Phase II Study of Adenovirus/PSA Vaccine in Men With Hormone - Refractory Prostate Cancer
Phase: PHASE2
Conditions: Hormone Refractory Prostate Cancer
Sponsor: David M Lubaroff

PROTOCOL #2 
 
 
PHASE II STUDY OF ADENOVIRUS/PSA VACCINE IN MEN WITH 
HORMONE - REFRACTORY PROSTATE CANCER 
Food and Drug Administration (FDA) Investigational New Drug (IND) #9706 
  Department of Defense, Prostate Cancer Research Program A-14059.2 
Version 42 – January 27, 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 
fax: 
319-353-4556 
 
 
 
 
fax: 
319-353-8377 
e-mail: david-lubaroff@uiowa.edu 
 
 
e-mail: daniel-vaena@uiowa.edu 
 
Co-Investigators 
 
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: 
319-35-2114 
e-mail: kenneth-nepple@uiowa.edu 
 
Pamela Zehr, RN, MA 
Holden Comprehensive Cancer Center 
tel: 
319-353-8914 
e-mail:  pamela-zehr@uiowa.edu 
 
Mary Schall, RN, BSN 
Holden Comprehensive Cancer Center 
tel: 
319-356-3516 
e-mail:  mary-schall@uiowa.edu 
 
1 Please send all communications to this investigator. 
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 
 
Lyse Norian, PhD 
Department of Urology 
Tel: 
319-335-3013 
e-mail: lyse-norian@uiowa.edu 
 
Nicole Miller, PA-C 
Department of Urology 
Tel:  319-356-3850 
e-mail:  nicole-miller@uiowa.edu 
 
Roseanne Rath, PA-C 
Department of Urology 
Tel:  319-353-6972 
e-mail:  roseanne-rath@uiowa.edu  
 

PROTOCOL #1 
Lubaroff, page 2 
Lubaroff 
 
 
 
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 a award from the Department of Defense 
Prostate Cancer Research Program.

Lubaroff Ad/PSA Phase II, protocol #2, page 3 
INDEX  
 
Section 
Pages 
 
 
Introduction 
4-14 
 
 
Objectives 
14 
 
 
Patient Selection 
14,15 
 
 
Registration Procedures 
15,16 
 
 
Research Intervention Plan 
16,17 
 
 
Adverse Events 
17-19 
 
 
Measurement of Clinical and Immunological Efficacy 
19-22 
 
 
Study Parameters 
23-26 
 
 
Drug Formulation and Procurement 
27,28 
 
 
Statistical plan 
28,29 
 
 
Addendum 
30 
 
 
Roles and Responsibilities of Study Personnel  
31,32 
 
 
Literature References 
32-36 
 

Lubaroff Ad/PSA Phase II, protocol #2, 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).  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.  Adenoviral gene transfer has been 

Lubaroff Ad/PSA Phase II, protocol #2, page 5 
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 (19-26). 
 
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). 
 

Lubaroff Ad/PSA Phase II, protocol #2, 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. 

Lubaroff Ad/PSA Phase II, protocol #2, 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 depletion, 
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.  The following Table 2 is a 
listing of all AEs, sorted by relationship to the vaccine injection. 
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. 

Lubaroff Ad/PSA Phase II, protocol #2, 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 

Lubaroff Ad/PSA Phase II, protocol #2, page 9 
AP020 
Metabolic 
14 
hyponatremia 
Possible 
AP020 
Metabolic 
14 
hyperglycemia 
Possible 
AP020 
Respiratory 
9 
viral symptoms 
Possible 
AP005 
Musculoskeletal 
1 
Left inguinal pain 
Possibly 
AP008 
GU 
21 
Proteinuria 
Possibly 
AP008 
Respiratory 
3 
Cold symptoms 
Possibly 
AP010 
Hematologic 
1 
Decrease in absolute neutrophils 
count 
Possibly 
AP027 
Liver 
21 
increased AST 
Possibly 
AP036 
Hematologic 
1 
Anemia 
Possibly 
AP036 
Hematologic 
63 
Anemia 
Possibly 
AP037 
Hematologic 
1 
Lymphopenia 
Possibly 
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 
 

Lubaroff Ad/PSA Phase II, protocol #2, 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 PSA 

Lubaroff Ad/PSA Phase II, protocol #2, page 11 
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 

Lubaroff Ad/PSA Phase II, protocol #2, page 12 
followed by vaccinia-PSA (rV-PSA) given by intradermal injection, or (C) rV-PSA followed 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 Folwlpox 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 with hormone-refractory metastatic disease into 
this Phase II clinical trial.  This is similar to the population that constituted the majority of patients 
in our Phase I toxicity trial of the Ad/PSA vaccine.  Patients in this trial will have low burden of 
disease, despite the fact that they are hormone refractory, i.e., have negative bone scans and/or 

Lubaroff Ad/PSA Phase II, protocol #2, page 13 
low serum PSA.  We will be comparing the clinical and immunologic response of these patients 
with two other patient populations in the trial.  The latter populations, described in a separate 
protocol, will be patients with newly recurrent disease, either hormone naïve or during androgen 
depletion therapy.  Although the patients with recent recurrences will have a smaller tumor burden 
than will the patients in this protocol that have metastatic disease, the observation period to detect 
a therapeutic effect of the vaccine will be shorter in the latter patient population than the former 
population.  It is for this important reason that we will enroll patients with metastatic disease on 
this protocol.  Patients will be eligible if they have recent evidence of hormone refractory disease 
(D3) and either (a) have a positive bone scan or CT scan with a PSA doubling time of >6 months, 
and total PSA of <10 ng/ml, and asymptomatic; or (b) have a negative bone scan and CT scan 
with any PSA doubling time, asymptomatic, and not a candidate for chemotherapy. 
 
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 
(ICVAHCS).  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 ICVAHCS meets to provide their approval for the study. 
 
Patient Recruitment:  The UIHC and ICVAHCS 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 ICVAHCS 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 Urology, Dr. Vaena from 
Internal Medicine, and Dr. Smith from Radiation Oncology.  Additional recruitment of patients will 
occur through a 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 ICVAHCS 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.  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 
ICVAHCS 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 

Lubaroff Ad/PSA Phase II, protocol #2, page 14 
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 response rates (PSA responses and changes in PSADT) following 
immunization with the Ad/PSA vaccine using a prime-boost immunization strategy, in 
patients with hormone refractory metastatic disease. 
 
2.2 Secondary objectives 
 
2.2.1  To evaluate the development of anti-PSA immune responses in study patients. 
 
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. 
 
3. SELECTION OF PATIENTS 
 
As described in Section 1.6 prostate cancer patients with hormone refractory metastatic disease 
will be enrolled in the study.   
 
 
3.1 Inclusion criteria: 
 
 
3.1.1 Men with prostate cancer who present with evidence of hormone refractory 
disease (D3). 
 
 
3.1.2 Men with a positive bone scan or a positive CT scan (with obvious soft tissue 
metastasis or lymph nodes >1 cm), a PSA doubling time of >6 months, and a total PSA of <10 
ng/ml, and asymptomatic OR men with a negative bone scan and a negative CT scan with 
any PSA doubling time and asymptomatic. 
 
 
3.1.3 Scans must be obtained within 6 weeks of initiation of treatment. 
 
 
3.1.4 Written informed consent. 
 
 
3.1.5 Age > 18 years. 
 
 
3.1.6 Required laboratory values (obtained within 2 weeks of initiation of treatment) 
 
 
3.1.6.1 Serum creatinine < 2.0 mg/dL 
 
 
3.1.6.3 Adequate hematologic function:  granulocytes > 1800 per mm3, platelets > 
100,000 per mm3, WBC >3700, and lymphocytes >590. 
 
 
3.1.6.4 Adequate hepatocellular function:  AST <3x ULN and total bilirubin <1.5 
mg/dl (unless bilirubin elevation is consistent with Gilbert’s syndrome). 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 15 
 
3.1.6.5 Castrate levels of testosterone of <50 ng/ml. 
 
3.1.7  PSA  used as an eligibility criterion must be drawn within 28 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 Active or unresolved clinically significant infection. 
 
 
3.2.2 Parenteral antibiotics <7 days prior to initiation of treatment. 
 
 
3.2.3 Evidence of prior or current CNS metastases. Specific imaging is not necessary 
in the absence of signs or symptoms. 
 
 
3.2.4 Co-morbid medical conditions which would result in a life expectancy 
(participation) of less than 1 year. 
 
 
3.2.5 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 3 months of initiation of treatment is not allowed. 
 
 
3.2.6 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 participation in any vaccine studies for non-infectious diseases. 
 
 
3.2.9 Prior chemotherapy, defined as prior cytotoxic chemotherapy for prostate cancer 
(or any cancer unless more than 5 years have elapsed).  Examples of cytotoxic 
chemotherapy are mitoxantrone/prednisone and taxanes.  Drugs such as Casodex or 
ketoconazole treatment must have been completed at least 6 weeks prior to initiation 
of treatment. 
 
 
3.2.10 The inability to understand the English language and the clinical protocol. 
 
 
3.2.11 Allergy or religious objection to pork products; Gelfoam is produced from pork. 
 
Patients that have been on bisphosphonate treatment for greater than 1 month prior to entry into 
the trial will be able to continue the medication.  Patients that have not been on bisphosphonate 
therapy will not start on the medication for at least 30 days after the first vaccination. 
 
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 (ICVAHCS).   
 
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. 

Lubaroff Ad/PSA Phase II, protocol #2, page 16 
 
4.3 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 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 have the opportunity to have questions answered to their satisfaction before signing 
the consent. 
 
4.4  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 at 319-353-8914.  Patients at the ICVAHCS 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. 
 
4.5  No patient may be enrolled or begin 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. 
 
4.6 
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. 
 
4.7 
Patients must begin the vaccine protocol within 30 days of registration (date the study 
committee approves patient eligibility). 
 
5.  RESEARCH INTERVENTION PLAN 
 
5.1 Administration Schedule 
 
Ad/PSA  
 
All patients will receive three injections of 0.16 ml. (0.125 ml. vaccine + 0.035 ml. Gelfoam) 
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 1, 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.   
 
 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 17 
5.2 5.2  Design and Stages  
 
The ideal design would be a two-stage design of Simon (1989) requiring 32 patients. But 
due to the complexity of the trial, a more conservative approach consisting of using 32 patients 
in a one-stage design at efficacy level and a two looks at toxicity level as means of stopping 
rules will be carried out. By using a one-stage design at efficacy endpoint, we are making the 
probability of early termination for efficacy purposes to be zero. The reason for this design is 
due to the nature of the research intervention as outlined in section 10. Stopping design based 
on toxicity is as follow: one look after 17 subjects are treated and another look after 25 subjects 
are treated. If 5 out of 17 show vaccine-related grade 3 toxicity or higher, the trial will stop. 
Otherwise, an additional 8 subjects will enter the trial. If 7 out of 25 show grade 3 toxicity or 
higher, the trial will stop otherwise we will proceed to full registration of 32 patients to assess 
efficacy. 
 
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-threateningB 
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.C 
 
B 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. 
C 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 

Lubaroff Ad/PSA Phase II, protocol #2, page 18 
 
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 
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.  
 
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. 

Lubaroff Ad/PSA Phase II, protocol #2, page 19 
In particular, the investigator must inform the agencies and committees by phone and fax 
using the timeline described above of occurrence of immediately life-threatening SAEs or 
SAEs with fatal outcome. SAEs must be reported to the site’s IRB according to the IRB’s 
requirements.  
 
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. 
 
7  MEASUREMENT OF CLINICAL AND IMMUNOLOGICAL EFFICACY 
 
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. 
 
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 follow-up examinations and 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 return to the clinic for two additional visits, at 
18 and 24 months, unless travel distance is a prohibitive factor.  Follow-up of patients for 
survival will occur every 6 months by telephone. 
 
7.2 Scans – Bone scans will be performed at 90 days, 6 months, and 12 months during the 
first year as well as the 24 month visit and annually until progression.  If there is 
measurable disease by CT scan at registration then CT scans will also be performed using 
the same schedule as the bone scans. 
 
7.3 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 (40).  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 

Lubaroff Ad/PSA Phase II, protocol #2, page 20 
with Hybritech, Inc. have been exploring hK2 and published a number of papers in recent 
years on the subject (67-70).  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. 
 
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. 
 
7.4 Experimental Evaluation of the Ad/PSA Vaccination 
 
7.4.1  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. 
 
7.4.2 Levels of PSA, hK2, anti-PSA antibodies, and anti-adenovirus antibodies will be 
measured in the serum. 
 
7.4.3  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. 
 
7.5 Definitions of Response – 
 
7.5.1 Primary Endpoint - PSA doubling-time response 
 
7.5.1.1 
Definition: a 50% increase in the PSADT compared to pre-enrollment 
PSADT. 
 
7.5.1.2 
PSADT will be calculated based on the MSKCC calculator, available at 
http://www.mskcc.org/mskcc/html/10088.cfm. 
 
7.5.1.3   
PSADT response will be measured at 9 and 18 months after initiation of 
the research intervention.  
 
7.5.1.4 
Three measurements of PSA, spaced at least 2 weeks apart, will be 
required prior to study enrollment. Post-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). 
 
7.5.2   Secondary Endpoint – PSA response 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 21 
7.5.2.1  Definition: a 50% reduction in the pre-research intervention PSA value, verified 
with a second measurement >28 days later. 
 
7.5.3 Progression: 
 
Progression of disease is defined for 3 different categories of disease:  measurable 
lesions; non-measurable/non-target lesions; and PSA-only disease.  The first category of 
disease to progress will meet the definition of progression in this trial, as follows: 
 
 
7.5.3.1 
Progression for measurable disease: 
 
Definition: Measurable lesions are defined as those that can be accurately measured 
in at least one dimension (longest diameter to be recorded) and is equal or greater 
than 20 mm with conventional techniques (CT, MRI, x-ray) or equal or greater than 10 
mm with spiral CT scan. All tumor measurements must be recorded in millimeters (or 
decimal fractions of centimeters) 
 
All measurable lesions (up to a maximum of 10) representative of all involved organs 
should be identified as target lesions and will be recorded and measured at baseline. 
Target lesions should be selected on the basis of their size (lesions with the longest 
diameter) and their suitability for accurate repetitive measurements (either by imaging 
techniques or clinically). A sum of the longest diameter (LD) for all target lesions will 
be calculated and reported as the baseline sum LD. The baseline sum LD will be used 
as reference to further characterize the objective tumor response of the measurable 
dimension of the disease. 
 
All other lesions, including non-measurable small lesions as defined above and bone 
metastasis, will be identified as non-target lesions (see definition of progression for 
non-target lesions below). 
 
Progression is defined if any of the criteria below are met: 
 
• 
at least a 20% increase in the sum of the LDs of target lesions (taking as a 
reference the smallest sum LD recorded since the treatment started) or the 
appearance of two or more new lesions 
• 
development of an indication for radiation therapy while on treatment 
 
 
7.5.3.2 
Progression for non-measurable disease/non-target lesions: 
 
Non-measurable disease/non-target lesions: progression of disease is defined as 
any of the following: 
 
7.5.3.2.1 - worsening bone scan as evidenced by the appearance of more than 
two new lesions, not felt to be consistent with a tumor flare; worsening 
of pre-existing lesions (increase in intensity or size of a lesion) may be 
difficult to interpret and in such a case will not be considered the 
evidence of progressive disease; or  
 
7.5.3.2.2 - appearance of new metastatic lesions outside the bone; or unequivocal 
progression of existing non-target lesions. Because a clear progression 

Lubaroff Ad/PSA Phase II, protocol #2, page 22 
of non-target lesions only is exceptional, such circumstances should be 
discussed with the study co-investigator's; or 
 
7.5.3.2.3 - development of an indication for radiation therapy while on treatment 
 
 
7.5.3.3 
Progression for patients with PSA-only disease 
 
PSA progression will be evaluated as follows: 
 
7.5.3.3.1  In patients that had any PSA decline from baseline: 
 
7.5.3.3.1.1 - increasing PSA by 25% above the nadir plus 
 
7.5.3.3.1.2 - an increase in PSA by a minimum of 2 ng/ml from the nadir plus 
 
7.5.3.3.1.3 - confirmation by second consecutive rising PSA >28 days later 
 
7.5.3.3.2 - in patients whose PSA has not declined, progression is defined as 
 
 
7.5.3.3.2.1 - an increase in PSA by 25% above the pretreatment level plus 
 
 
7.5.3.3.2.2 - an increasing PSA by a minimum of 2 ng/ml relative to the study 
entry PSA value, plus 
 
 
7.5.3.3.2.3 - confirmation by second consecutive rising PSA >28 days later.  
 
7.5.4 Onset of Response – In patients that present with radiologic evidence of 
metastases onset of response is the onset of PR or CR.  For patients with 
biochemical disease only the onset of response is defined by the first drop in serum 
PSA. 
 
7.5.5 Duration of Response – In patients that present with radiologic evidence of 
metastases, duration of response is time from onset of PR or CR, whichever 
occurs first, (even if the patient later has a CR) until objective evidence of 
progression.  In patients with PSA-only disease, duration of response is time 
from first drop in serum to objective evidence of progression.  Progression is 
defined in Section 7.5.3, beginning on page 20. 
 
7.6 Timing of Toxicity Assessments - Toxicity assessment will occur as stated in the 
calendar.  
 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 23 
8   STUDY PARAMETERS 
 
8.1 Scans or x-rays used to document measurable or evaluable disease must be done with 
6 weeks prior to the initiation of treatment. 
 
8.2 CBC with differential, LFT’s must be done <2 weeks before study entry.  Castrate levels 
of testosterone must be drawn at 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 the 
initiation of treatment. 
 
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 initiation of treatment. 
 
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, 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 17 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 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.  

Lubaroff Ad/PSA Phase II, protocol #2, page 24 
 
 
 
 
 
 
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 will be asked in the Informed Consent document 
whether they would return every three months up to 12 months from study entry 
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. 
 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 25 
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 
periodically 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 confidentially destroyed.  VAHCS records will be 
retained until disposition instructions are approved by National Archives and Records 
Administration.  
 
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 30 and 31).  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.   

Lubaroff Ad/PSA Phase II, protocol #2, page 26 
Table 6 
Study Design and Testing 
 
 
Std. of 
Care  
or 
Res. 
Prior 
to 
Reg.1 
Day 1 
First Inj. 
 
30 d. 
2nd Inj. 
 
44 d. 
 
60 d. 
3rd Inj. 
 
74 d. 
 
90 d. 
 
6 mo 
 
9 mo 
 
12 mo 
18 
mo. 
24 
mo. 
Annual 
to 
prog. 
Follow-
up7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Immunization2 
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 & PAP3 
S/R 
 
X 
X (R) 
 
X (R) 
 
X 
X 
X 
X 
X 
X 
 
 
anti-PSA Ab, and lymphocytes 
for cellular immunity 
R 
X 
X 
X 
X 
X 
X 
X  
X6 
X6 
X6 
X 
X 
 
 
CBC, differential 
R 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
 
AST, ALT, LDH, alkaline 
phosphatase, bilirubin 
R 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
Creatinine 
R 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
Urinalysis 
R 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
Serum testosterone 
R 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chest x-ray 
R 
X  
 
X4 
 
X4 
 
X4 
 
 
 
 
 
 
 
Bone scan 
S 
X 
 
 
 
 
 
X 
X 
 
X 
 
X 
X 
 
Abdominal/pelvic CT 
S 
X  
 
 
 
 
 
 
X5 
 
X5 
 
 
 
 
Survival assessment 
R 
 
 
 
 
 
 
 
 
 
 
 
 
 
X7 
 
  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 Androgen deprivation therapy must continue for the duration of study participation. 
3 If the PSA meets the definition of progression, partial or complete response, confirm the PSA results >/= 28 days. 
4 will be repeated only if abnormal at screening or if patient develops a fever. 
5  will be repeated only if abnormal at screening. 
6 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?).” 
7 Follow-up for survival every 6 months by telephone. 
 
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. 
 

Lubaroff Ad/PSA Phase II, protocol #2, 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 Clinical Research Unit.  Each patient will be injected with approximately 0.16 
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 is controlled by using the required volume from the dose vial.  The 
empty syringe will be disposed of in a biohazard container. 

Lubaroff Ad/PSA Phase II, protocol #2, page 28 
 
Patients will remain in the CRU for two hours after each injection to be monitored for signs 
of adverse events.   
 
9.8 Manufacturing 
 
9.8.1  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 (71) that 
encodes 262 amino acids with a predicted molecular weight of 28.8 kDa.  Using 
methods previously described (72), 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 (73).  Ad/lacZ used as a control was also 
obtained from the Gene Transfer Vector Core and is previously described (74).  
 
9.8.2 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. This trial is using a surrogate endpoint as a substitute to the clinically meaningful 
outcome since the tumors 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 
hormone—refractory metastatic prostate cancer—with the goal to induce anti-PSA T cells 
responses. Three injections of equal dose are proposed. The previous phase I trial consisting of 
a single injection in men with hormone-refractory metastatic prostate cancer was able to induce 
anti-PSA T cells responses. The Phase I consisting of a single injection using 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 serum 
PSADT. 
 
 
For efficacy purposes, we expect at least 50% of the patients to show 50% increase in PSADT. 
This proportion is judged clinically important and anything less than 30% can stop the trial for 
futility. The ideal design would be a two-stage design of Simon (1989) requiring 32 patients. After 
testing the research intervention on 12 patients, if 3 did not have a 50% increase in PSADT, the 
trial will be terminated. Otherwise, an additional 20 patients will be recruited for the trial. The 
expected sample size will be 19.73 and the probability of early termination, 0.72. If the research 
intervention is not effective, there is 0.1 probability of concluding that it is. If the research 
intervention is effective, there will be 0.2 probability of concluding that it is not. However, due to 
the complexity of the trial, we will not use Simon (1989) but a more conservative approach 
consisting of using 31 patients in a one-stage design at efficacy level and a two-stage design at 
toxicity level for stopping rules. By using a one-stage design at efficacy endpoint, we are making 

Lubaroff Ad/PSA Phase II, protocol #2, page 29 
the probability of early termination for efficacy purposes to be zero. Early termination will be based 
on toxicity only. The reason for this design is due to the nature of the research intervention and is 
explained below. Enrollment will not stop unless stopping rules based on toxicity are satisfied:  
“After the third vaccine dose is administered, toxicity will be evaluated 90 days into the trial”    
 
 
Due to the nature of the research intervention, anti-tumor activity will be potentially delayed 
and the primary efficacy endpoint will be determined 18 to 20 months after initial patient accrual. 
Since we will be able to assess PSADT after nearly 20 months into the study, it will be 
unreasonable to stop the study due to unsatisfactory results prior to that point. This is the main 
reason why we will carry a one-stage design for the primary endpoint and a two-stage design for 
toxicity. Criteria for proceeding with enrollment into a subsequent stage prior to the two-year 
efficacy evaluation will be based on assessment of toxicity in the patient cohorts since this can be 
done as early as 90 days into the trial.  
 
 
For toxicity purposes, we will test the null hypothesis that the toxicity level is less than 15% 
versus the alternative that it is greater than 35%. The trial will be stopped whenever the null 
hypothesis is rejected. We will conduct these tests in 2 different looks—one after an enrolment of 
17 subjects and the second after 25 subjects are enrolled. 
 
 
If 5 out of 17 show grade 3 vaccine-related toxicity or higher, the trial will stop (this will 
correspond to testing the toxicity hypotheses at level alpha=0.05 with a power 77%); otherwise, 
an additional 8 subjects will enter the trial. If 7 out of 25 show a grade 3 toxicity or if 2 out of 17 
show a grade 4 toxicity or higher, the trial will stop (this will be equivalent to testing the toxicity 
hypotheses at level alpha =0.05 with a power 82%); otherwise we will proceed to full registration. 
The overall type I error for this test is 0.09.  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. 
 
 
The reasons for using different looks at toxicity level is because we are using surrogate 
endpoints that had not been proven formerly to have an ease of predictability of an outcome of 
direct relevance to patients; also, it is not quite clear how theses surrogates relate to the pathway 
of the natural disease and to overall survival rate. 
 
Simon, Richard. “Optimal Two-Stage Designs for Phase II Clinical Trials,” Controlled Clinical 
Trials, 1989, Volume 10, pages 1-10. 
De Gruttola Victor et Al.,  “Considerations in the Evaluation of Surrogate Endpoints in Clinical 
Trials: Summary of NIH workshop” Controlled Clinical Trials, 2001, Volume 22, pages 485-502 
 

Lubaroff Ad/PSA Phase II, protocol #2, page 30 
Addendum 
PHASE II STUDY OF ADENOVIRUS/PSA VACCINE IN MEN WITH 
HORMONE - REFRACTORY PROSTATE CANCER 
Food and Drug Administration (FDA) Investigational New Drug (IND) #9706 
  Department of Defense, Prostate Cancer Research Program A-14059.2 
 
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 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. "  
 

Lubaroff Ad/PSA Phase II, protocol #2, page 31 
Roles and Responsibilities of Study Personnel: 
 
David M. Lubaroff, PhD, Principal Investigator – Dr. Lubaroff, along with Dr. Williams, 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 (ICVAHCS) 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 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 VAMC – 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. 

Lubaroff Ad/PSA Phase II, protocol #2, page 32 
Steven Varga, PhD, Collaborator – Dr. Varga is an immunologist and will assist in the 
immunologic evaluation of patient’s immune responses to the vaccine. 
 
Lyse Norian, PhD, Collaborator – Dr. Norian 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, 10:712-715, 
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