Feasibility, Safety and Efficacy Evaluation of Alpha-Type 1 Dendritic Cell(DC)-Based Vaccines Loaded With Allogeneic Prostate Cell Lines in Combination With Androgen Ablation in Patients With PSA Progression After Local Therapy for Prostate Cancer
Phase: PHASE2
Conditions: Cancer of Prostate, Cancer of the Prostate, Neoplasms, Prostate, Neoplasms, Prostatic, Prostate Cancer, Prostate Neoplasms, Prostatic Cancer
Sponsor: Roswell Park Cancer Institute

FEASIBILITY, SAFETY, AND EFFICACY EVALUATION OF ALPHA-TYPE-1 
DENDRITIC CELL (DC)-BASED VACCINES LOADED WITH ALLOGENEIC 
PROSTATE CELL LINE IN COMBINATION WITH ANDROGEN ABLATION IN 
PATIENTS WITH PSA PROGRESSION AFTER LOCAL THERAPY FOR PROSTATE 
CANCER 
UPCI Protocol #: 
06-070 
BB-IND: 
13,061 
Version date: 
06/02/2017 
Phase: 
2 
Agents: 
Alpha-type-1 Dendritic Cell Vaccine 
INVESTIGATOR: 
Leonard J. Appleman, MD, PhD 
Associate Professor, Hematology-Oncology 
UPMC Cancer Pavilion 
5150 Center Ave, 5th Floor 
Pittsburgh, PA  15232 
(412) 648-6538 phone 
(412) 648-6579 fax 
SPONSOR: 
Pawel Kalinski, MD, PhD 
Vice Chair for Translational Research,  
Department of Medicine 
Director of Cancer Vaccine and Dendritic Cell Therapies, 
Center for Immunotherapy 
Roswell Park Cancer Institute  
Elm and Carlton Sts. 
Buffalo, NY 14263 
Immunologic Monitoring and 
Cellular Products Laboratory: 
Lisa H. Butterfield, PhD, Director 
Research Pavilion, Rm 1.27 
Hillman Cancer Center 
5117 Centre Avenue 
Pittsburgh, PA 15213 
Biostatistician: 
William Gooding, MS, PCI Statistics 
Study Monitor: 
Education and Compliance Office for Human Subject Research 
Research Conduct and Compliance Office 
University of Pittsburgh 
3500 Fifth Avenue, Suite 205 
Pittsburgh, PA  15213 
Financial Support: 
University of Pittsburgh Cancer 
Institute
NCT#00970203 
UPCI 06-070 (BB-IND 13,061) 
1 
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ABBREVIATIONS 
 
αDC1 
alpha-type-1-polarized DC (monocyte-derived DC generated in the presence of GM-
CSF and IL-4 and matured under the influence of IL-1β/TNFα/p:IC/IFNγ/IFNα 
3 
Ag 
antigen 
CTL 
cytotoxic T lymphocyte 
DC 
dendritic cells  
GMP 
good manufacturing practice 
i.d. 
intradermal 
IL- 
interleukin- 
i.l. 
intralymphatic 
IFN 
interferon 
IMCPL 
Immunomonitoring and Cell Products Laboratory (cGMP facility of the UPCI, 
specialized in preparation of cell-based vaccines) 
IRB 
Institutional Review Board 
NK 
natural killer 
PAP 
prostatic acid phosphatase 
PCa 
prostate cancer 
PGE2 
prostaglandin E2 
pI:C 
polyinositic: polycystydylic acid 
PRC 
Protocol Review Committee 
PSA 
prostate specific antigen 
PSA-DT 
PSA doubling time  
PSMA 
prostate specific membrane antigen 
s.c. 
subcutaneous 
Th 
T helper 
TNF 
tumor necrosis factor  
UPCI 
University of Pittsburgh Cancer Institute 
 
 
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TABLE OF CONTENTS 
ABBREVIATIONS ..................................................................................................................................... 2 
SUMMARY ................................................................................................................................................. 5 
STUDY DESIGN ......................................................................................................................................... 6 
VACCINE SCHEMA ................................................................................................................................. 6 
1 
OBJECTIVES ....................................................................................................................................... 7 
1.1 
Primary Objectives .................................................................................................................... 7 
1.2 
Secondary Objectives ................................................................................................................ 7 
2 
BACKGROUND AND RATIONALE ................................................................................................ 8 
2.1 
Prostate Cancer .......................................................................................................................... 8 
2.2 
PSA Progression after Local Therapy ....................................................................................... 8 
2.3 
Dendritic Cells in Tumor Immunology ..................................................................................... 9 
2.4 
Type-1 Polarized DC ............................................................................................................... 10 
2.5 
Dendritic cell (DC) Immunotherapy for Prostate Cancer ........................................................ 12 
2.6 
Allogeneic Tumor Cells as a Source of Antigen ..................................................................... 13 
2.7 
Feasibility of Generating αDC1-based Vaccines for Prostate Cancer Patients ....................... 14 
2.8 
Prostate Cancer-Associated Antigens or Tumor-Associated Antigens (TAAs) ...................... 15 
2.9 
Experience with DC-based Vaccines and Cancer Vaccine Trials at UPCI ............................. 16 
3 
RATIONALE FOR TRIAL AND TARGET PATIENT POPULATION ..................................... 16 
4 
SUBJECT SELECTION .................................................................................................................... 18 
4.1 
Eligibility Criteria.................................................................................................................... 18 
4.2 
Exclusion Criteria .................................................................................................................... 19 
4.3 
Gender/Ethnicity ..................................................................................................................... 20 
4.4 
Subject Recruitment ................................................................................................................ 20 
5 
STUDY DESIGN AND METHODS ................................................................................................. 21 
6 
TREATMENT PLAN ........................................................................................................................ 22 
6.1 
Leukapheresis .......................................................................................................................... 22 
6.2 
DC Vaccine Administration .................................................................................................... 22 
6.3 
Dose Limiting Toxicities ......................................................................................................... 23 
6.4 
Study Discontinuation Criteria ................................................................................................ 23 
6.5 
Supportive Care Guidelines ..................................................................................................... 23 
6.6 
Duration of study therapy ........................................................................................................ 24 
7 
STUDY EVALUATIONS and STUDY CALENDAR ..................................................................... 24 
7.1 
Screening ................................................................................................................................. 24 
7.2 
Evaluation during Treatment ................................................................................................... 25 
7.3 
Post-Treatment ........................................................................................................................ 25 
7.4 
Long Term Follow-up ............................................................................................................. 25 
7.5 
Study Calendar ........................................................................................................................ 26 
8 
MEASUREMENT OF EFFECT ....................................................................................................... 28 
8.1 
Efficacy endpoints ................................................................................................................... 28 
8.2 
Safety endpoints ...................................................................................................................... 28 
8.3 
Evaluation of Clinical Responses ............................................................................................ 28 
8.4 
Evaluation of Safety ................................................................................................................ 29 
8.5 
Measurement of the Immunologic Effects .............................................................................. 29 
8.6 
Alpha-1 Polarized Dendritic Cells Loaded with Allogeneic Apoptotic Cells ......................... 31 
8.7 
LNCaP Cell Line ..................................................................................................................... 31 
8.8 
LHRH Analogue...................................................................................................................... 31 
8.9 
KLH Protein ............................................................................................................................ 32 
9 
STATISTICAL CONSIDERATIONS .............................................................................................. 32 
9.1 
Study Design and Endpoints ................................................................................................... 32 
9.2 
Number of Subjects ................................................................................................................. 32 
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9.3 
Characteristics of the Design ................................................................................................... 32 
9.4 
Safety Evaluation .................................................................................................................... 33 
9.5 
Data Analysis .......................................................................................................................... 34 
9.6 
Subjects Randomization .......................................................................................................... 34 
10 
DATA SAFETY MONITORING PLAN ................................................................................... 34 
11 
ADVERSE EVENT REPORTING ............................................................................................ 35 
11.1 
Definitions ............................................................................................................................... 35 
11.2 
Review of Safety Information: Sponsor Responsibilities ....................................................... 36 
11.3 
Review of Safety Information: Investigator Responsibilities .................................................. 37 
11.4 
IND safety reports ................................................................................................................... 37 
11.5 
Reporting adverse events to the responsible IRB .................................................................... 40 
12 
QUALITY CONTROL AND QUALITY ASSURANCE ......................................................... 40 
13 
DATA HANDLING AND RECORD-KEEPING ...................................................................... 40 
14 
ETHICS ........................................................................................................................................ 40 
14.1 
Institutional Review Board (IRB) approval ............................................................................ 40 
14.2 
Ethical and scientific conduct of the clinical research study ................................................... 41 
14.3 
Subject informed consent ........................................................................................................ 41 
15 
REFERENCES ............................................................................................................................. 42 
APPENDIX I: SUMMARY OF PRECLINICAL DATA ...................................................................... 48 
APPENDIX II: MAILLIARD ET AL, 2004 ........................................................................................... 58 
APPENDIX III: cGMP-GRADE ALLOGENEIC CELL LINE LNCAP ............................................ 62 
 
 
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SUMMARY 
This study will evaluate the feasibility, safety, and efficacy of intradermal vaccination of prostate 
cancer patients with alpha-type-1-polarized dendritic cells (αDC1) loaded with apoptotic 
allogeneic tumor (LNCap). The study will target men with recurrent prostate cancer, who failed 
local therapy, have no measurable metastasis, but have a rising PSA with a doubling time of less 
than 10 months. The selection of this study group enables us to evaluate time to PSA 
progression, a highly relevant, clinical primary endpoint of efficacy in this two arm study. In 
order to facilitate infiltration of vaccination-induced T cells into tumor site(s) and to reduce 
tumor-specific tolerance, subjects will receive the vaccine in combination with limited 
androgen ablation (AA) with a LHRH analogue for 3 months. Subjects will be randomly 
assigned to one of two cohorts. In cohort A subjects will be first treated with limited AA alone 
for 3 months, and at the time of PSA relapse (PSA ≥ 1 ng/dL) will receive the DC vaccine in 
conjunction with AA. In cohort B, the sequence of treatment will be reversed. Efficacy will be 
estimated as the within-subject difference in time to PSA relapse following the combination 
treatment as compared to the AA alone, thus, each subject will serve as his own control. All 
subjects will commence the αDC1-based vaccination 2 weeks prior to treatment with the LHRH 
analogue. Each subject will receive 1 intradermal (i.d.) dose of the vaccine at weeks 1, 5, 9, and 
13 for a total of 4 doses. Additional courses of vaccination may be administered to subjects 
without evidence of disease progression every 3 months (±1 month) for up to 12 months 
depending on the number of doses originally produced and available after the 4 intended protocol 
doses. All doses of the vaccine will be administered intradermally (i.d.). 
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STUDY DESIGN 
 
 
VACCINE SCHEMA 
 
Within 
2-4 weeks 
1 week 
after last 
of start 
Day 1a 
Day15a 
vaccine injection 
DTH 
Week 1 
Week5a 
Week 9a 
Week 13a 
DTH 
Testing 
3-5x106 DC 
3-5x106 DC 
3-5x106DC 
3-5x106DC 
Testing 
 
 
 
 
 
 
 
 
Study cohorts: 
 
• 
Cohort A: 
LHRH analogue or limited androgen ablation (AA) followed by Vaccine + AA at time of PSA 
relapse 
 
• 
Cohort B: 
Vaccine + AA followed by AA at time of PSA relapse 
 
a Vaccine injections may be delivered on a ± 2 workday schedule. 
 
AA 
AA 
AA 
+ 
VACCINE 
AA 
+ 
VACCINE 
PSA RELAPSE 
PSA progression 
PSA progression 
COHORT A 
COHORT B 
Lupron 22.5 mg i.m. or Zoladex 10.8 mg s.c. 
The LHRH analogue will be administered 
2 weeks after the 1st dose of the vaccine. 
 
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1 OBJECTIVES 
Dendritic Cell (DC)-based vaccination is a new treatment option for cancer patients. While the 
previous DC-based vaccination trials have shown the safety of this approach and its ability to 
induce objective clinical responses, the overall efficacy of DC-based vaccines is still 
disappointing (Rosenberg et al., 2004). Our preliminary in vitro data indicate that type-1-
polarized DC (αDC1) are 40-fold more potent than the current “gold standard” of clinically-
applied DC (sDC) in their ability to induce tumor-specific CTLs (see Appendix I for the 
Summary of the Preclinical Data and the Protocol and Appendix II for our original publication 
describing CDC1 (Mailliard et al., 2004)). Thus this trial will enroll subjects with prostate cancer 
who have failed local therapy and present with PSA-only disease. At the time of study entry, 
subjects must have a PSA doubling time of less than 10 months and an absolute PSA level of > 
2.0 ng/mL. This is a two-arm study, whereby subjects will either be randomized to limited 
androgen ablation (AA) for 3 months followed by the DC vaccine + AA at the time of PSA 
relapse (PSA > 1 ng/dL) or the converse sequence of treatment. Thus all subjects will be treated 
with both AA as well as AA + vaccine, with each subject, in essence, serving as his own control, 
in estimating the time to PSA recovery following limited AA. The αDC1 vaccine will be 
administered every 4 weeks x 4 and then, if vaccine is available from the original production, 
every 3 months (±1 month) in subjects who continue to respond. 
1.1 
Primary Objectives 
• 
Feasibility objective: the ability to successfully generate and administer the alpha-DC1 
vaccine. 
• 
Safety objective: assess the tolerability and toxicity of the alpha-DC1 vaccine. 
• 
Efficacy objective: evaluate the effect of the alpha-DC1 vaccine on time to PSA 
progression compared to AA alone. PSA progression is defined as a rise in the PSA value 
to ≥ 1.0 ng/mL. 
1.2 
Secondary Objectives 
• 
To determine the change in PSA velocity prior to and following the proposed treatment. 
• 
To evaluate (in all subjects) the vaccination-induced DTH responses to LNCap, the cell 
line vaccine, and to compare this with vaccination-induced responses to tumor-untreated 
antigen (KLH). 
• 
To evaluate the vaccination-induced changes of Th1/Th2 profiles of the responses to PAP 
and PSMA. 
• 
To evaluate the CTL responses in blood to the whole LNCap cells (in all subjects) and (in 
all subjects who are HLA-A2 positive) the CTL responses to HLA-A2.1 restricted 
peptides derived from PAP and PSMA. 
• 
To comprehensively evaluate the CD4+ and CD8+ T cell responses (fine specificity and 
Th1/Th2/Treg cytokine profile) to the previously-identified and novel immunogenic 
epitopes of PAP and PSMA, using the EPIMAX system. 
 
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2 BACKGROUND AND RATIONALE 
2.1 
Prostate Cancer 
In 2007, approximately 225,000 new patients will be diagnosed with prostate cancer (PCa) in the 
US, and about 31,000 will succumb to the disease (most from metastatic disease) (Jemal et al., 
2005). Standard treatment of organ-confined PCa includes surgery or radiation therapy, and is 
effective in the short term, but up to one third of patients relapse. Current systemic therapy 
(androgen ablation and chemotherapy) for PCa is limited, primarily palliative, and is associated 
with significant morbidity (Vogelzang, 1997). Most patients with advanced disease are treated 
with hormonal therapy and/or chemotherapy, and go on to develop resistance to both these 
modalities (DiPaola, 1999; Tannock et al., 2004). Prostate-specific vaccine therapy, if effective 
and if initiated early, i.e., at the time of initial relapse, offers the prospect of prolonging survival 
with minimal morbidity. Thus, we have chosen to focus on men with PCa, with a PSA relapse 
after either surgery and/or radiation therapy (Catalona and Smith, 1998; Dillioglugil et al., 1997; 
Roach et al., 1999). This population of patients has less tumor burden, less molecular 
mechanisms of drug resistance, and may be more likely to respond to immune approaches to 
therapy (Blades et al., 1995; Sanda et al., 1995). Men with PSA-only disease also represent the 
largest sub-population of patients with PCa, for whom there is no standard treatment. 
Furthermore, they represent a heterogeneous group, with widely varying PSA-doubling times 
(Bubley et al., 1999; Pound et al., 1999). 
2.2 
PSA Progression after Local Therapy 
Patients with PSA progression after local therapy represent an ideal population for the study of 
novel immune approaches. Current therapy for patients with PSA progression after local therapy 
is unclear, and a subset of patients can be selected with greater likelihood of progression to 
metastatic disease, supporting the need for novel approaches. Based on recent literature, men 
with a PSA-doubling time (PSA-DT) of less than 10 months are at the highest risk of rapid 
progression and are often offered androgen ablation therapy, either on an intermittent or a 
continuous schedule (Gulley et al., 2005; Pound et al., 1999). However, studies have not been 
completed to determine a benefit of early androgen ablation therapy in patients with PSA 
progression after local therapy (Gulley et al., 2005). Prior studies of adjuvant androgen ablation 
therapy have demonstrated a benefit in only select populations of patients (Hanks et al., 2003; 
Lawton et al., 2001; Messing et al., 1999). Two studies demonstrated a survival benefit of 
adjuvant androgen ablation in concert with definitive radiation therapy in patients with locally 
advanced prostate cancer (Hanks et al., 2003; Lawton et al., 2001). Another study demonstrated 
a survival benefit to androgen ablation therapy for stage D1 disease after surgery (Messing et al., 
1999). 
Predictive factors for a high risk of PSA-recurrence after local therapy have also been identified. 
Thus Pound et al. demonstrated that time to biochemical progression, Gleason score, and PSA 
 
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doubling time were predictive of the probability and time to the development of metastatic 
disease (Pound et al., 1999). Recently, D’Amico et al. also demonstrated the predictive value of 
PSA-DT, both pre-operatively as well as after definitive local therapy (D'Amico et al., 2004). 
These data support using Gleason score and PSA-DT as entry criteria to select and stratify 
patients at high risk of disease recurrence for novel approaches to systemic therapy. 
Our target population for treatment will be men with prostate cancer, who have relapsed 
following surgery and/or radiation, and now have a rising PSA with a PSA doubling time (PSA-
DT) of less than 10 months. This patient population is ideal for an immunologic intervention 
given that: i) this is the largest sub-population of men with prostate cancer in the U.S.; ii) there is 
no standard treatment for this sub-group; and iii) this sub-group has minimal systemic disease 
burden. We will carefully monitor immunological responses to vaccination at different time 
points, but the primary endpoint of vaccine efficacy will be time to PSA progression and 
stabilization of PSA levels. Several recent vaccine studies have validated failure of PSA 
progression as a good surrogate endpoint for lack of progression in clinical trials in prostate 
cancer (Bubley et al., 1999; Kelly et al., 1993; Scher et al., 2004). 
2.3 
Dendritic Cells in Tumor Immunology 
Several lines of evidence derived from both murine studies and human clinical trials suggest that 
cancer can be susceptible to immune-based therapies. Studies using dendritic cells (DCs), 
(Steinman, 1991; Steinman and Cohn, 1973) to stimulate tumor-specific immune responses have 
been particularly encouraging (Banchereau and Steinman, 1998; Nestle et al., 1998; Schuler and 
Steinman, 1997; Steinman, 1991). DCs are the most potent antigen presenting cells (APCs), 
capable of efficiently internalizing and presenting antigen in the context of co-stimulatory signals 
and cytokines essential to the induction of effective long-lasting T-cell mediated immunity. 
Animal models have demonstrated that DCs, pulsed ex vivo with defined tumor antigens or 
material derived from tumor cells, can induce protective tumor-specific immune responses and 
are capable of mediating the regression of established disease (Zitvogel et al., 1996). Human 
clinical trials, including our own, demonstrate that DCs pulsed with defined tumor-relevant 
antigenic peptides, or, alternatively, with tumor cell lysates or apoptotic bodies, can induce 
tumor-specific-specific immune responses and even occasional complete tumor regression in late 
stage cancer patients (Banchereau et al., 2001; Nestle et al., 1998). 
Several competing approaches to DC therapy are currently being evaluated in clinical trials. In 
this context, two emerging issues appear to be critical for the development of “optimal” DC-
based immunization strategies. First, defining a strategy for the delivery of tumor antigens that 
facilitates efficient DC presentation of a broad range of class I- and class II-restricted epitopes 
appears to be critical to the induction of effective antigen-specific T-cell immunity. Second, 
recent results suggest that in order to induce tumor regression and promote long-term disease-
free status, DC-based strategies need to drive the in vivo expansion and maintenance of 
 
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Th1/Tc1-type T-cells (both CD8+ and CD4+) effector function. Human DCs may be readily 
obtained in large numbers from peripheral blood by short-term in vitro culture in media 
containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor 
(GM-CSF) (Banchereau et al., 2001; Fong and Engleman, 2000; Fukao, 2002; Langenkamp et 
al., 2001; Lopez and Hart, 2002; Scher et al., 2004). 
2.4 
Type-1 Polarized DC 
(αDC1: see Appendix I for the summary of preclinical data and Appendix II for our published 
description of αDC1s) 
Two functions of dendritic cells are believed to be important for the ability of DCs to induce Th1 
cells and CTLs, (the types of immune cells most desirable in cancer immunotherapy): high co-
stimulatory activity and high production levels of anticancer cytokines, especially IL-12 (Romani 
et al., 2001; Schuler-Thurner et al., 2002; Trinchieri, 2003; Vieira et al., 2000). Clinical trials to 
date have relied on the use of either fully-matured DCs exhibiting high stimulatory function, but 
low IL-12 secretion, or immature DCs that display low stimulator/high IL-12 secretion functions. 
We have recently developed a novel culture method to generate mature DCs that are both highly 
stimulatory and produce exceedingly high levels of IL-12 (Vieira et al., 2000; Vieira et al., 
1998). Such DC will be referred to as type-1 polarized DC or αDC1. We have recently 
succeeded in adopting our original αDC1 protocols based on fetal bovine serum-supplemented 
cultures, to allow αDC1 generation in serum free media, allowing for the facilitated application 
of αDC1s in clinical trials of cancer immunotherapy. Our in vitro observations show that such 
type-1-polarized DCs (αDC1s) induce up to 40-fold higher frequencies of tumor-specific CTLs 
during in vitro sensitizations when compared to conventional mature DCs. Furthermore, these 
same αDC1s are highly effective inducers of tumor-specific Th1-type CD4+ T cell responses. 
Beyond merely exhibiting the unique combination of high immunostimulatory function and high 
production capacity for cytokines, αDC1s exhibit a stable phenotype that is resistant to tumor-
associated immunosuppressive factors, including IL-10 and PGE2 (Kalinski et al., 1997; Kalinski 
et al., 1999b; Vieira et al., 1998): these suppressive factors are overexpressed in many advanced 
cancers, including prostate cancer (Attiga et al., 2000; Chen and Hughes-Fulford, 2000; Chen et 
al., 1997; Gabrilovich and Pisarev, 2003; Gerosa et al., 2002; Goto et al., 1999; Kalinski et al., 
1997; Kalinski et al., 1999b; Liu et al., 2002). αDC1s can produce IL-12p70 upon interaction 
with CD4+ T cells that are unable to produce IFNγ or other IL-12 co-inducing factors. This 
suggests the possibility of using αDC1s to boost the clinical efficacy of cancer vaccines, despite 
the presumed immuno-suppressive environment of immunocompromised (Th2-, Th3- or Tr1-
dominated) cancer patients (Vieira et al., 2000). 
The objective of DC-based cancer immunotherapy is to induce tumor-specific cytotoxic T cells 
(CTLs), IFN-γ-producing T helper type-1 cells (Th1 cells), and to boost the antitumor activity of 
 
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natural killer (NK) cells. The past developments in the area of DC-based cancer vaccination 
focused on the selection of most appropriate cancer-specific antigens (delivery of “signal 1”) 
and generating the DCs that are fully mature and express high levels of co-stimulatory molecules 
(needed for the delivery of “signal 2”), the signals believed to be essential for the development 
of immune responses of high specificity and magnitude. In contrast, our own work concentrates 
on the development of the vaccination strategies allowing us to induce the right type of immunity 
(delivery of “signal 3”) (Kalinski et al., 1997; Kalinski et al., 1999a; Kalinski et al., 1999b; 
Mailliard et al., 2003; Mailliard et al., 2004; Vieira et al., 2000). Our goal is to selectively 
stimulate the responses of CTLs, Th1 cells and NK cells, without enhancing the ineffective, and 
often counterproductive, Th2- and B cell-dominated type-2 responses, known to spontaneously 
arise in patients with advanced cancer (Tatsumi et al., 2002). 
Based on the novel paradigm of polarization of dendritic cells (Kalinski et al., 1999a), we 
have recently developed the first clinically-applicable protocol to generate type-1 polarized DC 
(DC1 or αDC1). Such DC1s are the first clinically-applicable type of DCs that combine all 3 
properties deemed important for their in vivo activity as inducers of anticancer immunity. 
These are: 1) fully-mature status; 2) high migratory responsiveness to lymph node-produced 
chemokines; and 3) high IL-12p70-producing function. Such a combination was missing in all 
previously-applied protocols for preparing DC, where final DC maturation (needed to generate 
DC with high expression of co-stimulatory molecules and the ability to localize in the T cell 
areas of the lymph nodes) was associated with irreversible loss of their ability to produce the key 
Th1-, CTL-, and NK cell-activating cytokine with anti-cancer properties, IL-12 (Kalinski et al., 
1999b; Langenkamp et al., 2001). DC1s show a selectively enhanced ability to induce type-1 
anti-cancer responses, when compared to the current “gold standard” of clinically-used DCs. Our 
studies in the melanoma model have shown that peptide-pulsed DC1s induce up to 40-fold 
higher numbers of tumor-specific CTLs, during in vitro sensitization of peripheral blood T 
cells from the patients (see Appendix I for the summary of preclinical data and Appendix II for 
our published description of αDC1s). Importantly for clinical applications of DC-based 
vaccines, we could also show that type-1 DC polarization makes DC resistant to prostaglandin 
E2 (PGE2), an immunosuppressive factor produced by prostate cancer (Attiga et al., 2000; 
Boutemmine et al., 2002; Filella et al., 2000; Liu et al., 2002; Tjandrawinata et al., 1997). An 
additional consideration is the ability of DC1 to cross-present apoptotic tumor, an important 
factor allowing us to use as a source of tumor-relevant antigens, whole tumor cells, rather than 
defined peptide epitopes, broadening the spectrum of tumor-relevant targets for immunization 
and eliminating the need to restrict the study to patients with a defined MHC type (usually HLA-
A2). This last property of DC1s also facilitates the use of either autologous tumor or allogeneic 
prostate cancer cell lines as a source of antigenic material (Hrouda et al., 2000; Nouri-Shirazi et 
al., 2000; Pandha et al., 2004; Todryk et al., 2004). Allogeneic cell lines offer the advantage of 
standardized preparation of the vaccine, and a potential possibility to further boost the 
 
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immunogenic properties of vaccine-carrying DCs by promoting their interaction in vivo with 
high numbers of allo-specific CD8+ T cells and NK cells in tumor-bearing hosts (Mailliard et al., 
2002; Mailliard et al., 2003; Nouri-Shirazi et al., 2000). 
Our concurrent studies, performed in collaboration with the groups of Drs. David Bartlett and 
John Kirkwood, resulted in the design and FDA approval and recent implementation of αDC1-
based clinical trials in melanoma (UPCI 03-118; BB-IND 11,754) and colorectal cancer (UPCI 
05-063; BB-IND 13,234). Clinical activity of vaccination was observed in 2 of the initial 4 
melanoma subjects who completed the treatment (1PR 12M+ in a subject with Stage IIIb 
disease; 1 SD 11M+ in a subject with stage IV lung disease). The two first subjects with resected 
metastatic CRC are undergoing follow-up for time to recurrence and immunologic evaluations. 
No side effects were observed in either of these settings. In addition, our collaborators from the 
Departments of Dermatology (Drs. L. Geskin and L. Falo) and Neurosurgery (Dr. Hideho 
Okada) used this platform to vaccinate subjects with advanced cutaneous T cell lymphoma 
(CTCL) and recurrent glioma, observing lack of serious side effects and the occurrence of 
clinical responses in both of these settings. 
2.5 
Dendritic cell (DC) Immunotherapy for Prostate Cancer 
Prostate-specific immunotherapy and/or induction of autoimmune prostatitis are two related 
immunological approaches, which may offer the prospect of an effective treatment for prostate 
cancer (Fong et al., 1997; Liu et al., 1997). The recent identification of dendritic cells (DC) as 
powerful professional antigen-presenting cells capable of inducing primary T cell responses in 
vivo and in vitro has generated widespread interest in the application of DC to tumor 
immunology (Fong and Engleman, 2000; Schuler and Steinman, 1997; Steinman, 1991). DC also 
have the capacity to break peripheral tolerance and induce CTL responses to “self” antigens 
(Banchereau and Steinman, 1998). Prostate tumor cells express a variety of tissue-specific 
antigens, including prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), and 
prostate-specific membrane antigen (PSMA), all of which have been considered as potential 
targets for T cell immunotherapy, DC vaccination, or induction of therapeutic autoimmune 
prostatitis. PSA peptides containing HLA A*0201-binding motifs can induce CTL responses that 
lyse peptide-loaded target cells and prostate tumor cells (Correale et al., 1997; Correale et al., 
1998; Xue et al., 1997). Similarly, DC pulsed with HLA A*0201-binding PAP peptides are able 
to induce CTL responses against A*0201-positive prostate tumor cells (Machlenkin et al., 2005; 
Peshwa et al., 1998). These encouraging laboratory results have formed the basis of clinical trials 
for DC-based immunotherapy targeted against PSA, PAP, and PSMA (Burch et al., 2004; Fong 
et al., 2001; Heiser et al., 2002; Murphy et al., 2000). In a recent Phase I trial of PSA mRNA-
pulsed DC administered to subjects with metastatic prostate cancer, PSA-specific T cell 
responses were detected in all subjects, and vaccination was associated with a significant 
decrease in PSA in six of seven subjects (Heiser et al., 2002). No dose-limiting toxicity or other 
 
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adverse effects were noted. 
Working on the principle that xenogeneic antigens are more immunogenic than self antigens DC 
loaded with mouse PAP have been tested in a Phase I clinical trial in subjects with metastatic 
prostate cancer. All subjects developed T cell responses to the mouse PAP, and eleven of twenty-
one also developed T cell responses to the homologous human PAP (Fong et al., 2001). Both 
Phase I and Phase II trials have been conducted with DC loaded with a fusion protein consisting 
of PAP linked to granulocyte-macrophage colony-stimulating factor (GM-CSF) (Burch et al., 
2004; Small et al., 2000). The treatment was well tolerated, and 38% of subjects mounted 
immune responses to the PAP-GM-CSF fusion protein. Although, immunologic tolerance to the 
aforementioned proteins could be circumvented, the clinical benefit of these vaccines’ remains to 
be determined. Patients with PCa have been reported to have multiple immune defects: both in 
antigen presenting cells, as well as in tumor-reactive T cells (Kennedy-Smith et al., 2002). 
Functional defects in circulating T cells, have also been noted in patients with PCa, i.e., reduced 
expression of the T cell zeta-chain (associated with signaling). Interestingly, Meidenbauer et al. 
reported that 50% of the patients treated with PSA-based vaccination therapy, had normal 
recovery of the T cell zeta chain (Meidenbauer et al., 2000). Additionally, defects in the antigen 
processing machinery have also been described in PCa (Sanda et al., 1995). Furthermore, our 
own data also speak to the presence of PCa-associated immunosuppression: in particular, a) 
defects in dendropoiesis, and b) the propensity for DC to undergo apoptosis (Shurin et al., 2001; 
Tourkova et al., 2004). Thus generation of DCs resistant to immunosuppression and/or apoptosis 
(as proposed), may be of benefit (Kalinski et al., 1999a). 
2.6 
Allogeneic Tumor Cells as a Source of Antigen 
(See Appendix III for the data on DC-mediated cross-presentation of LNCap-associated epitopes to 
CTLs) 
Although there are a number of prostate-specific proteins amenable to immune targeting, the 
immunogenicity of these proteins is not well understood, and a priori, there is no perfect antigen. 
Given the theoretical appeal of targeting more than one cancer-associated protein, the in vivo 
efficacy of whole-tumor cell vaccines was initially demonstrated in preclinical animal studies 
(Suckow et al., 2005; Todryk et al., 2004; Vieweg et al., 1994). Several of these studies observed 
immunologic memory capable of protective immunity as well as curative effects against 
established subcutaneous (s.c.) tumors using Dunning rat prostatic carcinoma cells (MAT-LyLu) 
(Vieweg et al., 1994). Autologous whole cell vaccines for human prostate cancer were initially 
investigated by Simons et al (Simons et al., 1999). Surgically harvested prostate tumor cells were 
irradiated and engineered to secrete GM-CSF via a replication-defective retrovirus. A small 
Phase I study (n=11) demonstrated interesting immunologic response and confirmed the safety of 
this approach. The numerous technical difficulties involved in the preparation of autologous 
cells, however, represented a significant limitation. Thus the focus of research was shifted to 
 
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allogeneic vaccines, which are readily available from established prostate cancer cell lines. This 
type of vaccine may be manufactured on a larger scale for distribution. In addition, patients do 
not need to be HLA-matched for these vaccines as antigens can be presented by cross priming. A 
GM-CSF secreting vaccine, GVAX (Cell Genesys, South San Francisco, CA), is an admix of 
prostate cancer cell lines PC-3 and LNCaP, and is currently in two large Phase III clinical trials 
in hormone-refractory prostate cancer. The impetus for these studies was promising survival data 
from smaller Phase II studies (Michael et al., 2005; Simons et al., 1999). Another Phase II trial 
utilizing a different allogeneic whole cell, Onyvax-P (Onyvax Ltd, London, UK) consisting of 
irradiated prostate cancer cell lines OnyCap23, LNCaP, and P4E6, demonstrated improved 
median time to disease progression in patients with AIPC (Eaton et al., 2002). 
In this trial we also plan to use an allogenic cell line: LNCaP, as a source of tumor antigens to be 
loaded on each subject’s autologous αDC1s. We have verified that this cell line is a relevant 
source of DC-cros-presented CTL epitopes of PCa (Nouri Shirazi et al 2004: see Protocol 
Appendix III). In addition to circumventing the perceived problems with obtaining autologous 
tumor material in the current group of patients, the selection of allogeneic cell line has been 
supported on our data showing that human resting CD8+ T cells (such as allo-specific T cells 
promote the ability of DCs to induce Th1 and CTL responses (Mailliard et al 2002). Thus 
LNCap will act in our vaccine both as a source of both PCa-specific antigens and additional 
tumor-unrelated “heterologous” (helper) antigens (alloantigens). 
2.7 
Feasibility of Generating αDC1-based Vaccines for Prostate Cancer Patients 
(Also see Appendix I for the summary of preclinical results) 
We have successfully verified that αDC1 can be generated from the monocytes of prostate 
cancer patients. The current study will verify the ability to generate αDC-1 on a large scale, store 
them, and subsequently use them for vaccination. Compared to standard DCs, αDC1s from the 
same patients produce 10-100 times more IL-12p70, the known Th1-, CTL-, and NK cell-
activating factor (see Appendix I and II). In accordance with the resistance of polarized DCs to 
tumor-related suppressive factors, such as PGE2 (Vieira et al 2002), we observed that high IL-
12-producing mature CD83+/CCR7+ αDC1 are not negatively affected by the presence of 
apoptotic bodies from UV-irradiated LNCap cells during DC maturation (see Appendix III). The 
expansion, testing, and banking of the LNCaP master cell line that will be used as a source of 
prostate cancer antigens in the αDC1s vaccines has already been arranged with Bio_Reliance 
(see Appendix IV). The final documentation and the results of all tests of such master cell line 
will be delivered to the FDA prior to the accrual of any subjects to the current protocol. With 
regard to other vaccine components, KLH, a “heterologous CD4+ helper antigen”, has been 
approved for the use in a similar DC-based melanoma trial (UPCI 03-118; BB-IND 11,754) and 
is available in sufficient amount for the currently proposed trial. 
 
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All vaccines will be prepared by the experienced staff of the IMCPL (the cGMP-compliant 
facility of the UPCI), according to the procedures covered by the FDA master file 
(BB-MF 12,244) of the IMCPL. 
2.8 
Prostate Cancer-Associated Antigens or Tumor-Associated Antigens (TAAs) 
Over the past 10 years, numerous TAAs (PSA, PAP, PSMA, etc) and their derivative HLA-A2-
presented peptides have been identified in prostate cancer. These antigens can be used in the 
form of antigenic proteins and/or peptides. In addition, these and additional TAA, unique to 
individual tumors are present in whole apoptotic tumor cells and can be cross-presented by DC 
(Nouri-Shirazi et al., 2000) (see Appendix III). Our vaccine will consist of αDC1 loaded with 
allogeneic apoptotic tumor that is expected to contain all of these different classes of antigens. 
Thus vaccination-induced immune responses against allogeneic whole tumor are likely to be 
directed against both TAAs as well as allogeneic molecules. To validate that our αDC1-based 
vaccines are effectively presenting such tumor related epitopes in vivo, in vaccinated subjects, an 
important secondary endpoint in this study will be testing immune responses against HLA-A2 
restricted peptides derived from prostate TAAs (see Table 1 below), in any subjects who are 
HLA-A2 positive (expectedly 40-50%). 
TABLE 1. Prostate Cancer HLA-A2-restricted TAAs Used as Readouts (Secondary 
Efficacy Endpoints) 
Antigen 
Epitopes 
Sequence 
Reference 
PAP 
PAP-3 
NH2-ILLWQPIPV-COOH 
(Harada et al., 2004; Machlenkin et al., 
2005; Peshwa et al., 1998) 
PAP 
PAP-5 
NH2-ALDVYNGLL-COOH 
(Harada et al., 2004; Machlenkin et al., 
2005; Peshwa et al., 1998) 
PSA 
PSA147 
NH2-KLQCVDLHV-COOH 
(Alexander et al., 1998; Chakraborty et al., 
2003; Correale et al., 1997) 
PSMA 
PSMA27 
NH2-VLAGGFFLL-COOH 
(Harada et al., 2004; Kobayashi et al., 
2003) 
STEAP 
STEAP-3 
NH2-LLLGTIHAL-COOH 
(Machlenkin et al., 2005) 
 
KLH as a “heterologous” helper antigen: 
In general, most TAAs are considered to be “weak” antigens, inducing low-frequency, low-
avidity CD4+ T cell responses as a consequence of being “self” antigens that are subject to 
tolerance programming. In addition, recent reports have demonstrated an undesirable bias of 
tumor-specific CD4+ (“helper”) T cells towards the TH2 phenotype (Tatsumi et al., 2002), that is 
likely to be ineffective in providing helper signals for CTL generation. To circumvent the 
problem that Th1-type T cells responsive to TAAs are limiting in subjects receiving our 
vaccines, similar to the strategy adopted in our melanoma protocol UPCI 03-118 (BB-IND 
11,754), we propose to include “heterologous”, tumor-unrelated helper antigen KLH in our 
 
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vaccine formulation. In order to avoid preferential induction of KLH-specific responses and to 
avoid the risk of the strong DTH reactions to the booster doses of the vaccines, this protein will 
be included only in the first dose of the vaccine and will not be included in the vaccines used as 
booster doses. 
2.9 
Experience with DC-based Vaccines and Cancer Vaccine Trials at UPCI 
Dendritic cell based vaccines have been investigated extensively in a number of centers and to 
date, have not been reported to be associated with limiting toxicities or adverse side effects. 
UPCI has a long standing interest, a significant track record, and a robust infrastructure to 
support vaccine trials. 
3 RATIONALE FOR TRIAL AND TARGET PATIENT POPULATION 
Over the past decade, owing to aggressive PSA screening, patients with prostate cancer (PCa) are 
being diagnosed earlier and undergo both definitive treatment as well as androgen ablation (for 
those who relapse after surgery or radiation) early in the course of their disease. Consequently, 
the fastest growing subsets of patients with PCa include: i) men with a rising PSA following 
local therapy, i.e., irradiation and/or prostatectomy; and ii) men with a rising PSA following 
failure of hormonal therapy. These men with PSA only disease are virtually certain to develop 
clinically manifest prostate cancer, though the interval from detection of PSA increase to clinical 
disease may be very variable. In the hormone-naïve serologic failures, the mean time to 
development of metastasis may vary from 4 to 7 years (Dillioglugil et al., 1997; Pound et al., 
1999). Androgen deprivation is the standard of care for men with advanced prostate cancer but 
the optimal timing for such therapy is unclear. There is no evidence that continuous androgen 
deprivation for men with PSA elevation following local therapy improves quality or quantity of 
survival – and androgen deprivation is not without consequences: osteoporosis, diminished 
libido, loss of muscle mass, increase body fat and hot flashes. Hence alternatives to androgen 
deprivation are urgently needed – especially for this patient population. Likewise, there is no 
accepted standard of treatment for the non-metastatic androgen independent population, where 
the time to development of metastasis may vary from 9 to 24 months. Hence these two sub-
populations of men with PCa, are ideal for testing novel therapies. 
With respect to immunotherapy, since it is known that androgen ablation promotes an influx of T 
cells into the prostate (Mercader et al., 2001), in the proposed trial we plan on immunizing men 
in concert with limited (3 months) androgen ablation. We plan on introducing androgen ablation 
2 weeks after immunization, in order to focus the effector phase of prostate cancer-specific 
immune response on the existing prostate cancer sites. We anticipate that such timing of both 
treatments will allow the circulating inflammatory-type cancer-specific T cells (Th1 cells and 
CTLs) induced by the first dose of the αDC1-based vaccine, to enter the existing tumor sites 
damaged by the hormone therapy and to eliminate the surviving cancer cells. The second dose of 
the vaccine, administered 2 weeks after the hormone therapy and the 2 remaining vaccine doses, 
 
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are anticipated to re-enforce such an effect, cumulatively prolonging the time to disease 
progression, compared to what is typically observed after limited androgen ablation: PSA 
recurrence, as testosterone levels recover in 3 to 6 months. Thus, we hypothesize that the 
combination of the αDC1-based vaccine and limited androgen ablation, will significantly delay 
time to PSA recurrence, despite a normalization of testosterone levels. Finally, we also anticipate 
that the combination treatment proposed above, will further enhance the activity of vaccination 
by counteracting tumor-related, prostate-specific immune tolerance, that has been demonstrated 
in experimental murine models of prostate cancer (Drake et al., 2005). 
In order to allow the vaccine-constituting αDC1s to act as a medium of prolonged feedback 
interactions between the expanding populations of CD4+ and CD8+ T cells, the first priming dose 
of the αDC1-based vaccine will be administered intradermally over a period of four days, similar 
to our melanoma protocol UPCI 03-118 (see previous sections). We expect that such prolonged 
period of the presence of LN-immigrating DCs is particularly important during the priming cycle 
of vaccination, when the frequencies of functional tumor-specific CD4+ and CD8+ T cells are 
the lowest. Since we expect that the first cycle of vaccination will successfully increase the pools 
of functional CD4+ and CD8+ T cells, we expect that such prolonged period of vaccine delivery 
will be less important in case of the booster doses of vaccination. Therefore, for the feasibility 
reason, we plan to perform all booster doses as single injections. 
For the same reason (low-frequency of tumor specific T cells during the first priming cycle of 
vaccination), during this cycle alone, we also propose to use KLH as a “heterologous” helper 
antigen, given that most prostate tissue-derived antigens are considered to be “weak” antigens, 
inducing low-frequency, low-avidity CD4+ T cell responses as a consequence of being “self” 
antigens that are subject to tolerance programming. In addition, recent reports have demonstrated 
an undesirable bias of tumor-specific CD4+ (“helper”) T cells towards the Th2 phenotype 
(Tatsumi et al., 2002), that is likely to be ineffective in providing helper signals for CTL 
generation. To circumvent the problem that Th1-type T cells responsive to self are limiting in 
subjects receiving our vaccines, we propose to include “heterologous”, tumor-unrelated helper 
antigen, KLH (Nestle et al., 1998) in our formulation. 
Thus, our goal is to develop protocols to induce autoimmune prostatitis as a novel 
immunotherapy for prostate cancer, through dendritic cell-induced stimulation of CD8+ cytotoxic 
T cell (CTL) responses against prostate autoantigens. This is based on the premise that a 
vigorous autoimmune response against prostate-specific proteins, capable of destroying normal 
prostate tissue, will also destroy malignant prostate tissue, provided the malignant tissue 
expresses these proteins. Since the prostate gland is routinely removed or ablated as a part of the 
treatment for early prostate cancer, any remaining/recurring prostatic tissue could be destroyed 
immunologically. Three prostate tissue specific antigens are currently being evaluated in clinical 
trials: prostate specific antigen (PSA), prostatic acid phosphatase (PAP), and prostate specific 
 
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membrane antigen (PSMA). However the immunogenicity of these proteins is not well 
understood, and a priori, there is no perfect prostate-specific immunologic target. Hence our 
focus is on the allogenic tumor cell line - LNCaP. Additionally, we will also incorporate limited 
(3 months) androgen deprivation (with LHRH analogues) in the treatment protocol to enhance 
the immune-potentiating effects of this manipulation, as described above (Drake et al., 2005; 
Mercader et al., 2001). 
Our target population for treatment will be men with prostate cancer, who have relapsed 
following surgery and/or radiation, and now have a rising PSA with a PSA doubling time (PSA-
DT) of less than 10 months. This patient population is ideal for an immunologic intervention 
given that: i) this is the largest sub-population of men with prostate cancer in the U.S.; ii) there is 
no standard treatment for this sub-group; and iii) this sub-group has minimal systemic disease 
burden (Havranek et al., 2002; Kaufman et al., 2004). We will carefully monitor immunological 
responses to vaccination at different time points, but the primary endpoint of vaccine efficacy 
will be stabilization of PSA levels or failure of the PSA to progress. Several recent studies have 
validated failure of PSA progression as a good surrogate endpoint for lack of progression in 
clinical trials in prostate cancer (Scher et al., 2004). 
This is a 2 group crossover trial in which subjects are randomly assigned to one of two treatment 
arms: 
Cohort A: 3 months of androgen ablation (AA) to be followed at PSA progression by 3 months 
of the combination of AA and alpha-type 1 dendritic cell vaccine (DC1). 
Cohort B: 3 months of the combination of AA and alpha-type 1 dendritic cell vaccine followed 
at PSA progression by 3 months of AA. 
In this crossover trial each subject will serve as their own control. Following either therapy the 
time to PSA progression, defined as the time between treatment and the first instance of PSA 
increase to 1 ng/mL, will be estimated from monthly PSA monitoring. The endpoint is the 
difference between the time to PSA progression for the combination of AA + DC1 compared to 
AA alone. 
4 SUBJECT SELECTION 
4.1 
Eligibility Criteria 
• 
Patients with histologically proven prostate cancer and tumors limited to the prostate 
(including seminal vesicle involvement, provided all visible disease was surgically 
removed) who have completed local therapy and have an elevated PSA after surgery or 
rising PSA after radiation therapy, as defined below. 
• 
Age 18 years or older 
• 
Histologically confirmed diagnosis of prostate cancer. 
• 
Previous treatment with definitive surgery or radiation therapy or both. 
 
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• 
No evidence of metastatic disease on physical exam, CT/MRI/CXR (see Section 7.1 for 
radiologic imaging), and bone scan within 4 weeks prior to randomization. 
• 
Prior neoadjuvant/adjuvant hormonal, androgen deprovation therapy, or chemotherapy is 
allowed if it was last used > 12 months prior to first vaccination. 
• 
No therapy modulating testosterone levels (such as leuteinizing-hormone releasing-
hormone agonists/antagonists and antiandrogens) is permitted within 12 months prior to 
first vaccination. Agents such as 5α-reductase inhibitors, ketoconazole, megestrol acetate, 
systemic steroids (replacement doses of steroids are allowed), PC-SPES, and Saw 
Palmetto are not permitted at any time during the period that the PSA values are being 
collected. 
• 
Hormone-sensitive prostate cancer as evident by a serum total testosterone level > 150 
ng/dL or > 6 nmol/L at the time of enrollment within 4 weeks prior to randomization. 
• 
All patients must have evidence of biochemical progression as determined by a reference 
PSA value followed by 1 confirmatory rising PSA value, higher than the previous value, 
obtained at least 2 weeks apart. All of these PSA values must be obtained at the same 
reference lab, and all must be done within 6 months prior to enrollment. 
• 
The most recent of the PSA values must be ≥ 2.0 ng/mL. This measurement must be 
obtained within 1 month prior to enrollment. 
• 
The PSA doubling time (PSA-DT) must be less than 12 months. 
• 
ECOG performance status 0 or 1. 
• 
Patients must have normal organ and marrow function as defined below: 
o 
Absolute neutrophil count 
> 1,500/µL 
o 
Platelets 
> 100,000/µL 
o 
Total bilirubin 
1.5 x upper limit of normal (ULN) 
o 
SGOT (AST) and SGPT (ALT) 
< 2.5 x institutional ULN 
o 
Creatinine 
1.5 x ULN 
• 
The effects of dendritic cell vaccines on the developing human fetus are unknown. For 
this reason men must agree to use adequate contraception (hormonal or barrier method of 
birth control) prior to study entry and for the duration of study participation. 
4.2 
Exclusion Criteria 
• 
Patients must not be receiving other investigational agents or concurrent anticancer 
therapy. 
• 
No uncontrolled intercurrent illness including, but not limited to, ongoing or active 
infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit compliance with study 
requirements. 
• 
Patients must not have active eczema, atopic dermatitis, or other exfoliative skin 
conditions (e.g., burns, impetigo, varicella zoster, severe acne, contact dermatitis, 
psoriasis, herpes or other open rashes or wounds). 
• 
Presence of an active acute or chronic infection, including urinary tract infection, HIV or 
viral hepatitis. HIV patients are excluded based on immunosuppression which may render 
 
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them unable to respond to the vaccine; patients with chronic hepatitis are excluded 
because of concern that hepatitis could be exacerbated by the injections. If clinically 
indicate, HIV/viral hepatitis testing will be performed to confirm status. 
• 
Patients with a history of auto-immune disease such as, but not restricted to, 
inflammatory bowel disease, systemic lupus erythematosus, ankylosing spondylitis, 
scleroderma, or multiple sclerosis. Patients receiving replacement thyroid hormone would 
be eligible. 
• 
No concurrent use of systemic steroids, except for local (topical, nasal, or inhaled) steroid 
use. Adrenal replacement doses of corticosteroids are allowed. 
• 
Subjects with concurrent additional malignancy (with exception of non-melanoma skin 
cancers and superficial bladder cancer or malignancy within last 3 years). 
4.3 
Gender/Ethnicity 
Study entry is open to male patients of all ethnic backgrounds. The racial and ethnic 
characteristics of the proposed subject population will reflect the demographics of Pittsburgh and 
the surrounding area and/or the patient population of the UPMC Health Systems. We shall 
attempt to recruit subjects in respective proportions to these demographics. No exclusion criteria 
will be based on race, ethnicity, gender, or socioeconomic status. In recent Phase I/II trials at 
UPCI, approximately 11% of subjects were non-caucasian. Given the limited size of the patient 
population to be accrued on this trial, there will be limited power to determine differences in 
efficacy between different racial groups. However, we will describe any apparent differences in 
efficacy or toxicity. 
4.4 
Subject Recruitment 
Approximately 12-16 subjects are expected to enter the study. All of whom will be accrued 
through the University of Pittsburgh Cancer Institute. 
For the proposed trial, all the subjects will be accrued at UPCI Hillman Cancer Center. There is 
an extensive clinical practice in urologic cancers between the Medical Oncology offices and the 
Urology offices at UPCI/UPMC. In addition, UPCI has vast community links and a large clinical 
investigations program through its economically aligned network of over 60 medical and 
radiation oncologists who practice in community-based sites throughout western PA and 
adjacent areas of Ohio and West Virginia. In the UPMC/UPCI Network registry for 
Genitourinary Organs, 2004-2005, 1117 new cases of prostate cancer were diagnosed across the 
system hospitals. Across the entire state of Pennsylvania, 10,300 new cases of prostate cancer are 
expected to be diagnosed this year. At current rates, we expect one out of every 6 will be seen in 
the UPMC/UPCI network. Since most of these patients are referred to UPCI for novel clinical 
investigations, we expect rapid accrual to the above protocol. In the last 2 years, over 125 
subjects/year have been accrued to clinical trials in the UPCI prostate cancer program. 
Potential subjects will be approached by clinicians who are directly involved in their care. ”Cold-
 
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calling” will not be used to recruit subjects and “Finder’s fees” for referring a potential subject 
for participation in a research study are prohibited. Physicians and other health care professionals 
in the area are aware of active studies at UPCI by means of various publications including the 
World Wide Web. Such publications and Web listings are not advertisements for specific 
studies. Rather, they are public listings of trials available. Subjects will not receive any monetary 
compensation for participation. 
5 STUDY DESIGN AND METHODS 
This is a 2 group crossover trial in which subjects are randomly assigned to one of two treatment 
arms: 
Cohort A: 3 months of androgen ablation (AA) to be followed at PSA progression by 3 months 
of the combination of AA and alpha-type 1 dendritic cell vaccine (DC1). 
Cohort B: 3 months of the combination of AA and alpha-type 1 dendritic cell vaccine followed 
at PSA progression by 3 months of AA. 
In this crossover trial each subject will serve as their own control. Following either therapy the 
time to PSA progression is defined as the time between treatment and the first instance of PSA 
increase to 1 ng/mL. The endpoint is the difference between the time to PSA progression for the 
combination of AA + DC1 compared to AA alone. A total of 12 evaluable subjects (6 
subjects/arm) will be enrolled on the trial. Subjects who do not complete both courses (AA and 
AA+DCV) will be replaced. This schema will also help us better estimate the time to PSA 
recovery following 3 months of limited androgen ablation in our cohort of subjects. 
All subjects in Cohort B will commence αDC1-based vaccination 2 weeks prior to treatment 
with the LHRH analogue. Each subject will receive 1 intradermal (i.d.) dose of the vaccine at 
weeks 1, 5, 9, and 13 for a total of 4 doses. The first dose will be considered the ‘priming’ dose 
and injections on weeks 5, 9, and 13 are ‘booster’ doses. Depending upon cells/volume the 
injections may utilize 1-2 syringes being administered on a single day. Additional courses of 
vaccination, if available after the 4 intended protocol doses, can be administered to any subject 
without evidence of disease progression, every 3 months (±1 month) for up to 12 months. The 
schema for the vaccine can be found on page 6. 
The LHRH analogue (Lupron 22.5 mg or Zoladex 10.8 mg) will be administered 2 weeks after 
the 1st dose of the DC vaccine.  
We assume that 3 months of AA alone will be followed by PSA progression at a median of 6 
months with 90% of PSA recovery times between 5 and 7 months. We suspect that a second 
course of AA will delay time to testosterone recovery by approximately 10% and 
that testosterone recovery and PSA failure will likely correlate very closely. Therefore we 
anticipate that a second course of AA will delay time to PSA relapse by approximately 10%. The 
purpose of randomizing to one of two treatment sequences is to permit an unbiased estimate of 
 
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the improvement due to DC1 treatment effect in the presence of any 2nd course variability. In this 
study, on treatment PSA-relapse is defined as a PSA ≥ 1.0. We consider the clinically significant 
objective to be a within-subject delay in PSA progression time due to the addition of DC1 by 
30% or to 7.8 months. 
Our estimates of time to testosterone and PSA recovery are based on a recent study that we 
participated in (Figg et al, Lancet Oncology [in press]; Figg et al, ASCO Proceedings, 2008, 
Abstract #5015). 
6 TREATMENT PLAN 
Subjects will undergo a thorough pre-study evaluation (see below), and then undergo 
leukapheresis to generate the DC-based vaccine. Each subject will receive 1 dose of intradermal 
(i.d.) αDC1-based vaccine at weeks 1, 5, 9, and 13 for a total of 4 doses.  
6.1 
Leukapheresis 
Leukapheresis is a minimal risk procedure. All selected subjects will undergo a single 90-120 
minute-long limited leukapheresis before vaccination. The product is delivered immediately to 
the University of Pittsburgh Cancer Institute Immunologic Monitoring and Cellular Products 
Laboratory from the UPCI Outpatient Clinic pheresis area. Leukapheresed product will be 
immediately processed as described in the laboratory SOP. The product will be cryopreserved as 
described in the Chemistry, Manufacturing and Control section of the IND application. If 
cytopenia (WBC < 2000/mm3, or platelets < 40,000/mm3) develops during, or as a result of, 
leukapheresis, the procedure will be postponed until recovery. Samples from each cell product 
will be obtained for hemoglobin, hematocrit, total WBC and differential, and platelet count. 
6.2 
DC Vaccine Administration 
Each dose will consist of a single allogeneic tumor cell vaccine administered intradermally using 
1-2 1-mL syringe(s) and needle appropriate for intradermal injections and will hold a total of 
0.5 mL of the vaccine preparation. The allogeneic tumor dendritic cell vaccine will be injected in 
the vicinity of the major nodal basins of the thigh or arm. The nodal basin must not have been 
dissected. 
• 
Week 1: Both thighs - 1.5 to 2.5 x 106* cells on each side (target: 3-5 x 106 cells total) 
• 
Week 5: Both arms - 1.5 to 2.5 x 106* cells on each side (target: 3-5 x 106 cells total) 
• 
Week 9: Both thighs - 1.5 to 2.5 x 106* cells on each side (target: 3-5 x 106 cells total) 
• 
Week 13: Both arms - 1.5 to 2.5 x 106* cells on each side (target: 3-5 x 106 cells total) 
* If the cell count is lower, the total number of cells will be divided equally between the 2 sites. However, at the Investigators discretion, the 
vaccine may have to be repeated if the cell count is less than< 1.5 x 106/per site. 
 
In the event of a prior node dissection, the priming dose will be administered in the arms.  
The vaccine will be administrated by the Investigator/Sub-investigator or a member of the 
advanced practice staff. 
 
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Subjects may undergo additional vaccinations, every 3 months (± 1 month) if they demonstrate 
lack of disease progression on week 16 (or later) and if they have vaccine remaining. 
6.3 
Dose Limiting Toxicities 
This study will utilize the NCI CTCAE version 4.0 for toxicity and Adverse Event (AE) 
reporting (http://ctep.cancer.gov/forms/). 
The following, if they occur and are felt to be attributable to the investigational treatment, are 
considered to be dose limiting toxicities (DLT). Any subject experiencing one of these will be 
taken off study and no further injections will be given: 
• 
≥ Grade 2 or greater bronchospasm, 
• 
≥ Grade 2 or greater allergic reaction or generalized urticaria, 
• 
≥ Grade 2 or greater autoimmune disease 
• 
≥ Grade 3 injection site reaction due to vaccine, implanted port, or catheter 
• 
> Grade 3 hematologic and non-hematologic toxicities 
• 
Infection, local or systemic 
Management associated with the above adverse events is outlined below. Subject accrual will 
cease if 2 subjects have a DLT related to vaccine administration. The observation period for 
DLTs will be 1 week. 
6.4 
Study Discontinuation Criteria 
Subjects MUST be discontinued from study therapy AND withdrawn from the study for the 
following reasons: 
• 
Withdrawal of informed consent (subject’s decision to withdraw for any reason) 
• 
Any clinical adverse event, laboratory abnormality or intercurrent illness which, in the 
opinion of the Investigator/Sub-Investigator, indicates that treatment with study therapy 
is not in the best interest of the subject 
• 
Pregnancy: All WOCBP should be instructed to contact the Investigator/Sub-Investigator 
immediately if they suspect they might be pregnant (e.g., missed or late menstrual period) 
at any time during study participation. 
• 
Investigator decides to close the clinical trial prematurely. 
6.5 
Supportive Care Guidelines 
Toxicities of the vaccination itself are expected to be minimal. For fever, acetaminophen will be 
utilized (325 mg tabs, 1 or 2 by mouth every 4 hours). Fevers lasting more than 8 hours after 
treatment will be evaluated in terms of potential infection. For mild, local pain, oral opiates will 
be planned (oxycodone, 5-10 mg by mouth every 3-4 hours). Pain that is of more than mild-
moderate grade will be investigated for sources other than the therapy, and managed accordingly. 
In case of the occurrence of the acute hypersensitivity reactions, administration of IV 
antihistamines, corticosteroids, and epinephrine may also be required. Any cases of infection will 
 
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be treated with antibiotics, according to established guidelines. 
6.6 
Duration of study therapy 
In the absence of treatment delays due to adverse events, treatment will continue for at least 4 
doses or until one of the following criteria applies: 
• 
Disease progression, 
• 
Intercurrent illness that prevents further administration of treatment, 
• 
Unacceptable adverse event(s), 
• 
Severe reaction to the DTH testing with the tumor cell lysate prior to vaccination, 
• 
Subject decides to withdraw from the study, or 
• 
General or specific changes in the subject's condition rendering the subject unacceptable 
for further treatment in the judgment of the investigator/sub-investigator. 
• 
Dosing delays > 4 weeks 
7 STUDY EVALUATIONS AND STUDY CALENDAR 
7.1 
Screening 
Baseline evaluations are to be conducted within 14 days prior to the leukapheresis .. Scans and x-
rays must be done within six weeks prior to the start of therapy. 
• 
Complete physical examination (with ECOG performance status (PS), weight, height, 
and BSA) 
• 
Radiologic imaging to evaluate the status of disease (all scans must be obtained within 30 
days of starting therapy): Includes bone scan and CT scan of chest (or CXR), abdomen, 
and pelvis. 
• 
Chemistry profile including: electrolytes, creatinine, blood urea nitrogen, glucose, SGOT, 
SGPT, total bilirubin, calcium, and albumin. 
• 
Serum testosterone 
• 
PSA levels in the blood (as a tumor marker). To be eligible, subjects must have at least 
two PSA levels checked at least 2 weeks apart, shown to be rising, and with the the last 
PSA being ≥ 2.0 ng/dL 
• 
CBC, differential, platelet 
• 
ANA Testing (if clinically indicated) 
• 
Known history of HIV, HbsAg, and Anti-HCV. For subjects at high risk of HIV/viral 
hepatitis, HIV/viral hepatitis testing will be performed to confirm status. 
• 
Following confirmation of eligibility, based on the above tests and procedures, the 
following, additional pre-treatment procedures will be performed: 
o 
Leukapheresis – for vaccine production and a portion will be obtained for in vitro 
assays (immune parameters of CD4+ and CD8+ T cell responses against tumor-
associated and tumor-irrelevant control antigens). 
 
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o 
DTH placement within 48-72 hours before the day of first dendritic cell vaccine 
administration (baseline testing) to allow for it to be read on the day of and prior to 
the 1st vaccine injection. 
7.2 
Evaluation during Treatment 
During dendritic cell vaccine cycles (weeks 1, 5, 9, and 13) the following will be performed: 
• 
Complete physical examination (with ECOG performance status and weight) 
• 
Laboratory tests including: CBC, differential, platelet, and chemistry profile each visit 
(see Section 7.1 and Study Calendar). 
• 
Blood for in vitro assays will be obtained within 1 week after each cycle of vaccination. 
• 
PSA at week 13 
7.3 
Post-Treatment 
• 
Complete history and physical examination. 
• 
For subjects with suspected disease progression on therapy, radiographic imaging as 
indicated. 
• 
PSA at week 17, 21, and every 4 weeks thereafter. Confirmatory PSA should be 
performed 1 month post progression 
• 
Testosterone at week 17, 25, and every 2 months thereafter 
• 
CBC, differential, platelets at week 25 and every 3 months thereafter 
• 
Chemistry profile (see Section 7.1) at week 25 and every 3 months thereafter 
• 
Blood for in vitro assays (immune parameters of CD4+ and CD8+ T cell responses 
against tumor-associated and tumor-irrelevant control antigens) will be obtained 1 week 
and 6 weeks after completion of treatment. The assays will involve the evaluation of 
cytokine production, cytotoxicity, and changes in cell phenotype of T cell subsets. 
• 
Leukapheresis - a second limited leukapheresis will be performed during week 19 to 
facilitate immune-monitoring. 
• 
DTH testing (post-testing: 2 to 4 weeks after last vaccine, i.e., during week 15 through 
17). The subjects will be asked to agree to the biopsies being taken from the sites of the 
DTH tests, following their readout. 
• 
EPIMAX analysis may be used to define the magnitude and cytokine production profiles 
of CD4+ and CD8+ T cell responses to the overlapping peptide libraries and individual 
peptides covering the entire span of PAP and PSMA molecules  
• 
Subjects with lack of disease progression at 12 months after the last treatment may be 
monitored for disease free survival, as well as for overall survival. 
7.4 
Long Term Follow-up 
Patients will continue to be followed after the last vaccine injection and/or progression based 
upon the timing of their standard of care for their disease. 
 
 
 
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7.5 
Study Calendar 
Screening evaluations are to be conducted within 14 days prior to leukapheresis. Scans and x-rays must be done within 6 weeks prior to 
the start of therapy. 
There is a window of ±1 week available for scheduling treatment and/or procedures at the discretion of the Investigator/Sub-investigator. 
This applies also if a course is missed or a subject's treatment and/or testing day(s) need to be rescheduled due to the subject’s inability to 
comply with the study calendar (i.e., hospitalizations, business, vacation plans, travel from long distances for study treatment, in advance 
of the scheduled date to allow ready access to the result(s), reduce financial burden on the subject [i.e. non-UPMC insurance coverage] or 
reduce travel inconvenience, illness, transportation issues, holidays, family emergencies, etc.). 
 
Screening 
Baseline 
Treatment 
Post-treatment 
Parameter / Week # 
-4 to -1 
 
1 
3 
5 
9 
13 
14 
15 
16 
17 
19 
21 
25 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
History, PE, ECOG PS 
X 
 
X 
 
X 
X 
X 
 
 
 
X 
 
X 
X 
Serum PSAg - every 4 weeks 
after 12 weeks of hormones 
X 
 
 
 
 
 
X 
 
 
 
X 
 
X 
X 
Serum Testosterone k 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
ANA testing (if clinically 
indicated) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV/viral hepatitis testing 
Xf 
 
 
 
 
 
 
 
 
 
 
 
 
 
CT scan and Bone Scan i 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC, platelets, differential 
X 
 
X 
 
X 
X 
X 
 
 
 
 
 
 
Xh 
Chemistry Profile 
X 
 
X 
 
X 
X 
X 
 
 
 
 
 
 
Xh 
Leukapheresis (90-120 
mins)e 
 
X 
 
 
 
 
 
 
 
 
 
Xd 
 
 
Skin test (DTH) 
 
Xb 
 
 
 
 
 
 
Xc 
 
Xc 
 
 
 
Vaccinationa 
 
 
X 
 
X 
X 
X 
 
 
 
 
Xa 
 
 
Blood (70 cc) for in vitro 
assays j 
 
 
X 
 
X 
X 
X 
 
 
 
 
 
 
 
 
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Screening 
Baseline 
Treatment 
Post-treatment 
Parameter / Week # 
-4 to -1 
 
1 
3 
5 
9 
13 
14 
15 
16 
17 
19 
21 
25 
LHRH Analogue 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
a. Subjects may undergo additional vaccinations, every 3 months (±1 month) if they demonstrate lack of disease progression on week 16 (or later) and if they have vaccine remaining.  
b. DTH placement should be performed within 48-72 hours prior to the day of the first dendritic cell vaccine administration to ensure the test can be read on the day and prior to the first vaccine 
administration. 
c. DTH testing (post-testing: within 2 – 4 weeks after last vaccine, i.e. week 15 through 17). At the discretion of the investigator/sub-investigator, subjects may be asked to agree to biopsies being taken from 
the sites of the DTH tests, following their readout. 
d. A second limited leukapheresis will be performed at the end of the study to facilitate immune-monitoring. 
e. EPIMAX analysis may be performed on blood samples obtained with the leukapheresis procedure. 
f. When clinically indicated, HIV/viral hepatitis testing will be performed to confirm disease status. 
g. Confirmatory PSA should be performed 1 month post progression. 
h. CBC and Chemistry profile every 3 months after week 25. 
i. Scans must be obtained within 6 weeks of starting therapy. For subjects with suspected disease progression on therapy, radiographic imaging as indicated during post-treatment. 
j. Blood for in vitro assays will be obtained within one week after each cycle of vaccination. 
k. Testosterone at week 17, 25, and every 2 months thereafter. 
 
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8 MEASUREMENT OF EFFECT 
8.1 
Efficacy endpoints 
• 
The primary efficacy endpoint is to evaluate the effect of the alpha-DC1 vaccine on time 
to PSA progression. PSA progression is defined as a rise in the PSA value to 
> 1.0 ng/mL. 
• 
The secondary efficacy objectives include: 
o 
To determine the change in PSA velocity prior to and following the proposed 
treatment, 
o 
To estimate time to development of metastatic disease, 
o 
To evaluate the immunologic response to the vaccine in all subjects, 
o 
To evaluate the immune response to HLA-A2.1 restricted peptides derived from 
PAP and PSMA in subjects who are A2.1 positive, 
o 
EPIMAX analysis will be used to define the magnitude and cytokine production 
profiles of CD4+ and CD8+ T cell responses to the overlapping peptide libraries and 
individual peptides covering the entire span of PAP and PSMA molecules. 
8.2 
Safety endpoints 
The primary safety endpoint is to assess the tolerability and toxicity of the alpha-DC1 vaccine. 
8.3 
Evaluation of Clinical Responses 
The primary clinical endpoint is the time to PSA progression. Since all subjects will be treated 
with androgen ablation for 3 months, the expectation is that at the outset all subjects will have 
undetectable PSA levels. As subjects recover their testosterone levels (in 3 to 6 months), they are 
expected to develop a PSA relapse (5 to 7 months). 
Thus time to progression (TTP) will be defined as time from starting of androgen ablation (week 
2) to time of either developing PSA progression (see below) or the development of metastatic 
disease (whichever occurs sooner). Typically PSA progression will preceed the development of 
metastatic disease. 
Progressive Disease: All subjects are expected to have a PSA < 0.1 following androgen ablation. 
Thus PSA progression will be defined as a PSA rise of at least 1.0 ng/dL, measured at least 
twice, with the two values being at least 1 month apart. Changes in PSA below 1.0 ng/dL will be 
evaluated for the PSA slope and PSADT. 
PSA Slope:The change in PSA will be graphically depicted and a PSA slope calculated. The 
change in PSA slope (and PSADT) pre-treatment, during treatment, and off- treatment will be 
determined to see if vaccine therapy has any disease modifying effects, over and above 
castration. This end-point is exploratory, but may lead to continued interest in this agent if the 
PSA slope (or PSADT) is decreased during treatment. 
Clinical Progression: The appearance of new lesions on examination or radiographs (CT scan or 
 
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bone scan), or development of symptoms consistent with metastatic disease (i.e.bone pain). 
Identification of Lesions: All measurements should be taken and recorded in metric notation. All 
baseline evaluations should be performed as closely as possible to the beginning of treatment and 
never more than 4 weeks before randomization. A sum of the longest diameter (LD) for all target 
lesions will be calculated and reported as the sum LD. The sum LD is used as reference to assess 
progression.  
Clinical Progression free survival: This is the interval from entry to study to date of first 
documented lesion. 
8.4 
Evaluation of Safety 
The primary safety objective of this study is to evaluate the feasibility and safety of vaccination 
with type-1-polarized dendritic cells (αDC1) loaded with apoptotic allogeneic tumor and tumor-
unrelated “heterologous helper antigens”. Based on the past experience from the University of 
Pittsburgh Cancer Institute (UPCI) and other clinical centers performing DC-based vaccinations, 
our hypothesis is that the vaccination with DC will be well-tolerated, and not associated with 
serious adverse side effects. 
8.5 
Measurement of the Immunologic Effects 
The immunologic endpoints of this study will include an in vivo assessment of DTH responses 
against the allogeneic tumor cell lysate, and (on additional sites) responses to the “heterologous 
helper antigen” KLH. In addition, we will perform the in vitro assessment of the increase in the 
frequency of circulating tumor-specific T-cells as determined by IFNγ ELISPOT against the 
allogeneic tumor cells used in the vaccine and in case of HLA-A2+ subjects against the 
individual TAAs relevant to prostate cancer (Table 1). All of the subjects will be tested for 
PMBC responses to KLH. To allow for these evaluations, 70 cc of peripheral blood will be 
drawn 4 times (1 x pre- and 3 times post-vaccination, see study calendar). As additional tertiary 
endpoints, we will perform and IFNγ and IL-5 ELISPOT, and cytotoxicity assays. EPIMAX 
analysis will be used to define the magnitude and cytokine production profiles of CD4+ and 
CD8+ T cell responses to the overlapping peptide libraries and individual peptides covering the 
entire span of PAP and PSMA molecules. 
8.5.1 Immunologic Assays 
• 
DTH against the allogeneic tumor cell lysate and (on additional sites) to the 
“heterologous helper antigen” KLH. Following the readout, the subjects will be asked for 
a separate consent for a biopsy of the DTH test site to perform the in situ and ex vivo 
analyses of the DTH-infiltrating T cells. 
• 
Cytokine production in ELISPOT assay (IFNγ/IL-5), using the cells isolated from 70 cc 
of peripheral blood will be used to detect the frequencies of IFNγ-producing CTLs. 
 
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• 
In all HLA-A2 subjects we will also test the responses to all individual TAAs identified 
in Table 1. 
• 
All of the subjects will be tested for the responses against the whole tumor cells, against 
KLH and against recombinant PAP and PSA proteins, in order to evaluate the overall 
increase and the anticipated vaccination-induced Th2 to Th1 shift of CD4+ Th cell 
responses. 
• 
EPIMAX analysis will be used to define the magnitude and cytokine production profiles 
of CD4+ and CD8+ T cell responses to PAP and PSMA molecules. The reactivity will be 
first tested against the pooled batches of the overlapping peptide libraries. Any “positive” 
pools will be subdivided into individual peptides, in order to define fine specificity of the 
responses. This analysis will allow us to cover all length of PSMA and PAP, in any 
subject, independently on HLA type, allowing us to detect immune responses to the 
previously-identified and potentially novel epitopes.  
• 
We will also analyze CTL cytotoxic activity [to additionally confirm the results of 
ELISPOT assays],  
• 
We will also monitor the overall numbers of CD4+ and CD8+ T cells, as well as the 
frequencies of undesirable CD4+/CD25+/FoxP3/GITR+/ Treg cells. In case of success, 
these additional novel parameters may be then included in the overall evaluation of this 
clinical study as additional tertiary end-points. 
8.5.2 DTH Testing 
Delayed-type hypersensitivity is an in vivo assessment of inflammatory Th1-type T-lymphocyte 
reactivity against specific antigens. It is the only in vivo assay currently available for the 
measurement of cellular immunity in humans. A positive DTH response against antigen at 
cutaneous injection sites will be defined as an increase of 3 mm of induration post dendritic cell 
vaccine over pre-dendritic cell vaccine. Vehicle only will be used as a negative control in both 
pre- and post- dendritic cell vaccine evaluations. At each time-point, individual separate sites 
will be injected with the allogeneic tumor cells used in the vaccine (100 µL in lysate form) and 
with KLH (100 µg KLH in 200 µL 0.9% sodium chloride). Intradermal injection of saline alone 
will be used as the negative controls (100 µL 0.9% sodium chloride).  Place 3 intradermal 
injections containing the following (from IMCPL lab): 
1)  100 µL of allogeneic tumor cells vaccine (cell line) 
2) 100 µg KLH in 200 µL 0.9% sodium chloride 
3) 100 µL 0.9% sodium chloride for Negative Control 
All testing will be performed on the anterior forearm separated by at least 2 cm in the vertical 
and horizontal axis.Tattooing of the DTH sites will be done using permanent marker to assure 
that the site in not injected twice. Skin tests will be read at 48-72 hours by trained clinical 
personnel. 
 
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DRUG FORMULATION AND PROCUREMENT 
8.6 
Alpha-1 Polarized Dendritic Cells Loaded with Allogeneic Apoptotic Cells 
Manufacture of the vaccine will take place in the IMPCL, accordingly to the cGMP-compliant 
procedures. 
8.7 
LNCaP Cell Line 
The GMP grade cell line (See Appendix IV and the letter from our collaborator, Dr. Palucka) 
received from Baylor Institute is maintained by the UPCI IMCPL. 
8.8 
LHRH Analogue 
We will plan on using the q 3 month depot preparation of either Goserelin Acetate 10.8 mg 
(Zoladex) (NSC-606864) or ELIGARD® 22.5 mg (Lupron) for once every 3 month dosing (NDA 
#21-379). 
8.8.1 Goserelin Acetate 
Chemical Structure: pyro-Glu-His-Trp-Ser-Tyr-D-Ser (But)-Leu-Arg-Pro-Azgly-NH2 
Molecular Weight: 1269 daltons (as base) 
Solubility: Soluble in water, dimethylformamide, dimethylsulfoxide, 0.1 M hydrochloric acid and 
0.1 M sodium hydroxide 
Pharmacological Class: LHRH agonist 
Zoladex™ is supplied as a 10.8 mg solid depot formulation preloaded in a disposable syringe 
device mounted on a #14 gauge hypodermic needle. It should be administered subcutaneously 
every 12 weeks into the upper abdominal wall using sterile technique under the supervision of a 
physician. While a delay of a few days is permissible, every effort should be made to adhere to 
the q 12 week schedule. 
8.8.2 ELIGARD® 22.5 mg 
Active ingredient: Leuprolide acetate 
Chemical name: 5-oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-leucyl-L-leucyl-L-
arginyl-N-ethyl-L-prolinamide acetate 
Relative Molecular Weight: 1,209 
Physical Appearance: Lyophilized, white to off-white powder 
Solubility: Soluble in water and in acetic acid 
Pharmacological Class: Synthetic non-peptide GnRH agonist 
Eligard is provided as a powder and solvent for solution/suspension for injection (prolonged 
release), provided as 2 sterile, pre-filled, polypropylene syringes. One syringe contains the LA 
powder and the other syringe contains the Atrigel® Delivery System. When reconstituted, 
 
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ELIGARD® 22.5 mg injection is a white to off-white, opaque, viscous liquid. It should be 
administered once every 3 months. 
8.9 
KLH Protein 
This clinical-grade KLH (Immucothel), used as a “heterologous helper antigen”, is being 
provided by Biosyn. It has previously been approved for use in our concurrent melanoma trial: 
UPCI 03-118/BB-IND 11,754, IRB# IRB0507089). KLH will be loaded only on αDC1s used in 
the first cycle of vaccination (first vaccine) and will be used as a one of the readouts of the 
ability of the vaccines to induce DTH responses (see secondary endpoints): the strategy used in 
our currently active melanoma protocol UPCI 03-118. 
9 STATISTICAL CONSIDERATIONS 
9.1 
Study Design and Endpoints 
This is a 2 group crossover trial in which subjects are randomly assigned to one of two treatment 
arms: 
• 
3 months of androgen ablation (AA) to be followed at PSA progression by 3 months of 
the combination of AA and alpha-type 1 dendritic cell vaccine (DCV) 
• 
3 months of the combination of AA and alpha-type 1 dendritic cell vaccine followed at 
PSA progression by 3 months of AA. 
The primary objectives of this novel trial are to evaluate feasibility, safety, and a preliminary 
estimate of efficacy. Following either treatment arm the time to PSA progression, defined as the 
time between treatment and the first instance of PSA increase to 1.0 ng/mL, will be estimated 
from monthly PSA monitoring. The efficacy endpoint is the difference between time to PSA 
progression for the combination of AA + DCV compared to AA alone. 
9.2 
Number of Subjects 
For the feasibility objective, the goal is to evaluate the production of bulk vaccine, storage of 
vaccine, and subsequent administration. If this is successful in the first 4 subjects, we will go on 
to accrue a total of 12 subjects, 6 per arm, for safety testing, with 6 subjects per arm being 
consistent with a standard Phase I design. 
9.3 
Characteristics of the Design 
The difference in PSA progression between AA and AA + DCV will be calculated for each 
individual subject. We note that if the standard deviation of this difference is 1.5 months, a one 
tailed signed rank test at α = .05 will have 98% power to detect a improvement of 1.8 months, 
equivalent to a hypothesized 30% improvement in time to PSA recovery (6 months vs 7.8 
months). 
 
 
 
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9.4 
Safety Evaluation 
Serious adverse advents are uncommon in cancer vaccine trials. For example, no treatment-
related deaths or grade 4 toxicities have occurred among 652 subjects enrolled in Phase I studies 
by CTEP, NCI’s Cancer Therapy Evaluation Program (Horstmann et al., 2005). Rather than a 
separate phase of the study to characterize vaccine safety, we will use continual Bayesian 
monitoring of SAEs (Berry, 2006) with a stopping rule that permits the investigator to suspend 
the trial for review by the Data and Safety and Monitoring Committee (DSMC). We have 
selected prior probabilities of SAEs to conform to our prior beliefs that the probability of a 
treatment-related SAE, caused by αDC1 vaccine is as low as 5%. The prior probability 
distribution was selected to permit at most a 10% chance that this prior probability could be 
double the expected prior probability. Assuming the prior probability follows a beta distribution, 
the stopping rule will be based on the updated posterior probability of an SAE, which also has a 
beta distribution. As each subject is observed for an SAE, the posterior probability of an SAE 
can be calculated. If at any time during the trial the probability is ≥ .50 that the treatment-related 
SAE rate exceeds 5%, the study will be suspended pending review by the DMSC. In order to 
permit continuous monitoring of serious toxicities, a look-up table has been provided (below). 
This table is based upon cumulative observed SAEs at any point in the trial and can be used to 
signal when the therapy should be suspended and the trial results reviewed by the DSMC. 
TABLE 4. Number of Observed Treatment-Related SAEs and Associated Posterior 
Probabilities Needed to Suspend the Study 
Subjects 
Total 
Observed 
SAE’s 
Pr(π ≥ .05)* 
B(SAE)** 
1 
1 
.690 
.05 
2 
1 
.675 
.098 
3 
1 
.660 
.143 
4 
1 
.645 
.186 
5 
1 
.630 
.226 
6 
1 
.615 
.264 
7 
1 
.601 
.302 
8 
1 
.586 
.337 
9 
1 
.572 
.370 
10 
1 
.558 
.401 
11 
1 
.543 
.431 
12 
1 
.529 
.460 
π is the posterior probability of a treatment-related SAE. Pr(π ≥.05) is the chance that the posterior probability of a treatment-related SAE given 
the observed number of subjects treated and observed SAEs, exceeds .05. 
** B is the binomial probability of observing at least the number of SAEs in column 2 if the underlying rate of SAE is 5%. 
 
This table presents the minimum number of SAEs that dictate suspension of the trial in 
accordance with the stopping rule. According to this table a single SAE due to the αDC1-based 
vaccine among any of the 10 subjects will trigger suspension and review by the DSMC. 
 
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9.5 
Data Analysis 
Subjects will be required to have monthly PSA monitoring. This will enable linear interpolation 
between PSA samples to detect the time at which PSA progression occurs following each course 
of therapy. The efficacy endpoint is estimated by the difference between time to PSA 
progression for the AA+DCV arm and the AA arm. This difference will be tested for 
significance with a one tailed signed rank test. Other conventional estimates of clinical efficacy 
will also be calculated including PSA doubling time and PSA velocity. The PSA-DT will be 
calculated as follows: 
The logarithm of a minimum of two PSA measurements will be plotted against the time of each 
PSA measurement. If 3 or more measurements are available a linear regression equation will be 
estimated. Two sequential PSA measurements (actual or estimated by linear regression) will be 
denoted as PSA1 and PSA2. Denote their times of blood samples as t1 and t2, respectively. Thus 
the PSA doubling time is: log (2)[t2 - t1] / [log(PSA2) - log(PSA1)]. 
Further analysis will involve testing the immunologic response of subjects receiving the αDC1-
based vaccine. The principal measure of improved immunogenicity will be the comparison of pre 
to post αDC1-based vaccine T cell response to prostatic acid phosphatase (PAP) as measured by 
the IFN-γ Elispot assay. We expect that prostate cancer subjects undergoing androgen ablation in 
the absence of immune stimulation will have a negative or very low Elispot count for measuring 
reaction to PAP. Even with a nominal count of 5 spots in the group, a sample of 7 subjects with 
androgen ablation alone and 7 subjects treated with androgen ablation + αDC1-based vaccine 
will provide 90% power to detect a difference of double the Elispot count (average of 5 to 10 
spots) using a one tailed alpha level Wilcoxon test if the Elispot counts have a Poisson 
distribution. Additional immunologic testing will conducted by analyzing subject profiles before 
treatment and after each course of therapy. Immunologic endpoints include T cell reactivity by 
IFNγ or IL-5 Elispot, cytokine release assays for cytotoxicity and DTH tests (as measured by 
induration in mm). Changes over time will be tested with the Friedman Test or, if there are 
missing values, the Mack-Skillings procedure. 
9.6 
Subjects Randomization 
Subjects will be randomized through the email randomization program of the Biostatistics 
Facility of the UPCI. 
10 DATA SAFETY MONITORING PLAN 
Investigator/Sub-investigators, regulatory, CRS management, clinical research coordinators, 
clinical research associates, data managers, and clinic staff meet monthly in disease center Data 
Safety Monitoring Boards (DSMB) to review and discuss study data to include, but not limited 
to, the following: 
• serious adverse events 
 
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• subject safety issues 
• recruitment issues 
• accrual 
• protocol deviations 
• unanticipated problems 
• breaches of confidentiality 
 
All toxicities encountered during the study will be evaluated on an ongoing basis according to 
the NCI Common Toxicity Criteria version 4. All study treatment associated adverse events that 
are serious, at least possibly related and unexpected will be reported to the IRB. Any 
modifications necessary to ensure subject safety and decisions to continue, or close the trial to 
accrual are also discussed during these meetings. If any literature becomes available which 
changes the risk/benefit ratio or suggests that conducting the trial is no longer ethical, the IRB 
will be notified in the form of an Unanticipated Problem submission and the study may be 
terminated. 
All study data reviewed and discussed during these meetings will be kept confidential. Any 
breach in subject confidentiality will be reported to the IRB in the form of an Unanticipated 
Problem submission. The summaries of these meetings are forwarded to the UPCI DSMC which 
also meets monthly following a designated format. 
For all research protocols, there will be a commitment to comply with the IRB’s policies for 
reporting unanticipated problems involving risk to subjects or others (including adverse events). 
DSMC progress reports, to include a summary of all serious adverse events and modifications, 
and approval will be submitted to the IRB at the time of renewal. 
Protocols with subjects in long-term (survival) follow-up or protocols in data analysis only, will 
be reviewed twice a year rather than monthly by the disease center DSMB. 
Both the UPCI DSMC as well as the individual disease center DSMB have the authority to 
suspend accrual or further investigate treatment on any trial based on information discussed at 
these meetings. 
All records related to this research study will be stored in a locked environment. Only the 
researchers affiliated with the research study and their staff will have access to the research 
records. 
11 ADVERSE EVENT REPORTING 
11.1 
Definitions 
Adverse event: Any untoward medical occurrence associated with the use of a drug in humans, 
whether or not considered drug related. 
Life-threatening adverse event or life-threatening suspected adverse reaction: An adverse event 
or suspected adverse reaction is considered "life-threatening" if, in the view of either the 
 
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investigator or sponsor, its occurrence places the patient or subject at immediate risk of death. It 
does not include an adverse event or suspected adverse reaction that, had it occurred in a more 
severe form, might have caused death. 
Serious adverse event or serious suspected adverse reaction: An adverse event or suspected 
adverse reaction is considered "serious" if, in the view of either the investigator or sponsor, it 
results in any of the following outcomes: death, a life-threatening adverse event, inpatient 
hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity or 
substantial disruption of the ability to conduct normal life functions, or a congenital 
anomaly/birth defect. Important medical events that may not result in death, be life-threatening, 
or require hospitalization may be considered serious 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. Examples of such medical 
events include allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
Suspected adverse reaction: 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. Suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
Unexpected adverse event or unexpected suspected 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, as amended. For example, 
under this definition, hepatic necrosis would be unexpected (by virtue of greater severity) if the 
investigator brochure referred only to elevated hepatic enzymes or hepatitis. Similarly, cerebral 
thromboembolism and cerebral vasculitis would be unexpected (by virtue of greater specificity) 
if the investigator brochure listed only cerebral vascular accidents. "Unexpected," as used in this 
definition, 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. 
11.2 
Review of Safety Information: Sponsor Responsibilities1 
The sponsor must promptly review all information relevant to the safety of the drug obtained or 
1 21CFR Sec.312.50 
 
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otherwise received by the sponsor from foreign or domestic sources, including information 
derived from any clinical or epidemiological investigations, animal or in vitro studies, reports in 
the scientific literature, and unpublished scientific papers, as well as reports from foreign 
regulatory authorities and reports of foreign commercial marketing experience for drugs that are 
not marketed in the United States. 
11.3 
Review of Safety Information: Investigator Responsibilities2 
An investigator shall promptly report to the sponsor of the IND application any adverse effect 
that may reasonably be regarded as caused by, or probably caused by, the drug. If the adverse 
effect is alarming, the investigator shall report the adverse effect immediately. An investigator 
shall provide the sponsor with an adequate report shortly after completion of the investigator's 
participation in the investigation. 
11.4 
IND safety reports 
The sponsor must notify FDA and all participating investigators (i.e., all investigators to whom 
the sponsor is providing drug under its INDs or under any investigator's IND) in an IND safety 
report of potential serious risks, from clinical trials or any other source, as soon as possible, but 
in no case later than 15 calendar days after the sponsor determines that the information qualifies 
for reporting under Sections 12.4.1 to 12.4.4 below. In each IND safety report, the sponsor must 
identify all IND safety reports previously submitted to FDA concerning a similar suspected 
adverse reaction, and must analyze the significance of the suspected adverse reaction in light of 
previous, similar reports or any other relevant information. 
11.4.1 Serious and unexpected suspected adverse reaction 
The sponsor must report any suspected adverse reaction that is both serious and unexpected. The 
sponsor must report an adverse event as a suspected adverse reaction only if there is evidence to 
suggest a causal relationship between the drug and the adverse event, such as: 
• 
A single occurrence of an event that is uncommon and known to be strongly associated 
with drug exposure (e.g., angioedema, hepatic injury, Stevens-Johnson Syndrome); 
• 
One or more occurrences of an event that is not commonly associated with drug 
exposure, but is otherwise uncommon in the population exposed to the drug (e.g., tendon 
rupture); 
• 
An aggregate analysis of specific events observed in a clinical trial (such as known 
consequences of the underlying disease or condition under investigation or other events 
that commonly occur in the study population independent of drug therapy) that indicates 
those events occur more frequently in the drug treatment group than in a concurrent or 
historical control group. 
 
 
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11.4.2 Findings from other studies 
The sponsor must report any findings from epidemiological studies, pooled analysis of multiple 
studies, or clinical studies (other than those reported under section 12.4.1), whether or not 
conducted under an IND, and whether or not conducted by the sponsor, that suggest a significant 
risk in humans exposed to the drug. Ordinarily, such a finding would result in a safety-related 
change in the protocol, informed consent, investigator brochure (excluding routine updates of 
these documents), or other aspects of the overall conduct of the clinical investigation. 
11.4.3 Findings from animal or in vitro testing 
The sponsor must report any findings from animal or in vitro testing, whether or not conducted 
by the sponsor, that suggest a significant risk in humans exposed to the drug, such as reports of 
mutagenicity, teratogenicity, or carcinogenicity, or reports of significant organ toxicity at or near 
the expected human exposure. Ordinarily, any such findings would result in a safety-related 
change in the protocol, informed consent, investigator brochure (excluding routine updates of 
these documents), or other aspects of the overall conduct of the clinical investigation. 
11.4.4 Increased rate of occurrence of serious suspected adverse reactions 
The sponsor must report any clinically important increase in the rate of a serious suspected 
adverse reaction over that listed in the protocol or investigator brochure. 
11.4.5 Submission of IND safety reports 
The sponsor must submit each IND safety report in a narrative format or on FDA Form 3500A or 
in an electronic format that FDA can process, review, and archive. FDA will periodically issue 
guidance on how to provide the electronic submission (e.g., method of transmission, media, file 
formats, preparation and organization of files). The sponsor may submit foreign suspected 
adverse reactions on a Council for International Organizations of Medical Sciences (CIOMS) I 
Form instead of a FDA Form 3500A. Reports of overall findings or pooled analyses from 
published and unpublished in vitro, animal, epidemiological, or clinical studies must be 
submitted in a narrative format. Each notification to FDA must bear prominent identification of 
its contents, i.e., "IND Safety Report," and must be transmitted to the review division in the 
Center for Drug Evaluation and Research or in the Center for Biologics Evaluation and Research 
that has responsibility for review of the IND. Upon request from FDA, the sponsor must submit 
to FDA any additional data or information that the agency deems necessary, as soon as possible, 
but in no case later than 15 calendar days after receiving the request. 
11.4.5.1 
Unexpected fatal or life-threatening suspected adverse reaction reports 
The sponsor must also notify FDA of any unexpected fatal or life-threatening suspected adverse 
reaction as soon as possible but in no case later than 7 calendar days after the sponsor's initial 
receipt of the information. 
 
 
 
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11.4.5.2 
Reporting format or frequency 
FDA may require a sponsor to submit IND safety reports in a format or at a frequency different 
than that required under this paragraph. The sponsor may also propose and adopt a different 
reporting format or frequency if the change is agreed to in advance by the director of the FDA 
review division that has responsibility for review of the IND. 
11.4.5.3 
Investigations of marketed drugs 
A sponsor of a clinical study of a drug marketed or approved in the United States that is 
conducted under an IND is required to submit IND safety reports for suspected adverse reactions 
that are observed in the clinical study, at domestic or foreign study sites. The sponsor must also 
submit safety information from the clinical study as prescribed by the post marketing safety 
reporting requirements (e.g., 310.305, 314.80, and 600.80 of this chapter). 
11.4.5.4 
Reporting study endpoints 
Study endpoints (e.g., mortality or major morbidity) must be reported to FDA by the sponsor as 
described in the protocol and ordinarily would not be reported under paragraph (c) of this 
section. However, if a serious and unexpected adverse event occurs for which there is evidence 
suggesting a causal relationship between the drug and the event (e.g., death from anaphylaxis), 
the event must be reported under Serious and unexpected suspected adverse reaction as a serious 
and unexpected suspected adverse reaction even if it is a component of the study endpoint (e.g., 
all-cause mortality). 
11.4.6 Follow-up 
• 
The sponsor must promptly investigate all safety information it receives. 
• 
Relevant follow-up information to an IND safety report must be submitted as soon as the 
information is available and must be identified as such, i.e., "Follow-up IND Safety 
Report." 
• 
If the results of a sponsor's investigation show that an adverse event not initially 
determined to be reportable under Section 12.4 is so reportable, the sponsor must report 
such suspected adverse reaction in an IND safety report as soon as possible, but in no 
case later than 15 calendar days after the determination is made. 
11.4.7 Disclaimer 
A safety report or other information submitted by a sponsor under this part (and any release by 
FDA of that report or information) does not necessarily reflect a conclusion by the sponsor or 
FDA that the report or information constitutes an admission that the drug caused or contributed 
to an adverse event. A sponsor need not admit, and may deny, that the report or information 
submitted by the sponsor constitutes an admission that the drug caused or contributed to an 
adverse event. 
 
 
 
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11.5 
Reporting adverse events to the responsible IRB 
In accordance with applicable policies of the University of Pittsburgh Institutional Review Board 
(IRB), the Sponsor-Investigator will report, to the IRB, any observed or volunteered adverse 
event that is determined to be 1) associated with the investigational drug or study treatment(s); 
2) serious; and 3) unexpected. Adverse event reports will be submitted to the IRB in accordance 
with the respective IRB procedures. 
Applicable adverse events will be reported to the IRB as soon as possible and, in no event, later 
than 10 calendar days following the sponsor-investigator’s receipt of the respective information. 
Adverse events which are 1) associated with the investigational drug or study treatment(s); 2) 
fatal or life-threatening; and 3) unexpected will be reported to the IRB within 24 hours of the 
Sponsor-Investigator’s receipt of the respective information. 
Follow-up information to a reported adverse event will be submitted to the IRB as soon as the 
relevant information is available. If the results of the Sponsor-Investigator’s follow-up 
investigation show that an adverse event that was initially determined to not require reporting to 
the IRB does, in fact, meet the requirements for reporting; the Sponsor-Investigator will report 
the adverse event to the IRB as soon as possible, but in no event later than 10 calendar days, after 
the determination was made. 
12 QUALITY CONTROL AND QUALITY ASSURANCE 
Independent monitoring of the clinical study for protocol and GCP compliance will be conducted 
periodically (i.e., at a minimum of annually) by qualified staff of the Education and Compliance 
Office – Human Subject Research, Research Conduct and Compliance Office, University of 
Pittsburgh. 
The Sponsor-Investigator and the University of Pittsburgh and UPMC will permit direct access 
of the study monitors and appropriate regulatory authorities to the study data and to the 
corresponding source data and documents to verify the accuracy of this data. 
13 DATA HANDLING AND RECORD-KEEPING 
The Investigator will maintain records in accordance with Good Clinical Practice guidelines. 
The Investigator will retain the specified records and reports for up to 2 years after the marketing 
application is approved for the investigational drug; or, if a marketing application is not 
submitted or approved for the investigational drug, until 2 years after investigations under the 
IND have been discontinued and the FDA so notified. 
14 ETHICS 
14.1 
Institutional Review Board (IRB) approval 
The Investigator will obtain, from the University of Pittsburgh Institutional Review Board (IRB), 
prospective approval of the clinical protocol and corresponding informed consent form(s); 
 
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modifications to the clinical protocol and corresponding informed consent forms, and 
advertisements (i.e., directed at potential research subjects) for study recruitment.  
The only circumstance in which a deviation from the current IRB-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective IRB approval is to 
eliminate an apparent immediate hazard to the research subject(s). In such circumstances, the 
Investigator will promptly notify the University of Pittsburgh IRB of the deviation.  
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 CFR Parts 
50 and 21 CFR 56, and in conformance with applicable International Conference on 
Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
In the event that the University of Pittsburgh IRB requires, as a condition of approval, substantial 
changes to a clinical protocol submitted under an FDA-accepted IND application, or in the event 
of the Sponsor’s decision to modify the previously accepted clinical protocol: 
The Investigator will submit (i.e., in advance of implementing the change) a Protocol 
Amendment to the IND describing any change to the Phase I clinical protocol that significantly 
affects the safety of the subjects. For changes that do not affect critical safety assessments, the 
revisions to the clinical protocol will be addressed in the Annual Report to the IND. 
14.2 
Ethical and scientific conduct of the clinical research study 
The clinical research study will be conducted in accordance with the current IRB-approved 
clinical protocol; ICH GCP Guidelines adopted by the FDA; and relevant policies, requirements, 
and regulations of the University of Pittsburgh IRB, University of Pittsburgh and UPMC, 
Commonwealth of Pennsylvania, and applicable federal agencies. 
14.3 
Subject informed consent 
The Investigator will make certain that an appropriate informed consent process is in place to 
ensure that potential research subjects, or their authorized representatives, are fully informed 
about the nature and objectives of the clinical study, the potential risks and benefits of study 
participation, and their rights as research subjects. The Investigator, or a sub-investigator(s) 
designated by the Investigator, will obtain the written, signed informed consent of each subject, 
or the subject’s authorized representative, prior to performing any study-specific procedures on 
the subject. The date and time that the subject, or the subject’s authorized representative, signs 
the informed consent form and a narrative of the issues discussed during the informed consent 
process will be documented in the subject’s case history. The Investigator will retain the original 
copy of the signed informed consent form, and a copy will be provided to the subject, or to the 
subject’s authorized representative. 
The Investigator will make certain that appropriate processes and procedures are in place to 
ensure that ongoing questions and concerns of enrolled subjects are adequately addressed and 
 
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that the subjects are informed of any new information that may affect their decision to continue 
participation in the clinical study. In the event of substantial changes to the clinical study or the 
risk-to-benefit ratio of study participation, the Investigator will obtain the informed consent of 
enrolled subjects for continued participation in the clinical study 
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APPENDIX I: SUMMARY OF PRECLINICAL DATA 
Based on a novel paradigm of polarization of dendritic cells 1,2, we have recently developed the first 
clinically-applicable protocol (serum-free conditions) to generate alpha-type-1 polarized DC (αDC1) 3. 
Such αDC1s are the first clinically-applicable type of DCs that combine all 3 properties deemed 
important for their in vivo activity as inducers of anticancer immunity. These are: 1) fully-mature 
status; 2) high migratory responsiveness to lymph node-produced chemokines; and 3) high IL-12p70-
producing function (Fig 1). Such combination was missing in all previously-applied protocols of 
preparing DC, where final DC maturation (needed to generate DC with high expression of costimulatory 
molecules and the ability to localize in the T cell areas of the lymph nodes) was associated with 
irreversible loss of their ability to produce the key Th1-, CTL-, and NK cell-activating cytokine with anti-
cancer properties, IL-12 4,5.  
 
Compared to the current “gold standard” of clinically-used DCs, αDC1s show selectively enhanced 
ability to induce type-1 anti-cancer responses, when. Our studies in the melanoma model have shown that 
peptide-pulsed DC1s induce up to 40-fold higher numbers of tumor-specific CTLs, during in vitro 
sensitization of peripheral blood T cells from the patients, and induce strong antitumor activity of NK 
cells (Ref 3 and  Fig. 2). 
 
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Importantly for wider clinical applications of DC-based vaccines, we could also show high ability of 
αDC1 to cross-present apoptotic tumor (Fig. 3), an important factor allowing to use as a source of 
tumor-relevant antigens whole tumor cells, rather than defined peptide epitopes, broadening the spectrum 
of tumor-relevant targets of immunization and eliminating the need to restrict the study to patients with a 
defined MHC type (usually HLA-A2). This last property of DC1s also facilitates the use of either 
autologous tumor or allogeneic prostate cancer cell lines as a source of antigenic material. Allogeneic cell 
lines offer the advantage of standardized preparation of the vaccine, and a potential possibility to further 
boost the immunogenic properties of vaccine-carrying DCs by promoting their interaction in vivo with 
high numbers of allo-specific CD8+ T cells 6 and NK cells 7 in tumor-bearing hosts. 
 
Until recently, our effort concentrated on using this strategy to develop more effective vaccines against 
skin cancers: malignant melanoma and cutaneous T cell lymphoma. These studies, performed in 
collaboration with the groups of Dr. Walter Storkus, Dr. Michael Lotze, Dr. Luis Falo, and Dr. John 
Kirkwood, resulted in the design and FDA approval (BB-IND 11,754) a clinical protocol in melanoma 
(UPCI 03-118). In the meantime, our collaborators from the Department of Dermatology (Drs. L. Geskin 
and L. Falo) used our platform to vaccinate patients with advanced cutaneous T cell lymphoma (Sezary 
Syndrome), observing lack of serious side effects and the ability of tumor-loaded αDC1s to induce 
clinical responses. While our past attempts concentrated on the development of the clinical trials with the 
immunologic primary efficacy endpoint, the recently established collaboration between our two groups 
(of Dr. Pawel Kalinski and Dr. Gurkamal Chatta), allow us to extend our unique vaccination platform 
(developed and currently used exclusively in the University of Pittsburgh Cancer Institute) in prostate 
cancer: the leading cause of cancer-related deaths in men in the US.  
0%
10%
20%
30%
40%
50%
60%
70%
80%
pre-act.
(sDC)
pre-act.
(aDC1)
NK alone
% of maximal chromium release
Figure 2. αDC1s are potent inducers of tumoricidal activities in immune cells. 
A, B,: Superior induction of CTL responses. Control DC (IL-1/TNF/IL-
6/PGE2-matured or αDC1 (generated in serum-free AIM-V and matured by IL-
1β/TNFα/pI:C/IFNα/IFNγ) were pulsed with melanoma-associated antigenic 
peptides (MART-1, gp100, tyrosinase)and used to sensitize autologous CD8+ T 
cells from HLA-A2+ melanoma patients. The induction of peptide-specific CTL 
responses was evaluated after 28 days, using IFN-g ELISPOT, using peptide 
pulsed T2 cells (A) or chromium release assay (B) using 51Cr-labeled T2 cells 
either pulsed with the mixture of all of the vaccine-relevant peptides (top) or with 
irrelevant peptide (Flu, bottom). Similar data was obtained  using whole A2+ 
FEM-X melanoma cells as targets. C. Superior NK cell-activating potential of 
αDC1s. Cytotoxic activity of NK cells against chromium-labeled target cells 
(Daudi), either without prestimulation or prestimulated overnight by αDC1 and 
DCs. 4 hour-long chromium release assay at 10:1 T:T ratio. Data from one of two 
experiments that both showed similar results. 
0
10
20
30
40
5
15
50
0
10
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30
40
5
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cDC
DC1
% Target Lysis
CD8 T cell : Target Ratio
relevant peptide-pulsed T2
irrelevant peptide-pulsed T2
Numbers of IFNγ-producing CD8+ T cells
Donor 51
0
250
500
750
1000
0
1000
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0
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Donor 52
Donor 53
Induced by cDC      induced by αDC1
C.
A.
B.
0%
10%
20%
30%
40%
50%
60%
70%
80%
pre-act.
(sDC)
pre-act.
(aDC1)
NK alone
% of maximal chromium release
Figure 2. αDC1s are potent inducers of tumoricidal activities in immune cells. 
A, B,: Superior induction of CTL responses. Control DC (IL-1/TNF/IL-
6/PGE2-matured or αDC1 (generated in serum-free AIM-V and matured by IL-
1β/TNFα/pI:C/IFNα/IFNγ) were pulsed with melanoma-associated antigenic 
peptides (MART-1, gp100, tyrosinase)and used to sensitize autologous CD8+ T 
cells from HLA-A2+ melanoma patients. The induction of peptide-specific CTL 
responses was evaluated after 28 days, using IFN-g ELISPOT, using peptide 
pulsed T2 cells (A) or chromium release assay (B) using 51Cr-labeled T2 cells 
either pulsed with the mixture of all of the vaccine-relevant peptides (top) or with 
irrelevant peptide (Flu, bottom). Similar data was obtained  using whole A2+ 
FEM-X melanoma cells as targets. C. Superior NK cell-activating potential of 
αDC1s. Cytotoxic activity of NK cells against chromium-labeled target cells 
(Daudi), either without prestimulation or prestimulated overnight by αDC1 and 
DCs. 4 hour-long chromium release assay at 10:1 T:T ratio. Data from one of two 
experiments that both showed similar results. 
0
10
20
30
40
5
15
50
0
10
20
30
40
5
15
50
cDC
DC1
% Target Lysis
CD8 T cell : Target Ratio
relevant peptide-pulsed T2
irrelevant peptide-pulsed T2
0
10
20
30
40
5
15
50
0
10
20
30
40
5
15
50
cDC
DC1
% Target Lysis
CD8 T cell : Target Ratio
relevant peptide-pulsed T2
irrelevant peptide-pulsed T2
Numbers of IFNγ-producing CD8+ T cells
Donor 51
0
250
500
750
1000
0
1000
2000
3000
4000
0
250
500
750
1000
Donor 52
Donor 53
Induced by cDC      induced by αDC1
Numbers of IFNγ-producing CD8+ T cells
Donor 51
0
250
500
750
1000
0
1000
2000
3000
4000
0
250
500
750
1000
0
250
500
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1000
0
1000
2000
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0
250
500
750
1000
0
250
500
750
1000
Donor 52
Donor 53
Induced by cDC      induced by αDC1
C.
A.
B.
IFNγ-spots / 100,000 CD8+ T cells (IVS)
0
250
500
750
0
1000
2000
3000
αDC1
iDC
T2/MART-1
FEM-X 
Figure 3. αDC1 effectively cross-present apoptotic tumor cells to 
CD8+ T cells αDC1 matured in the presence of UV-irradiated 
apoptotic tumor cells (FEM-X) were used to sensitize CD8+ T cells 
from HLA-A2+ donor Priming with immature DC was used as a 
negative control. Frequencies of CD8+ T cells responsive to FEM-X 
cells, or to MART-1 (27-37 epitope) pulsed on T2 cells were determined 
by IFNγ ELISPOTs. 
IFNγ-spots / 100,000 CD8+ T cells (IVS)
0
250
500
750
0
1000
2000
3000
0
250
500
750
0
1000
2000
3000
αDC1
iDC
T2/MART-1
FEM-X 
Figure 3. αDC1 effectively cross-present apoptotic tumor cells to 
CD8+ T cells αDC1 matured in the presence of UV-irradiated 
apoptotic tumor cells (FEM-X) were used to sensitize CD8+ T cells 
from HLA-A2+ donor Priming with immature DC was used as a 
negative control. Frequencies of CD8+ T cells responsive to FEM-X 
cells, or to MART-1 (27-37 epitope) pulsed on T2 cells were determined 
by IFNγ ELISPOTs. 
 
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Our data demonstrate the feasibility of inducing αDC1s from the monocytes of prostate cancer 
patients. As shown in Fig. 4, αDC1s from such patients share fully mature status (CD86high, CD83+, 
CCR7+ phenotype) with the elevated ability to produce high levels of IL-12p70 (bioactive form of IL-12). 
These features positively distinguish αDC1s from both immature DCs (CD86low, CD83-, CCR7- and IL-
12p70 intermediate) and from (CD86high, CD83+, CCR7+ but IL-12p70low) sDCs (see Fig 4). 
Numerous tumor-produced factors, including prostate-produced prostaglandin E2 (PGE2), an 
immunosuppressive factor produced by prostate cancer 8-18, are known to suppress the IL-12-producing 
capacity of maturing DCs2,19-21. On the other hand, our data with the non-clinical grade DC1s obtained in 
the presence of serum demonstrated their resistance to PGE2 
2, suggesting that such cells may be able to 
resist prostate cancer-associated immune suppression. In order to verify that the loading of αDC1s  with 
apoptotic LNCap will not prevent the development of high IL-12 producing function of αDC1s (generated 
under the clinically-desirable serum-free condition), we compared the expression of DC maturation 
markers and IL-12p70 producing capacity in αDC1s generated in the absence and in the presence of UV-
irradiated apoptotic LNCap cells. As shown in Fig 5, in accordance with our expectations, the presence of 
apoptotic LNCap cells did not prevent the development of αDC1s characterized by mature phenotype and 
sgtrongly elevated ability to produce bioactive IL-12p70. xpression to induce autoimmune prostatitis as a 
novel immunotherapy for prostate cancer, through dendritic cell-induced stimulation of CD8+ cytotoxic T 
cell (CTL) responses against prostate autoantigens. This is based on the premise that a vigorous 
autoimmune response against prostate-specific proteins, capable of destroying normal prostate tissue, will 
also destroy malignant prostate tissue, provided the malignant tissue expresses these proteins. Since the 
prostate gland is routinely removed or ablated as a part of the treatment for early prostate cancer, any 
remaining/recurring prostatic tissue could be destroyed immunologically. Three prostate tissue specific 
antigens are currently being evaluated in clinical trials: prostate specific antigen (PSA), prostatic acid 
phosphatase (PAP) and prostate specific membrane antigen (PSMA). However the immunogenicity of 
these proteins is not well understood, and a priori, there is no perfect prostate-specific immunologic 
target. Hence our focus on either autologous tumor (where available) or allogenic tumor cell lines. 
Additionally, since advanced prostate cancer in humans, is initially treated with androgen ablation, we 
will also incorporate limited (3 to 6 months) androgen deprivation (with LHRH analogues) in the 
treatment protocol. It has recently been reported that androgen ablation22 in patients with prostate cancer, 
induces a T cell infiltrate in to the prostate, and could potentially augment the immune response, 
following immunization.  
Our target population for treatment will be men with prostate cancer, who have relapsed following 
surgery and/or radiation, and now have a rising PSA with a PSA doubling time (PSA-DT) of less than 12 
 
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months. This patient population is ideal for an immunologic intervention given that: i) this is the largest 
sub-population of men with prostate cancer in the U.S.; ii) there is no standard treatment for this sub-
group; and iii) this sub-group has minimal systemic disease burden. We will carefully monitor 
immunological responses to vaccination at different time points, but the primary endpoint of vaccine 
efficacy will be stabilization of PSA levels or failure of the PSA to progress. Several recent studies have 
validated failure of PSA progression as a good surrogate endpoint for lack of progression in clinical trials 
in prostate cancer. 
Within the 12 months funding period of the currently-proposed CTDA, we will 1) develop the clinical 
protocol to test the safety and efficacy of αDC1-based vaccines for patients against advanced prostate 
cancer, will 2) discuss the trial design with our PRC and with FDA (in form of a pre-IND meeting), and 
will subsequently 3) obtain the FDA and IRB approvals for the developed protocol. As the part of the 
proposed trial development process, we will 4) develop and submit a Clinical Trial Award DOD 
application or an R21-type NIH application (PAR 03-005: Quick Funding of Clinical Trials) to request 
the appropriate funding for the clinical implementation of the developed protocol. 
B. Relevance 
DC-based immunotherapy of cancer has demonstrated immunologic efficacy, ability to induce clinical 
responses in individual patients 23-44 and can result in an improved survival benefit in a subset of patients 
with hormone refractory prostate cancer 37. 
Despite these encouraging observations, the overall clinical efficacy of this type of immunotherapy is still 
below expectations. Based on the high ability of DC-based vaccines to produce IL-12, the factor critically 
important for the induction and persistence of the tumoricidal activities of CTLs, TH1 and NK cells 45 
should  boost the effectiveness of DC-based vaccination, and our preliminary data showing superior 
activation by DC1s of CTLs,Th1 cells, and NK cells, in melanoma models and in healthy volunteers (2,3,6,7 
and Figs 1-3), and preliminary clinical results observed in CTCL patients, we expect that the introduction 
of the FDA-approved αDC1s (BB-IND 11,754) in the immunotherapy of prostate cancer will strongly 
increase its clinical efficacy.  
We expect that the established resistance of polarized DC1 against the immunosuppressive activity of 
PGE2 
2 makes these cells particularly suitable as a therapeutic tool against prostate cancer (PCa), 
associated with overproduction of PGE2 8-18, and the pronounced immunosuppressive impact on 
endogenous DC 46. In addition, the availability of PSA as a marker of prostate cancer progression, makes 
prostate cancer a good system to monitor the biologic, rather than immunological responses to 
vaccination as a primary endpoint of the vaccine efficacy, facilitating the overall progress in the field of 
cancer immunotherapy.  
C. INTERVENTIONS 
The core of the proposed immunointervention (αDC1-based vaccine)3 will be similar to the current FDA 
(BB-IND 11,754)- and IRB-approved protocol UPCI 03-118 (see Part 12 of this application) recently 
developed for melanoma patients. The study will evaluate 2 patient treatment groups in a phase I/II 
evaluation of DC-based intralymphatic (a single priming cycle) and intradermal (four booster cycles) 
immunizations against metastatic prostate cancer. The patients will be vaccinated with tumor-loaded 
αDC13, used as a single treatment, or followed up (at 6 weeks) by subsequent limited androgen 
ablation (for 3 months). The rationale for the latter intervention lies in the ability of androgen ablation22 
to induce both apoptosis as well as a T cell infiltrate in the prostate of patients with prostate cancer. Thus 
we predict that the proposed sequence of therapeutic interventions will result in their synergistic activity 
by concentrating the αDC1-induced CTLs and Th1 cells into the prostate cancer tissue. 
Based on our preliminary discussions and the previously-established collaboration with Cell Genesys Inc 
(San Francisco, CA), we will prioritize the use of allogeneic prostate cancer cell lines PC-3 cells 
(CG1940) and LNCaP cells (CG8711), as a source of apoptotic bodies loaded on αDC1. These cellular 
vaccines are generated in a FDA-approved GMP facility by Cell Genesys Inc., and to date approximately 
 
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182 patients with prostate cancer have been treated under BB-IND 7285 and BB-IND 8041. This feasible 
source of prostate-cancer antigen showed promise both in preclinical murine studies 47,48, or in previously-
performed clinical trials, either used as a self-standing vaccine, or pulsed on or fused with conventional 
dendritic cells 33,36,49. Dr. Chatta is the PI on the currently ongoing UPCI 04-149 trial, involving both 
cell lines as a stand-alone vaccine (rather than as a component of the DC-based vaccine). The use of such 
standardized antigenic material has emerged during our discussions as not only being convenient and 
standardized, but also being superior to the patients autologous tumor material, HLA-A2-restricted 
peptide antigens, or recombinant PSA and/or PMSA proteins, that would respectively, restrict our study 
to the patients with available tumor tissue, patients with a defined HLA-A2+ allotype, or to targeting of 
mostly CD4+ T cell responses against PCa. 
In order to maximize the efficacy of vaccine delivery, to assure the optimal timing of their interaction 
with lymph node-based T cells, and to allow them to act as a medium of prolonged feedback interactions 
between the expanding populations of CD4+ and CD8+ T cells, the first priming dose of the αDC1-based 
vaccine will be administered via the intralymphatic route, over the period of four days, similar to our 
melanoma protocol UPCI 03-118 (see section E below and Part 12). This priming cycle of immunization 
will be administered in our GCRC on an inpatient basis, and will be followed by four doses of intradermal 
administration of the vaccine, alone, or in combination with androgen depletion (performed on outpatient 
basis; see section E below). 
D. PATIENT POPULATION  
220,000 men are newly diagnosed with prostate cancer (PCa) each year, and 30,000 men die of the 
disease annually1. The risk for PCa increases with age. A number of plausible scenarios exist that could 
explain this: i) hormones, growth factors, and cytokines within the prostate tissue itself may become 
deregulated with age; and ii) aging maybe associated with changes in immune surveillance. Standard 
treatment of organ-confined PCa includes surgery or radiation therapy, and is effective in the short term, 
but up to one third of patients relapse. Current systemic therapy (androgen ablation and chemotherapy) 
for PCa is limited, primarily palliative 502, and is associated with significant morbidity. Prostate-specific 
vaccine therapy, if effective and if initiated early, i.e., at the time of initial relapse, offers the prospect of 
prolonging survival with minimal morbidity. Thus, we have chosen to focus on men with PCa, with a 
PSA relapse after either surgery and/or radiation therapy. Men with PSA-only disease, represent the 
largest sub-poulation of patients with PCa, for whom there is no standard treatment. Furthermore, they 
also represent a heterogenous group, with widely varying PSA-doubling times. Once again the focus in 
this trial will be on patients with a PSA-doubling time of less than 12 months.  
E. CLINICAL PROTOCOL: 
The core of the proposed trial will be similar to our currently PRC- FDA- and IRB-approved protocol 
UPCI 03-118 (BB-IND 11,754: see part 12), recently developed for melanoma patients. 
We propose to evaluate two patient treatment groups in a phase I/II evaluation of DC-based 
intralymphatic (a single priming cycle) and intradermal (four booster cycles) immunizations against 
metastatic prostate cancer. The patients will be vaccinated with tumor-loaded alpha-type-1-polarized DC 
(αDC1), used as a single treatment, or followed up (at 6 weeks) by the subsequent androgen-depleting 
therapy. The rationale for limited androgen ablation is described above.22 We expect that the proposed 
sequence of therapeutic interventions will result in their synergistic activity by concentrating the 
αDC1-induced CTLs and Th1 cells into the prostate cancer tissue.  
Study schema (may be modified in the course of the protocol development): 
Group 1 (up to 14 patients): αDC1 vaccine alone:  
4 day long intralymphatic priming 
course (week 1), and four booster 
intradermal single doses of αDC1: 
weeks 6, 10, 14, 18)  
 
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Group 2 (up to 14 patients): αDC1 vaccine (see group 1)  
plus limited (for 3 months) androgen 
ablation 
(initiated 
at 
week 
6 
of 
treatment) 
Any patient with a demonstrable clinical response (first assessed at 8 weeks) will have an option of 
undergoing re-treatment with αDC1 only for up to 10 rounds of treatment. The priming cycle of 
immunization (four day long intralymphatic administration of αDC1-based vaccines) will be administered 
on an inpatient basis in our GCRC. The rationale for the prolonged administration of the first cycle of 
vaccine is to allow for the prolonged DC-mediated interaction between the expanding populations of the 
vaccine induced tumor-specific CD4+ and CD8+ T cells, as well as to assure the efficient and rapid 
delivery of αDC1s to the T cell areas of the lymph nodes, in order to assure their optimal IL-12-producing 
activity during the interaction with T cells (see the comprehensive justification of this approach in our 
approved melanoma protocol UPCI 03-118; see part 12). The above vaccination strategy has been 
extensively discussed and eventually approved in case of our melanoma protocol UPCI 03-118. We 
expect that it will also be accepted by our PRC and IRB, as well as by the FDA, in which case we plan to 
request a pre-IND meeting, to discuss our proposal and to receive the FDA feedback, prior to the formal 
IND filing. 
Overall Design and Endpoints: We propose to conduct a 2-arm clinical trial to evaluate the administration 
of tumor-loaded alpha-type-1-polarized DC (αDC1) with or without androgen ablation in men with PSA-
only disease. Patients will be sequentially accrued to receive either DC alone or DC followed by 
aandrogen ablation, 6 weeks later. The primary endpoint will be the stablization of PSA levels, and lack 
of PSA progression, as defined by the PSA working group.51 The secondary (immunologic) endpoint of 
efficacy will be the induction of Ag-specific IFNγ-producing CD8+ T cells against the tumor cell lines 
used in the vaccine. IFNγ ELISPOT assays will be employed as a principal readout system (performed as 
immunomonitoring by the Lab of Dr T. Whiteside: IMCPL. The selection of this endpoint is based on our 
(and the others) observations, that they (in contrast to e.g. cytotoxic tests) can be reliably performed with 
frozen aliquots of the cells, allowing for the simultaneous analysis of the samples from multiple 
donors/time points, and eliminating the problem of inter-assay variability. As tertiary endpoints of 
efficacy, we will analyze (using tumor-pulsed autologous DCs) the changes in the frequencies of the 
IFNγ versus IL-5-producing (Th1/Th2 profile) tumor-specific CD4+ T cells, the cytotoxicity of 
circulating tumor-specific CTLs, and NK cells, against the cell lines used in vaccines, and in any patients 
with available tumor tissue, also against autologous tumor cells.  
Sample Size: To test the hypothesis of sufficient PSA response, we will apply a Simon two stage design 
to each cohort. A response probability of .05 was selected as too low to be of further interest (and 
comparable to the lack of activity). In order to declare that this vaccine strategy warrants sufficient 
additional study we would like to detect a PSA response rate of 40%. In the first stage we will accrue 5 
patients. If none of the patients demonstrate an immunologic response accrual will cease at that dose tier. 
If we observe a single response among the first 5 patients we will continue to the second stage accruing 9 
more patients or a total of 14. The same two stage design will be applied to both the arms. Therefore a 
minimum of 10 and a maximum of 28 patients are required to successfully screen this vaccination 
strategy. 
Characteristics of the Design: Each of the sequential two stage designs have been selected to reject a 
low immunologic response rate of 5% at 
 = .05. The d
if the underlying response rate is 40%. We note 1) that the probability of rejecting the strategy at the first 
stage is 77% if the response rate is no better than 5%, and 2) the probability of falsely declaring either 
strategy to be effective is at most 10%.  
Data Analysis: We anticipate completing the clinical trial within 12 months of starting. The proportion of 
patients with a PSA response will be calculated with a 95% confidence interval. With regard to the 
secondary endpoints of the efficacy CTL responses, we will compute a two-sample Wilcoxon test using 
the variable net increase in IFN-γ spots to compare the 14 patients who received αDC1 administration 
alone and the 14 patients who also received androgen-depleting therapy. We will perform a one-sided test 
 
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at significance level 0.05 of a hypothesis of no difference vs. an alternative of an improvement for the 
combination therapy. The test will have power 0.80 to detect a difference in means of 1 standard 
deviation (if the data are normally distributed). The analysis of the tertiary endpoints will be performed in 
an exploratory manner. Most of these indicators are quantitative or ordered categorical data that will be 
analyzed for change from pre to post vaccine by either the signed rank test or McNemars’ test. The step-
down Bonferroni method will be used to control for overall error rate in reporting the results of several 
tests.  
F. MANAGEMENT PLAN: 
The proposed trial will be performed under the direct supervision of Dr. Chatta (the clinical PI on the 
developed protocol), the PI on several ongoing clinical trials in prostate cancer and other genito-urinary 
tumors,  in a close collaboration with Dr. Kalinski (laboratory/scientific co-PI, playing the same role on 
the melanoma protocol UPCI 03-118), who, jointly with Dr. Storkus (co-I), will be also responsible for 
the evaluation of the secondary (immunologic) endpoints of efficacy. The priming cycle of immunization 
(four day long intralymphatic administration of αDC1-based vaccines) will be administered on an 
inpatient basis in our GCRC by Dr. Howard Edingon (co-I), who plays a similar role on our melanoma 
protocol (UPCI 03-118), while the follow-up booster vaccinations and anti-androgen therapy will be 
administered on an outpatient basis at our GCRC at the Hillman Cancer Center. The vaccine will be 
generated in the cGMP facility of the UPCI (IMCPL), directed by Dr. Whiteside, the co-investigator on 
the current application with extensive expertise in cell therapy, who is also a co-investigator on several or 
our currently-ongoing clinical trials.  
The investigators and clinical research coordinator meet weekly to review and discuss study data from 
this site, to review subject safety issues and subject recruitment, accrual and retention. Decisions to 
continue or close the trial to accrual are also discussed during these meetings. Any modifications 
necessary to ensure patient safety are discussed and modifications will be submitted to the IRB. The IRB 
will be notified of any change in the risk/benefit ratio which would affect whether the study should 
continue. If any literature becomes available which suggests that conducting this trial is no longer ethical, 
the study will be terminated and the IRB will be notified of the new findings. All serious adverse events 
will be reported to the IRB according to the established guidelines. Serious adverse events will also be 
reported to the sponsor and /or other regulatory agency as per their requirements. All study data reviewed 
and discussed during these meetings will be kept confidential. In addition, the University of Pittsburgh 
Cancer Institute’s Data and Safety Monitoring Committee (DSMC) will provide oversight for this study. 
The DSMC will ensure that the investigator is assessing and reporting all serious adverse events to the 
FDA and IRB in accordance with their respective guidelines. The DSMC meets monthly to review all 
active protocols. Findings of these meetings are reported to the IRB at the time of renewal. 
F. INSTITUTIONAL SUPPORT: The University of Pittsburgh Cancer Institute (UPCI) is one of the 
first centers in the world attempting immunotherapy of cancer with dendritic cells. For this reason, our 
center is both particularly committed to and is in a special position to test and implement novel strategies 
of DC-based vaccination against cancer. This environment is supportive both for the logistics of the 
proposed protocol (αDC1-based vaccines are being currently prepared within the Hillman Cancer Center 
in the cGMP facility (IMCPL) directed by Dr. Whiteside, the co-investigator on current application), as 
well as for obtaining the funding for the clinical implementation of the developed protocol (multiple PO1-
type and SPORE-type programs, existing and under development, or the institutional bridge funds which 
were used in the past by the PI of this application to obtain the clinical grade peptides for our melanoma 
protocol, prior to the subsequent award of the NIH funding (1R21CA114131). Any developments in this 
field can be rapidly utilized in our center to also treat patients with other types of malignancies, 
solidifying the position of Pittsburgh and the University of Pittsburgh Cancer Institute as the leading 
center in cancer treatment, and can be widely disseminated by the network of our collaborations. 
 
 
 
 
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44. 
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APPENDIX II: MAILLIARD ET AL, 2004 
α-Type-1 Polarized Dendritic Cells: A Novel Immunization Tool with Optimized CTL-
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APPENDIX III: CGMP-GRADE ALLOGENEIC CELL LINE LNCAP 
BioReliance Test Results 
 
 
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