Phase II Study of Adenovirus/PSA Vaccine in Men With Recurrent Prostate Cancer After Local Therapy
Phase: PHASE2
Conditions: Recurrent Prostate Cancer
Sponsor: David M Lubaroff

PROTOCOL #1 
 
 
PHASE II STUDY OF ADENOVIRUS/PSA VACCINE IN MEN WITH RECURRENT 
PROSTATE CANCER AFTER LOCAL THERAPY 
Food and Drug Administration (FDA) Investigational New Drug (IND) #9706 
  Department of Defense, Prostate Cancer Research Program #A-14059.1 
Version 50 – January 23, 2015 
 
Co-Principal Investigators 
 
David M. Lubaroff, Ph.D.1 
 
 
 
Daniel Vaena, MD 
Department of Urology 
 
 
 
 
Department of Internal Medicine 
University of Iowa 
 
 
 
 
University of Iowa 
375 Newton Road 
 
 
 
 
200 Hawkins Drive 
Iowa City, IA  52242 
 
 
 
 
Iowa City, IA 52242 
tel: 
319-335-8423 
 
 
 
 
tel: 
319-356-3944 
e-mail: david-lubaroff@uiowa.edu 
 
 
e-mail: daniel-vaena@uiowa.edu  
 
Co-Investigators 
 
 
1 Please send all communications to this investigator. 
James A. Brown, MD 
Department of Urology 
Tel:    
319-356-2273 
e-mail: james-brown-2@uiowa.edu 
 
Mark C. Smith, MD 
Department of Radiation Oncology 
Tel:  
319-384-6135 
e-mail: mark-c-smith@uiowa.edu 
 
Michael O’Donnell, MD 
Department of Urology 
Tel: 
319-384-6981 
e-mail: michael-odonnell@uiowa.edu 
 
Sundeep Deorah, MD 
Department of Urology 
Tel: 
319-384-5418 
e-mail: sundeep-deora@uiowa.edu 
 
Amit Gupta, MD 
Department of Urology 
Tel: 
319-384-5251 
e-mail: amit-gupta-1@uiowa.edu 
 
Kenneth Nepple, MD 
Department of Urology 
Tel: 
319-356-2114 
e-mail: kenneth-nepple@uiowa.edu 
 
Mary Schall, RN, BSN 
Holden Comprehensive Cancer Center 
Tel: 
319-356-3516 
e-mail:  mary-schall@uiowa.edu 
 
Karen Clark Griffith, RN, PhD 
Iowa City Veterans Affairs Health Care System  
Tel: 
319-338-0581, ext. 7519 
e-mail: karen.clarkgriffith@va.gov 
  
Erica Brown, ARNP 
Department of Urology 
Tel: 
319-467-5072 
e-mail: erica-brown@uiowa.edu 
 
Gideon Zamba, PhD 
Department of Biostatistics 
Tel: 
319 384-5020 
e-mail: gideon-zamba@uiowa.edu 
 
Steven Varga, PhD 
Department of Microbiology 
Tel: 
319-335-774 
e-mail: steven-varga@uiowa.edu 
 
 
Pamela Zehr, RN, MA 
Holden Comprehensive Cancer Center 
Tel: 
319-353-8914 
e-mail:  pamela-zehr@uiowa.edu 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

PROTOCOL #1 
Lubaroff, page 2 
 
 
 
Investigational Pharmacist 
Kristine Johnson, BS, RPh 
Pharmacy Department  
Tel: 
319-353-7077 
e-mail: kristine-johnson@uiowa.edu 
 
Medical Monitor 
Daniel Berg, MD 
Holden Comprehensive Cancer Center 
Tel: 
319-353-7800 
e-mail: daniel-j-berg@uiowa.edu 
 
 
 
The Principal Investigator is funding this clinical trial through an award from the Department of Defense 
Prostate Cancer Research Program.

PROTOCOL #1 
Lubaroff, page 3 
 
 
INDEX  
 
 
Section 
Pages 
 
 
Introduction 
4-14 
 
 
Objectives 
14 
 
 
Patient Selection 
15,16 
 
 
Registration Procedures 
16,17 
 
 
Research Intervention Plan 
17,18 
 
 
Adverse Events 
18-20 
 
 
Measurement of Clinical and Immunological Effect 
20-22 
 
 
Study Parameters 
22-26 
 
 
Drug Formulation and Procurement 
27-29 
 
 
Statistical plan 
29,30 
 
 
Addendum 
30,31 
 
 
Roles and Responsibilities of Study Personnel 
31,32 
 
 
Literature References 
32-36 
 
Footnote 
 
36,37 

PROTOCOL #1 
Lubaroff, page 4 
 
 
1. INTRODUCTION 
 
1.1 Background- Immunotherapy in Prostate Cancer 
 
Prostate cancer is the second leading cause of cancer death among males in the United 
States. There will be an estimated 234,460 new diagnoses of prostate cancer made in the 
United States in 2006 (1) (the estimates for 2006 were not available at the time of the protocol 
completion).  Treatments for organ-confined prostate cancer include radical prostatectomy and 
radiation therapy.  When the cancer presents de novo, or recurs outside the prostate, first-line 
systemic treatments typically include hormonal blockade (with LHRH agonists or bilateral 
orchiectomy), which suppress testosterone levels, limit the growth of androgen-dependent 
cancer cells, and result in clinical tumor control. After a median time of 2 years, patients 
progress into a clinical hormone-refractory state, when the prostate specific antigen (PSA) 
levels rise despite castration, there is proliferation of androgen-independent cancer cells, and 
there is continued clinical tumor growth that becomes fatal. Therapeutic measures in this 
situation include further hormonal manipulations or the use of systemic chemotherapy, which 
has recently shown a small survival benefit in phase III trials. Approximately 30,000 Americans 
die from prostate cancer each year. 
 
Immunotherapeutic approaches against prostate cancer have been investigated for several 
years.  Most of these studies have concentrated on active non-specific therapy and adoptive or 
passive therapy, with only recent focus on the induction of antigen-specific immune responses.  
Viral vectors have been used successfully in both gene transfer and vaccine therapy studies (2).  
Replication-competent and replication-deficient adenoviruses expressing foreign proteins have 
been used to elicit immune responses to a variety of tumor antigens (3-7). 
 
We have demonstrated that immunizations with adenovirus, carrying the human PSA gene, 
can induce vigorous anti-PSA T-cell responses and cause the destruction of PSA-secreting 
tumors in a pre-clinical mouse model of prostate cancer (8,9).  Such active immunization 
against prostate-cancer associated antigens might be more effective than active non-specific or 
adoptive/passive immunotherapy.  Therefore, we have pursued a vaccination strategy based on 
an adenovirus that carries the gene for prostate specific antigen (PSA).  Results from our Phase 
I trial of adenovirus/PSA (Ad/PSA) vaccine (section 1.4, below) demonstrated that a single 
immunization of men with metastatic prostate cancer was able to induce anti-PSA T cell 
responses.  The trial design was a dose escalation study with the vaccine administered 
subcutaneously (sc) either in an aqueous solution or in a collagen matrix (Gelfoam®).  We now 
propose a Phase II clinical trial using the Ad/PSA vaccine, administered in multiple injections to 
prostate cancer patients with minimal disease burden. 
 
1.2 Adenovirus vectors 
 
Recombinant adenoviral vectors transduce a wide range of dividing and nondividing cells 
types, making this gene delivery system valuable as a tool for studying diseases, for vaccine 
therapy, and for potential clinical use (10). Recombinant adenovirus can be prepared and 
purified in high titers.  In addition, wild-type adenovirus infections are extremely common in the 
general population, giving adenovirus a well-documented safety record (11).  Moreover, 
adenoviruses are structurally stable and no adverse effects have been reported following the 
vaccination of US military recruits with wild types, demonstrating their safety for human use 
(11).  Adenoviral vectors for gene therapy and vaccine therapy are adenoviruses which have 
been genetically modified to allow insertion of foreign genes and to render the virus replication-
defective.  Current vectors have a deletion in the E1 region or in both the E1 and E2 regions.  

PROTOCOL #1 
Lubaroff, page 5 
 
 
Adenoviral gene transfer has been used in a variety of experimental conditions that include 
transfers to the liver (12), lung (13), central nervous system (14,15), and to cancer cells (16). 
 
There is evidence that the introduction of foreign transgenes by adenovirus induces immune 
responses to the transgene product, which become ultimately responsible for the elimination of 
the virus (17,18).  While this is disadvantageous for insertion of functional genes into host cells, 
it is advantageous in the use of viruses carrying foreign genes as immunogens.  In the vaccine 
therapy of cancer, active immunization against a murine colon cancer, breast cancer, and 
melanoma antigens have been induced by adenoviral vaccines (19-25). 
 
The Ad/PSA vaccine used our laboratory and in our Phase I clinical trial was produced by 
inserting the gene for the full length pre-pro form of human PSA into a replication deficient 
adenovirus serotype 5.  Replication deficiency was induced by deletion of the E1a and E1b 
genes of the virus.  Details of the vaccine can be found in section 9 of this protocol.  Approval 
for the use of the vaccine in the Phase I trial was obtained from the FDA under IND #9706. 
 
In pre-clinical studies, our group has demonstrated that the Ad/PSA vaccine was able to 
induce stronger anti-PSA immune responses than other viral PSA vaccines.  These include 
vaccinia viruses, both replication competent and replication deficient, and to a canarypox 
vaccine (Table 1), The frequency of PSA-specific CD8+ cells T cells generated by the Ad/PSA 
vaccine was greater than were generated by any of the other vaccines tested.. In addition to the 
superior immunizing property of the Ad/PSA, the incorporation of Gelfoam, a collagen matrix 
(section 1.3), has been shown in pre-clinical studies to enhance the ability of the vaccine to 
induce strong anti-PSA immune responses (8). Lastly, immunization of mice with Ad/PSA in 
matrix can induce anti-PSA responses even in the presence of high titer anti-adenovirus 
antibodies (8).  This latter finding is important in light of the fact that most humans have pre-
existing levels of anti-adenovirus antibodies as a result of prior natural exposure to the virus. 
 
Table 1 
Effector Cell Frequency Analysis (ELISPOT) 
 
Vaccine 
Virus 
Frequency of PSA-Specific 
CD8+ T Cells 
 
 
 
  Ad/PSA*  
   Replication deficient adenovirus 
   1/455 
  Prostvac 
   Replication competent vaccinia 
  1/2028 
  NYVAC/PSA 
   Replication deficient vaccinia 
  1/3597 
  ALVAC/PSA 
   Canarypox 
1/35,714 
 
1.3 Gelfoam® Matrix 
 
Gelfoam (Pharmacia & Upjohn Company, Kalamazoo, MI) is a medical device intended for 
application to bleeding surfaces as a hemostatic agent.  It is a water-insoluble, off-white, non-
elastic, porous, pliable product prepared from purified pork skin. The Gelfoam gelatin 
preparation is available either as a cross-linked sponge or as non-cross linked beads.  It is able 
to absorb and hold within its interstices approximately 45 times its weight of blood and other 
fluids (26). The absorptive capacity of Gelfoam is a function of its physical size, increasing with 
increasing gelatin volume (27). 
 

PROTOCOL #1 
Lubaroff, page 6 
 
 
The mechanism of action of surface-mediated hemostatic devices is supportive and 
mechanical (27). Surface-acting devices, when applied directly to bleeding surfaces, arrest 
bleeding by the formation of an artificial clot and by producing a mechanical matrix that 
facilitates clotting (28).  Jenkins et al have theorized that the clotting effect of Gelfoam may be 
due to release of thromboplastin from platelets, occurring when platelets entering the Gelfoam 
become damaged by contact with its myriad of interstices (29).  Thromboplastin interacts with 
prothrombin and calcium to produce thrombin, and this sequence of events initiates the clotting 
reaction.  The authors suggest that the physiologic formation of thrombin in Gelfoam is sufficient 
to produce formation of a clot, by its action on the fibrinogen in blood (29).  The spongy physical 
properties of Gelfoam hasten clot formation and provide structural support for the forming clot 
(28,30). 
 
Gelfoam has been used experimentally for the delivery of soluble proteins and drugs, 
including insulin, antibiotics, and growth factors (31-33). Gelfoam was used for sustained 
release of insulin in an ocular implant device (31). Delivery of insulin in solution had no effect on 
blood glucose levels.  In contrast, the use of Gelfoam as a sustained release delivery agent 
provided measurable insulin activity for up to 10 hours after implantation.  Glucose levels in the 
blood stabilized at 60% of the original value, whereas administration of insulin in eye drops had 
no effect. 
 
MacDonald and Mathews (34) studied Gelfoam implants in canine kidneys and reported that 
it assisted in healing, with no marked inflammatory or foreign-body reactions. Jenkins and 
Janda (35) studied the use of Gelfoam in canine liver resections and noted that Gelfoam 
appeared to offer a protective cover and provide structural support for the reparative process. 
Correll et al (36) studied the histology of Gelfoam when implanted in rat muscle and reported no 
significant tissue reaction.  
 
Gelfoam has been used as a hemostatic agent in dog prostate (37).  In these studies no 
gross histological evidence of tissue damage or calcification was induced.  In addition, these 
investigators demonstrated that placement of Gelfoam into the lumen of the bladder resulted in 
liquefaction of the Gelfoam without any evidence of calculogenesis.  Finally, Bischoff and 
Goerttler (38) used Gelfoam in human prostate therapeutic embolization with success.  
 
Our laboratory, in collaboration with Dr. Timothy Ratliff, has demonstrated that 
administration of the Ad/PSA vaccine in Gelfoam induces a stronger anti-PSA immune response 
(Figure 1).  In our pre-clinical studies, immunization with the vaccine in an aqueous suspension 
induces strong immunity with 109 pfu with weaker immunity induced with 108 and 107 pfu.  Use 
of Gelfoam permits the induction of strong responses at the lower dose of 108 pfu.  In addition, 
strong anti-PSA T cell responses could be induced by immunization with the Ad/PSA vaccine in 
Gelfoam even in mice pre-immunized to adenovirus (Figure 2).  In the Phase I clinical trial 
(section 1.4), the addition of Gelfoam to the vaccine immunization did not result in excess 
serious adverse events. 
 
1.26
3.125
6.25
12.5
25
0
20
40
60
80
100
Ad5-PSA 109
Ad5-PSA 108
Ad5-PSA 107
Ad5-lacZ
Fluid Phase
Effector Cells x 104/104
Target Cells
% 51Cr Release
1.56
3.125
6.25
12.5
25
0
20
40
60
80
100
Ad5-PSA 109
Ad5-PSA 108
Ad5-PSA 107
Ad5-lacZ
Gelfoam
Effector Cells x 104/104
Target Cells
% 51Cr Release
Figure 1 – Anti-
PSA cytotoxic 
activity of 
spleen cells 
obtained from 
mice 
immunized with 
Ad/PSA in 
aqueous (fluid 
phase) versus 
Gelfoam. 

PROTOCOL #1 
Lubaroff, page 7 
 
 
 
 
1.4 Phase I study 
 
A Phase I clinical trial of the Ad/PSA vaccine has been completed in men with measurable 
metastatic prostate cancer, with the primary objectives of determining the toxicity profile and 
maximal tolerated dose (MTD).  The ability of the vaccine to induce anti-PSA immune 
responses and any clinical responses was also evaluated. Funding for the Phase I trial was 
provided by multiple sources that include the Holden Comprehensive Cancer Center, the 
Department of Urology, and the Carver College of Medicine at the University of Iowa. 
 
Eligible patients consisted of men with prostate cancer that had measurable metastatic 
disease, 90% of whom were stage D3. Prior therapies had included androgen withdrawal, 
ketaconazole, prednisone, Casodex, Taxotere, and external beam radiation, but the initiation of 
vaccine therapy was equal to, or greater than, 30 days after the most recent therapy. Patients 
were treated in successive dose levels and aqueous vs. matrix cohorts, according to the 
protocol plan. We were able to administer the maximum permitted dose of 108 pfu without any 
serious adverse events by vaccination of the first of 18 patients. These initial 18 patients were 
followed throughout the one-year period after injection. An additional 14 patients were treated at 
the MTD dose level, as planned in the protocol and confirmed by a letter to the FDA.  The 
purpose of the additional patients was to have sufficient numbers of patients in the groups to 
statistically evaluate the anti-PSA immune responses induced by the Ad/PSA vaccine. In 
summary, 32 patients were treated in the study followed through the one-year period.  Two 
additional patients were enrolled in the study (total number of enrolled patients, 34) but never 
received the vaccine and were therefore not evaluable.  One patient chose to have radiation 
therapy instead of participating in the trial and the second was diagnosed with a second 
malignancy (melanoma) shortly after his enrollment in the phase I study. 
 
1.4.1 Phase I study results 
 
 The median age of the patients was 70.2 years (range, 52 to 89). The vaccine was 
administered as an aqueous suspension or in a collagen (Gelfoam) matrix to 32 patients.  
Sixteen (16/32) or 50% of the patients exhibited grade 1 vaccine-related adverse events (AE), 
1/32 (3.1%) that exhibited a grade 2 AE, and one patient exhibited a grade 3 AE which was a 
decrease in neutrophil count.  There were no vaccine-related grades 4 or 5 AEs. 
Figure 2. Effect of the presence 
of anti-adenovirus Ab on Ad5-
PSA immunization with and 
without Gelfoam. Mice were 
injected i.p. with 109 PFU of Ad5-
lacZ or PBS 2 wk before 
immunization with 109 PFU Ad5-
PSA delivered s.c. in the fluid 
phase (PBS) or Gelfoam. CTL 
were tested against RM-11psa 
and RM-11neo to demonstrate 
PSA specificity. Cytolytic activity 
of each group against RM-11neo 
targets was <10% for all E:T 
ratios. 

PROTOCOL #1 
Lubaroff, page 8 
 
 
Table 2 
Ad/PSA Phase I Trial 
Adverse Events – by Vaccine Relationship 
 
Patient 
System 
Day of 
onset* 
Event 
Vaccine-
related 
 
AP005 
Neural 
21 
Agitation 
No 
AP006 
Musculoskeletal 
13 
Left hip and thigh pain 
No 
AP007 
Musculoskeletal 
22 
Left back pain 
No 
AP007 
Neural 
14 
Situational depression 
No 
AP009 
GI 
21 
Constipation 
No 
AP009 
Musculoskeletal 
14 
Joint aches, secondary to fall 
No 
AP009 
Neural 
11 
Fall, causing head lacerations & loss 
of consciousness 
No 
AP010 
GU 
21 
Proteinuria 
No 
AP010 
Skin 
21 
Edema 
No 
AP014 
Constitutional 
22 
Fatigue 
No 
AP014 
Musculoskeletal 
14 
Leg pain (bone – femur) 
No 
AP015 
Respiratory 
1 
Cough 
No 
AP016 
GU 
172 
Left nephrolithiasis 
No 
AP016 
GU 
183 
New primary tumor – papillary bladder 
No 
AP016 
Immune 
8 
Flu-like symptoms 
No 
AP016 
Musculoskeletal 
4 
Bone pain – left leg 
No 
AP016 
Musculoskeletal 
21 
Decreased left leg strength 
No 
AP018 
Musculoskeletal 
8 
Bilateral rib pain 
No 
AP018 
Neural 
8 
Insomnia 
No 
AP020 
Neural 
168 
cord compression 
No 
AP022 
GI 
2 
Constipation 
No 
AP022 
Hematologic 
14 
decreased lymphocyte count 
No 
AP022 
Hematologic 
63 
decreased WBC & neutrophils 
No 
AP022 
Musculoskeletal 
5 
increased bone pain 
No 
AP022 
Neural 
16 
Depression 
No 
AP023 
Musculoskeletal 
20 
pain in left buttocks 
No 
AP025 
Musculoskeletal 
16 
bone pain 
No 
AP026 
Cardiovascular 
14 
Lower extremity edema 
No 
AP027 
GI 
1 
GI cramping 
No 
AP027 
GI 
1 
Constipation 
No 
AP034 
Respiratory 
prior 
Edema, wheezing (change in inhaler 
in same time period) 
No 
AP035 
Constitutional 
64 
Weight loss 
No 
AP035 
GI 
50 
Constipation 
No 
AP035 
GU 
22 
Proteinuria 
No 
AP035 
Hematologic 
15 
Lymphopenia 
No 
AP035 
Musculoskeletal 
22 
Increased alkaline phosphatase 
No 
AP037 
GI 
73 
Nausea 
No  
AP018 
Skin 
1 
Injection site tenderness 
Possible 
AP019 
Cardiovascular 
1 
Hypotension 
Possible 
AP019 
Constitutional 
1 
Fever 
Possible 
AP019 
Constitutional 
14 
Fatigue 
Possible 
AP019 
GU 
1 
Proteinuria 
Possible 
AP020 
GI 
21 
increased alkaline phosphatase 
Possible 
AP020 
GU 
21 
Ketonuria 
Possible 

PROTOCOL #1 
Lubaroff, page 9 
 
 
AP020 
Metabolic 
14 
Hyponatremia 
Possible 
AP020 
Metabolic 
14 
Hyperglycemia 
Possible 
AP020 
Respiratory 
9 
viral symptoms 
Possible 
AP005 
Musculoskeletal 
1 
Left inguinal pain 
Possible 
AP008 
GU 
21 
Proteinuria 
Possible 
AP008 
Respiratory 
3 
Cold symptoms 
Possible 
AP010 
Hematologic 
1 
Decrease in absolute neutrophils 
count 
Possible 
AP027 
Liver 
21 
increased AST 
Possible 
AP036 
Hematologic 
1 
Anemia 
Possible 
AP036 
Hematologic 
63 
Anemia 
Possible 
AP037 
Hematologic 
1 
Lymphopenia 
Possible 
AP002 
Musculoskeletal 
3 
Groin pain 
Unlikely 
AP002 
Neural 
3 
Headache 
Unlikely 
AP002 
Skin 
1 
Itching 
Unlikely 
AP003 
Pulmonary 
21 
Pleural effusion 
Unlikely 
AP006 
Constitutional 
14 
Chills 
Unlikely 
AP006 
Neural 
14 
Migraine headache 
Unlikely 
AP012 
Cardiovascular 
21 
Hypotension 
Unlikely 
AP012 
GI 
0 
Heartburn 
Unlikely 
AP012 
Neural 
21 
Dizziness 
Unlikely 
AP014 
Hematologic 
82 
Anemia 
Unlikely 
AP015 
Cardiovascular 
137 
Myocardial infarction 
Unlikely 
AP018 
Respiratory 
43 
DOE? 
Unlikely 
AP021 
Hematologic 
1 
decreased lymphocytes 
Unlikely 
AP021 
Hematologic 
63 
thrombocytopenia 
Unlikely 
AP023 
Immune 
16 
urinary tract infection 
Unlikely 
AP023 
Liver 
21 
elevated AST 
Unlikely 
AP024 
Hematologic 
14 
Anemia 
Unlikely 
AP024 
Hematologic 
1 
Anemia 
Unlikely 
AP025 
GI 
85 
abdominal distention, constipation, 
apparently related to metastatic 
disease in the periaortic lymph nodes 
and periprostatic tumor mass 
Unlikely 
AP025 
Hematologic 
14 
thrombocytopenia 
Unlikely 
AP025 
Neural 
122 
Headache 
Unlikely 
AP027 
Immune 
21 
Infection 
Unlikely 
AP034 
Cardiovascular 
14 
Hypotension/dizziness 
Unlikely 
AP034 
Musculoskeletal 
14 
Back pain 
Unlikely 
AP036 
Cardiovascular 
63 
Edema – bilateral ankles 
Unlikely 
AP002 
Skin 
0 
hematoma at injection site 
Yes 
AP003 
Skin 
0 
Bruising at injection site 
Yes 
AP004 
Skin 
0 
Ecchymosis at injection site 
Yes 
AP005 
Skin 
0 
Ecchymosis and erythema at injection 
site 
Yes 
AP008 
Skin 
0 
Ecchymosis at injection site 
Yes 
AP009 
Skin 
0 
Ecchymosis at injection site 
Yes 
AP018 
Skin 
0 
Ecchymosis 
Yes 
AP025 
Skin 
0 
Ecchymosis at injection site 
Yes 
AP026 
Skin 
0 
Erythema at injection site 
Yes 
AP034 
Skin 
0 
Pain at injection site 
Yes 
* day 0 = day of injection; day 1 = day after injection 
 

PROTOCOL #1 
Lubaroff, page 10 
 
 
We measured the anti-PSA immune responses, both antibody and T cell, in all patients 
enrolled in the study.  Antibody responses to PSA were measured by the binding to PSA-
secreting cell lines using the method adapted from Cavacini, et al. (39). Results of those 
analyses demonstrated that 57% of men immunized with the Ad/PSA vaccine developed 
measurable anti-PSA antibodies.  ELISPOT assays were utilized to measure anti-PSA T cell 
responses.  The results, depicted in Table 3, demonstrate that of the 32 patients, 18 (56.3%) 
developed anti-PSA T cell responses.  The addition of Gelfoam did not appear to affect the 
development of anti-PSA responses, but in this Phase I study the numbers of patients in each 
group was too small to make statements of statistical significance of the data. These results 
demonstrate the ability of men with late stage metastatic prostate cancer, injected one time with 
Ad/PSA, to respond to the vaccine with the production of anti-PSA T cells. 
 
Table 3 
ELISPOT Analysis of Anti-PSA T Cell Immune Responses 
Patient 
Number 
Dose/ 
Vehicle 
Response 
Frequency 
 
 
 
Pre-Immunization 
Post- Immunization 
AP-002 
106-aqueous 
- 
1/985,000 
1/258,571 
AP-004 
106-aqueous 
- 
0 
1/311,111 
AP-007 
106-aqueous 
+ 
1/46,901 
1/14,804 
AP-003 
106-matrix 
+ 
1/90,000 
1/8075 
AP-005 
106-matrix 
+ 
1/1.2x106 
1/152,353 
AP-006 
106-matrix 
+ 
1/93,103 
1/15,762 
AP-008 
107-aqueous 
- 
1/100,000 
1/97,419 
AP-010 
107-aqueous 
- 
0 
0 
AP-013 
107-aqueous 
+ 
0 
1/60,000 
AP-009 
107-matrix 
+ 
1/52,535 
1/30,566 
AP-012 
107-matrix 
- 
1/13,488 
1/254,286 
AP-014 
107-matrix 
+ 
1/562,500 
1/120,000 
AP-015 
108-aqueous 
+ 
1/1.4x106 
1/780 
AP-016 
108-aqueous 
+ 
0 
1/130,000 
AP-017 
108-aqueous 
+ 
0 
1/124,375 
AP-025 
108-aqueous 
- 
0 
0 
AP-026 
108-aqueous 
- 
0 
1/560,000 
AP-027 
108-aqueous 
+ 
0 
1/26,364 
AP-029 
108-aqueous 
+ 
0 
1/333 
AP-032 
108-aqueous 
+ 
1/250,000 
1/3793 
AP-018 
108-matrix 
- 
0 
0 
AP-019 
108-matrix 
+ 
1/14,235 
1/336 
AP-020 
108-matrix 
+ 
1/8824 
1/1802 
AP-021 
108-matrix 
+ 
1/689 
1/431 
AP-022 
108-matrix 
+ 
0 
1/180,000 
AP-023 
108-matrix 
- 
1/320,000 
0 
AP-030 
108-matrix 
+ 
1/666.667 
1/32,000 
AP-034 
108-matrix 
+ 
1/80,000 
1/15,152 
AP-035 
108-matrix 
+ 
1/5295 
1/2459 
AP-036 
108-matrix 
+ 
1/62,000 
1/9487 
AP-037 
108 matrix 
- 
1/2.1x106 
1/965,000 
 
The effects of vaccination on serum PSA and on patient survival were also evaluated as a 
secondary endpoint in the phase I trial. Although there was no sustained decline in individual 
PSA levels, the PSA doubling times (PSADT, calculated based on 3 pre-enrollment consecutive 

PROTOCOL #1 
Lubaroff, page 11 
 
 
PSA measurements) were reduced in 54 percent of the patients compared to pre-vaccine 
administration, with the best responses occurring in patients immunized with the highest dose of 
the vaccine (Table 4). In addition, published survival nomograms for patients with hormone 
refractory prostate cancer were applied to patients in this phase I trial (40,41). Table 5 shows 
that 57% of all patients at all doses, whether injected with the vaccine as an aqueous 
suspension or in the collagen matrix, had a survival time longer than that predicted by the 
nomogram. The range of increased survivals in the different groups was 33% to 100%.   
 
Table 4 
PSA Doubling Time (DT) in Phase I Patients 
Vaccine Dose & Vehicle 
Percent Increased PSA DT 
 
 
106 aqueous 
33% 
106 matrix 
33% 
107 aqueous 
67% 
107 matrix 
67% 
108 aqueous 
25% 
108 matrix 
62% 
Total 
54% 
 
Table 5 
Ad/PSA Phase I Trial 
Predicted and Actual Patient Survival 
Vaccine Dose 
Vehicle 
Percent with Longer 
Than Predicted Survival 
 
 
 
106  
Aqueous 
  33% 
106  
Matrix 
  67% 
107  
Aqueous 
100% 
107  
Matrix 
  33% 
108  
Aqueous 
  56% 
108  
Matrix 
  50% 
          Overall  
  57% 
 
1.5 Other immunotherapy clinical trials for prostate cancer 
 
The last several years have seen an increase in the number of clinical trials using vaccine 
immunotherapy for the treatment of prostate cancer.  The trials have used a variety of target 
antigens that have been shown to be associated with prostate and prostate cancer cells.  These 
include PSA (39,42-49), prostatic acid phosphatase (PAP) (50-53), prostate specific membrane 
antigen (PSMA) (54-56), telomerase (hTERT) (57,58), Thomsen-Friedenreich antigens (59), 
mucins (60), carbohydrates (61), and HLA-associated peptides (62).  A variety of vectors have 
been used in the immunization process that include dendritic cells (45,50-58,63), vaccinia virus 
(39,42,43,47,49), fowlpox virus (39,47), liposomes (44), plasmids, (48), and chemical 
conjugates (59-61).   
Recently, Kaufman et al. recently reported the results of a phase II trial (ECOG 7897) with a 
prime/boost vaccine using vaccinia virus and fowlpox virus expressing human PSA in patients 
with hormone-dependent prostate cancer (64). Sixty-four eligible patients with biochemical 
progression after local therapy were randomly assigned to three treatment arms: (A) fowlpox-
PSA (rF-PSA) by intramuscular injection every six weeks for four doses, (B) rF-PSA for three 
doses followed by vaccinia-PSA (rV-PSA) given by intradermal injection, or (C) rV-PSA followed 

PROTOCOL #1 
Lubaroff, page 12 
 
 
by three rF-PSA vaccines.  Dreicer et al. reported a randomized phase II study with a 
recombinant Modified Vaccinia Ankara virus which expresses both MUC1 and IL2 (TG4010) 
(65). MUC1 is a glycoprotein associated with several malignancies. Eligible patients were 
required to have no evidence of metastatic disease following curative intent local therapy, 
evidence of PSA failure (PSA over 2 ng/ml) and PSA doubling time (PSA-DT) less than 10 
months. Arm 1 had TG4010 injected sc weekly, at a dose of 108 pfu, for six weeks, then every 
three weeks. Arm 2 had 108 pfu TG4010 injected sc every three weeks. Therapy was continued 
to disease progression or to a maximum of 36 weeks.  Lastly, Small et al. recently presented the 
results of a phase III trial with APC8015, an immunotherapy cellular product consisting of 
autologous peripheral blood mononuclear cells enriched for a dendritic cell fraction pulsed with 
PA2024, a Prostatic Acid Phosphatase (PAP)-GM-CSF construct (66). Patients with 
asymptomatic, metastatic hormone-refractory prostate cancer were randomized (2:1) to receive 
APC8015 (n=82) or placebo (n=45) every 2 weeks x 3. 
 
ECOG is currently planning a phase III trial using the Vaccinia virus (PROSTVAC-
V/TRICOM) followed by Fowlpox virus vaccination (PROSTVAC-F/TRICOM) with GM-CSF, 
compared with placebo vaccine plus GM-CSF in patients with hormone-refractory prostate 
cancer with absence of metastatic disease (ECOG 1805, PARADIGM). 
 
In summary, the results from these trials vary in terms of patient populations studied 
(hormone dependent vs. independent) and in levels of positive results, which include the 
induction of antigen-specific immune responses, decreases in levels of serum PSA and in rates 
of change in PSA velocity, and measures of clinical responses. Thus far no single vaccine 
immunotherapy has proven to be definitely superior to others in terms of clinical benefit, and 
other phase II and III trials continue to be planned or conducted. The results of some of these 
vaccine trials raise the question that an increase in PSADT may in the future represent a 
possible surrogate marker for increased time to progression, or overall survival in 
immunotherapy studies, and that absolute PSA responses may not constitute an obligatory step 
for the ultimate demonstration of clinical benefit of immunotherapy approaches in prostate 
cancer. Furthermore, the T-cell stimulation index may have important correlation with clinical 
vaccine efficacy, as seen in the phase III trial by Small et al.(66).  These developing notions 
further support the current proposal for clinical development of our Ad/PSA vaccine, also based 
on the results of our prior phase I trial. 
 
1.6 Proposed phase II clinical trial: rationale 
 
Based on the significant pre-clinical activity of the Ad/PSA vaccine in generating tumor-
specific T cells, and the encouraging safety and efficacy results from our phase I study, we 
propose to continue the clinical development of the Ad/PSA vaccine with the performance of the 
current phase II trial. It is our contention that the vaccine product and the method of 
immunization set this therapy apart from other ongoing prostate cancer investigational 
immunotherapeutic approaches.  Specifically, the incorporation of Gelfoam, not present in other 
vaccine preparations, enhances the induction of strong anti-PSA responses. Immunization of 
mice with Ad/PSA in Gelfoam matrix was able to induce anti-PSA responses even in the 
presence of high-titer anti-adenovirus antibodies. Notably, most humans naturally possess high 
titers of anti-adenovirus antibodies due to natural exposure to adenoviruses. 
 
We plan to enroll prostate cancer patients into one of two arms (A & B) of the Phase II 
clinical trial.  The ideal patient population to determine a therapeutic benefit of a new treatment, 
particularly immunotherapy, is one with minimal disease burden.  The low tumor burden should 
allow therapies, particularly those relying on antigen-specific effector T lymphocytes, to destroy 

PROTOCOL #1 
Lubaroff, page 13 
 
 
all of the cancerous tissues and cells.  The first therapeutic arm (Arm A) will enroll men with 
recent evidence of recurrence following surgery or radiation therapy for their primary tumor.  
Patients in the population will be eligible if they exhibit at least three separate rises in serum 
PSA, at least one month apart with differences >0.03 ng/ml and a total PSA of >0.2  ng/ml; have 
a PSA doubling time of >6 months; are not at high risk or if they refuse treatment with Taxotere 
or radiation.  A high risk patient is defined below.  All patients will be hormone naïve, meaning 
that they may not have had prior systemic therapies for prostate cancer (hormonal therapy, 
including but not limited to LHRH agonists, antiandrogens, ketoconazole or chemotherapy – 
mitoxantrone/taxanes/estramustine, etc.) unless they stopped hormone therapy two or more 
years prior to enrollment and currently have normal testosterone levels..  Since standard 
therapy for these patients would be to postpone androgen ablation therapy until such time as 
there is a high serum PSA level (>20 ng/ml), enrolling patients into this Phase II trial does not 
withhold accepted treatment.  Patients will be excluded from the trial if they refuse salvage 
radiation therapy or had greater than two positive margins at surgery, or positive lymph nodes. 
 
The second therapeutic arm (Arm B) will enroll men with recurrent disease who will undergo 
androgen depletion therapy.  The choice of this additional patient population is based upon 
published documentation that inflammation and the generation of immune responses are 
augmented by hormone withdrawal (67-69).  Mercader, et al., in attempts to demonstrate an 
enhanced termination of tolerance to prostate associated antigens documented CD4+ and 
CD8+ T cell infiltrates in benign prostates and in prostate tumors of men undergoing androgen 
withdrawal (67).  Roden and co-workers published data demonstrating that T cell levels and T 
cell proliferation were increased in mice following castration (68) while Drake, et al. reported 
breaking tolerance to antigens associated with the TRAMP prostate tumors in mice (69).  
Therefore, we propose to vaccinate men beginning 14 days after the initiation of androgen 
depletion therapy using the same three injection protocol.   
 
As part of consideration for enrollment, the study will only enroll subjects for whom androgen 
depletion therapy or deferral of such treatment are considered standard of care options.  In 
addition, in order to be considered for the study, patients must be willing to be in either study 
group (Arm A or Arm B).  Randomization of patients to Arm A (vaccine only) or Arm B (ADT plus 
vaccine) will be 1:1.  Fifty (50) patients will be enrolled, 25 in each Arm of the study.  Twenty five 
(25) cards will be printed for Arm A and 25 cards printed for Arm B and all 50 cards will be 
placed in a container.  Following the determination of eligibility of each patient and 
administration of the Informed Consent document, a card will be drawn to determine which Arm 
of the study the patent is entered. 
 
We will enroll and treat patients at two affiliated and adjacent medical centers, the University 
of Iowa Hospitals and Clinics (UIHC) and the Iowa City Veterans Affairs Health Care System (IC 
VAHCS).  There is a single Institutional Review Board (IRB) that approves protocols for both 
institutions.  After the University of Iowa’s IRB has approved the protocols the Research and 
Development Committee of the IC VAHCS meets to provide their approval for the study. 
 
Patient Recruitment:  The UIHC and IC VAHCS draw from a large catchment area that will 
ensure the enrollment of sufficient numbers of men into the protocol.  We will use recruitment 
methods similar to the methods used to obtain subjects for our Phase I clinical trial.  That 
includes contacting patients that are being or have been treated in the Departments of Urology, 
Internal Medicine, and Radiation Oncology at the UIHC and IC VAHCS and letters written to 
urologists, oncologists, and medical oncologists in Iowa and the neighboring states of 
Wisconsin, Illinois, Missouri, Nebraska, Minnesota, and South Dakota.  Drs. J. Brown and M. 
O’Donnell and ARNP E. Brown will assist in the recruitment of patients from the Department of 

PROTOCOL #1 
Lubaroff, page 14 
 
 
Urology, Dr. Vaena from Internal Medicine, and Dr. Smith from Radiation Oncology.  Additional 
recruitment of patients will occur through a referral collaboration with Mercy Medical Center, 
Des Moines, IA (Hereafter referred to as DMMCC).  Once accrual has been completed, no 
further recruitment methods will be utilized. 
 
A physician from outside the UIHC or IC VAHCS may inform patients of the study.  If a 
patient expresses interest in learning more about the trial, the physician will provide him with 
contact information for the clinical trial team.  When a patient contacts the clinical trial team, a 
member of the trial team will briefly describe the study and request the patient’s mailing address 
in order to mail him a copy of the study informed consent form.  
 
After the patient has had time to become familiar with the study and discuss it with his 
family, a member of the research team will follow up with a telephone call to discuss the study 
and answer any questions the patient may have.  Of particular importance is the patient’s 
understanding that he will be assigned to one of two arms in the protocol, vaccine alone or 
androgen deprivation therapy followed by vaccine.  At this time, if the patient is still interested in 
participating, he will be asked to provide verbal consent to allow the research team to review 
information from his medical record to evaluate his eligibility for the trial.  The researcher will 
document the verbal consent process in the research file, and will contact the patient’s 
physician to request the medical record for review. The patient will be required to sign a HIPAA 
privacy form and a Release of Medical Information form at his physician’s office prior to the 
physician releasing his records to the research team.  
 
  After reviewing the relevant portions of the medical record, a member of the research team 
will again contact the patient.  Eligible individuals will be scheduled for a visit to the UIHC or IC 
VAHCS to undergo additional eligibility testing.  At that visit, informed consent for participation in 
the clinical trial will be obtained.  The subject will then be further evaluated for study eligibility 
through a medical history and physical exam, blood tests, and scans.  Final determination of 
eligibility will occur at a weekly meeting of the clinical trial team. 
 
2. OBJECTIVES 
 
2.1 Primary Objective 
 
To evaluate the development of anti-PSA immune responses in study patients, of 
particular importance is the comparison of immune responses generated in the hormone 
naïve and ADT patients. 
 
2.2 Secondary objectives 
 
2.2.1  To evaluate the response rates (PSA responses and changes in PSADT) of the 
Ad/PSA vaccine using a prime-boost immunization strategy, in patients with 
recurrent disease, either hormone naïve or during androgen deprivation therapy 
(ADT). 
 
2.2.2 To evaluate biochemical (PSA recurrence) and radiographic (bone scans) time to 
progression and overall survival in evaluable patients receiving the Ad/PSA 
vaccine. 

PROTOCOL #1 
Lubaroff, page 15 
 
 
 
3. SELECTION OF PATIENTS 
 
As described in Section 1.6 prostate cancer patients will be enrolled in one of two arms of 
the study.  All men will have recurrent disease and who are considered hormone naïve, having 
received no prior hormonal therapy except as allowed in section 3.2.9.  Following the 
determination of eligibility to participate in the protocol, one group of patients will receive the 
Ad/PSA vaccine alone (Arm A) and a second group of patients will begin androgen deprivation 
therapy with vaccination beginning fourteen days later (Arm B).   
 
3.1 Inclusion criteria: 
 
 
3.1.1 Men with prostate cancer who have received prior local therapy (radical 
prostatectomy or definitive radiation therapy) and have biochemical (PSA) relapse without 
evidence of radiographic or clinical metastatic disease. 
 
 
3.1.2 For men who had prior prostatectomy, the surgery must have occurred at least 6 
months prior to initiation of treatment. 
 
 
3.1.3 For men who had prior definitive radiation therapy, radiation must have been 
completed at least 1 year prior to initiation of treatment. 
 
 
3.1.4 Exhibit at least three separate rises in serum PSA, at least one month apart with 
differences >0.03 ng/ml and a total PSA of >0.2 ng/ml. 
 
 
3.1.5 Have a PSA doubling time of >6 months if the baseline serum PSA was >2 
ng/ml. 
 
 
3.1.6 Negative bone scans. 
 
3.1.7 
Negative CT scans of abdomen and pelvis (no evidence of soft tissue lesions >1 
cm). 
 
3.1.8 
Scans must be obtained within 6 weeks of entry into the trial (initiation of 
treatment). 
 
 
3.1.9 Written informed consent. 
 
 
3.1.10 Age > 18 years. 
 
 
3.1.11 Required laboratory values [obtained within 2 weeks of study entry (initiation of 
treatment)] 
 
 
3.1.12.1 
Serum creatinine < 2.0 mg/dL 
 
 
3.1.12.2 
Adequate hematologic function:  granulocytes > 1800 per mm3, 
platelets > 100,000 per mm3, WBC >3700, and lymphocytes >590. 
 
 
3.1.12.3 
Adequate hepatocellular function:  AST <3x ULN and total bilirubin 
<1.5 mg/dl (unless bilirubin elevation is consistent with Gilbert’s syndrome). 
 

PROTOCOL #1 
Lubaroff, page 16 
 
 
3.1.13  PSA used as an eligibility criterion must be drawn within  42 days prior to injection 
number 1 and will be redrawn on Day 1 for use as a baseline value. 
 
 
3.2 Exclusion criteria: 
 
 
3.2.1 Candidates for salvage radiation therapy unless the patient refuses. 
 
 
3.2.2 Active or unresolved clinically significant infection. 
 
 
3.2.3 Parenteral antibiotics <7 days prior to initiation of treatment. 
 
 
3.2.4 Evidence of prior or current CNS metastases. Specific imaging is not necessary 
in the absence of signs or symptoms. 
 
 
3.2.5 Co-morbid medical conditions which would result in a life expectancy 
(participation) of less than 1 year. 
 
 
3.2.6 Patients with compromised immune systems; congenital, acquired, or drug-
induced (immunosuppressive agents) will be excluded from the study. Use of prednisone 
at doses higher than 10 mg daily (or equipotent steroid doses) for more than 7 days 
within the last 3 months is not allowed. 
 
 
3.2.7 Pre-existing malignancies that required treatment within the past 5 years except 
for basal or squamous cell cancers of the skin. 
 
 
3.2.8 Prior systemic therapies for prostate cancer not allowed (hormonal therapy, 
including but not limited to LHRH agonists, antiandrogens, ketoconazole or 
chemotherapy – mitoxantrone/taxanes/estramustine, etc.) except when patients stopped 
hormone therapy two or more years prior to enrollment and currently have normal 
testosterone levels; patients in Arm B, undergoing androgen depletion therapy during the 
vaccination study will be eligible. 
 
 
3.2.9 Prior participation in any vaccine studies for non-infectious diseases. 
 
 
3.2.10 The inability to understand the language and the clinical protocol. 
 
 
3.2.11 Allergy or religious objection to pork products; Gelfoam is produced from pork. 
 
4.  Registration Procedures  
 
 
4.1 All patients will be registered through the Department of Urology at the University of 
Iowa Hospitals and Clinics (UIHC) or the Urology Service at the Iowa City Veterans Affairs 
Health Care System (IC VAHCS).   
 
4.2  Patients who are candidates for enrollment into the study will be evaluated for eligibility 
by the clinical investigators to ensure that the criteria outlined in Section 3 have been 
satisfied and that the patient is eligible for participation in this clinical investigation.  The 
University of Iowa will provide a patient eligibility case report form for this evaluation. 
 
4.3 Following determination of eligibility patients will be assigned to receive the vaccine 
alone (Arm A) or ADT followed by the vaccine (Arm B). 

PROTOCOL #1 
Lubaroff, page 17 
 
 
 
4.4 Informed Consent - Signed informed consent for enrollment in this protocol will be 
obtained from eligible patients by the attending physician, study coordinator, or clinical trial 
coordinator before the start of the research intervention.  At the preadmission consultation, 
patients will be fully informed of the purpose and potential risks and benefits of participating 
in the study.  Patients will have the opportunity to have questions answered to their 
satisfaction before signing the consent. 
 
4.5  Eligible patients must be registered Monday through Friday between 8:00 a.m. and 
4:30 p.m. (Central Time).  Patients at the University of Iowa Clinical Cancer Center will be 
registered by calling Pamela Zehr, RN, MA  319-353-8914.  Patients at the IC VAHCS will 
be registered by calling Karen Clark Griffith, RN, PhD at 319-338-0581, ext. 7519.  
Information from the eligibility form will be provided by the investigator or the investigator's 
research staff to the University of Iowa Cancer Center at this time, and the patient will be 
registered and assigned a unique patient number. 
 
4.6 No patient may be enrolled or begin the research intervention prior to registration and 
assignment of a patient number.  As a follow-up, University of Iowa Cancer Center will 
provide the investigator with written confirmation of each patient's registration. 
 
4.7 
All investigators will be notified by the Chair of the Protocol Review and Monitoring 
Committee or by the trial’s Data and Safety Monitoring Board if the study is placed on 
administrative hold, and when the study is completed or closed to further patient enrollment. 
 
4.8 
Patients must begin the vaccine protocol, either the vaccine alone (Arm A) or begin 
ADT (Arm B) within 30 days of registration (date the study committee approves patient 
eligibility). 
 
5.  RESEARCH INTERVENTION PLAN 
 
5.1 Administration Schedule 
 
Ad/PSA  
Patients with recurrent disease after surgical treatment or radiation therapy will be 
randomized to either Arm A (vaccine only) or Arm B (androgen depletion therapy plus 
vaccine).  All patients will receive three injections of 0.125 ml. of the Ad/PSA 
subcutaneously in the right thigh unless patient condition precludes the use of that leg, in 
which case the left thigh will be used for all three injections.  The dose of the vaccine, based 
upon our results from the Phase I trial, will be 1 x 108 pfu (4.4 x 109 particles) in the Gelfoam 
matrix.  The Gelfoam comes in sterile patient-ready packages.  The virus will be suspended 
in sterile saline and the Gelfoam powder added in a ratio of 30 mg of powder per ml. of virus 
suspension.  Injections will be spaced apart by 30 days, such that each patient will receive 
the vaccine on days 0, 30, and 60.  The use of the matrix has been shown in collaborative 
pre-clinical experiments to enhance infection of host cells by the virus.  Results from the 
Phase I trial indicated that the injection of the vaccine in Gelfoam did not produce any 
adverse events greater than those produced by the vaccine in an aqueous suspension.   
The vaccine induced anti-PSA immune response in patients injected as an aqueous or 
Gelfoam vaccination.  Injections will be carried out in the University of Iowa Clinical 
Research Unit (CRU).  Each subject will be required to stay in the CRU for two hours and 
observed for early signs of toxicities following each of the three vaccinations.   
 

PROTOCOL #1 
Lubaroff, page 18 
 
 
5.2 Design and Stages  
 
 
Subjects will enter the study according to a phase II two-stage design Simon (1989). We 
anticipate a total of 25 subjects (per arm) if the stopping rules do not signal futility. After 
testing the research intervention on 10 patients, if 3 do not have the desired increase in anti-
PSA T cells, the trial will be terminated. Otherwise, an additional 15 patients will be allowed 
to enter the trial for a total of 25 patients with the goal that more than 13 will show the 
desired increase. For both arms, we will need 50 subjects.  For statistical considerations and 
the power reached under this study design consideration, we refer the reader to Section 10. 
 
6.  Adverse Events 
 
Toxicity will be graded according to the NCI common toxicity criteria “Common 
Terminology Criteria for Adverse Events” (NCI-CTCAE v3.0 can be accessed at website:  
http://ctep.cancer.gov/forms/CTCAE_Index.pdf).  Non-hematologic and Hematologic dose 
limiting toxicity (DLT) will be defined as described in Section 8.5.1. 
 
6.1 Definitions 
 
Adverse Event (AE) is any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and that does not 
necessarily have a causal relationship with this research intervention. An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not related to the medicinal (investigational) 
product. 
This will also include intercurrent diseases and accidents observed during the research 
intervention period as well as corresponding events during drug-free, pre- and post-
intervention periods, under placebo or in a reference group receiving drug or non-drug 
therapy.  
Serious adverse event (SAE) is any untoward medical occurrence that:  
a. results in death 
b. is life-threateningi 
c. requires inpatient hospitalization or prolongation of existing hospitalization 
d. results in persistent or significant disability or incapacity 
e. is a congenital anomaly / birth defect or 
f. 
is another medically important condition.ii 
 
6.2 
Procedures of documentation of AEs 
 
All AEs occurring during the study must be documented, regardless of the assumption of 
a causal relationship, on the respective AE CRF. All events, which occurred after signed 
informed consent, should be documented. The investigator should ensure that all events 
are recorded that occurred within at least 4 weeks after the last exposure to the study 
drug (through Day 90).  
Documentation of AEs includes: date of onset and offset, intensity, frequency, 
seriousness, related interventions and outcome. The investigator will also evaluate the 

PROTOCOL #1 
Lubaroff, page 19 
 
 
probability of a causal relationship of the adverse event to the study medication as 
being: “definite, probable, possible, unlikely, or unrelated.” 
All unanticipated problems involving risk to subjects or others, serious adverse events 
related to participation in the study and subject deaths related to participation in the 
study 
will 
be 
promptly 
reported 
by 
phone 
(301-619-2165), 
by 
email 
(hsrrb@det.amedd.army.mil), or by facsimile (301-619-7803) to the USAMRMC, Office 
of Research Protections, Human Research Protection Office.  A complete written report 
will follow the initial notification.  In addition to the methods above, the complete report 
will be sent to the U.S. Army Medical Research and Materiel Command, ATTN: MCMR-
ZB-PH, 504 Scott Street, Fort Detrick, Maryland 21702-5012. 
The medical monitor is required to review all unanticipated problems involving risk to 
volunteers or others, serious adverse events, and all volunteer deaths associated with 
the protocol and provide an unbiased written report of the event.  At a minimum, the 
medical monitor should comment on the outcomes of the event or problem, and in the 
case of a serious adverse event or death, comment on the relationship to participation in 
the study.  The medical monitor should also indicate whether he/she concurs with the 
details of the report provided by the study investigator.  Reports for events determined 
by either the investigator or medical monitor to be possibly or definitely related to 
participation and reports of events resulting in death should be promptly forwarded to the 
HSRRB. 
Expedited reporting 
The investigator must immediately report serious adverse events (SAE) occurring or 
observed during the course of the study and within 4 weeks of last administration of the 
study drug to the FDA, IRB, OBA, and CRU.  
The order of reporting of SAEs is the following:  reported to the Principal Investigator 
within 24 hours of the event; the PI will then report the event to the FDA, OBA, 
Institutional Biosafety Committee (IBC), and DOD within 7 days of the event and to the 
IRB within 10 days of the event.  After notifying the proper agencies by telephone of an 
SAE the “Serious Adverse Event Report ”must also be sent by fax to the agencies 
whether or not complete information is available at the time. If complete information is 
unavailable the investigator must provide follow-up information to the agencies as soon 
as it is known.  
In particular, the investigator must inform the agencies and committees by phone and 
fax using the timeline described above of immediately life-threatening SAEs or SAEs 
with fatal outcome.  
 
Important: 
The investigator must report any SAE to the FDA, IRB, OBA, and 
CRU, regardless of causality. 
Reports will be evaluated by the Medical Monitor/Sponsor. FDA/HPB and investigators 
will be informed as required by the regulations. The same information will also be made 
available to all participating investigators as well as to other investigators participating in 
different clinical trials utilizing the same study medication.  
It should be noted that, although the Ad/PSA vaccine contains the gene for PSA, there is 
no need for patients to utilize contraceptive practices.  The adenovirus will not be 
transmitted from patient to his partner. 
In addition, the side effects of androgen depletion therapy, fluid retention, hair loss, hot 
flashes, nausea, vomiting, bone pain, memory changes, and depression, were not 

PROTOCOL #1 
Lubaroff, page 20 
 
 
observed in vaccinated men our Phase I trial.  Therefore, there should not be any 
confusion as to which side effects will be attributed to ADT and which to the Ad/PSA 
vaccine.  However, subjects will be closely monitored by the research team for the 
development of ADT-related side effects.  If side effects occur, investigators will take 
necessary steps to treat them, including discontinuation of the medication and/or 
discontinuation from the study. 
 
7  MEASUREMENT OF CLINICAL AND IMMUNOLOGICAL EFFECT 
 
7.1 Methods of Malignant Disease Evaluation  - Each patient will have a baseline evaluation 
prior to the injection of the Ad/PSA vaccine.  The measurements will include physical 
exam, temperature, weight, serum PSA, blood chemistries, urinalysis, a quantitative 
bone scan for bone metastases, chest x-ray, and CT for soft tissue metastases, and 
performance status for quality of life.  PSA measurements, CT and bone scans are 
routinely used to follow disease recurrence and/or progression in individual prostate 
cancer patients and as such would be considered standard of care.  Laboratory 
measurements such as hematology, liver function and kidney function chemistries, while 
are routinely used to follow the health of a prostate cancer patient, would not normally be 
performed at the frequency proposed in this trial to assess possible vaccination 
toxicities.  Therefore, they would be considered part of the research protocol. 
 
Patients will be seen in the Urology and Oncology Clinics (see Table 6 for schedule).   
The injection site will be examined for evidence of erythema, induration and necrosis 
and patients will have their temperature and weight recorded and the patients will be 
interviewed to determine whether they experienced any adverse reactions.  Blood 
samples will also be taken for standard laboratory testing and measurement of PSA and 
anti-PSA immune responses (see Table 6).  Patients recruited from the DMMCC will 
return to that facility for their laboratory testing on days 44, 74, and at 6, 9, 12 months 
and all subsequent visits.  At the 6 month, 9 month, 12 month, and subsequent semi-
annual visits each patient will be evaluated comparing the baseline visit measurements 
to those obtained at the specific visit. 
 
Because changes to the clinical status of the patients will be prolonged, continued follow 
up will be important.  Therefore, patients will be requested to return to the clinic every six 
months following the 12 month post-vaccination visit.  The visits will continue until the 
patient demonstrates signs of progressive disease.  Follow-up of patients for survival will 
occur every 6 months by telephone following progressive disease. 
 
7.2 Use of Serum PSA for Disease Evaluation – Based upon our pre-clinical experiments 
and the results from the Phase I clinical trial we expect the immunized men to produce 
anti-PSA antibodies.  The levels of antibody will be measured by a flow cytometry assay 
as described by Cavacini, et al. used in our Phase I clinical trial (39).  We will also 
explore the use of a second serum marker for prostate cancer, hK2 in collaboration with 
Donald Tindall, Charles Young, and George Clee at the Mayo Clinic.  Investigators at 
Mayo, along with Hybritech, Inc. have been exploring hK2 and published a number of 
papers in recent years on the subject (70-73).  Patient sera from each clinic visit will be 
sent to Mayo where they will measure the levels of hK2.  We will use the data to 
evaluate the effect of anti-PSA antibodies on both PSA and hK2 in the sera of 
vaccinated patients. 

PROTOCOL #1 
Lubaroff, page 21 
 
 
7.3 Experimental Evaluation of the Ad/PSA Vaccination 
 
7.3.1  Blood will be collected prior to each injection of the Ad/PSA vaccine and at return 
dates indicated in Table 6, page 26.  Two separate samples will be collected; one 
in red top tubes to allow collection of serum from coagulated blood and a second 
in heparinized tubes to permit collection of lymphocytes.  
 
7.3.2 Levels of PSA, hK2, anti-PSA antibodies, and anti-adenovirus antibodies will be 
measured in the serum. 
 
7.3.3  Anti-PSA T cell immune responses will be measured by ELISPOT analysis using 
the methods developed for, and used in, our Phase I clinical trial.  In addition to 
measuring the anti-PSA T cell activity, we will also measure anti-adenovirus T 
cell activity as well as reactivity to stimulation with cytomegalovirus (CMV).  A 
non-specific stimulus will be provided by PMA and ionomycin for each patient’s 
lymphocytes. 
 
7.4 Definitions of Response – 
 
7.4.1  Arm A – Hormone Naïve –  
 
 
7.4.1.1   Primary Endpoint - Development of Anti-PSA Immune Responses 
 
 
7.4.1.1.1 Immunologic response – Definition: an increase of >200% 
above pre-immunization levels of anti-PSA T cells as measured by 
ELISPOT analysis, measured at any point after vaccination.  
 
 
7.4.1.2 Secondary Endpoints – PSA Responses 
 
 
7.4.1.2.1   PSA response - Definition: a 50% reduction in the pre-research 
intervention PSA value, verified with a second measurement >/= 28 days 
later 
 
 
7.4.1.2.2   A 50% increase in the PSADT compared to pre-enrollment 
PSADT. 
 
 
7.4.1.2.3 
PSADT will be calculated based on the MSKCC online 
calculator, available at http://www.mskcc.org/mskcc/html/10088.cfm.  
 
 
7.4.1.2.4  PSADT response will be measured at 9 and 18 months after 
initiation of the research intervention. 
 
 
7.4.1.2.5  Three measurements of PSA, spaced at least 2 weeks apart, 
will be required prior to study enrollment. Post-research intervention 
PSADT will be based on PSA levels at 3, 6 and 9 months (9 month 
PSADT calculation) and 3, 6, 9,12,15,18 month levels (18 month PSADT 
calculation). 
 
7.4.2  Arm B – Androgen-Deprivation Therapy – 
 
 
7.4.2.1  Primary Endpoint - Immunologic response 

PROTOCOL #1 
Lubaroff, page 22 
 
 
 
 
7.4.2.1.1  Definition: an increase of >200% above pre-immunization levels 
of anti-PSA T cells as measured by ELISPOT analysis, measured at any 
point after vaccination. 
 
 
7.4.2.2   Secondary Endpoint - PSA response - Definition: a 50% reduction in the 
pre-research intervention PSA value, verified with a second measurement >28 
days later 
 
7.5 Definition of Progression 
 
7.5.1  Development of positive bone scan.B 
 
7.5.2 Development of rising PSA above baseline, if existent.  A rising PSA will be 
defined as a 50% increase above baseline measurement on two consecutive 
measurements, >28 days apart. 
 
7.6 Timing of Toxicity Assessments - Toxicity assessment will occur as stated in the calendar. 
We will wait for the last patient of stage I to reach the 90 day toxicity assessment date (and 
review of safety data) prior to proceeding with stage II as outlined in the statistical plan. 
 
8   STUDY PARAMETERS 
 
8.1 Scans or x-rays must be done within 6 weeks prior to the initiation of treatment. 
 
8.2 CBC with differential, LFT’s must be done <2 weeks before the initiation of treatment. 
 
8.3 All chemistries must be done <2 weeks before the study entry, unless specifically 
required on day 1 as per protocol.  If abnormal, they can be repeated within 48 hours 
prior to study entry. 
 
8.4 Hgb, Hct, WBC, Plt must be done <2 weeks before study entry but, if abnormal, they can 
be repeated <48 hours prior to study entry. 
 
8.5 REMOVAL OF PATIENTS FROM STUDY (Criteria for discontinuation of a patient's 
study participation) 
 
8.5.1 Adverse events:  Study subjects will be monitored for adverse events for 30 days 
beyond the final study injection, or longer if existing unresolved AEs are being 
monitored.  In the event of a vaccine-associated unmanageable or irreversible 
toxicity, that would include hematological and non-hematological toxicities (see 
Section 8.5.1.1), the investigator will withdraw a patient from further research 
intervention and notify the Study Chair immediately.  In addition, the FDA and the 
IRB and other agencies listed on page 18 will be notified of the adverse events.  
If unmanageable or irreversible toxicities occur the patients will receive the best 
possible medical care according to the recommendations of the treating 
physicians.  The treatment plan will depend upon what unmanageable or 
irreversible toxicities may occur.  In the event of an emergency the patient will be 
 
B Only patients with negative bone scans are eligible for the study 

PROTOCOL #1 
Lubaroff, page 23 
 
 
instructed to seek immediate care by calling 911, his local physician, the 
University of Iowa or Iowa City VA Health Care System urology staff on call. 
 
 
8.5.1.1    Management of Toxicities 
 
 
 
8.5.1.1.1 
Hematological Toxicity Management 
 
8.5.1.1.1.1 
Patients with grade 2 or 3 hematological toxicity will 
not receive the subsequent dose of vaccine until there is 
normalization of cbc parameters until grade 1 or less toxicity is 
reached. An exception to withholding a dose of vaccine would be 
if the cbc parameters were stable (at or near the pre-vaccination 
values).  
 
8.5.1.1.1.2 
In case of grade 2 or 3 hematological toxicity 
detected any time during protocol therapy, CBC with differential 
will be checked on a weekly basis until grade 1 or less toxicity is 
reached.  
 
8.5.1.1.1.3 
If the subsequent vaccine dose needs to be 
postponed for more than 2 weeks, those patients will be removed 
from protocol therapy.  
 
 
 
8.5.1.1.1.4 
Patients with grade 4 hematological toxicity at any 
time will be permanently removed from protocol therapy. 
 
 
 
8.5.1.1.2 
Non-Hematological Toxicity Management 
 
8.5.1.1.2.1 
Patients with grade 2 or 3 non-hematological 
toxicity will not have the subsequent dose of vaccine administered 
until the toxicity ameliorates down to a grade 1 level.  
 
8.5.1.1.2.2 
Patients will be evaluated once a week with study 
team assessments if a grade 3 non-hematological toxicity occurs.  
 
8.5.1.1.2.3 
If the subsequent dose of vaccine needs to be 
postponed for more than 2 weeks, those patients will be 
permanently removed from protocol therapy.  
 
8.5.1.1.2.4 
Patients with grade 4 non-hematological toxicity at 
any time will be permanently removed from protocol therapy. 
 
 
Patients may also be removed from protocol therapy at any time based on 
assessment of any other risks, at the discretion of the study investigators. 
 
8.5.2  Disease Progression:   Patients will be taken off-study if they have progressive 
disease (PD) or clinically significant deterioration at any time during the study if 
the investigator feels that (a) alternative prostate cancer therapy might benefit the 
patient, or (b) to continue on study might be unsafe for the patient.  Patients 
receiving alternative prostate cancer therapies will be followed for toxicity for 30 
days beyond the third AdPSA injection only unless existing unresolved AEs are 
being monitored.  Patients were asked in the Informed Consent document 
whether they would return every three months up to 12 months from study entry 

PROTOCOL #1 
Lubaroff, page 24 
 
 
for immunologic evaluations.  Patients who are being treated with prednisone at 
doses higher than 10 mg daily (or equipotent steroid doses) will be excluded from 
these every three month returns. 
 
8.5.3  Allergic Reactions:  Patients must be removed from the study should they 
develop grade II allergic reactions. 
 
8.5.4  Personal Reasons:  As stated in the informed consent, patients may withdraw 
from the study at any time. 
 
8.5.5  Clinical Judgment:  A patient may be withdrawn from the study, if, in the opinion 
of the investigators, it is not in the patient's best interest to continue (e.g. an 
adverse experience, intercurrent illness, non-compliance, etc.) 
 
8.5.6  The date of discontinuation and the reason(s) for patient discontinuation from the 
study will be recorded in the CRF.  All efforts will be made to conduct evaluations 
that are required at the follow-up for each patient who discontinues research 
intervention, regardless of the reason. 
 
Regulatory and Reporting Requirements 
The Data and Safety Monitoring Committee (DSMC) of the Holden Comprehensive Cancer 
Center will provide data and safety monitoring for this study. “The Data and Safety Monitoring 
Plan of the Holden Comprehensive Cancer Center” provides standard operating procedures to 
monitor all clinical cancer trials at the UIHC. All investigator-initiated trials are automatically 
monitored by the DSMC. A detailed data and safety monitoring plan for this study is on file with 
the DSMC and the Clinical Research Safety Officer (CRSO). 
Data Management, Quality Control and Data Security  
In order to protect confidentiality the subject will be assigned an identification number.  This 
number will be used on all specimens from the subject and will be used for documentation 
purposes.  
Data management for the optimal entry, processing, storage, and retrieval for this protocol's 
data will be accomplished by the principal investigator. The database will be located on a 
computer or in a locked cabinet in a locked office. This computer will be secured, accessible 
only by the research team. There will be more than one copy of the database. The second, 
secured, copy of the protocol data will be stored in a locked room accessible only by the 
research team. For quality control, auditing, and checking data for integrity, there will be a 
regular accounting of data performed.  The medical record and research record will be linked by 
the study identification number.  The data managers in this trial will be responsible for 
verification of the accuracy of all data transferred from the medical record to the research 
record.  These records will be audited quarterly by the Data Safety Monitoring Committee.  All 
Data Safety Monitoring Committee reports will be provided to the USAMRMC Office of 
Research Protections, Human Research Protection Office as they become available. 
 
Data will be kept on file on each patient for at least two years past the termination of that 
patient’s participation in the trial.  All records will be stored long-term in a secure location.  UIHC 
records will be kept for at least 15 years and then be confidentially destroyed.  VAHCS records 
will be retained until disposition instructions are approved by National Archives and Records 
Administration. 
 

PROTOCOL #1 
Lubaroff, page 25 
 
 
Information from the medical records of patients referred by physicians outside the University of 
Iowa and the VA Health Care System required to determine eligibility for this protocol will be 
placed in a research folder, identified by the patient code and will be available only to members 
of the clinical trial team (identified on pages 28 and 29).  The signed HIPAA forms will be kept in 
the patient’s folder in the office of the referring physician.  The records of such patients that are 
deemed ineligible following a screening procedure will have their signed Release of Information 
forms, informed consent document, and eligibility form kept in a research folder under similar 
security conditions.  The records for ineligible subjects will be stored with the other research 
records as indicated above.   

PROTOCOL #1 
Lubaroff, page 26 
 
 
Table 6 
Study Design and Testing 
 
 
Std. of 
Care  
or 
Res. 
Prior 
to 
Reg.1 
Day  
-14 
Day 1 
First Inj. 
 
30 d. 
2nd Inj. 
 
44 d. 
 
60 d. 
3rd Inj. 
 
74 d. 
 
90 d. 
 
6 mo 
 
9 mo 
 
12 
mo 
Every 
6 mo. 
to 
prog. 
Follow-
up8 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Immunization 
R 
 
 
X 
X 
 
X 
 
 
 
 
 
 
 
Physical Examination 
S 
 
 
X 
X 
 
X 
 
X 
 
 
 
 
 
Adverse Event Assessments 
R 
 
 
X 
X 
 
X 
 
X 
 
 
 
 
 
Performance Status 
S 
 
 
X 
X 
 
X 
 
X 
X 
X 
X 
 
 
Vital Signs 
S 
 
 
X 
X 
 
X 
 
X 
X 
X 
X 
 
 
Weight 
S 
 
 
X 
X 
 
X 
 
X 
X 
X 
X 
 
 
Blood for PSA & PAP2 
S/R 
 
 
X 
X (R) 
 
X (R) 
 
X 
X 
X 
X 
X 
 
anti-PSA Ab, and 
lymphocytes for cellular 
immunity 
R 
 
 
X 
X 
X 
X 
X 
X 
X7 
X7 
X7 
X9 
 
CBC, differential 
R 
X 
 
 
X 
 
X 
 
X 
X 
X 
X 
X9 
 
AST, ALT, LDH, alkaline 
phosphatase, bilirubin 
R 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
 
Creatinine 
R 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
 
Urinalysis 
R 
X 
 
 
X 
 
X 
 
X 
 
 
 
 
 
Chest x-ray 
R 
X5 
 
 
X5 
 
X5 
 
X5 
 
 
 
 
 
Bone scan 
S 
X 
 
 
 
 
 
 
X6 
X6 
X6 
X6 
X6 
 
Abdominal/pelvic CT 
S 
X 
 
 
 
 
 
 
X6 
X6 
X6 
X6 
X6 
 
Serum testosterone3 
R 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
Begin ADT3, 4 
S 
 
X 
 
 
 
 
X 
 
 
 
 
 
 
Survival assessment 
R 
 
 
 
 
 
 
 
 
 
 
 
 
X8 
  S =  Procedures considered “Standard of Care” for prostate cancer patients. 
  R =  Procedures considered “Research” as part of this Clinical Trial. 
 
1 Reg. = Registration, the date patient eligibility approved by the study team. 
2 If the PSA meets the definition of progression, partial or complete response, confirm the PSA results >/= 28 days. 
3 For Arm B only 
4  First ADT injection 14 days prior to vaccine injection #1.  Second dose 12-16 weeks later (depending on formulation; see package insert) 
5 Will be repeated only if patient develops a fever. 
6 Will be repeated only if the patient demonstrates a rise in PSA (50% increase above baseline on 2 consecutive measurements, 30 days apart) 
7 To be performed even if there is disease progression (up to 12 months from study entry) if the patient has consented to this in the informed consent (question in 
section “How long will I be on the study?).” 
8 Follow-up for survival every 6 months by telephone. 
9 Obtained at 18 and 24 months only 
 
In the event of holidays or other extenuating circumstances the times on the table may vary by + 4 days for the first 90 days.  Beyond that every effort will be made 
to see the patient within 2 weeks of the expected date of return, but the investigator is willing to allow for other arrangements for the convenience of the patient. 

PROTOCOL #1 
Lubaroff, page 27 
 
 
9  DRUG FORMULATION AND PROCUREMENT 
 
9.1 Drug Name 
 
Adenovirus/PSA (Ad/PSA) 
 
9.2 Classification 
 
Vaccine 
 
9.3 Mode of Action 
 
The adenovirus is a replication-deficient virus unable to produce virus progeny in the 
infected cells.  The virus will infect cells in the location of the injection site, the PSA gene 
will produce the protein product which will be recognized as an antigen by the immune 
system and produce anti-PSA immune responses.  Based upon our pre-clinical studies 
in an animal model of human prostate cancer, these responses, mainly the CD8+ CTL 
response, will cause the destruction of PSA-secreting prostate tumors. 
 
9.4 Dose Specifics and Route of Administration 
 
The route of injection, vehicles for the vaccine, and dose schedules have been 
outlined in Section 5.1 of this protocol. 
 
9.5 Availability 
 
Produced and provided by Molecular Medicine, LLC, San Diego, CA 
 
9.6 Storage 
 
The vaccine is stored in the UIHC’s Pharmacy Department in a -70oC temperature-
monitored and controlled access freezer.  Only the Investigational Pharmacist will 
remove the vaccine from the freezer and enter the amount removed in a trial-specific 
log. 
 
9.7 Injection Procedures & Observation 
 
At the weekly meetings of the clinical trial team at which the eligibility of the patients 
is decided, orders for the initial vaccine injection will be written by clinician members of 
the team.  Orders for the second and third vaccine injections will be written at the 
meetings as close to the date of the scheduled injections as possible.  On the 
designated days of injections and immediately prior to vaccination, the Investigational 
Pharmacist will mix the Ad/PSA (108 pfu or 4.4 x 109 particles) with the Gelfoam powder 
(30 mg/ml).  Each vial of the vaccine contains twice the volume needed for each 
injection.  One half of the vial will contain the proper pfu and particles required for the 
vaccination.  The injection material will be taken from the pharmacy in a 1 ml. syringe 
with 25 gauge needle and presented to staff in the General Clinical Research Center.  
Each patient will be injected with approximately 0.125 ml. of the vaccine/Gelfoam 
material subcutaneously in the thigh.  The exact volume of the vaccination mixture 
(Ad/PSA in Gelfoam) is not as important as the exact number of pfu or particles and that 

PROTOCOL #1 
Lubaroff, page 28 
 
 
is controlled by using the required volume from the dose vial.  The empty syringe will be 
disposed of in a biohazard container. 
 
 Patients will remain in the CRU for 2 hours after each injection to be monitored for 
signs of adverse events. 
 
9.8 Assignment of Patients to Arms A or B 
 
Randomization of patients to Arm A (vaccine only) or Arm B (ADT plus vaccine) will be 
1:1.  Fifty (50) patients will be enrolled, 25 in each Arm of the study.  Twenty five (25) cards 
will in printed for Arm A and 25 cards printed for Arm B and all 50 cards will be placed in a 
container.  Following the administration of the Informed Consent document and 
determination of eligibility of each patient, a card will be drawn to determine which Arm of 
the study the patent is entered.  Patients that choose not to be placed on ADT will not be 
enrolled in the study.  For this study, ADT will consist of LHRH or GNRH agonists, such as 
leuprolide (Lupron) or goserelin (Zoladex).  ADT will be continued for the first nine months of 
the study.  Anti-androgens will be used initially to avoid testosterone flair, but then 
discontinued.  Aromatase inhibitors will not be used.  Patients in Arm B will be vaccinated 14 
days after the initiation of ADT.  To insure that all patients in Arm B reach castrate levels of 
testosterone (T) (<50 ng/ml), serum T levels will be measured on their 60 day visit, prior to 
their third vaccination.  This will be approximately 10 weeks after the initiation of androgen 
withdrawal, a time at which T levels should reach their nadir.  
 
9.9 Manufacturing 
 
9.9.1  The PSA cDNA provided by Donald Tindall, Mayo Clinic, Rochester, MN, was 
placed 3’ to the CMV promoter in a shuttle vector containing Ad5 DNA.  The 
sequence inserted was the pre-pro form of PSA described by Lundwall (75) that 
encodes 262 amino acids with a predicted molecular weight of 28.8 kDa.  Using 
methods previously described (75), the shuttle vector and E1a-E1b deletion 
mutant Ad5 DNA were transfected into HEK 293 cells, and recombination 
between the DNA species was allowed to occur.  The amplification and 
purification of Ad/PSA was performed by the University of Iowa Gene Transfer 
Vector Core as previously described (76).  Ad/lacZ used as a control was also 
obtained from the Gene Transfer Vector Core and is previously described (75).  
 
9.9.2 The Principal Investigator provided the Ad/PSA vaccine used for the pre-clinical 
studies to Molecular Medicine, LLC of San Diego, CA for the production of the 
clinical grade product.  Information on the manufacturing of the GMP Vaccine by 
Molecular Medicine, LLC is found in the accompanying documents supplied by 
the company. 
10  STATISTICAL CONSIDERATIONS 
 
 
The ideal endpoint would be a clinical outcome that is of particular relevance to the patient 
such as increased time to tumor progression, increased time to death, or reducing the 
proportion of death. The trial is using a surrogate endpoint as a substitute to the clinically 
meaningful outcome since the tumor cannot be accessed directly. The association between the 
surrogate and survival rate had not been clearly established by any phase I & II trial. The trial 
consists of using Ad/PSA vaccine administered in multiple injections to prostate cancer patients 
with minimal disease burden—with the goal to induce anti-PSA T cells responses. Three 
injections of equal dose are proposed. A previous phase I trial consisting of a single injection in 

PROTOCOL #1 
Lubaroff, page 29 
 
 
men with metastatic prostate cancer was able to induce anti-PSA T cells responses. The Phase 
I consisting of a single injection using a dose escalation protocol of the vaccine in an aqueous or 
matrix delivery vehicle did not show any significant AE. Additional pre-clinical pharmacology 
/toxicology studies required by the FDA did not show any significant side effects using the three-
injection schedule. The primary endpoint is the development of anti-PSA immune response—
defined as an increase of greater than 200% above pre-immunization level of anti-PSA T cells 
as measured by ELISPOT analysis, measured 14 days after the 3rd immunization. This endpoint 
will be the same for both arms considered in the protocol—the vaccine only and the vaccine 
plus androgen deprivation therapy arms. 
 
 
During the Phase I trial consisting of vaccine alone, patients that developed anti-PSA T cell 
responses had an increase in the frequency of anti-PSA T cells.  We decided to express the 
anti-T cell response as percent change in antigen-specific T cell frequency compared to the 
frequency prior to immunization. Based on our phase I results, we judge 70% of the patients 
developing anti-PSA immune response as being clinically important while anything less than 
40% in both arms will be judged unworthy.  
 
 
The ideal design would be a two-stage design of Simon (1989). After testing the research 
intervention on 10 patients, if 3 did not have the desired increase in anti-PSA T cells, the trial 
will be terminated. Otherwise, an additional 15 patients will be recruited for the trial for total of 
25 patients with the goal that more than 13 will show the desired increase. If 8 or more show the 
desired increase, then the research intervention will be judged worthy for further considerations. 
In case the number is less than 8, the research intervention will be rejected. 
 
The expected sample size will be 15.14 and the probability of early termination, 0.63. If the 
research intervention is not effective, there is 0.05 probability of concluding that it is. If the 
research intervention is effective, there will be 0.09 probability of concluding that it is not. 
 
 
We will be conservative and use the same design and sample size considerations for both 
arms (25 per arm for a total of 50 patients) even though we expect the vaccine plus androgen 
deprivation therapy arm to yield better results. This brings a natural question of why two 
different research interventions and how one would assess the superiority of one research 
intervention over the other.  We postulate that men in Arm B, those receiving ADT plus vaccine, 
will generate a stronger anti-PSA immune response, as measured by the production of PSA-
specific T cells, than will men in Arm A, those receiving the vaccine alone. For patients in Arm 
B, there will be a quantitative and/or qualitative difference in the generation of anti-PSA T cells.  
That is, a higher percentage of men will demonstrate antigen specific T cells and/or the 
magnitude of the T cell response, as indicated by the precursor frequency, will be greater in this 
population.  The differences will be most evident 14 days after the third and last vaccination. A 
test for comparison of these two proportions will reveal the superiority of ADT plus Vaccine over 
Vaccine alone.  
 
 
The secondary endpoint is the PSADT that will be assessed 18 to 20 months after initial 
patient accrual. Points estimate and exact confidence interval for the proportion of subjects who 
show 50% increase in PSADT will be reported. 
 
Monitoring toxicity:  Stopping rules for toxicity purpose are based on testing the null that toxicity 
level is less than 15% versus the alternative that it is actually greater than 35%. In this setting, 
we will carry two different but dependent looks. If 3 out of 10 show grade 3 vaccine-related 
toxicity or higher, the trial will stop (this will correspond to testing the toxicity hypotheses at level 
alpha=0.1 with a power of 68%); otherwise, another test for toxicity after we have a total of 17 

PROTOCOL #1 
Lubaroff, page 30 
 
 
subjects will be considered. If 5 out of 17 show a grade 3 vaccine-related toxicity or higher, or if 
2 out 17 show a grade 4 toxicity the trial will stop (this will be equivalent to testing the toxicity 
hypotheses at level alpha =0.1 with a power 77%); otherwise we will proceed to full registration. 
The overall type I error for testing toxicity level is around 0.19.  In addition, the trial will stop if 
any vaccine-related deaths occur.  The determination of a death related to the vaccine will be 
made by the clinical trial team and an audit by the Data and Safety Monitoring Board. 
 
Randomization to clinical arm: one-to-one. 
Simon, Richard. “Optimal Two-Stage Designs for Phase II Clinical Trials,” Controlled Clinical 
Trials, 1989, Volume 10, pages 1-10. 
 
Addendum 
PHASE II STUDY OF ADENOVIRUS/PSA VACCINE IN MEN WITH RECURRENT 
PROSTATE CANCER AFTER LOCAL THERAPY 
Food and Drug Administration (FDA) Investigational New Drug (IND) #9706 
Department of Defense, Prostate Cancer Research Program #A-14059.1 
 
The following are reporting requirements and responsibilities of the Principal Investigator to 
the United States Army Medical Research and Materiel Command’s (USAMRMC) Office of 
Research Protections (ORP), Human Research Protection Office (HRPO). 
 
1. The protocol will be conducted in accordance with the protocol submitted to and 
approved by the USAMRMC ORP HRPO and will not be initiated until written notification 
of approval of the research project is issued by the USAMRMC ORP HRPO.   
 
2. Accurate and complete study records will be maintained and made available to 
representatives of the U.S. Army Medical Research and Materiel Command as a part of 
their responsibility to protect human subjects in research.  Research records will be 
stored in a confidential manner so as to protect the confidentiality of subject information.   
 
3. All unanticipated problems involving risk to subjects or others, serious adverse events 
related to participation in the study and subject deaths related to participation in the 
study 
should 
be 
promptly 
reported 
by 
phone 
(301-619-2165), 
by 
email 
(hsrrb@det.amedd.army.mil), or by facsimile (301-619-7803) to the USAMRMC, Office 
of Research Protections, Human Research Protection Office.  A complete written report 
will follow the initial notification.  In addition to the methods above, the complete report 
will be sent to the U.S. Army Medical Research and Materiel Command, ATTN: MCMR-
ZB-PH, 504 Scott Street, Fort Detrick, Maryland 21702-5012.   
 
4. Any deviation to the protocol that may have an effect on the safety or rights of the 
subject or the integrity of the study must be reported to the USAMRMC ORP HRPO as 
soon as the deviation is identified.   
 
5. Major modifications to the research protocol and any modifications that could potentially 
increase risk to subjects must be submitted to the USAMRMC ORP HRPO for approval 
prior to implementation.  All other amendments will be submitted with the continuing 
review report to the USAMRMC ORP HRPO for acceptance. 
 
6. A copy of the approved continuing review report and the local IRB approval notification 
will be submitted to the USAMRMC ORP HRPO as soon as these documents become 
available.  A copy of the approved final study report and local IRB approval notification 

PROTOCOL #1 
Lubaroff, page 31 
 
 
will be submitted to the USAMRMC ORP HRPO as soon as these documents become 
available.   
 
7. The knowledge of any pending compliance inspection/visit by the FDA, OHRP, or other 
government agency concerning clinical investigation or research, the issuance of 
Inspection Reports, FDA Form 483, warning letters or actions taken by any Regulatory 
Agencies including legal or medical actions and any instances of serious or continuing 
noncompliance with the regulations or requirements will be reported immediately to 
USAMRMC ORP HRPO. "  
 
Roles and Responsibilities of Study Personnel: 
 
David M. Lubaroff, PhD, Principal Investigator – Dr. Lubaroff, along with Dr. Vaena, will 
manage all aspects of the trial, from assisting in patient recruitment, co-chairing the clinical trial 
meetings, to supervising the immunologic testing of the patients’ sera and lymphocytes for anti-
PSA immune responses.  He will work together with Dr. Williams the Co-Principal Investigator 
on all important clinical issues for the trial. 
 
Daniel Vaena, MD; Co-Principal Investigator – Dr. Vaena is a Medical Oncologist who cares 
for prostate cancer patients following recurrences of their disease.  He will manage all patient 
care activities associated with this proposal and will work together with Dr. Lubaroff on 
managing the trial.   Dr. Vaena will function as co-chair of the weekly trial team meetings.  He 
will be a major participant in the clinical management of prostate cancer patients that includes 
recruitment, patient management, assessing clinical response and any adverse events and 
long-term follow-up during the trial. 
 
James Brown, MD, Co-Investigator – Dr. Brown, a clinical urologist on the clinical trial team, 
will also participate in the recruitment and clinical management of the patients.  As Chief of 
Urology at the Iowa City Veterans Affairs Health Care System (IC VAHCS) Dr. Brown will be an 
active participant in the treatment and follow-up of patients at that hospital.  He will work closely 
with Dr. Vaena in that capacity. 
 
Mark C. Smith, MD, Co-Investigator – Dr. Smith is a Radiation Oncologist who is responsible 
for radiation therapy of men with prostate cancer.  He will assist in the recruitment and care of 
patients in the trial. 
 
Michael O’Donnell, MD, Co-Investigator – Dr. O’Donnell is an urologist at the UIHC who will 
contribute to the recruitment of new patients and care of enrolled patients in the study. 
 
Sundeep Deorah, MD, Co-Investigator – Dr. Deorah is an urologist at the UIHC who will 
contribute to the recruitment of new patients and care of enrolled patients in the study. 
 
 
Amit Gupta, MD, Co-Investigator – Dr. Gupta is an urologist at the UIHC who will contribute to 
the recruitment of new patients and care of enrolled patients in the study. 
 
Kenneth Nepple, MD, Co-Investigator – Dr. Nepple is an urologist at the UIHC who will 
contribute to the recruitment of new patients and care of enrolled patients in the study. 
 
Pamela Zehr, RN, MA - Clinical Trial Coordinator – Ms. Zehr participates in clinical trials in 
the Holden Comprehensive Cancer Center at the University of Iowa.  She has and will continue 

PROTOCOL #1 
Lubaroff, page 32 
 
 
to guide the clinical protocol through the institutional and federal other regulatory approval 
processes.   She will coordinate follow-up visits of the patients. 
 
Mary Schall, RN – Clinical Trial Coordinator - Ms. Schall also participates in clinical trials in 
the Holden Comprehensive Cancer Center at the University of Iowa.  She will act as a backup 
for Ms. Zehr. 
 
Karen Clark Griffith, RN, PhD - Clinical Trial Coordinator VAHCS – Ms. Griffith will be 
responsible for trial for veteran patients enrolled in the clinical trial at the VA Health Care 
System.  She will work closely with all members of the trial team and attend the weekly 
meetings to discuss eligibility and follow-up of patients. 
 
Steven Varga, PhD, Collaborator – Dr. Varga is an immunologist and will assist in the 
immunologic evaluation of patient’s immune responses to the vaccine. 
 
 
Gideon Zamba, PhD – Biostatistician – Dr. Zamba participated in the discussions pertinent to 
the development of the clinical protocols, performed the power analysis, and constructed the 
section on statistical considerations for the trial.  He will assist in the data analysis and statistical 
interpretation of the patient data. 
 
 
LITERATURE REFERENCES 
 
 
1. Jemal, A, Siegel, R Ward, E, Murray, T, Xu, J, Smigal, C, and Thun, MJ.  Cancer Statistics 
2006.  CA Cancer J Clin, 56:106-130, 2006. 
2. Koostra, NA and Verma, IM.  Gene therapy with viral vectors.  Ann. Rev. Immunol, 43:413-
439, 2003. 
3. Connolly, JB.  Conditionally replicative viruses in cancer therapy.  Gene Therapy, 10:712-
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i The term “life-threatening” in the definition of “serious” refers to an event in which the patient is at risk of 
death at the time of the event; it does not refer to an event, which hypothetically might have caused death 
if it were more severe. 
ii Medically important conditions that may not result in death, be life-threatening or require hospitalization 
may be considered as SAE when, based upon appropriate medical judgment, they may jeopardize the 
patient or may require intervention to prevent one of the outcomes listed in the definition above. 
Examples of such events are intensive treatment in an emergency room or at home for allergic 
bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of 
drug dependency or drug abuse.  N.B.: The term “severe” is often used to describe the intensity (severity) 
of an event (such as: mild, moderate, or severe e.g., pain). The event itself may be of relatively minor 
medical significance (such as severe headache). This is not the same as “serious”, which is based on 
patient/event outcome or action criteria usually associated with events that pose a threat to patient’s life 
or vital functions. Seriousness (not severity) serves as a guide for defining regulatory reporting 
obligations.