 cell line was 
developed from a bone metastasis and expresses significant levels of proteases 
associated with metastatic prostate cancer. While LNCaP is androgen-sensitive, PC3 is 
considered androgen insensitive. Together, these 2 cell lines are likely to serve as a 
diverse source of prostate- and prostate tumor-specific antigens. 
Two phase II studies have been performed using this product, officially registered as 
PC3/LNCaP immunotherapy for prostate cancer (or GVAX). In one of these studies, 41 
hormone-naïve patients were primed with a single 500 million-cell immunotherapy 
dose, then boosted with 12 doses of 100 million cells at 2 week intervals. No 
autoimmunity was detected, and no life-threatening toxicities were observed. The most 
common adverse event was a reaction at the injection site. One patient had a partial PSA 
response (Simons et al 2006). In a second phase II trial, 55 hormone-refractory patients 
were treated with a 500 million-cell immunotherapy dose, then boosted with 12 doses 
of either 100 million or 300 million cells at 2 week intervals. One patient had a PSA 


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response, and there was a non-significant trend towards a longer median time to bone 
scan progression (140 days vs. 85 days, P = 0.095) in the high-dose arm (Small et al 
2007). Taken together, these phase II data demonstrate the safety of this 
immunotherapy approach, but suggest a relatively low overall response rate. The most 
common adverse events in both studies were injection-site erythema, fever, fatigue, 
malaise, myalgias, and arthralgias. 
Two large randomized phase III studies of GM-CSF–secreting cell-based immunotherapy 
for prostate cancer (GVAX) have been conducted: VITAL-1 and VITAL-2. The first study, 
VITAL-1, enrolled 626 men with asymptomatic chemotherapy-naïve castration-resistant 
prostate cancer, and randomized them to receive GVAX or docetaxel/prednisone, with 
the primary endpoint being overall survival (Higano et al 2009). The second study, 
VITAL-2, was planned initially to enroll 600 patients with symptomatic metastatic 
castration-resistant prostate cancer, randomizing them to docetaxel/prednisone or 
docetaxel/GVAX (Small et al 2009). Both trials were terminated early because of data 
observed at the time of interim analyses suggesting that the survival improvements 
initially hypothesized were unlikely to be observed if the trials were to be continued 
(Higano et al 2009; Small et al, 2009). In both studies, overall survival was not 
statistically superior in patients receiving GVAX. While a large number of potential 
reasons for this low activity may be postulated, perhaps the simplest is the well-known 
immunological observation that persistent antigen exposure tends to induce immune 
tolerance (Ramsdell and Fowlkes 1992; Rocha et al 1993; den Boer et al 2004). In 
addition, cancer immunotherapy might prove more efficacious in a minimal disease 
state, as has been observed in multiple experimental systems (Migita and Ochi 1993). 
2.2.2 
Cyclophosphamide as a potent enhancer of immune responses to GVAX 
Efficient immunization against cancer requires a vaccine capable of eliciting a potent 
CD4+ and CD8+ T cell immune response. However, tumors have evolved several 
mechanisms to escape immune surveillance, including immune tolerance mediated by 
immunosuppressive T lymphocytes (Drake et al 2006). To this end, tumors have been 
shown to induce rapid expansion of CD4+CD25+ regulatory T cells (Tregs) in humans and 
mice, leading to delayed rejection of immunogenic cancers (Liyanage et al 2002; Woo et 
al 2001; Curiel et al 2004). Conversely, elimination of these Tregs, which constitute about 
2-5% of the peripheral CD4+ T cells in naïve mice, elicits potent antitumor immune 
responses leading to tumor eradication (Shimizu et al 1999; Sutmuller et al 2001). For 
many years, cyclophosphamide has been studied for its role in breaking immune 
tolerance. It is known that low-dose and high-dose cyclophosphamide have different 
biological activities (Motoyoshi et al 2006). High-dose cyclophosphamide has cytotoxic 
activity, whereas low-dose cyclophosphamide shows predominantly immune 
modulating effects. Cyclophosphamide, as an FDA-approved cytotoxic chemotherapy 
agent, is used in treating multiple types of cancer including breast cancer, lymphoma, 
leukemia, and sarcoma. In addition, cyclophosphamide is used as an immune suppressor 
in many autoimmune disorders. 
Immune modulating doses of cyclophosphamide-containing chemotherapy have been 
studied in mouse tumor models in conjunction with vaccination. For example, Dr. Jaffee 
and colleagues have studied a HER-2/neu transgenic mouse model of mammary tumors, 
representing a clinically relevant model of human breast cancer. These mice develop 
spontaneous HER-2/neu–expressing mammary tumors due to activation of the 
transgene by the murine mammary tumor virus (MMTV) promoter in normal mammary 
tissue. It has been demonstrated that vaccination of these mice induces weak HER-
2/neu–specific B cell and T cell responses that are not capable of controlling mammary 


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tumor growth regardless of the vaccine type. However, treatment of these mice with 
low-dose cyclophosphamide prior to vaccination resulted in tumor rejection in 10-30% 
of animals, while mice receiving vaccination alone all became tolerant to tumor growth 
(Ercolini et al 2005). In this study, it was shown that the immune enhancing effect of 
cyclophosphamide was mediated through selective inhibition of the cycling population 
of CD4+CD25+ Treg cells and recruitment of high-avidity antitumor effector T cells. These 
findings were confirmed in a separate study using the same HER-2/neu transgenic 
mouse model of breast cancer. In that study, combined treatment with immune-
modulating doses of cyclophosphamide and a GM-CSF–secreting whole-cell vaccine 
delayed tumor growth by overcoming immune tolerance and inducing antigen-specific 
antitumor immune responses (Machiels et al 2001). Importantly, because 
cyclophosphamide exerts its effect on the regulatory T cells of the immune system 
rather than on the cancer cells themselves, this approach can be applied to treat any 
type of cancer. 
These animal studies have led to a phase I/II clinical trial testing the feasibility, safety, 
and immunogenicity of cyclophosphamide when given in sequence with a GM-CSF–
secreting cell-based vaccine in breast cancer patients (Emens et al 2004). In this study, 
21 women with metastatic breast cancer received one of three doses of 
cyclophosphamide (200, 250, or 350 mg/m2) in conjunction with doxorubicin and an 
allogeneic, HER-2/neu+, GM-CSF–secreting breast tumor vaccine. In addition, 6 women 
received immunotherapy alone. Patients on the combined-modality arms received three 
monthly immunizations and a booster 6 to 8 months later according to the following 
treatment schedule: cyclophosphamide was given on day –1, vaccination on day 0, and 
doxorubicin on day +7. Total Her-2–specific immune responses were observed in 33%, 
50%, 21%, and 25% of patients receiving 0, 200, 250, and 350 mg/m2 of 
cyclophosphamide respectively (Emens et al 2008). These clinical data suggest that 
addition of cyclophosphamide enhances Her-2–specific vaccine responses in patients 
with breast cancer, but that increasing doses of cyclophosphamide may abrogate these 
immune responses. 
A second pilot study borrowing from these principles has recently been conducted in 
patients with metastatic pancreatic cancer (Laheru et al 2008). In this open-label study, 
30 patients received up to six doses of a GM-CSF–secreting cell-based pancreatic cancer 
vaccine at 21-day intervals, while 20 patients received 250mg/m2 of intravenous 
cyclophosphamide 1 day prior to the same vaccine. After 3 cycles of therapy, pancreatic 
cancer-specific (mesothelin-specific) CD8+ T cell responses were augmented in only 
50% of patients treated with vaccine alone compared to 90% of patients treated with 
cyclophosphamide plus vaccine. Mean immune response augmentation (as judged by 
mesothelin-specific CD8+ T cell effects) after 3 cycles of treatment was 39% in patients 
receiving immunotherapy alone and 131% in patients receiving cyclophosphamide plus 
immunotherapy (Laheru et al 2008). In addition, median survival in patients on the 
vaccine arm was 2.3 months while survival on the cyclophosphamide/vaccine arm was 
4.3 months. The administration of this cell-based immunotherapy in sequence with 
cyclophosphamide showed minimal treatment-related toxicity, although all patients 
developed erythema, induration and/or pain, soreness at the treatment sites following 
immunotherapy. Other common adverse events included fever, chills, fatigue, malaise, 
skin rash, arthralgias, and nausea. 
2.2.3 
Cyclophosphamide combined with GVAX in prostate cancer 
Recently, data evaluating the effect of cyclophosphamide on anti-tumor immune 
responses to a cell-based GM-CSF–secreting prostate cancer vaccine (GVAX) was 


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published by Dr. Drake and colleagues (Wada et al 2009). In this study, an 
autochthonous animal model was created using the transgenic adenocarcinoma of the 
mouse prostate (TRAMP) mouse crossed with analogous transgenic mice expressing the 
model antigen hemagglutinin (HA) in a prostate-restricted manner (ProHA). This 
clinically relevant murine model allowed investigation of immune responses to tissue-
tumor–restricted HA as a function of immunotherapy and/or cyclophosphamide 
administration. In this model (ProHA × TRAMP), giving cyclophosphamide 1 day prior to 
GVAX immunotherapy induced maximal augmentation of HA-specific CD8+ T cell 
responses both in the prostate and the prostate-draining lymph nodes (Figure 2a). 
Administering cyclophosphamide on day –4, day –2, or day +1 in relation to GVAX was 
not as effective. Using this treatment sequence, the optimal dose of cyclophosphamide 
with respect to antitumor immune responses was found to be 50 mg/kg given 
intraperitoneally. This dose produces serum levels equivalent to those achieved in 
humans using a cyclophosphamide dose of 200 mg/m2 given intravenously. 
Importantly, higher doses of cyclophosphamide in this animal model resulted in a loss of 
immunotherapy efficacy and a suppression of specific CD8+ T cell responses. 
When comparing the antitumor immune responses of different permutations of GVAX 
and cyclophosphamide in ProHA × TRAMP mice (Wada et al 2009), it was shown that 
the combination of these two agents produced markedly superior HA-specific CD8+ T 
cell responses in both the prostate and prostate-draining lymph nodes than either agent 
used alone (Figure 2b). This finding is particularly important, as it further demonstrates 
the weak antitumor immunogenicity of GVAX when used alone against prostate cancer. 
Further mechanistic experiments showed that immune augmentation by 
cyclophosphamide was associated with a transient depletion of regulatory 
(CD4+FOXP3+) T cells in the prostate and prostate-draining lymph nodes (Figure 2c) but 
not in the peripheral circulation. In addition, combined treatment resulted in increased 
ratios of CD4+ and CD8+ effector T cells to regulatory T cells (Teff/Treg ratio) in the 
prostate glands of these mice (Figure 2d). Alterations in Treg function with 
cyclophosphamide use were not seen. Finally, combination of GVAX and 
cyclophosphamide resulted in increased expression of dendritic cell maturation 
markers, although a dramatic change in dendritic cell numbers was not observed. Taken 
together, these data provide a strong rationale for a strategy combining low-dose 
cyclophosphamide with allogeneic GVAX immunotherapy in patients with prostate 
cancer.  


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Figure 2  
Cyclophosphamide Augments Antitumor Immunity Produced by GVAX 
 
2.2.4 
Immunological effects of androgen deprivation 
It is now appreciated that androgen deprivation therapy (ADT) has profound effects 
on the host immune system, resulting in enhancement of antitumor immune 
responses (Aragon-Ching et al 2007). Initial observations in this regard came from a 
study demonstrating that ADT administered before prostatectomy resulted in 
profuse CD4+ T cell infiltration into human prostate glands, and that these T cells 
were oligoclonal suggesting an antigen-specific response (Mercader et al 2001). In a 
similar study of androgen-deprived prostate cancer patients, an increased CD8+ T 
cell infiltrate was noted as well (Gannon et al 2009). Also supporting a pro-
immunogenic role for ADT are recent data demonstrating induction of new antibody 
specificities in castrated patients (Morse et al 2010). In the laboratory, using an 
autocthonous prostate cancer mouse model, our group has previously shown that 
ADT decreases CD4+ T cell tolerance to prostate cancer-associated antigens, 


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mitigating immune evasion (Drake et al 2005). Intriguingly, additional studies in 
both mice and humans have shown that ADT reverses age-related thymic involution, 
resulting in renewed thymopoiesis and increased output of naïve T cells (Sutherland 
et al 2005). Finally, expansion of the pre-B cell population is observed in the bone 
marrow after castration in mice, a process that may improve antigen presentation 
and T cell responses (Wilson et al 1995). Taken together, these data support the 
notion that ADT, through its augmentation of prostate-specific immune responses, 
may act synergistically with immunotherapy in the treatment of prostate cancer. 
2.2.5 
Combining androgen deprivation with immunotherapy 
The addition of ADT to immunotherapy has been examined in several preclinical 
and clinical studies. Using our autocthonous prostate cancer mouse model, our 
group noted prostate-specific effector T cell expansion when a recombinant vaccinia 
virus vaccine was administered to castrated tumor-bearing mice, but this did not 
occur in non-castrated mice (Drake et al 2005). In a more recent study of tumor-free 
mice, vaccination with a prostate stem cell antigen (PSCA)-containing DNA-based 
vaccine produced greater numbers of PSCA-specific IFN-γ-secreting T cells in mice 
that were castrated compared to those that were not castrated (Koh et al 2009). 
Notably, this expansion of effector T cells was only seen when vaccination was 
performed prior to castration, and not when this treatment order was reversed. 
Another study using a PSA-expressing transgenic mouse model demonstrated 
proliferation of PSA-specific cytotoxic T cell in response to immunization with a 
PSA-based recombinant vaccinia virus but this effect was only noted in castrated 
mice (Arredouani et al 2010).  
A small randomized clinical trial in men with non-metastatic castration-resistant 
prostate cancer examining the combination of antiandrogen therapy and a PSA-
based recombinant poxviral vaccine showed a higher frequency of PSA-specific T 
cell responses in men who received vaccination before antiandrogen than in those 
receiving antiandrogen before vaccination (Arlen et al 2005). Intriguingly, longer 
follow up of this same study revealed that overall survival was increased in men 
who received vaccine followed by antiandrogen than in those who received this 
combination in the reverse order (median survival 6.2 vs 3.7 years, P = 0.045) 
(Madan et al 2008). Finally, a larger randomized study of sipuleucel-T versus 
placebo combined with ADT in men with non-castrate PSA-recurrent prostate 
cancer showed a slower post-treatment PSA doubling time (PSADT) in men 
receiving the combination therapy than in those receiving ADT alone (PSADT 5.1 vs 
3.4 months, P = 0.038) (Beer et al 2011). In summary, these studies suggest that 
combined immuno-hormonal therapy may provide additive clinical benefit and is 
worthy of further evaluation. 
2.3 
Rationale 
2.3.1 
Rationale for conducting the study 
In both animal models and humans with cancer (including prostate cancer), allogeneic cell-
based GM-CSF–secreting immunotherapy is often unable to break specific T cell tolerance 
when this modality is used alone. Preclinical studies in an autochthonous prostate cancer 
mouse model have shown that giving low-dose cyclophosphamide prior to GVAX abrogates 
immune tolerance through enhancement of CD8+ T cell infiltration into the prostate, 
transient depletion of regulatory T cells (Tregs), and increased expression of dendritic cell 


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maturation markers. Augmentation of antitumor immunity has also been observed in early 
human clinical trials where cyclophosphamide has been given in sequence with GM-CSF–
secreting vaccines for the treatment of breast and pancreatic cancers. 
Furthermore, emerging evidence suggests that androgen deprivation therapy (ADT) itself 
has profound effects on the host immune system, resulting in thymic regeneration and 
enhancement of antitumor immunity. In addition, preclinical and clinical studies 
demonstrate that ADT augments prostate cancer-specific immune responses induced by 
immunotherapy, suggesting that ADT may act synergistically with immunotherapy. Based on 
data from mouse models as well as human clinical trials, it has been suggested that prostate 
cancer immunotherapy may be most effective when administered in the setting of an 
androgen-suppressed environment. 
The sequencing of androgen-ablation after immunotherapy is supported by preclinical data  
(Koh et al 2009)and more importantly by clinical data(Aragon-Ching et al 2007).  In this 
sequence, the additive effect of these two treatment modalities likely represents a classical 
prime-boost regimen in which the well-established immune effects of androgen-ablation 
boost T cell responses initiated by vaccination.  It should be noted that other sequences are 
possible; in fact a randomized phase II of sipuleucel-T testing two alternative sequences 
(NCT01431391) is currently accruing patients. 
Building on these findings, we have designed a study to assess the use of ADT given alone or 
in sequence with low-dose cyclophosphamide and prostate GVAX, in patients scheduled to 
undergo radical prostatectomy. We aim (1) to determine whether ADT is immunogenic in 
men with localized prostate cancer by evaluating T-cell infiltration in harvested prostate 
glands; (2) to determine whether administering ADT in sequence with low-dose 
cyclophosphamide and prostate GVAX augments immune infiltration into the prostate gland; 
and (3) to investigate whether this combinatorial immuno-hormonal approach is safe and 
feasible. We hypothesize that the combination of ADT and cyclophosphamide/GVAX will 
produce significantly greater antitumor immune responses than ADT used alone. 
If this study shows significant enhancement of antitumor immunity when using ADT in 
sequence with low-cyclophosphamide and prostate GVAX, and this combination is feasible 
and safe, then future studies would aim to use this combinatorial approach in both the 
adjuvant and the salvage settings. For example, a trial could be designed to assess the 
efficacy of ADT combined with cyclophosphamide/GVAX given before and after radical 
prostatectomy, using PSA recurrence as the primary endpoint. Alternatively, a trial of ADT 
combined with cyclophosphamide/GVAX for patients with PSA recurrence could be 
designed, with metastatic progression as the primary endpoint. 
2.3.2 
Rationale for dosage selection 
The dose of prostate GVAX in this study will be the same as the priming dose used in two 
large phase III studies evaluating this immunotherapy in patients with castration-resistant 
metastatic prostate cancer (Higano et al 2009; Small et al, 2009). Five 0.8-mL intradermal 
injections of PC3 (2.5 × 108 cells) and five 0.5-mL intradermal injections of LNCaP (1.6 × 108 
cells) will be administered, for a total dose of 4.1 × 108 cells. In contrast to previous trials, 
the present study will use only a single dose of immunotherapy rather than a series of doses 
spread out over time. A single dose should be sufficient since we will be evaluating T cell 
infiltration in resected prostate glands (harvested three weeks later) as our primary 
endpoint.  
Cyclophosphamide will be administered at 200 mg/m2, as a single intravenous infusion. In 
the cyclophosphamide/GVAX treatment arm, cyclophosphamide will be given one day prior 
to GVAX, which itself will be administered two weeks prior to prostatectomy. We 
hypothesize that 200 mg/m2 of cyclophosphamide will be the optimal immune priming dose 
in humans because this dose produces serum levels equivalent to those achieved using 50 


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mg/kg intraperitoneally in the ProHA × TRAMP mouse model, the dose which produced the 
largest antitumor immune responses (Wada et al 2009). In addition, the 200 mg/m2 dose of 
cyclophosphamide was recently used in a human pancreatic cancer study, where it was 
shown to augment the immune effects of an allogeneic cell-based GM-CSF–secreting 
pancreatic cancer vaccine (Laheru et al 2008). In another study of breast cancer patients, 
200 mg/m2 of cyclophosphamide given in sequence with a cell-based GM-CSF–secreting 
breast cancer vaccine seemed to be the most immunogenic dose, while doses of 250 mg/m2 
and 350 mg/m2 appeared to suppress HER-2–specific immune responses (Emens et al 2008). 
Degarelix acetate will be administered as three 8  mg subcutaneous injections, for a total 
dose of 240 mg. This is the standard loading dose of degarelix, and is expected to induce 
castrate levels of serum testosterone for at least 4 weeks. Degarelix, an LHRH antagonist, 
was chosen rather than an LHRH agonist in order to produce rapid testosterone suppression 
without causing an initial testosterone surge. This rapid testosterone ablation more closely 
resembles the effect of surgical castration in preclinical animal models. 
2.3.3 
Rationale for immunobiologic endpoints 
The primary goal of this study is to assess the degree of immune infiltration into the prostate 
after administering the protocol therapy, and to determine whether the combination of ADT 
and cyclophosphamide/GVAX may enhance intraprostatic immune infiltration compared to 
ADT administered alone. In studies using the ProHA × TRAMP mouse model, the primary 
endpoint was hemagglutinin (HA)-specific CD8+ T cell responses in harvested prostate 
glands (Wada et al 2009). However, in humans with prostate cancer receiving GVAX, the 
exact immunogenic antigen is not known. Therefore, the primary endpoint for the present 
study will be assessment of total CD8+ T cell infiltration into the prostate. Our previous 
studies have shown that the mean staining percentage for CD8+ T cells in human prostate 
tumor tissue is 0.42% (standard deviation 0.36%, interquartile range 0.07%–0.71%). In the 
present study, we seek to augment the intratumoral CD8+ T cell response by 100% in the 
experimental arm compared to the control arm. A 100% increase in tumor-infiltrating CD8+ 
T cells was sought because in patients with advanced melanoma, adoptive T cell transfer 
studies have shown that a 100% increase in tumor-infiltrating CD8+ T cells was required to 
cause regression of metastatic melanoma lesions (Rosenberg et al 2011); increases in tumor-
infiltrating CD8+ T cells that were less than 100% did not result in tumor regressions. 
In the present study, CD8+ T cell density will serve as a surrogate measure of prostate-
/prostate cancer-specific T cell responses. By comparison, the human breast cancer trial 
used Her-2–specific immune responses as its primary endpoint (Emens et al 2008), while the 
pancreatic cancer trial used mesothelin-specific T cell responses as its primary endpoint 
(Laheru et al 2008). One of our goals in the future will be to identify a useful tumor-
associated antigen in prostate cancer patients receiving GVAX, and to use this as a more 
rational marker of immunologic effects.  
In addition to augmenting antitumor immune responses in ProHA × TRAMP mice, the 
combination of cyclophosphamide/GVAX also decreased the wet weight of the urogenital 
tract (a gross surrogate for tumor burden) compared to either agent used alone. Moreover, 
histologic tumor scores were lower and apoptotic bodies were more frequent in the 
combined treatment group compared with either single treatment alone (Wada et al 2009). 
To evaluate potential direct antitumor effects in the present study, we will measure tumor 
apoptosis and proliferation using validated markers such as activated caspase 3 (Marcelli et 
al 2000) and Ki-67 (Rubin et al 2002). These will be assessed by immunohistochemical 
staining of formalin-fixed tissue samples. Ki-67 is a nuclear antigen that is found in 
proliferating cells, whose expression correlates with the degree of apoptosis and cellular 
turnover in prostate cancer (Berges et al 1995). Apoptotic cells will also be identified 
histologically and quantified by the method of terminal deoxynucleotidyl transferase (TdT)-
mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL assay) (Furuya et al 


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1995). We will also evaluate the proportion of patients achieving a pathological complete 
response (pCR) in each of the 2 treatment groups. 
3. 
PATIENT SELECTION 
3.1 
Target Population 
Subjects will include men with multifocal, Gleason 7 or greater, clinically localized prostate 
cancer for whom the decision has been made to perform radical prostatectomy at Johns Hopkins 
Hospital. Subjects will be identified and recruited through the Outpatient Clinic of the Department of 
Urology and from the Multidisciplinary Prostate Cancer Clinic. 
3.2 
Inclusion Criteria 
To be eligible for this study, patients must meet all of the following criteria: 
• 
Histologically confirmed adenocarcinoma of the prostate (clinical stage T1c–T3b, 
N0, M0) without involvement of lymph nodes, bone, or visceral organs 
• 
Initial prostate biopsy is available for central pathologic review, and is confirmed to 
show at least 2 positive cores and a maximum Gleason sum of ≥7 
• 
Radical prostatectomy has been scheduled at Johns Hopkins Hospital 
• 
Age ≥21 years 
• 
ECOG performance status 0-1, or Karnofsky score ≥ 70% (see Appendix A) 
• 
Adequate bone marrow, hepatic, and renal function: 
o WBC >3,000 cells/mm3 
o ANC >1,500 cells/mm3 
o Hemoglobin >9.0 g/dL 
o Platelet count >100,000 cells/mm3 
o Serum creatinine <2.0 mg/dL 
o Serum bilirubin <2 mg/dL 
o ALT <2 × upper limit of normal (ULN) 
o AST <2 × ULN 
o Alkaline phosphatase <2 × ULN 
• 
Willingness to provide written informed consent and HIPAA authorization for the 
release of personal health information, and the ability to comply with the study 
requirements (note: HIPAA authorization will be included in the informed consent) 
• 
Willingness to use barrier contraception from the time of cyclophosphamide and/or 
GVAX administration until the time of prostatectomy. 
3.3 
Exclusion Criteria 
To be eligible for this study, patients should not meet any of the following criteria: 
• 
Presence of known lymph node involvement or distant metastases 
• 
Other histologic types of prostate cancers such as ductal, sarcomatous, lymphoma, 
small cell, and neuroendocrine tumors 
• 
Prior radiation therapy, hormonal therapy, biologic therapy, or chemotherapy for 
prostate cancer 


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• 
Prior immunotherapy/vaccine therapy for prostate cancer 
• 
Previous or concurrent use of cyclophosphamide 
• 
Concomitant treatment with other hormonal therapy or 5α-reductase inhibitors 
• 
Current use of systemic corticosteroids or use of corticosteroids within 4 weeks of 
enrollment (inhaled corticosteroids for asthma or COPD are permitted) 
• 
Use of experimental agents for prostate cancer within the past 3 months 
• 
Known allergy to cyclophosphamide or G-CSF/GM-CSF 
• 
Known hypersensitivity to materials of bovine origin (e.g. fetal bovine serum), or 
other components of GVAX which include DMSO and hydroxyethyl starch as well as 
small amounts of porcine trypsin and DNase 
• 
History or presence of autoimmune disease requiring systemic immunosuppression 
(including but not limited to: inflammatory bowel disease, systemic lupus 
erythematosus, vasculitis, rheumatoid arthritis, scleroderma, multiple sclerosis, 
hemolytic anemia, Sjögren syndrome, and sarcoidosis) 
• 
 Other concurrent malignancies, with the exception of non-melanoma skin cancers 
and superficial bladder cancer 
• 
Uncontrolled major active infectious, cardiovascular, pulmonary, hematologic, or 
psychiatric illnesses that would make the patient a poor study candidate 
• 
Known prior or current history of HIV and/or hepatitis B/C  
3.4 
Inclusion of Minorities 
Men of all races and ethnic groups will be considered for study participation. Candidates must 
conform to all eligibility criteria to be accepted into the study. Minority patients who meet entry 
criteria will be actively recruited, although the trial is not designed to measure differences in 
outcomes between ethnic groups. The estimated breakdown of the target population by race and 
ethnicity is: 80% white/Caucasian, 15% black/African American, and 5% comprised of other ethnic 
minorities. 
3.5 
Prohibited Concomitant Medications 
Concurrent use of other anticancer agents or therapies including other experimental treatments is 
not permitted. Patients may not currently be taking any other form of androgen deprivation therapy, 
antiandrogens, 5α-reductase inhibitors, chemotherapy, radiation therapy, biologic therapy, 
immunosuppressive medications, or systemic corticosteroids. 
Within 30 days of administration of study drug(s), patients should ideally not receive other cancer 
immunotherapies or vaccines including but not limited to the live rotavirus vaccine, the live BCG 
(bacillus Calmette-Guerin) vaccine, the live influenza vaccine, the live measles vaccine, the live 
mumps vaccine, the live poliovirus vaccine, the live rubella vaccine, the smallpox vaccine, the typhoid 
vaccine, the varicella vaccine, and the yellow fever vaccine. 
The following list of medications should generally be taken with caution by patients on chronic 
cyclophosphamide: allopurinol, cyclosporine, etanercept, hydrochlorothiazide, nevirapine, 
ondansetron, pentostatin, St John’s wort, tamoxifen, trastuzumab, and warfarin. In the present study, 
because only a single low-dose administration of cyclophosphamide will be given (in treatment arm 
B), these medications are not prohibited. However, the principal investigator must be notified if a 
patient is taking any of these listed agents. 


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Because of the potential for unknown drug-drug interactions, concurrent use of all other agents, 
over-the-counter medications, herbal remedies, vitamins/minerals, and alternative therapies must 
be documented on the case report form (CRF).
4. 
REGISTRATION AND ENROLLMENT PLAN 
4.1 
Registration Procedure 
Patients who are considered candidates for the study will first be evaluated for eligibility by one of 
the principal investigators, co-investigators, or the research nurse. After screening for eligibility, 
patients who are eligible to participate in the trial must be registered with the Sidney Kimmel 
Comprehensive Cancer Center (SKCCC) according to the instructions below. A record of patients who 
fail to meet entry criteria (i.e., screen failures) will also be maintained. Registration must be 
completed before beginning any study-related activities. 
To initiate registration at SKCCC, study personnel should forward copies of the signed informed 
consent form (with embedded research authorization/HIPAA form), the institutional registration 
form, plus any required pathology information/laboratory tests to the project manager by fax or 
email. Once eligibility is confirmed, each subject will be assigned a unique patient study identification 
number. Treatment must not commence until the patient has received his identification number. 
Prior to protocol enrollment and initiation of treatment, subjects must sign and date an IRB-
approved consent form. Authorized study personnel should fully explain the scope of the study to 
each patient before obtaining informed consent. Patients should be advised of any known risks 
inherent in the planned treatments/procedures, any alternative treatment options, their right to 
withdraw from the study at any time and for any reason, and their right to privacy. When obtaining 
informed consent, study personnel should: first, confirm that the patient has received and has had 
time to read the informed consent form (including the research authorization/HIPAA form); next, 
confirm eligibility as defined in Sections 3.2 and 3.3 (inclusion and exclusion criteria); and finally, 
obtain dated and signed informed consent. A copy of the signed informed consent should be supplied 
to the project manager. 
To register a patient, the following documents must be completed and faxed (410-614-7287) or 
emailed (hcao7@jhmi.edu) to the study coordinator, Harry Cao: 
• 
signed patient consent form 
• 
institutional registration form 
• 
copies of the prostate cancer pathology report and baseline laboratory studies including CBC 
with differential, liver and kidney function tests. Other materials may also be sent if considered 
pertinent for confirming patient eligibility. 
The principal investigator and/or other authorized study personnel will then review these 
documents to confirm eligibility. To complete the registration process, the project manager will 
assign a patient study number (i.e., protocol patient number). This number is unique to the patient 
and must be written on all data and correspondence for the patient. The project manager will also 
register the patient with SKCCC’s Clinical Research Office (CRO). 
4.2 
Expected Enrollment 
A total of 32 patients (16 in each arm) will be included in this study. The first patients are expected to 
be enrolled in October 2012, once the protocol has been approved by the IRB. With an estimated 
enrollment rate of 4 patients per month, accrual is expected to be completed in 8 months. 
4.3 
Study Centers 
This is a single-institution study that will be conducted only at Johns Hopkins through a collaboration 
between the Brady Urological Institute and the Sidney Kimmel Comprehensive Cancer Center. 


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4.4 
Recruitment 
Subjects will be recruited from the outpatient facilities of the Brady Urological Institute, and from the 
outpatient Multidisciplinary Prostate Cancer Clinic of the SKCCC. Patients will not receive payment or 
reimbursement for participation. Every effort will be made to include patients of racial and ethnic 
minorities who fulfill the eligibility criteria. 
4.5 
Randomization  
Patients will be randomized in a 1:1 ratio to one of 2 treatment arms (see Study Schema). Because our 
data showed that T cell function is associated with the tumor grade, the randomization will be 

ǣζ͹ͺ-10. Treatment allocation will be determined by a computer-
generated permuted-block randomization scheme developed and maintained by the Department of 
Oncology Biostatistics at Johns Hopkins. Randomization will be performed by the study statistician, 
Marianna Zahurak, MS. This pre-generated randomization scheme has been approved by the Johns 
Hopkins Research Pharmacy, who will also maintain a copy of the scheme.
5. 
STUDY PLAN 
5.1 
Overview and Schema 
This is a single-center, randomized, 2-arm, open-label, prospective clinical trial evaluating the 
immunogenicity and safety of a single administration of LHRH antagonist (degarelix) used alone, or 
degarelix administered in sequence with low-dose cyclophosphamide and GVAX, given 3 weeks 
prior to radical prostatectomy in men with localized prostate cancer. 
Patients will be recruited from the outpatient Urology clinic. Eligible patients will be randomized in 
ͳǣͳȋ
ǣζ͹ͺ-10) to receive one of two treatment regimens (see 
Schema below), each beginning 28 (±3) days prior to radical prostatectomy. In Arm B, 
Cyclophosphamide will be given at  a dose of 200 mg/m2 as a single intravenous infusion. 1 day 
later, prostate GVAX will be administered as five 0.8-mL intradermal injections of PC3 (2.5 × 108 
cells) and five 0.5-mL intradermal injections of LNCaP (1.6 × 108 cells), for a total dose of 4.1 × 108 
cells.  On day 14, Degarelix acetate will be administered as three 80  mg subcutaneous injections, for 
a total dose of 240 mg. Prostate glands will be harvested 14  (±3) days later, at the time of radical 
prostatectomy, and prostate tissue will be examined for the primary endpoint. In Arm A, an 
identical dose of degarelix acetate will be administered 14 (±3) days prior to surgery.  A telephone 
follow-up interview (or an in-person clinic visit) to evaluate for adverse events will occur 28 (±21) 
days after prostatectomy. Patients will then be followed by their urologists according to standard 
institutional practices, but will require PSA evaluations every 3 (±1) months during year 1 and 
every 6 (±2) months during years 2-3. 
                                                          Treatment Schema: 
.


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Table 1A   STUDY CALENDAR for ARM A (Androgen-Ablation Alone) 
 
Every effort should be made to keep visits, tests, and procedures on schedule. Acceptable 
deviations are listed below. 
 
ARM A 
Screening 
Evaluation a 
Day 
14  
Radical 
Prostatectomy, 
Day 28 (±3) 
Follow-up,  28 Days (±21) 
Post-op l m 
Informed consent 
X 
 
 
 
Medical history 
X 
 
X 
 
Review of medications 
X 
 
X 
 
Physical examination 
X 
 
X 
 
Vital signs 
X 
 
X 
 
Height and weight 
X 
 
 
 
Performance status 
X 
 
X 
X 
CT and bone scan b 
(X) 
 
 
 
Hematology labs c 
X 
 
X 
X 
Chemistry labs d 
X 
 
X 
X 
Serum PSA e 
X 
 
X 
 
HLA typing f 
 
X 
 
 
Sera for immunoassays g 
 
X 
X 
X 
Blood (200 mL) for PBLs h 
 
X 
 
X 
Degarelix acetate dose i 
 
X 
 
 
Toxicity assessment 
         X------------------------------------------------------------------X 
Vaccine site assessment 
         X------------------------------------------------------------------X 
Surgical specimen j 
 
 
X 
 
Pathologic review k 
X 
 
X 
 
a: 
The screening (pre-treatment) evaluation should be conducted within 3 weeks (±4 days) of starting protocol therapy. 
b: 
Staging CT (if allergic to CT scan contrast, obtain MRI with contrast) and bone scans should only be performed if clinically 
indicated, and are not mandatory. 
c: 
Hematology laboratories include hemoglobin, hematocrit, white blood cell count with differential (including absolute eosinophil 
count), and platelets. In addition, prothrombin time (PT/INR) and activated partial thromboplastin time (APTT) should be 
checked as clinically indicated. 
d: 
Chemistry laboratories include sodium, potassium, chloride, bicarbonate, urea nitrogen, creatinine, glucose, calcium, albumin, 
total protein, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase. 
e: 
Serum PSA should be obtained pre-treatment and on the day of surgery, and as clinically indicated thereafter according to 
institutional practices. 
f: 
At baseline (day 14), collect 10 mL of blood into a 10-mL ACD (acid citrate dextrose) tube [test code 4470; CPT code 86813]. 
g: 
Blood samples will be collected at day 14 and again on the day of prostatectomy and at follow up visit. At each time point, 2 
plain (red top) or SST (gold top) tubes should be collected, each containing ≥5mL of blood. After centrifugation, serum should be 
transferred into plastic cryovials and frozen at –20°C or colder until the time of analysis (see Section 5.4). 
h: 
Peripheral blood lymphocytes (PBLs) will be obtained by collecting 200 mL of blood into 50-mL heparinized conical tubes. PBLs 
will be prepared by Ficoll-Hypaque density gradient centrifugation according to standard protocols, and will be cryopreserved in 
a liquid nitrogen freezer at –140°C for further batched analyses. 
i: 
Degarelix acetate may be administered 2-14 days after the screening visit, and that day is designated as day + 14 for future 
reference. See also the treatment schema for further information on dosage and timing of degarelix acetate. 
j: 
Harvested prostate gland to be evaluated for CD8
+ T cell infiltration, as well as other secondary endpoints. 
k: 
Archival prostate core biopsies to be centrally reviewed at baseline prior to study entry; radical prostatectomy specimens to be 
processed at Johns Hopkins according to standard procedures. 
l: 
The post-operative evaluation (28 days after prostatectomy) may take place over the telephone or in person, but patients are 
required to have hematology and chemistry labs collected at this time-point for safety purposes. 
m: 
In addition, PSA should be measured every 3 (±1) months in the first year after prostatectomy, and every 6 (±2) months in the 
second and third years after prostatectomy. 


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Table 1B   STUDY CALENDAR for ARM B (Experimental Arm) 
Every effort should be made to keep visits, tests, and procedures on schedule. Acceptable 
deviations are listed below. 
ARM B 
Screening 
Evaluation a 
Day 1 Day 2 Day 
14 
Radical 
Prostatectomy, 
Day 28 (±3) 
Follow-up,  
28 Days (±21) 
Post-op k l 
Informed consent 
X 
 
 
 
 
 
Medical history 
X 
 
 
 
X 
 
Review of medications 
X 
 
 
 
X 
 
Physical examination 
X 
 
 
 
X 
 
Vital signs 
X 
 
 
 
X 
 
Height and weight 
X 
 
 
 
 
 
Performance status 
X 
 
 
 
X 
X 
CT and bone scan b 
(X) 
 
 
 
 
 
Hematology labs c 
X 
 
 
 
X 
X 
Chemistry labs d 
X 
 
 
 
X 
X 
Serum PSA e 
X 
 
 
 
X 
 
HLA typing f 
 
X 
 
 
 
 
Sera for immunoassays g 
 
X 
 
 
X 
X 
Blood (200 mL) for PBLs h 
 
X 
 
 
 
X 
Cyclophosphamide dose 
 
X 
 
 
 
 
GVAX dose 
 
 
X 
 
 
 
Degarelix acetate dose 
 
 
 
X 
 
 
Toxicity assessment 
X---------------------------------------------------------------------------X 
Vaccine site assessment 
X---------------------------------------------------------------------------X 
Surgical specimen i 
 
 
 
 
X 
 
Pathologic review j 
X 
 
 
 
X 
 
a: 
The screening (pre-treatment) evaluation should be conducted within 3 weeks (±4 days) of starting protocol therapy. 
b: 
Staging CT (if allergic to CT scan contrast, obtain MRI with contrast) and bone scans should only be performed if clinically 
indicated, and are not mandatory. 
c: 
Hematology laboratories include hemoglobin, hematocrit, white blood cell count with differential (including absolute eosinophil 
count), and platelets. In addition, PT/INR and APTT should be checked as clinically indicated. 
d: 
Chemistry laboratories include sodium, potassium, chloride, bicarbonate, urea nitrogen, creatinine, glucose, calcium, albumin, 
total protein, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase. 
e: 
Serum PSA should be obtained pre-treatment and on the day of surgery, and as clinically indicated thereafter according to 
institutional practices. 
f: 
At baseline (day 1), collect 10 mL of blood into a 10-mL ACD (acid citrate dextrose) tube [test code 4470; CPT code 86813]. 
g: 
Blood samples will be collected at day 1 and again on the day of prostatectomy and at follow up visit. At each time point, 2 plain 
(red top) or SST (gold top) tubes should be collected, each containing ≥5mL of blood. After centrifugation, serum should be 
transferred into plastic cryovials and frozen at –20°C or colder until the time of analysis (see Section 5.4). 
h: 
Peripheral blood lymphocytes (PBLs) will be obtained by collecting 200 mL of blood into 50-mL heparinized conical tubes. PBLs 
will be prepared by Ficoll-Hypaque density gradient centrifugation according to standard protocols, and will be cryopreserved in 
a liquid nitrogen freezer at –140°C for further batched analyses. 
i: 
Harvested prostate gland to be evaluated for CD8
+ T cell infiltration, as well as other secondary endpoints. 
j: 
Archival prostate core biopsies to be centrally reviewed at baseline prior to study entry; radical prostatectomy specimens to be 
processed at Johns Hopkins according to standard procedures. 
k: 
The post-operative evaluation (28 days after prostatectomy) may take place over the telephone or in person, but patients are 
required to have hematology and chemistry labs collected at this time-point for safety purposes. 
l: 
In addition, PSA should be measured every 3 (±1) months in the first year after prostatectomy, and every 6 (±2) months in the 
second and third years after prostatectomy. 


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5.2 
Screening/Pretreatment Evaluation  
Before initiating any screening activities, the scope of the study should be explained to each patient. 
Patients should be advised of any known risks inherent in the planned procedures, any alternative 
treatment options, their right to withdraw from the study at any time and for any reason, and their 
right to privacy. After this explanation, patients should be asked to sign and date an IRB-approved 
informed consent form that meets the requirements of the Code of Federal Regulations (Federal 
Register Vol. 46, No. 17, January 27, 1981, part 50). 
The pretreatment/screening visit will determine patient eligibility according to the inclusion and 
exclusion criteria. All subjects must undergo a number of baseline evaluations as part of this 
screening visit, as detailed below. All of these evaluations should be conducted within 21 ± 4 days of 
starting the protocol. This information is also summarized in the Study Calendar (Tables 1A and 1B). 
• 
informed consent (including research authorization) 
• 
demographic information 
• 
medical history, including review of systems 
• 
performance status, using ECOG or Karnofsky scales (Appendix A) 
• 
physical examination 
• 
vital signs: temperature, pulse, sitting blood pressure, respiratory rate 
• 
height and weight 
• 
current medication list, including drug allergies/adverse events 
• 
hematological laboratories (hemoglobin, hematocrit, white blood cell count with 
differential [including absolute eosinophil count], platelets); coagulation profile (INR, 
aPTT) if clinically indicated 
• 
serum chemistry profile (sodium, potassium, chloride, bicarbonate, urea, creatinine, 
glucose, calcium, albumin, total protein, bilirubin, ALT, AST, alkaline phosphatase) 
• 
serum PSA level 
• 
sera for immunoassays (Pre-treatment) (see Section 5.4) 
• 
CT (If allergic to CT scan contrast, obtain MRI with contrast) and/or bone scan, only if 
clinically indicated (not mandatory) 
• 
central pathologic review of prostate core biopsies 
After all relevant screening information is documented, registration should be finalized and 
appropriate documents (i.e., signed informed consent, supporting source documentation for 
eligibility) should be faxed or emailed to the program manager. 
Information on patients who do not meet eligibility criteria to participate in this study (i.e., screening 
failures) should also be captured at the pretreatment visit.
5.3 
Radical Prostatectomy 
The following must be performed on the day of radical prostatectomy, which itself should occur 28 
(±3) days after the administration of degarelix acetate. 
• 
medical history 
• 
performance status 
• 
physical examination, including vital signs 


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• 
review of medication list 
• 
review of toxicity/adverse events 
• 
hematological laboratories and serum chemistry profile 
• 
serum PSA 
• 
sera for immunoassays (see Section 5.4) 
• 
collection of prostatectomy tissue for analysis of study endpoints 
• 
pathological processing of prostatectomy specimen according to standard procedures 
5.4 
Sera for Immunoassays 
At each time point (Pre-treatment, at prostatectomy, and at follow-up visit), the following research 
blood samples should be collected and processed as outlined below: 
• 
Draw approximately 10 mL of peripheral blood into 2 plain (red top) or SST (gold top) tubes, 
each containing ≥5 mL of blood per vacutainer. 
• 
Allow blood to coagulate for 20 minutes, then centrifuge at 25oC, 1500 x g (2700-3000 rpm), for 
15 minutes. 
• 
Pipette the serum into 10 cryotubes (about 0.5 mL/tube). 
• 
Store cryotubes frozen, below –20oC (–70oC preferred), until the time of analysis. 
5.5 
Follow-Up Evaluations 
A follow-up visit scheduled for 28 (±21) days after radical prostatectomy may occur in the outpatient 
clinic or by telephone interview. Required evaluations during this follow-up visit are listed below. 
Patients should then continue to be followed by their treating urologist according to standard 
institutional practices. 
• 
performance status 
• 
review of toxicity/adverse events 
• 
assessment for vaccine site reactions 
Patients withdrawing from the study early because of adverse events should be followed until the 
adverse event has either resolved or stabilized. Reasons for premature withdrawal should be 
determined and documented. 
In addition, patients will be required to have a structured assessment of post-operative PSA 
measurements for 3 years. In the first year after prostatectomy, PSA will be measured every 3 
months (±1 mo). In the second and third years after prostatectomy, PSA will be measured every 6 
months (±2 mo). These PSA measurements may be obtained outside of Johns Hopkins, but the results 
need to be made available to the study team. 
Long-Term Follow-Up Protocol.  All patients that have received GVAX will also be encouraged to 
enter a separate long-term follow-up protocol to assess for potential late toxicities of GVAX (e.g. 
autoimmune disease, second malignancies). This long-term follow-up protocol will involve annual 
visits for 3 years. 
5.6 
Duration of Therapy 
Participation in this study will be terminated for any of the following reasons listed below: 
• 
the patient decides to withdraw from the study (withdrawal of consent) due to unacceptable 
toxicities or for any other reason 
• 
the patient completes all of the protocol procedures and follow-up requirements 


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• 
there are adverse events that, in the judgment of the investigator, may cause severe or 
permanent injury or are incompatible with continuation on study 
• 
there are major violations to the study protocol or the patient is noncompliant with treatments, 
as judged by the investigator 
• 
the patient experiences concurrent illness or a change in his condition that, in the judgment of 
the investigator, renders him unacceptable for further treatment on study 
• 
the patient dies 
• 
the patient is lost to follow-up 
• 
the study is prematurely terminated for safety or feasibility concerns or other reasons 
Patients should be removed from the study when any of the above criteria are met. Because an 
excessive rate of withdrawals can render the study uninterpretable, unnecessary withdrawal of 
patients should be avoided if possible. When a patient leaves the study early, the investigator should 
make every effort to contact the patient and to perform the final follow-up evaluation (even by 
telephone interview). The reason for removal of a patient from the study, and the date of removal, 
must be appropriately documented. 
Patients will be replaced if they are removed from the study after signing the informed consent but 
before receiving degarelix, cyclophosphamide, or GVAX. Patients receiving at least one dose of the 
study drugs will be included in safety analyses, and those also undergoing prostatectomy will be used 
for the efficacy analyses. 
6. 
THERAPEUTIC AGENTS 
6.1 
Prostate GVAX Immunotherapy  
6.1.1  
Dosage, Formulation, and Administration  
The prostate GVAX immunotherapy will consist of 2.5 × 108 cells from the AAV 
genomic GM-CSF–transduced PC3 Clinical Lot and 1.6 × 108 cells from the AAV 
genomic GM-CSF–transduced LNCaP Clinical Lot.  GVAX will only be given to 
patients in arm B. 
Prostate GVAX will be administered as five 0.8-mL intradermal injections of PC3 (2.5 
× 108 cells) and five 0.5-mL intradermal injections of LNCaP (1.6 × 108 cells), for a 
total dose of 4.1 × 108 cells. In the case of PC3, each vile is filled with 1mL of cells in 
suspension. The contents of five such vials should be distributed into 5 syringes. 
Because it is not possible to remove a full 1mL from each vial after thawing, a 
minimum of 0.8-mL should be drawn into each of the 5 syringes. In the case of 
LNCaP, each vile is also filled with 1mL of cells in suspension. The contents of three 
such vials should be distributed into 5 syringes. Because it is not possible to remove 
a full 1mL from each vial after thawing, approximately 0.5-mL should be drawn into 
each of the 5 syringes. Every attempt should be made to use the entire content of 
three vials of LNCaP cells, distributed among these 5 syringes (i.e. none of the LNCaP 
cells should be discarded). 
The immunotherapy clinical lots were manufactured and released by the Cell 
Processing and Gene Therapy Facility, a cGMP-compliant facility, at the Sidney 
Kimmel Comprehensive Cancer Center at Johns Hopkins. The manufacturing and 
product release data for the PC3 and LNCaP cell lines were submitted to the FDA 
prior to initiating this study. 


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The transduced cells are irradiated prior to cryopreservation and stored frozen in 
the vapor phase of liquid nitrogen until the day of use. The cryoprotectant is in an 
injectable formulation: 6% hetastarch (in 0.9% sodium chloride) supplemented 
with 2% human serum albumin and 5% DMSO. 
The cell lines used for the vaccine preparation have undergone extensive regulatory 
testing and have been determined to be sterile and free of viral contamination. 
The details of production, irradiation, freezing, and preparation for administration 
are described in detail in the Investigational New Drug (IND) submission. 
Released vials of the AAV genomic GM-CSF–transduced PC3 Clinical Lot and the AAV 
genomic GM-CSF–transduced LNCaP Clinical Lot will be transferred from the Cell 
Processing and Gene Therapy Facility to the CTL in a ‘dry’ liquid nitrogen shipper. 
The vials will be stored in a vapor-phase liquid nitrogen freezer until use, in 
accordance with existing standard operating procedures. The freezer is alarmed and 
monitored using an R & D Datatron System. 
Administration of the cells will be given as intradermal injections using a 22-gauge 
needle and must be administered within 75 minutes from the time that the vials 
have thawed. Each cell line should be administered on a separate leg. Injections 
should use the upper portion of the thigh. Injections should be placed randomly, 
approximately 2 inches apart from each other. If the patient is unable to receive an 
injection in the legs, an acceptable alternative injection site is the abdomen. Do not 
place injections into skin that is ulcerated or infected. Patients must remain in the 
clinic for observation for ≥60 minutes following vaccination. 
A lidocaine-based topical anesthetic cream (e.g. EMLA or ELA-MAX) will be 
recommended to be applied to each injection site approximately 1-2 hours (up to 
2.5 hours) prior to vaccination to diminish the discomfort associated with the intra-
dermal injections. The topical anesthetic is optional and participants are allowed to 
decline its use. Self-application of the topical anesthetic is allowed after the research 
participant receives instruction. If the research participant is non-compliant with 
topical anesthetic administration instructions, the topical anesthetic will be applied 
by the research nurse. If the subject is allergic to lidocaine, the topical anesthetic 
should not be used. All research participants will be seen in the Oncology Outpatient 
Center for immunotherapy administration and monitoring.  
6.1.2  
Potential Toxicities 
Toxicities will be assessed using the most recent National Cancer Institute Common Toxicity 
Criteria for Adverse Events (NCI CTCAE version 4.0 – see Appendix B). 
6.1.2.1 
Potential Toxicities of the Storage Solutions 
Dimethyl Sulfoxide (DMSO)  
The prostate GVAX vaccine is cryopreserved in a solution containing approximately 
5% DMSO. DMSO is a cryoprotectant used to store a variety of infused cell products 
including hematopoietic stem cells and bone marrow. After cells are thawed, DMSO 
contained in the freezing solution is infused along with the cells. Toxicity associated 
with DMSO infusion is usually limited to minor changes in heart rate, blood 
pressure, fever, chills, sweats, nausea, vomiting and headaches. These are usually 
self-limited, lasting no more than a few hours. The most notable effect is a garlicky 


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odor due to exhalation of a DMSO metabolite that may last up to one day. More 
severe adverse effects include severe acute allergic reactions including 
anaphylactoid reactions, pulmonary embolism, respiratory failure, renal failure, 
cardiac arrhythmias, seizures, and death. These side effects are rarely observed and 
occur primarily in patients receiving much higher volumes of cryopreserved cell 
products than being infused in the present study. Nevertheless, patients will be 
carefully monitored for any adverse effects as outlined in Section 7. Any adverse 
events should be documented and recorded on the appropriate Case Report Form.  
Human Serum Albumin (HSA) 
The prostate GVAX vaccine is formulated in a cryoprotectant containing 2% human 
serum albumin. Albumin solutions are FDA-approved for volume resuscitation 
(treatment of hypovolemia, shock, and burns). Although hypersensitivity reactions 
are possible, reactions are not anticipated to the small amount of albumin 
administered in this study.  
Hetastarch (6%) 
The prostate GVAX vaccine is formulated in a cryoprotectant containing 6% 
hetastarch in 0.9% sodium chloride. Toxicities associated with the intravenous 
administration of 6% hetastarch include circulatory overload, abnormalities of 
coagulation (prolonged prothrombin, activated partial thromboplastin and clotting 
times), and hypersensitivity reactions characterized by wheezing, urticaria, 
hypotension, and anaphylactic/ anaphylactoid reactions.  
6.1.2.2 Potential Toxicities of the Johns Hopkins Prostate GVAX Immunotherapy  
The Johns Hopkins prostate GVAX immunotherapy will consist of two allogeneic 
prostate cell lines (PC3 and LNCaP). GM-CSF–secreting vaccines have been 
administered to over 300 patients at Johns Hopkins and toxicities have been limited 
to localized vaccine site reactions involving erythema and tenderness. Rarely, 
systemic reactions have been reported such as low-grade fevers and Grover’s 
syndrome (transient acantholytic dermatosis).   
The GM-CSF released by the PC3/LNCaP cells after injection may also cause side 
effects. Redness at old injection sites may return long after the administration of 
GVAX. The following side effects have also been reported by people taking GM-CSF: 
fever, chills, weakness, headache, bone pain, and muscle aches. Other side effects 
reported less often are difficulty breathing, rash, fluid overload/anasarca, 
tachycardia, arrhythmia, leukopenia, pain (e.g. abdomen, back, chest, and joint pain), 
venous thrombosis, abnormal liver function tests, and pleural/pericardial effusions. 
GVAX contains porcine trypsin, fetal bovine serum, dextran sulfate and DNase and 
could cause an allergic reaction in sensitized individuals. There is a possible risk of a 
severe allergic reaction (anaphylaxis) to GVAX. 
The LNCaP cell line contains very small amounts of fetal bovine serum. There is a 
remote possibility that bovine serum may contain known or unknown infectious 
agents that may cause human disease, such as Creutzfeldt-Jakob (mad cow) disease. 
There is a slight possibility that GVAX could cause autoimmune disease. This has 
only been observed when GVAX was combined with the CTLA4-blocking monoclonal 
antibody, ipilimumab. 
There is a theoretical concern that people who receive gene therapy may develop a 
new cancer. 


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A single dose of GVAX given 2 weeks prior to prostatectomy is not expected to cause 
side-effects that would potentially delay surgery or negatively affect surgical 
outcomes. Nonetheless, its influence on prostatectomy will be closely monitored. 
GVAX is an experimental product that may have other side effects that are unknown 
and have not been predicted. These side effects may be serious. 
6.1.2.3 Potential Toxicities Observed with the Cell Genesys Prostate GVAX® 
Cell Genesys Prostate GVAX® has been given to at least 1200 men with hormone-
refractory prostate cancer in prior clinical studies. Nearly all men developed 
swelling and redness where CG1940 (PC3) and CG8711 (LNCaP) were injected. Most 
of the men also had itching and pain at the injection site. In addition, other reported 
side effects were fatigue, malaise, headache, myalgias, arthralgias, fever, 
chills/rigors, rash, anemia, weight loss, nausea, decreased appetite, flu-like 
symptoms, weakness, constipation, general body pains, dizziness, back pain, and 
hives. 
In a trial comparing Cell Genesys GVAX® to docetaxel, an increase in serious urinary 
tract obstructions was noticed in patients that received GVAX® as compared to 
those that received docetaxel. In that study, three patients had serious side effects 
that may have been related to GVAX®, and later died. An 82 year-old man died in his 
sleep four days after his first GVAX® treatment; the cause of his death was unknown. 
After a fall, another man had a subdural hematoma, thrombocytopenia, and later 
died. A third man had a stroke which resulted in death. 
In another trial comparing docetaxel/GVAX® to docetaxel/prednisone, adverse 
events on the vaccine arm included infections, anemia, metabolic disturbances, 
altered mental status, abnormal coordination, seizure, cardiovascular complications, 
respiratory complications, abnormal taste, abdominal pain, chest pain, cough, 
hypertension, flushing, gastrointestinal disturbance, musculoskeletal effects, renal 
impairment, and fractures. It is unclear whether or not these side effects were 
related to GVAX®, docetaxel, or due to the omission of prednisone in that treatment 
arm. In addition, there were 8 deaths in the GVAX® arm in that trial that were not 
attributable to prostate cancer progression. These deaths were caused by cardiac 
arrest (2), respiratory arrest (1), myocardial infarction (1), intracranial bleed (1), 
ischemic encephalopathy (1), pneumonia (1), and failure to thrive (1). 
Additional serious side effects have been reported as being related to GVAX® when 
given alone: anemia, deep venous thrombosis, sensorineural hearing loss, difficulty 
walking, weakness in the lower legs, severe joint pain, severe muscle pain, 
worsening of existing arrhythmia, dehydration resulting in kidney failure, 
hypercalcemia, nausea, vomiting, weight loss, pulmonary embolism, shortness of 
breath, fever, diarrhea, and malaise. Also, GVAX® may possibly be related to 
hypothyroidism and adrenal insufficiency. 
6.1.3  
Treatment Modifications and Dosing Delays 
Since only a single dose of prostate GVAX will be administered to each patient (arm B only), 
dose modifications or treatment delays will not be permitted. 
6.1.4  
Supportive Care and Concomitant Medications 
Injection site reactions: EMLA cream (or a similar product) should be applied to the site of 
vaccination at a dose of approximately 2.5 grams per site approximately one hour prior to 
vaccination to mitigate injection-site pain. 


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Nausea/vomiting: GVAX does not typically induce nausea or vomiting. However, 
prochlorperazine may be given for this indication at a dose of 10 mg by mouth or 
intravenously as frequently as every 6 hours. Oral or intravenous corticosteroid use is not 
permitted. However, prophylactic antiemetic therapy at the discretion of the treating 
physician is allowed. 
During treatment (and for at least 60 minutes afterwards), patients should be observed for 
adverse reactions. Treatment areas should have ACLS-certified personnel on site, as well as 
access to appropriate supportive care modalities including but not limited to oxygen 
administration, corticosteroids, antihistamines and catecholamines. Any or all of these 
modalities may be used in the case of hypersensitivity reactions or anaphylactic reactions. 
The interaction of PC3/LNCaP and other vaccines has not been studied, and there is a 
possibility that PC3/LNCaP may affect the immune response to other vaccines and vice versa. 
Whenever possible, patients should not receive another vaccine within 30 days of a dose of 
GVAX. However, medically indicated vaccines should not be delayed if doing so may be 
detrimental to the patient. 
6.2 
Low-Dose Cyclophosphamide 
6.2.1  
Dosage and Schedule 
Immunomodulatory cyclophosphamide will be given in the outpatient setting as a single 
administration, about 4 weeks prior to prostatectomy. It will be given at a dose of 200 
mg/m2, one day before administration of prostate GVAX.  It will only be given to patients in 
arm B. 
6.2.2  
Formulation and Preparation 
Cyclophosphamide is a sterile, white, lyophilized powder containing 75 mg mannitol per 100 
mg cyclophosphamide (anhydrous) supplied in 100 mg, 200 mg, 500 mg, 1000 mg, and 2000 
mg vials for single-dose intravenous administration.  
Lyophilized cyclophosphamide should be prepared for parenteral (intravenous) use by 
adding sterile water for injection (USP) to the vial, and shaking to dissolve. Vials containing 
100 mg, 200 mg, 500 mg, 1000 mg, and 2000 mg should be reconstituted with 5 ml, 10 ml, 
20 ml, 50 ml, and 100 ml respectively. Cyclophosphamide may be infused intravenously 
using the following solutions: 5% dextrose, 5% dextrose and 0.9% sodium chloride, 5% 
dextrose and Ringer’s injection USP, lactated Ringer’s injection USP, or sodium lactate 
injection USP. 
Vials of cyclophosphamide are stable at room temperature and at temperatures less than 
32°C. Reconstituted parenteral solutions are stable for 24 hours at room temperature (RT), 
or for 14 days if refrigerated at 2-8°C. After 24 hours at RT, or 14 days at 2-8°C, reconstituted 
cyclophosphamide must be discarded. 
6.2.3  
Drug Administration 
Cyclophosphamide will be obtained from a commercial supplier and prepared as per the 
institutional standard procedures. Cyclophosphamide will be administered by intravenous 
infusion over 30 min in the outpatient setting. Cyclophosphamide should not be mixed or 
diluted with other drugs. 
6.2.4  
Potential Toxicities 
Toxicity will be assessed using the most recent National Cancer Institute Common Toxicity 
Criteria for Adverse Events (NCI CTCAE version 4.0 – see Appendix B). 


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The dose of cyclophosphamide proposed in this trial is below those commonly used when 
this agent is employed as a cytotoxic drug (e.g. typically 600 mg/m2 of cyclophosphamide is 
used per treatment cycle in breast cancer chemotherapy). In addition, a 200 mg/m2 dose of 
cyclophosphamide is at about 1/9th the dose used for pre-conditioning during bone marrow 
transplant or for primary management of some autoimmune disorders. 
Therefore, we anticipate that the risk of toxicity related to the use of a single dose of 
cyclophosphamide at 200 mg/m2 is very small. Based on the toxicity evaluations of higher 
dose of cyclophosphamide (600 mg/m2), the most common adverse events with this agent 
include fatigue, alopecia, nausea, vomiting, diarrhea, fluid retention, skin rash, cytopenias, 
and infections. In addition, hemorrhagic cystitis, interstitial pneumonitis, and 
cardiomyopathy have been observed. However, these side effects have not been reported 
with the low doses of cyclophosphamide that will be used in this study. With standard-dose 
cyclophosphamide, secondary leukemia and myelodysplastic syndrome have also been 
reported as rare late occurrences. 
Allergic and anaphylactic reactions to cyclophosphamide have been reported. In addition, 
rare cases of Stevens-Johnson syndrome/toxic epidermal necrolysis have occurred. 
A single dose of cyclophosphamide given 3 weeks prior to prostatectomy is not anticipated 
to cause side-effects that would potentially delay surgery or negatively affect the outcomes 
of surgery. 
6.2.5 
Treatment Modifications and Dosing Delays 
Since only a single dose of cyclophosphamide will be administered to each patient (arm B 
only), dose modifications or treatment delays will not be permitted. 
6.2.6  
Supportive Care and Concomitant Medications 
Nausea/vomiting: Low-dose cyclophosphamide does not typically induce nausea or 
vomiting. However, prochlorperazine may be given for this indication at a dose of 10 mg by 
mouth or intravenously as frequently as every 6 hours. Oral or intravenous corticosteroid are 
not permitted. However, prophylactic antiemetic therapy at the discretion of the treating 
physician is allowed. 
During treatment (and for at least 60 minutes afterwards), patients should be observed for 
adverse reactions. Treatment facilities should have ACLS-certified personnel on site, as well 
as access to appropriate supportive care modalities including but not limited to oxygen 
administration, corticosteroids, antihistamines and catecholamines. Any or all of these 
modalities may be used in the case of hypersensitivity reactions or anaphylactic reactions. 
The following medications should generally be taken with caution by patients on chronic 
cyclophosphamide: allopurinol, cyclosporine, etanercept, ondansetron, hydrochlorothiazide, 
nevirapine, pentostatin, St John’s wort, tamoxifen, warfarin, and trastuzumab. In the present 
study, because only a single administration of low-dose cyclophosphamide will be given, 
these medications are not prohibited. However, the Principal Investigator should be notified 
if a patient is taking any of these listed agents. 
6.3 
Degarelix Acetate 
6.3.1  
Dosage and Schedule 
Degarelix will be given in the outpatient setting as three 80 mg subcutaneous injections, for a 
total dose of 240 mg, approximately 2 weeks prior to radical prostatectomy. It will be given 
to patients in both treatment arms, A and B. 


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6.3.2  
Formulation and Preparation 
Degarelix is supplied as a powder for subcutaneous injection. One vial of degarelix contains 
80 mg of degarelix. Each vial is to be reconstituted with 2 mL of sterile water for injection, 
USP. 2 mL is withdrawn to deliver 80 mg degarelix at a concentration of 40 mg/mL. One dose 
of degarelix comprises 240 mg given as three 2 mL injections of 80 mg each. 
6.3.3  
Drug Administration 
Degarelix is administered as a subcutaneous injection in the abdominal region. As with other 
drugs administered by subcutaneous injection, the injection site should vary periodically. 
Injections should be given in areas of the abdomen that will not be exposed to pressure, e.g. 
not close to waistband or belt. 
Degarelix is supplied as a powder to be reconstituted with sterile water for injection, USP. 
The reconstitution procedure needs to be carefully followed. Administration of other 
concentrations is not recommended. See “Instructions for Proper Use” below. 
Instructions for Proper Use 
• Gloves should be worn during preparation and administration. 
• Reconstituted drug must be administered within one hour after addition of sterile water 
for injection, USP. 
• Keep the vial vertical at all times 
• Do not shake the vials 
• Follow aseptic technique 
Preparation 
• Draw up 2 mL of sterile water for injection, USP (WFI) using a reconstitution needle 
(21G/2 in) 
• Inject the WFI slowly into the degarelix 80 mg vial. To keep the product and syringe sterile, 
do not remove the syringe and the needle. 
• Keeping the vial in an upright position, swirl it very gently until the liquid looks clear and 
without undissolved powder or particles. If the powder adheres to the vial over the liquid 
surface, the vial can be tilted slightly to dissolve powder. Avoid shaking to prevent foam 
formation. A ring of small air bubbles on the surface of the liquid is acceptable. The 
reconstitution procedure may take up to 15 minutes. 
• Tilt the vial slightly and keep the needle in the lowest part of the vial. Withdraw 3 mL of 
degarelix 80 mg without turning the vial upside down. 
• Exchange the reconstitution needle with the administration needle for deep subcutaneous 
injection (27G/1.25 in). Remove any air bubbles. 
• Inject 2 mL of degarelix 80 mg subcutaneously immediately after reconstitution. Grasp the 
skin of the abdomen, elevate the subcutaneous tissue. Insert the needle deeply at an angle of 
not less than 45 degrees. Gently pull back the plunger to check if blood is aspirated. If blood 
appears in the syringe, the reconstituted product can no longer be used. Discontinue the 
procedure and discard the syringe and the needle (reconstitute a new dose for the patient). 
• Repeat reconstitution procedure for the second dose. Choose a different injection site and 
inject 3 mL. 
6.3.4  
Potential Toxicities 
The most commonly observed adverse reactions with degarelix therapy include injection 
site reactions (e.g. pain, erythema, swelling or induration), hot flashes, increased weight, 


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fatigue, and increases in serum levels of transaminases and glutamyltransferase (GGT). The 
majority of the adverse reactions are grade 1 or 2, with grade 3/4 adverse reactions 
occurring at incidences of ≤1%. 
The most frequently reported adverse reactions at the injection sites are pain (28%), 
erythema (17%), swelling (6%), induration (4%) and nodule (3%). These adverse reactions 
are mostly transient, of mild to moderate intensity, occur primarily with the starting dose 
and lead to few discontinuations (<1%). Grade 3 injection site reactions occur in ≤2% of 
patients receiving degarelix. Hepatic laboratory abnormalities are primarily grade 1 or 2 and 
are generally reversible. Grade 3 hepatic laboratory abnormalities occur in <1% of patients. 
Other potential toxicities of degarelix include fatigue, asthenia, chills, night sweats, 
hypertension, arthralgias, back pain, diarrhea, constipation, erectile dysfunction, 
gynecomastia, hyperhidrosis, headache, dizziness, and insomnia. 
6.3.5 
Treatment Modifications and Dosing Delays 
Since only a single dose of degarelix acetate will be administered to each patient, dose 
modifications or treatment delays will not be permitted. 
6.3.6  
Concomitant Medications 
Degarelix is not a substrate for the human CYP450 enzyme system. Degarelix is not an 
inducer or inhibitor of the CYP450 system in vitro. Therefore, clinically significant CYP450 
pharmacokinetic drug-drug interactions are unlikely. 
6.4 
Concomitant Medications and Supportive Care 
Because of the potential for drug-drug interactions, the concurrent use of all other drugs, over-the-
counter medications, and alternative therapies must be documented on the case report form (CRF). 
Concurrent use of other therapies (including other experimental modalities) intended for the 
treatment of prostate cancer is not permitted. Patients may not currently be taking any other form of 
androgen deprivation therapies, antiandrogens, 5α-reductase inhibitors, chemotherapy, radiation 
therapy, biologic therapy, or immunosuppressive drugs. 
Because glucocorticoids (e.g. dexamethasone) may contribute to immunosuppression, patients 
should generally not initiate steroid therapy while on study. Accordingly, intermittent use of 
dexamethasone as an antiemetic agent is discouraged. The Principal Investigator must be notified if 
steroid use is being considered. Subjects may receive other forms of non-cancer-directed endocrine 
replacement therapy (e.g. thyroxine replacement and insulin replacement). 
Injection site reactions: For patients receiving GVAX, EMLA cream (or a similar product) should be 
applied to the site of vaccination at a dose of approximately 2.5 grams per site approximately one 
hour prior to vaccination to mitigate injection-site pain. 
Nausea/vomiting: GVAX and low-dose cyclophosphamide do not typically induce nausea or vomiting. 
However, prochlorperazine may be given for this indication at a dose of 10 mg by mouth or 
intravenously as frequently as every 6 hours. Prophylactic antiemetic therapy at the discretion of the 
treating physician is allowed. 
During treatment (and for at least 60 minutes afterwards), patients should be observed for adverse 
reactions. Treatment areas should have ACLS-certified personnel on site, as well as access to 
appropriate supportive care modalities including but not limited to oxygen administration, 
corticosteroids, antihistamines and catecholamines. Any or all of these modalities may be used in the 
case of hypersensitivity reactions or anaphylactic reactions. 
Diet will not be regulated on this study, except as required by standard pre-operative protocols. The 
use of vitamins, nutritional supplements, homeopathic medications, and herbal remedies is 


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discouraged and must be approved by the principal investigator. Multivitamin tablets and calcium 
supplementation are generally permitted. 
Patients should receive full supportive care as appropriate, including transfusions of blood and blood 
products, erythropoietin, antibiotics, antiemetics, analgesics, electrolyte replacement, and other 
supportive measures. The indications, doses, and dates of such treatments should be recorded on the 
CRFs. Use of growth factors such as filgrastim/peg-filgrastim (G-CSF) or sargramostim (GM-CSF) is 
discouraged. 
6.5 
Drug Interactions/Cautions 
Within 30 days of administration of study drug(s), patients should not receive other vaccines 
including but not limited to the live rotavirus vaccine, the live BCG (bacillus Calmette-Guerin) 
vaccine, the live influenza vaccine, the live measles vaccine, the live mumps vaccine, the live 
poliovirus vaccine, the live rubella vaccine, the smallpox vaccine, the typhoid vaccine, the varicella 
vaccine, and the yellow fever vaccine. 
The following list of medications should generally be taken with caution by patients on 
chronic cyclophosphamide: allopurinol, cyclosporine, etanercept, hydrochlorothiazide, 
nevirapine, ondansetron, pentostatin, St John’s wort, tamoxifen, trastuzumab, and warfarin. 
In the present study, however, because only a single low-dose administration of 
cyclophosphamide will be given, these medications are not prohibited. Nevertheless, the 
principal investigator must be notified if a patient is taking any of these listed agents. 
6.6 
Drug Dispensing Log 
Study site personnel should record all study drugs administered during this trial on the drug-
dispensing log. This log may also be reviewed by the research pharmacist. The drug dispensing log 
will contain the following information: 
• 
patient study identification number 
• 
date(s) of study drug administered 
• 
doses/quantities of study drug(s) administered 
• 
signature of the investigator or authorized research nurse 
6.7 
Pregnancy 
Men with partners of child-bearing potential must agree to use adequate contraception 
(hormonal and/or barrier methods; or abstinence) for the duration of the study and for 3 
months after the last dose of study drug(s). 
7. 
ADVERSE EVENTS AND REPORTING REQUIREMENTS 
An adverse event (AE) is defined as any untoward medical occurrence (symptom, sign, illness or 
experience) that develops or worsens in a research patient during a clinical study or within 30 days 
post-treatment, regardless of causality. This includes adverse clinical or laboratory findings, any 
adverse drug reaction (ADR), an illness with onset during the study, or an exacerbation of a 
preexisting illness or condition. Cancer progression should not be considered an AE, unless the 
investigator believes that the study treatment exacerbated the patient’s condition. Exceptions are if 
disease progression results in death or hospitalization while a patient is on study, in which case the 
disease progression is considered a serious AE. Abnormal findings on physical examination or 
diagnostic procedures are also considered AEs if: they are associated with clinical signs or 
symptoms; they require therapeutic intervention or additional diagnostic testing; they lead to dose 
modifications/termination of the study drug; or they are considered clinically significant by the 


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investigator. All observed or reported AEs, regardless of their suspected causal relationship to the 
study drug, should be recorded on the Case Report Form (CRF). 
 The NCI CTCAE version 4.0 will be used for adverse event descriptions and grading 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf). These 
criteria are summarized in Appendix B. The type and severity of an AE, as well as its potential link to 
the study drug(s), will determine whether the event requires expedited reporting in addition to 
routine reporting. For all AEs, the investigator must pursue and obtain information to adequately 
determine the causality and outcome of the event, and to assess whether it meets criteria for a 
serious AE. In addition, follow-up of AEs should continue until the event and any sequelae resolve or 
stabilize at a level acceptable to the investigator and/or the medical monitor. 
7.1 
Recording and Grading 
7.1.1 
Recording 
All observed or volunteered adverse events, regardless of treatment group, severity, 
suspected causality, expectedness, or seriousness will be documented on the CRF. 
A clinically significant change in a physical examination finding or an abnormal test result 
(i.e., laboratory value) should be recorded as an AE, if it: 
• 
is associated with accompanying symptoms 
• 
requires additional diagnostic testing or medical or surgical intervention 
• 
leads to a change in study dosing or discontinuation from the study 
• 
requires additional concomitant drug treatment or other therapy, or 
• 
is considered clinically significant by the investigator 
An abnormal test result that is subsequently determined to be an error does not require 
recording as an AE even if it originally met one or more of the above criteria. 
7.1.2 
Grading severity 
All adverse events will be graded for intensity on a scale of 0 to 5, according to the NCI 
CTCAE version 4.0 (see Table 2 and Appendix B). These criteria can be found at 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/ctcaev3.pdf 
Table 2 Intensity of Adverse Event 
Grade 
Description 
0 (none) 
No adverse event or within normal limits. 
1 (mild) 
Transient or mild discomfort; generally non-progressive; no limitation in 
daily activities; no medical intervention required. 
2 (moderate) 
Mild/moderate limitation in daily activities; some assistance may be 
required; no/minimal medical intervention required. 
3 (severe) 
Marked limitation in daily activities; some assistance usually required; 
medical intervention is required. 
4 (life-threatening) 
Extreme limitation in daily activities; major assistance required; significant 
medical intervention required. 
5 (death) 
Fatal adverse event. 


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7.1.3 
Attributing causality 
After grading for severity, the investigator must evaluate all clinical AEs and abnormal 
laboratory values for possible causal relationship to the study drug(s). Causality attribution 
will be decided using the criteria outlined in Table 3. 
Table 3 Relationship of Adverse Event to Study Drug 
Relationship 
Description 
Unrelated 
AE is clearly not related to the study drug (An event that does not meet any 
of the criteria below). 
Unlikely 
AE is doubtfully related to the study drug (An event that follows a 
reasonable temporal sequence after drug administration; that follows a 
known or expected response pattern; but that could more reasonably be 
explained by other characteristics of the patient’s clinical state). 
Possible 
AE may be related to the study drug (An event that follows a reasonable 
temporal sequence after drug administration; that follows a known or 
expected response pattern; but that could just as readily be attributed to a 
number of other factors). 
Probable 
AE is likely related to the study drug (An event that follows a reasonable 
temporal sequence after drug administration; that follows a known or 
expected response pattern; that is confirmed by stopping/reducing the 
drug dose; and that could not be reasonably explained by the 
characteristics of the patient’s clinical state). 
Definite 
AE is clearly related to the study drug (An event that follows a reasonable 
temporal sequence after drug administration; that follows a known or 
expected response pattern; and that is confirmed by improvement on 
stopping/reducing the drug dose and reappearance on repeated exposure). 
 
Abnormal laboratory values of clinical significance that were present at baseline and did not 
change in severity or frequency during experimental therapy and those that can obviously 
be attributed to underlying disease will be recorded as unrelated and will not be considered 
when evaluating study drug toxicity. 
7.2 
Unexpected Adverse Events 
An unexpected adverse event is any event not associated by nature or intensity with the 
investigational agent(s) under study. A comprehensive list of adverse events and potential risks 
related to prostate GVAX and cyclophosphamide are provided in this Protocol and in the Consent 
form. Both of these agents have been reported to cause allergic reactions in very rare instances. A 
severe allergic reaction could be life-threatening. Examples of allergic reactions include: rash; 
shortness of breath; wheezing; sudden drop in blood pressure; swelling around the mouth, throat, or 
eyes; fast pulse; and sweating. 
7.3 
Serious Adverse Events and Serious Adverse Drug Reactions 
The investigator must assess each event to determine if it meets the criteria for classification as a 
serious adverse event (SAE) or serious adverse drug reaction (ADR). An SAE/ADR is defined in the 
Code of Federal Regulations (21CFR312.32) as any event that: 


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• 
results in death 
• 
is life-threatening 
• 
results in inpatient hospitalization or prolongation of existing hospitalization 
• 
results in persistent or significant disability or incapacity 
• 
results in congenital anomaly or birth defect 
• 
is medically significant in the opinion of the investigator 
All SAEs that occur any time while a patient is on study (i.e., as soon as the informed consent has been 
signed) or within 30 days of the last dose of study drug administration must be documented, 
regardless of the suspected relationship to the investigational agent(s). Any SAE occurring more than 
30 days after the last dose of the study drug(s) must be recorded if a causal relationship to the 
investigational agent(s) is suspected. 
7.3.1 
Progression of malignancy 
Progression of a patient’s malignancy should not be considered an AE unless, in the 
investigator’s opinion, study treatment resulted in an exacerbation of the patient’s condition. 
If disease progression results in death or hospitalization while on study or within 30 days of 
the last dose of study drug administration, progressive disease will be considered an SAE. 
7.3.2 
Life-threatening events 
A life-threatening event is any AE that places the patient at immediate risk of death from the 
reaction as it occurs. It is not a reaction that, had it occurred in a more severe form, might 
have caused death. 
7.3.3 
Hospitalization or prolongation of hospitalization 
Hospitalization encompasses any inpatient admission (even if < 24 hours) resulting from a 
precipitating treatment-emergent adverse event. For chronic or long-term patients, 
inpatient admission also includes transfer within the hospital to an acute or intensive care 
inpatient unit. Hospitalizations for administrative reasons or a non-worsening preexisting 
condition should not be considered AEs (e.g., admission for workup of a persistent 
pretreatment laboratory abnormality, yearly physical exam, protocol-specified admission, or 
prostatectomy surgery). Hospitalization because of an unplanned event will be deemed an 
SAE. Preplanned treatments or surgical procedures should be noted in the baseline 
documentation. In the case of this study, all patients will have a preplanned prostatectomy 
surgery. 
Prolongation of hospitalization is any extension of an inpatient hospitalization beyond the 
stay anticipated or required for the original reason for admission. 
7.3.4 
Significant disability 
This is defines as a substantial disruption of the patient’s ability to conduct normal life 
functions and activities of daily living. 
7.3.5 
Congenital anomaly 
If the female partner of a male patient becomes pregnant during the course of the study, the 
treating physician must be notified immediately. All confirmed pregnancies must be 
immediately reported to the principal investigator and the medical monitor, and recorded in 
the CRF. All pregnancies will be followed until resolution (i.e., voluntary or spontaneous 
termination or birth) and assessed for congenital anomalies and birth defects. 


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7.3.6 
Medical significance 
An event that is not fatal or life-threatening and that does not necessitate hospitalization 
may be considered serious if, in the opinion of the investigator, it jeopardizes the patient’s 
status and might lead to medical or surgical intervention to prevent any of the above 
outcomes. Such medically significant events could include allergic bronchospasm requiring 
intensive treatment in the emergency room or at home, blood dyscrasias that do not result in 
inpatient hospitalization, or the development of drug dependency or abuse. 
7.4 
Perioperative Adverse Events 
Complications of surgery or those occurring in the early post-operative period will be recorded. 
These include wound complications, estimated blood loss, post-operative infections, delayed wound 
healing, abnormal laboratory values, etc. Duration of hospital stay will be recorded. Additional 
medical examinations will be allowed at the request of patients during drug administration or at the 
discretion of the principal investigator for the evaluation of new adverse events that warrant 
physical examination. During and following completion of the study, patients should notify the study 
staff of any problems that occur between visits or following study termination by telephone and, if 
necessary, will be evaluated by the investigator or study personnel at an unscheduled interim visit. 
Operative/perioperative events will be recorded as described in Section 7.1, and their severities will 
be categorized using the NCI CTCAE version 4.0 criteria. The relationship of these events to the 
investigational drug(s) will be determined by the principal investigator together with urologist co-
investigators and, if necessary, with the primary urologic surgeon. Reporting of these events will 
follow the same guidelines described in Section 7.5. 
7.5 
Reporting Adverse Events 
7.5.1 
Reporting serious adverse events (SAEs) 
All SAEs and unknown/unexpected reactions should be reported to the PI (Emmanuel 
Antonarakis, MD) within 24 hours. In addition, all SAEs must be reported by the PI to the 
IND Sponsor (Charles Drake, MD, PhD) within 24 hours.  
Harry Cao (phone 443-287-6882, fax 410-614-7287, hcao7@jhmi.edu) should be contacted 
when reporting an SAE or death. If this person cannot be reached within 24 hours, the PI 
(Emmanuel Antonarakis MD) should be contacted at 443-287-0553 or at 
eantona1@jhmi.edu. Alternatively, the co-PI (Edward Schaeffer MD, PhD) can be contacted 
at (410) 502-2733 or at eschaef4@jhmi.edu. 
The initial report for each SAE or death should include the following information (see also 
Appendix C for SAE Reporting Form): 
• 
protocol # and title 
• 
patient initials, study identification number, sex, age 
• 
date the event occurred 
• 
description of the SAE 
• 
investigational agent received (and dose level) at the time the SAE occurred 
• 
description of the patient’s condition 
• 
indication of whether the patient remains on study 
Follow-up information including severity, causality, action taken, concomitant medications, 
and outcome should be communicated to the principal investigator and IND Sponsor as soon 
as possible with an indication whether an amendment will need to be made to the protocol, 
the consent form, or both, as a result of this event. 


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7.5.2 
Reporting requirements for the Sidney Kimmel Comprehensive Cancer Center (SKCCC) 
The principal investigator will notify the appropriate regulatory agencies of any SAEs 
occurring during the study period, regardless of causality. These agencies include the Sidney 
Kimmel Comprehensive Cancer Center (SKCCC) Data and Safety Monitoring Committee 
(DSMC), and the Institutional Review Board (IRB) and the Institutional Biosafety Committee 
(IBC) of the Johns Hopkins Medical Institutions (JHMI). Expedited reporting to the IND 
Sponsor by the PI within 24 hours is required for all SAEs (see Section 7.4.1). For SAEs that 
are fatal, life-threatening, or treatment-related but non-life threatening: IRB/IBC reporting 
by the PI is required within 3 days. For unrelated SAEs, IRB/IBC reporting by the PI is 
required within 15 days. All other AEs should be documented on CRFs and submitted 
according to the standard data management guidelines. 
Adverse event information will be collected continuously throughout the duration of the 
study. Participants will be instructed to notify their treating provider of any new signs or 
symptoms, and providers will actively assess patients for adverse events at each visit 
(including by evaluation of laboratory studies). The investigator will assess each AE for its 
severity and for its relationship to the study drug, and all events Grade 1 or higher will be 
documented on CRFs and then reported as described above within the required time frame. 
Any AE occurring while a patient is on study (i.e. after informed consent has been signed) or 
within 30 days of study termination requires reporting. AEs occurring later than this must 
still be reported if a causal relationship with the study drug is suspected. 
For all AEs, the investigator must pursue and obtain information to adequately determine 
the causality and outcome of the event, and to assess whether it meets criteria for a SAE. In 
addition, follow-up of an AE is required until the event either resolves or stabilizes. Initial 
reporting of an AE should include at a minimum the patient number, age, the dose at which 
the event occurred, and the type and severity of the event. Follow-up information including 
causality, duration, outcome, action taken, and concomitant medications should be reported 
soon thereafter. 
The principal investigator must keep copies of all CRFs and other AE information, including 
correspondence with the IRB and/or FDA, for as long as required to comply with national 
and international regulations (generally at least 3 years after study completion). 
7.5.3 
FDA reporting requirements 
Annual IND Report 
All SAEs are reported to the FDA via the IND annual report per 21 CFR 312.33. SAEs deemed 
unexpected and related to the investigational product qualify for expedited reporting and 
must be submitted by the IND Sponsor-Investigator to the FDA per 21 CFR 312.32 as shown 
immediately below. 
 
7 Calendar-day telephone or fax report   
The IND Sponsor (Charles Drake, MD, PhD) is required to notify the FDA of any fatal or life-
threatening adverse event that is unexpected and assessed by the investigator to be possibly 
related to the use of prostate GVAX. An unexpected adverse event is one that is not already 
described in this Protocol or the Consent form. Such reports are to be telephoned or faxed 
(301-827-9796) to the FDA within 7 calendar days of first learning of the event. Each 
telephone call or fax transmission should be directed to the FDA new drug review division in 
the product review division for the Center for Biologics Evaluation and Research (CBER), 
whichever is responsible for the review of the IND. 
15 Calendar-day written report   
The IND Sponsor (Charles Drake, MD, PhD) is also required to notify the FDA and all 
participating investigators, in a written IND Safety Report, of any serious, unexpected AE 


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that is considered reasonably or possibly related to the use of prostate GVAX. An unexpected 
adverse event is one that is not already described in this Protocol or the Consent form. 
Written IND Safety Reports should include an “Analysis of Similar Events” in accordance 
with regulation 21 CFR 312.32. All safety reports previously filed with the IND concerning 
similar events should be analyzed. The new report should contain comments on the 
significance of the new event in light of the previous similar reports.  
Written IND safety reports with “Analysis of Similar Events” are to be submitted to the FDA 
and all participating investigators within 15 calendar days of first learning of the event. The 
FDA prefers these reports on the FDA 3500A Form but alternative formats are also 
acceptable (e.g. summary letter). 
Long-Term Follow-Up Protocol 
All patients that have received GVAX will also be encouraged to enter a separate long-term 
follow-up protocol to assess for potential late toxicities of GVAX (e.g. autoimmune disease, 
second malignancies). This long-term follow-up protocol will involve annual visits for 3 
years after the last dose of GVAX has been administered to a given patient. 
7.5.4 
RAC reporting requirements 
All AEs and SAEs will be reported to the Johns Hopkins Institutional Biosafety Committee 
(IBC) per current institutional standards, and to the Recombinant DNA Advisory Committee 
(RAC) as described in Section 7.5.3. These reporting procedures will be in accordance with 
Appendix M of the NIH Guidelines for Research Involving Recombinant DNA Molecules 
(http://oba.od.nih.gov/oba/rac/guidelines_02/APPENDIX_M.htm#_Toc7255846). 
7.6 
Pregnancy 
Pregnancy is not an AE unless it results in congenital anomaly or birth defect, in which case it is a 
SAE. If the partner of a male patient should become pregnant while he is participating in the study, 
the patient should inform his treating physician immediately. All pregnancies must be reported at 
once to the principal investigator and medical monitor. All confirmed pregnancies will be followed 
until birth or until voluntary or spontaneous termination.
8. 
OUTCOME ASSESSMENT 
8.1 
Radical Prostatectomy Specimen 
Most of the study outcomes in this immunologic trial will depend on collection of prostate tissue 
from prostatectomy specimens. All pathologic specimens will be handled in routine fashion by the 
operating room (OR) staff, except that as soon as the specimen is removed the OR nurse will directly 
page a tissue harvesting technician that is part of the Brady Urological Research Institute Prostate 
Specimen Repository Team. Accessioning of pathology specimens will be coordinated in the OR areas 
by the Departments of Urology and Pathology. A study technician will be available to the pathologist 
receiving the specimen to assure that the tissue is handled appropriately for the intended bioassays. 
At the time of harvesting, the pathologist will apply ink and cut the prostate specimen in transverse 
sections. The specimen will be fixed in 10% phosphate buffered formalin, and tissue blocks will be 
paraffin-embedded and sectioned at 4 μm thickness for routine histologic evaluation and for 
immunohistochemical determinations. Fixation should occur as soon as possible after operative 
removal of the prostate, and ideally within 30-60 minutes. Assessment of index tumors for Gleason 
grade, nodal involvement, and pathologic staging will be conducted in usual fashion and will be 
provided to the patient for prognostic information. After the pathology report is available, a database 
will be established and all information from the pathology reports of all samples will be included. 
Tumor blocks and/or additional unstained slides will be collected for study-specific quantitative 
immunohistochemical evaluations. 


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8.2 
Primary Endpoints 
8.2.1 
CD8+ T cell infiltration 
The primary measure of treatment effect in this study will be achieved by quantifying the 
extent of CD8+ T cell infiltration into the prostate from harvested prostate glands in men 
with high-risk localized prostate cancer receiving neoadjuvant ADT alone or in sequence 
with low-dose cyclophosphamide and GVAX (administered three weeks prior to radical 
prostatectomy).  
This will be done using immunohistochemical staining methods. We will also attempt to 
quantify prostatic CD4+ T cell infiltration and Treg infiltration, as well as to determine the 
CD8/Treg ratio and the CD4/Treg ratio. The primary endpoint will be expressed as the mean 
CD8+ T cell staining percentage in harvested tumor tissue, and will be analyzed separately 
for each treatment arm. 
Analysis of the primary endpoint will be achieved by preparing tissue microarrays (TMAs) 
using the highest-grade/largest tumor per patient and sampling it with 100-fold 
redundancy. All tissues will first be fixed in 10% neutral buffered formalin and processed 
into paraffin blocks. For each immunohistochemical stain (e.g. CD8+ T cell stain, CD4+ T cell 
stain, Treg stain), TMA slides will be scanned using the Aperio ScanScope® CS instrument 
(Aperio Technologies, Vista, CA) and TMA cores will first be assigned a diagnosis by the 
study pathologist and will then be  subjected to semi-automated image analysis using the 
Aperio system. For each biomarker, we will divide the area of brown DAB staining by the 
area of epithelial cells on the TMA core, obtaining a staining percentage. The area of 
epithelium will be obtained on each TMA core by staining with cytokeratin-8 and using 
automated image analysis. Cores with both tumor and normal tissue will be excluded if they 
contained >10% of the other component. 
For CD8 staining, slides will be steamed for 20 minutes in citrate antigen retrieval solution 
(Vector Laboratories, Burlingame, CA) followed by  incubation with a mouse monoclonal 
anti-CD8 antibody for 45 minutes at room temperature (Dako, Carpinteria, CA). For CD4 
staining, slides will be steamed for 40 minutes in high pH antigen retrieval solution (Dako, 
Carpinteria, CA) and then incubated with a mouse monoclonal anti-CD4 antibody for 45 
minutes at room temperature (Serotec, Kidlington, UK). For Treg analysis, cells will be stained 
for the FoxP3 protein by steaming slides for 40 minutes in high pH antigen retrieval solution 
(Dako, Carpinteria, CA) and incubating them with a mouse monoclonal anti-FoxP3 antibody 
for 45 minutes at room temperature (eBioscience, San Diego, CA, 1:1000 dilution). In all 
cases, poly-HRP-conjugated anti-mouse IgG Ab (Dako, Carpinteria, CA) will be used as the 
secondary antibody. Staining will be visualized with diaminobenzidine (Sigma, Saint Louis, 
MO) and slides will be counterstained with hematoxylin. 
Images of each TMA core will be captured by automated scanning of TMA slides using the 
Aperio ScanScope® CS instrument (Aperio Technologies, Vista, CA). Captured images will be 
imported into the TMAJ Images Application program (http://tmaj.pathology.jhmi.edu). 
Histological diagnoses (normal, atrophy, prostatic intraepithelial neoplasia, cancer) will be 
applied to all images used for the analyses by a pathologist. In addition, for TMA spots 
containing more than one type of lesion, the percentage of each diagnosis will be noted. All 
images and data will be available for viewing/downloading at 
http://demarzolab.pathology.jhmi.edu/Pubs.html. For image analysis, we will use a custom 
open source software package, FRIDA (Framework for Image Dataset Analysis; 
http://sourceforge.net/projects/fridajhu), for the evaluation of red-green-blue (RGB) color 
image datasets, including those generated from scanning of tissue microarray slides. To 
analyze CD4, CD8 and FoxP3 (Treg) staining, hue-saturation-brightness (HSB) segmentation 
ranges for brown DAB staining will be defined from the tissue microarray image set, and the 
total number of pixels in every image that fall within the defined parameters for brown DAB 


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staining will be calculated, reflecting the total area of brown DAB staining for each spot. For 
every spot, a “staining ratio” for each of the three proteins will be calculated by dividing the 
total area (in pixels) of brown DAB staining by the average TMA spot area. 
Previous TMA studies conducted by Angelo DeMarzo, MD PhD and Charles G. Drake, MD PhD 
have defined the extent of T cell infiltration into human prostate glands using prostatectomy 
specimens. In normal prostate tissue, the mean staining percentage for CD8+ T cells is 0.29% 
(interquartile range, IQR, 0.13% - 0.39%) while in tumor tissue, the mean percentage of 
CD8+ T cells is 0.42% (IQR 0.07% - 0.71%). The corresponding values for CD4+ T cell 
infiltration in normal and tumor tissue are 0.16% (IQR 0.04% - 0.18%) and 0.25% (IQR 
0.01% - 0.32%), respectively. The corresponding values for Treg infiltration in normal and 
tumor tissue are 0.03% (IQR 0.02% - 0.04%) and 0.06% (IQR 0.02% - 0.08%), respectively 
(Gurel and DeMarzo, unpublished data). These values are helpful for the determination of 
sample size/power calculations in the present study (see Section 10.1). 
The above analyses will be performed in the laboratory of Angelo DeMarzo, MD PhD. A 
detailed description of these methodologies has previously been published (Zha et al 2001; 
Faith et al 2004; Gurel et al 2008). The contact information for the DeMarzo laboratory is 
listed below: 
Angelo DeMarzo, MD, PhD 
CRB1 - Room 151 
1650 Orleans Street 
Baltimore, MD 21231 
Phone:  (410) 614-5686 
Fax: (410) 502-9817 
Email: ademarz@jhmi.edu 
8.2.2 
Rate of adverse events 
All subjects receiving at least one dose of the study drug(s) will be evaluated for safety by 
monitoring symptoms, physical examinations, and laboratory tests. Adverse events will be 
classified and graded according to the NCI Common Toxicity Criteria version 4.0 (see 
Appendix B). The absolute number and frequency of each adverse event will be reported, 
and subdivided according to toxicity grade. A description of adverse events by treatment 
arm will also be reported. A particular adverse event occurring more than once in the same 
subject will be counted only once and at its worse grade. 
8.3 
Secondary Endpoints 
8.3.2 
Regulatory T cell (Treg) infiltration 
The method for quantifying Treg density from harvested prostate tissue is as described in 
Section 8.2.1. This endpoint will be expressed as the mean staining percentage in tumor 
tissue, and will be reported separately for each treatment arm. 
8.3.1 
CD4+ T cell infiltration 
The method for quantifying CD4+ T cell density from harvested prostate tissue is as 
described in Section 8.2.1. This endpoint will be expressed as the mean staining percentage 
in tumor tissue, and will be reported separately for each treatment arm. 
8.3.3 
Tissue androgen concentrations 
Tissue concentrations of testosterone and 5α-dihydrotestosterone (DHT) will be measured 
using a highly sensitive liquid chromatography–electrospray ionization tandem mass 
spectrometry method using a high proton affinitive derivatization of the 17β-hydroxyl group 
of testosterone and DHT with picolinic acid, and a mobile phase consisting of MeCN–MeOH–


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H2O–formic acid and a conventional octadecylsilica (ODS) column (Yamashita et al 2009). 
Purification of the derivatives will be carried out using solid-phase extraction with the ODS 
cartridge. By this method, testosterone and DHT will be determined simultaneously with 
limits of quantification of 0.5 pg and 1 pg/3 mg of prostate tissue, respectively. Tissue 
androgen concentrations will be reported separately for each treatment arm. 
8.3.4 
Androgen receptor (AR) quantification 
The method for quantifying androgen receptor (AR) density from harvested prostate tissue 
is similar to that described in Section 8.2.1, and will rely on immunohistochemical staining 
for the AR protein. This endpoint will be expressed as the mean staining percentage in tumor 
tissue, and will be reported separately for each treatment arm. 
8.3.5 
Markers of apoptosis 
TMAs from formalin-fixed tumor samples will be analyzed for the degree of tumor apoptosis 
using immunohistochemical staining for activated caspase 3 (Marcelli et al 2000), or by 
using the method of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 
triphosphate (dUTP) nick end labeling (i.e. TUNEL assay) (Furuya et al 1995). The in situ Cell 
Death Detection Kit (Roche, Indianapolis, IN) may be used to perform TUNEL reactions. 
Quantification of staining percentage will be achieved using the Aperio ScanScope® CS 
instrument (Aperio Technologies, Vista, CA) as described in Section 8.2.1. This endpoint will 
be expressed as the mean staining percentage in tumor tissue, and reported separately for 
each treatment arm. Analyses will be performed in the laboratory of Dr Angelo DeMarzo. 
8.3.6 
Markers of cell proliferation 
TMAs from fixed tumor samples will be analyzed for the degree of tumor cell proliferation 
using the validated marker, Ki-67. This will be achieved by immunohistochemical staining 
(Berges et al 1995; Rubin et al 2002), using the Ki-67 monoclonal antibody (Dako North 
America, Carpineteria, CA). Quantification of staining percentage will be performed using the 
Aperio ScanScope® CS instrument (Aperio Technologies, Vista, CA) as described in Section 
8.2.1. This endpoint will be expressed as the mean staining percentage in tumor tissue, and 
reported separately for each treatment arm. Assays will be performed in the laboratory of Dr 
DeMarzo. 
8.3.7 
Pathological complete responses (pCR) 
This will be defined as the absence of tumor identification by the study pathologist on 
standard histological analysis of the resected prostate specimens. The proportion of men 
achieving a pCR will be reported separately for each treatment arm. 
8.3.9 
Novel anti-prostate antibodies 
Patient sera will be cryopreserved and archived at the Johns Hopkins Specimen Acquisition 
Core (SAC) facility. Novel induced antibody specificities will be investigated by interrogating 
patient sera using the high-throughput immunoblot (HTI) assay developed by our 
collaborator, Dr. Douglas McNeel, as previously described (Smith et al 2011; Zabransky et al 
2012). Briefly, λ-phage encoding 126 unique prostate-associated antigens will spotted in 
triplicate in a 16×24 array onto E. Coli bacterial lawns using a Biomek FX liquid handling 
robot. These individual antigens include 29 cancer-testis antigens, 40 antigens identified in 
patients with chronic prostatitis, and 57 antigens identified in individual patients, some of 
whom were treated with androgen ablation or other immunomodulatory therapies.  
Encoded proteins will be transferred to nitrocellulose membranes, washed, blocked, and 
probed with sera from patients pre- or post-treatment, diluted 1:100 in isotonic buffer. 
Human IgG will then be detected with an IgG-specific secondary antibody conjugated to 
alkaline phosphatase and immunoreactivity will be detected by development with 0.3 
mg/mL nitro blue tertazolium chloride (NBT) (Fisher Biotech, Hampton, NH) and 0.15 


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mg/mL 5-bromo 4-chloro 3-indoylphosphate (BCIP) (Fisher Biotech). Membranes will be 
digitally scanned and then assessed visually, with individual plaques scored “positive” or 
“not positive” by 3 independent observers, blinded to the treatment arm, timing of sample 
acquisition, and membrane layout.  Triplicate samples will be evaluated for each antigen, and 
immunoreactivity to individual antigens will be scored positive if there is concordance 
among 2 of 3 observers, and if immunoreactivity is scored positive in at least two of the 
three replicates. Novel antibody specificities with potential biomarker and/or therapeutic 
potential will be independently confirmed by developing individual ELISA assays for those 
particular antigens. 
8.3.9 
PSA response rates 
This will be defined as the proportion of patients who achieve an undetectable PSA (<0.1 
ng/mL) by 3 months after prostatectomy. The proportion of men achieving a PSA response 
will be reported separately for each treatment arm. 
8.3.10 
Time to PSA recurrence  
This will be defined as the interval from the time of prostatectomy to the time when the 
serum PSA is ≥0.2 ng/mL. PSA will be measured every 3 (±1) months during the first post-
operative year and every 6 (±2) months during the second and third post-operative years. 
For subjects who have not yet demonstrated PSA relapse at the time of censoring, patients 
will be censored at the date of the last assessment that shows a lack of PSA recurrence. This 
outcome will be expressed as a median for each treatment arm, and will be determined using 
the Kaplan-Meier method. 
9. 
REGULATORY AND REPORTING REQUIREMENTS 
Contact details for personnel connected with this study are provided on the title page at the front of 
this protocol. Patient registration procedures are described in Section 4. 
9.1 
Regulatory Responsibilities 
9.1.1 
Protocol chair 
The protocol chair (=PI), Emmanuel Antonarakis is responsible for the following tasks: 
• 
Coordinating, developing, writing, submitting, and obtaining IRB-approval for the 
protocol as well as its subsequent amendments. 
• 
Assuring that all study personnel are using the correct version of the protocol. 
• 
Taking responsibility for the overall conduct of the study and for monitoring the 
progress of the study. 
• 
Reviewing and ensuring reporting of serious adverse events (SAEs). 
• 
Reviewing data from all patients. 
9.1.2 
Study Coordinator 
The study coordinator, Harry Cao, is responsible for the following tasks: 
• 
Ensuring that IRB approval has been obtained prior to patient registration, and 
maintaining copies of IRB approvals (including approval of amendments). 
• 
Managing patient registration. 
• 
Collecting and compiling data from each patient. 
• 
Establishing procedures for documentation, reporting, and submission of AEs/ SAEs to 
the protocol chair (Charles G. Drake, MD PhD) and other applicable parties. 


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• 
Facilitating audits by securing selected source documents and research records from 
participating patients for audit. 
9.1.3 
Study personnel 
Study personnel (co-investigators, research nurses) are responsible for these tasks: 
• 
Following the protocol as written, and Good Clinical Practice (GCP) guidelines. 
• 
Submitting data to the project manager. 
• 
Registering all patients by submitting the patient registration form and signed informed 
consent form promptly. 
• 
Providing sufficient experienced clinical and administrative staff and adequate facilities 
and equipment to conduct the trial according to the protocol. 
• 
Maintaining regulatory binders and providing copies of all required documents to the 
project manager. 
• 
Collecting/submitting data according to the schedule specified by the protocol. 
9.2 
Data Management 
Data collected during this study will be entered into a secure database. The study coordinator will be 
responsible for the initial study configuration and setup of the database and for any future changes. 
9.2.1 
Case report forms 
Case report forms (CRFs) will be generated by the study coordinator for the collection of all 
study-related data. Investigators and study personnel will be responsible for ensuring that 
the CRFs are kept up-to-date. 
9.2.2 
Source documents 
Investigators and study personnel will record clinical data in each patient’s source 
documents (i.e., the patient’s medical record). Source documentation will be made available 
to support the patient research record. Study monitors will review entries on the CRFs at 
regular intervals, comparing the content with the source documents. 
9.2.3 
Record retention 
The investigator will maintain adequate and accurate records to enable the conduct of the 
study to be fully documented and the study data to be subsequently verified. After study 
closure, the investigator should maintain all source documents, study-related documents, 
and the CRFs. Because the length of time required for retaining records depends upon a 
number of regulatory and legal factors, documents should be stored until the investigator is 
notified that the documents may be destroyed. In this study, records are to be retained and 
securely stored for a minimum of 3 years after the completion of all study-related activities. 
9.3 
Study Monitoring and Quality Assurance 
This is a DSMP Level III study under the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center 
(SKCCC) Data Safety Monitoring Plan (herein after referred to as either JHU SKCCC DSMP or DSMP; 
please see Appendix E for complete plan). The protocol will be internally monitored by the principal 
investigator. External data monitoring will be performed by the SKCCC Clinical Research Office 
Quality Assurance Program (CRO QA) at least quarterly, but may occur more or less frequently 
depending on the rate of accrual (per the DSMP).  Additional data and safety monitoring oversight 
will also be performed by the SKCCC Safety Monitoring Committee (SMC - as defined in the DSMP) 
and a Medical Expert Committee (MEC) as detailed below. 


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Authorized representatives of SKCCC are permitted to perform audits or inspections, including 
source data verification. The purpose of these audits or inspections is to systematically and 
independently examine all trial-related activities and documents to determine whether these 
activities were conducted and data were recorded, analyzed, and accurately reported according to 
the protocol, Good Clinical Practice (GCP) guidelines, and any applicable regulatory requirements. 
Regularly scheduled registration reports will be generated to monitor patient accrual and the 
completeness of registration data. Routine data quality reports will be generated to assess missing 
data and inconsistencies. Accrual rates and the extent and accuracy of evaluations and follow-up will 
be monitored periodically throughout the study period, and potential problems will be brought to the 
attention of the principal investigator for discussion and action. 
The Medical Expert Committee (MEC) for this clinical study contains two medical oncologists and one 
urologist (see below) from other disciplines who are not affiliated with this clinical trial protocol. The 
MEC will review safety data on at least a semi-annual basis. The MEC will provide a written summary 
of each assessment to the IND Sponsor-Investigator after each meeting. In turn, the study team will 
forward these summaries to the JHU IRB and JHU SKCCC SMC. The operating plan of the MEC will be 
as follows:   
• 
Meetings will be held at least semi-annually, and potentially more frequently if needed.  
• 
Meetings will be conducted in-person or via video/teleconference, with a participant sign-in 
sheet collected at each meeting.  
• 
Approximately one week prior to each MEC meeting, the study team will submit the 
following items to MEC personnel for review and discussion at the meeting (the PI may join 
the MEC meeting in order to answer any questions the MEC might have):  
 
A summary of the clinical trial’s progress to date; 
 
The latest IRB-approved consent document;  
 
A summary of all AEs, SAEs, deaths, and withdrawals to date 
Note that the MEC reserves the right to halt trial accrual or all study activity if, after review, serious 
safety concerns warrant this action. If the MEC halts study accrual or all study activity, then the study 
team must notify the JHU SKCCC SMC, JHU IRB, FDA, and NIH RAC immediately. 
The MEC consists of the following members: 
 
Dr Dung Le (medical oncology); phone: 443-287-0002; email: dle@jhmi.edu  
 
Dr Evan Lipson (medical oncology); phone: 410-502-5977; email: elipson2@jhmi.edu 
 
Dr Ronald Rodriguez (urology); phone: 410-614-6662; email: rrodrig@jhmi.edu 
All clinical work conducted under this protocol is subject to Good Clinical Practice (GCP) guidelines. 
This includes inspection of study-related records by the principal investigator, project manager, 
sponsor or its designee, or health authority representatives at any time. 
10. 
STATISTICAL CONSIDERATIONS 
10.1 
Study Design and Sample Size 
This is a single-center, randomized, 2-arm, open-label, prospective clinical trial evaluating the 
immunogenicity and safety of ADT used alone (arm A), or ADT given in sequence with low-dose 
cyclophosphamide and prostate GVAX (arm B), each administered 3 weeks prior to radical 
prostatectomy in men with high-risk localized prostate cancer. The primary goal of this study is to 
quantify the extent of CD8+ T cell infiltration into the prostate gland from patients in the two 
treatment arms, and to compare the two arms. Our primary hypothesis is that men on the 
combination arm receiving cyclophosphamide/GVAX in addition to ADT (arm B) will have a 100% or 


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greater CD8+ T cell infiltration of the prostate than will men receiving ADT alone (arm A). A 
difference smaller than this in the primary endpoint would not be considered biologically 
meaningful. 
In the absence of any intervention prior to prostatectomy, we have previously determined that the 
mean staining percentage for CD8+ T cells in prostate tumor tissue is 0.42% (standard deviation 
0.36%) (Gurel, De Marzo and Drake, unpublished data). We hypothesize that men on the control arm 
(arm A) would have similar CD8+ T cell staining percentages as those quoted above. In the 
combination arm (arm B), a biologically meaningful treatment effect would be achieved if prostatic 
CD8+ T cell responses were augmented by 100% or more compared to arm A. Therefore, with 16 
patients per arm, and assuming an 86% coefficient of variation for the average CD8 area/spot area 
ratios, a one-sided 0.05 α-level t-test of the logarithms of these ratios would have 82% power to 
detect a 2-fold (100%) increase of CD8+ T cells in the degarelix plus cyclophosphamide/GVAX arm 
(arm B) compared to the degarelix alone arm (arm A). 
The safety of ADT monotherapy and the combination of ADT with cyclophosphamide/GVAX 
delivered before prostatic surgery is an important co-primary endpoint. We hypothesize that 
combining ADT with low-dose cyclophosphamide and prostate GVAX in the neoadjuvant setting will 
be feasible and safe. 
Patients will be replaced if they are removed from the study after signing the informed consent but 
before receiving the study drug(s). Patients receiving at least one dose of the study drug(s) will be 
included in both safety and efficacy analyses. 
10.2 
Study Endpoints 
10.2.1 
Analysis of the primary endpoints 
10.2.1.1  CD8+ T cell infiltration 
The primary efficacy objective of this study is to quantify the extent of CD8+ T cell infiltration 
into the prostate gland in the two treatment arms, and to compare them. This outcome will 
be expressed as the mean CD8+ T cell staining percentage in harvested prostate tissues, and 
will be reported separately for each treatment arm. The standard deviation, 95% confidence 
interval, median, and range of values will also be reported where appropriate. Since the CD8+ 
T cell quantity is a ratio variable and the distribution is skewed, the log transformation will 
be used for the analysis. 
Tissue microarrays (TMAs) using the highest-grade/largest tumor per patient and sampling 
it with 100-fold redundancy will be used for CD8+ T cell quantification.  We expect that 3-
50% of the spots per patient will be assigned a carcinoma diagnosis by the study pathologist.  
Cores with both tumor and normal tissue will be excluded if they contain >10% of the other 
component.  The percent positive staining score for CD8+ T cells in the spots classified as 
tumor will be used to quantify the primary outcome.  The mean will be used to pool multiple 
spot measurements for each patient. Following a log transformation, means will be 
compared between treatment arms using a two-way analysis of variance (ANOVA) with the 
stratification variable (Gleason score) treated as a block factor. Boxplots and descriptive 
summaries by treatment arm will also be provided as appropriate. To account for co-
morbidities and other immunosuppressive factors as potential confounding factors for 
treatment comparisons, multivariable regression analysis will be performed. The model will 
consider Gleason score and disease stage as covariates. 
10.2.1.2  Safety and feasibility 
An additional co-primary endpoint is safety. To this end, the frequency of adverse events will 
be described separately for each treatment arm using summary statistics. The proportion of 
patients in each arm with an adverse event will be reported with an exact binomial 95% 


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confidence interval. All subjects receiving at least one dose of the study drug(s) will be 
evaluable for toxicity. 
Early stopping rules for safety:  Safety will be monitored closely in the degarelix plus 
cyclophosphamide/GVAX arm of the study (arm B). The stopping rule for safety is based on 
the posterior probability that the adverse event (AE) rate is too high.  Any systemic 
symptoms (fevers, rash, myelosuppression) compromising the planned surgery or any 
grade-3/4 local reactions (erythema, swelling, pain) attributable to the cyclophosphamide or 
GVAX immunotherapy will be considered AEs for purposes of this stopping rule. We expect 
that the adverse event rate will be low. Trial enrollment will be suspended for a safety 
evaluation if the AE rate convincingly exceeds 25%. This stopping rule will halt enrollment if 
the posterior probability of the AE rate exceeding 0.25 is 70% or higher. The prior 
probability for this toxicity monitoring rule will be a Beta-distribution with parameters of 2 
and 8, assuming that 2 of 8 patients may experience an AE. This distribution corresponds to 
assuming a 1-in-4 chance that the risk of AEs is 25% or higher and 90% certainty that the 
risk is between 4% and 43%. The stopping rule applies this prior distribution to the 
observed number of patients experiencing an AE and computes the resulting probability that 
the rate is too high. If the posterior certainty that the rate is too high based on Bayes’ rule 
and these assumptions is 70% or higher, the study should stop. The following table shows 
the resulting stopping rules. 
Study termination if:  
2 AEs 
3 AEs 
4 AEs 
5 AEs 
6 AEs 
And number of 
patients between: 
2 
3 - 5 
6 - 9 
10 - 12 
13 - 16 
For example, the rule will call for stopping the study if 4 patients out of the first 6 experience 
AEs.  The next table shows the percentage of the time that the stopping rule will terminate 
the study under different hypothetical risks of AEs, along with the average sample size 
(based on 5000 simulations).  
Risk of AE 
0.10 
0.20 
0.25 
0.30 
0.35 
0.40 
% of Time study stops 
2.2% 
16% 
29% 
45% 
60% 
75% 
Expected sample size 
15.8 
14.6 
13.5 
12.3 
11.0 
9.5 
 
Early stopping rule for feasibility:  Androgen deprivation therapy (ADT) does not 
independently alter the surgical approach to prostatectomy and we do not anticipate an 
increase in the surgical difficulty with the use of neoadjuvant degarelix. Men who have had 
acute (post-biopsy) and prolonged issues (chronic prostatitis) with prostate 
infection/inflammation are fairly routine in urologic surgical practice, and we anticipate that 
the immune infiltrate potentially induced by GVAX would not pose a substantial increase in 
surgical difficulties. However, in addition to safety, feasibility will be monitored separately in 
both arms of the study. The feasibility rule for this study will be based on a change in 
surgical outcomes beyond what may be expected for patients without presurgical 
interventions and that may be attributable to the study drugs (degarelix, or the combination 
of degarelix plus cyclophosphamide/GVAX treatment). These events would include: (1) 
average blood loss in excess of 2500 mL, (2) average operative time in excess of 3.5 hours, 
and (3) average hospital stay in excess of 4 days. These values are approximately 2 standard 
deviations above the average surgical outcomes for men undergoing radical prostatectomy 
at Johns Hopkins Hospital. 
We require that the probability that the surgery will not be complicated by these events to 
be high (≥90%). We will monitor this endpoint after every patient. If it becomes apparent 
that the surgeries are being negatively impacted by the pre-surgical investigational 
treatments, then the study will be suspended for review. This stopping rule will halt 


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enrollment if the posterior probability of no complications <0.90 is greater than 80%. The 
prior probability for this rule has a Beta-distribution with parameters of 9 and 1, based on 
the expectation that these treatments will not impact the feasibility of surgery. Instances 
where the study would be temporarily suspended are listed below.    
Stopping rules: 
•  Not feasible if both of the first 2 patients in an arm have surgical complications. 
•  Not feasible if two of the first 3 patients in an arm have complications. 
•  Not feasible if 2 out of the first 4 patients in an arm have complications. 
•  Not feasible if 3 out of the first 6 patients in an arm have complications. 
•  Not feasible if 4 out of the first 8 patients in an arm have complications. 
•  Not feasible if 5 out of the first 10 patients in an arm have complications. 
•  Not feasible if 6 out of the first 12 patients in an arm have complications. 
•  Not feasible if 7 out of the first 14 patients in an arm have complications. 
•  Not feasible if 8 out of 16 patients in an arm have complications. 
 
10.2.2 
Analysis of the secondary endpoints 
The secondary endpoints of this study have previously been defined (see Section 8.3). The 
statistical analysis of these endpoints is described below. Data transformation will be 
performed when they are not normally distributed. 
• 
Regulatory T cell (Treg) density. The methods for analyzing Treg infiltration will be similar 
to those described for the primary endpoint. Descriptive statistics and graphical summaries 
by treatment arm for these outcomes will be provided. In addition, the CD8+/Treg ratio and 
the CD4+/Treg ratio will be computed, and reported using descriptive statistics. Comparisons 
between treatment arms will be made using two-way ANOVA. 
• 
CD4+ T cell density. The methods for quantifying and analyzing CD4+ T cell infiltration will 
be similar to those described for the primary endpoint (i.e. CD8+ T cell infiltration). 
Descriptive statistics and graphical summaries by treatment arm for these outcomes will be 
provided. Comparisons between treatment arms will be made using two-way ANOVA. 
• 
Tissue androgen concentrations. Intraprostatic androgen concentrations (i.e. testosterone 
and dihydrotestosterone) will be summarized descriptively by study arm, and comparisons 
across arms will be made using two-way ANOVA. 
• 
Androgen receptor (AR) quantification. Androgen receptor (AR) staining concentrations 
will be summarized descriptively by study arm, and comparisons across arms will be made 
using two-way ANOVA. 
• 
Apoptotic markers. Caspase 3 staining (and/or TUNEL assay results) will be expressed as 
the mean staining percentage in tumor samples, and reported separately for each treatment 
group. Standard deviations, 95% confidence intervals, and ranges will also be reported 
where appropriate. Means will be compared across treatment arms using two-way ANOVA. 
• 
Proliferation markers. Ki-67 staining will be expressed as the mean staining percentage in 
tumor samples, reported separately for each treatment arm. Standard deviations, 95% 
confidence intervals, and ranges will also be reported where appropriate. Means will be 
compared across treatment arms using two-way ANOVA. 
• 
Pathological complete responses (pCR). This will be defined as an absence of tumor 
identification on standard histological analysis of the resected prostate specimens. The 
proportion of patients achieving a pCR will be compared between treatment arms using the 
Mantel-Haenszel test. 


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• 
Novel anti-prostate antibodies. Induction of novel anti-prostate antibodies will be 
assessed with a high-throughput immunoblot (HTI) assay, before and after treatment 
initiation. The change in immunoreactivity will be evaluated for each antigen and scored as a 
gain, loss, or no change. These data will be visualized per patient for both treatment groups 
using heatmaps. A comparison of the proportions of patients with these ordered outcomes 
between the treatment arms will be made using an exact version of the Cochran-Armitage 
test for trend (Armitage 1955). Antibody responses will be dichotomized as “gain” versus 
“loss or no change” and correlated with PSA response and pathologic complete response 
(pCR) using χ2-tests or Fisher’s exact tests. Antibody responses will be correlated with time-
to-PSA-recurrence using Kaplan-Meier curves and log-rank tests. The correlation of antibody 
responses with CD8 concentrations, CD4 concentrations, and Treg concentrations will be 
explored using Spearman’s correlation coefficient (ρ). 
• 
PSA response rates. This will be defined as an undetectable PSA (<0.1 ng/mL) at 3 months 
after prostatectomy. The proportion of patients achieving a PSA response will be compared 
between treatment arms using the Mantel-Haenszel test. 
• 
Time to PSA recurrence. This will be defined as the interval from prostatectomy to the time 
when the serum PSA is ≥0.2 ng/mL. For subjects who have not yet demonstrated PSA 
recurrence at the time of censoring, patients will be censored at the date of the last 
assessment that shows a lack of PSA recurrence. For each treatment arm, the median time to 
PSA recurrence after prostatectomy (i.e., the median PSA-recurrence-free survival) will be 
estimated with 95% confidence intervals using Kaplan-Meier survival analysis. Comparisons 
will be sought using the log-rank test, stratified by Gleason score. 
10.3 
Analysis Populations 
10.3.1 
Intention-to-treat population 
All patients who meet eligibility criteria and receive at least one dose of the study drug(s) 
will be included in the analysis of the primary and secondary endpoints, even if there are 
subsequent protocol deviations. However, in cases where prostatectomy is not performed or 
if adequate surgical tissue is not collected, then determination of the primary endpoint 
(tissue CD8+ T cell analysis) and some secondary endpoints (apoptosis/proliferation marker 
analysis) will not be possible. Patients will be replaced if they are removed from the study 
after signing the informed consent but before receiving the study drug(s). 
10.3.2 
Safety population 
All patients enrolled in the study will be included in the safety analysis population and 
considered evaluable for toxicity from the time of their first dose of the study drug(s). 
Patients never receiving any of the study drugs will not be included in this analysis. 
Demographic and baseline characteristics for the safety population will be summarized by 
number and percent for categorical data and by descriptive statistics for continuous data. 
10.4 
Safety Analysis 
10.4.1 
Evaluation of adverse events 
Treatment-emergent adverse events will be translated from investigator terms to MedDRA 
version 6.0 terminology and summarized (number and percentage of patients) for all 
patients who receive at least one dose of the study drug(s). Adverse event summaries will be 
organized by body system, frequency of occurrence, intensity (i.e., severity grade), and 
causality or attribution. Patients who experience an adverse event more than once will be 
counted only once. The occurrence with the maximum severity will be used to calculate 
intensity. 


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10.4.2 
Evaluation of serious adverse events and premature withdrawals 
Adverse events deemed serious and those resulting in early treatment withdrawal or death 
will be summarized separately. Narrative paragraphs will be generated to describe the 
circumstances surrounding each SAE and each death. 
10.4.3 
Evaluation of laboratory parameters and assays 
Abnormal laboratory parameters (e.g. electrolyte levels, liver function tests, renal function 
tests, complete blood counts) will be summarized, and clinically significant changes from 
baseline will be discussed. 
10.5 
Statistical Procedures 
10.5.1 
General 
Most study outcomes will be reported using descriptive statistics: number of observations, 
means, standard deviations, medians, minimum, and maximum values. 95% confidence 
intervals will be provided where appropriate. A P-value of ≤0.05 will be used to denote 
statistical significance. 
10.5.2 
Sample size calculation 
Our primary hypothesis is that men on the combination arm receiving cyclophosphamide/ 
GVAX in addition to ADT (arm B) will have a 100% or greater CD8+ T cell infiltration of the 
prostate than will men receiving ADT alone (arm A). A difference smaller than this in the 
primary endpoint would not be considered biologically meaningful. In the absence of any 
intervention, we have previously determined that the mean staining percentage for CD8+ T 
cells in tumor tissue is 0.42% (standard deviation 0.36%) (Gurel and DeMarzo, unpublished 
data). We hypothesize that men on the control arm (arm A) would have similar CD8+ T cell 
staining percentages as those quoted above. In the combination arm (arm B), a biologically 
meaningful treatment effect would be achieved if prostatic CD8+ T cell responses were 
augmented by 100% or more compared to arm A. Therefore, with 16 patients per arm, and 
assuming an 86% coefficient of variation for the average CD8 area/spot area ratios, a one-
sided 0.05 α-level t-test of the logarithms of these ratios would have 82% power to detect a 
2-fold (100%) increase of CD8+ T cells in the degarelix plus cyclophosphamide/GVAX arm 
(arm B) compared to the degarelix alone arm (arm A). Treating Gleason score as a block 
factor reduces the variance, which would be adequate to offset the loss of power due to the 1 
degree of freedom associated with the blocking factor. 
In summary, 16 patients per arm will yield a total sample size of 32 men. With an estimated 
enrolment rate of 4 men per month, accrual is expected to last 8 months. 
10.5.3 
Stratification factors 
The randomization will be stratified by Gleason score: ≤7 vs 8-10. 
10.5.4 
Statistical analysis of primary and secondary endpoints 
For a description of statistical methods used to evaluate the primary and secondary study 
outcomes, please see Section 10.2. 
11. 
PROTECTION OF HUMAN SUBJECTS 
11.1 
Ethical Considerations 
This study will be conducted in compliance with the protocol, Good Clinical Practice (GCP) guidelines 
established by the International Conference on Harmonization (ICH), and the ethical standards set 


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forth in the Declaration of Helsinki of 2004 (these documents may be found at 
www.wma.net/e/policy/b3.htm and www.laakariliitto.fi/e/ethics/helsinki.html). Review of this 
protocol by the Institutional Review Board (IRB)/Ethics Committee (EC), and the performance of all 
aspects of the study including acquisition of informed consent, must also be in accordance with the 
principles elaborated in the Declaration, as well as the ICH guidelines (Code of Federal Regulations 
(CFR), Title 21: Part 50 and Part 312). The principal investigator will be responsible for submitting 
documents to the IRB/EC, and obtaining written approval for the protocol prior to study initiation. 
The approval of both the protocol and the informed consent must specify the date of approval, 
protocol number and version, and amendment number. The principal investigator is also responsible 
for notifying the IRB/EC of any serious deviations from the protocol, or other circumstances that may 
result in added risk to participating patients. 
11.2 
Protocol Amendments 
Before starting the study, the protocol must be approved by the IRB/EC, the JHU Institutional 
Biosafety Committee (IBC), the FDA, and the Recombinant DNA Advisory Committee (RAC). 
Amendments to the protocol are subject to IRB approval before instituting. Any amendments made 
after IRB/EC approval is granted must be resubmitted to the IRB/EC for new approval. 
11.3 
Written Informed Consent 
Before obtaining consent, members of the study team must review the rationale for the treatment 
program with the patient. The discussion will review the alternatives available, the potential benefits 
of this program, the risks and the probability of their occurrence, and the procedures to minimize 
these risks. Should an adverse event occur, the provisions available to ensure medical intervention 
must also be reviewed. Why the risks are reasonable in relation to the anticipated benefits, 
incentives, or costs that will/may be incurred as a result of participating in the study, as well as the 
efforts to maintain confidentiality, should also be discussed with the patient. 
Patients will be required to sign and date (in triplicate) a statement of informed consent that meets 
the requirements of the Code of Federal Regulations (Federal Register Vol. 46, No. 17, January 27, 
1981, part 50) and the IRB. The consent form should be submitted with the protocol for review and 
approval by the IRB/EC. The medical record should include a statement that written informed 
consent was obtained (and should document the date that it was obtained) before the patient is 
enrolled in the study. The original signed consent document will become part of the patient’s medical 
record, a copy will be forwarded to the project manager pursuant to registration, and a copy will be 
sent home with each patient. 
The consent form must include the following information: 
• 
the nature and objectives, potential toxicities, and benefits of the intended study 
• 
the length of therapy and follow-up required 
• 
alternatives to the proposed therapy (including standard and investigational therapies) 
• 
the name of the investigator(s) responsible for the protocol 
• 
the right of the patient to accept or refuse treatment and to withdraw from participation 
in the study at any time 
11.4 
Protection of Privacy 
Patients will be informed of the extent to which their confidential health information generated from 
this study may be used for research purposes. After this discussion, they will be asked to sign a 
Notice of Privacy Practice research authorization/HIPAA form. This may be embedded within the 
informed consent document. The original signed documents will become part of the patient’s medical 
records, and each patient will receive a copy of the signed documents. The use and disclosure of 
protected health information will be limited to the individuals described in the research 


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authorization form. The research authorization form must be prepared by the principal investigator 
and approved by the IRB.  
In compliance with US federal regulations, the investigator is required to permit representatives of 
the US Food and Drug Administration (FDA) or other regulatory authorities to review and/or copy 
any medical records relevant to the study in accordance with local laws. Patients will be informed of 
the extent to which their confidential health information generated from this study may be 
disseminated to other parties. Should direct access to medical records require a waiver or 
authorization separate from the subject’s statement of informed consent, it is the responsibility of the 
investigator to obtain such permission in writing from the patient. 
11.5 
Study Termination or Modification 
Adverse events and laboratory data from this trial will be assessed by the principal investigator 
and/or medical monitor on an ongoing basis. At least quarterly, data from the clinical database will 
be reviewed. The results of this review will be shared with all investigators either in writing or as 
part of a teleconference. SAEs will be reviewed as they are reported to the principal investigator or 
project manager, and the medical monitor will make an assessment regarding the safety of 
continuing or modifying the study. This assessment will be shared with the investigators either in 
writing or as part of a teleconference. Should the assessment of either the principal investigator or 
the medical monitor be that the trial should be terminated, the study will then be closed to further 
accrual. Follow-up safety assessments will be performed for all patients who are taken out of the 
study prematurely.


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12. 
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41. Mercader M, Bodner BK, Moser MT, et al. T cell infiltration of the prostate induced by androgen 
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45. Pardoll DM. Spinning molecular immunology into successful immunotherapy. Nat Rev Immunol 
2002; 2: 227-238. 
46. Pound CR, Partin AW, Eisenberger MA, et al. Natural history of progression after PSA elevation 
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47. Ramsdell F, Fowlkes BJ. Maintenance of in vivo tolerance by persistence of antigen. Science 
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APPENDIX A:  PERFORMANCE STATUS CRITERIA 
            ECOG Performance Status Scale 
              Karnofsky Performance Scale 
Grade 
Description 
% 
Description 
0 
Normal activity. Fully active, able to 
continue all pre-disease performance 
without restriction. 
100 
Normal, no complaints, no evidence of 
disease 
90 
Able to carry on normal activity, minor 
signs or symptoms of disease 
1 
Symptoms, but ambulatory. Restricted in 
physically strenuous activity but 
ambulatory and able to carry out work of a 
light or sedentary nature (e.g., light 
housework, office work). 
80 
Normal activity with effort, some signs or 
symptoms of disease 
70 
Cares for self, unable to carry on normal 
activity or to do active work 
2 
In bed < 50% of the time. Ambulatory and 
capable of all self-care but unable to carry 
out any work activities. Up and about more 
than 50% of waking hours. 
60 
Requires occasional assistance but is able 
to care for most needs 
50 
Requires considerable assistance and 
frequent medical care 
3 
In bed > 50% of the time. Capable of only 
limited self-care, confined to bed or chair  
> 50% of waking hours. 
40 
Disabled, requires special care and 
assistance 
30 
Severely disabled, hospitalization indicated. 
Death not imminent. 
4 
100% bedridden. Completely disabled, 
cannot carry on any self-care, totally 
confined to bed or chair. 
20 
Very sick, hospitalization indicated. Death 
not imminent. 
10 
Moribund, fatal processes progressing 
rapidly 
5 
Dead 
0 
Dead 
 


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APPENDIX B:  COMMON TOXICITY CRITERIA, VERSION 4.0 
 
Adverse events will be described and graded using the NCI Common Toxicity Criteria (Version 4.0). 
A copy of this document can be downloaded from the CTEP website (http://ctep.cancer.gov/forms). 
All treatment areas must have a copy of this document, or must be able to access a copy. 
 
In general, the grading system can be summarized as follows: 
 
 
Grade: 
Severity: 
Description: 
Grade 1 
Mild 
Transient or mild discomfort; generally non-progressive; no limitation in 
daily activities; no medical intervention required. 
Grade 2 
Moderate 
Mild/moderate limitation in daily activities; some assistance may be 
required; no/minimal medical intervention required. 
Grade 3 
Severe 
Marked limitation in daily activities; some assistance usually required; 
medical intervention is required. 
Grade 4 
Life-threatening 
Extreme limitation in daily activities; major assistance required; 
significant medical intervention required. 
Grade 5 
Death 
Death related to an adverse event. 
 
 


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APPENDIX C:  SERIOUS ADVERSE EVENT (SAE) REPORTING FORM 
 
 
Please notify Dr. Charles Drake within 24 hours  
Protocol Title: 
 
 
A neoadjuvant immunologic study of androgen deprivation therapy 
combined with a GM-CSF–secreting allogeneic prostate cancer vaccine and 
low-dose cyclophosphamide in men with high-risk localized prostate 
cancer undergoing radical prostatectomy 
Protocol 
Number: 
 
J12XX 
Principal  
Investigator: 
 
Dr. Emmanuel Antonarakis 
Signature  
of PI: 
 
 
 
Date: 
 
 
 
Report Date: 
 
Hospital Admission Date: 
Date of Discovery of 
Event: 
 
 
Report Type: 
  Initial    
  Follow-up 
  Final Follow-up 
  Death 
  Addendum to: 
 
Section A: Subject Information 
Subject ID: 
Subject Initial: 
 
Subject Gender: 
  Male 
  Female 
 
Section B: Event Information 
Event diagnosis or 
symptoms: 
 
Date of CY Dose: 
 
Action taken regarding the study drugs: 
 
 None 
 Interrupted 
 Discontinued 
 
Date of GVAX Dose: 
 
Event Onset Date: 
 
Event End Date: 
Relationship to: 
Cyclophosphamide 
Prostate GVAX 
Underlying Disease 
Unrelated 
 
 
 
Probably Unrelated 
 
 
 
Possible Related 
 
 
 
Probably Related 
 
 
 
Definitely Related 
 
 
 


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Section C: Brief Description of the Event: 
 
 
 
 
 
 
 
Section D: Relevant Medical History 
 
 
 
 
Section E: Concomitant Drug (Not related to SAE) 
Name of the Drug 
Start Date 
Stop Date 
Route 
Dose 
Frequency 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Section F: Comments 
 
Additional Documents: 
 Please specify  
 
 
 
 
 
 
 


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APPENDIX D:  ABBREVIATIONS AND ACRONYMS 
AAV 
adeno-associated virus 
ACLS 
Advanced Cardiac Life Support 
ADR 
adverse drug reaction 
ADT 
androgen deprivation therapy 
AE 
adverse event 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
ANOVA 
analysis of variance 
APTT 
activated partial thromboplastin time 
AR 
androgen receptor 
AST 
aspartate aminotransferase 
BCG 
bacillus Calmette-Guerin vaccine 
bid 
bis in die (twice a day) 
BP 
blood pressure 
BSA 
body surface area 
BUN 
blood urea nitrogen 
⁰C 
degrees Celsius 
Ca++ 
calcium 
CBC 
complete blood count 
CD4+ T cells 
cluster determinant 4–positive T lymphocytes 
CD8+ T cells 
cluster determinant 8–positive T lymphocytes 
CFR 
Code of Federal Regulations 
CG1940 
PC3-derived prostate cancer cell line expressing human GM-CSF (Cell Genesys, Inc.) 
CG8711 
LNCaP-derived prostate cancer cell line expressing human GM-CSF (Cell Genesys, Inc.) 
CI 
confidence interval 
Cl− 
chloride 
cm 
centimeter 
COPD 
chronic obstructive pulmonary disease 
CR 
complete response 
CRO 
Clinical Research Office 
CRF 
case report form 
CRPC 
castration resistant prostate cancer 
CRRMC 
Clinical Research Review and Monitoring Committee 
CT 
computerized tomography 
CTL 
Cell Therapy Laboratory 
CTLA-4 
cytotoxic T lymphocyte-associated antigen 4 
CTCAE 
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 


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DAB 
3,3′-diaminobenzine tetrahydrochloride 
dL 
deciliter 
DLT 
dose-limiting toxicity 
DMSO 
dimethyl sulfoxide 
DNA 
deoxyribonucleic acid 
DNase 
deoxyribonuclease 
DPBS 
Dubelcco's phosphate buffered saline 
DSMC 
data and safety monitoring committee 
DTH 
delayed-type hypersensitivity 
EC 
ethics committee 
ECOG 
Eastern Cooperative Oncology Group 
EMLA 
eutectic mixture of local anesthetics 
FDA 
Food and Drug Administration 
GCP 
good clinical practice 
G-CSF 
granulocyte-colony stimulating factor 
GM-CSF 
granulocyte-macrophage colony-stimulating factor 
GnRH 
gonadotropin-releasing hormone  
GVAX 
allogeneic cell-based GM-CSF–secreting immunotherapy for prostate cancer 
HA 
hemagglutinin 
HIPAA 
Health Insurance Portability and Accountability Act 
HR 
heart rate 
HRPC 
hormone-refractory prostate cancer 
HSA 
human serum albumin 
HSB 
hue-saturation-brightness 
IBC 
Institutional Biosafety Committee 
ICH 
International Conference on Harmonisation 
IHC 
Immunohistochemistry 
ID 
intradermal 
IND 
investigational new drug 
INR 
international normalized ratio 
IQR 
interquartile range 
IRB 
Institutional Review Board 
IV 
intravenous 
JHMI 
Johns Hopkins Medical Institutions 
K+ 
potassium 
LDH 
lactate dehydrogenase 
LHRH 
luteinizing hormone releasing hormone 
LNCaP 
AR-positive human prostate cancer cell line derived from a lymph node metastasis 
LOI 
letter of intent 
MedDRA 
Medical Dictionary for Regulatory Activities 


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MMTV 
murine mammary tumor virus 
MTD 
maximum tolerated dose 
NA 
not applicable 
N/A 
not available 
NCI 
National Cancer Institute 
OR 
operating room 
PC3 
androgen-independent human prostate cancer cell line derived from a bone metastasis 
PI 
principal investigator 
PO 
per os (by mouth) 
PSA 
prostate-specific antigen 
PSMA 
prostate-specific membrane antigen 
PT 
prothrombin time 
PTT 
partial thromboplastin time 
qd 
quaque die (every day) 
RAC 
Recombinant DNA Advisory Committee 
RP 
radical prostatectomy  
RT 
room temperature 
SAE 
serious adverse event 
SC 
Subcutaneous 
SD 
standard deviation 
SKCCC 
Sidney Kimmel Comprehensive Cancer Center 
TdT 
terminal deoxynucleotidyl transferase 
tid 
ter in die (3 times a day) 
TMA 
tissue microarray  
TRAMP 
transgenic adenocarcinoma of the mouse prostate(mouse model) 
Tregs 
regulatory T lymphocytes 
TUNEL 
TdT-mediated deoxy uridine triphosphate (UTP) nick end-labeling assay 
ULN 
upper limit of normal 
USP 
United States Pharmacopeia (sterile, hypotonic, nonpyrogenic water for injection) 
WBC 
white blood cell 


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APPENDIX E:  DATA AND SAFETY MONITORING PLAN (DSMP), Version 4.0 (9/22/11)