Phase 1 Trial of Oncolytic Adenovirus-Mediated Cytotoxic and Interleukin 12 Gene Therapy for Locally Recurrent Prostate Cancer After Definitive Radiotherapy
Phase: PHASE1
Conditions: Prostate Cancer
Sponsor: Henry Ford Health System

Page 1 of 47 
 
 
 
 
 
 
 
 
 
 
 
 
PHASE 1 TRIAL OF ONCOLYTIC ADENOVIRUS-MEDIATED CYTOTOXIC 
AND INTERLEUKIN 12 GENE THERAPY FOR LOCALLY RECURRENT 
PROSTATE CANCER AFTER DEFINITIVE RADIOTHERAPY 
 
NCT02555397 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
version 7; revised 02/16/2016 

Page 2 of 47 
TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  Page 
 
 
 
Face Page 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1 
 
 
 
Table of Contents 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 
 
 
 
Eligibility Checklist  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 - 5 
 
1.0  
Background  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 - 18 
 
2.0  
Objectives 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
19 
 
3.0  
Subject Selection 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
19 - 21 
 
4.0  
Registration Procedures 
 
 
 
 
 
 
 
 
 
 
 
 
 
22 
 
5.0  
Study Therapy and Protocol 
 
 
 
 
 
 
 
 
 
 
 
 
22 - 29 
 
6.0  
Subject Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
30 - 36 
 
7.0  
Data Collection  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
36 - 39 
 
8.0  
Statistical Considerations  
 
 
 
 
 
 
 
 
 
 
 
 
39 - 41 
 
9.0  
Data and Safety Monitoring Plan  
 
 
 
 
 
 
 
 
 
 
41 
 
10.0 
Inclusion of Minorities  
 
 
 
 
 
 
 
 
 
 
 
 
 
42 
 
11.0 
Process of Informed Consent  
 
 
 
 
 
 
 
 
 
 
 
42 
 
12.0 
Literature Cited  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
43 - 46 
 
 

Henry Ford Health System 
Phase 1 Locally Recurrent Prostate Cancer Gene Therapy Trial 
 
Eligibility Checklist 
 
 
 
 
 
 
Page 3 of 47 
 
Subject Study Number: ___ ___ ___  
 
 
 
 
 
 
 
Subject Initials: ___ ___ ___ 
 
Eligibility Checklist – Please refer to primary source documents for this information. 
 
Questions 1 - 29 must all be answered Yes for the subject to be eligible to participate. 
 
1. The subject has biopsy-proven adenocarcinoma of the prostate at least 1 year 
 
 
0[ ] No  
1[ ] Yes 
 
after completion of definitive radiation therapy and within 180 days of registration? 
 
 
When was the biopsy performed? ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 
 
 
    M    M 
 D    D     Y    Y     Y    Y 
 
 
When was the last day of definitive radiation therapy? ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   M    M 
D    D    Y     Y   Y    Y 
 
2. The subject has evidence of biologically active disease as demonstrated by an 
 
 
0[ ] No  
1[ ] Yes 
 
unequivocally rising serum PSA level that is ≥ 2 ng/mL above the nadir? 
 
 
Last PSA value: ___ ___ . ___ ng/mL assessed on ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 
 
 
 
 
 
 
      M    M   D    D      Y     Y    Y     Y 
 
 
PSA nadir: ___ ___ . ___ ng/mL assessed on ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 
 
 
 
 
 
    M    M 
 D    D     Y    Y    Y     Y 
 
3. The last PSA is < 100 ng/mL? 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
4. The subject is ≥ 18 years of age? 
 
 
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Age: ___ ___ years 
 
5. The subject has a Karnofsky performance status ≥ 70?.  
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Karnofsky performance status ___ ___ 
 
6. The subject has negative lymph nodes within 90 days of registration? 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
7. The subject has no evidence of metastatic disease within 90 days of registration? 
 
0[ ] No  
1[ ] Yes 
 
8. The subject has adequate renal function with serum creatinine ≤ 1.5 mg/dL 
 
 
 
0[ ] No  
1[ ] Yes 
or creatinine clearance > 50 mL/min/m2? 
 
 
Serum creatinine: ___ . ___ mg/dL or Creatinine clearance: ___ ___ . ___ mL/min/m2 
 
9. The subject has a platelet count > 100,000/μL (100 K/µL)? 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Platelet count: ___ ___ ___ K/µL 
 
 
 
 
 

Henry Ford Health System 
Phase 1 Locally Recurrent Prostate Cancer Gene Therapy Trial 
 
Eligibility Checklist 
 
 
 
 
 
 
Page 4 of 47 
 
Subject Study Number: ___ ___ ___  
 
 
 
 
 
 
 
Subject Initials: ___ ___ ___ 
 
10. The subject has an absolute neutrophil count > 1,000/µL(1 K/µL)? 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Neutrophil count: ___ ___ . ___ K/µL 
 
11. The subject has a hemoglobin > 10.0 g/dL?  
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Hemoglobin: ___ ___ . ___ g/dL 
 
12. The subject has a bilirubin < 1.5 mg/dL?  
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Bilirubin: ___ . ___ mg/dL 
 
13. The subject has a SGOT ≤ 3.0 x ULN?  
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
SGOT: ___ ___ . ___ IU/L 
 
14. The subject has a SGPT ≤ 3.0 x ULN?  
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
SGPT: ___ ___ . ___ IU/L 
 
15. The subject is willing to use effective contraception while on treatment and for at  
 
0[ ] No  
1[ ] Yes 
least 3 months afterwards? 
 
16. The subject’s prostate volume is not > 100 cc?  
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Prostate volume: ___ ___ . ___ cc 
 
17. The subject has not had a prior invasive malignancy except for non-melanoma 
 
 
0[ ] No  
1[ ] Yes 
 
skin cancer within the past 5 years? 
 
18. The subject has not had prior radical prostatectomy, cryosurgery or bilateral  
 
 
0[ ] No  
1[ ] Yes 
 
orchiectomy? 
 
19. If the subject had prior androgen deprivation therapy (ADT), the subject did not exhibit 
 
0[ ] No  
1[ ] Yes 
biochemical (PSA) failure while on ADT? 
 
20. The subject has not had prior chemotherapy for the study cancer? 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
21. Major surgery is not planned within 3 months of registration?  
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
22. The subject does not have any of the following co-morbidities? 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
-New York Heart Association Class II or greater congestive heart failure 
 
  or active ventricular arrhythmia requiring medication. 
 
-Chronic obstructive pulmonary disease (COPD) exacerbation or other respiratory illness 
  requiring hospitalization within last 3 months or precluding study therapy at the time of registration. 
 
-Acute infection.  Acute infection is defined by any viral, bacterial, or fungal infection that 
  requires specific therapy within 72 hours of initiation of the study therapy. 
 

Henry Ford Health System 
Phase 1 Locally Recurrent Prostate Cancer Gene Therapy Trial 
 
Eligibility Checklist 
 
 
 
 
 
 
Page 5 of 47 
 
Subject Study Number: ___ ___ ___  
 
 
 
 
 
 
 
Subject Initials: ___ ___ ___ 
 
 
23. The subject does not have a previous history of liver disease including hepatitis?  
 
0[ ] No  
1[ ] Yes 
 
24. The subject is not taking immunosuppressive therapy including systemic corticosteroids? 0[ ] No  
1[ ] Yes 
 
Note: Use of inhaled and topical corticosteroids is permitted. 
 
25. The subject does not have impaired immunity or susceptibility to viral infections?  
 
0[ ] No  
1[ ] Yes 
 
26. The subject does not have a known allergy to any product used in the protocol? 
 
(If the subject has an allergy to Ciproflaxin, another antibiotic can be substituted 
 
at the discretion of the treating physician). 
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
27. The subject has no serious medical or psychiatric illness or concomitant medication,  
0[ ] No  
1[ ] Yes 
 
which, in the judgment of the investigator, might interfere with the subject’s ability to 
 
respond to or tolerate the treatment or complete the trial? 
 
28. The subject has the ability to give informed consent and has expressed a willingness  
0[ ] No  
1[ ] Yes 
 
to meet all of the expected requirements of the protocol for the duration of the study? 
 
29. The subject gave informed consent? 
 
 
 
 
 
 
 
 
 
 
 
 
0[ ] No  
1[ ] Yes 
 
 
Date the Informed Consent Form was signed: ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 
 
 
 
 
 
   M    M       D    D       Y    Y     Y    Y 
 
 
 
Completed By Initials: ___ ___ ___ 
 
Form Completion Date: ___ ___ / ___ ___ / ___ ___ ___ ___ 
 
 
 
 
 
 
 M   M       D    D       Y    Y     Y     Y 
 
 

 
Page 6 of 47 
1.0  
BACKGROUND 
 
An estimated 221,000 men will be diagnosed with prostate cancer in the United States in 
2015 and approximately one-third will elect radiation therapy (external beam and/or 
brachytherapy) as their primary treatment (1).  For men with organ-confined, low-risk 
disease (Stage T1/T2, Gleason ≤ 6, PSA < 10 ng/mL), radiation therapy results in excellent 
long-term survival and disease control (2).  However, it is less effective against more 
aggressive forms of the disease (Stage ≥ T3, Gleason ≥ 7, PSA > 10 ng/mL) and a 
significant fraction of patients develop disease recurrence within 10 years.  Fortunately, the 
rate of occult distal failure following definitive radiotherapy is < 10% at 10 years (3).  Hence, 
there is a substantial window of opportunity after initial recurrence in which the disease may 
still be largely localized and amenable to local salvage therapies. 
 
Men with locally recurrent prostate cancer after definitive radiotherapy have few therapeutic 
options that have a high likelihood of eradicating the tumor with a reasonable degree of 
safety.  Other than expectant management and systemic therapy (i.e., androgen deprivation 
therapy, ADT), there are four available local treatment options for locally recurrent prostate 
cancer: 1) salvage radical prostatectomy (RP), 2) salvage cryoablation (CA), 3) salvage 
brachytherapy (BT), and 4) salvage high-intensity focused ultrasound (HIFU).  Although 
some have demonstrated encouraging 5-year disease-free survival rates in single institution 
trials, most are associated with significant morbidity and there are no prospective 
randomized studies demonstrating their long-term effectiveness. 
 
1.1  
Local Salvage Therapies for Radio-Recurrent Prostate Cancer 
 
1.1.1 Radical Prostatectomy 
 
Salvage RP following definitive radiotherapy has resulted in disease-free 
survival of 55% - 71% at 5 years and 30% - 43% at 10 years (4, 5).  Patients 
with organ-confined disease, who represent 20% - 50% of cases, have an 
excellent prognosis.  However, the complication rate associated with salvage 
RP is high.  Urinary incontinence is observed in 20% - 68% of patients, 
anastomotic stricture in 9% - 32%, and erectile dysfunction in over 72%. 
 
1.1.2 Cryoablation 
 
Recent technical innovations have led to a re-emergence in the interest in 
CA as a minimally invasive alternative to salvage RP.  In patients treated 
with third-generation cryotechnology, 5-year disease-free survival is 44% - 
73% (5).  Incontinence is observed in 4% - 40% of cases, 
obstruction/retention in 3% - 21%, and erectile dysfunction in over 72%.  
Hence, the results with salvage CA are comparable to salvage RP. 
 
1.1.3 Brachytherapy 
 
Five-year disease-free survival following salvage BT is 34% - 87% (4, 5), 
although many patients also received short-term hormone therapy 
confounding the results.  As expected, most of the complications associated 
with salvage BT were genitourinary (GU) or gastrointestinal (GI) in nature.  
Grade 3/4 GU toxicity (incontinence, frequency/urgency, hesitancy, nocturia) 
is 14% - 47% and grade 3/4 GI toxicity (rectal ulcers, bleeding) is 0% - 24%. 

 
Page 7 of 47 
Only one study reported erectile dysfunction, which was 100% grade 2. 
 
1.1.4 HIFU 
 
There have been only three published reports on salvage HIFU and the 
follow-up is relatively short (< 3 years).  The largest study (167 patients) 
reported 3-year disease-free survival of 53%, although 18% of patients also 
received hormone therapy.  Complications associated with salvage HIFU 
include incontinence (7% - 50%), urinary obstruction/retention (9% - 36%), 
urinary stricture (17% - 20%), recto-urethral fistula (3% - 7%).  Only one 
study reported erectile dysfunction, which was 72%. 
 
1.2  
Oncolytic Adenovirus-Mediated Cytotoxic Gene Therapy (OAMCGT) 
 
1.2.1 Preclinical Studies 
 
Suicide gene therapy is an investigational cancer therapy that is currently 
being evaluated in clinical trials (6).  It is based on the premise that cycling 
tumor cells expressing the suicide gene will be rendered sensitive to specific 
pharmacologic agents thereby providing a therapeutic index.  Delivery of the 
suicide gene to the tumor is usually accomplished by direct intratumoral or 
systemic injection of a viral vector containing the suicide gene. 
 
Two suicide genes that have been evaluated in preclinical models and in the 
clinic are the E. Coli cytosine deaminase (CD) and herpes simplex virus 
thymidine kinase (HSV-1 TK), which confer sensitivity to 5-fluorocytosine (5-
FC) and ganciclovir (GCV), respectively (7-25).  The mechanism of CD/5-FC 
killing is mediated through its ability to inhibit thymidylate synthase (TS) 
resulting in the depletion of dTMP, which is required for DNA synthesis.  The 
mechanism of HSV-1 TK/GCV killing is mediated through its ability to inhibit 
DNA chain elongation.  Both the CD/5-FC and HSV-1 TK/GCV suicide gene 
systems exhibit a bystander effect, which results in the destruction of 
neighboring tumor cells not expressing the suicide genes (7-9). 
 
The concept of using CD and HSV-1 TK suicide gene therapies to improve 
the effectiveness of EBRT was first proposed by Kim et al. (26-28).  The 
product of the CD/5-FC reaction, 5-FU, is a well established 
chemotherapeutic and radiosensitizer in the clinic.  The mechanism of 5-FU 
radiosensitization is believed to be mediated through the inhibition of TS (by 
5-FdUMP), resulting in the depletion of dTMP pools and increased DNA 
strand breaks as well as redistribution of cells in early S phase, a 
radiosensitive phase of the cell cycle.  It was hypothesized that the HSV-1 
TK/GCV system may also result in tumor cell radiosensitization by 
preventing the repair of radiation-induced DNA damage.  Both suicide gene 
systems demonstrated marked tumor cell radiosensitization in vitro resulting 
in sensitization enhancement ratios (SER) in the range of 1.8 to 2.2 (26-28). 
 
Because the CD/5-FC system increases DNA strand breaks and the HSV-1 
TK/GCV system may inhibit the repair of those breaks, Rogulski et al. were 
the first to propose combining the two suicide gene systems (29).  They 
generated a novel CD/HSV-1 TK fusion gene that could confer both 5-FC 

 
Page 8 of 47 
and GCV sensitivity onto cells.  Implementation of both suicide gene 
systems simultaneously without, or with, EBRT proved to be far superior 
than either system alone in vitro and in vivo (29-32). 
 
To further enhance the effectiveness of CD and HSV-1 TK suicide gene 
therapies in combination with EBRT, Freytag et al. generated a replication-
competent oncolytic adenovirus (Ad5-CD/TKrep) expressing the prototype 
CD/HSV-1 TK fusion gene (33).  Not only do replication-competent 
adenoviruses provide a therapeutic effect via their cytolytic activity (oncolytic 
viral therapy), they also result in a greater efficiency of gene transduction in 
vivo relative to replication-defective adenoviruses.  Moreover, the 
multiplicative property of replication-competent adenoviruses results in a 
greater therapeutic gene copy number per infected cell as well as a greater 
number of tumor cells infected due to local viral spread.  The merit of 
oncolytic replication-competent adenovirus-mediated suicide gene therapy in 
combination with EBRT (trimodal therapy) has been evaluated in several 
preclinical tumor models (34, 35).  In an orthotopic prostate tumor model, 
oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) 
significantly improved both local and metastatic tumor control relative to 
EBRT alone (35). 
 
1.2.2 Clinical Trials 
 
The toxicity and efficacy of OAMCGT has been evaluated in five clinical 
trials of prostate cancer (36-44), including a prospective randomized phase 2 
study (Table 1). 
 
Table 1.  Gene Therapy Trials Conducted by the Sponsor 
Trial 
# Subjects 
Oncolytic Adenovirus 
Phase 
Indication1 Radiation2 
Median FU3 
1 
16 
Ad5-CD/TKrep 
1 
LRPC 
N 
12.9 yrs 
2 
15 
Ad5-CD/TKrep 
1 
NDPC 
Y 
11.5 yrs 
3 
  9 
Ad5-yCD/mutTKSR39rep-ADP 
1 
NDPC 
Y 
8.2 yrs 
4 
18 
Ad5-yCD/mutTKSR39rep-hNIS 
1 
NDPC 
Y 
6.8 yrs 
5 
44 
Ad5-yCD/mutTKSR39rep-ADP 
2 
NDPC 
Y 
5.2 yrs 
1LRPC, locally recurrent prostate cancer; NDPC, newly diagnosed prostate cancer. 
2N, no; Y, yes. 
3Median PSA follow-up of surviving subjects as of 05/15/15.  Subjects lost to follow-up were excluded. 
 
Three different (but related) oncolytic adenoviruses armed with therapeutic 
genes have been used: 
 
1) Trials 1 & 2- used the first-generation Ad5-CD/TKrep adenovirus, which 
contains a bacterial CD (bCD)/wild-type HSV-1 TK fusion gene 
(bCD/HSV-1 TK).  Trial 1 enrolled 16 subjects with locally recurrent 
prostate cancer (36, 38).  Trial 2 enrolled 15 subjects with newly 
diagnosed intermediate- to high-risk prostate cancer and included 
radiation therapy (37). 
 
2) Trials 3 & 5- used the second-generation Ad5-yCD/mutTKSR39rep-ADP 
adenovirus, which contains an improved yeast CD (yCD)/mutant HSV-1 
TKSR39 fusion gene (yCD/mutTKSR39) and the adenovirus death protein 

 
Page 9 of 47 
(ADP) gene (45).  Both the yCD and mutant HSV-1 TKSR39 genes have 
better catalytic properties than the bCD and wild-type HSV-1 TK genes 
contained in Ad5-CD/TKrep (46, 47).  ADP increases the cytolytic activity 
of replication-competent adenoviruses (48, 49).  Trial 3 enrolled 9 
subjects with newly diagnosed intermediate- to high-risk prostate cancer 
and included radiation therapy (39).  Trial 5 was a prospective 
randomized phase 2 study that compared the toxicity and efficacy of 
combining OAMCGT with contemporary dose (80 Gy) intensity 
modulated radiotherapy (IMRT) versus IMRT alone and enrolled 44 
subjects with intermediate-risk prostate cancer (44). 
 
3) Trial 4- used the second-generation Ad5-yCD/mutTKSR39rep-hNIS 
adenovirus, which contains the improved yCD/mutTKSR39 fusion gene 
and the human sodium iodide symporter (hNIS) reporter gene.  hNIS 
allows for non-invasive imaging of recombinant adenoviruses using 
single photon emission computer tomography (SPECT) or positron 
emission tomography (PET) when coupled with radioactive tracers such 
as sodium pertechnetate (Na99mTcO4) or iodide (123I, 125I or 131I), 
respectively (50, 51).  This trial enrolled 18 subjects with newly 
diagnosed intermediate- to high-risk prostate cancer and included 
radiation therapy (42, 43). 
 
The results of trial 1 (phase 1 study in locally recurrent setting), trial 5 
(randomized phase 2 study in newly diagnosed setting), and trial 4 (phase 1 
study in newly diagnosed setting with non-invasive imaging) are described. 
 
1.2.2.1  
Locally Recurrent Prostate Cancer (Trial 1) 
 
1.2.2.1.1 Description 
 
Sixteen subjects with locally recurrent prostate 
cancer after definitive radiotherapy were enrolled at 
the Henry Ford Health System between January 
2000 and December 2001 (36).  All men received a 
single intraprostatic injection of the first-generation 
Ad5-CD/TKrep adenovirus on day 1.  The adenovirus 
dose was escalated from 1 x 1010 vp (cohort 1) to 1 x 
1012 vp (cohorts 3 & 4) in four cohorts.  5-FC + GCV 
prodrug therapy was administered for one (cohorts 1 
- 3) and 2 (cohort 4) weeks. 
 
The primary endpoint was acute toxicity through day 
30.  Secondary endpoints included PSA response, 
transgene expression and tumor destruction in post-
treatment biopsies, adenoviral DNA in blood, 
infectious adenovirus in blood and urine, and 
neutralizing antibodies (NAB) to adenovirus. 
 
 
 
 

 
Page 10 of 47 
 
1.2.2.1.2 Short-term Results 
 
There were no dose-limiting toxicities (DLTs) or 
serious adverse events (SAEs).  Ninety-four percent 
of the AEs observed were mild or moderate (grade 
1/2) in nature.  There were nine (6%) grade 3 events 
including four events of hyperglycemia, and one 
event each of cardiac ischemia, dyspnea, hyper-
magnesemia, lymphopenia, and neutropenia.  All 
subjects exhibiting grade 3 hyperglycemia were 
diabetics.  The one event of grade 3 cardiac ischemia 
occurred in a subject with a history of heart disease.  
The one event of grade 3 dyspnea occurred in a 
subject with a history of chronic obstructive 
pulmonary disease.  The grade 3 events of 
hypermagnesemia, lymphopenia, and neutropenia 
each occurred in a single subject and were transient. 
There were no grade 4 events. 
 
Seven of 16 (44%) subjects demonstrated a > 25% 
decrease, and 3 of 16 (19%) demonstrated a > 50% 
decrease, in serum PSA lasting < 120 days.  There 
was excellent correlation between persistence of the 
Ad5-CD/TKrep adenovirus, as determined by viral 
DNA in blood, and PSA response.  Transgene 
expression and tumor destruction at the injection site 
were confirmed by needle biopsy at 2 weeks.  Two 
subjects were negative for adenocarcinoma at 1 year. 
 
Ad5-CD/TKrep DNA was detected in blood as far out 
as day 76.  No infectious adenovirus was detected in 
serum or urine at any time point.  All subjects 
developed high titers of NAB to adenovirus. 
 
1.2.2.1.3 Long-term Results 
 
Long-term outcome was assessed at 5 and 7 years. 
At 5 years, PSA doubling time (PSADT) and delay in 
the administration of additional salvage therapy was 
examined (38).  At 7 years, overall (OS) and disease-
specific (DSS) survival were compared to well-
matched historical controls (41). 
 
When considering all evaluable subjects (n = 14), 
PSADT increased from a mean of 17 months (prior to 
gene therapy) to 31 months (after gene therapy) (P = 
0.014) (Table 2).  A greater percentage of subjects 
who received the highest adenovirus dose exhibited 
an increase in PSADT suggesting a dose-dependent 
effect.  Assuming that salvage androgen deprivation 

 
Page 11 of 47 
therapy (ADT) would have been initiated at a PSA of 
15 ng/mL, administration of the gene therapy delayed 
the projected onset of salvage ADT by a mean of 2.2 
years in all evaluable subjects and 2.6 years in 
subjects receiving the highest adenovirus dose. 
 
1PSADT after the gene therapy could not be determined in two subjects owing to a non-treatment-related 
death, and implementation of salvage ADT 2 months after gene therapy at subject’s request. 
2The delay in administration of salvage ADT was determined by calculating when the PSA would have 
crossed a threshold of 15 ng/mL using pre-gene therapy versus post-gene therapy PSADT. 
 
OS and DSS were examined at 7 years and 
compared to well-matched historical controls (Figure 
1 and Table 3).  The control cohort included 154 
patients with locally recurrent prostate cancer treated 
at the University of Michigan between 1986 and 2000 
(52).  At 7 years, OS was 69% vs. 44% (P = .07), and 
DSS was 94% vs. 55% (P = .03), in the gene therapy 
vs. the control cohort, respectively.  Median survival 
was 8.6 vs. 5.9 years in the gene therapy vs. the 
control cohort, respectively. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1.  Overall and disease-specific survival.  The control cohort included 154 patients with locally 
recurrent prostate cancer treated at the University of Michigan (52).  For the gene therapy cohort, men that 
expired with evidence/symptoms of metastatic disease or were lost to follow-up were declared a death due to 
prostate cancer.  Ticks marks indicate where subjects were censored.  Red dotted line indicates 7 years. 
 
Table 2.  5-Year Analysis in Locally Recurrent Setting 
 
Mean PSADT 
Delay in Salvage ADT2 
Subject cohort 
Before GT 
(months) 
After GT 
(months) 
 
All evaluable subjects (n = 14)1 
17 
31 
26 months (2.2 years) 
Highest Ad dose subjects (1012 vp; n = 6) 
18 
32 
31 months (2.6 years) 
 
 
 
 
Subject cohort 
Subjects who exhibited 
increase in PSADT 
 
Two lowest Ad dose cohorts (1010 and 1011 vp) 
3 of 8 (38%) 
 
Highest Ad dose cohort (1012 vp; n = 6) 
5 of 6 (83%) 
 

 
Page 12 of 47 
 
Table 3.  Overall and Disease-Specific Survival 
 
Gene Therapy Cohort 
Control Cohort 
7-year OS 
  69% 
  44% 
7-year DSS 
  94% 
  55% 
Median survival (years) 
8.6 
5.9 
 
1.2.2.1.4 Summary of Results in Locally Recurrent Setting 
 
OAMCGT was associated with minimal toxicity when 
administered to men with locally recurrent prostate 
cancer.  There was suggestive evidence of long-term 
benefits as demonstrated by a lengthening of 
PSADT, a delay in when additional salvage therapy is 
indicated, and possible improvements in overall and 
disease-specific survival. 
 
1.2.2.2  
Newly Diagnosed Prostate Cancer In Combination With 
Contemporary Dose IMRT (Trial 5) 
 
1.2.2.2.1 Description 
 
Forty-four men with newly diagnosed intermediate-
risk prostate cancer were enrolled at the Henry Ford 
Health System and Johns Hopkins Medical Institute 
between January 2008 and July 2010 (44).  Subjects 
were randomly assigned to receive either OAMCGT 
plus 80 Gy IMRT (arm 1; n = 21) or 80 Gy IMRT only 
(arm 2; n = 23).  Subjects were stratified into three 
groups based on Gleason score, PSA, and percent 
positive biopsy cores at baseline.  Men in arm 1 
received a single intraprostatic injection (1 x 1012 vp) 
of the second-generation Ad5-yCD/mutTKSR39rep-
ADP adenovirus on day 1 followed by 2 weeks 
(weekdays only) of 5-FC + valganciclovir (vGCV) 
prodrug therapy.  IMRT (40 x 2 Gy for 80 Gy) 
commenced with the initiation of the prodrug therapy. 
Men in arm 2 received IMRT (40 x 2 Gy for 80 Gy) 
only.  Hormone therapy was not allowed until 
biochemical recurrence was documented. 
 
The primary phase 2 endpoint was acute (≤ 90 days) 
toxicity.  Secondary endpoints included freedom from 
biochemical/clinical failure (FFF), prostate biopsy (12 
core) positivity at 2 years, disease-specific and 
overall survival, and quality of life (QOL), 
 
1.2.2.2.2 Results 
 
Men in arm 1 exhibited a greater incidence of flu-like 
symptoms, 
transaminitis, 
neutropenia 
and 

 
Page 13 of 47 
thrombocytopenia relative to men in arm 2.  These 
events were expected and attributable to the 
oncolytic 
adenovirus 
(flu-like 
symptoms, 
transaminitis) and prodrug therapy (neutropenia, 
thrombocytopenia).  The vast majority (98%) of AEs 
were grade 1/2 and transient.  Less than 10% of men 
in each arm exhibited acute (≤ 90 days) ≥ grade 2 GI 
toxicity.  While acute ≥ grade 2 GU events were more 
prevalent (arm 1- 43%, arm 2- 31%), no significant 
difference in acute GI or GU toxicity was noted 
between the two arms.  There was no significant 
difference in QOL between the two treatment arms. 
 
Prostate biopsy outcome ≥ 2 years after radiotherapy 
is highly prognostic for long-term outcome (53-55).  
Hence, short-term efficacy was assessed by a 12 
core prostate biopsy 2 years after completion of 
IMRT.  Two-year biopsies were obtained on 37 (84%) 
men.  There was a 42% (P = 0.13) and 34% (P = 
0.14) relative reduction in biopsy positivity on arm 1 
based on actual biopsies and intent-to-treat (ITT), 
respectively (Table 4).  This type 1 error is within the 
accepted range for randomized phase 2 trials (56).  
There was a 60% (P = 0.07) and 48% (P = 0.08) 
relative reduction in biopsy positivity on arm 1 in men 
with < 50% positive biopsy cores at baseline based 
on actual biopsies and ITT, respectively.  Owing to 
the adenovirus injection algorithm that skewed the 
adenovirus dose distribution to the positive sextants, 
men in arm 1 with < 50% positive cores received 2.5 
times the adenovirus dose/positive core than men 
with ≥ 50% positive cores. 
 
To date, 1 subject in arm 1 (4.8%) and 2 subjects in 
arm 2 (8.7%) have exhibited biochemical failure.  No 
subject has developed hormone-refractory or 
metastatic disease and none have died from prostate 
cancer. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Page 14 of 47 
 
Table 4.  2-Year Prostate Biopsy Results 
 
 
Subjects with Positive Biopsy1 
 
 
 
Arm 1 
Arm 2 
P value 
All subjects 
Biopsied 
  6/18 (33%) 
11/19 (58%) 
0.13 
 
ITT2 
  9/21 (43%) 
15/23 (65%) 
0.14 
 
 
 
 
 
Stratification Group3 
 
 
 
 
1 
Biopsied 
  0/1 (0%) 
  1/1 (100%) 
> 0.99 
ITT 
  0/1 (0%) 
  1/1 (100%) 
> 0.99 
2 
Biopsied 
  3/13 (23%) 
  8/14 (57%) 
0.07 
ITT 
  5/15 (33%) 
11/17 (65%) 
0.08 
3 
Biopsied 
  3/4 (75%) 
  2/4 (50%) 
0.47 
ITT 
  4/5 (80%) 
  3/5 (60%) 
0.49 
 
 
 
 
 
% Positive biopsy cores 
 
 
 
 
< 50% 
Biopsied 
  3/13 (23%) 
  8/14 (57%) 
0.07 
ITT 
  5/15 (33%) 
11/17 (65%) 
0.08 
≥ 50% 
Biopsied 
  3/5 (60%) 
  3/5 (60%) 
> 0.99 
ITT 
  4/6 (67%) 
  4/6 (67%) 
> 0.99 
1Number of subjects with a positive 2-year biopsy over the total number of subjects with percentage in 
parentheses. 
2Intent-to-treat (ITT) includes all subjects whether or not a 2-year biopsy was obtained.  Those subjects 
that were not biopsied were given a positive score. 
3Group 1- Gleason score 5/6 and PSA < 10 ng/mL and ≥ 50% positive biopsy cores.  Group 2- (Gleason 
score 5/6 and PSA 10 - 20 ng/mL) or (Gleason score 7 and PSA 0 - 20 ng/mL) and < 50% positive biopsy 
cores.  Group 3- (Gleason score 5/6 and PSA 10 - 20 ng/mL) or (Gleason score 7 and PSA 0 - 20 ng/mL) 
and ≥ 50% positive biopsy cores. 
 
1.2.2.2.3 Summary of Results in Newly Diagnosed Setting 
In Combination With Contemporary Dose IMRT 
 
There was no significant difference in GI/GU events 
and QOL between the two arms.  There was a 42% 
relative reduction in 2-year biopsy positivity on the 
gene therapy arm (arm 1).  There was a 60% relative 
reduction in 2-year biopsy positivity on the gene 
therapy arm in men who had < 50% positive biopsy 
cores at baseline.  The results indicate that 
combining OAMCGT with contemporary dose IMRT 
does not exacerbate the most common side effects of 
prostate radiotherapy and results in a clinically 
meaningful reduction in positive biopsies at 2 years in 
men with intermediate-risk prostate cancer. 
 
1.2.2.3  
Newly Diagnosed Prostate Cancer In Combination With 
Conformal Radiotherapy and Non-Invasive Imaging (Trial 4) 
 
1.2.2.3.1 Description 
 
Eighteen men with newly diagnosed intermediate- to 
high-risk prostate cancer were enrolled at the Henry 

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Ford Health System between May 2006 and January 
2010 (42, 43).  All men received a single 
intraprostatic injection of the second-generation Ad5-
yCD/mutTKSR39rep-hNIS adenovirus on day 1.  The 
adenovirus dose was escalated from 1 x 1011 vp 
(cohort 1) to 5 x 1012 vp (cohort 3) in three cohorts.  
5-FC + vGCV prodrug therapy was administered for 2 
weeks (weekdays only).  Following the adenovirus 
injection, subjects were administered Na99mTcO4 on 
various days and subjected to SPECT imaging. 
 
The primary endpoint was acute toxicity through day 
90.  Secondary end points included feasibility of using 
hNIS as a reporter gene to monitor adenoviral gene 
therapy vectors non-invasively in vivo, volume and 
magnitude of gene expression in the prostate, 
kinetics and persistence of reporter gene expression, 
whole body distribution of 99mTcO4 uptake, PSA 
response, and 2-year prostate biopsy positivity. 
 
1.2.2.3.2 Results 
 
hNIS reporter gene expression (i.e., 99mTcO4 uptake) 
was detected in 0 of 3 (0%) subjects at 1 x 1011 vp, 6 
of 8 (75%) subjects at 1 x 1012 vp, and 6 of 6 (100%) 
subjects at 5 x 1012 vp.  Gene expression was readily 
detected in the prostate following injection of 1 x 1012 
vp and 5 x 1012 vp (Figure 2).  Serial imaging on 
consecutive days demonstrated that gene expression 
in the prostate lasted ≤ 7 days (Figure 3). 
 
Importantly, whole body planar scans demonstrated 
that no reporter gene expression was detected 
outside of the prostate in any subject (Figure 4). 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.  Non-invasive imaging of gene expression in the prostate.  The subject received an 
intraprostatic injection of 5 x 1012 vp of the Ad5-yCD/mutTKSR39rep-hNIS  adenovirus on day 1.  16 mCi 
Na99mTcO4 was administered on days 2 and 3 and SPECT was performed 4 hours later.  Day 3 images are 
shown.  Radiotracer uptake is indicated by the colors (yellow > red > green).  Activity in the urinary bladder  
(UB) is due to excretion of the radiotracer through that organ.  Activity in the penis (Pe) is due to blood flow 

Stricker et al. 
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through that organ.  Activity in the bowel (Bo) is due to trapping.  L, left; R, right; P, posterior; A, anterior. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.  Persistence of gene expression in the prostate.  A subject with intermediate-risk prostate 
cancer received an intraprostatic injection (right side only) of Ad5-yCD/mutTKSR39rep-hNIS (1 x 1012 vp) on 
day 1.  Na99mTcO4 (16 mCi) was administered on days 2, 3, 4 and 8 and SPECT imaging (whole body and 
pelvic planar scans, pelvic SPECT) commenced 4 h later.  hNIS gene expression (i.e., 99mTcO4- uptake) was 
detected in the right side of the prostate on all days.  L, left; R, right; P, posterior; A, anterior. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 4.  Whole body distribution of 99mTcO4 uptake.  A subject with intermediate-risk prostate cancer 
received an intraprostatic injection of Ad5-yCD/mutTKSR39rep-hNIS (1 x 1012 vp) on day 1.  Na99mTcO4 (16 
mCi) was administered on day 4 and SPECT imaging commenced 4 h later.  Shown are whole body planar 
scans prior to (pre-injection) and three days after (post-injection) the adenovirus injection.  Activity in the 
salivary glands (Sal), lachrymal glands (Lac), thyroid gland and stomach are due to endogenous hNIS 

Stricker et al. 
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expression.  Na99mTcO4 is secreted through the urinary tract, accounting for the activity in the urinary bladder. 
 The activity observed in the penis (Pen) and testicles (Tes) is due to blood flow through those structures.  
The intravenous 99mTcO4 injection site can be seen in the post-injection scan. 
1.2.2.3.3 Summary of Results in Newly Diagnosed Setting 
In Combination With Conformal Radiotherapy and 
Non-Invasive Imaging 
 
Gene expression was readily detected in the prostate 
at adenovirus doses ≥ 1 x 1012 vp.  Gene expression 
peaked on days 2 or 3 and last ≤ 7 days in most 
subjects as determined by SPECT.  No gene 
expression was detected outside the prostate in any 
subject.  At an adenovirus dose of 5 x 1012 vp, on 
average, 45% of the prostate volume was covered 
with gene expression (43). 
 
1.3  
Interleukin 12 (IL-12) Gene Therapy 
 
To enhance our investigational approach further, we have added IL-12 to our 
therapeutic platform (57).  IL-12 was selected for evaluation for several reasons.  IL-
12 plays an essential role in the interaction between innate and adaptive immunity 
(58-60).  It is normally produced by phagocytes (monocytes/macrophages, 
neutrophils) and dendritic cells in response to pathogens, and directly activates cells 
of the innate (NK and NK-T cells) and adaptive (CD4+ and CD8+ cells) arms of 
immunity.  IL-12 helps to prime T cells and increases their survival, promotes Th1 
differentiation, enhances T cell, NK cell, and NK-T cell effector functions, and 
induces the secretion of IFN-γ, which mediates many of IL-12’s anti-tumor effects.  
IFN-γ acts directly on tumor cells as well as stromal and endothelial cells within the 
tumor microenvironment.  IFN-γ signaling results in 1) increased MHC class I 
processing and presentation (enhancing recognition of tumor by T cells); 2) 
induction of chemokines (IP-10 and MIG) that may recruit innate and adaptive 
immune effectors, which in turn lead to 3) alterations in extracellular matrix 
remodeling, including inhibition of matrix metalloproteinase expression, that reduces 
angiogenesis and tumor invasion; and 4) decreased expression of adhesion 
molecules by endothelial cells that may further limit angiogenesis.  Hence, the 
rationale behind IL-12 immunotherapy lies in its ability to induce IFN-γ expression, 
promote immune cell survival and activation, enhance productive antigen 
presentation, and inhibit tumor angiogenesis. 
 
IL-12 has demonstrated significant anti-tumor activity in preclinical models including 
prostate cancer (61).  However, its activity in the clinic has been modest when 
administered as a recombinant protein or expressed from retrovirally-transduced 
fibroblasts (59).  Furthermore, IL-12 has resulted in severe toxicity including two 
treatment-related deaths when administered systemically as a recombinant protein 
(62, 63).  We believe that IL-12 produced by a locally administered, oncolytic 
adenovirus may exhibit better activity in the clinic without excessive toxicity relative 
to previous approaches.  Administering the adenovirus intraprostatically (rather than 
systemically) should result in a high, local concentration of IL-12 within the prostate. 
This should limit systemic toxicity and promote IL-12’s anti-tumor effects.  IL-12 has 
been well-tolerated in humans when administered locally (59).  Moreover, we 
believe our multi-modal approach may function like an autologous, whole tumor 

Stricker et al. 
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vaccine.  All modalities (oncolytic viral therapy, cytotoxic gene therapy, IL-12 gene 
therapy, and radiotherapy when combined with radiation) result in significant tumor 
cell destruction and the release of tumor antigens.  This may facilitate antigen cross-
presentation by professional APCs, which, when coupled with IL-12’s ability to 
promote Th1 differentiation, may lead to the development of antigen-specific, cell-
mediated immunity. 
 
We have demonstrated in a preclinical model of prostate cancer that the addition of 
IL-12 to our therapeutic platform improves both local and metastatic tumor control 
that is mediated by both the innate and adaptive arms of immunity (57). 
 
1.4  
Health-Related Quality of Life (HRQOL) and Utilities 
 
1.4.1 Expanded Prostate Index Composite (EPIC) 
 
The Expanded Prostate Cancer Index Composite (EPIC) is a comprehensive 
instrument designed to evaluate patient function and bother after prostate 
cancer treatment.  EPIC is a robust prostate cancer HRQOL instrument that 
measures a broad spectrum of urinary, bowel, and sexual symptoms related 
to radiotherapy (64). The domains (e.g., urinary, bowel, sexual) were 
validated separately; since each domain will be used intact there is no threat 
to validity (65). 
 
1.4.2 Utility as Measured by the EQ-5D 
 
The EQ-5D is a two-part, self-assessment questionnaire that takes 
approximately 5 minutes to complete (66).  The first part consists of 5 items 
covering 5 dimensions including: mobility, self care, usual activities, 
pain/discomfort, and anxiety/depression.  Each dimension has an index 
score of 1- no problems, 2- moderate problems, or 3- extreme problems.  
The second part is a visual analogue scale (VAS) valuing current health 
state, measured on a 20-cm 10-point interval scale.  Worst imaginable 
health state is scored as 0 at the bottom of the scale, and best imaginable 
health state is scored as 100 at the top.  Both the 5-item index score and the 
VAS score are transformed into a utility score between 0 “Worst health state” 
and 1 “Best health state”.  The EQ-5D will be completed by the patient. 
 
 

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2.0  
OBJECTIVES 
 
2.1  
Primary 
 
The primary purpose of this phase 1 study is to determine the dose-dependent 
toxicity and maximum tolerated dose (MTD) of oncolytic adenovirus-mediated 
cytotoxic and IL-12 gene therapy in men with locally recurrent prostate cancer after 
definitive radiotherapy. 
 
2.2  
Secondary 
 
To assess: 
 
2.2.1 PSA response. 
 
2.2.2 Freedom from biochemical/clinical failure (FFF). 
 
2.2.3 PSA doubling time (PSADT) before and after administration of the study 
therapy. 
 
2.2.4 Disease-specific and overall survival. 
 
2.2.5 Quality of life (QOL). 
 
2.3  
Exploratory 
 
2.3.1 Possible association between the primary and secondary outcomes and 
immunological endpoints including serum IL-12 and IFN-γ levels, and NK cell 
cytolytic activity. 
 
3.0  
SUBJECT SELECTION 
 
3.1  
Number of Subjects 
 
Fifteen to 30 men with locally recurrent prostate cancer after definitive radiotherapy. 
 
3.2  
Criteria for Subject Eligibility 
 
To be eligible, the subject must have: 
 
3.2.1 Biopsy-proven local recurrence of prostate cancer at least one year after the 
completion of definitive radiation therapy.  The biopsy must be performed 
within 180 days of registration. 
 
3.2.2 Evidence of biologically active disease as demonstrated by an unequivocally 
rising serum PSA level that is ≥ 2 ng/mL above the nadir. 
 
3.2.3 PSA < 100 ng/mL. 
 
3.2.4 Age ≥ 18 years 

Stricker et al. 
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3.2.5 Karnofsky performance status ≥ 70. 
 
3.2.6 Negative lymph nodes as established by imaging (pelvic CT or pelvic MRI) 
within 90 days of registration. 
 
3.2.6.1  
Subjects with lymph nodes equivocal or questionable by imaging 
are eligible if the nodes are ≤ 1.0 cm. 
 
3.2.6.2  
Subjects with positive lymph nodes by capromab pendetide 
(ProstaScint) scans are eligible provided a corresponding lymph 
node identified by CT or MR imaging is ≤ 1.0 cm. 
 
3.2.7 No evidence of metastatic disease, as evaluated by bone scan and CT scan 
of the abdomen and pelvis within 90 days prior to registration.  Equivocal 
bone scan findings are allowed if plain films are negative for metastasis. 
 
3.2.8 Subjects must have adequate baseline organ function, as assessed by the 
following laboratory values, within 30 days before initiating the study therapy: 
 
3.2.8.1  
Adequate renal function with serum creatinine ≤ 1.5 mg/dL or 
creatinine clearance >50 mL/min/m2. 
 
3.2.8.2  
Platelet count > 100,000/μL. 
 
3.2.8.3  
Absolute neutrophil count > 1,000/μL. 
 
3.2.8.4  
Hemoglobin > 10.0 g/dL. 
 
3.2.8.5  
Bilirubin < 1.5 mg/dL 
 
3.2.8.6  
AST/SGOT and ALT/SGPT < 3.0 times upper limit of normal 
(ULN). 
 
3.2.9 Men of child-producing potential must be willing to consent to use effective 
contraception for at least 3 months after the gene therapy. 
 
3.2.10 Subjects must possess the ability to give informed consent and express a 
willingness to meet all of the expected requirements of the protocol for the 
duration of the study. 
 
3.3  
Criteria for Subject Exclusion 
 
Subjects with the following conditions will be excluded from the study: 
 
3.3.1 PSA ≥ 100 ng/mL. 
 
3.3.2 Prostate volume > 100 cc. 
 

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3.3.3 Pathologically positive lymph nodes or nodes > 1.0 cm on imaging.  Note: 
nodes > 1.0 cm but biopsy negative are allowed. 
 
3.3.4 Evidence of M1 metastatic disease. 
 
3.3.5 Prior invasive malignancy except for non-melanoma skin cancer within 5 
years of enrollment.  Subjects must be disease-free for > 5 years. 
 
3.3.6 Prior radical prostatectomy, cryosurgery for prostate cancer, or bilateral 
orchiectomy for any reason. 
 
3.3.7 If the subject had prior androgen deprivation therapy (ADT), the subject 
exhibited biochemical failure while on ADT. 
 
3.3.8 Prior systemic chemotherapy for the study cancer.  Note that prior 
chemotherapy for a different cancer is allowed; however, subjects must be > 
2 years post-completion of chemotherapy at time of registration.  Subjects 
on Proscar therapy must stop to be eligible. 
 
3.3.9 Major surgery planned within 3 months of registration. 
 
3.3.10 Severe, active co-morbidity defined as: 
 
3.3.10.1 
New York Heart Association Class II or greater congestive heart 
failure or active ventricular arrhythmia requiring medication. 
 
3.3.10.2 
Chronic obstructive pulmonary disease (COPD) exacerbation or 
other respiratory illness requiring hospitalization within last 3 
months or precluding study therapy at the time of registration. 
 
3.3.10.3 
Acute infection.  Acute infection is defined by any viral, bacterial, 
or fungal infection that requires specific therapy within 72 hours 
of initiation of the study therapy. 
 
3.3.11 Previous history of liver disease including hepatitis. 
 
3.3.12 Immunosuppressive therapy including systemic corticosteroids.  Use of 
inhaled and topical corticosteroids is permitted. 
 
3.3.13 Impaired immunity or susceptibility to serious viral infections. 
 
3.3.14 Allergy to any product used in the protocol.  If the subject has an allergy to 
Ciproflaxin, another antibiotic can be substituted at the discretion of the 
treating physician. 
 
3.3.15 Serious medical or psychiatric illness or concomitant medication, which, in 
the judgment of the principal investigator, might interfere with the subject’s 
ability to respond to or tolerate the treatment or complete the trial. 
 
 

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4.0  
REGISTRATION PROCEDURES 
 
4.1  
Registration and Enrollment 
 
4.1.1 Subjects can be registered to the trial only after the following conditions have 
been met: 
 
4.1.1.1  
All inclusion/exclusion criteria of the study are met. 
 
4.1.1.2  
The Informed Consent has been signed and witnessed. 
 
The following registration procedures will be used: 
 
4.1.2 Once an eligible subject who meets the inclusion/exclusion criteria of the 
study has signed the Informed Consent, and eligibility case report form 
(CRF) will be completed by the clinical research staff.  The Data Collection 
Center (DCC) in the Department of Health Services (PHS) will be notified 
with tentative treatment start date through the eligibility CRF. 
 
4.1.3 The subject is considered to be enrolled into the trial if/when he receives the 
adenovirus injection. 
 
4.1.4 A subject schedule will be generated by the DCC for clinical 
assessments/visits and medications when the subject’s enrollment 
information is entered by the clinical staff. 
 
4.2  
Study Drug Accountability 
 
Dr. Freytag will be responsible for the preparation, allocation and disposal of the 
Ad5-yCD/mutTKSR39rep-hIL12 adenovirus.  He will also be responsible for 
maintaining the study drug inventory and log.  The study drug inventory will be 
reviewed by the DCC on a regular basis for accountability. 
 
5.0  
STUDY THERAPY AND PROTOCOL 
 
5.1  
Treatment Plan 
 
5.1.1 Pretreatment Evaluations 
 
5.1.1.1  
History and physical examination including digital rectal exam 
(DRE). 
 
5.1.1.2  
Hematology- CBC/DP. 
 
5.1.1.3  
Blood chemistries- glucose, Na, K, Cl, Ca, Mg, P, CO2, BUN, 
creatinine, AST/SGOT, ALT/SGPT, ALKP, bilirubin, serum 
protein, albumin, globulin, GFR, CPK, LDH, GGT, testosterone. 
 
5.1.1.4  
Diagnostic serum PSA. 
 
5.1.1.5  
Urinalysis, urine culture, and sensitivity. 

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5.1.1.6  
Chest radiographs (PA and lateral). 
 
5.1.1.7  
Diagnostic imaging- bone scan and CT scan of the abdomen and 
pelvis within 90 days of registration. 
 
5.1.1.8  
Biopsy/pathology reviews- a TRUS-guided needle biopsy of the 
prostate (≥ 6 core) must be performed within 180 days of 
registration to confirm the presence of adenocarcinoma and to 
determine prostate volume.  Biopsy cores should be kept 
separate, coded and submitted to Department of Pathology for 
analysis. 
 
5.1.1.9  
Molecular studies- presence of Ad5-yCD/mutTKSR39rep-hIL12 in 
blood by polymerase chain reaction (PCR).  This is a baseline 
control for the post-treatment PCR assays and does not have to 
be completed before the subject can be enrolled. 
 
5.1.1.10 
Immunological studies- serum IL-12 and IFN-γ will be measured 
by enzyme-linked immunosorbent assay (ELISA).  NK cell 
cytolytic activity will be measured by 51Cr release or non-
radioactive cytotoxicity assay.  These are baseline controls for 
the post-treatment evaluations and do not have to be completed 
before the subject can be enrolled. 
 
5.1.1.11 
EPIC and EQ-5D instruments. 
 
5.1.1.12 
The subject will be required to meet with a urologic surgeon to 
discuss other treatments options such as salvage prostatectomy. 
This meeting must be documented in the patient’s records. 
 
5.1.2 Oncolytic Adenovirus-Mediated Gene Therapy 
 
5.1.2.1  
Description of Ad5-yCD/mutTKSR39rep-hIL12 Adenovirus 
 
Ad5-yCD/mutTKSR39rep-hIL12 is a replication-competent, type 5 
adenovirus (Ad5) containing a yeast CD (yCD)/mutant SR39 
HSV-1 TK (HSV-1 TKSR39) fusion gene in the E1 region and 
human IL-12 coding sequences in the E3 region (57).  
Expression of the yCD/HSV-1 TKSR39 fusion gene renders tumor 
cells sensitive to 5-FC and vGCV.  Expression of IL-12 stimulates 
the innate and adaptive arms of immunity, inhibits angiogenesis, 
and mitigates immune suppression in the tumor milieu. 
 
5.1.2.2  
Supply of Ad5-yCD/mutTKSR39rep-hIL12 Adenovirus 
 
Clinical-grade Ad5-yCD/mutTKSR39rep-hIL12 adenovirus was 
manufactured at the Baylor College of Medicine Vector 
Production Facility (Houston, TX) using GMP procedures.  The 
adenovirus was supplied as a sterile, frozen liquid in vials 
containing 1.1 mL at a concentration of 1 x 1012 viral particles 
(vp) per milliliter (mL). 

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Page 24 of 47 
5.1.2.3  
Storage of Ad5-yCD/mutTKSR39rep-hIL12 Adenovirus 
 
The Ad5-yCD/mutTKSR39rep-hIL12 adenovirus is stored in a 
locked ultralow (-80oC) freezer located in a room with restricted 
access.  An inventory log is maintained by Dr. Freytag. 
 
Stability testing of the Ad5-yCD/mutTKSR39rep-hIL12 adenovirus 
will be performed on a semi-annual basis.  This will involve: 1) 
determination of titer, 2) genetic stability as determined by 
restriction digest analysis of adenoviral DNA, 3) quantification of 
the adenovirus cytopathic effect following infection of human 
prostate adenocarcinoma cells, 4) ability of the adenovirus to 
confer 5-FC and GCV sensitivity onto infected prostate 
adenocarcinoma cells, and 5) IL-12 expression following 
infection of human prostate adenocarcinoma cells. 
 
5.1.2.4  
Handling and Dilution of Ad5-yCD/mutTKSR39rep-hIL12 
Adenovirus 
 
Human adenoviruses are Risk Group 2 (RG2) agents and should 
be handled using Biosafety Level 2 (BL2) procedures (67). 
 
Thirty minutes prior to each injection, a new vial of Ad5-
yCD/mutTKSR39rep-hIL12 adenovirus will be removed from the 
storage freezer, thawed rapidly and placed on ice.  The 
adenovirus will be diluted with sterile saline to the prescribed 
concentration.  The diluted adenovirus will be loaded into 1 mL 
disposable syringes and placed on ice in a Styrofoam container.  
The container will be sealed with tape and transported to the 
treatment room. 
 
The manufacturer, lot number and expiration date of the sterile 
saline used to dilute the adenovirus will be recorded. 
 
5.1.2.5  
Ad5-yCD/mutTKSR39rep-hIL12 Adenovirus Dose 
 
The treatment schema is depicted in Figure 5. 
 
Dosing of the Ad5-yCD/mutTKSR39rep-hIL12 adenovirus is shown 
in Table 5.  The starting dose level will be 1 x 1010 vp.  The 
interval between dosing of the first two subjects in the first cohort 
should be ≥ 2 weeks. 
 
All subjects in each cohort will be evaluated for ≥ 30 days before 
proceeding to the next cohort.  If one of three subjects 
experiences a dose-limiting toxicity (DLT) (defined in Section 
6.3.2.3), three additional subjects will be enrolled at that dose 
level.  If only one of six subjects experiences a DLT at a given 
dose level, the dose will escalate to the next cohort of three 
subjects.  If two subjects experience a DLT at a given dose level, 
the maximum tolerated dose (MTD) is exceeded.  Three 

Stricker et al. 
Page 25 of 47 
additional subjects will be enrolled at the previous dose level if 
there were only three subjects at that dose. The adenovirus dose 
will be escalated to a maximum of 1 x 1012 vp. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 5.  Treatment Schema.  Subjects will undergo a series of pre-treatment evaluations 
and must meet all of the eligibility criteria to be enrolled in the study.  Subjects will receive a 
single intraprostatic injection of the Ad5-yCD/mutTKSR39rep-hIL12 adenovirus at one of five 
dose levels (see Table 5) on day 1.  Two days later (day 3), subjects will receive a one week 
(7 day) course of 5-FC (150 mg/kg/day) + vGCV (1,800 mg/day) prodrug therapy.  Toxicity 
assessments will occur twice a week during the first two weeks and then at every scheduled 
follow-up visit.  The primary endpoint is toxicity through day 30. 
 
5.1.2.6  
Injection Volume 
 
The total injection volume will be 3.0 - 4.0 mL depending on the 
size of the prostate. 
 
5.1.2.7  
Intraprostatic Injection Procedure 
 
Subjects will be seen as outpatients the day of the adenovirus 
injection.  The injection procedure will be performed in a private 
treatment room with restricted access.  Biosafety Level 2 
procedures will be followed throughout. 
 
Table 5.  Ad5-yCD/mutTKSR39rep-hIL12 Dosing 
Cohort 
# Subjects 
Adenovirus Dose (vp) 
1 
3 to 6 
1 x 1010 
2 
3 to 6 
3 x 1010 
3 
3 to 6 
1 x 1011 
4 
3 to 6 
3 x 1011 
5 
3 to 6 
1 x 1012 

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Page 26 of 47 
 
Adenovirus injections will be performed transrectally under 
transrectal ultrasound guidance (TRUS) typically using 20 gauge 
needles.  The subject will be placed in the left lateral decubitus 
position with the legs flexed and the knees brought up toward the 
chest.  The buttocks are placed over the edge of the examining 
table to allow for the appropriate manipulation of the ultrasound 
probe.  Prior to the adenovirus injection, the subject will receive a 
local injection of 1% lidocaine into the area surrounding the 
prostatic capsule.  Two needle passes (one medial, one lateral) 
will be made in the left and right sides of the prostate starting at 
the base and proceeding toward the apex until all of the 
adenovirus is delivered. 
 
Used syringes and needles will be disposed of properly.  After 
the procedure, the treatment room will be cleaned with an 
approved disinfectant. 
 
5.1.2.8  
Toxicity 
 
Injection procedure- Possible side effects of the injection 
procedure include infection, gross hematuria and urinary 
retention.  Infection occurs in less than 1% of cases.  Antibiotic 
therapy will be given as prophylaxis prior to and after the 
adenovirus injection.  Gross hematuria following prostate biopsy 
occurs in less than 1% of cases and is usually self-limited. 
Injection of the adenovirus into the prostate may cause local 
swelling that could lead to acute urinary retention.  A Foley 
catheter will be placed, if needed. 
 
Ad5-yCD/mutTKSR39rep-hIL12 adenovirus- Based on the results 
of five phase 1/2 trials with similar oncolytic adenoviruses lacking 
IL-12 (36-44), likely adverse events attributable to the oncolytic 
adenovirus include transient flu-like symptoms (37 of 79 subjects 
or 47%) and transaminitis (32 of 79 subjects or 41%).  The vast 
majority (> 94%) of these events were mild (grade 1) to moderate 
(grade 2).  The flu-like symptoms typically resolve within a few 
days and can be alleviated with analgesics.  The transaminitis is 
self-limiting and typically resolves within 2 weeks. 
 
The toxicities associated with IL-12 administration are well-
established (59, 62, 63), some of which overlap those associated 
with the adenovirus and prodrug therapy.  It is possible/likely that 
IL-12 will exacerbate flu-like symptoms (e.g., chills/rigor, fever, 
malaise), hepatic toxicity (e.g., transaminitis, ALKP, bilirubin), 
and 
hematological 
events 
(e.g., 
anemia, 
leukopenia, 
lymphopenia, neutropenia, thrombocytopenia).  Other adverse 
events possibly/probably related to IL-12 administration include: 
ascites, confusion, capillary leak syndrome, cytokine release 
syndrome, dehydration, delirium, diarrhea, dizziness, dyspnea, 
edema, headache, hypotension, nausea, pleural effusion, 

Stricker et al. 
Page 27 of 47 
tachycardia, and vomiting. 
5.1.2.9  
Dose Modification 
 
Subjects will receive only a single adenovirus injection.  No dose 
modifications are necessary. 
 
5.1.3 Prodrug Therapy 
 
5.1.3.1  
Description 
 
5-fluorocytosine (5-FC, Ancobon®) is approved for the treatment 
of serious infections caused by susceptible strains of Candida 
and Cryptococcus.  Valganciclovir (vGCV, Valcyte®) is approved 
for the treatment of CMV retinitis in people with AIDS. 
 
5.1.3.2  
Supply of 5-FC and vGCV Prodrugs 
 
Both 5-FC (Ancobon®) and vGCV (Valcyte®) are commercially 
available.  5-FC (Ancobon®) is manufactured by Valeant 
Pharmaceuticals and is available as 250 and 500 mg capsules 
for oral administration.  vGCV (Valcyte®) is manufactured by 
Hoffman La-Roche and is available as 450 mg tablets for oral 
administration. 
 
5.1.3.3  
Storage of 5-FC and vGCV Prodrugs 
 
5-FC (Ancobon®) and vGCV (Valcyte®) should be stored as 
directed by the supplier. 
 
5.1.3.4  
Duration of Prodrug Therapy 
 
All subjects will receive a one week (7 day) course of 5-FC 
(Ancobon®) + vGCV (Valcyte®) prodrug therapy beginning two 
days after the adenovirus injection (Figure 5). 
 
5.1.3.5  
5-Fluorocytosine (5-FC, Ancobon®) Dose 
 
The 5-FC (Ancobon®) dosage will be 150 mg/kg per day given in 
four equally divided doses.  Due to 5-FC (Ancobon®) only being 
available in 250 and 500 mg capsules, the dose may be rounded 
up or down to the nearest possible amount.  The capsules will be 
taken a few at a time over a 15 minute period. 
 
5.1.3.6  
Valganciclovir (vGCV, Valcyte®) Dose 
 
The vGCV (Valcyte®) dosage will be 1,800 mg/day given twice 
daily (2 x 450 mg tablets per dose) with food. 
 
 

Stricker et al. 
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5.1.3.7  
Toxicity 
 
The most common side effects associated with 5-FC (Ancobon®) 
+ vGCV (Valcyte®) prodrug therapy include neutropenia and 
thrombocytopenia.  In five previous phase 1/2 trials representing 
79 subjects (36-44), 19 of 79 (24%) subjects exhibited grade 1 - 
3 neutropenia and 26 of 79 (33%) subjects exhibited grade 1/2 
thrombocytopenia.  The prodrugs may also contribute to anemia 
(32 of 79 subjects or 41%) and possibly lymphopenia (69 of 79 
subjects or 87%).  Except for lymphopenia, the vast majority of 
these events are mild (grade 1) to moderate (grade 2).  Sixty-four 
percent (64%) of the events of lymphopenia were moderate 
(grade 2) and 25% were severe (grade 3).  All hematologic 
events are transient and self-limiting, and typically resolve within 
two 
(anemia, 
neutropenia, 
thrombocytopenia) 
to 
six 
(lymphopenia) weeks. 
 
Other possible side effects associated with 5-FC (Ancobon®) + 
vGCV (Valcyte®) prodrug therapy include nausea, vomiting, 
diarrhea and headache. 
 
5.1.3.8  
Dose Modifications 
 
5-FC and vGCV doses will be modified based on the guidelines 
below. 
 
5.1.3.8.1 Cytopenia 
 
5.1.3.8.1.1  
Absolute neutrophil count < 1,000/μL 
or platelet count < 100,000/μL- reduce 
5-FC and vGCV doses to 75%. 
 
5.1.3.8.1.2  
Absolute neutrophil count < 500/μL or 
platelet 
count 
< 
50,000/μL- 
discontinue 5-FC and vGCV, and 
resume the drugs at 50% of the 
original dose when the neutrophil 
count is > 500/μL and platelets > 
50,000/μL. 
 
 

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5.1.3.8.2 Creatinine Clearance 
 
If creatinine clearance (CrCl) is < 50 mL/min/m2, 
follow the guidelines in Table 6 and check the 
creatinine every other day (except weekends).  
Creatinine clearance should be calculated based on 
the most recent creatinine level as follows: 
 
CrCl = (140 - age [years]) x  (body weight [kg]) 
           ---------------------------------------------------- 
              (72)  x   (serum creatinine [mg/dL]) 
 
Table 6.  Ancobon and Valcyte Dose Modifications 
Creatinine Clearance 
Ancobon 
Valcyte 
≥ 50 
No modifications 
No modifications 
≥ 40 and < 50 
Reduce to 75 mg/kg/day 
Reduce to 450 mg b.i.d. 
≥ 25 and < 40 
Reduce to 37.5 mg/kg/day 
Reduce to 450 mg q.d. 
< 25 
Suspend 
Suspend 
 
In all cases, if creatinine clearance improves, 
Ancobon and Valcyte doses can be increased 
(resumed) once according to guidelines in Table 6.  If 
creatinine clearance drops a second time, Ancobon 
and Valcyte dosage should be reduced accordingly, 
but may not be increased if creatinine clearance 
improves.  In all cases, the subject should take no 
more than a maximum of 7 days of prodrug 
medication (regardless of the dose received).  If 
prodrugs are suspended continuously for > 2 weeks, 
the subject should not receive any further prodrugs. 
 
5.1.4 Other Medications 
 
5.1.4.1  
Antibiotics 
 
To reduce the risk of infection, subjects will be given ciprofloxacin 
(500 mg, p.o., b.i.d.) the evening before, and the morning of, the 
adenovirus injection, continuing the antibiotic twice daily for a 
total of 3 days.  They will also receive gentamicin (120 mg, I.M.) 
just prior to the adenovirus injection.  If the subject is allergic to 
ciprofloxacin, another antibiotic can be substituted at the 
discretion of the treating physician. 
 

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6.0  
SUBJECT ASSESSMENTS 
 
Subject assessments are summarized in Table 7. 
 
Table 7.  Subject Assessments 
Parameters 
Pre-Study Therapy 
First 2 Weeks 
Follow Up 
History, Physical Examination, DRE 
Xa 
 
Xi 
EPIC and EQ-5D Instruments 
Xa 
 
Xj 
Bone Scan 
Xa 
 
Xk 
CT Scan 
Xa 
 
Xk 
On-site Physician Assessment 
Xb 
Xe 
Xi 
Hematology (CBC/DP) 
Xb 
Xf 
Xi 
Blood Chemistries 
Xb 
Xf 
Xi 
Urinalysis 
Xb 
 
 
Diagnostic Serum PSA 
Xb 
Xe 
Xi 
Toxicity Assessment 
Xb 
Xf 
Xi 
Molecular Studies 
Xb 
Xg 
Xf 
IL-12 and IFN-γ ELISA 
Xb 
Xg 
Xf 
NK Cytolytic Assay 
Xb 
Xh 
 
Chest Radiographs 
Xc 
 
 
TRUS-Guided Needle Biopsy 
Xd 
 
 
Pathology Assessment 
Xd 
 
 
aWithin 90 days of registration.  bWithin 30 days of registration.  cWithin 90 days of registration if the subject has 
history of pulmonary disease; otherwise, within 180 days.  dWithin 180 days of registration.  eOnce a week for the 
first two weeks.  fTwice a week for the first 2 weeks.  gAt every blood draw until not detected in two consecutive 
measurements.  hAt every blood draw during the first two weeks.  iAt every scheduled follow-up visit at 1, 3, 6, 9, 12, 
18, 24 months and then annually thereafter.  jAt 6, 12, and 24 months.  kIn subjects who experience bone pain or at 
PSA relapse and then annually thereafter. 
 
6.1  
Assessments During First Two Weeks After Adenovirus Injection 
 
The following assessments will be performed: 
 
6.1.1 An on-site physician assessment will be performed once a week. 
 
6.1.2 CBC/DP and blood chemistries will be measured twice a week, or as 
clinically indicated. 
 
6.1.3 Diagnostic serum PSA. 
 
6.1.4 Ad5-yCD/mutTKSR39rep-hIL12 viral DNA in blood will be measured at every 
scheduled blood draw until not detected in two consecutive measurements. 
 
6.1.5 Serum IL-12 and IFN-γ will be measured at every scheduled blood draw until 
not detected in two consecutive measurements. 
 
6.1.6 NK cytolytic activity will be measured at every scheduled blood draw during 
the first two weeks after the adenovirus injection. 
 
6.1.7 Toxicity assessments will be performed twice a week using protocol-specific 
Case Report Forms (CRFs).  Toxicity assessments will be based on physical 
examinations, diagnostic evaluations, and laboratory tests. 

Stricker et al. 
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6.2  
Assessments Following the Study Therapy 
 
Follow-up visits will occur at 1, 3, 6, 9, 12, 18 and 24 months after the adenovirus 
injection and then at least annually thereafter.  Evaluations will include: 
 
6.2.1 History and physical examination including DRE. 
 
6.2.2 CBC/DP and blood chemistries. 
 
6.2.3 Diagnostic serum PSA. 
 
6.2.4 EPIC and EQ-5D instruments at 6, 12, and 24 months. 
 
6.2.5 Ad5-yCD/mutTKSR39rep-hIL12 viral DNA in blood will be measured at every 
scheduled blood draw until not detected in two consecutive measurements. 
 
6.2.6 Serum IL-12 and IFN-γ will be measured at every scheduled blood draw until 
not detected in two consecutive measurements. 
 
6.2.7 A bone scan will be performed on any subject with complaints of bone pain 
that cannot be attributed to any intercurrent disease.  Discretionary plain 
films and/or MRI may be needed to evaluate lesions seen on bone scan to 
confirm the diagnosis of metastatic disease. 
 
6.2.8 In subjects with biochemical failure, a bone scan and CT scan of the 
abdomen and pelvis should be performed at that time and then annually 
and/or as clinically indicated. 
 
6.2.9 Toxicity assessments will be performed at every scheduled follow-up visit 
using CRFs specifically designed for this trial.  Toxicity assessments will be 
based on physical examinations, diagnostic evaluations, and laboratory test 
results. 
 
6.2.10 It is understood that the performance of an individual study or test as 
specified in this protocol is subject to factors such as subject compliance, 
schedule difficulties, equipment malfunction, or the judgment of the principal 
investigator or patient care physicians, and that a test may not be done in an 
individual instance without violating the protocol.  However, any systematic 
modification of the original protocol in this regard, whether related to subject 
safety or not, will be submitted to the IRB for approval. 
 
 

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6.3. 
Toxicity Assessments 
 
6.3.1 Adverse Event Grading Criteria 
 
Adverse events will be graded according to NCI’s Common Terminology 
Criteria for Adverse Events (CTCAE) v4.03 (68).  Adverse events will be 
documented using CRFs specifically designed for this trial. 
 
6.3.2 Definitions 
 
6.3.2.1  
Definition of Adverse Event (AE) 
 
An adverse event is defined as any undesirable experience 
associated with the use of a medical product (69).  Adverse 
events will be categorized as depicted in Table 8 (70). 
 
Table 8.  Categorization of Adverse Events 
Relationship 
Attribution 
Description 
Unrelated to investigational 
agent/intervention 
Unrelated 
The AE is clearly NOT related to 
agent/intervention 
Unlikely 
The AE is doubtfully related to 
agent/intervention 
Related to investigational 
agent/intervention 
Possible 
The 
AE 
may 
be 
related 
to 
agent/intervention 
Probably 
The 
AE 
is 
likely 
related 
to 
agent/intervention 
Definite 
The AE is clearly related to 
agent/intervention 
 
6.3.2.2  
Definition of Serious Adverse Event (SAE) 
 
A SAE is defined as any undesirable experience associated with 
the use of a medical product that results in any of the following 
outcomes: 
 
6.3.2.2.1 Death. 
 
6.3.2.2.2 A life-threatening experience. 
 
6.3.2.2.3 Hospitalization 
or 
prolongation 
of 
existing 
hospitalization. 
 
6.3.2.2.4 Disability or permanent damage. 
 
6.3.2.2.5 A congenital anomaly/birth defect. 
 
6.3.2.2.6 Required 
intervention 
to 
prevent 
permanent 
impairment or damage. 
 

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6.3.2.2.7 Other serious events that may jeopardize the subject 
and may require medical or surgical intervention 
(treatment) to prevent one of the other outcomes. 
6.3.2.3  
Definition of Dose-Limiting Toxicity (DLT) 
 
Dose-limiting toxicity is defined as any of the following adverse 
events considered possibly/probably/definitely related to the 
study treatment. 
 
6.3.2.3.1 ≥ Grade 2 allergic reaction or generalized urticaria. 
 
6.3.2.3.2 ≥ Grade 2 cardiovascular or neurologic toxicity. 
 
6.3.2.3.3 ≥ Grade 3 hematologic or non-hematologic toxicity of 
any duration except for: 
 
6.3.2.3.3.1  
Grade 3 lymphopenia with recovery 
within 2 weeks. 
 
6.3.2.3.3.2  
Grade 3 anemia, leukopenia, or 
neutropenia that is reversible within 
72 hours. 
 
6.3.2.3.3.3  
Grade 3/4 fever or flu-like symptoms 
that are reversible within 72 hours. 
 
6.3.2.3.3.4  
Grade 
3 
SGOT/SGPT 
that 
is 
reversible within 72 hours. 
 
6.3.2.3.3.5  
Grade 3 hyperglycemia that can be 
explained by patient's baseline co-
morbidity (i.e., diabetes). 
 
6.3.2.3.3.6  
Grade 3 hypermagnesemia that is 
reversible within 72 hours. 
 
6.3.2.3.3.7  
Grade 3 hypophosphatemia that is 
reversible within 72 hours. 
 
6.3.2.4  
Definition of Maximum Tolerated Dose (MTD) 
 
MTD is defined as the highest Ad5-yCD/mutTKSR39rep-hIL12 
dose at which < 33% (< 1 of 3 or < 2 of 6) of any cohort develops 
DLT. 
 
If 1 of 3 subjects in a cohort develops DLT, the cohort will be 
expanded to 6 subjects.  If ≥ 2 subjects out of 6 in a cohort 
develop DLT, MTD will have been exceeded at that dose.  If 
there is a lower dose and if the cohort at that dose has not 
already been expanded to 6, the cohort will be expanded to 6 at 
the lower dose.  If ≥ 2 subjects develop DLT, MTD will have been 

Stricker et al. 
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exceeded and the trial will stop. 
 
 

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6.3.3 Adverse Event Reporting Guidelines 
 
Guidelines for adverse event reporting are depicted in Table 9.  Adverse 
events that require reporting will be reported using the Adverse Event 
Reporting CRF specifically designed for this study. 
 
 
6.4  
Subject Removal and Study Stopping Rules 
 
6.4.1 Subject Removal Rules 
 
Every attempt will be made to obtain follow-up on subjects who have 
received the study medication. 
 
6.4.1.1  
The subject may withdraw from the study upon request of the 
participating subject or person with the power of attorney. 
 
6.4.1.2  
Noncompliance. 
 
6.4.1.3  
Intercurrent medical or psychiatric problem or institution of a 
concomitant medication that interferes with compliance with the 
protocol or places the subject at increased risk. 
 
6.4.1.4  
Dose-limiting toxicity (DLT). 
 
6.4.1.5  
Investigator’s discretion, to the extent that subsequent clinical 
study requirements place the subject at unacceptable risks. 
 
6.4.2 Study Stopping Rules 
 
6.4.2.1  
DLT in the first dose cohort. 
 
6.4.2.2  
If > 33% of enrolled subjects develop ≥ grade 3 hematuria or 
urinary retention, the study will be stopped and the injection 
procedure re-evaluated. 
 
6.4.2.3  
Transmission of the Ad5-yCD/mutTKSR39rep-hIL12 adenovirus to 
any subject contacts, including health care personnel. 
 
6.4.2.4  
Lack of availability of the Ad5-yCD/mutTKSR39rep-hIL12 
Table 9.  Adverse Event Reporting Requirements 
Not Serious 
Serious AND unrelated to 
the agent/intervention 
Serious AND possibly, probably or 
definitely related to the 
agent/intervention 
Reporting is NOT required. 
Reporting is NOT required 
Report by phone/FAX/email to IRB 
within 24 hours of discovery of event. 
 Written report to IRB, IBC, OBA and 
FDA within 10 calendar days.  This 
includes deaths within 30 days of 
adenovirus injection. 

Stricker et al. 
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adenovirus. 
6.4.2.5  
Insufficient accrual. 
 
6.5  
Measurement of Treatment Effect 
 
Treatment effect will be based on post-treatment digital rectal examinations (DRE), 
PSA evaluations, bone and CT/MRI scans.  DRE and PSA will be assessed at every 
scheduled follow-up visit.  Bone and CT/MRI scans will be performed as clinically 
indicated. 
 
6.6  
Definition of Response Parameters 
 
6.6.1 Freedom from Biochemical (PSA) Failure- The time to PSA failure will be 
measured from the date of the adenovirus injection to the date of a rise of ≥ 
2 ng/mL above the nadir PSA (71).  Nadir PSA is defined as the lowest PSA 
value after the adenovirus injection and before the call date of PSA failure.  
The time of failure will be the date of the first PSA that is ≥ 2 ng/mL above 
the post-treatment nadir. 
 
6.6.2 Freedom from Clinical Failure- The time to clinical failure (local and/or 
regional/distant) will be measured from the date of the adenovirus injection 
to the date that clinical failure is first documented. 
 
Local clinical failure is defined as having any ONE of the following: 
 
6.6.2.1  
Local progression, as measured by a ≥ 25% increase in the 
product of the two largest perpendicular diameters of the 
prostate tumor at any time. 
 
6.6.2.2  
Failure of regression of palpable tumor by 2 years. 
 
6.6.2.3  
Redevelopment of a palpable abnormality after complete 
disappearance of previous abnormalities. 
 
Note: In all above cases, unless clinically contraindicated, needle biopsy 
verification is required to document the presence of local tumor. 
 
Regional/distant clinical failure is defined as having any ONE of the 
following: 
 
6.6.2.4  
Radiographic (CT or MRI) evidence of new or increased pelvic 
lymphadenopathy (measuring >1.5 cm on short axis) and/or 
histological confirmation of tumor. 
 
6.6.2.5  
The development of a new area of isotope uptake on bone scan 
relative to baseline that cannot be attributed to trauma and is 
clinically consistent with metastatic disease.  Any questionable 
findings must be confirmed by other imaging modalities (e.g., 
MRI) and/or biopsy. 
 
6.6.2.6  
The development of a clinical and/or radiographic finding 

Stricker et al. 
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consistent with metastatic disease (e.g., a pulmonary 
metastasis). 
6.6.3 Disease-Specific Survival- The time to disease-specific death will be 
measured from the date of the adenovirus injection to the date of death due 
to prostate cancer.  Causes of death may require review by the study PI or 
designee.  Death due to prostate cancer will be defined as: 
 
6.6.3.1  
Primary cause of death certified as due to prostate cancer. 
 
6.6.3.2  
Death in association with any ONE of the following conditions: 
 
6.6.3.2.1 Further clinical tumor progression occurring after 
initiation of additional salvage therapy such as ADT. 
 
6.6.3.2.2 A rise in serum PSA that exceeds 1.0 ng/mL on at 
least two consecutive occasions during or after 
salvage ADT. 
 
6.6.3.2.3 Disease progression in the absence of any prostate 
cancer therapy. 
 
6.6.3.2.4 Death from a complication of prostate cancer therapy, 
irrespective of disease status. 
 
6.6.4 Survival- The survival time will be measured from the date of the adenovirus 
injection to the date of death.  All subjects will be followed for survival.  Every 
effort should be made to document the cause of death.  Post-mortem 
examination will be carried out when feasible and a copy of the final autopsy 
report should be sent to the DCC. 
 
7.0  
DATA COLLECTION 
 
7.1  
Data Collection and Management 
 
The Data Coordinating Center (DCC) is headed by Jingfang Cheng, RN, and is 
located in the Department of Radiation Oncology, Henry Ford Health System 
(HFHS).  Ms. Chang has over 10 years experience in managing clinical trial 
databases.  The DCC will use a Microsoft Access database for data collection using 
a set of Standard Operating Procedure (SOPs) developed by Ms. Cheng and the 
study coordinator. 
 
7.2  
Standard Operating Procedures 
 
Standard Operating Procedures (SOPs) have been developed by Ms. Cheng and 
the study coordinator to be compliant with FDA regulations and implemented at the 
DCC for FDA regulated clinical trials.  SOPs were developed using the FDA Good 
Clinical Practice Guidelines. 
 
7.3  
Case Report Forms 
 
Case Report Forms (CRFs) have been developed specifically for this trial.  The 

Stricker et al. 
Page 38 of 47 
database will be created to match with CRFs so that the data structure provides fast, 
efficient, and accurate data processing. 
7.4  
Database and Data Management 
 
The database for this study will be implemented using a Microsoft Access database 
housed on a secure departmental drive.  Access is a relational database with 
security options that limit the ability to access the data to specified people.  The 
database structure will be set up according to the CRFs.  Specific queries can be 
run to check for data discrepancies and missing fields.  An independent monitor will 
conduct audits of the data for completion and accuracy no less than one time per 
year. 
 
7.5  
Plan for the Site Initialization 
 
Subjects will be enrolled at the Henry Ford Health System only.  After receiving all 
required federal (FDA, OBA) and local (IRB, IBC) approvals, Drs. Stricker, Freytag 
and Cheng will conduct the site initiation.  They will meet with the study nurse and 
clinical coordinator to discuss the following: study drug, study protocol, subject 
enrollment, drug accountability, CRFs and instructions for completion of CRF and 
CRF submission, and adverse event (AE) reporting.  A follow-up meeting(s) will be 
conducted, if one meeting is not sufficient.  During subject enrollment and follow-up, 
Drs. Stricker and Freytag, and Ms. Cheng, will be available to answer questions 
regarding administrative issues, enrollment, CRF data collection and quality 
assurance.  The key investigators will meet on a regular basis to go over issues 
encountered during the study. 
 
7.6  
Subject Registration 
 
Subject registration is described in Section 4.0.  After receiving the registration 
information, the DCC will generate the subject’s schedule for follow-up. 
 
7.7  
CRF Data Collection and Data Management 
 
CRF data is collected based on the sources of truth (e.g., medical records).  Data 
sources will include subject interviews (subject history, adverse events), physical 
examination, histology/pathology reports, all laboratory data, and medical records.  
Subjects will be identified by study subject number and initials (first, middle, last); if 
there is no middle initial, a hyphen will be used (first-last).  Last names with 
apostrophes will be identified by the first letter of the last name.  Completed CRFs 
will be entered into the study database by study staff on a regular basis. 
 
Data entry is summarized in Table 10. 
 
 
 
 
 
 
 
 
 

Stricker et al. 
Page 39 of 47 
 
 
Table 10.  Summary of Data Submission 
Case Report Form 
Due 
Eligibility Checklist 
At registration 
Registration Form 
Within 2 weeks of registration 
Pre-Treatment Conditions 
Within 2 weeks of registration 
Concomitant Medications 
Within 2 weeks of registration 
Adenovirus Injection Summary 
Within 2 weeks of adenovirus injection 
Prodrug Therapy 
Within 2 weeks of completion of prodrug 
therapy regimen 
Blood Labs 
Within 2 weeks of blood draw 
Toxicity Assessment 
Within 2 weeks of toxicity assessment 
SAE That Require Reporting 
-24 h by phone/FAX/email 
-Written report within 10 calendar days 
Adenoviral DNA in Blood 
Within 90 days of adenovirus injection 
On-site Physician Assessment 
Within 2 weeks of assessment 
Discontinuation of Study Therapy 
Within 2 weeks of discontinuation 
Death Report 
If ≤ 30 days from adenovirus injection: 
-24 h by phone/FAX/email 
-Written report within 10 calendar days 
If > 30 days from adenovirus injection: 
-Within 2 weeks of learning of event 
Long-Term Follow-Up 
Within 2 weeks of assessment 
EPIC and EQ5D 
Within 2 weeks of assessment 
 
7.8  
Quality Assurance Plan to Ensure Data Quality 
 
Data quality assurances (QA) are applied both at the DCC and clinical sites.  
Features of the QA plan at the DCC include: 
 
7.8.1 Initial review of forms 
 
7.8.2 Check for possible missing forms 
 
7.8.3 CRF data field checks for missing, miscodes and outliers 
 
7.8.4 Code for tracking person who completed forms 
 
7.8.5 Data consistency checks (within or across the forms) 
 
7.8.6 Generation of queries and an electronic audit trail 
 
All QA queries will be documented by the data manager with respect to: 
 
7.8.7 Query generation date 
 
7.8.8 Date query sent to clinical site for resolution 
 
7.8.9 Identification information for the form 
 
7.8.10 Old (or original) value 

Stricker et al. 
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7.8.11 Error message(s) 
7.8.12 Code of person who completed the form 
 
7.8.13 New value (may not represent a change) 
 
7.8.14 Reason for new value 
 
7.8.15 ID of site staff member responsible for new value 
 
7.8.16 Date change made to database 
 
Report forms are produced from a query table and sent to the PI for resolution.  
Data are processed as they are collected to ensure the smooth and systematic flow 
of activities, and to avoid backlogs and uneven workloads. 
 
7.9  
Confidentiality 
 
Throughout the data collection period, the data of individual subjects will be kept 
confidential by DCC staff.  The clinical site only holds names or other personal 
identifiers for conducting the subject’s follow-up.  Subjects’ names and other 
personal identifiers can not appear on any data collection form, CRFs and analysis 
reports.  The DCC must keep these strictly confidential among staff. 
 
8.0  
STATISTICAL CONSIDERATIONS 
 
This phase 1 trial is designed to determine the toxicity of oncolytic adenovirus-mediated 
cytotoxic and IL-12 gene therapy in locally recurrent prostate cancer.  Endpoints are 
classified as primary, secondary and exploratory. 
 
8.1  
Endpoints, Hypotheses and Analyses 
 
8.1.1 Primary Endpoint 
 
8.1.1.1  
Hypothesis- Oncolytic adenovirus-mediated cytotoxic and IL-12 
gene therapy is associated with acceptable toxicity. 
 
Adverse events will be graded according to NCI’s Common 
Terminology Criteria for Adverse Events (CTCAE) v4.03 (68).  
Adverse events will collected through day 30 (day 1 being the 
day of the adenovirus injection). 
 
8.1.2 Secondary Endpoints 
 
8.1.2.1  
Hypothesis 1- Oncolytic adenovirus-mediated cytotoxic and IL-12 
gene therapy has an effect on serum PSA levels. 
 
Serum PSA will be assessed according to Table 7 and compared 
to the subject’s baseline value. 
 
 

Stricker et al. 
Page 41 of 47 
 
 
8.1.2.2  
Hypothesis 2- Oncolytic adenovirus-mediated cytotoxic and IL-12 
gene therapy has an effect on PSADT. 
 
Serum PSA will be assessed according to Table 7.  PSADT will 
be determined by logarithm regression analysis as described 
previously (38).  PSADT prior to the study therapy will be 
calculated using a minimum of three PSA values including the 
last PSA prior to initiation of the study therapy.  Subjects who do 
not have at least three pre-treatment PSA values will be 
excluded from the analysis.  PSADT after the study treatment will 
be calculated from the nadir and will use all PSA values prior to 
the implementation of additional salvage therapy such as ADT. 
 
PSADT will be calculated assuming first-order kinetics using the 
following equation: 
 
Ln (PSA) = βo + β1t 
 
where βo is the point where the regression line crosses the 
ordinate at t = 0 and β1 is the number of units that Ln (PSA) 
changes for every one-unit change in time (i.e., the slope).  
PSADT is defined as Ln (2) / β1.  Data are evaluated for 
normality the ranked data are considered if data are not normal. 
 
8.1.2.3  
Hypothesis 3- Oncolytic adenovirus-mediated cytotoxic and IL-12 
gene therapy has no significant effect on quality of life. 
 
We will use two instruments to measure QOL: EPIC and EQ-5D. 
QOL instruments will be collected on all subjects at baseline, 6, 
12 and 24 months after the adenovirus injection. 
 
EPIC questionnaires will be completed by the subject.  The 
questionnaires will be summarized into three endpoints of 
function and bother in urinary, bowel and sexual domains with a 
score of 0 to 100.  EQ-5D will have a self-report score of 1 to 3 in 
five domains (mobility, self-care, usual activity, pain/discomfort 
and anxiety/depression), and an overall health score 10 to 100.  
If a subject death is related to prostate cancer or the study 
treatment, the worst score will be given and the subject will be 
included in the analysis. 
 
Data will be evaluated for normality, data transformation or 
nonparametric approach will be considered if data are not 
normal. 
 
For each QOL domain variable, analysis of variance and 
covariance (ANCOVA) will be used.  Analysis will include the 
baseline covariates of pre-treatment domain score, covariate of 
time (of assessment post-treatment) and the independent 

Stricker et al. 
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treatment variable.  The analysis will start testing for treatment by 
time interaction, followed by testing the treatment effect at each 
time point if the interaction is detected at critical value 0.10, or 
testing the overall treatment effect or time effect at critical value 
of 0.05 if otherwise. 
 
8.1.3 Exploratory Hypothesis 
 
8.1.3.1  
Hypothesis- There is a possible association between the primary 
and secondary outcomes and immunological endpoints including 
serum IL-12 and IFN-γ levels, and NK cell cytolytic activity.  
Immunological measurements, as continuous variables, will be 
collected from blood at baseline and at specified times after the 
adenovirus injection. 
 
Each variable will be evaluated for the proportional hazard 
assumption.  Data will be categorized if the assumption is not 
met.  Cox regression models will be used to study the 
association 
of 
baseline 
and 
post-treatment 
variables, 
respectively, with outcome.  The analysis will begin testing the 
individual variable effect including testing for treatment by 
individual variable interaction, followed by multivariable analysis. 
The variable with individual effect at 0.05 level or variable 
interaction at 0.10 will remain in the final multivariable model with 
assessment of goodness-of-fit (e.g. ROC). 
 
8.2  
Sample Size Calculation/Power Justification for the Primary Endpoint 
 
This is a MTD determination study.  Therefore, the power calculation is not relevant. 
A 3 to 6 subject per dose cohort with a total of 15 to 30 subjects is standard for a 
phase 1 trial (72). 
 
8.3  
Statistical Analysis 
 
Analysis for this phase 1 trial will be descriptive.  We will report all toxicity results 
taking into account any baseline abnormalities. 
 
9.0  
DATA AND SAFETY MONITORING PLAN 
 
A Data and Safety Monitoring Plan has been developed according to FDA guidelines (49592 
Federal Register / Vol. 63, No. 179 / Wednesday, September 16, 1998 / Notices). 
 
 

Stricker et al. 
Page 43 of 47 
10.0 INCLUSION OF MINORITIES 
 
Based on subject demographics of five phase 1/2 gene therapy trials conducted by the 
sponsor, the Targeted/Planned Enrollment for this clinical trial is shown in Table 11.  Forty-
five of the 102 (44%) subjects enrolled were minorities. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
11.0 PROCESS OF INFORMED CONSENT 
 
The Principal Investigator and Co-Principal Investigators will ensure that the study is 
conducted in full conformance with FDA standards for human research.  An IRB-approved 
Informed Consent will be obtained for each subject.  The form must be signed, witnessed, 
and dated.  Copies of the signed document will be given to the subject and filed in the study 
Master File.  All reports and subject samples will be identified only by a coded number to 
maintain subject confidentiality.  All records will be kept confidential to the extent permitted 
by law. 
 
 
Table 11.  Targeted/Planned Enrollment (assuming 15 subjects enrolled) 
TARGETED/PLANNED ENROLLMENT: Number of Subjects 
ETHNIC CATEGORY 
Gender 
FEMALES 
MALES 
TOTAL 
Hispanic or Latino 
0 
  0 
  0 
Not Hispanic or Latino 
0 
15 
15 
Ethic Categories: Total of All Subjects 
0 
15 
15 
RACIAL CATEGORIES 
 
American Indian/Alaskan Native 
0 
  0 
  0 
Asian 
0 
  0 
  0 
Native Hawaiian or Other Pacific Islander 
0 
  0 
  0 
Black or African American 
0 
  7 
  7 
White 
0 
  8 
  8 
Ethic Categories: Total of All Subjects 
0 
15 
15 

Stricker et al. 
Page 44 of 47 
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