GYNECOLOGIC ONCOLOGY 64, 378–385 (1997)ARTICLE NO. GO964566

Transductional Efficacy and Safety of an Intraperitoneally DeliveredAdenovirus Encoding an Anti-erbB-2 Intracellular Single-Chain

Antibody for Ovarian Cancer Gene Therapy

JESSY DESHANE,* GENE P. SIEGAL,†,‡,§ MINGHUI WANG,* MARCI WRIGHT,* R. PAT BUCY,‡RONALD D. ALVAREZ,§ AND DAVID T. CURIEL*,†,§,1

*Gene Therapy Program, †Department of Pathology, ‡Departments of Cell Biology and Surgery, §Department of Gynecologic Oncology,University of Alabama at Birmingham, Birmingham, Alabama 35294

Received June 18, 1996

to achieve abrogation of this transforming oncogene, weWe have previously shown that adenoviral-mediated delivery of an have developed a novel strategy based upon the intracellular

anti-erbB-2 intracellular single-chain antibody (sFv) causes specific expression of a single chain antibody (sFv) targeted againstcytotoxicy in erbB-2-overexpressing ovarian carcinoma cells. Further- the erbB-2 oncoprotein. Our previous studies demonstratedmore, intraperitoneal delivery of the anti-erbB-2 sFv enhances sur- that an endoplasmic reticulum (ER)-directed version of thevival and reduces tumor burden in a xenograft model of human anti-erbB-2 sFv could exert a specific cytocidal effect inovarian carcinoma in SCID mice. These findings have led to an

erbB-2-overexpressing tumor cells [11–14]. To validate thisRAC-approved Phase I clinical trial for patients with ovarian cancer.strategy in an in vivo model system, we also showed thatIn this report, we show that expression of the anti-erbB-2 sFv couldadenoviral-mediated intraperitoneal delivery of the anti-be readily detected in target tumor cells by in situ hybridizationerbB-2 sFv gene could achieve selective tumor killing andmethodology. PCR analysis of DNA extracted from various murineenhance survival in a murine model of human ovarian carci-tissues demonstrated that the anti-erbB-2 sFv remained localized to

the peritoneum. Delivery of the sFv to the non-erbB-2-overexpressing noma [13, 14].REN mesothelial and Hep G2 hepatocellular carcinoma cell lines Based upon these findings, a strategy for human ovarianwas not deleterious to either one, affirming the tumor specificity of carcinoma has been developed to achieve direct in vivo deliv-this gene therapy strategy. In addition, histopathological analysis of ery of an adenoviral vector encoding the anti-erbB-2 sFvvarious tissues showed that adenoviral-mediated delivery of the anti- gene in a clinical setting. Use of the anti-erbB-2 sFv-encod-erbB-2 sFv to immunocompetent mice with either primary exposure ing adenovirus in a human context requires the careful analy-or previous vector challenge at different doses produced no abnormal

sis of safety issues related to the adenoviral vector as wellchanges when compared to untreated animals. These findings suggestas the biologic utility of this gene therapy intervention. Thisthat adenoviral-mediated delivery of the anti-erbB-2 sFv in a humananalysis would include studies designed to determine vectorcontext can be effectively assayed, is potentially free of vector-associ-efficacy in accomplishing in situ tumor cell transduction inated toxicity, and retains biologic utility based on tumor specificity.addition to studies of antitumor efficacy. The present reportq 1997 Academic Press

describes the development of assay systems and methodsin animal models which will be employed to obtain this

INTRODUCTION information in our planned human clinical gene therapy trial.

A direct correlation has been noted in carcinoma of theMATERIALS AND METHODSovary between overexpression of erbB-2 and reduced overall

patient survival [1–3]. Based on this concept, strategies de-signed to ablate the function of erbB-2 have been developed Cell lines. The human ovarian carcinoma cell line,

SKOV3.ip1, was kindly provided by Dr. J. Price (Universityas therapeutic modalities. These include a number of anti-body-based and molecular strategies designed to modulate of Texas M. D. Anderson Cancer Center, Houston, TX). Hep

G2, a human hepatocellular carcinoma cell line, REN, aerbB-2 levels in tumor targets [4–10]. As an alternate meanshuman mesothelial cell line, and HeLa, a human cervicalcarcinoma cell line, were obtained from the American Type

1 To whom correspondence should be addressed at Gene Therapy Pro-Culture Collection (Rockville, MD). SKOV3.ip1 and HeLagram, University of Alabama at Birmingham, 1824 6th Avenue South, WTIcells were maintained in DMEM/F12 media supplemented620, Birmingham, AL 35294. Fax: (205) 975-7476. E-mail: david.curiel@

cc.uab.edu. with 10% heat-inactivated fetal calf serum (FCS) (PAA Lab-

3780090-8258/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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379EFFICACY AND SAFETY OF ANTI-erbB-2 sFv-ENCODING ADENOVIRUS

oratories, Newport Beach, CA), L-glutamine (200 mg/ml), dominal fat) employing the QIA amp tissue Kit (Qiagen,Inc., Chatsworth, CA). PCR (polymerase chain reaction)penicillin (100 U/ml), and streptomycin (25 mg/ml). Hep G2

was maintained in Eagle’s MEM supplemented with nones- primers which amplify the 5* flanking region of the sFv anti-erbB-2, including the KpnI linker (AGGGGTACCATG-sential amino acids and sodium pyruvate. All cell lines were

maintained at 377C in a humidified 5% CO2 atmosphere. AAGTCACATTCCCAGGT) and the XbaI site, (GCTCTA-GATTAGGAGACGGTGACCGTGGTCC) were used forIn situ gene transfer to ovarian cancer cells. HumanPCR-based detection of the sFv gene in harvested tissues.ovarian carcinoma cells SKOV3.ip1 cells were plated at aPrevious studies have shown that target sequence was detect-density of 1 1 106 in 6-cm dishes. Cells were infected withable at the level of one gene copy per 10 pg of total DNA.recombinant adenoviral vectors to accomplish in situ gene

Specificity of anti-erbB-2 sFv-mediated cytotoxicity. Tar-transfer to tumor cells. Vectors included AdCMVLacZ, anget cells, including immortalized human cell lines derivedE1A/B-deleted replication incompetent adenovirus encodingfrom mesothelium (REN), liver (Hep G2), as well as anan Escherichia coli b-galactosidase (LacZ) reporter geneerbB-2-positive ovarian control (SKOV3.ip1), and an erbB-kindly provided by Dr. D. Tang (University of Alabama at2-negative control (HeLa), were plated at a density of 5000Birmingham), and Ad21, encoding an endoplasmic reticu-cells/well in 96-well plates and transfected with the adenovi-lum (ER)-directed anti-erbB-2 single chain antibody (sFv)rus–polylysine (AdpL) conjugate complexed with a controlgene, previously described (15). Cells were infected withplasmid, pcDNA3, or a plasmid encoding the ER-directedeither recombinant adenoviral vector at a multiplicity of in-form of the anti-erbB-2 sFv gene, pGT21, as described else-fection (m.o.i.) of 100 plaque-forming units (PFU) per cell.where (11). Cells were analyzed for cell viability 96 hr afterTwenty hours postinfection the cells were analyzed for intransfection using the XTT assay (17).situ gene transfer by in situ hybridization techniques. Cells

were also analyzed for the presence of the erbB-2 protein Determination of in vivo safety and potential toxicity ofby immunohistochemistry. adenoviral vector encoding the anti-erbB-2 sFv. Safety

and toxicity studies were carried out with the Ad21 adenovi-In situ hybridization and immunohistochemical analysis.ral vector in an immunocompetent murine model to deter-The general methodology for in situ hybridization has beenmine any acute toxicity associated with vector delivery ofdescribed in detail (16). Briefly, the probes used for in situanti-erbB-2 sFv gene. For these studies, female C57BL/6hybridization were RNA molecules produced by transcrip-mice, ages 4–6 weeks, were injected with either 1 ml oftion from a Bluescript plasmid containing an anti-erbB-2serum-free medium or 1 ml of serum-free medium con-sFv cDNA insert. For use as transcription templates, thetaining the adenoviral vector. In group 1, primary viral chal-plasmids were linearized downstream of the insert and puri-lenges to the animals (n Å 4) were performed with varyingfied with a spin column (Clonetech, Palo Alto, CA). Digoxi-doses of Ad21 (108, 109, 1010 PFU/mouse). After 7 days,genin-UTP (Boehringer Mannheim, Indianapolis, IN) wasanimals were anesthetized with ketamine/xyline (10 mg /directly incorporated at one-third molar ratio.15 mg/100 mg, respectively) and sacrificed by cervical dislo-For in situ hybridization, the cells were cytocentrifugedcation. In a second group, the effects of preimmunizationand fixed in 3% paraformaldehyde for 1 hr at room tempera-on vector associated toxicity were determined. This groupture. The slides were acetylated with 0.1 M triethanolamine,(n Å 4) received 107 PFU of Ad21 via the intraperitonealwashed, and hybridized with 1 fmole/ml of digoxigenin-la-route. After 10 days, animals then received an additionalbeled RNA probe at 507C overnight. The slides were thenAd21 inoculation intraperitoneally at one of three doses (108,washed in high stringency buffers, treated with RNase, and109, or 1010 PFU/mouse). Seven days postchallenge, animalsstained with anti-digoxigeninrF (ab)2 antibody that was con-were sacrificed as described above and necropsy was per-jugated to alkaline phosphatase(Boehringer-Mannheim). Theformed. The major organs of the chest, abdominal cavity,reaction was then visualized by overnight incubation withpelvis, and retroperitoneum were analyzed microscopicallyNBT/BCIP(Boehringer-Mannheim). For immunohistochem-following fixation, paraffin embedding, and hematoxylin–istry, after appropriate blocking steps, a rabbit anti-humaneosin staining for histopathologic evidence of tissue toxicity.anti-erbB-2 antibody (Oncogene Sciences, Uniondale, NY)All animal protocols were approved by the institutional Ani-was employed at the manufacturer’s prediluted concentrationmal Resource Committee in compliance with the Guide forand an ABC peroxidase system was utilized(Vector Labs,the Care and Use of Laboratory Animals provided by theBurlingame, CA) to detect cell surface erbB-2 in tumor cells.National Institutes of Health.

In vivo localization of adenoviral vector. The recombi-nant adenovirus encoding the anti-erbB-2 sFv was delivered

RESULTSintraperitoneally (ip) into SCID mice at 2 1 1010 PFU. Ani-mals (n Å 4) were sacrificed at either 7 or 40 days afterviral administration and DNA was extracted from various In situ tumor cell transduction by the anti-erbB-2 sFv-organ sites (heart, liver, spleen, lung, kidney, brain, uterus, encoding adenoviral vector. Mutation compensation gene

therapy strategies require direct in vivo transduction of tumorthymus, tail, omentum, body wall, diaphragm, gut, and ab-

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380 DESHANE ET AL.

targets. In this regard, our anti-erbB-2 sFv approach man- In vivo localization of the anti-erbB-2 sFv gene after intra-dates a vector technology which can achieve efficient in situ peritoneal vector delivery. We next sought to study thetransduction of tumor cells in the context of malignant asci- localization of transduced cells after intraperitoneal vectortes. In previous work, we have shown that recombinant ade- delivery. For in vivo localization studies, CB-17 SCID micenoviral vectors represent a gene transfer system appropriate (n Å 4) were injected with 1010 PFU of the recombinantfor this application [13, 14, 18, 19]. Thus, our planned gene adenovirus encoding the anti-erbB-2 sFv. DNA was ex-therapy trial employs a replication-defective adenovirus en- tracted from the visceral organs of animals sacrificed at ei-coding the anti-erbB-2 sFv. The studies reported here evalu- ther 7 or 40 days after viral administration. PCR analysisate this vector for safety as well as gene transfer efficacy. was used to detect the presence of the anti-erbB-2 sFv-en-To this end, methods were developed to specifically identify coding DNA sequences. In the group sacrificed at 7 days,transduced tumor cells to establish that we could accomplish DNA harvested from mesothelial cells lining the peritonealeffective gene transfer. For this analysis, an in situ assay cavity and uterine-derived cellular elements demonstratedsystem was developed to specifically detect vector DNA the presence of the anti-erbB-2 gene (Fig. 2A). All othersequences in transduced tumor cells. Control experiments organs failed to demonstrate the sFv sequences at the limitsestablished that anti-erbB-2 sFv-specific PCR primers could of detection. At 40 days postinjection, the omentum wasamplify target DNA sequences from Ad21 genomic DNA uniquely positive for the presence of the anti-erbB-2 sFv(data not shown). (Fig. 2B). There was no evidence of anti-erbB-2 localization

To validate the utility of this assay in a system paralleling in any other tissue analyzed in the animal groups. Thus, afterour planned trial, either the anti-erbB-2 sFv-encoding adeno- intraperitoneal administration of the adenovirus encoding thevirus (Ad21) or a control adenovirus (AdCMVLacZ) was anti-erbB-2 sFv, the virus was localized exclusively to intra-employed to transduce human ovarian carcinoma cells. Im- peritoneal tissues. However, by 40 days postexposure onlymunohistochemical analysis of cells infected with Ad- the omentum demonstrated residual evidence of the sFv.CMVLacZ or Ad21 demonstrated the presence of cell sur-

Effect of the ectopic expression of the anti-erbB-2 sFvface erbB-2 20 hr postinfection (Fig. 1B). Analysis by in situgene on human cell targets. Previous studies by Smythehybridization, however, demonstrated that only the Ad21-et al. have shown that recombinant adenovirus delivered byinfected tumor cells contained anti-erbB-2 sFv DNA se-the intraperitoneal route to tumor targets may also transducequences (Fig. 1A). Further, these transduced cells weremesothelial cells, and to a much lesser extent, cells of thereadily detected by this assay system. AdCMVLacZ-trans-liver [20]. To evaluate any potential deleterious effects ofduced cells did not demonstrate the presence of sFv se-the anti-erbB-2 sFv expression at these sites, correspondingquences by in situ analysis. Thus, this assay is capable ofhuman cell lines (REN and Hep G2) were transfected withspecifically detecting transduced tumor cells in the context

of ovarian carcinoma for demonstration of vector efficiency. the plasmid construct encoding the anti-erbB-2 sFv. Control

FIG. 1. Determination of in situ gene transfer to ovarian carcinoma cells. SKOV3. ip1 cells were infected with AdCMVLacZ or Ad21 and analyzedfor gene transfer and the presence of tumor marker 20 hr after infection. (A) Analysis of cells by in situ hybridization (B) Immunohistochemical analysis.

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381EFFICACY AND SAFETY OF ANTI-erbB-2 sFv-ENCODING ADENOVIRUS

upon analogous FDA-directed studies for adenoviral deliveryto the peritoneum. In group I, animals were injected intraperito-neally with serum-free medium in the presence or absence of108, 109, or 1010 PFU of Ad21. In group II, animals werechallenged with 107 PFU of virus and 10 days post-initialinoculation, given a second dose of 108, 109, or 1010 PFU ofAd21. This is an inoculum known to induce antivector immu-nity in this mouse strain. All animals underwent autopsy. Uponpathologic examination, the pelvic and peritoneal cavities inboth groups were found to be grossly unremarkable and withoutevidence of ascites. None of the animals, in either the controlor experimental groups showed any detectable pathologicchanges, all organs were appropriately placed and there wasno evidence of atrophy or dysgenesis. None of the animalsshowed any gross zones of necrosis, abscess formation, orinfarction. One animal from each group was retained for in situphotographic documentation (Figs. 4a and 4b). Microscopicsectioning was performed of the major organs of the remaininganimals.

Animals treated with a viral inoculation of 108 PFU wereessentially identical to the control animals and showed no evi-dence of pathologic abnormalities. The paraganglia, adrenal

FIG. 2. Determination of the presence of adenoviral vector encoding glands, ovaries, fallopian tubes, uterus, and pelvic lymph nodesthe sFv gene in vivo. (A) PCR analysis of DNA from tissues 7 days after were sampled in addition to the major organs of the abdominal,intraperitoneal delivery of the adenoviral vector. (B) PCR analysis of DNA retroperitoneal, and thoracic cavities and found to be histologi-from tissues 40 days after adenoviral-mediated delivery of the anti-erbB-2

cally within normal limits. Importantly, histologic findings insFv gene.the animals treated with a viral inoculation of 1010 PFU wereagain without significant histopathology and, therefore, similar

experiments had established that each of these cell lines was to those seen in the control animals (Figs. 4a and 4b). Evidencetransduced at ú95% frequency (data not shown). Trans- of significant acute or chronic inflammation, necrosis, or acutefected cells were analyzed at 96 hr posttransfection for cellviability employing the XTT assay. Expression of anti-erbB-2 sFv had no cytocidal effects on these cellular targets (Fig.3). This is consistent with our previous findings that anti-erbB-2 sFv-mediated cytotoxicity is specific for erbB-2-overexpressing tumor targets [11–15]. On the basis of thestudies summarized in Fig. 2, significant ectopic levels ofintraperitoneally delivered adenovirus are not expected out-side the peritoneal cavity. As an additional level of safety,however, our genetic intervention study is highly specificfor killing only tumor cells within the transduced cell sub-sets. Thus, in the context whereby some level of ectopictransduction has been shown to occur, expression of anti-erbB-2 sFv does not appear to be deleterious.

Safety of intraperitoneal delivery of an adenovirus encod-ing anti-erbB-2 sFv. As an initial step toward evaluating thesafety of this gene therapy strategy, we utilized an immunocom-petent mouse model (C57BL/6) because this would moreclosely parallel employment in humans. Furthermore, previous

FIG. 3. Determination of the effect of ectopic expression of the anti-reports demonstrated that differences exist between immuno-erbB-2 sFv gene on human cell targets. Transfected cells were assayed forsuppressed/immunodeficient and immunocompetent hosts incell viability by the XTT assay at 96 hr after transfection. Target cellstheir ability to respond to an adenoviral vector challenge [21,included a human hepatocellular carcinoma cell line, Hep G2; a human

22]. Our studies thus employed an immunocompetent model mesothelial cell line, REN; a human cervical carcinoma cell line, HeLa;to allow for analysis of acute toxicity in the preimmune state and human ovarian carcinoma cell line, SKOV3.ip1. Experiments were

performed five times each and data represent means { SEM.as well as postvector immunization. These studies were based

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organs including the lung, liver, gall bladder, muscle, and uri-toxicity was not appreciated histomorphologically at any viralnary bladder for a variety of gene therapy applications [18,dose tested for either group. In summary, no animal suffered23–26]. In addition, these vehicles have been employed forany untoward clinical effects related to the viral vector in thesethe in situ transduction of tumor in the context of cancer genestudies as determined by both gross and histologic morphology.therapy approaches. As a gene therapy strategy for humanIn addition, no extracerebral pathology could be attributed toovarian carcinoma, adenoviral-mediated delivery of an intracel-the anti-erbB-2-encoding recombinant adenovirus.lular single chain antibody against erbB-2 requires similar high

DISCUSSION efficiency in situ transduction of tumor. There are thus twoRecombinant adenoviral vectors have been utilized for the potential safety issues relating to this strategy. The first issue

relates to potential ectopic localization of the intraperitoneallydirect in vivo delivery of genes into a variety of tissue and

FIG. 4. Determination of the safety of intraperitoneal delivery of adenovirus encoding anti-erbB-2 sFv. Immunocompetent C57BL/6 mice wereinjected intraperitoneally with adenoviral doses 108, 109, or 1010 PFU. (a) Non-immunized mice: (A) control lung, (B) control liver, (C) 108 PFU kidney,(D) 109 PFU adrenal gland, (E) 1010 PFU uterus, (F) 1010 PFU ovary. (b) Animals preimmunized with 107 PFU of virus and 10 days postinoculationreceived a second dose of virus 108, 109, or 1010 PFU: (A) control lung, (B) control ovary, (C) 108 PFU abdominal wall muscle, (D) 109 PFU uterus,(E) 1010 PFU kidney, (F) 1010 PFU lung.

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384 DESHANE ET AL.

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