A phase I study of a tropism-modified conditionally replicative adenovirus for recurrent malignant gynecologic diseases.

Abstract

To determine the maximum tolerated dose (MTD), toxicity spectrum, clinical activity, and biological effects of the tropism-modified, infectivity-enhanced conditionally replicative adenovirus (CRAd), Ad5-Δ24-Arg-Gly-Asp (RGD), in patients with malignant gynecologic diseases.
Cohorts of eligible patients were treated daily for 3 days through an i.p. catheter. Vector doses ranged from 1 × 10(9) to 1 × 10(12) viral particles per day. Toxicity was evaluated using CTCv3.0. CA-125 and Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used to determine clinical efficacy. Corollary biological studies included assessment of CRAd replication, wild-type virus generation, viral shedding, and neutralizing antibody response.
Twenty-one patients were treated. Adverse clinical effects were limited to grade 1/2 fever, fatigue, or abdominal pain. No vector-related grade 3/4 toxicities were noted. No clinically significant laboratory abnormalities were noted. The maximum tolerated dose was not reached. Over a 1 month follow-up, 15 (71%) patients had stable disease and six (29%) had progressive disease. No partial or complete responses were noted. Seven patients had a decrease in CA-125; four had a >20% drop. RGD-specific PCR showed the presence of study vector in ascites of 16 patients. Seven revealed an increase in virus after day 3, suggesting replication of Ad5-Δ24-RGD. Minimal wild-type virus generation was detected. Viral shedding studies showed insignificant shedding in the serum, saliva, and urine. Anti-adenoviral neutralizing antibody effects were prevalent.
This study, the first to evaluate an infectivity-enhanced CRAd in human cancer, shows the feasibility, safety, potential antitumor response, and biological activity of this approach in ovarian cancer. Further evaluation of infectivity enhanced virotherapy approaches for malignant gynecologic diseases is warranted.

Full text

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Cancer Therapy: Clinical
A Phase I Study of a Tropism-Modified Conditionally
Replicative Adenovirus for Recurrent Malignant
Gynecologic Diseases
Kristopher J. Kimball1, Meredith A. Preuss1, Mack N. Barnes1, Minghui Wang1, Gene P. Siegal1, Wen Wan1,
Huichien Kuo1, Souheil Saddekni1, Cecil R. Stockard1, William E. Grizzle1, Raymond D. Harris2,
Rosemarie Aurigemma2, David T. Curiel1, and Ronald D. Alvarez1
Abstract
Purpose: To determine the maximum tolerated dose (MTD), toxicity spectrum, clinical activity, and
biological effects of the tropism-modified, infectivity-enhanced conditionally replicative adenovirus
(CRAd), Ad5–Δ24–Arg-Gly-Asp (RGD), in patients with malignant gynecologic diseases.
Experimental Design: Cohorts of eligible patients were treated daily for 3 days through an i.p.
catheter. Vector doses ranged from 1 × 109to 1 × 1012viral particles per day. Toxicity was evaluated
using CTCv3.0. CA-125 and Response Evaluation Criteria in Solid Tumors (RECIST) criteria were used
to determine clinical efficacy. Corollary biological studies included assessment of CRAd replication,
wild-type virus generation, viral shedding, and neutralizing antibody response.
Results: Twenty-one patients were treated. Adverse clinical effects were limited to grade 1/2 fever, fa-
tigue, or abdominal pain. No vector-related grade 3/4 toxicities were noted. No clinically significant
laboratory abnormalities were noted. The maximum tolerated dose was not reached. Over a 1 month
follow-up, 15 (71%) patients had stable disease and six (29%) had progressive disease. No partial or
complete responses were noted. Seven patients had a decrease in CA-125; four had a >20% drop.
RGD-specific PCR showed the presence of study vector in ascites of 16 patients. Seven revealed an increase
in virus after day 3, suggesting replication of Ad5-Δ24-RGD. Minimal wild-type virus generation was
detected. Viral shedding studies showed insignificant shedding in the serum, saliva, and urine. Anti-
adenoviral neutralizing antibody effects were prevalent.
Conclusions: This study, the first to evaluate an infectivity-enhanced CRAd in human cancer, shows
the feasibility, safety, potential antitumor response, and biological activity of this approach in ovarian
cancer. Further evaluation of infectivity enhanced virotherapy approaches for malignant gynecologic
diseases is warranted. Clin Cancer Res; 16(21); 5277–87. ©2010 AACR.
In recent years, advances in cancer therapy have
brought about modest improvement in outcomes for pa-
tients with malignant gynecologic diseases. Although
many patients with advanced ovarian, fallopian tube, or
peritoneal carcinoma will initially respond to surgical
debulking and cytotoxic chemotherapy, most will eventu-
ally recur and ultimately succumb to their disease. Like-
wise, only a few patients with advanced or recurrent
endometrial will experience long term overall survival
(1, 2). Clearly, novel treatment strategies are needed
for patients affected with these devastating malignant
gynecologic diseases.
One such novel approach, oncolytic virotherapy, in-
volves the development of replication competent viruses
that specifically infect targeted cancer cells, proliferate in
and induce cancer cell oncolysis, and subsequently release
progeny viral particles that will infect and lyse surrounding
cancercells.Adenovirusesarewellsuitedtodevelopmentas
a virotherapy agent due to excellent stability, unparalleled
infectivity, efficient gene transfer, and biological plasticity
compared with other viruses (3). Conditionally replicating
adenoviruses(CRAd)arerendered conditionallyreplicative
by deleting viral genes that become extraneous in many tu-
mor cells such as genes involved in replication through p53
and Rb pathways (4). In normal cells, p53 and Rb serve as
tumor suppressor genes modulating cell cycle andinducing
Authors' Affiliations:1The University of Alabama at Birmingham,
Birmingham, Alabama and2SAIC-Frederick, Inc., National Cancer
Institute at Frederick, Frederick, Maryland
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
The content of this publication does not necessarily reflect the views or
policies of the Department of Health and Human Services nor does the
mention of trade names, commercial products, or organizations imply
endorsement by the U.S. government.
Corresponding Author: Ronald D. Alvarez, Room 538, Old Hillman
Building, 619 South 20th Street, Birmingham, AL 35249-7333. Phone:
205-934-4984; Fax: 205-975-6174; E-mail: ronald.alvarez@ccc.uab.edu.
doi: 10.1158/1078-0432.CCR-10-0791
©2010 American Association for Cancer Research.
Clinical
Cancer
Research
www.aacrjournals.org
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apoptosis if cellular DNA damage is incurred. However,
many cancers, such as ovarian cancer, are known to have
very low rates of functional p53 and Rb due to mutations
in the aforementioned genes (5, 6). As such, these tumors
lend themselves well to potential CRAd therapy.
One such CRAd, ONYX 015, has been used previously
in clinical trials of gynecologic and other malignant dis-
eases but with limited success (7, 8). Limited efficacy has
been, in part, attributed to inefficient gene transfer due to
a relative paucity of the primary adenovirus receptor,
known as CAR, on cancer cells (9). Our group has previ-
ously shown that genetic manipulation of the adenoviral
capsid proteins to incorporate an Arg-Gly-Asp (RGD)
sequence in the HI loop of the fiber knob accomplishes
enhanced infectivity of tumor cells through CAR indepen-
dent pathways (10, 11). RGD modification had been
shown to exhibit a degree of specificity for ovarian cancer
cells over normal tissue because of the upregulation of ανβ
integrins in ovarian cancer cells and infectivity enhance-
ment was shown to dramatically improve antitumor po-
tency of various gene therapy approaches in vitro and in
animal models of ovarian cancer (11).
In keeping with these data, we constructed a novel
infectivity-enhanced CRAd, designated Ad5-Δ24-RGD,
that uses a 24-bp deletion in the E1A gene known to be
necessary for host cell Rb protein binding, thereby confer-
ring conditional replication only in cells that are deficient
in the Rb/p16 pathway. Incorporation of the RGD capsid
modification also allows Ad5-Δ24-RGD to achieve en-
hanced tumor cell infectivity through integrin binding
and relative increased infection specificity. Preclinical stud-
ies on Ad5-Δ24-RGD have shown enhanced infectivity,
oncolytic capacity, tumor specificity, and therapeutic effi-
cacy in ovarian cancer cell lines, primary ovarian cancer
cells, and in a well established murine model for ovarian
cancer (12). In vivo biodistribution and toxicity studies
noted appropriate viral clearance and no significant per-
manent pathologic or laboratory abnormalities associated
with i.p. administration to cotton rats, which are permis-
sive to adenovirus serotype 5 replication (13).
These preclinical efficacy and safety studies provided
justification for a phase I clinical trial designed to deter-
mine the maximum tolerated dose (MTD) and spectrum
of toxicities encountered with i.p. delivery of the tropism
modified CRAd, Ad5-Δ24-RGD, in patients with recurrent
ovarian and other select gynecologic cancers. Secondary
objectives included determination of potential clinical ac-
tivity, biological effects of, and the immunologic response
to i.p. administration of Ad5-Δ24-RGD. Importantly, this
infectivity enhanced adenovirus represents the first ever
tropism-modified CRAd applied in the context of human
cancer clinical trials.
Materials and Methods
Patient eligibility
This study was conducted by a 3 + 3 dose-escalation
strategy at a single institution following Institutional
Review Board, Institutional Biosafety Committee, Recom-
binant DNA Advisory Committee, and Food and Drug
Administration approval. Participants were enrolled from
July 2007 to April 2009. Eligible patients originally includ-
ed histologically documented persistent or recurrent epi-
thelial ovarian or primary peritoneal adenocarcinoma
and eventually were expanded to include fallopian tube
and endometrial carcinoma. All patients were required to
have previous treatment with conventional surgery and
chemotherapy and have evidence of intra-abdominal dis-
ease. Patients were required to have adequate organ labo-
ratory function defined as white blood cells, >3,000 μL;
granulocyte count, >1,500 μL; platelets, >100,000; creati-
nine clearance, >80 mg/dL; creatinine, <2.0; aspartate ami-
notransferase or alanine aminotransferase, <2.5×, the
upper limit of the reference range; bilirubin, <2.0; and pro-
thrombin time/partial thrombopastin time/international
normalized ratio (PT/PTT/INR), <1.5×, the upper limit of
the reference range. Patients were required to have an ejec-
tion fraction >55% on echocardiogram and an O2satura-
tion>92%.Patientswererequiredtobe≥19yearsofage,have
a gynecologic oncology group performance status of 0 to 2,
have a life expectancy of >3 months, and sign an informed
consent document. Patients with low malignant potential
epithelial, stromal, or germ cell ovarian tumors were
excluded. Patients with active heart disease, pulmonary
disease, or coagulation disorders were excluded.
Ad5-Δ24-RGD manufacturing
The Ad5-Δ24 mutant adenovirus containing the 24-
nucleotide deletion from Ad5 bp 923 to 946 was original-
ly provided by Dr. Juan Fueyo (MD Anderson Cancer
Center, Houston, TX). An E1 fragment containing the
24-bp deletion from this plasmid was cloned through ho-
mologous recombination into a ClaI-digested plasmid
pVK503 containing the RGD fiber as previously described
(14). Following PacI digestion, the resulting genome was
released from the plasmid backbone, transfected into
A549 cells, and rescued. RGD presence and Δ24 absence
were verified through PCR.
Ad5-Δ24-RGD was manufactured with the support of
the National Cancer Institute (NCI) Rapid Access to
Intervention Development program at the Cell and
Gene Therapy Center at Baylor College of Medicine and
Translational Relevance
Novel treatment strategies are needed for patients
with advanced or recurrent gynecologic cancers. One
suchnoveltherapeuticapproach, oncolytic virotherapy,
isapromisingtherapyforthesemalignantdiseases.This
studyisthefirsttoevaluateaninfectivity-enhancedcon-
ditionally replicative adenovirus in the context of hu-
man cancer and shows the feasibility, safety, potential
antitumor response, and biological activity of this vir-
otherapy approach in malignant gynecologic diseases.
Kimball et al.
Clin Cancer Res; 16(21) November 1, 2010
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at the Biopharmaceutical Development Program/SAIC
at NCI-Frederick. All viral doses were administered in
250 mL of 0.9% sodium chloride and kept refrigerated
until administration.
General treatment plan and Ad5-Δ24-RGD dose cohorts
Pretreatment evaluation consisted of: history and phys-
ical, toxicity grading, performance status assignment, com-
pletebloodcount,chemistrypanel,liverprofile,coagulation
profile, CA-125, determination of ejection fraction by echo-
cardiogram, O2saturation, and computed tomography of
the abdomen and pelvis. Patients completing pretreatment
evaluation and meeting all eligibility criteria were enrolled
and had an i.p. Quinton Curl, 22.4-inch, double-cuffed,
Tenchkhoff-type catheters (Tyco Healthcare) placedbyinter-
ventional radiology at least 1 week before use.
Patients were then enrolled in successive escalating dose
cohorts such that cohort 1 received 1 × 109vp/d (viral
particles per day) and each successive cohort dose
increased by 1/2 log vp/d. The seventh and final cohort
received 1 × 1012vp/d. Assigned doses were instilled
through the i.p. catheter daily for three consecutive days
in an inpatient setting. On the 4th day, the patient was
discharged. Dose escalation occurred 4 weeks after the final
patient in the previous cohort was treated. No individual
patient dose escalation was done.
On days 0 to 3, 7, 14, and 28, patients were evaluated
through history and physical examination, performance
status assignment, toxicity grading, complete blood count,
and chemistry profile. Peritoneal aspirates for biological
ancillary studies and urine, saliva, and serum specimens
for viral shedding studies were obtained immediately pre-
ceding Ad5-Δ24-RGD administration on day 0, 3, 7, 14,
and 28. Serum CA-125 and a computed tomography of
the abdomen and pelvis were repeated on day 28. All
samples were processed to assure anonymity; individuals
doingthebiologicalstudieswereblindedtopatientidentity.
Toxicity evaluation
Toxicity grading was done using NCI Common Toxicity
Criteria v3.0. MTD was defined as the dose exceeded by the
dose at which at least two patients experience dose-limiting
toxicity (DLT). DLT was defined as any vector related grade
3nonhematologictoxicity,notincludingnausea,vomiting,
or fatigue. Dose-limiting hematologic toxicities were
definedasanyadmissionforneutropenicfever,absoluteneu-
trophil count of <500 for >5 days, or platelet count of
<20,000. Any patient experiencing vector related grade 3/4
toxicitybeforecompletionofscheduledtreatmenthadsubse-
quentdaysoftreatmenthelduntilresolutionoftoxicity.Ifno
resolutionoccurredwithin 72hours orifa second doseinter-
ruption occurred, patients received no further study drug.
Evaluation of clinical efficacy
RECIST criteria version 1.0 was used to define patients'
best radiographic response to treatment. Measurable dis-
ease was defined as at least one lesion that could be accu-
rately measured in one dimension and >1 cm. Up to five
lesions per organ or 10 lesions total were identified as tar-
get lesions. Complete response required disappearance
of all target lesions and normalization of CA-125. Partial
response was defined as >30% decrease in the sum of tar-
get lesions' recorded dimensions. Progressive disease was
defined as >20% increase in sum of target lesions recorded
dimensions. Stable disease was a condition that did not
qualify for partial response or progressive disease.
Evaluation of viral infection, CRAd replication,
generation of wild-type (WT) virus in
peritoneal fluid cells
Genomic DNA of tumor cells in peritoneal fluid was
isolated using a QIAamp DNA Mini Kit (QIAGEN) accord-
ing to the manufacturer's protocols. Real-time quantitative
PCR was used to evaluate gene transfer and CRAd replica-
tion. RGD copies in genomic DNA were determined by
amplification of the RGD gene with forward primer CACA‐
CTAAACGGTACACAGGAAACA, reverse primer ATGCA-
GATGGGCAGAAACAGT, and probe 6-FAM-AGACA-
CAACTTGTGACTGCCGCGG-BHQ-1. Resultant RGD
copies were normalized to a human cellular housekeeping
gene (human β-actin), which was amplified with forward
primer CCAGCAGATGTGGATCAGCA, reverse primer CTA-
GAAGCATTT-GCGGTGGAC, and probe 6-HEX-AGGAG-
TATGACGAG-TCCGGCCCCTC-BHQ-1.
TodeterminewhetherpotentialcontaminatingWTadeno-
virus were replicating, the WT E1 gene was amplified from
cells in the ascites fluid with forward primer TGCCAAACC‐
TTGTACCGGA, reverse primer CGTCGTCACTGGGGTG-
GAAA, and probe 6-FAM-ATCGATCTTACCTGCCAC-
GAGGCTGG-BHQ. Resultant WT E1 copies were normalized
to housekeeping genes to allow for comparison between
patients and at different time points and varying amount
of cellular material.
All primers and probes were designed by the Primer Ex-
press 1.5 software and synthesized by Sigma-Aldrich. Fas-
tStart TaqMan Probe Master (Roche Applied Science) was
used for duplexing the PCR on a LightCycler 480 (Roche
Applied Science). Thermal cycling conditions began with
8 minutes at 95°C followed by 45 cycles of 10 seconds at
95°C and 40 seconds at 60°C. Data were analyzed with
LightCycler 480 1.5.0 SP1 software. WT and RGD specific
primers were confirmed to only amplify the virus being
tested (data not shown).
Immunohistochemistry
After being transported to the laboratory, DMSO (Sigma
Aldrich) was added to ascites specimens containing tumor
cells to reach 5% and immediately frozen at -80°C until
assays and analysis commenced. Upon thawing, cells were
suspended in cell culture media (Sigma Aldrich). Cell sus-
pensions of ascites were used to prepare at least two cytos-
pinslidespersampleusingtheThermoShandon3Cytospin
at 2,000 rpm for 10 minutes at room temperature. After the
cytospin, slides were removed and fixed in 70% ethanol
overnight (Sigma Aldrich). Slides were incubated with rab-
bitpolyclonalanti-hexonantibody(Abcam)atadilutionof
Modified CRAd for Malignant Gynecologic Diseases
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1:2,000 and CC-49 anti-Tag72 antibody (provided by M.B.
Khazaeli, PhD, University of Alabama at Birmingham) at a
dilution of 5 μg/μL for 1 hour. CC-49 anti-Tag72 antibody
isanantibody toatumor associated glycoprotein known to
be expressed by ovarian cancer cells (15). Anti-hexon is an
anti-adenovirus antibody. Negative controls were done by
omitting primary antibodies. Ascites cells from patients un-
treated with adenovirus were used to confirm that, in the
absence ofadenovirus, staining withanti-hexon isnegative.
Ascites cells from patients not in this trial were treated
ex vivo with adenovirus and stained with anti-hexon anti-
body to confirm that anti-hexon will detect present adeno-
virus (supplementary data; Fig. 1). Secondary antibodies
Alexafluor 488 (Invitrogen) and Alexafluor 594 (Invitrogen)
were incubated at 1:100 for 1 hour in PBS. Slides were then
mounted with 0.25% Propyl Gallate in a 9:1 (v/v) glycerol:
PBS solution to prevent photobleaching (Sigma Aldrich).
Fluorescence microscopy was done with an inverted IX-70
microscope (Olympus) equipped with a Magnifire digital
CCD camera (Optronics) or a DP71 digital camera (Olym-
pus). All images were at 40× magnification. The images of
fluorescent signals for tumor cells and adenovirus were
merged using Adobe Photoshop CS.
Evaluation of viral shedding
Viral DNA from urine specimens was isolated with a
QIAamp Viral RNA Mini Kit (QIAGEN) after being concen-
trated with Millipore Amicon Ultra-4 and Amicon Ultra-15
Centrifugal Filter Units. Viral DNA from saliva specimens
was isolated using a QIAampMinElute Virus Spin Kit
(QIAGEN), following the manufacturer's instructions.
Viral DNA from sera specimens was isolated with the
DNeasy Tissue Kit's blood protocol (QIAGEN). One micro-
liter from resultant samples were used as a template for
Fig. 1. Quantification of Ad5-Δ24-RGD in ascites/peritoneal lavage samples (A) and immunohistochemical evidence of colocalization of Ad5-Δ24-RGD
andovariancancercells(B).A,usingreal-timequantitativePCR,Ad5-Δ24-RGDcopynumberswerequantifiedineachpatient'slavagesamples(intriplicate)on
days 0, 3, 7, 14, and 28. P, patient. B, representative immunohistochemical stains of ascites from selected patients are depicted. Original magnification,
×40. Top, localization of Tag72, an antibody to a tumor associated glycoprotein known to be expressed by ovarian cancer cells. Middle, localization of
anti-hexon antibody, an anti-adenovirus antibody. Bottom, a digital overlay of the two previous images. On day 0, green fluorescence shows the presence of
ovarian cancer cells, whereas the anti-adenovirus staining shows only background red fluorescence. Some red background fluorescence is present in
adenovirus negative cells. Representative images shown after treatment contain cells that stain for the tumor marker and adenovirus.
Kimball et al.
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Clinical Cancer Research
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real-time quantitative PCR because many of these samples
lacked genomic DNA for normalization of results. We
amplified a fragment of RGD with forward primer CACAC-
TAA‐ACGGTACACAGGAAACA, reverse primer ATGCA-
GATGGGCAGAAACAGT, and probe 6-FAM-
AGACACAACTTGTGACTGCCGCGG-BHQ-1 (Sigma
Aldrich). Real-time quantitative PCR conditions were
similar to those previously described to evaluate viral
infection, CRAd replication, and generation of WT virus.
Evaluation of an anti-adenovirus neutralizing
antibody response
For evaluation of induced anti-adenovirus neutralizing
antibodies response in serum and ascites specimens after
treatment, a nonreplicative luciferase expressing virus, Ad5-
RGD-Luc1, was neutralized by either serum or ascites before
infection of SKOV3.ip1 cells. SKOV3.ip1 cells are a cell line
derived from the implantation of SKOV3 cells (American
Type Culture Collection) in nude mice. These cells were last
tested and authenticated for epithelial staining through
pooledAE1/AE3 antibodyinDecember2009. Theseantibo-
dies stain normal and neoplastic cells of epithelial origin.
Following neutralization in respective samples, Ad5-
RGD-Luc1 transduction efficacy was determined by a lucif-
erase assay. Triplicates of SKOV3.ip1 cells were plated into
96-well plates (10,000 per well) and allowed to grow
overnight before infection. A 1:2 dilution of serum or asci-
tes of each day point specimen was prepared in Opti-MEM
(Media Preparation Shared Facility, University of Alabama
at Birmingham) in a normalized volume. Nonreplicative
Ad5-RGD-Luc1 at 100 plaque-forming unit per cell was
mixed with each dilution for 30 minutes at room tempera-
ture before adding to appropriate wells. This infection
was allowed to proceed for 48 hours. A luciferase assay
was carried out using a luciferase assay system (Promega)
on an Orion microplate luminometer (Berthold) reading
Culturplate-96 (Research Parkway) according to manufac-
turer's protocols.
Statistical analysis
Demographic and baseline characteristics of the treated
patients are summarized descriptively. The incidence of
adverse events and laboratory tests are also briefly summa-
rized respectively. For the analysis of biological effects, a
repeated measures ANOVA was used to compare baseline
values to the other study day values. The raw data were
transformed into logarithms tothe base 10tomeet thenor-
mality assumption before statistical testing.
Results
Patient demographics
From 2007 to 2009, 26 patients were consented to
participate. Five of these patients were not treated with
Table 1. Selected demographics of treated patients
Pt IDAge RaceGOG scoreCancerOriginal stageHistologyPrevious
regimens
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
65
77
54
52
54
70
47
71
67
72
83
75
64
57
65
77
68
61
66
53
72
C
C
C
C
C
C
C
C
C
C
C
A
C
C
C
A
C
C
C
C
C
0
0
0
0
0
1
0
1
2
0
1
2
1
0
1
1
0
0
0
0
1
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Endometrial
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Ovarian
Endometrial
Peritoneal
3c
3c
3b
3c
3c
3c
3c
3c
3c
3c
3c
3a
3b
2c
3c
3c
3b
4
3c
4
3c
Serous
Serous
Endometrioid
Serous
Serous
Serous
Serous
Serous
Serous
Serous
Serous
Endometrioid
clear cell
Endometrioid
Serous
Serous
Serous
Serous
Serous
Endometrioid
Serous
2
3
3
7
5
4
2
5
7
1
3
2
3
2
3
6
4
2
4
2
2
NOTE: GOG score is a performance score system.
Abbreviations: Pt, patient; C, Caucasian, A, African American.
Modified CRAd for Malignant Gynecologic Diseases
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Ad5-Δ24-RGD: three patients were ineligible due to leuco-
penia, thrombocytopenia, or abnormal liver function tests
on screening, respectively. Two additional patients had
i.p. catheter placement complications that did not allow
for i.p. administration of the study CRAd. A total of 21
patients were successfully treated in seven dose levels per
study guidelines.
Study demographics for treated patients are provided
in Table 1. In summary, the median and mean age of
the treated patients were 66 and 65.2 years, respectively
(range, 47-83 y). Ninety percent were Caucasian, and
86% had recurrent ovarian cancer. The median and mean
number of previous chemotherapy treatments was 3 and
3.4, respectively (range, 1-7).
Toxicity associated with i.p. delivery of Ad5-Δ24-RGD
One consented patient experienced grade 3 abdominal
pain and grade 3 infection related to her i.p. catheter but
was not treated with Ad5-Δ24-RGD and not included in
final analysis of reagent specific toxicities. Table 2 provides
a summary of clinical and laboratory adverse events by se-
verity as reported on scheduled study visits for patients
treated with Ad5-Δ24-RGD. No Ad5-Δ24-RGD–related
grade 3 or 4 clinical or laboratory toxicities were observed.
The most common clinical toxicities listed as “possible,”
“probable,” or “definitely” attributable to the Ad5-Δ24-RGD
werelimitedtograde1or2constitutionalsymptoms(feveror
fatigue) and gastrointestinal/pain symptoms (abdominal
pain). The most common laboratory abnormalities included
anemia and abnormalities of glucose not thought to be asso-
ciated with viral administration. Four treated patients experi-
enced a total of 10 grade 3 toxicities. One patient had grade 3
shortness of breath and grade 3 chest pain due to a disease
related pleural effusion. Two patients experienced grade 3
nausea and vomiting, dehydration, and bowel obstruction
symptoms related to their underlying disease. One patient ex-
perienced grade 3 hypokalemia related to nausea and vomit-
ing. Listed grade 3 toxicities were “not” or “unlikely” to be
attributedtothestudyvector.Therewereno grade4or5toxi-
citiesencountered.Novector-associatedDLTswerenotedand
the MTD of Ad5-Δ24-RGD was not identified.
Clinical efficacy associated with i.p. delivery of
Ad5-Δ24-RGD
Of the 21 treated patients, 19 had measureable disease
and were evaluable for best response using RECIST criteria
one month following Ad5-Δ24-RGD treatment. Fourteen
patients had stable disease, and five patients had progressive
disease. There were no partial or complete responses noted.
Two additional patients had evaluable but nonmeasurable
disease; one had stable disease and one had progressive dis-
ease 1 month following Ad5-Δ24-RGD treatment (Table 3).
Seven of 20 evaluable patients had decrease in CA-125
from pretreatment values to day 29 values; one patient did
nothaveaposttreatmentCA-125leveldrawn(Table3).Four
of these patients had a >20% drop in CA-125 levels. Five of
thesesevenpatientshadstablediseasebyRECISTcriteria;the
other two patients were noted to have progressive disease.
Viral infection and replication
At time points specified in the methods, before and after
Ad5-Δ24-RGD administration, peritoneal lavage samples
were obtained from each patient and evaluated for the
Table 2. Clinical (A) and laboratory (B) toxicity
in patients treated with Ad5-Δ24-RGD by
category and grade
A
Toxicity
grades (NCI
CTC V.3)
12
Total
occurrences
34
Body system
Gastrointestinal
Constitutional
Pain
Neurology
Infection
Dermatology/skin
Cardiac general
Lymphatics
Musculoskeletal
Allergy/immunology
Syndromes
Respiratory
Renal/genitourinary
Blood/bone marrow
Cardiac arrhythmia
61 34
18
10
4
1
0
2
3
3
3
0
0
0
1
1
6
0
1
0
0
1
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
101
24
19
6
8
4
6
4
2
1
1
0
2
0
1
0
0
8
7
5
4
4
4
3
2
1
1
1
1
B
Laboratory tests
Hemoglobin
Hematocrit
Glucose
Potassium
LDH
Creatinine
Gamma-glutamyl
transpeptidase
WBC
Sodium
Alkaline phosphatase
Calcium
Phosphorous
Bicarbonate
Chloride
Blood urea nitrogen
Total bilirubin
Uric acid
Platelet
42
34 12
24
11
8
6
4
70
0
0
1
0
0
0
0
0
0
0
0
0
0
49
46
29
14
10
5
1
2
0
1
6
5
5
2
5
2
2
4
0
2
1
0
0
0
3
0
2
2
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
5
5
4
4
4
2
2
1
0
0
Abbreviations: LDH, lactate dehydrogenase; WBC, white
blood cells.
Kimball et al.
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Page 7

presence of Ad5-Δ24-RGD through real-time quantitative
PCR. All patient specimens were positive for cellular DNA.
Any value >1 RGD copy number per nanogram of cellular
DNA was considered positive. By day 3 (after treatment),
16ofthe21patientshadRGD-specificDNAdetected(Fig.1A)
after Ad5-Δ24-RGD treatment was completed. In seven pa-
tients (patients 5, 6, 7, 8, 9, 11, and 20), the detected copy
number of RGD specific virus increased at time points after
Ad5-Δ24RGDtreatment.Although rigorousstatisticaleval-
uation was not feasible given the limited sample numbers,
these data suggest that replication of Ad5-Δ24-RGD may
have occurred in these patients. Qualitative evidence of vi-
ral localization within cancer cells is depicted in represen-
tative ascites samples using immunohistochemistry.
Images of select patient samples depict the presence of
Ad5-Δ24-RGD even at day 28 (Fig. 1B).
Generation of WT virus
Ingeneral,generationofreplicationcompetentWTadeno-
virus (RCA) was minimal. WT E1 was detected in ascites
specimens of eight patients, four of which had detectable
WT E1 before Ad5-Δ24-RGD administration. Only one
patient had >25 copies of WT E1 DNA per nanogram of
DNA following administration of Ad5-Δ24-RGD. Specifi-
cally,patient20,inthehighestdosecohort,hadanelevated
level of WT E1 DNA (62 copies per nanogram DNA) de-
tected before administration of Ad5-Δ24-RGD and had
549copiespernanogramDNAdetectedonday28.Interest-
ingly, this patient also reported upper respiratory symp-
toms before vector administration.
Viral shedding
At specified time points before and after Ad5-Δ24-RGD
administration, serum, saliva, and urine were obtained from
each patient to assess for viral shedding (Fig. 2). These data
were normalized to day 0 RGD copies, and any value <1 copy
RGD per microliter was considered negative. RGD-specific
virus was detected in serum samples from 10 patients as
showninFig.2A.Figure2BshowsthepresenceofRGDcopies
in saliva samples from 10 patients. Viral shedding was
detectedinurinesamplesfromninepatients(Fig.2C).Ingen-
eral,viralsheddingwasnotedmorefrequentlyand,toagreater
extent, in patients in higher dose cohorts and in the saliva.
Anti-adenovirus neutralizing antibody
At specified time points before and after Ad5-Δ24-RGD
administration, an anti-adenoviral neutralizing antibody
response was determined in serum and ascites (Fig. 3).
Overall, neutralizing antibody effects in serum and ascites
were consistent and dose dependent. Following exposure
to day 14 sera and ascites samples, infection of Ad5-RGD-
luc was, in general, significantly limited, and by day 28,
evidence of transduction was minimal. Neutralizing anti-
body effects were present in all patients, and when all data
points from all patients for days 0 and 3 were compared
with all data points for all patients for days 14 and 28, the
effects of neutralizing antibody were significantly higher
by day 14 and beyond (P < 0.0001).
Discussion
This report serves as one of the first published reports
evaluating an infectivity enhanced virotherapy approach
for the treatment of patients with cancer. Specifically, this
study evaluated i.p. administration of the infectivity en-
hancedCRAdAd5-Δ24-RGDinapreviouslyheavilytreated
cohort of patients with recurrent ovarian or endometrial
cancer. A single 3-day cycle of Ad5-Δ24-RGD in dosages
ranging from 1 × 109to 1 × 1012vp/d was administered
to treated patients. The most commonly noted Ad5-Δ24-
RGD–related toxicity consisted of primarily grade 1 and 2
constitutionalandgastrointestinalsymptoms.Mostgrade3
toxicities noted were in general disease related and not
associated with Ad5-Δ24-RGD treatment. No grade 4 toxi-
cities were reported. Manufacturing constraints limited the
ability to administer higher dosages of Ad5-Δ24-RGD.
Thus, the MTD was not identified and the maximum feasible
doseinthisstudywasidentifiedtobe1×1012vp/dfor3days.
Although not a primary aim of this study, there was
some suggestion of potential antitumor activity associated
with Ad5-Δ24-RGD treatment. Specifically, 14 of the 21
Table 3. Best response by RECIST criteria and
CA-125 values
Pt IDDose
cohort
(vp/d)
Best
response
CA-125
(units/mL)
pre/post
CA-125
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
1 × 109
1 × 109
1 × 109
5 × 109
5 × 109
5 × 109
1 × 1010
1 × 1010
1 × 1010
5 × 1010
5 × 1010
5 × 1010
1 × 1011
1 × 1011
1 × 1011
5 × 1011
5 × 1011
5 × 1011
1 × 1012
1 × 1012
1 × 1012
SD
SD
SD
PD
SD
SD
PD
SD
PD
SD
PD†
SD
PD
PD
SD
SD
PD
SD
SD
SD
SD†
1390/1060
164/278
52/56
505/1148
1540/2261
914/424
618/854
112/231
429/286
11/24
1103/1662
1092/967
1339/1128
47/79
187/131
166/247
3044/5774
46/90
64/153
158/129
992/NA
Decrease*
Increase
Increase
Increase
Increase
Decrease*
Increase
Increase
Decrease*
Increase
Increase
Decrease
Decrease
Increase
Decrease*
Increase
Increase
Increase
Increase
Decrease
N/A
Abbreviations: SD, stable disease; PD, progressive disease.
*>20% decrease in CA-125.
†Nonmeasurable disease by RECIST criteria.
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patients were noted to have stable disease by RECIST
criteria over the month of observation following adminis-
tration of Ad5-Δ24-RGD. Of note, four patients experi-
enced a >20% decline in CA-125 levels, a common
marker of overall disease burden for advanced ovarian
and endometrial cancers. There did not seem to be a dose
related response.
Our ancillary studies provided potential evidence of
Ad5-Δ24-RGD replication and other important insights
about the biological effects of i.p. administration of Ad5-
Δ24-RGD. Specifically, Ad5-Δ24-RGD specific DNA was
detected at various time points after treatment in 16 of
the 21 patients. In seven of these patients, higher quanti-
ties of Ad5-Δ24-RGD specific DNA was noted after day 3
of Ad5-Δ24-RGD treatment and could potentially be at-
tributed to replication of the virus within the abdominal
cavity. Ad5-Δ24-RGD specific viral shedding was noted in
serum, urine, and saliva. Shedding was highest in saliva
and seemed to be dose dependant. This noted level of
shedding and tropism for the upper respiratory tract is
not inconsistent with what has been documented in other
adenoviral-based, gene therapy-based trials (15–17). In
addition, the generation of WT adenovirus (>25 copies
per nanogram DNA) was minimal and was not detected in
Ad5-Δ24-RGD–treated patients at a dose of <5 × 1010vp/d.
OnepatienthadelevatedlevelsofWTadenovirus(>50copies
before administration and on day 28); this patient was noted
tohaveclinicalevidenceofanupperrespiratorytractinfection
at the time. Lastly, anti-adenovirus neutralizing antibodies
were uniformly present in serum and ascites samples from
most patients and noted to be significant 14 days after
Ad5-Δ24-RGDtreatment.Thisneutralizinganti-adenoviral
antibody response is similar to what has been noted in
other adenoviral based trials (18, 19). However, evidence
of potential Ad5-Δ24-RGD replication noted in this study
and in previous preclinical studies may point toward
diminished interaction of neutralizing antibodies with
RGD-modified adenoviruses. These findings may also
show the potential ability of this infectivity-enhanced
modified CRAd to abrogate immunologic interactions that
Fig. 2. Quantification of Ad5-Δ24-RGD in serum (A), saliva (B), and urine (C). Using RT-PCR, Ad5-Δ24-RGD copy numbers were quantified in each
patient's serum (A), saliva (B), and urine (C) samples on days 0, 3, 7, 14, and 28.
Kimball et al.
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Page 9

would mitigate potential antitumor activity (20). Additional
studies evaluating the effect of Ad5-Δ24-RGD on other
immunologic variables are planned and a clinical trial eval-
uating intracranial administration of Ad5-Δ24-RGD in pa-
tients with malignant glioblastoma is currently in progress.
Two other virotherapy approaches have been evaluated
in the context of recurrent ovarian cancer. ONYX-015, the
first CRAd to be evaluated in this disease context, has a E1B
gene deletion that allows for conditional replication in p53
deficient tumor cells but does not have any modification
that allow for improved ovarian cancer cell transfection
(7). In this phase I study, patients were treated through IP
ONYX-015 in dose cohorts up to 1 × 1011plaque-forming
unit daily for 5 days every 4 weeks. A total of 35 cycles was
administered to 16 treated patients with a median of two
cycles per patient. Only one DLT (grade 3 abdominal pain
and diarrhea) was noted, and the MTD was not identified.
Four of the 16 patients exhibited stable disease as a best re-
sponse and one patient exhibited a significant drop in her
serum CA-125. Five of eight evaluable patients had PCR
detectable ONYX-015 DNA in peritoneal lavage specimens
10daysfollowingthelastadministration.Interestingly,one
patient had specific DNA detectable 354 days following her
last treatment. Despite this unique finding, the capacity to
document any evidence of replication was limited because
of the inability to quantitatively show increased ONYX-
015–specific DNA remote from administration. All eight
tested patients had undetectable levels of ONYX-015
Fig. 3. Assessment of anti-adenoviral
neutralizing antibody response in serum (A)
and ascites (B). Assessment of induced
anti-adenovirusneutralizingantibodyresponse
after treatment was carried by exposing a
nonreplicative luciferase expressing virus,
Ad5-RGD-Luc1, to either patient serum (A)
or ascites (B) before infection of SKOV3.ip1
cells. Following neutralization in respective
samples, Ad5-RGD-Luc1 transduction
efficacy was determined by a luciferase
assay.Eachtreatmentcohort'smeanresponse
is documented here in addition to a negative
controlrepresentedby“Nosera”or“Noascites.”
RLU, relative light units.
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DNAintheirserum.Asnotedinthecurrentstudy,nearlyall
patients had very high levels of neutralizing antibodies de-
velop over the course of the evaluation period (7).
In a similar approach, Galanis et al. (21) evaluated a rep-
licative-competent Edmonston B measles vaccine strain,
MV-CEA, in a phase I trial for patients with recurrent ovar-
iancancer.MV-CEAcapitalizesontheoverexpressionofthe
measlesvirusreceptorCD46intumorcells andismodified
to express the soluble marker peptide carcinoembryonic
antigen (CEA). Eligible ovarian cancer patients were treated
i.p. with MV-CEA monthly for up to six doses in dosages
ranging from 103to 109TCID50 (median tissue culture
infective dose). A total of 126 cycles were administered to
atotalof21patientswithamediannumberofsixcyclesper
patient.Minimaltoxicitieswerenoted,andtheMTDwasnot
identified. Fourteen of the 21 patients experienced durable
stable disease as a best response. An apparent dose depen-
dant response was noted with higher dose cohorts having
higher rates of stable disease. CA-125 levels were noted to
have decreased >30% in 5 of 21 patients. CEA elevation
was detected in the peritoneal fluid of one patient and in
the serum of three patients in the highest dose cohort. Evi-
dence of MV-CEA viral DNA in the serum was detected in
four patients by quantitative RT-PCR. No MV-CEA specific
shedding was noted in urine or saliva. No development of
anti-measles or anti-CEA antibodies was detected (21).
The results of these previous studies show the feasibility
to deliver relatively high dosages of conditionally replica-
tive viruses i.p. in patients with recurrent ovarian cancer
with good tolerance. The current study shows for the first
time the ability to deliver an infectivity enhanced CRAd
(Ad5-Δ24-RGD)athighdosagesinasimilarcohort.Clinical
trials evaluating Ad5-Δ24-RGD in the context of multiple
cycles of Ad5-Δ24-RGD at the maximum feasible dose
(1 × 1012vp/d × 3 d) and evaluating Ad5-Δ24-RGD in
combination with chemotherapy in patients with recurrent
ovarian cancer are in development. Preclinical studies (22)
and ongoing clinical trials support the feasibility of a
combination virotherapy and chemotherapy approach (23).
The importance of the rationale for the use of infectivity
enhancement and the consequent improvement in overall
antitumor potential cannot be understated because we
strive to forward the utility of adenoviral virotherapy.
The culmination of rigorous preclinical work (9–12) in a
RAC-guided preclinical safety trial (13) led to a fully vetted
tropism-modified and conditionally replicative virus for
use in this unique clinical trial. Hopefully, this previous
research, in concert with the findings of the current trial,
serves to establish a precedent for the safety of tropism-
modified adenoviral vectors and will open the door for
clinical use of future enhancements in CRAd therapy.
Other strategies designed to improve the potential ther-
apeutic index of adenoviral based virotherapy have been
evaluated in vitro and in vivo. These include, for example,
the arming of CRAds (24), incorporation of tissue specific
promoters(25),novelmeanstoassessvirustrafficking(26),
thedevelopmentofnewserotypechimericmodificationsto
further enhance infectivity (27, 28), and the assessment of
stem cell delivery mechanisms for CRAds (29). Clinical
translation of several of these approaches into early phase
clinical trials is ongoing or in development.
In summary, this study serves as the first clinical trial
to evaluate an infectivity-enhanced CRAd in patients with
recurrent ovarian and other selected gynecologic cancers
and provides guidance for the future development of
Ad5-Δ24-RGD and other infectivity-enhanced virothera-
peutics intended for the treatment of cancer.
Disclosure of Potential Conflicts of Interest
D.T. Curiel has ownership interests and consultative roles in
VectorLogics, Inc.
Acknowledgments
We thank Jolane Gable, RN; Michael Ann Markiewicz, PharmD; and the
staff of the Participant and Clinical Interactions Resources unit of the
Center for Clinical and Translational Science at the University of
Alabama at Birmingham for their care of patients with malignant
gynecologic diseases and support of this research.
Grant Support
Federal funds from the NCI, NIH, under contract no. N01-CO-12400
and the Developmental Therapeutics Program in the Division of Cancer
Treatment and Diagnosis of the NCI (R.D. Harris); NIH grants
5R01CA121187, NCI R21 CA128222, and NCI P50-CA83591 (D.T. Curiel);
and NIH grants NCI R21 CA128222 and NCI P50-CA83591 (R.D. Alvarez).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Received 04/01/2010; revised 06/11/2010; accepted 06/25/2010;
published OnlineFirst 10/26/2010.
References
1.
Brinton LA, Lacey JV, Sherman ME. Epidemiology of gynecologic
cancers. In: Hoskins WJ, Perez CJ, Young RC, et al, editors. Princi-
ples and practice of gynecologic oncology. 4th ed Philadelphia (PA):
Lippincott-Raven; 2005, p. 3–32.
LiuTC,GalanisE,KirnD.Clinicaltrialresultswithoncolyticvirotherapy:a
centuryofpromise,adecadeofprogress.NatClinPract2007;4:101–17.
Kimball KJ, Numnum TM, Rocconi RP, Alvarez RD. Gene therapy for
ovarian cancer. Curr Oncol Rep 2006;6:441–7.
Alemany R, Balague C, Curiel D. Replicative adenovirus for cancer
therapy. Nat Biotechnol 2000;18:723–7.
Agarwal R, Linch M, Kaye SB. Novel therapeutic agents in ovarian
cancer. Eur J Surg Oncol 2006;32:875–86.
2.
3.
4.
5.
6.
Niederacher D, Yan HY, An HX, Bender HG, Beckman MW. CDKN2
gene inactivation in epithelial sporadic ovarian cancer. Br J Cancer
1999;80:1920–6.
Vasey PA, Schulman LN, Campos S, et al. Phase I trial of intraperi-
toneal injection of the E1-55kd-gene deleted adenovirus ONYX-015
(dl1520) given on days 1 through 5 every 3 weeks in patients with
recurrent/refractory epithelial ovarian cancer. J Clin Oncol 2002;20:
1562–9.
Neumunaitis J, Cunningham C, Buchanan A, et al. Intravenous infusion
of a replication-selective adenovirus (ONYX-015) in cancer patients:
safety, feasibility, and biological activity. Gene Ther 2001;8:746–59.
Douglas JT, Kim M, Sumerel LA, Carey DE, Curiel DT. Efficient
7.
8.
9.
Kimball et al.
Clin Cancer Res; 16(21) November 1, 2010
Clinical Cancer Research
5286

Page 11

oncolysis by a replicating adenovirus (ad) in vivo is critically depen-
dant on tumor expression of primary Ad receptors. Ca Res 2001;61:
813–7.
10. Dmitriev I, Krasnykh V, Miller CR, et al. An adenovirus vector with
genetically modified fibers demonstrates expanded tropism via
utilization of a coxsackievirus and adenovirus receptor-independent
cell entry mechanism. Virol 1999;72:9706–13.
11. Vanderkwaak T, Wang M, Navarro J, et al. An advanced generation
of adenoviral vectors selectively enhances gene transfer for ovarian
cancer gene therapy approaches. Gynecol Oncol 1999;74:227–34.
12. Bauerschmitz G, Lam J, Kanerva A, et al. Treatment of ovarian cancer
withatropismmodifiedoncolyticadenovirus.CARes2002;62:1266–9.
13. Page JG, Tian B, Schweikart K, et al. Identifying the safety profile
of a novel infectivity-enhanced conditionally replicative adenovirus,
Ad5-24-RGD, in anticipation of a phase I trial for recurrent ovarian
cancer. Am J Obstet Gynecol 2007;196:389e1–10.
14. Suzuki K, Alemany R, Yamamoto M, Curiel DT. The presence of the
adenovirus E3 region improves the oncolytic potency of conditionally
replicative adenoviruses. Clin Cancer Res 2002;8:3348–59.
15. Ponnusamy MP, Venkatraman G, Singh AP, et al. Expression of
TAG-72 in ovarian cancer and its correlation with tumor stage and
patient prognosis. Cancer Lett 2007;251:247–57.
16. Schenk-Braat EA, van Mierlo MM, Wagemaker G, Bangma CH,
Kaptein LC. An inventory of shedding data from clinical gene therapy
trials. J Gene Med 2007;9:910–21.
17. Kawahira H, Matsushita K, Shiratori T, et al. Viral shedding after p53
adenoviral gene therapy in 10 cases of esophageal cancer. Cancer
Sci 2010;101:289–91.
18. Alvarez RD, Barnes MN, Gomez-Navarro J, et al. A cancer gene
therapy approach utilizing an anti-erbB-2 single-chain antibody-
encoding adenovirus (AD21): a phase I trial. Clin Can Res 2000;6:
3081–7.
19. Barnett BG, Crews CJ, Douglas JT. Targeted adenoviral vectors.
Biochim Biophys Acta 2002;1575:1–14.
20. Blackwell J, Li H, Navarro J, et al. Circumventing inhibitory factors in
ascites fluid using a tropism modified adenoviral vector. Hum Gene
Ther 2000;11:1657–69.
21. Galanis E, Hartmann LC, Cliby WA, et al. Phase I trial of intraperito-
neal administration of an engineered strain of measles virus, modified
to express carcinoembryonic antigen for recurrent ovarian cancer.
Can Res 2010;70:875–82.
22. Hemminki A, Belousova N, Zinn K, et al. An adenovirus with
enhanced infectivity mediates molecular chemotherapy of ovarian
cancer cells and allows imaging of gene expression. Mol Ther
2001;4:223–31.
23. Opyrchal M, Aderca I, Galanis E. Phase I clinical trial of locoregional
administration of the oncolytic adenovirus ONYX-015 in combination
with mitomycin-C, doxorubicin, and cisplatin chemotherapy in pa-
tients with advanced sarcomas. Methods Mol Biol 2009;542:705–17.
24. Cody JJ, Douglas JT. Armed replicating adenoviruses for cancer
virotherapy. Cancer Gene Ther 2009;16:473–88.
25. Rocconi RP, Zhu ZB, Stoff-Khalili M, et al. Gynecol Oncol 2007;105:
113–21.
26. Kimball KJ, Rivera AA, Zinn KR, et al. Novel infectivity-enhanced on-
colyticadenoviruswithacapsid-incorporateddual-imagingmoietyfor
monitoringvirotherapyinovariancancer.MolImaging2009;8:264–77.
27. Bauerschmitz GJ, Guse K, Kanerva A, et al. Triple-targeted oncolytic
adenoviruses featuring the Cox2 promoter, E1A transcomplementa-
tion, and serotype chimerism for enhanced selectivity for ovarian
cancer cells. Mol Ther 2006;14:164–74.
28. Stoff-Khalili MA, Rivera AA, Glascow JN, et al. A human adenoviral
vector with a chimeric fiber from a canine adenovirus type 1 results in
novel expanded tropism for cancer therapy. Gene Ther 2005;12:
1696–706.
29. Yong RL, Shinojima N, Fueyo J, et al. Human bone marrow-derived
mesenchymal stem cells for intravascular delivery of oncolytic
adenovirus Delta24-RGD to human gliomas. Cancer Res 2009;69:
8932–40.
Modified CRAd for Malignant Gynecologic Diseases
Clin Cancer Res; 16(21) November 1, 2010 www.aacrjournals.org
5287