A Pilot Study of Vaccination With Epitope-Enhanced TARP Peptide and TARP Peptide-Pulsed Dendritic Cells in the Treatment of Stage D0 Prostate Cancer
Phase: PHASE1
Conditions: Prostate Specific Antigens, Prostate Neoplasms
Sponsor: National Cancer Institute (NCI)

Abbreviated Title: TARP Peptide Vaccine Prostate Cancer 
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Abbreviated Title: TARP Peptide Vaccine Prostate Cancer 
CC Protocol #: 09-C-0139  L 
Version Date: January 10, 2019 
NCT Number: NCT00972309 
 
Title: A Pilot Study of Vaccination with Epitope-Enhanced TARP Peptide and TARP Peptide 
Pulsed Dendritic Cells in the Treatment of Stage D0 Prostate Cancer 
 
NCI Principal Investigator: Hoyoung Maeng, M.D 
 
Vaccine Branch  
 
Center for Cancer Research (CCR) 
National Cancer Institute (NCI) 
Building 10, Room B2L312 
9000 Rockville Pike 
Bethesda, MD 20892 
Phone: 240-781-3253  
Email: hoyoung.maeng@nih.gov  
 
 
 
Investigational Agents: 
Drug Name: 
TARP 27-35 Peptide 
(Native Peptide) 
NSC #740703 
Tarp 29-37-9V Peptide 
(Epitope Enhanced Peptide) 
NSC # 740704 
IND Number: 
13925 
13925 
Sponsor: 
CCR 
CCR 
Manufacturer: 
NeoMPS, Inc 
NeoMPS, Inc. 
 
Commercial Agents:  
Interleukin-4, CellGenix, BB-MF 11269 
Montanide ISA 51 VG, Seppic, NSC #737063 
Hemocyanin from Keyhole Limpet, Sigma –Aldrich, DMF 21067 
Leukine, Sanofi-Aventis 
FluMist, MedImmune 
Elutra Cell Separation System, Gambro BCT, Inc., BB-MF11922 
LPS (Endotoxin, E. Coli), NIH Clinical Center, BB-MF-7294  
 
 
 

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PRÉCIS 
Background  
• T-cell receptor alternate reading frame protein (TARP) is expressed by both normal and 
malignant prostate cancer tissue and is found in about 95% of prostate cancer specimens. 
TARP is immunogenic and hence is a target antigen for vaccination. 
• The immunogenicity of TARP peptides can be augmented through epitope enhancement that 
is achieved through amino acid substitutions resulting in increased peptide binding affinity. 
• Two HLA-A*0201 TARP peptide epitopes are associated with generation of catalytic T-cell 
responses: TARP27-35 and TARP29-37. Substitution of Val for Leu at position 9 in 
TARP29-37, results in a peptide with increased binding affinity (TARP29-37-9V) that 
induces antigen specific T cells able to recognize wild type and multiple modified TARP 
peptides. The affinity of the TARP 27-35 peptide, corresponding to a distinct but overlapping 
epitope, is high enough that no enhancement was required. 
• Stage D0 prostate cancer patients have no evidence of visceral or bony metastatic disease but 
have persistently elevated or rising PSA levels (biochemical progression) and are at 
increased risk for disease progression.  Since they lack much of the immune dysfunction 
associated with the high tumor burden characteristic of end-stage metastatic disease, they are 
an ideal population in which to study therapeutic vaccination to slow or prevent disease 
recurrence and progression. 
• Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and 
are being studied extensively for anti-tumor activity in a broad spectrum of cancer patients. 
• As the optimal method for therapeutic immunization with peptide vaccines in patients with 
cancer is unclear, vaccination with TARP peptides in Montanide® ISA 51 VG adjuvant plus 
Sargramostim will be studied in a randomized fashion with autologous, TARP peptide-
pulsed DCs in HLA-A*0201 Stage D0 prostate cancer patients.   
 
Objectives 
• Determine the safety and toxicity of TARP peptide and TARP peptide-pulsed dendritic cell 
vaccination in patients with Stage D0 prostate cancer. 
• Determine the T-lymphocyte immune responses to TARP peptide vaccination with 
Montanide® ISA 51 VG plus Sargramostim or autologous dendritic cells as measured by 
tetramer staining, IFN- ELISPOT and 51Cr release CTL assays. 
 
Eligibility 
• Males > 18 years of age with histologically confirmed adenocarcinoma of the prostate. 
• Must have completed and recovered from all prior definitive therapy (surgery, 
brachytherapy, cryotherapy or radiotherapy) for the primary tumor, or other definitive-intent 
local therapy. 
• Stage D0 disease with documented biochemical progression documented by rising PSA and 
no evidence of metastatic disease by physical examination, CT scan or bone scan.  
• PSADT > 3 months and < 15 months: 

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• Patients must have > 3 PSA measurements over > 3 months. 
• The interval between PSA measurements must be > 4 weeks.  
• For patients following definitive radiation therapy or cryotherapy: a rise in PSA of > 2ng/mL 
above the nadir (per RTOG-ASSTRO consensus criteria). 
• For patients following radical prostatectomy: 2 absolute PSA values > 0.3ng/ml (per NCCN 
guidelines). 
• Non-castrate level of testosterone: > 50 ng/dL (prior ADT allowed; must be > 6 months since 
last dose of ADT). 
• HLA-A*0201 positive. 
• Performance Status: ECOG 0-2 or Karnofsky 70-100% and life expectancy > 1 year. 
• Hemoglobin > 10.0 gm/dL, WBC > 2,500/mm3, ALC > 500/ mm3, ANC > 1,000/mm3, 
platelet count > 100,000/mm3, and PT/PTT < 1.5X ULN unless receiving clinically indicated 
anticoagulant therapy; SGPT/SGOT < 2.5X ULN, total bilirubin < 1.5X ULN; creatinine < 
1.5X ULN and estimated GFR (eGFR) > 60 ml/min. 
• Hepatitis B and C negative (unless the result is consistent with prior vaccination or prior 
infection with full recovery); HIV negative. 
• No use of investigational agents within 4 weeks of study enrollment or use of 
immunosuppressive or immunomodulating agents within 8 weeks of study entry. 
• No other concurrent anticancer therapy or prior prostate cancer vaccines expressing TARP or 
HLA A2. 
• No alternative medications known to alter PSA (e.g. phytoestrogens and saw palmetto).  
Note: patients receiving medications for urinary symptoms such as Flomax or 5-alpha 
reductase inhibitors (finasteride and dutasteride) on a chronic stable dose for at least 3 
months are allowed.  
 
Study Design 
• This is a randomized, prospective, pilot study of vaccination with a mixture of wild type 
(TARP27-35) and epitope-enhanced (TARP29-37-9V) TARP peptides in HLA-A*0201 
patients with stage D0 prostate cancer. 
• Vaccination with TARP peptides admixed with Montanide® ISA 51 VG  plus Sargramostim 
administered by deep subcutaneous injection will be compared with vaccination with TARP 
peptide-pulsed autologous dendritic cells (DCs) administered intradermally. 
• Autologous dendritic cells will be matured from peripheral blood monocytes with 
Sargramostim, IL-4, IFN- and LPS and pulsed with wild type and epitope-enhanced TARP 
peptides. 
• Apheresis will be performed on all patients at weeks 0, 24, and 48. 
• Randomization and assignment to received TARP peptide vaccine with Montanide® ISA 51 
VG  plus Sargramostimgiven by deep subcutaneous injection or TARP peptide-pulsed 
autologous DCs given ID will be performed at week 0. 

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• All patients will receive live, attenuated influenza vaccine (FluMist) when seasonally 
available at the very end of their week 0 visit as a control vaccine to assess cytotoxic T 
lymphocyte responses. 
• TARP Peptide vaccines will be administered every three weeks at weeks 3, 6, 9, 12, and 15, 
with a sixth and seventh booster dose of vaccine at Week 48 and 96.  Follow-up will be 
through 144 weeks on study. 
• The trial uses an optimal 2-stage design targeting an immunologic response between 10 and 
40%.  We will initially accrue 9 patients in each arm.  If 0-1 patients develop an 
immunologic response, then no further patients will be enrolled.  If 2 or more of these 
patients develop an immunologic response, we will accrue 11 additional patients for a 
maximum total of up to 20 patients in each arm. A stopping rule for excessive toxicity will 
be incorporated. 

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TABLE OF CONTENTS 
PRÉCIS ...........................................................................................................................................2 
TABLE OF CONTENTS .................................................................................................................5 
1 
INTRODUCTION ..................................................................................................................7 
1.1 
Study Objectives ............................................................................................................. 7 
1.2 
Background and Rationale .............................................................................................. 7 
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT .......................................................13 
2.1 
Eligibility Criteria ......................................................................................................... 13 
2.2 
Research Eligibility Evaluation .................................................................................... 15 
2.3 
Registration Procedures ................................................................................................ 15 
3 
STUDY IMPLEMENTATION ............................................................................................15 
3.1 
Study Design ................................................................................................................. 15 
3.2 
Vaccine Administration ................................................................................................ 17 
3.3 
Treatment Modifications and Immunization Stopping Rules ....................................... 18 
3.4 
On Study Evaluation ..................................................................................................... 19 
3.5 
Concurrent Therapies .................................................................................................... 20 
3.6 
Criteria for Removal from Protocol Therapy and Off Study Criteria ........................... 21 
4 
SUPPORTIVE CARE ...........................................................................................................22 
5 
DATA COLLECTION AND EVALUATION .....................................................................22 
5.1 
Data Collection ............................................................................................................. 22 
5.2 
Response Criteria .......................................................................................................... 22 
5.3 
Toxicity Criteria ............................................................................................................ 25 
6 
STATISTICAL CONSIDERATIONS ..................................................................................25 
6.1 
Overall Statement of Objectives ................................................................................... 25 
6.2 
Primary Objective and Statistical Justification for Sample Size .................................. 25 
7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN ....................................................................................................................................27 
7.1 
Definitions .................................................................................................................... 27 
7.2 
NIH Intramural IRB and Clinical Director Reporting .................................................. 29 
7.3 
IND Sponsor Reporting Criteria ................................................................................... 29 
7.4 
Data and Safety Monitoring Plan .................................................................................. 30 
8 
HUMAN SUBJECTS PROTECTION..................................................................................31 
8.1 
Rationale for Subject Selection ..................................................................................... 31 
8.2 
Evaluation of Benefits and Risks/Discomforts ............................................................. 31 
8.3 
Consent and Assent Processes and Documentation ...................................................... 31 
9 
PHARMACEUTICAL INFORMATION .............................................................................32 
9.1 
Sargramostim ................................................................................................................ 32 

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9.2 
Interleukin-4 CellGenix ................................................................................................ 33 
9.3 
TARP 29-37-9V Peptide (Epitope-Enhanced) NSC #740704 ...................................... 33 
9.4 
TARP 27-35 (Wild Type) NSC#740703 ...................................................................... 34 
9.5 
Influenza Virus Vaccine Live Intranasal ...................................................................... 36 
9.6 
MONTANIDE® ISA 51 VG (NSC 737063) Classification:  Adjuvant ....................... 37 
9.7 
KLH (Keyhole Limpet Hemocyanin) ........................................................................... 39 
10 
REFERENCES .....................................................................................................................40 
11 
STUDY APPENDICES ........................................................................................................44 

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1 
INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objectives   
1.1.1.1 Determine the safety and toxicity of TARP peptide and TARP peptide-pulsed dendritic 
cell vaccination in patients with Stage D0 prostate cancer naïve to androgen deprivation 
therapy (ADT). 
1.1.1.2 Determine the T-lymphocyte immune responses to TARP peptide vaccination with 
Montanide® ISA 51 VG plus GM-CSF or autologous dendritic cells as measured by 
tetramer staining, IFN- ELISPOT and 51Cr release CTL assays. 
1.1.2 Secondary Objectives   
1.1.2.1 Determine the effect of TARP peptide vaccination on serum prostate specific antigen 
doubling time (PSADT).  
1.1.2.2 Determine the effect of TARP peptide vaccination on PSA growth rate and regression 
rate constants. 
1.2 
BACKGROUND AND RATIONALE 
1.2.1 Peptide Vaccines 
Elucidation of the crystal structure of the MHC and of the peptides bound 1 , and discovery of 
anchor-residue sequence motifs accounting for binding specificity of peptides to MHC 
molecules2 provided the visual and mechanistic answer to how T cells recognize antigens in the 
form of short peptides. The observation that short peptide segments (8-10 amino acids) fit into a 
groove in the MHC molecule, combined with knowledge of the amino acid sequences of tumor 
epitopes, prompted the use of peptides as therapeutic agents in the treatment of cancer. These 
observations were followed by cloning of the first human tumor associated antigen and 
identification of its nonamer peptide sequence 3. Several strategies have been developed both to 
improve immunogenicity and to steer the immune system toward desired types of responses. 
Peptides have been administered loaded on autologous DCs in an attempt to enhance their 
immunogenicity and improve on peptide vaccination efficacy. However, individual peptides will 
each be useful only in patients with appropriate HLA molecules presenting that peptide. 
In a study of vaccination with p53 peptide-pulsed dendritic cells in HLA-A2+ patients with 
progressive advanced breast cancer, a significant fraction of breast cancer patients (8/19 
evaluable patients) obtained stable disease (SD) or minor regression4.  The effect of p53-specific 
immune therapy was supported by: positive correlation between p53 expression of tumor and 
observed SD; therapy–induced p53 specific T cells in 4/7 patients with SD but only 2/9 patients 
with PD; and significant response- associated changes in serum YKL-40 and IL-6 levels.  In a 
trial of patients with metastatic melanoma vaccinated with melanoma peptide-pulsed DCs, 
longer survival was associated with induction of melanoma peptide-specific immunity to at least 
two of the four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100) used in 
the vaccine5.  Similarly, in a phase I study of HLA-A2-bindingMUC1 peptide pulsed DC 

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vaccination in patients with metastatic renal cell carcinoma, development of MUC1 peptide-
specific T-cell responses in peripheral blood mononuclear cells was seen in the six (of twenty) 
patients with objective responses6.  Consistent with the observed correlations between induction 
of peptide-specific immune responses following DC vaccination and clinical outcomes is a trial 
of CA9-peptide-pulsed mature dendritic cells in patients with renal carcinoma7: none of the 
immunomonitoring assays documented evidence of CA9-peptide-specific immunity and no 
clinical responses were observed. 
1.2.2 Epitope Enhancement  
Modification of the amino acid sequence of epitopes, commonly referred to as epitope 
enhancement, can improve the efficacy of vaccines through several means: 1) increasing affinity 
of peptide for MHC molecules 8-10, 2) increasing T cell receptor (TCR) triggering 11-13, or 3) 
inhibiting proteolysis of the peptide by serum peptidases 8,14,15.  Whenever the peptide sequence 
is altered, it is important to demonstrate that the T cells induced still recognize the native peptide 
sequence.  There is precedent for epitope-enhanced peptides showing greater efficacy in clinical 
trials10. Epitope-enhanced subdominant peptides can bypass self tolerance because subdominant 
epitopes do not generally induce tolerance but can be made more immunogenic by epitope 
enhancement16. 
1.2.3 Clinical Trials of Peptide Vaccines  
The best-studied clinical model of peptide vaccination is malignant melanoma.  Rosenberg and 
colleagues evaluated vaccination with native gp100 peptide 209-217 and found that it produced 
only low levels of T cell reactivity in 2 of 8 melanoma patients analyzed whereas an epitope-
enhanced gp100 (209-2M) peptide generated strong T cell reactivity in 10 of 11 patients 
immunized 10.  Nevertheless, only a single objective clinical response was reported.   
Immunization with the epitope-enhanced peptide, 209-2M, combined with high dose IL-2 
treatment, produced objective clinical anti-tumor responses in 42% of patients, although T cell 
reactivity was observed in <10% of patients. In the adjuvant setting, Smith et al. 17 found a 
vaccination frequency of every two or three weeks resulted in a median frequency of CD8+ cells 
binding 209-2M tetramers of 0.34% after 6 months compared with 0.02% before vaccination, 
whereas less frequent vaccination gave substantially lower responses of 0.03%. The impact of 
age was striking.  In patients under 60 years old the median number of tetramer positive CD8+ 
cells induced by vaccination was 0.64%, whereas in those over 60 it was 0.08%.  In other studies 
using this peptide, addition of either IL-12 18 or GM-CSF 19 increased this percentage slightly.    
Immunization with tyrosinase peptide has been significantly less effective despite epitope 
enhancement. Immunization with the tyrosinase 370D peptide in Incomplete Freund’s Adjuvant 
with or without cytokines, including IL-12 18  or GM-CSF 19, only rarely resulted in low numbers 
of tetramer positive CD8 cells or cytokine production. The levels of 370D-responsive cells has 
been low (0.01-0.03% of input cells) by ELISPOT assay 20.  The use of peptide vaccines may be 
additionally complicated by the choice of adjuvants.  Most studies used IFA but Schaed et al.20 
showed no response with 370D peptide in IFA, whereas almost 50% of patients showed low-
level ELIPSOT responses with QS21 or GM-CSF as adjuvants.  
Recent peptide-pulsed DC-based vaccines have yielded more promising and clinically significant 
results against a variety of tumors when associated with the induction of peptide specific 
immune responses and include: disease stabilization in pts with p53 expressing advanced breast 
cancer4; prolonged survival in subsets of patients with metastatic malignant melanoma 

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vaccinated with melanoma peptides5; and objective responses and disease stabilization in 
patients with metastatic renal cell carcinoma6.  In addition, HER2/neu peptide vaccination alone 
has been associated with reduced recurrence rates in disease-free, conventionally treated, high-
risk breast cancer patients21.  These results must be balanced against clinical trials with negative 
outcomes: a trial of CA9-peptide-pulsed mature dendritic cells failed to show any clinical benefit 
in patients with progressive renal cell carcinoma7, while a randomized trial of autologous DC 
vaccines pulsed with MHC class I and II-restricted peptides could not be demonstrated to be 
more effective than dacarbazine (DTIC) in patients with stage IV metastatic melanoma22.  These 
contrasting outcomes reflect the evolving understanding of dendritic cell biology and delivery, a 
refinement of tumor antigens and peptides selected, and the optimization of patient populations 
that are candidates for therapeutic dendritic cell vaccination. 
The importance of epitope enhancement is supported by the promising results of vaccination 
with the epitope enhanced carcinoembryonic antigen (CEA) peptide13, using peptide-pulsed 
FLT3 ligand-expanded DCs to immunize patients with advanced colorectal cancer.  Five of 12 
patients immunized developed > 1% tetramer-positive CD8+ T cells and two had clinical 
responses.  
Despite the small sample sizes and the variable populations treated, some principles emerge.  
Immunization with native peptide sequences is often insufficient to generate reactive T cells and 
clinical responses in most patients.  Epitope-enhanced peptides can generate T cell responses, 
but not always clinical tumor responses. Adjuvants, including cytokines and costimulatory 
molecules, improve the immunogencity of peptide vaccination.  Paradoxically, combining 
peptide vaccination with IL-2 significantly reduced detection of specific T cells in blood, but 
nearly half the patients showed objective cancer regressions10, possibly due to IL-2-induced 
innate immunity combined with vaccine immunity.  In addition, preclinical studies indicate that 
high avidity CTL to self-peptides tumor antigens may be deleted by self tolerance 16.  Epitope 
enhancement of subdominant epitopes allows induction of protective high avidity CTL responses 
to these tumor antigen epitopes that are not limited by self-tolerance.  This provides an important 
rationale for use of epitope enhanced peptides. 
1.2.4 Previous Vaccine Branch Studies of Peptide-Pulsed Dendritic Cells 
Although there has been significant progress in the development of peptide vaccines for the 
treatment of melanoma, for other solid malignancies progress has lagged.  We have evaluated 
mutated ras and p53 peptides in patients with lung and colon cancer.   
In study 94-C-0031, a phase I/II trial of mutant ras and p53 peptides in a variety of cancers, both 
CTL responses and interferon-gamma responses to the peptide vaccine were statistically 
significantly correlated with median survival times (393 vs 98 days, p = 0.04, and 470 vs 88 
days, p = 0.02, for the two types of immune response, respectively)23. 
In study 99-C-0023, a phase I/II trial of vaccination with mutant Ras peptide-pulsed DCs in HLA 
A2.1 patients with colorectal cancer, two groups of patients were eligible for study participation: 
patients with evidence of metastatic disease and patients without evidence of disease but at high 
risk for recurrence.  One of 15 patients enrolled in the metastatic arm developed a detectable 
immunologic response but there were no objective tumor responses, although occasional patients 
showed regression of individual metastatic lesions.  There were no immunologic responses 
among the four patients accrued to the adjuvant arm without metastatic disease. 

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In study 99-C-0142, a phase II trial of individualized mutant p53 peptide-pulsed DCs in the 
treatment of patients with locally advanced non-small cell lung cancer after standard therapy, 
twelve patients were screened for the study and two patients were eligible for enrollment.  Of the 
two patients enrolled, only one was evaluable for immune responses to p53 vaccination and there 
was no evidence of vaccine-induced immunologic reactivity.  The other patient experienced 
rapid disease progression following completion of vaccination and could not be evaluated for 
immune response. 
These trials of peptide-pulsed dendritic cells were not successful in demonstrating an acceptable 
level of immunization with mutated ras or p53.  This may be due to the relatively low binding 
affinity of the mutated ras peptides for HLA-A*0201; successful immunization of patients with 
peptides appears to require epitope-enhanced peptides as discussed earlier.  Another challenge 
that has made use of ras and p53 peptides particularly difficult is the need to treat patients whose 
tumor cells express the mutated sequences for this approach to be beneficial.  Only one in three 
patients screened for eligibility for the ras trial was eligible, increasing the amount of screening 
required to identify potential candidates.  In addition, in both these studies, the dendritic cell 
vaccines were administered intravenously.  It has become clear from the emerging literature on 
therapeutic dendritic cell cancer vaccination that intradermal delivery results in optimal 
immunogenicity. For this reason we have looked for other peptides that are more universally 
expressed that would be attractive targets for immunization and are administering the TARP 
peptide-pulsed dendritic cell vaccines in this study intradermally.   
1.2.5 TARP 
A novel protein expressed in patients with prostate and breast cancer has recently been described 
24 .  This 58 amino acid protein, T-cell receptor γ alternate reading frame protein (TARP), was 
identified with the expressed sequence database.  The mRNA is initiated in the Jγ 1 exon of the 
TCR γ and the protein expressed is initiated in an alternative reading frame than the TCR γ 
coding sequence. In their initial description of TARP in the human prostate, Pastan et al 
demonstrated that it originated from epithelial cells and not from infiltrating T lymphocytes, and 
that it is expressed in normal prostate epithelium, adenocarcinoma of the prostate, and the 
prostatic adenocarcinoma cell line LNCaP25.  They subsequently showed that TARP was also 
expressed in three breast cancer cell lines and breast cancer tissues26 and determined that TARP 
is expressed in androgen-sensitive (LNCaP) but not in androgen-independent (PC3) prostate 
cancer cell lines, implicating the role of TARP in prostate cancer progression27.  The protein is 
expressed both by normal and malignant prostate cancer tissue with about 95% of prostate 
cancer specimens positive for its expression.  Oh et al determined two HLA-A2 epitopes that 
produce cytolytic T cell responses 28.  These sequences map to amino acids 27-35 and 29-37.  
TARP27-35 was found to bind with an affinity that was 10 times greater than that of TARP29-
37.   These peptides were demonstrated to be immunogenic by immunizing A2Kb transgenic 
mice (expressing human HLA-A*0201) with dendritic cells pulsed with these peptides or with 
DNA encoding the peptide.  Dendritic cell immunization produced a higher level of immunity 
than DNA immunization and as expected due to its higher binding affinity, TARP27-35 
produced a higher level of CD8+ T cell response than TARP29-37. 
Epitope enhancement of the TARP peptides was performed to increase the level of immunity 
that could be generated with these peptides.  Amino acid substitutions in the TARP27-35 peptide 
did not increase binding affinity but two amino acid substitutions in TARP29-37 did produce 

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higher binding affinity peptides.  For TARP29–37, Arg at position 3 and Leu at position 9 were 
substituted with Ala (TARP29–37-3A) and Val (TARP29–37-9V), respectively.  Substitution at 
position 3 with Ala in TARP29–37 resulted in the greatest increase in the binding affinity of the 
peptide.  Although TARP29–37-9V showed a lower binding affinity to HLA-A2 than TARP29–
37-3A did, substitution of Leu at position 9 with Val did enhance the binding affinity compared 
with the wild-type peptide, TARP29–37.  When the immunogenicity of these peptides was 
evaluated in A2Kb transgenic mice both of the epitope-enhanced peptides produced a higher 
percentage of CD8+ T cells specific than the wild type sequence.  It was also shown that T cells 
generated with the epitope enhanced TARP29-37 sequences reacted with targets pulsed with the 
wild type TARP29-37 peptide in the mouse. 
Although immunogenicity of these peptides was demonstrated in the mouse it is important to 
confirm their immunogenicity and cross reactivity in humans.  Studies of these peptides in 
human cells showed that TARP29-37, TARP29–37-3A, and TARP29–37-9V were immunogenic 
in human T cells.  TARP29–37-9V specific T cells recognize targets pulsed with all three 
peptides equally well whereas TARP29–37-3A specific T cells recognized only targets pulsed 
with TARP29–37-3A, and that TARP29–37 specific T cells recognized targets pulsed with the 
epitope enhanced peptides less well.  This would suggest that the TARP29–37-3A peptide would 
not be appropriate for immunization in humans whereas the TARP29–37-9V would be more 
likely to generate T cells that recognize the wild type sequence.  Human T cells specific for 
TARP27-35 recognized targets pulsed with that sequence as anticipated.  In addition to their 
ability to kill targets pulsed with TARP peptides, CD8+ T cells specific for TARP peptides were 
able to kill human tumor targets that were HLA A2 positive and that expressed TARP sequences.  
The availability of tetramers that react with CD8+ T cells specific for TARP provide a simple 
means of evaluating the ability to stimulate immunity to the TARP peptides.  In a limited survey 
tetramer positive cells ranged from 0.66% to 3.9% of the CD8+ T cells in prostate and breast 
cancer patients compared with .01-.6% in normal controls. 
1.2.6 Dendritic Cell Vaccination with TARP 
This antigen will be presented on the patient's own HLA-A*0201 positive dendritic cells.  
Dendritic cells are very potent antigen presenting cells.  They have several distinct features 
making them ideal vehicles for presentation of these tumor specific peptides.  Dendritic cells 
express a high level of both MHC class I and II molecules, as well as, a variety of adhesion and 
costimulatory molecules that contribute to their ability to effectively stimulate T cells 29 .  
Dendritic cells have been shown to be more effective in the stimulation of the immune response 
than B cells or macrophages 30 .  In addition, ex vivo maturation of dendritic cells may, in part, 
overcome the inability of cancer patients to direct the immune response toward their own tumor.  
Dendritic cells within tumor bearing mice have been found to be marginally effective when 
presenting peptides to the animal’s immune system.  This is due to the presence of vascular 
endothelial growth factor, present in many cancers, including colorectal cancers bearing ras 
mutations, that inhibits dendritic cell maturation.  However, cells from the same tumor bearing 
animals, matured ex vivo, are extremely effective in presentation to the immune system 31,32.  
Dendritic cells can be matured ex vivo from human peripheral blood mononuclear cells using 
interleukin 4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF) followed 
by CD40 ligand treatment 33,34.  We have conducted pilot experiments along with the Department 
of Transfusion Medicine using peripheral blood monocytes cells (PBMCs). We have measured 

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the up regulation of CD83, costimulatory and adhesion molecules as surrogate markers and have 
measured the function of dendritic cells in a mixed lymphocyte reaction 29,35.  
 
Figure 1 
Dendritic Cells
Monocytes
 
 
We also plan to further improve our dendritic 
cell manufacturing process utilizing IFN- 
and LPS for dendritic cell maturation.  
Labeur et al have compared both the cell 
surface markers and the functional activity of 
dendritic cells cultured with GM-CSF and IL-
4 (immature dendritic cells) and those 
cultured with GM-CSF and IL-4 plus CD40L 
or LPS (mature dendritic cells) 26.  The 
mature dendritic cells when compared to 
immature dendritic cells demonstrated greater 
expression of most cell surface markers 
thought to be important in T cell interaction. 
In addition, the mature dendritic cells produced significantly more interleukin 12 than immature 
dendritic cells.  More importantly, in a tumor challenge model, six of six mice that were 
immunized with antigen pulsed mature dendritic cells did not develop tumors while six of six 
mice that were immunized with antigen pulsed immature dendritic cells did develop tumors.  
Further, in an established tumor model, tumor growth was significantly inhibited in mice 
vaccinated with mature dendritic cells when compared to mice vaccinated with immature 
dendritic cells 36.  The ability of CD40 ligand to induce the maturation of dendritic cells that are 
better able to stimulate the T cell response has also been evaluated ex vivo in humans.  Morse et 
al were able to show that dendritic cells treated with CD40 ligand were more effective at 
stimulating cytotoxic T cells than were immature dendritic cells grown in GM-CSF and IL-4 37.  
1.2.7 Vaccination with Peptides or Peptide-Pulsed Dendritic Cells 
There are limited studies that address the most effective means for induction of cytotoxic T cell 
responses in patients with cancer.  Slingluff compared the use of peptide in adjuvant with 
peptides pulsed on dendritic cells in a small study in patients with melanoma.  This study 
0
5000
10000
15000
3H-THYMIDINE INCORPORATION
1:1
1:0.1
1:0.01
1:0.005
1:0
0:1
EFFECTOR TO STIMULATOR RATIO
 COMPARISON OF DENDRITIC CELLS VS. MONOCYTES
AS STIMULATORS IN A MIXED LYMPHOCYTE REACTION
   MLR PULSED W/ TRITIATED THYMIDINE ON DAY 7
DENDRITIC CELLS ALONE
MONOCYTES ALONE
EFFECTORS ALONE
DENDRITIC CELLS
MONOCYTES

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showed a superior response with peptides in adjuvant compared with peptides pulsed on 
dendritic cells.  T cell response was monitored in peripheral blood and draining lymph nodes.  In 
patients vaccinated with peptide in adjuvant T cell responses were seen in 42% of peripheral 
blood samples and 80% of lymph nodes whereas in patients vaccinated with peptides pulsed on 
dendritic cells 11% showed response in the blood and 13% in the nodes.  Despite the disparity in 
immunologic response rates objective responses were observed in both arms of the study.  This 
study used immature dendritic cells and previous studies have shown that immature dendritic 
cells are ineffective in producing a cytolytic T cell response compared with mature dendritic 
cells.  Another possible disadvantage with immature dendritic cells is their ability to induce 
tolerance.  This study will address in a randomized fashion the immunogenicity of TARP 
peptides in adjuvant compared with TARP peptides pulsed on mature dendritic cells.  
2 
ELIGIBILITY ASSESSMENT AND ENROLLMENT 
2.1 
ELIGIBILITY CRITERIA 
2.1.1 
Inclusion Criteria 
2.1.1.1 
Males > 18 years of age with histologically confirmed adenocarcinoma of the 
prostate. 
2.1.1.2 
HLA-A*201 positive 
2.1.1.3 
Patients must have 
2.1.1.3.1 
Completed and recovered from all prior definitive therapy (surgery, brachytherapy, 
cryotherapy or radiotherapy) for the primary tumor, or other definitive-intent local 
therapy. 
2.1.1.3.2 
Stage D0 disease with documented biochemical progression documented by a rising 
PSA. 
2.1.1.3.3 
No evidence of metastatic disease by physical examination, CT scan or bone scan. 
2.1.1.3.4 
For patients following definitive radiation therapy or cryotherapy: a rise in PSA of 
>2 ng/mL above the nadir (per RTOG-ASTRO consensus criteria). 
2.1.1.3.5 
For patients following radical prostatectomy: 2 absolute PSA values > 0.3 ng/mL 
(per NCCN guidelines) 
2.1.1.3.6 
Non-castrate level of testosterone: > 50 ng/dL (prior ADT allowed; must be > 6 
months since last dose of ADT). 
2.1.1.3.7 
A Pre-Enrollment/Baseline PSADT > 3 months and < 15 months 
2.1.1.3.7.1 Patients must have > 3 PSA measurements over > 3 months 
2.1.1.3.7.2 The interval between PSA measurements must be > 4 weeks 
2.1.1.3.7.3 For patients receiving 5-alpha reductase inhibitors (5ARI) e.g. finasteride or 
dutasteride, only PSA values obtained after at least 3 months on therapy may be used 
to calculate PSADT. 
2.1.1.3.8 
 Performance Status: ECOG 0-2 or Karnofsky 70-100% 
2.1.1.3.9 
Life expectancy > 1 year. 
2.1.1.3.10 Hemoglobin > 10.0 gm/dL, WBC > 2,500/mm3, ALC > 500/mm3, ANC > 
1,000/mm3, platelet count > 100,000/mm3. 

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2.1.1.3.11 
PT/PTT < 1.5X ULN unless receiving clinically indicated anticoagulant therapy. 
2.1.1.3.12 
SGOT/SGPT <2.5X ULN, total bilirubin < 1.5X ULN, Cr < 1.5X ULN, estimated 
GFR (eGFR) > 60 ml/min. 
2.1.1.4 
Hepatitis B and C negative, unless the result is consistent with prior vaccination or 
prior infection with full recovery. 
2.1.1.5 
HIV negative  
2.1.1.6 
No use of investigational agents within 4 weeks of study enrollment. 
2.1.1.7 
No use of immunosuppressive (cytotoxic chemotherapy, systemic steroids) or 
immunomodulating agents (including IVIG) within 8 weeks of study entry.  Note: 
topical and intranasal steroid therapy is permitted. 
2.1.1.8 
No other concurrent anticancer therapy. 
2.1.1.9 
No alternative medications known to alter PSA (e.g. phytoestrogens and saw 
palmetto). Note: patients receiving medications for urinary symptoms such as 
Flomax or 5-alpha reductase inhibitors (finasteride and dutasteride) on a chronic 
stable dose for at least 3 months are allowed.  
2.1.1.10 
No prior prostate cancer vaccines expressing TARP or HLA A2. 
2.1.1.11 
Able to understand and provide Informed Consent. 
2.1.2 
Exclusion Criteria 
2.1.2.1 
HLA-A*201 negative 
2.1.2.2 
Patients with an active second malignancy other than adequately treated squamous 
or basal cell carcinoma of the skin, or superficial bladder carcinoma.   
2.1.2.3 
Patients with active infection. 
2.1.2.4 
Patients with brain, visceral or bony metastatic disease. 
2.1.2.5 
Patients in who live attenuated intranasal influenza vaccine (FluMist) is 
contraindicated38 including individuals with asthma or reactive airways disease, 
cardiovascular or pulmonary disease, chronic metabolic diseases (including diabetes 
mellitus), renal dysfunction or hemoglobinopathies. 

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2.2 
RESEARCH ELIGIBILITY EVALUATION 
2.2.1 Protocol screening evaluation, other than the determination of HLA typing, will be 
performed within 2 weeks of study entry. 
2.2.2 All patients will undergo a baseline history and physical examination including review of 
systems, performance status and life expectancy assessment.  Radiologic evaluation 
(performed within 8 weeks of study) will include a, CT scan of the, chest, abdomen and 
pelvis, and a bone scan and may be performed at outside institutions.  Laboratory studies 
will include a CBC with differential, platelet count, PT/PTT, chemistries (Acute Care 
Panel, Mineral Panel, Hepatic Panel, total protein, CK, Uric acid, LDH), PSA, 
testosterone, 25 hydroxy vitamin D, HIV-1 antibody, Hepatitis B Surface antigen 
(HBSAg) and antibody to Hepatitis B Surface antigen (anti-HBSag), Hepatitis C 
antibody (anti-HCV) and HTLV-1 antibody (anti-HTLV-1). 
2.2.3 Refer to Appendix 1 for the Schedule of Study Clinical, Laboratory and Radiographic 
Evaluations. 
2.3 
REGISTRATION PROCEDURES 
Authorized staff must register an eligible candidate with NCI Central Registration Office (CRO) 
within 24 hours of signing consent.  A registration Eligibility Checklist from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) must be completed and faxed to 301-
480-0757.  After confirmation of eligibility at Central Registration Office, CRO staff will call 
pharmacy to advise them of the acceptance of the patient on the protocol prior to the release of 
any investigational agents.  Verification of Registration will be forwarded electronically via e-
mail to the research team. A recorder is available during non-working hours. 
Randomization Procedures  
Once registration is confirmed, enrolled patients will be randomized 1:1 to receive TARP 
peptides with Montanide® ISA 51 VG adjuvant plus GM-CSF administered by deep 
subcutaneous (SC) injection or autologous, TARP peptide-pulsed dendritic cells administered 
intradermally (ID):  
Arm A (TARP peptides with Montanide® ISA 51 VG plus GM-CSF): study numbers 
101-120 
Arm B (TARP peptide pulsed dendritic cells): study numbers 201-220 
All patients will undergo apheresis for immunologic monitoring.  The apheresis will also be used 
for preparation of dendritic cells by elutriation of monocytes for dendritic cell culture, 
maturation and vaccine generation for those randomized to receive peptide-pulsed dendritic 
cells.   
3 
STUDY IMPLEMENTATION 
3.1 
STUDY DESIGN 
This is a randomized, prospective pilot study of TARP peptide vaccination in HLA-A*201 
patients with Stage D0 prostate cancer.  HLA-A*201 positive eligible patients with no evidence 

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of brain, visceral or bony metastatic disease, a non-castrate testosterone level > 50 ng/dL (prior 
ADT allowed; must be > 6 months since last dose of ADT), and a PSADT > 3 months and < 15 
months will be will be randomized to receive TARP peptides with Montanide® ISA 51 VG 
adjuvant plus GM-CSF administered by deep subcutaneous (SC) injection or autologous, TARP 
peptide-pulsed dendritic cells administered intradermally (ID).   
TARP peptides or TARP-pulsed dendritic cells will be administered every three weeks at weeks 
3, 6, 9, 12, and 15 for a total of five vaccinations with a booster dose of TARP vaccine given at 
Weeks 48 and 96.   PSADT will be calculated in months at the following study time points: Pre-
Enrollment/Baseline, weeks 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144.  PSADT will 
be calculated using the Memorial Sloan-Kettering Cancer Center cancer information prostate 
nomogram for PSA doubling time found at: 
http://www.mskcc.org/mskcc/shared/forms/Nomograms/flash/load.cfm?type=Prostate&width=5
86&height=505&title=Prostate%20Nomogram as recommended in the Prostate Specific Antigen 
Working Group Guidelines.39  Cumulative PSA values within 12 months inclusive of the study 
time point will be used for calculation of the baseline/Week 0 PSADT. 
Exploratory comparisons of percent change in PSADT will be done among the following time 
points: 
➢ Pre-Enrollment/Baseline PSADT vs. Week 12 PSADT 
➢ Pre-Enrollment/Baseline PSADT vs. Week 24 PSADT 
➢ Pre-Enrollment/Baseline PSADT vs. Week 36 PSADT 
➢ Pre-Enrollment/Baseline PSADT vs. Week 48 PSADT 
➢ Week 24 PSADT vs. Week 36 PSADT 
➢ Week 36 PSADT vs. Week 48 PSADT 
➢ Additional exploratory analyses of PSADT will be performed of Pre-Enrollment/Baseline 
PSADT vs. Weeks 60, 72 and 96 and Week 48 PSADT vs. Weeks 96, 108 and 144 
PSADT. 
Week 24 PSADT responders will be allowed to receive an additional dose of TARP peptide 
vaccine at Week 36.  All patients will receive a booster of vaccine at Weeks 48 and 96. 
Assessment of humoral and cellular immune responses to TARP peptide vaccination will be 
performed at weeks 0, 12, 18, 24, 36, 48, 60, 96, 108, 120, 132, and 144.  Antigen-specific T-
lymphocyte responses will be assessed by tetramer staining, IFN- ELISPOT and 51Cr release 
CTL assays following exposure to native wild type and epitope-enhanced TARP peptides. 
The schema for treatment is detailed below. 

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3.1.1 Study Schema  
Patients with Prostate Cancer 
 
Assessment for HLA-A*0201 Positivity 
                            
HLA-A*0201 Positivity Confirmed 
 
HLA-A*0201 Negative  INELIGBILE for Study 
Study Eligibility Confirmed, Informed Consent Obtained 
 
RANDOMIZATION 
                   
TARP Peptide-Pulsed DCs 
TARP Peptide w/ Montanide® ISA 51 VG and GM-CSF 
APHERESIS 
Dendritic Cell Maturation 
 
APHERESIS (Apheresis performed at weeks 0, 24, 48.) 
 
 
Vaccination q 3 Wks X 5 at weeks 3, 6, 9, 12 and 15 with booster dose at Weeks 48 and 96 
 
 
Assessment of Immunologic Responses to TARP peptides at weeks 0, 12, 18, 24, 36, 48, 60, 
96, 108, 120, 132, and 144. 
Assessment of PSADT at Pre-Enrollment/Baseline and weeks 12, 24, 36, 48, 60, 72, 84, 96, 
108, 120, 132, and 144. 
3.2 
VACCINE ADMINISTRATION 
Following apheresis to remove peripheral blood monocytes for dendritic cell preparation as well 
as peripheral blood mononuclear cells for flow cytometry and immunologic studies at their week 
0 visit, all patients will undergo intranasal vaccination with live attenuated influenza vaccine 
(FluMist) when seasonally available.  Attenuated influenza vaccine is being administered as a 
control vaccine to characterize the patient’s innate ability to generate immune responses to 
vaccination.  Conserved HLA-A*0201 restricted cellular and humoral responses to flu matrix 
peptides have been extremely well characterized regarding antibody titers and the range of 
ELISPOT responses that are typically observed in normal healthy individuals.  Following receipt 
of the intranasal flu vaccine, patients will be immediately discharged to home and precautions 
will be taken to minimize the risk of potential transmission to other Clinical Center patients as 
outlined in Section 9.5 
Because live attenuated influenza viruses replicate in the nasopharynx of recipients and can be 
shed in respiratory secretions, the possibility exists that the vaccine viruses can be transmitted to 
susceptible individuals who have not received the vaccine.  Studies in children 8-36 months of 
age attending daycare who were randomized to receive a single dose of influenza virus vaccine 
live intranasal or placebo indicated that 80% of vaccine recipients shed at least 1 vaccine strain; 
the mean duration of shedding was 7.6 days (range: 1-21 days).38  The frequency and duration of 
shedding of vaccine virus strains by individuals 5-49 years of age who receive influenza virus 

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vaccine live intranasal have not been established.   Consequently, study patients will return to the 
CC at study week 3 which should provide an adequate window to ensure that recipients are 
unlikely to be shedding attenuated viral vaccine strains. 
Patients in Arm A will receive an admixture of the wild-type and epitope-enhanced TARP 
peptides emulsified with Montanide® ISA 51 VG and GM-CSF administered by deep 
subcutaneous (SC) injection.  Patients in Arm B will receive peptide-pulsed dendritic cells 
administered intradermally in two vaccination sites.  Each peptide will be pulsed on dendritic 
cells separately in order to assure adequate binding of the peptide and cells will not be washed to 
remove free peptide after pulsing.  Following verification of mature dendritic cell validation 
markers and release standards, the separately peptide-pulsed dendritic cells will be recombined 
for administration.  Refer to Appendix 2 for details concerning the production of peptide –pulsed 
dendritic cells by the NIH Clinical Center Department of Transfusion Medicine under the 
supervision of Drs. Marianna Sabatino, Hanh Khuu, Susan Leitman, & David Stroncek. 
For both groups vaccinations will be administered at weeks 3, 6, 9, 12 and 15 for a total of five 
vaccinations with a booster dose of vaccine at Weeks 48 and 96. Patients will be monitored for 
immediate adverse event vaccine reactions (VS, clinical assessment) for 1 hours following their 
first TARP peptide vaccine dose.  If no adverse reactions are observed with the first vaccination, 
patients will be monitored for 15 minutes for all subsequent vaccinations.  If adverse reactions 
are observed following the first vaccine, the duration of post-vaccination monitoring with 
subsequent vaccinations will be determined by the Principal Investigator and Lead Associate 
Investigator as clinically indicated depending on the severity of the initial vaccine reaction.    
Since this protocol involves TARP vaccination in humans for the first time, enrollment will be 
staggered to allow for safety monitoring.  For the first 3 patients on each study arm, enrollment 
of the next patient can only proceed until the preceding study subject is 2 weeks status-post their 
first vaccination.  If no adverse events are observed in the two week window following the first 
vaccination in these 6 patients, enrollment of additional patients may proceed as quickly as is 
logistically feasible.  Cells used for subsequent dendritic cell maturation will be derived from 
monocytes frozen during the initial apheresis. 
3.3 
TREATMENT MODIFICATIONS AND IMMUNIZATION STOPPING RULES 
No dose modifications will be made in patients receiving vaccination.  Subjects will cease to 
receive immunization if they experience dose-limiting toxicity (DLT). Toxicity will be graded 
according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v3.0 
(http://ctep.info.nih.gov/reporting/ctc_v30.html)  
3.3.1 Immunization-Related Dose Limiting Toxicity (DLT)  
Immunization-related DLT is defined by the parameters outlined below attributed by the 
Principal Investigator or Protocol Chairperson as possibly, probably or definitely related to 
vaccine administration: 
3.3.1.1 Any Grade 2 or Grade 3 or greater allergic/hypersensitivity reaction 
3.3.1.2 Any Grade 2 or greater rash consistent with erythema multiforme. 
3.3.1.3 Grade 3 or greater hematologic or non-hematologic toxicity, excluding lymphopenia.  
Abnormal laboratory studies will be repeated to verify toxicity 
3.3.1.4 Grade 3 or greater acute vascular leak syndrome: respiratory compromise or fluids 
indicated. 

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3.3.1.5 The following Grade 3 reactions commonly associated with immunization will be dose-
limiting: 
➢ Injection site reactions: ulceration or necrosis that is severe; operative 
intervention indicated. 
➢ Skin rash/desquamation: severe, generalized erythroderma or macular, papular 
or vesicular eruption; desquamation covering > 50% BSA. 
➢ Urticaria: intervention indicated for > 24 hrs. 
3.3.1.6 The following Grade 3 reactions commonly associated with immunization will not be 
dose-limiting: 
➢ Pruritis/itching: intense or widespread and interfering with ADL lasting < 72 
hrs 
➢ Fatigue: severe fatigue interfering with ADL lasting < 72 hrs 
➢ Fever: > 40.0 C for < 24hrs 
➢ Local lymphadenopathy lasting < 1 week 
3.3.2 Stopping for Excessive toxicity 
If more than one of the first nine subjects in either arm of the trial experiences dose limiting 
toxicity, enrollment to that arm will cease pending discussion and review with the FDA and the 
IRB. 
3.3.3 Delay in Vaccination 
Patients who are unable to receive their vaccine injection as scheduled due to adverse events, 
toxicity or unforeseen personal or medical circumstances and who are otherwise eligible to 
continue on protocol, may be continued on vaccine therapy provided that their next vaccine is 
within three weeks of the previous vaccine.  Patients will be advised of their options concerning 
alternative treatments before proceeding with the completion of their study vaccinations.  The 
reason for the change in vaccination schedule will be documented in the patient's chart. 
3.4 
ON STUDY EVALUATION 
Refer to Appendix 1 for the Schedule of Clinical, Laboratory and Radiographic Evaluations.  
3.4.1 Clinical Evaluation 
(weeks 0, 3, 6, 9, 12, 15, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132 and 144.) 
3.4.1.1 Review of systems and documentation of symptoms 
3.4.1.2 Assessment of ECOG Performance Status 
3.4.1.3 Vital signs and physical examination 
3.4.1.4 Assessment of injection site reactions 
3.4.1.5 Provision of Vaccination Report Card to study subject (when TARP vaccine 
administered) 
3.4.2 Laboratory Evaluation  
(weeks 0, 3, 6, 9, 12, 15, 18, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144.) 
3.4.2.1 CBC with differential and platelets 
3.4.2.2 Chemistries (Acute Care Panel, Mineral Panel, Hepatic Panel, total protein, CK, Uric 
acid, LDH), 

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3.4.2.3 PSA, testosterone 
3.4.3 Radiographic Evaluation  
(Weeks 0, 48, 96 and 144.  No further restaging unless clinically indicated. ) 
3.4.3.1 CT Scan of the chest/abdomen/pelvis 
3.4.3.2 Bone Scan 
3.4.4 Immunologic Responses  
(Weeks 0, 12, 18, 24, 36, 48, 60, 96, 108, 120, 132, and 144.) 
3.4.4.1 Lymphocyte Subsets: CD4 and CD8 percent and absolute counts (done at Clinical 
Center) 
3.4.4.2 Anti-TARP antibody responses 
3.4.4.3 NKT Cell analysis (weeks 0, 12, 18 and 24 only) 
3.4.4.4 IFN- ELISPOT of PBMCs 
3.4.4.5 Tetramer staining and 51Cr release CTL assay of PBMCs 
3.4.5 Apheresis  
Weeks 0, 24, 48. 
3.4.6 Research Specimens for Assessment of Immunologic Responses 
3.4.6.1 Cryopreserved PBMCs (6 tubes sodium heparin 10ml GTT) for tetramer, IFN-g 
ELISPOT and CTL assays.  Weeks 0, 3 (FluMist responses), 12, 18, 24, 36, 48, 60, 72, 
84, 96, 108, 120, 132 and 144.  Note: PBMCs are for storage only at Weeks 72 and 84.  
3.4.6.2 Serum Storage (10ml RTT) for anti-TARP antibody responses. Weeks 0, 12, 18, 24, 36, 
48, 60, 72, 84, 96, 108, 120, 132 and 144. 
3.4.6.3 NKT Cell Analysis (2 tubes 8ml blue tiger top sodium citrate tubes). Weeks 0, 12, 18, 
24,  
3.5 
CONCURRENT THERAPIES 
3.5.1 Contraindicated Therapies 
3.5.1.1 Patients may not receive chemotherapy, hormonal therapy or radiation therapy while on 
study. 
3.5.1.2 Systemic administration of steroids is not allowed with the exception of oral 
corticosteroids for management of acute allergic reactions or contact hypersensitivity of 
< 14 days duration in patients who require them. Topical steroids as well as intranasal 
steroids are permitted. Patients must not have received systemic steroids within 14 days 
to TARP peptide vaccination. 
3.5.1.3 Pharmacologic doses of immune modulating agents including IVIG, recombinant 
cytokines or growth factors. 
3.5.1.4 Immunosuppressive agents such as cytotoxic chemotherapy. 
3.5.1.5 Other investigational agents. 

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3.5.2 Allowed Therapies 
Study subjects will be allowed to take multivitamins, analgesics/antipyretics/antihistamines for 
local and systemic injection site reactions, and other medications as clinically indicated.  Use of 
herbal or nutritional supplements, known to alter PSA e.g. phytoestrogens or saw palmetto is not 
allowed.  
3.6 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY CRITERIA 
3.6.1 Criteria for removal from protocol therapy 
3.6.1.1 Patient experiences a Grade 3 or greater toxicity attributed as possibly, probably or 
definitely related to vaccine as described in Section 3.3 Treatment Modifications and 
Immunization Stopping Rules. 
3.6.1.2 Patient is taken off treatment by the Principal Investigator or Lead Associate 
Investigator for reasons other than toxicity e.g. insufficient recovery of dendritic cells to 
generate required vaccine dose, failure to adhere to scheduled study visits, etc. 
3.6.2 Post Treatment Evaluation 
This study is 144 Weeks in duration.   
3.6.2.1 Clinical Evaluation: physical examination and assessment of performance status 
3.6.2.2 Laboratory Evaluation: CBC with differential, platelets, PT/PTT, chemistries (Acute 
Care Panel, Mineral Panel, Hepatic Panel, total protein, CK, Uric acid, LDH), PSA. 
3.6.2.3 Radiographic Evaluation: CXR, CT scan of the brain/chest/abdomen/pelvis, bone scan. 
3.6.2.4 Immunologic Evaluation: CD4 and CD8 percent and absolute counts. 
3.6.3 Off Study Criteria 
3.6.3.1 Patient elects to withdraw from participation in the study at any time. 
3.6.3.2 Patient is removed from study after resolution of Grade 3 or greater toxicities attributed 
as possibly, probably or definitely related to vaccine as described in Section 3.3 
Treatment Modifications and Immunization Stopping Rules. 
3.6.3.3 Patient develops evidence of brain, visceral or bony metastatic disease. 
3.6.3.4 Patients with a calculated PSADT that has decreased by > 50% compared to their Pre-
Enrollment/Baseline PSADT. PSADT is calculated as specified in Section 3.1. 
3.6.3.5 Patients with a calculated PSADT of < 3 months at any of the outlined PSADT 
assessment time points i.e. weeks 12, 24, 36 or 48. PSADT is calculated as specified in 
Section 3.1. 
3.6.3.6 For those patients receiving them, a discontinuation or dose reduction of their Flomax 
or 5-alpha reductase inhibitors. 
3.6.3.7 Patient completed study 
3.6.4 Off-Study Procedure 
Authorized staff must notify Central Registration Office (CRO) when a subject is taken off-
study. An off-study form from the web site 
(http://home.ccr.cancer.gov/intra/eligibility/welcome.htm) main page must be completed and 
faxed to 301-480-0757. 

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4 
SUPPORTIVE CARE 
We do not anticipate significant systemic or organ toxicities associated with TARP peptide 
vaccination using either Montanide® ISA 51 VG plus GM-CSF or peptide- pulsed dendritic 
cells.  We have not observed any clinically significant toxicities in our previous trials 
investigating other class I peptides in patients with lung or colon cancer.  Non-steroidal anti-
inflammatory medication or other analgesics and possibly antihistamines may be needed for 
local injection site reactions to vaccination.  
5 
DATA COLLECTION AND EVALUATION 
5.1 
DATA COLLECTION 
The Vaccine Branch clinical research team will perform real time data collection. The Principal 
Investigator will be responsible for oversight of the protocol. 
All patients enrolled on study will be centrally registered at the NCI Central Registration Office.  
All on study evaluations must be performed within one week of their scheduled time point as 
outlined in the study schema (Appendix 1).  Delay in vaccine administration is allowed for up to 
3 weeks as outlined in Section 3.3.3.  Modifications in this policy can be discussed on an 
individual basis with the Lead Associate Investigator.  Research data will be collected in the NCI 
C3D database. 
 
All AEs, including clinically significant abnormal findings on laboratory evaluations, regardless 
of severity, will be followed until satisfactory resolution. AEs should be reported up to 30 days 
following the last dose of study drug.   
An abnormal laboratory value will be considered an AE if the laboratory abnormality is 
characterized by any of the following: 
• 
Results in discontinuation from the study 
• 
Is associated with clinical signs or symptoms  
• 
Requires treatment or any other therapeutic intervention 
• 
Is associated with death or another serious adverse event, including hospitalization.  
• 
Is judged by the Investigator to be of significant clinical impact 
 
End of study procedures: Data will be stored according to HHS, FDA regulations, and NIH 
Intramural Records Retention Schedule as applicable.  
Loss or destruction of data: Should we become aware that a major breach in our plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified. 
5.2 
RESPONSE CRITERIA 
5.2.1 Immunologic Response Criteria  
Immunologic responses to TARP peptide vaccination will be assessed at weeks 0, 12 (after 3 
vaccines), 18 (after 5 vaccines), 24, 36, 48, 60 and 96; Note: apheresis will be performed at 
weeks 0, 24, 48 and 96.   Refer to Appendix 3 for details of specimen tube, collection and 

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processing and to Appendix 4 for descriptions of the immunology assays.  Additional 
exploratory analyses of TARP immunologic responses will be performed at Weeks 60, 72, 96, 
108, 120, 132 and 144.  
 
A positive immunological response to class I TARP peptides is defined as: 
➢ A three-fold increase over baseline in the number of positive cells by tetramer staining 
OR 
➢ A three-fold increase over baseline in the number of positive cells by IFN- ELISPOT 
assay. 
➢ Positive assays must be confirmed at two study time points to be considered a definitive, 
vaccine-induced positive result. 
➢ Week 24 immunologic responders will be allowed to receive an additional dose of TARP 
peptide vaccine at Week 36. 
➢ The study time points at which a positive immunologic response is documented will be 
noted for every patient enrolled on the study.   
 
Hence a patient will be considered a responder if they demonstrate a positive response as 
described above in either the tetramer or the IFN- ELISPOT assays.  51Cr release cytotoxic T 
lymphocyte (CTL) assays using T2 cells as targets will only be performed in patients 
documented to have a positive tetramer or ELISPOT response and will not be utilized in the 
assessment of immunologic response. 
 
A positive 51Cr release CTL assay will be defined as:   
➢ The percent lysis observed against TARP peptides is at least 2 standard deviations greater 
than the percent lysis observed against control peptides AND  
➢ Greater than or equal to a 10% difference in the TARPT peptide percent lysis vs. control 
peptide percent lysis at more than one effector to target cell ratio.   
5.2.2 PSADT Response Criteria  
PSADT was chosen as a surrogate marker for biochemical response to vaccination based on the 
Prostate-Specific Antigen Working Group’s consensus Guidelines on PSADT.39 Utilization of 
PSADT as a predictive factor of disease progression and prostate cancer death is based on level 
II evidence derived from cohort- or case-controlled analytic studies or controlled, non-
randomized trials that have been validated in multiple data sets,40-42  PSADT will be calculated 
using the Memorial Sloan-Kettering Cancer Center cancer information prostate nomogram for 
PSA doubling time found at: 
http://www.mskcc.org/mskcc/shared/forms/Nomograms/flash/load.cfm?type=Prostate&width=5
86&height=505&title=Prostate%20Nomogram recommended in the Prostate Specific Antigen 
Working Group Guidelines.  The PSADT is calculated assuming an exponential increase in 
serum PSA and first order kinetics.  The formula takes into account the natural logarithm of 2 
divided by the slope obtained from fitting a linear regression of the natural log of PSA on time.  
All PSA values used in the calculation should be 0.20 ng/ml or greater and follow an increasing 
trend.  PSA values need not be consecutively increasing and all values obtained during a 
maximum period of 12 months prior to a given protocol time point will be included in the 
calculation.  The maximum period of the last 12 months is recommended to reflect current 
disease activity because in some men PSADT may change over time. Multiple studies concur 
that men with a PSADT < 3 months are at extremely high risk for metastatic disease progression 

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and death with prostate cancer mortality approaching 100%; for this reason they have been 
excluded from participation in this study. In contrast, men with a slow PSADT i.e. > 15 months 
have an extremely low risk of death from prostate cancer. 
The following general guidelines39 regarding PSA determinations and calculation of PSADT 
used in this protocol will be adhered to (refer to Appendix 5, PSADT Calculation Guidelines) 
 
Pre-Enrollment/Baseline and On Study PSADT: 
➢ PSADT will be calculated using the Memorial Sloan-Kettering Cancer Center cancer 
information prostate nomogram for PSA doubling time found at: 
http://www.mskcc.org/mskcc/shared/forms/Nomograms/flash/load.cfm?type=Prostate&w
idth=586&height=505&title=Prostate%20Nomogram 
➢ Minimum requirements for PSADT include > 3 PSA measurements over > 3 months. 
➢ The interval between PSA measurements must be > 4 weeks. 
➢ For patients receiving 5-alpha reductase inhibitors (5ARI) e.g. finasteride or dutasteride, 
only PSA values obtained after at least 3 months on therapy may be used to calculate 
PSADT. 
➢ Calculated PSADT values will be reported in months 
➢ The same laboratory must have performed PSA values used in the calculation of PSADT. 
➢ All PSA values used in the calculation should be > 0.20 ng/mL and follow a rising trend 
although all values need not be consecutively rising. 
➢ All values obtained over a maximum period of 12 months prior to any given protocol 
time point assessment should be included in the PSADT calculation to reflect the 
patient’s current disease activity. 
➢ PSA values obtained within < 2 weeks of instrumentation, documented infection 
(prostatitis), or radiation proctopathy should be omitted from inclusion in PSADT 
calculations. 
On Study PSADT will be calculated at baseline and weeks 12, 24, 36, 48 60, 72, 84, 96, 108, 
120, 132, and 144. 
Exploratory comparisons of percent change in PSADT will be done among the following time 
points: 
➢ Pre-Enrollment/Baseline PSADT vs. Week 3-12 PSADT/ 
➢ Pre-Enrollment/Baseline PSADT vs. Week 24 PSADT 
➢ Pre-Enrollment/Baseline PSADT vs. Week 36 PSADT 
➢ Pre-Enrollment/Baseline PSADT vs. Week 48 PSADT 
➢ Week 24 PSADT vs. Week 36 PSADT 
➢ Week 36 PSADT vs. Week 48 PSADT 
➢ Additional exploratory analyses of PSADT will be performed of Pre-Enrollment/Baseline 
PSADT vs. Weeks 60, 72 and 96 and Week 48 PSADT vs. Weeks 96, 108 and 144 
PSADT. 
 
5.2.2.1 PSADT Response & Failure  
(assessed in all patients at weeks 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144.) 
➢ PSADT response is defined as: 
o a > 50% increase in calculated PSADT OR 

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o a PSADT > 15 months 
➢ Patient’s whose PSADT is decreased by > 50% will be considered PSADT failures. 
➢ Week 24 PSADT responders will be allowed to receive an additional dose of TARP 
peptide vaccine at Week 36. 
5.3 
TOXICITY CRITERIA 
This study will utilize NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
3.0, published June 10, 2003, for toxicity and adverse event monitoring and reporting.  (Refer to 
http://ctep.info.nih.gov/reporting/ctc_v30.html). All clinical treatment areas will have access to a 
copy of CTCAE version 3.0. 
6 
STATISTICAL CONSIDERATIONS   
6.1 
OVERALL STATEMENT OF OBJECTIVES 
The primary objective of this study, on which the sample size will be based is to determine the 
proportion of patients who will generate a specific immune response following vaccination as 
tested in tetramer, ELISPOT and cytotoxicity assay, and to evaluate if this is adequate for future 
consideration. Determination of safety of the vaccination is also an important endpoint.  It is also 
of interest to assess risk of progression among patients with metastatic Stage D0 prostate cancer 
naïve to androgen deprivation therapy vaccinated with these peptides using PSA doubling time 
as a surrogate marker of this risk. 
6.2 
PRIMARY OBJECTIVE AND STATISTICAL JUSTIFICATION FOR SAMPLE SIZE 
This study will be conducted as a prospective, randomized two arm, pilot trial to explore the 
utility of TARP peptide vaccination alone or vaccination with TARP peptide-pulsed dendritic 
cells in patients with Stage D0 prostate cancer.  
 
The primary objective is to obtain a reliable estimate of the immunologic response rate to 
vaccination with either peptide vaccine or dendritic cells in patients with biochemical 
progression and to determine if the rate of immunologic response in either arm is adequate to 
warrant further investigation in subsequent studies.  Evaluation of safety and toxicity will also be 
a primary goal. The study is not designed to have adequate power to identify if one arm is 
associated with a significantly improved immunologic response rate as compared to the other, as 
such detection may require very large numbers of patients given the potential similarity of the 
arms. Furthermore, there is no requirement that only one arm be selected for further 
development. Should both arms demonstrate adequate immunologic responses, then future 
studies will consider either or both arms depending on immune responses, feasibility, toxicity 
and other outcomes such as PSADT. 
 
Patients will initially be screened for HLA-A*0201 positivity.  HLA-A*0201 patients will then 
undergo comprehensive protocol eligibility screening and if found to be eligible, will be enrolled 
in the study.  Upon enrollment patients will be randomized in a 1:1 manner to receive TAPR 
peptide vaccination with Montanide ISA51 VG plus GM-CSF (Arm A) or TARP peptide-
pulsed dendritic cells (Arm B).   
 

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For each arm, using an acceptable immunologic response rate of 40% (p1=0.40) and an 
unacceptable rate of 10% (P0=0.10) and using a two-stage optimal design (Simon R, Controlled 
Clinical Trials 10:1-10, 1989) with alpha = 0.05 and beta =0.10, we will initially accrue 9 
patients in each arm. An unacceptable response rate of 10% (or less) and an acceptable response 
rate of 40% were determined based on historical clinical trial response rates to vaccines and 
immune-based therapies for cancer.4-7  If 0-1 patients develop an immunologic response as 
defined in Section 5.2.1, then no further patients will be enrolled.  If 2 or more of these 9 patients 
develop an immunologic response, we will accrue 11 additional patients for a maximum total of 
20 patients per arm.  If 2-4 of 20 has an immunologic response, then the results from this arm 
will be considered inadequate for further investigation. If 5 or more of these 20 has an immune 
response, we will be able to conclude that the vaccine on the given arm is worthy of further 
development in this population on the basis of immune response. Under the null hypothesis 
(p0=0.20), the probability of early termination is 77%. 
 
There is no requirement that only one arm be selected for further development; should both arms 
demonstrate adequate immunologic responses, then future studies will consider either or both 
arms depending on immune responses, feasibility, toxicity and other outcomes such as PSADT. 
 
As stated in Section 3.3.2, an early stopping rule will be used to ensure that the study subjects do 
not have exposure to excessive toxicity: If more than one of the first nine subjects on a given arm 
experiences dose limiting toxicity (defined in Section 3.3.1), enrollment will cease for that arm 
and revisions to the treatment will be discussed with the IRB and FDA. This is because 1/9 has 
an upper one-sided 90% confidence interval limit of 37%, while 2/9 has an upper one-sided 90% 
confidence interval limit of 49%; the former would be marginally acceptable as a rate of toxicity 
while the latter is not. In addition, any unusually severe toxicity noted may result in temporary 
suspension of accrual pending re-evaluation of the agent. All toxicities noted will be tabulated by 
type and grade, for each arm. Any such toxicities that appear in at least 5 patients on either arm 
will also have the distribution of grades of that toxicity compared between the arms using an 
exact Cochran-Armitage test.  
 
The percent of patients who meet PSADT response criteria as outlined in section 5.2.2 will also 
be evaluated as a secondary endpoint.  In addition, exploratory comparisons of PSADT among 
study time points will be performed as outlined in Sections 3.1 and 5.2.2.  Given that there will 
be 6 separate comparisons of PSADT, we will only report differences with p<0.01 as being 
statistically significant while other differences with 0.01 < p <0.05 will be considered to be 
trends. Appropriate 95% confidence intervals will be constructed and reported which takes into 
consideration the two-arm nature of the design.  No formal comparison will be undertaken since 
the study does not have adequate power to address this comparison properly. 
 
It is expected that 1-2 patients per month may be enrolled onto this study. Based on accrual of 
18-20 patients per year, accrual should be completed in approximately 2 to 2.5 years if all 40 
patients were to be required. To allow for the possibility of inevaluable patients, an accrual 
ceiling of 43 will be used. 

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7 
SAFETY REPORTING REQUIREMENTS/DATA AND SAFETY MONITORING 
PLAN 
At each study visit, patients will be evaluated for new or evolving AEs.  Symptoms will be 
elicited through the use of open-ended questions, followed by appropriate questions that clarify 
the patient’s verbatim description of AEs or changes in concomitant medications.  A review of 
systems will be performed.   
 
The Principal Investigator or Lead Associate Investigator will assess whether the AE or SAE is 
associated with the administration of TARP peptide vaccine:   
➢ Unrelated, Unlikely, Possible, Probably, Definite 
 
7.1 
DEFINITIONS 
7.1.1 Adverse Event 
Any untoward medical occurrence in a human subject, including any abnormal sign (for 
example, abnormal physical exam or laboratory finding), symptom, or disease, temporally 
associated with the subject’s participation in research, whether or not considered related to the 
subject’s participation in the research 
• 
If any abnormal laboratory result is considered clinically significant, the investigator will 
provide details about the action taken with respect to the test drug and about the patient’s 
outcome. 
 
7.1.2 Suspected adverse reaction 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
 
7.1.3 Unexpected adverse reaction 
An adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in 
the investigator brochure or is not listed at the specificity or severity that has been observed; or, 
if an investigator brochure is not required or available, is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current application. 
"Unexpected”, also refers to adverse events or suspected adverse reactions that are mentioned in 
the investigator brochure as occurring with a class of drugs or as anticipated from the 
pharmacological properties of the drug, but are not specifically mentioned as occurring with the 
particular drug under investigation. 
7.1.4 Serious  
An Unanticipated Problem or Protocol Deviation is serious if it meets the definition of a Serious 
Adverse Event or if it compromises the safety, welfare or rights of subjects or others. 

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7.1.5 Serious Adverse Event 
An adverse event or suspected adverse reaction is considered serious if in the view of the 
investigator or the sponsor, it results in any of the following: 
• 
Death, 
• 
A life-threatening adverse drug experience 
• 
Inpatient hospitalization or prolongation of existing hospitalization 
• 
Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• 
A congenital anomaly/birth defect. 
• 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the patient or subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. 
7.1.6 Disability 
A substantial disruption of a person’s ability to conduct normal life functions. 
7.1.7 Life-threatening adverse drug experience 
Any adverse event or suspected adverse reaction that places the patient or subject, in the view of 
the investigator or sponsor, at immediate risk of death from the reaction as it occurred, i.e., it 
does not include a reaction that had it occurred in a more severe form, might have caused death. 
7.1.8 Protocol Deviation (NIH Definition) 
Any change, divergence, or departure from the IRB-approved research protocol. 
7.1.9 Non-Compliance (NIH Definition) 
The failure to comply with applicable NIH Human Research Protections Program (HRPP) 
policies, IRB requirements, or regulatory requirements for the protection of human research 
subjects. 
7.1.10  Unanticipated Problem 
Any incident, experience, or outcome that: 
•  Is unexpected in terms of nature, severity, or frequency in relation to  
(a) the research risks that are described in the IRB-approved research protocol and 
informed consent document; Investigator’s Brochure or other study documents, and 
(b) the characteristics of the subject population being studied; AND 
• Is related or possibly related to participation in the research; AND 
• Suggests that the research places subjects or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 

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7.2 
NIH INTRAMURAL IRB AND CLINICAL DIRECTOR REPORTING 
7.2.1 NIH Intramural IRB and NCI CD Expedited Reporting of Unanticipated Problems and 
Deaths 
The Protocol PI will report in the NIH Problem Form to the NIH Intramural IRB and NCI 
Clinical Director: 
• All deaths, except deaths due to progressive disease  
• All Protocol Deviations 
• All Unanticipated Problems 
• All non-compliance 
Reports must be received within 7 days of PI awareness via iRIS. 
7.2.2 NIH Intramural IRB Requirements for PI Reporting at Continuing Review 
The protocol PI will report to the NIH Intramural IRB:  
 
1. A summary of all protocol deviations in a tabular format to include the date the deviation 
occurred, a brief description of the deviation and any corrective action. 
2. A summary of any instances of non-compliance 
3. A tabular summary of the following adverse events: 
• 
All Grade 2 unexpected events that are possibly, probably or definitely related to 
the research;  
• 
All Grade 3 and 4 events that are possibly, probably or definitely related to the 
research;  
• 
All Grade 5 events regardless of attribution;  
• 
All Serious Events regardless of attribution.  
NOTE: Grade 1 events are not required to be reported.  
7.2.3 NIH intramural IRB Reporting of IND Safety Reports 
Only IND Safety Reports that meet the definition of an unanticipated problem will need to be 
reported to the NIH Intramural IRB. 
7.3 
IND SPONSOR REPORTING CRITERIA  
An investigator must immediately report to the sponsor, using the mandatory MedWatch form 
3500a, any serious adverse event, whether or not considered drug related, including those listed 
in the protocol or investigator brochure and must include an assessment of whether there is a 
reasonable possibility that the drug caused the event.  
• 
All Grade 5 (fatal) events (except death due to progressive disease) must be 
reported via email within 24 hours.  A complete report must be submitted within one 
business day.  
• 
All other serious adverse events including deaths due to progressive disease must 
be reported within one business day 

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Study endpoints that are serious adverse events (e.g. all-cause mortality) must be reported in 
accordance with the protocol unless there is evidence suggesting a causal relationship between 
the drug and the event (e.g. death from anaphylaxis).  In that case, the investigator must 
immediately report the death to the sponsor. 
Events will be submitted to Center for Cancer Research (CCR) at: CCRsafety@mail.nih.gov and 
to the CCR PI and study coordinator. 
7.4 
DATA AND SAFETY MONITORING PLAN 
7.4.1 Principal Investigator/Research Team  
The Principal Investigator and Lead Associate Investigator will monitor this pilot, prospective 
randomized study, as required by the FDA as part of the Sponsor Investigator IND held by the 
Principal Investigator.  Based on the minimal risk and the limited number of subjects to be 
studied, monitoring by a Data Safety Monitoring Board is not warranted for this trial.  There will 
be continuous close monitoring of protocol-related toxicity by the Lead Associate Investigator 
and Principal Investigator.  The study research nurse coordinator and/or data manager will 
generate a bi-weekly (or weekly) reports of all on-study laboratory data with correspondent 
toxicity grading according to CTCAE v3.0 as well as a summary of all clinical and Vaccine 
Report Card (VRC) surveillance adverse event data for review by the Principal Investigator and 
The Lead Associate Investigator in conjunction with Associate Investigators.  In addition, study 
summaries will be generated every 6 months and forwarded to the FDA.  Adverse events will be 
reported to the IRB as described in section 7.2. Decisions regarding revision of the protocol or 
consent will be promptly communicated to the IRB and FDA.      
7.4.2 Sponsor Monitoring Plan 
As a sponsor for clinical trials, FDA regulations require the CCR to maintain a monitoring 
program. The CCR’s program allows for confirmation of: study data, specifically data that could 
affect the interpretation of primary study endpoints; adherence to the protocol, regulations, and 
SOPs; and human subjects protection. This is done through independent verification of study 
data with source documentation focusing on: 
• Informed consent process 
• Eligibility confirmation 
• Drug administration and accountability 
• Adverse events monitoring 
• Response assessment. 
The monitoring program also extends to multi-site research when the CCR is the coordinating 
center. 
This trial will be monitored by personnel employed by an NCI contractor. Monitors are qualified 
by training and experience to monitor the progress of clinical trials. Personnel monitoring this 
study will not be affiliated in any way with the trial conduct. 

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8 
HUMAN SUBJECTS PROTECTION  
8.1 
 RATIONALE FOR SUBJECT SELECTION 
All subjects enrolled in this study will be male as the disease under study does not affect women 
or children.  We will make every effort to accrue patients from all racial and ethnic groups.  
Since our referral population is the nation, we should obtain referrals representative of the 
national composition of races.  In addition, HLA-A*0201 positivity is not restricted to a 
particular racial or ethnic group: it is present in 49.54% of Caucasians, 33.74% of African-
Americans, and 55.87% of Orientals. Hence the requirement for HLA-A*0201 positivity should 
not preclude our ability to accrue a racially and ethnically diverse population representative of 
individuals affected by prostate cancer.15  
Participants will be accrued through web-based advertisements, community-based contacts and 
referrals of patients from other studies at the Clinical Center.  This protocol will also be 
advertised on the NCI PDQ Clinical Trials database as well as through ClinicalTrials.gov.  
8.2 
EVALUATION OF BENEFITS AND RISKS/DISCOMFORTS 
The benefit of vaccination with peptides is unknown.  Although several patients in our previous 
Phase I peptide vaccine studies mounted immune responses to various peptides, the benefit or 
clinical significance of these immune responses remains unknown.  However, in animal models, 
immune responses have been correlated with both a decrease in measurable disease and a 
subsequent resistance to tumor challenge (Ref).  In this study, we will determine the impact of 
vaccination on PSADT, a well established surrogate marker that is a predictive factor of longer 
time to metastases, prostate-cancer death and all-cause adverse clinical outcomes.30-33  The risks 
of vaccination are also unknown.  However, no severe or systemic toxicity in response to 
vaccination with peptides alone has been observed and the most frequent adverse event is local 
injection site reactions, an observation common to all vaccines.   
8.3 
CONSENT AND ASSENT PROCESSES AND DOCUMENTATION 
Consent forms are attached.  Two consent forms will be used for this study.  Participants will 
initially agree to allow their blood to be tested for HLA-A*0201 positivity.  The process for 
obtaining patients’ consent for HLA typing may involve telephone contact only.  The study will 
be explained to the prospective patient and any questions answered.  The consent form will be 
signed and witnessed and returned with the blood sample for HLA typing to the NIH by FEDEX.  
If the patient is HLA-A*0201 positive they will be invited for a screening visit at the NCI to be 
evaluated for protocol eligibility. Following eligibility screening, each patient will be discussed 
with the Principal Investigator or the Lead Associate Investigator.  Eligible patients will be 
presented with a detailed description of the study protocol plan and treatment.  The specific 
requirements, objectives, risks, alternatives, time commitments and potential benefits will be 
reviewed with the patient.  A copy of the Informed Consent will be provided to the patient for 
their review.  The patient will be reassured that participation in this study is entirely voluntary 
and that they may withdraw or decide against receipt of vaccination at any time without adverse 
consequences.  The Principle Investigator, Lead Associate Investigator or their designee will 
summarize the screening results and answer any questions prior to obtaining Informed Consent. 

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9 
PHARMACEUTICAL INFORMATION 
9.1 
SARGRAMOSTIM 
9.1.1 Product Description  
The GM-CSF to be used in this study is glycosylated, recombinant human Granulocyte-
Macrophage Colony Stimulating Factor (rhu GM-CSF) (brand name Leukine).  This rhu GM-
CSF is an altered form of the native molecule: the position 23 arginine has been replaced with a 
leucine to facilitate expression of the protein in yeast (Saccharomyces cerevisiae) and the 
carbohydrate moiety may be different from the native protein.  The Clinical Center Pharmacy 
will purchase commercially available sargramostim. Rhu GM-CSF will be used both as an 
ancillary product for the maturation of dendritic cells and for administration to patients with 
peptide vaccination.  
9.1.2 Formulation and Preparations  
A.) Lyophilized LEUKINE is a sterile, white, preservative-free powder (250 mcg) that 
requires reconstitution with 1 mL Sterile Water for Injection, USP or 1 mL Bacteriostatic 
Water for Injection, USP.  Reconstituted lyophilized LEUKINE is a clear, colorless 
liquid suitable for subcutaneous injection or intravenous infusion. The vial of lyophilized 
LEUKINE contains 250 mcg (1.4 x 106 IU/vial) sargramostim, 40 mg/mL mannitol, 
USP; 10 mg/mL sucrose, NF; and 1.2 mg/mL tromethamine, USP, as excipients. 
Biological potency is expressed in International Units (IU) as tested against the WHO 
First International Reference Standard. The specific activity of LEUKINE is 
approximately 5.6 x 106 IU/mg. 
B.) Sargramostim solution is a sterile, preserved (1.1% benzyl alcohol) injectable 500 
mcg/ml solution.  
9.1.3 Stability and Storage   
A.) Lyophilized LEUKINE should be refrigerated at 2 to 8 C (36 to 46 F) and is stable for 
at least eighteen months. It should not be frozen, shaken or used beyond the expiration 
date printed on the vial.  LEUKINE reconstituted with 1.0 ml of Sterile Water for 
Injection, USP (without preservative) should be administered as soon as possible and 
within 6 hours following reconstitution.  Reconstituted solutions prepared with 
Bacteriostatic Water for Injection USP (0.9% benzyl alcohol) may be stored for up to 20 
days at 2 to 8 C prior to use. The contents of vials reconstituted with different diluents 
should not be mixed together. 
B.) Sargramostim solution: store intact vials in the refrigerator (2C-8C). Do not 
freeze or shake the vial. 
9.1.4 Administration Procedures  
A.) Lyophilized LEUKINE will be used in vitro by the Department of Transfusion Medicine 
for the generation of dendritic cells by the Department of Transfusion Medicine. 
B.) Sargramostim solution (110 mcg or 0.22ml) will be given with Montanide ISA 51with 
TARP peptides as described in Sections 3.2 Vaccine Administration and Sections 9.3.4 
and 9.4.4.  

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9.1.5 Incompatibilities:  
Refer to the package insert/PDR/Formulary.  
9.2 
INTERLEUKIN-4 CELLGENIX 
9.2.1 Product Description  
Interleukin-4 (IL-4) used in this study is investigational.  It is manufactured and supplied by 
CellGenix (Master File cross reference BB-MF 11269).  It will be used as an ancillary product to 
mature dendritic cells in vitro and will not be administered directly to patients. IL-4 exerts 
important effects on B cells, T cells, macrophages, eosinophils, hematopoietic progenitor cells, 
endothelial cells and promotes the maturation of dendritic cells.  The complementary DNA clone 
(cDNA), when expressed in E.coli yields a 129 amino acid protein with a molecular weight of 
14,957 daltons. IL-4 is a highly purified ( 95% chromatographically pure), sterile, water-
soluble protein. 
9.2.2 Formulation and Preparation  
RhIL-4 Sterile Powder for Injection is supplied in 100 mcg and 200 mcg vials (containing a total 
of 120mcg and 240mcg of IL-4, respectively) as a sterile lyophilized powder formulated with 
glycine, human serum albumin, citric acid, and sodium citrate. Un-reconstituted IL-4 should be 
kept refrigerated at 2-8C. 1.2 mL of Sterile Water for Injection USP should be added to each 
vial of rhIL-4 Sterile Powder for Injection. The vial should be gently agitated to completely 
dissolve the powder and should be inspected visually for discoloration and particulates prior to 
use. 
9.2.3 Stability and Storage  
The reconstituted product should be refrigerated at 2-8C and should be used within 24 hours. 
9.2.4 Administration Procedures  
To be used in dendritic cell culture, not administered directly to patients.  
9.2.5 Incompatibilities:  
None known in culture.  
 
9.3 
TARP 29-37-9V PEPTIDE (EPITOPE-ENHANCED) NSC #740704 
9.3.1 Product Description  
TARP 29-37-9V is investigational. TARP 29-37-9V is a synthetic HLA-A2-restricted 9-amino 
acid epitope of the tumor associated protein TARP, with a single amino acid substitution (valine 
at position 37, instead of leucine) to increase its binding affinity and immunogenicity.    Amino 
acid sequence: Phenylalanine-Leucine-Arginine-Asparagine-Phenylalanine-Serine-Leucine-
Methionine-Valine (FLRNFSLMV) 
Molecular Weight:  1126.4 
9.3.2 Formulation and Preparation 
The peptide is manufactured by NeoMPS, Inc., 9395 Cabot Drive, San Diego, CA 92126. The 
peptide is vialed by the Clinical Center Pharmacy as a 5 mL siliconized sterile amber type 1 
glass vial with a Teflon-lined stopper containing 0.5 mL of a sterile clear solution.  Each mL 

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contains 2.2 mg of TARP:29-37(37V) Peptide and 0.5 mcL of trifluoroacetate 0.05% v/v. 
9.3.3 Stability and Storage  
Store the finished injectable dosage forms in the freezer (-70˚C) for long-term storage. Intact 
vials are stable for at least 6 months when stored at controlled room temperature (15ºC – 30ºC), 
at least 9 months when stored in the refrigerator (2ºC – 8ºC), and for at least 36 months when 
stored in the freezer (-10ºC to -25ºC and -70ºC). The peptide vial contains no preservatives; once 
the peptide vial is entered, discard unused peptide solution after 3 hours.  Vaccine emulsions 
consisting of peptide admixed with Montanide ISA 51 VG are stable for up to 3 hours, but 
should be administered as soon as possible following preparation. 
9.3.4 Administration Procedures   
Preparation and administration of Epitope-Enhanced TARP with Montanide® ISA 51 VG and 
sargramostim: Each vial of TARP 29-37-9V peptide is in aqueous solution containing 0.5ml of 
peptide at a concentration of 2.2 mg/ml, for a total of 1.1mg peptide per vial. Sargramostim 
solution, 110 micrograms, (0.22 mL) and 0.1 mL of 10x saline will be added to the vial of 
peptide for a total aqueous peptide volume of 0.82 ml.  Prepare the peptide emulsion by injecting 
the 0.82ml aqueous TARP 29-37-9V peptide solution into 0.8 mL of Montanide ISA 51 VG as 
outlined in Section 9.6.6 Syringe Method of Emulsification.  The total volume of peptide 
emulsion will be 1.62 ml.  Withdraw a total of 1.5 mL (~1.0 mg of peptide) for administration by 
deep subcutaneous injection in one or two equal volumes into the anterior thigh.   
Administration:  Injections will preferentially be administered in the anterior thigh. The upper 
arm may be used if necessary. The same extremity will be used for each dose of peptide 
administered on any given day but the extremities may be rotated. 
9.3.5 Incompatibilities   
None known.    
9.3.6 Reported Adverse Events and Potential Risks  
Common adverse events reported with peptides emulsified with Montanide® ISA 51 VG have 
consisted of fatigue, fever, flushing, injection site reactions including pain, erythema and 
induration; localized swelling, rare granuloma formation.  Other reported possible adverse events 
include chills/rigors, transient increases in liver transaminases, headache, rash, and pruritis. 
TARP is a 58 amino acid protein found on cells of prostate origin and is over expressed in over 
90% of patients with prostate cancer and about half of patients with breast cancer. Because the 
peptide mimics portions of a prostate protein found naturally in the body, there is a chance for 
development of an autoimmune reaction to it and may result in the possible development of 
inflammation in the prostate gland.   
9.3.7 Special Handling  
The peptide is NOT a cytotoxic or infectious agent and requires no special handling. 
 
9.4 
TARP 27-35 (WILD TYPE) NSC#740703 
9.4.1 Product Description  
TARP 27-35 is a synthetic HLA-A2-restricted 9-amino acid epitope of the tumor-associated 
protein TARP. 

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Amino acid sequence: Phenylalanine-Valine-Phenylalanine-Leucine-Arginine-Asparagine-
Phenylalanine-Serine-Leucine (FVFLRNFSL) 
Molecular Weight:  1142.4 
9.4.2 Formulation and Preparation  
The peptide is manufactured by NeoMPS, Inc., 9395 Cabot Drive, San Diego, CA 92126. The 
peptide is vialed by the Clinical Center Pharmacy as a 5 mL siliconized sterile amber molded 
glass vial containing a sterile white lyophilized powder.  Each vial contains 1.1 mg of TARP:27-
35 peptide and Mannitol. 
9.4.3 Stability and Storage  
Store the finished injectable dosage forms in the freezer (-70˚C) for long-term storage.  Intact 
vials are stable for at least 6 months when stored at controlled room temperature (15ºC – 30ºC) 
or in the refrigerator (2ºC – 8ºC), and for at least 36 months when stored in the freezer (-10ºC to 
-25ºC and -70ºC). The peptide vial contains no preservatives; once the peptide vial is entered, 
discard unused peptide solution after 3 hours.  Vaccine emulsions consisting of peptide admixed 
with Montanide ISA 51 are stable for up to 3 hours, but should be administered as soon as 
possible following preparation. 
9.4.4 Administration Procedures 
Preparation and administration of WT TARP with Montanide® ISA 51 VG and sargramostim: 
Each vial of WT TARP 27-35 peptide contains 1.1 mg as a lyophilized powder.  It is 
reconstituted  in 0.5 mL of Sterile Water for Injection, USP to yield a final concentration of 2.2 
mg/mL. Sargramostim solution, 110 micrograms, (0.22 mL) and 0.1 mL of 10x saline will be 
added to the vial of peptide for a total aqueous peptide volume of 0.82 ml.  Prepare the peptide 
emulsion by injecting the 0.82ml aqueous WT TARP 27-35 peptide solution into 0.8 mL of 
Montanide ISA 51 as outlined in Section 9.6.6 Syringe Method of Emulsification. The total 
volume of peptide emulsion will be 1.62 ml.  Withdraw a total of 1.5 mL (~1.0 mg of peptide) 
for administration by deep subcutaneous injection in one or two equal volumes into the anterior 
thigh.   
Administration:  Injections will preferentially be administered in the anterior thigh. The upper 
arm may be used if necessary. The same extremity will be used for each dose of peptide 
administered on any given day but the extremities may be rotated. 
9.4.5 Incompatibilities  
None known.    
9.4.6 Reported Adverse Events and Potential Risks  
Common adverse events reported with peptides emulsified with Montanide® ISA 51 VG have 
consisted of fatigue, fever, flushing, injection site reactions including pain, erythema and 
induration; localized swelling, rare granuloma formation.  Other reported possible adverse events 
include chills/rigors, transient increases in liver transaminases, headache, rash, and pruritis. 
TARP is a 58 amino acid protein found on cells of prostate origin and is over expressed in over 
90% of patients with prostate cancer and about half of patients with breast cancer. Because the 
peptide mimics portions of a prostate protein found naturally in the body, there is a chance for 
development of an autoimmune reaction to it and may result in the possible development of 
inflammation in the prostate gland.   

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9.4.7 Special Handling 
The peptide is NOT a cytotoxic or infectious agent and requires no special handling. 
9.5 
INFLUENZA VIRUS VACCINE LIVE INTRANASAL 
9.5.1 Product Description  
The influenza virus vaccine live intranasal (FluMist )is a live trivalent nasally administered 
vaccine intended for active immunization for the prevention of influenza.  The product is 
manufactured by MedImmune and will be purchased by the Clinical Center Pharmacy.  The 
previous season’s vaccine might be used until the supply for the current influenza season 
becomes available.  
9.5.2 How Supplied  
This vaccine contains live attenuated, cold adapted virus from both influenza A and B. Each 0.2 
mL dose is formulated to contain 10
7.0+0.5 TCID50 (median tissue culture infectious dose) of live 
attenuated influenza virus reassortants of the strains recommended by the U.S. Public Health 
Service (USPHS).     
9.5.3 Stability and Storage  
Influenza virus vaccine live intranasal can be stored for extended periods at 2-8 ºC.  Influenza 
virus vaccine live intranasal is a clear to slightly cloudy, colorless to pale yellow liquid; some 
proteinaceous particulates may be present.  The thawed vaccine should not be refrozen. 
9.5.4 Administration Procedures  
Patients should receive 0.2 ml intranasally of influenza virus vaccine (0.1ml in each nostril) 
during vaccination. Under no circumstances should the vaccine be administered parenterally.  If 
the vaccinee sneezes after administration of the intranasal vaccine, the dose should not be 
repeated. Influenza virus vaccine live intranasal is contraindicated in patients with known 
hypersensitivity to any component of the vaccine, including egg or egg products, individuals 
with a history of Guillan-Barre Syndrome, a known or suspected immunodeficiency 
disease/condition and individuals with asthma or reactive airway disease or other chronic 
pulmonary, cardiovascular or metabolic conditions. Vaccine administration should be postponed 
for a least 72 hours after the acute phase of a febrile and /or respiratory illness. 
9.5.5 Special Precautions  
Because live attenuated influenza viruses replicate in the nasopharynx of recipients of influenza 
virus vaccine live intranasal and can be shed in respiratory secretions, the possibility exists that 
the vaccine viruses can be transmitted to susceptible individuals who have not received the 
vaccine.  The manufacturer states that individuals receiving influenza virus vaccine live 
intranasal should avoid close contact with immunocompromised individuals for at least 21 days 
after vaccination.  To minimize risk of transmission of attenuated vaccine virus to other Clinical 
Center patients the following procedures will be adhered to: 
➢ influenza virus vaccine live intranasal will be administered immediately prior to the 
patient’s departure from the Clinical Center. 
➢ The patient will be asked to wear a mask in the process of exiting the building and 
will be immediately discharged to home. 

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➢ Transportation by private taxi to the airport will be authorized to eliminate exposure 
to other CC patients traveling on the NIH shuttle.  
➢ Patients will be provided with a hand hygiene product as well as tissues. 
➢ Patients will not be authorized to return to the CC until at least 21 days following 
receipt of their vaccine.   
➢ While away from the CC they will be advised to avoid contact with severely 
immunosuppressed patients for 7 days after vaccination. 
9.6 
MONTANIDE® ISA 51 VG (NSC 737063) CLASSIFICATION:  ADJUVANT 
9.6.1 Product Description   
Montanide® ISA 51 VG (vegetable-grade) is an oil-based adjuvant composed of a highly refined 
mannide monooleate (Montanide 80), a surfactant, in a mineral oil solution (Drakeol 6VR). The 
mannide monooleate is synthesized from raw materials of vegetable origin.  Montanide® ISA 51 
VG, when mixed with an equal part of an aqueous solution, is designed to form an injectable 
water-in-oil emulsion. Comparative analysis of the chemical compositions of the previous 
animal-grade formulation (Montanide® ISA 51) and current vegetable-grade formulation 
(Montanide® ISA 51 VG) oleic acid sources shows a final product with similar chemical and 
applicative properties (e.g., emulsion stability and viscosity).  
9.6.2 How Supplied   
Montanide® ISA 51 VG is supplied in 5 mL amber glass vials containing 3 mL of a sterile, clear 
yellow, oil solution.  The agent is manufactured by Seppic, Inc. and will be supplied through the 
Pharmaceutical Development Service, Clinical Center Pharmacy Department. 
9.6.3 Storage   
Store intact vials at controlled room temperature. 
9.6.4 Stability  
Montanide® ISA 51 VG is stable at room temperature until the expiration date printed on the 
agent label. The solution contains no preservatives; once the vial is entered, discard any 
remaining solution after 3 hours.  Withdraw and administer peptide vaccine emulsions 
immediately after preparation. 
9.6.5 Toxicity   
Toxicities related to Montanide® ISA 51 VG have included erythema at the injection site, rare 
granuloma formation and low-grade fever.   
9.6.6 Peptide Vaccine Preparation   
To prepare a water-in-oil emulsion vaccine, consisting of the peptide (aqueous phase) and the 
adjuvant Montanide ISA 51 VG (oil phase), combine equal parts of the peptide and the 
adjuvant and subject the mixture to high sheer forces using the following method: 
 
Syringe Method of Emulsification (Investigator’s Preference): Arm A Patients 101-110 
For preparation of emulsions of water-soluble peptide antigens in an adjuvant oil phase for 
immunization a modification of the "double hubbed needle method" will be employed.  In this 
method two sterile 2.5 ml glass Hamilton syringes (TLL (PTFE Luer Lock) w/o slots #81420, 
Hamilton Co., Reno,Nev) with Teflon plungers and male Luer Lock tip connectors are used.  

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The materials are kept sterile at all times.  The adjuvant, Montanide® ISA 51 VG (0.8 ml) is 
drawn up completely in one syringe using a sterile 23 gauge needle.  The needle is removed and 
discarded and any air bubbles displaced. A sterile double ended female Teflon connector (3/4 
inch, 24 gauge, #86510, Hamilton Co., Reno,Nev) is attached to the adjuvant syringe and the 
plunger is pushed carefully to allow the adjuvant to fill the connector space. The peptide aqueous 
solution (0.82 ml total) is then drawn up into the other glass syringe with a sterile 23 gauge 
needle.  The needle is discarded and any air bubbles displaced.  The syringe containing the 
aqueous peptide solution is securely attached to the free female end of the Teflon connector.   
 
It is very important that the first pass be aqueous peptide antigen into oil adjuvant to 
produce the correct water-in oil-emulsion.  A water-in-oil emulsion is obtained by briskly 
forcing the aqueous peptide solution through the connector into the adjuvant and passing the 
material back and forth between the two syringes 40-50 times. This is best achieved by holding 
the connector between thumb and forefinger of one hand with the syringe held securely in the 
palm of the hand and the other syringe held securely in the palm of the other hand and firmly 
pressing the plunger of the syringe on a non-slippery surface or blue diaper. The material is 
passed back and forth between the two syringes by a rocking motion in which the plungers are 
alternately pushed in and out by forced pressure of the plunger on the bench top. As the emulsion 
forms, increased resistance should be noted, the emulsion should appear homogeneous (opaque 
white) with no trapped air bubbles. Following emulsification, almost the entire emulsion (1.5 ml) 
should be drawn up in one syringe, detached from the Teflon connecter assembly and a sterile 23 
gauge needle attached to the syringe. Gently push the emulsion into the needle until a small drop 
is visible at the tip.  The peptide-adjuvant emulsion is now ready for immunization. 
 
Modified Syringe Method of Emulsification: Arm A Patients 111-120 
For preparation of emulsions of water-soluble peptide antigens in an adjuvant oil phase for 
immunization, a modification of the "double hubbed needle method" will be employed.  In this 
method two sterile 3 ml Norm-Ject plastic Luer Lock tip syringes (manufactured by Henke Sass 
Wolf in Tuttlingen, Germany) are used.  The materials are kept sterile at all times.  The adjuvant, 
Montanide® ISA 51 VG (0.8 ml) is drawn up completely in one syringe using a sterile 23 gauge 
needle.  The needle is removed and discarded and any air bubbles displaced. A sterile Discofix, 
3-way stopcock (manufactured by B. Bruan Medical, Inc., Bethlehem, PA) is attached to the 
adjuvant syringe and the plunger is pushed carefully to allow the adjuvant to fill the connector 
space. The peptide aqueous solution (0.82 ml total) is then drawn up into the other plastic 
syringe with a sterile 23 gauge needle.  The needle is discarded and any air bubbles displaced.  
The syringe containing the aqueous peptide solution is securely attached to the opposite end of 
the 3-way stopcock.   
 
It is very important that the first pass be aqueous peptide antigen into oil adjuvant to 
produce the correct water-in oil-emulsion.  Peptide will then be emulsified as follows: 
➢ 20 “pre-emulsion cycles” will be performed (a cycle is defined as one bass back and 
forth between the two syringes).  The pre-emulsion cycles are to be performed slowly, 
such that 20 cycles are performed over approximately 1 minute.  The pre-emulsion 
cycles are a necessary and important step in the quality of the final emulsion. 
➢ Following the pre-emulsion cycles, 100 additional cycles will be performed such that 
the additional 100 cycles are performed over approximately 1 minute. 

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➢ Withdraw a total of 1.5 ml (~1.0mg of peptide) for administration into one of the 
syringes, disconnect from the 3-way stopcock and cap the syringe. 
➢ Label the syringe with the CRIS label (a “butterfly” label may be used). 
9.6.7 Administration Procedures   
Divide peptide vaccine emulsion dose volumes of greater than 1.5 mL into two or more 
injections. Administer peptide vaccine emulsions prepared with Montanide® ISA 51 VG by deep 
subcutaneous injection using a 23 gauge needle.  Injections will be preferentially given into the 
anterior thigh but the upper arm may be used if necessary.  The same extremity will be used for 
each dose of peptide administered on any given day but the extremities may be rotated.   
9.7 
KLH (KEYHOLE LIMPET HEMOCYANIN) 
9.7.1 Product Description  
Stellar Biotechnology’s KLH is a potent form of clinical grade KLH that is manufactured by 
Sigma-Aldrich. It is purified from the hemocyanin of the giant keyhole limpet, Megathura 
crenulata. The denatured subunit of KLH is a glycoprotein with a molecular weight of 400-
450,000 daltons. The native form of KLH is a dodecamer, which consists of twenty (20) subunits 
of KLH with a molecular weight of 6-9000.000 daltons. In the hemocyanin, at least 50% of the 
KLH exists as a dodecamer and the remainder can be found as dodecamer aggregates. Stellar 
Biotechnology’s KLH is purified as native molecules with high molecular weight and designated 
as KLH-HMW. 
9.7.2 Formulation and Preparation  
Stellar Biotechnology’s KLH is provided in soluble form in a buffer solution that is composed of 
10mM sodium phosphate, 135mM NaCl, 1mM CaCl2 and 0.5mM MgCl2. It is provided by the 
manufacturer in 600mg containers at 5mg/mL. It has been received by the Clinical Center 
Pharmacy Department and re-vialed into single use vials at 2mg/mL, 250 µcL/vial. 
9.7.3 Drug Procurement  
Stellar Biotechnology’s KLH will be purchased from Stellar Biotechnology and then vialed by 
Clinical Center Pharmacy Development Service (PDS). It will be dispensed by PDS to DTM for 
use in dendritic cell culture.  
9.7.4 Stability and Storage  
KLH-HMW is stable for at least 12 months when stored at 2 to 8C. 
9.7.5 Administration Procedures  
KLH-HMW will be used in vitro by the Department of Transfusion Medicine at a concentration 
of 10mcg/mL for the generation of dendritic cells. Cells will be extensively washed before 
administration. 
9.7.6 Incompatibilities  
Refer to the package insert.  

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(27) Wolfgang CD, Essand M, Lee B, Pastan I. T cell receptor gamma chain alternate reading 
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Cancer Res. 2004;64:2610-2618. 
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(30) Steinman RM. The dendritic cell system and its role in immunogenicity. Ann Rev 
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(31) Gabrilovich DI, Chen HL, Girgis KR et al. Production of vascular endothelial growth 
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(32) Gabrilovich DI, Nadaf S, Corak J, Berzofsky JA, Carbone DP. Dendritic cells in antitumor 
immune responses. II. Dendritic cells grown from bone marrow precursors, but not mature 
DC from tumor-bearing mice, are effective antigen carriers in the therapy of established 
tumors. Cell Immunol. 1996;170:111-119. 
(33) Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human 
dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus 
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1994;179:1109-1118. 
(34) Romani N, Gruner S, Brang D et al. Proliferating dendritic cell progenitors in human 
blood. J Exp Med. 1994;180:83-93. 
(35) Freudenthal PS, Steinman RM. The distinct surface of human blood dendritic cells, as 
observed after an improved isolation method. Proc Natl Acad Sci U S A. 1990;87:7698-
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(36) Labeur MS, Roters B, Pers B et al. Generation of tumor immunity by bone marrow-derived 
dendritic cells correlates with dendritic cell maturation stage. J Immunol. 1999;162:168-
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(37) Morse MA, Lyerly HK, Gilboa E, Thomas E, Nair SK. Optimization of the sequence of 
antigen loading and CD40-ligand-induced maturation of dendritic cells. Cancer Res. 
1998;58:2965-2968. 
(38) MedImmune Vaccines. FluMist (influenza virus vaccine live, intranasal) prescribing 
information. Gaithersburg, MD.  September 2007. 
(39) Arlen PM, Bianco F, Dahut WL et al.  Prostate-Specific Antigen Working Group's 
Guidelines on PSA Doubling Time. J Urol 2008;179:2181-86. 
(40) Loberg RD, Fielhauer JR, Pienta BA et al.  Prostate-specific antigen doubling time and 
survival in patients with advanced metastatic prostate cancer.  Urology 2003;62 Suppl 
1:128-133. 

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(41) Semeniuk RC, Venner PM and North S.  Prostate-specific antigen doubling time is 
associated with survival in men with hormone-refractor prostate cancer.  Urology 
2006;68:565-569. 
(42) Freedland SJ, Humphreys EB, Mangold LA, et al. Death in patients with recurrent prostate 
cancer after radical prostatectomy: prostate -specific antigen doubling time subgroups and 
their associated contributions to all-cause mortality.  J Clin Oncol 2007:25:1765-1771.  
 

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11 STUDY APPENDICES 
Appendix 1 
Schedule of Study Clinical, Laboratory and Radiographic Evaluations .............. 45 
Appendix 2 
Standard Operating Procedure for Dendritic Cell Vaccine Preparation ............... 47 
Appendix 3 
Handling and Processing of Research Specimens ................................................ 49 
Appendix 4 
Description of Immune Response Assays ............................................................. 51 
Appendix 5: 
Guidelines and Worksheet for Calculations of PSADT ....................................... 55 
Appendix 6: 
TARP Peptide Vaccination Report Card .............................................................. 57 
Appendix 7:  
 Exploratory Correlative Studies ................................................................... 58 
 

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Appendix 1 
Schedule of Study Clinical, Laboratory and Radiographic Evaluations 
 
 
Schedule study Events Weeks 60-144 
 
Wk 60 
Wk 72 
 Wk 84 
Wk 96 
Wk 108 
Wk 120 
Wk 132 
Wk 144 
HLA-A*0201 Testing 
Prior to Screening 
 
 
 
 
 
 
 
 
Informed Consent 
 
 
 
 
 
 
 
 
Medical History & ROS 
X 
X 
X 
X 
X 
X 
X 
X 
Schedule study Events Weeks 0-48 
 
Screen 
D-14 to D-1 
Wk 0  
D1 
Wk 0 
D3 or D5 
Wk 
3 
Wk 
6 
Wk 
9 
Wk 
12 
Wk 
15 
Wk 
18 
Wk 
24 
Wk 
36 
Wk48 
HLA-A*0201 
Testing 
Prior to Screening 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical History & 
ROS 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical 
Examination 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status      
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Height 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Weight 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Apheresis 
 
 
X 
 
 
 
 
 
 
X 
 
X 
FluMist Flu 
Vaccine 
 
 
X 
 
 
 
 
 
 
 
 
 
TARP Peptide 
Vaccine 
 
 
 
X 
X 
X 
X 
X 
 
 
X* 
X 
Vaccine Report Card  
 
 
 
X 
X 
X 
X 
X 
 
 
X* 
X 
CBC/differential, 
plts 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PT / PTT 
X 
 
 
 
 
 
 
 
 
 
 
 
Acute Panel 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Hepatic Panel 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Mineral Panel 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lipid Panel 
X 
 
 
 
 
 
 
 
 
 
 
X 
Amylase / Lipase 
X 
 
 
 
 
 
 
 
 
 
 
X 
Vitamin D, 25 
hydroxy 
X 
 
 
 
 
 
 
 
 
 
 
 
PSA 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
PSADT Calculation 
X 
 
 
 
 
 
X 
 
 
X 
X 
X 
Serum Testosterone 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
X 
 
 
 
 
 
X 
 
 
X 
 
X 
HIV Serology 
X 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis B Serology 
X 
 
 
 
 
 
 
 
 
 
 
 
Hepatitis C Serology 
X 
 
 
 
 
 
 
 
 
 
 
 
HTLV-1 Serology 
X 
 
 
 
 
 
 
 
 
 
 
 
VDRL 
X 
 
 
 
 
 
 
 
 
 
 
 
EKG 
X 
 
 
 
 
 
 
 
 
 
 
 
CT Scan 
Chest/Abd/Pelvis 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Bone Scan 
X 
 
 
 
 
 
 
 
 
 
 
X 
Lymphocyte Subsets 
X 
 
 
 
 
 
X 
 
 
X 
 
X 
Anti-TARP 
Antibody 
 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
NKT Cell 
Assessment 
 
X 
 
 
 
 
X 
 
X 
X 
 
 
Tetramer Staining 
 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
IFN- ELISPOT 
 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
51Cr CTL Assay# 
 
X 
 
 
 
 
X 
 
X 
X 
X 
X 
PBMCs for Storage 
 
X 
 
X 
 
 
X 
 
X 
X 
X 
X 
Serum for Storage 
 
X 
 
 
 
 
X 
 
X 
X 
X 
X 

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Physical Examination 
X 
X 
X 
X 
X 
X 
X 
X 
Performance Status                                
X 
X 
X 
X 
X 
X 
X 
X 
Height 
X 
X 
X 
X 
X 
X 
X 
X 
Weight 
X 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
Apheresis 
 
 
 
 
 
 
 
 
FluMist Flu Vaccine 
 
 
 
 
 
 
 
 
TARP Peptide Vaccine 
 
 
 
X 
 
 
 
 
Vaccine Report Card  
 
 
 
X 
 
 
 
 
CBC/differential, plts 
X 
X 
X 
X 
X 
X 
X 
X 
PT / PTT 
 
 
 
 
 
 
 
 
Acute Panel 
X 
X 
X 
X 
X 
X 
X 
X 
Hepatic Panel 
X 
X 
X 
X 
X 
X 
X 
X 
Mineral Panel 
X 
X 
X 
X 
X 
X 
X 
X 
Lipid Panel 
 
 
 
X 
 
 
 
X 
Amylase / Lipase 
 
 
 
X 
 
 
 
X 
Vitamin D, 25 hydroxy 
 
 
 
 
 
 
 
 
PSA 
X 
X 
X 
X 
X 
X 
X 
X 
PSADT Calculation 
X 
X 
X 
X 
X 
X 
X 
X 
Serum Testosterone 
X 
X 
X 
X 
X 
X 
X 
X 
Urinalysis 
 
 
 
X 
 
 
 
X 
HIV Serology 
 
 
 
 
 
 
 
 
Hepatitis B Serology 
 
 
 
 
 
 
 
 
Hepatitis C Serology 
 
 
 
 
 
 
 
 
HTLV-1 Serology 
 
 
 
 
 
 
 
 
VDRL 
 
 
 
 
 
 
 
 
EKG 
 
 
 
 
 
 
 
 
CT Scan /Chest/Abd/Pelvis 
 
 
 
 
 
 
 
X 
 
 
 
X 
Bone Scan 
 
 
 
X 
 
 
 
X 
Lymphocyte Subsets 
X 
 
 
X 
 
 
 
X 
Anti-TARP Antibody 
X 
 
 
X 
 
 
 
X 
NKT Cell Assessment 
 
 
 
 
 
 
 
 
Tetramer Staining 
X 
X 
X 
X 
X 
X 
 
X 
IFN- ELISPOT 
X 
X 
X 
X 
X 
X 
 
X 
51Cr CTL Assay# 
X 
X 
X 
X 
X 
X 
 
X 
PBMCs for Storage 
X 
X 
X 
X 
X 
X 
 
X 
Serum for Storage 
X 
X 
X 
X 
X 
X 
 
X 
 
Notes: 
➢ 
Screening CT scans of the Chest/Abd/Pelvis and Bone Scans may be performed up to 8 weeks prior to enrollment and at 
outside institutions.  
➢ 
All on study assessments can be obtained within + / - one week of scheduled measurement to allow for holidays and 
unscheduled delays. 
➢ 
Screening for HLA-A*0201 positivity will be completed before protocol eligibility screening is initiated.  Only HLA-
A*0201 positive patients are brought to NIH for further screening and eligibility assessment.  
➢ 
FluMist vaccination will be given with the seasonal influenza preparation when it is available at the time of enrollment. 
➢ 
X*: a 6th dose of TARP peptide vaccine will be administered to those patients with a > 50% increase in PSADT at week 24 
or who have confirmed immunologic responses to vaccination by tetramer or IFN-g ELISPOT assays as outlined in Section 
5.2.1 Immunologic Response Criteria.  A booster dose of TARP peptide vaccine will be administered to all patients at Week 
48 and 96 who elect to continue on the extension phase of the study through Week 144.. 
➢ 
#: 51Cr CTL Assay will be performed at the time points indicated only in those patients with evidence of immunologic 
reactivity by tetramer staining and/or IFN- ELISPOT assays.  

Abbreviated Title: TARP Peptide Vaccine Prostate Cancer 
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Appendix 2 
Standard Operating Procedure for Dendritic Cell Vaccine Preparation 
 
Autologous Cell Harvest  
 
Blood collection shall be by standard lymphapheresis. 8 to 15 liters of whole blood will be 
processed in order to collect peripheral blood mononuclear cells (MNC) with a target number of 
at least 2.2 ×109 monocytes. Lymphocytes will also be cryopreserved. Apheresis will be 
performed in the Clinical Center (CC) Department of Transfusion Medicine (DTM) using 
approved standard operating procedures. Bilateral peripheral venous access will be used for 
apheresis whenever possible. Alternatively, a temporary femoral central venous catheter (CVL) 
will be placed for collection. If this is necessary, the patient will be admitted to the hospital for 
CVL placement prior to starting apheresis. The CVL will be inserted by appropriately trained 
personnel (e.g., Critical Care, Interventional Radiology, General Surgery) with the assistance of 
anesthesiology as indicated. The patient will remain hospitalized until the CVL is removed. 
Prophylactic intravenous CaCl2 and MgSO4 infusions may be administered during apheresis to 
treat or prevent citrate toxicity at the discretion of the DTM physician per routine.  
 
Patient Cell Processing 
 
All cell processing will be conducted in accordance with approved DTM policies and 
procedures. 
 
TARP Peptide-Pulsed Dendritic Cells 
 
Background: Autologous dendritic cells prepared from peripheral blood monocytes will be 
loaded with 2 different TARP-derived peptides.  These peptides are made of amino acids 27-35 
and 29-37, namely TARP 27-35 and TARP 29-37-9V, comprised of HLA-A2-restricted epitopes 
that are involved in the cytolytic T cell response. TARP 29-37-9V contains a substitution that 
has been shown to enhance its binding affinity. Different fractions of autologous dendritic cells 
will be pulsed individually with only one of these peptides and the two fractions will be 
combined before administration to the patient. 
 
Formulation and Preparation: Autologous peptide-pulsed dendritic cell vaccines will be 
prepared under GMP conditions from cryopreserved patient monocytes. Autologous monocytes 
for dendritic cell culture will be enriched from peripheral blood MNC apheresis collections by 
counter-flow elutriation, aliquoted into at least 6 vials with ~ 333 x 106 cells/vial and 
cryopreserved for future preparation of the dendritic cell products. After thaw, the monocytes 
will be placed into a 5 day culture with rhIL-4 and rhGM-CSF to generate immature dendritic 
cells, followed by pulse with KLH and maturation with LPS and IFN-, and pulsed with TARP 
peptide. After removing peptide-pulsing media, dendritic cells will be concentrated down at 40 x 
106 cells/ml in infusion media (Plasma-Lyte A containing 10% autologous heat inactivated 
plasma). The final peptide-loaded, volume-reduced mature dendritic cell product will be 
prepared in sterile syringes for fresh administration intradermally.  A validated manufacturing 
process described in the Department of Transfusion Medicine, Clinical Center, NIH standard 
operating procedures will prepare the dendritic cell vaccine product. Detailed standard operating 
procedures for processing, labeling, storage, and quality assays are available on site in the Cell 
Processing Section of the Department of Transfusion Medicine.   
 

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Stability and Storage:  Autologous peptide-pulsed dendritic cell vaccines will be harvested from 
the 5-day culture product and packaged for fresh administration on the same day according to 
Standard Operating Procedures of the Department of Transfusion Medicine. A fixed autologous 
peptide-pulsed dendritic cell vaccine dose of 20 X 106 cells/ in 0.25ml or 0.5ml will be 
administered immediately upon receipt in the clinical setting.  Post packaging tests indicated that 
the product was stable for at least 2hours. 

Abbreviated Title: TARP Peptide Vaccine Prostate Cancer 
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Appendix 3 
Handling and Processing of Research Specimens 
 
The majority of the testing on this study will be done in the NIH Clinical Center clinical 
laboratory following their guidelines for blood collection and tube type.  The appropriate tube 
for uncommon laboratory tests and immunologic research specimens and where they should be 
sent are as follows: 
 
HLA Typing:  
1 Yellow Top to HLA Laboratory, Department of Transfusion Medicine  
 
FACS Analysis for Lymphocyte Subsets:  1 Purple Top to the Clinical Laboratory for 
lymphocyte subsets 
 
NOTE: FACS of dendritic cells utilized for TARP peptide-pulsed dendritic cell vaccine 
preparations will be done in the cell processing section of the blood bank. 
 
Tetramer & IFN- ELISPOT Assays:  
3 10ml Green Top Heparinized Tubes (30 ml total)  
Send via Frederick Courier to NCI Frederick Clinical Support 
Laboratory (Dr. William Kopp) for specimen processing and 
freezing. 
Tetramer Assay:  
Will be performed by the flow cytometry unit in the laboratory of 
Dr. William Kopp. 
IFN- ELISPOT Assay:  Will be performed in the Laboratory of Cell Mediated Immunity- 
Dr. Anatoly Malaguine 
 
51Cr Release CTL Assay:  
3 10ml Green Top Heparinized Tubes (30 ml total) 
Send via Frederick Courier to NCI Frederick Clinical Support 
Laboratory (Dr. William Kopp) for specimen processing and 
freezing. 
Assay will be performed in the Laboratory of Cell Mediated 
Immunity- Dr. Anatoly Malaguine 
NKT Cell Analysis:  
2 8mlBlue Tiger Top Sodium Citrate Tubes (16 ml total) 
 
 
 
 
Deliver to laboratory of Dr. Jane Trepel Bldg.10, Rm. 12N230 
301-496-1547 
 
NOTE: DTM will send aliquots of PBMCs following apheresis at weeks 0, 24, 48 and 96, 108, 
120, 132 and 144 and shipped to NCI Frederick as described above.  Multiple (N=6) heparinized 
green top tubes for immune assays will be drawn at non-apheresis time points (weeks 12, 18, 36, 
60, 72, 84, 108, 120, 132 and 144) and processed in an identical manner. 
 
The Clinical Support Laboratory, processes and cryopreserves samples in support of IRB-
approved, NCI clinical trials.  The laboratory is located in a controlled access building and 
laboratory doors are kept locked at all times.  Upon specimen receipt, each sample is assigned a 
unique, sequential laboratory accession ID number.  All products generated by the laboratory that 
will be stored either in the laboratory freezers or at a central repository facility are identified by 
this accession ID.  An electronic database is used to store information related to patient samples 
processed by the laboratory.  Vial labels do not contain any personal identifier information.  
Samples are stored inventoried in locked laboratory freezers and are routinely transferred to the 
NCI-Frederick repository facilities for long-term storage.  Access to stored clinical samples is 

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restricted.  Investigators establish sample collections under “Source Codes” and the investigator is 
responsible for the collections, typically the protocol Principal Investigator who has access to the 
collection.  Blood and tissue specimens collected in the course of this research project may be 
banked and used in the future to investigate new scientific questions related to this study. 
However, this research may only be done if the risks of the new questions were covered in the 
consent document, the patient has provided consent for the research use of their clinical 
specimens, and the proposed research has undergone prospective IRB review and approval.  The 
NIH Intramural IRB will be notified when samples are destroyed. 

Abbreviated Title: TARP Peptide Vaccine Prostate Cancer 
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Appendix 4 
Description of Immune Response Assays 
 
Monitoring of humoral and cellular immunologic responses will be performed at study weeks 0, 
12, 18, 24, 36, 48, 60, 96, 108, 120, 132, and 144.  Apheresis to ensure adequate numbers of 
cells for dendritic cell vaccine preparation (subjects randomized to Arm B) and for assessment of 
immunologic responses (all subjects) will be performed at weeks 0, 24, 48 and 96.  The 
procedures involved in each assay are outlined below. 
 
Separation of PBMCs 
 
At week 0 and subsequent non-apheresis time points (weeks 12, 18, 36, 60, 72, 84, 108, 120, 
132, and 144) blood will be drawn for immunological assays in 6 10ml green top tubes 
containing heparin.  This will be divided into aliquots, diluted with PBS 1:2 and 12 mls of 
Ficoll-Hypaque will be layered under 30 ml of the diluted patient sample.  The cells will be spun 
at 2000 rpm for 30 minutes.  The cells at the interface of the ficoll and the serum will then be 
collected and washed X 2. 
 
Tetramer Assay (All Patients) 
 
Utilizing HLA A2.1 tetramers containing TARP peptide sequences, cells obtained prior to 
vaccination and those obtained post vaccination will be stained with these tetramers and the 
precursor frequency (if any) will be determined at baseline and following TARP vaccination. 
 
IFN- ELISPOT Assay (All Patients) 
 
Lymphocytes will be separated from heparinized blood using density gradient centrifugation and 
then be placed in human AB serum with 10% DMSO for storage in liquid nitrogen.  At week 24, 
samples from weeks 0, 12, 18 and 24 will be assessed concurrently utilizing the ELISPOT 
assay.  The ELISPOT measures the number of IFN-
cells and will be used for determination 
of CTL precursor frequency to TARP peptides in both pre- and post-vaccination peripheral 
blood mononuclear cells (PBMC).  Additional ELISPOT assays will be preformed using samples 
from weeks 0, 24 and 48 as well as extension year (48-96 and 96-144) timepoints. 
 
Briefly, 96-well MultiScreenTM -HA (nitrocellulose) plates are coated with capture mAb against 
human IFN-  overnight at RT.  Effectors will be thawed and cultured overnight in RPMI 1640 
plus 10% human AB serum.  Plates are washed and blocked for 2 h with RPMI 1640 plus 10% 
human AB serum.  C1R.A2, antigen presenting cells (APC), are irradiated with 20,000 rad and 
pulsed for 2 h at 37ºC with either TARP peptide, Flu matrix peptide, a negative control peptide 
(TAX peptide) or a positive control peptide (CEF peptide pool- see notes below).  Non-pulsed 
C1R.A2 APC will be incubated in tandem and will serve as a negative control.  1 x 105 PBMC 
are added to each well followed by pulsed- or non-pulsed C1R.A2 cells at an effector:APC ratio 
of 1:1.  Each sample is run in six replicates to control for variability.  HLA-A2 binding Flu 
matrix peptide 59-66 serves as a positive control for inherent cellular reactivity.  In addition, 
PBMC from two "normal" HLA-A2-positive individuals with previously determined levels of 
FMP- or CMV-peptide T-cell precursors are run as assay controls.  Cells are incubated for 24h 
and lysed with phosphate buffered saline (PBS)-Tween (.05%).   Biotinylated antihuman IFN- 

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antibody in PBS-Tween containing 1% bovine serum albumin (BSA) is added and the plates are 
incubated overnight at 4°C. Plates are then washed and incubated with streptavidin-alkaline 
phosphatase for 2 h at RT.  Spots are visualized with BCIP-NBT substrate and subjected to 
automated evaluation using the ImmunoSpot Imaging system (Cellular Technology LTd, 
Cleveland, OH).  ELISPOT assays will be performed by the Laboratory of Cell Mediated 
Immunity (Dr. Anatoly Malyguine) at NCI Frederick.   
 
NOTE:  The CEF peptide pool from Mabtech may be used as HLA.A2 positive control peptides 
as alternatives to flu matrix peptide (FMP) as an HLA.A2 positive control.  The CEF peptide 
pool is a pool of 23 HLA class I restricted T cell epitopes from human cytomegalovirus, Epstein-
Barr virus and influenza virus. The peptide has been shown to stimulate the release of IFN-γ 
from CD8+ T-cells in individuals with certain highly frequent HLA types (HLA: A1, A2, A3, 
A11, A24, A68, B7, B8, B27, B35, B44).   Chosen epitopes are presented by the most common 
Caucasian HLA types, whose cumulative frequencies represent >100% of Caucasian individuals. 
The 23 peptides cover 11 HLA types. The CEF peptide pool has proven useful as a peptide 
specific positive control in ELISPOT, CTL and intracellular cytokine assays using human 
PBMC. Original pool described by Currier et. al  
 
51Cr Release CTL Assay (Patients with Evidence of Positive Tetramer and IFN- ELISPOT 
Responses) 
 
3 X 106 cells/well in a 24 well plate will be in vitro pulsed for 7 days in the presence of the 
following conditions:   
1) 10 M of the TARP 27-35 peptide (Native/WT peptide) 
2) 10 M of the TARP 29-37-9V peptide (Epitope Enhanced Peptide) 
3)  
  flu matrix peptide (lower concentration due to potent stimulation 
peptide).   
 
On day one 1000 U/ml IL-7 will be added and on day three cultures will be supplemented with 
20 IU IL-2/ml.  On day 7, cells will be harvested and plated to result in fixed effector to target 
cell ratios.  Cells initially cultured with native or epitope enhanced TARP peptides will be tested 
against targets pulsed with 50 µM of their respective TARP peptide, 50 µM of TAX negative 
control peptide, and 50 µM of CEF positive control peptide.  Cells initially cultured with flu 
matrix peptide will be tested against targets pulsed with 1 µM flu matrix peptide and 50 µM of 
TAX negative control peptide, and 50 µM of CEF positive control peptide. Targets will be 
labeled with 150 Ci [51Cr] sodium dichromate.  All target cells will then be washed three times 
and 3,000 labeled targets aliquoted into each well in a 96-well tissue culture plate that has 
already been plated with effector cells.  At this point, both the effector and target cells will be 
suspended in complete T cell medium with 10% fetal calf serum and with a 10 fold excess of 
K562s (over targets).  The plate will be incubated at 37o C for four hours.  Supernatants will be 
harvested using a Skatron supernatant harvesting device and the samples counted on a gamma 
counter.  Spontaneous release values will be obtained by incubating targets alone and maximum 
release values obtained by lysing the targets with Triton X-100.   
 
The percent specific lysis is determined, on triplicate samples, by the following formula:  percent 
specific lysis = 100 X (experimental release - spontaneous release)/(maximum release - 

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spontaneous release).  The percent specific lysis of targets with TARP  and control peptide will 
be compared to the percent specific lysis of targets without peptide.  If these are significantly 
different using student’s t test and if there is a greater than 10% difference at more than one 
effector:target ratio, we will consider the CTL assay to be positive.   
 
NOTE Regarding Immunologic Response Testing:  Assays will be run in batch for specimens 
from weeks 0. 12, 18 and 24.  Samples with reactivity at week 24 will be used as a comparator 
control for batch assays for specimens run from weeks 36, 48 60 and 96. 
 
Restimulation Assay Following Apheresis at Weeks 0, 24, 48 and 96 (All Patients) 
 
The restimulation assay will be performed by the lab of Cell Mediated Immunity at NCI 
Frederick.  Once apheresis is collected, prior to monocyte elutriation (week 0 only), 10 ml of 
apheresed PBMCs should be sent to the NCI Frederick Clinical Support Laboratory for 
processing.  These cells will be placed in a three week cytotoxicity assay in which they are 
stimulated repeatedly with the mixture of TARP peptides with which they were vaccinated and 
flu matrix peptides as a control. A fraction will also be cryopreserved for future immunologic 
assays.  Following these repeated stimulations, cells with then be tested on targets pulsed with 
each of these three peptides and on HLA A2.1 positive tumor cells expressing TARP.  
 
Lymphocyte Subsets for FACS Analysis 
 
Peripheral blood mononuclear cells will be tested for the following markers via flow cytometry; 
CD3, CD4, CD8, CD3-/CD16/56+, CD3+/CD16/CD56+.  Additional testing will be done on all 
patients in the research laboratory.  We plan to use these markers to determine whether 
vaccination has caused a change in the number of these cells circulating in the peripheral blood 
and in the expression of cell surface markers.  This assay will require three purple top tubes. 
 
Multiparameter Flow Cytometric Analysis of NKT Cells 
 
Analysis will be performed by the laboratory of Dr. Jane Trepel. NKT cells will be identified by 
mulitparameter flow cytometry.  Markers may include, but are not limited to, Va24, Vb11, CD3, 
CD1d tetramer, CD4, CD8, and Hoechst 33258 for identification of viable cells.  If sample 
permits, PBMC may be placed in culture for NKT cell activation, i.e. with -galactosylceramide, 
incubated at 37C, and intracellular cytokines such as IFN-, IL-4, IL-13 may be evaluated 
using multiparameter flow cytometry.  If sample permits, immune cell subsets that contribute to 
regulation of the NKT axis, i.e. Tregs, may be analyzed. 
 
When the patient is scheduled please notify the Trepel Lab by email: 
Yeong Sang Kim; kimye@mail.nih.gov and cc: to  
Jane Trepel: trepel@helix.nih.gov   
 
When the blood is drawn: 
Please label the tubes including week number 
Contact the Trepel lab at 301-496-1547 

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Leave the blood for pickup at room temperature in the soiled utility room, in a container labeled 
“Trepel Lab”. 
 

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Appendix 5: Guidelines and Worksheet for Calculations of PSADT 
Appendix 5A Guidelines and Worksheet for Calculation of PSADT 
Pre-Enrollment/Baseline and On Study PSADT Calculation: 
 
PSADT will be calculated using the Memorial Sloan-Kettering Cancer Center cancer 
information prostate nomogram for PSA doubling time found at: 
http://www.mskcc.org/mskcc/shared/forms/Nomograms/flash/load.cfm?type=Prostate&width
=586&height=505&title=Prostate%20Nomogram 
 
Minimum requirements for PSADT include > 3 PSA measurements over > 3 months. 
 
The interval between PSA measurements must be > 4 weeks. 
 
For patients receiving 5-alpha reductase inhibitors (5ARI) e.g. finasteride or dutasteride, only 
PSA values obtained after at least 3 months on therapy may be used to calculate PSADT. 
 
PSA values used in the calculation of PSADT must have been performed by the same 
laboratory, when possible. 
 
All PSA values used in the calculation should be > 0.20 ng/mL and follow a rising trend 
although all values need not be consecutively rising. 
 
All values obtained over a maximum period of 12 months prior to a given protocol time point 
will be included in the PSADT calculation to reflect the patient’s current disease activity. 
 
Date Range of PSA Values (should not exceed 12 months):  
 
 
 
 
 
 
 
Cumulative Total Months of PSA Values (should not exceed 12 months): 
 
 
 
 
 
Receiving Flomax: 
 YES 
 NO 
Start Date: 
 
 On at least 3 Months: 
 YES 
 NO  
 
Receiving 5ARI: 
 YES 
 NO 
Start Date: 
 
 On at least 3 Months: 
 YES 
 NO  
 
 
DATE 
PSA ng/mL 
TESTOSTERONE ng/dL  
PSADT  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Record PSA values with a maximum of 2 digits after the decimal point. 
NIH PSA values in blue. 
Calculated Pre-Enrollment/Baseline PSADT (in months): 
 
 
 
Value is > 3.0 Months and <15 Months: 
  YES- Eligible 
 
 
 
 
 
 
  NO- Ineligible for Study 

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Appendix 5B Guidelines and Worksheets for Calculation of PSADT 
 
Calculated Pre-Enrollment/Baseline PSADT (in months): 
 
 
 
 
PSADT Response is Defined As: 
➢ A >50% increase over baseline PSADT OR 
➢ A PSADT > 15 months 
 
PSADT Response will be assessed at weeks 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144. 
On Study PSADT Calculated at Weeks 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144 
DATE 
STUDY 
WEEK 
PSA 
ng/mL 
TESTOSTERONE 
ng/dL 
Calculated 
PSADT 
PSADT 
Responder? 
 
0 
 
 
 
 
 
3 
 
 
 
 
 
6 
 
 
 
 
 
9 
 
 
 
 
 
12 
 
 
 
 
 
15 
 
 
 
 
 
18 
 
 
 
 
 
24 
 
 
 
 
 
36 
 
 
 
 
 
48 
 
 
 
 
 
60 
 
 
 
 
 
72 
 
 
 
 
 
84 
 
 
 
 
 
96 
 
 
 
 
 
108 
 
 
 
 
 
120 
 
 
 
 
 
132 
 
 
 
 
 
144 
 
 
 
 
 
Record PSA values with a maximum of 2 digits after the decimal point. 
 
On Study Sequential PSADT Calculated for Statistical Analysis 
Analysis Window 
Study Wk PSA Values Included  
Calculated 
PSADT/Slope Log 
Percent Change 
PSADT 
Pre-NIH 
Outside PSAs -12 Months to Entry 
 
 
-12Months to Week 3 
-12 Months plus Wks 0, 3 
 
 
Week 0 to Week  3 
NIH PSAs only Wks 0, 3  
 
 
Week 3 to Week 12 
Wks 3, 6, 9, 12 
 
 
Week 3 to Week 24 
Wks 3, 6, 9, 12, 15, 18, 24 
 
 
Week 3 to Week 48 
Wks 3, 6, 9, 12, 15, 18, 24, 36, 48 
 
 
Week 24 to Week 48 
Wks 24, 36, 48 
 
 
Week 48 to Week 72 
Wks 48, 60, 72 
 
 
Week 48 to Week 96 
Wks 48, 60, 84, 72, 96 
 
 
Week 96 to Week 120 
Wks 96, 108, 120 
 
 
Week 96 to Week 144 
Wks 96, 108, 120, 144 
 
 
 

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Appendix 6: TARP Peptide Vaccination Report Card 
 

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Appendix 7:   Exploratory Correlative Studies 
 
Measurement of Function-Associated mRNAs in Whole Blood: Dr. Masato Mitsuahashi 
Hitachi Chemical. 
 
To be performed on the last 10 subjects enrolled in the 09-C-0139 TARP vaccine study. 
 
Function-Associated mRNAs:  
One 10ml green top sodium heparin tube 
(Weeks 0, 12, 18 and 24) 
 
➢ Blood will be drawn into one 10ml green top tube. 
➢ Add 60 L each of blood into three 8-well strips (provided by Dr. Mitsuhashi). Strips 
contain PHA, HAG, PBS, rIL2, rIFN, CEF, LPS/ZA, TARP27-35 and TARP EE-29-
37). 
➢ Incubate at 37C for 4 hours. 
➢ Store in -80C freezer until ready for shipment to Dr. Mitsuhashi  
➢ Note: specimen should be drawn before 1pm to allow adequate time for specimen 
processing and four hour incubation by Trepel lab staff.