[CANCER RESEARCH 60, 1009–1013, February 15, 2000]

ONYX-015 Works Synergistically with Chemotherapy in Lung Cancer Cell Linesand Primary Cultures Freshly Made from Lung Cancer Patients

Liang You, Cheng-Ta Yang, and David M. Jablons1

Thoracic Oncology Laboratory, University of California, San Francisco, California 94143-1674

ABSTRACT

p53 mutations and loss of heterozygosity (LOH) have been detected in>50% of lung cancers. Wild-type p53 can prevent replication of damagedDNA and promote apoptosis of cells with abnormal DNA. A humanadenovirus, ONYX-015, which has a deletion in the E1B region, has showntumor-specific cytolytic effect in tumor cells with nonfunctional p53 andantitumor efficacy that can be augmented by chemotherapeutic agents. Arecent report from an independent group, however, indicates that wild-type p53 is necessary for the infection of this replicating virus, and it is indirect contradiction to previous observations of the ONYX group. In thisstudy, we carried out cytopathic effect (CPE) assays using ONYX-015 onfive human lung cancer cell lines with known p53 status. Two of these celllines, NCI-H522 and NCI-H1703, have mutations and LOH in theirp53gene. Both lines were lysed in a dose-dependent manner and showed 100%cytolysis at a multiplicity of infection of 0.1. Two additional cell lines,NCI-H2347 and NCI-H838, both of which have wild-type p53 gene,showed near complete lysis at a multiplicity of infection of 1. We demon-strate here that the lung cancer cells with nonfunctional p53 are at least 10times more sensitive to ONYX-015 cytolysis than the lung cancer cells withwild-type p53. In addition, standard chemotherapeutic agents (paclitaxoland cisplatin) showed a synergistic effect when combined with ONYX-015,and this effect was p53 mutant dependent. Furthermore, we tested thecytolytic effect of ONYX-015 on a panel (n5 7) of primary first-passagecultures made from freshly resected lung cancers. ONYX-015 lysed pri-mary lung cancer cells in six of seven (86%) primary cultures. Two of fourprimary cultures treated with chemotherapeutic agents had a synergisticeffect with ONYX-015. Our data indicate that wild-type p53 is not re-quired for the infection of this replicating virus, and also we demonstratethat ONYX-015 is effective alone and works synergistically with chemo-therapeutic agents in lung cancer cell lines and primary cultures. Thisstudy suggests that ONYX-015 may be effective, especially in combinationwith conventional chemotherapy, in the treatment of patients with lungcancer.

INTRODUCTION

Lung cancer is the leading cause of cancer deaths in the UnitedStates among both men and women, with.170,000 newly diagnosedcases each year (1). Despite aggressive approaches made in thetherapy of lung cancer in the past decades, the 5-year survival rate forlung cancer remains,15% (1). Surgery, chemotherapy, and radiationhave been used with generally unsatisfactory results in advanceddisease. Improvement in the efficacy of lung cancer treatment is amajor public health goal. New therapies based on better understandingof the biology of lung cancer are needed for the treatment of patientswith lung cancer. Lung cancers are divided into two major histologicalclasses: NSCLC2 and SCLC. NSCLC (75% of all lung cancers)consists of three major types: adenocarcinoma, squamous cell carci-

noma, and large cell carcinoma (2). Lung adenocarcinomas and squa-mous cell carcinomas represent 60–70% of all lung cancers.

Extensive studies have revealed several types of genetic alterationsthat contribute to lung cancer:ras gene mutation,mycgene amplifi-cation,Her-2/neuoverexpression, and LOH in chromosome regions3p, 5q, 8p, 9p, 11p, 13q, 17p, and 18q. LOH is highly suggestive ofthe presence of a tumor suppressor gene at the deleted chromosomesite (3).p53, Rb,and the pathways in which they are involved havebeen described in lung cancer. There are a number of chromosomeregions,e.g., 3p, 5q, 9p, and others., thought to harbor currentlyundiscovered tumor suppressor genes (2). p53 mutations and LOHhave been detected in.50% of lung cancers (4). Loss or mutation ofp53 tumor suppressor gene is a very common genetic abnormality inmany human cancers (5). Germ-line inheritance of one defectivep53gene is responsible for a cancer family syndrome, the Li-Fraumenisyndrome, associated with a greatly increased risk of developing awide range of cancers (6). The human p53 protein is a 393-amino acidnuclear phosphoprotein. It binds both specifically and nonspecificallyto DNA and has specific interactions with a number of viral andcellular proteins that can affect its activity as a transcription factor.The viral proteins include viral oncogene products, SV40 large Tantigen, the adenovirus E1B 55K protein, and the human papillomavirus E6 protein (5). The host proteins include the TATA box-bindingprotein and the MDM-2 protein. The p53 protein is a tumor suppressorthat responds to DNA damage. The protein activates one of the twopathways in a cell with DNA damage: either the growth-arrest path-way in which cell division is halted until the DNA damage has beenrepaired, or the apoptotic pathway in which heavily damaged cellsundergo programmed cell death (7). Whenp53 genes are deleted ormutated, these two protective pathways in the cells become nonfunc-tional, and the cells become prone to DNA damage and unregulatedcell growth.

The better understanding of the p53 protein and its role in lungcancer development has madep53-based gene therapy possible. Clin-ical studies of adenovirus- and retrovirus-mediated wild-type genetransfer to lung cancer patients have resulted in tumor regression insome patients (7, 8). More recently, ONYX Pharmaceuticals, Inc.(Richmond, CA) has genetically designed a tumor-targeting adenovi-rus, ONYX-015, that only replicates in cells that lack functionalp53gene and therefore kills tumor cells specifically (9). This geneticdesign takes advantage of the fact that adenovirus E1B 55K binds(and inactivates) wild-type p53 protein. This binding is essential tovirus replication. The newly designed mutant adenovirus ONYX-015contains an 827-bp deletion in the E1B region and a point mutation atcodon 2022 that generates a stop codon. ONYX-015 cannot replicatein normal cells but can replicate in tumor cells lacking functional p53(9). ONYX-015 has been shown to kill cervical carcinoma cells, coloncarcinoma cells, glioblastoma cells, and pancreatic adenocarcinomacells lacking functional p53 with an efficiency comparable with thatof wild-type adenovirus (9). The virus has no effect on normal cellswith functional p53 at a MOI of up to 1 pfu/cell, whereas the cellswith nonfunctional p53 were killed at MOI of 0.01 pfu/cell. Thus, p53inactivation increases sensitivity to ONYX-015 by a factor of 100(10). It was also demonstrated that this virus caused a significantreduction in tumor size and complete regression of 60% of the tumors

Received 8/5/99; accepted 12/15/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Section of General ThoracicSurgery, 1600 Divisadero Street, Suite C322, University of California, San Francisco, CA94143-1674. Phone: (415) 885-3882; Fax: (415) 353-9525.

2 The abbreviations used are: NSCLC, non-small cell lung cancer; SCLC, small celllung cancer; LOH, loss of heterozygosity; MOI, multiplicity of infection; pfu, plaque-forming unit; CPE, cytopathic effect; SSCP, single-strand conformation polymorphism.

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induced by p53-deficient human cervical carcinoma cells in nudemice (10). This virus has entered Phase II clinical trials for thetreatment of squamous cell cancers of the head and neck and adeno-carcinomas of the pancreas (11).

Recently, Hallet al. (12) showed that dl1520 (or ONYX-015) canonly generate a CPE in cells with wild-type p53 but not in the cellswith mutant p53. They claimed that the virus can only kill cells withwild-type p53, and the cells with nonfunctional p53 are resistant tovirus-induced cell death. Their data raised serious questions on theusefulness of ONYX-015 to kill tumor cells.

To determine the potential therapeutic role of ONYX-015 on lungcancer patients, primary human lung cancer cultures and lung cancercell lines with known p53 status were treated with the virus in thisstudy. Additional experiments testing the potential efficacy of ONYX-015 and conventional chemotherapy (cisplatin and paclitaxol) to lyselung cancer cells were also performed.

MATERIALS AND METHODS

Cell Lines. Five NSCLC cell lines (NCI-H522, NCI-H1703, NCI-H157,NCI-H838, and NCI-H2347) were obtained from American Type CultureCollection. NCI-H1703 is a lung adenocarcinoma cell line with a missensemutation at codon 285 (GAG3AAG), and NCI-H522 is a lung adenocarci-noma cell line with a single base deletion at codon 191 (CCT3CT) of p53gene. LOH of thep53 gene was found in both NCI-H522 and NCI-H1703cells. NCI-H157 is a lung squamous carcinoma cell line with a nonsensemutation at codon 298. NCI-H838 and NCI-H2347 are lung adenocarcinomacell lines with wild-typep53gene sequence. The p53 status of all five cell lineswas independently confirmed by DNA sequencing at the University of Cali-fornia at San Francisco Cancer Center core facility.

Primary Tumor and Normal Peripheral Lung Cell Cultures. Lungtumors and adjacent normal lung tissues of patients with lung cancer weretaken from the operating room at room temperature immediately after resec-tion. The experiments were performed after approval by the Committee ofHuman Research at the University of California and in accordance with anassurance filed with and approved by the Department of Health and HumanServices. The specimens were dissected with scalpels into,5-mm cubes. Thepieces of tumor were placed in triple enzyme medium (13collagenase, 13hyaluronidase, and 13DNase; Sigma, St. Louis, MO; Ref. 13) in HBSS (LifeTechnologies, Inc., Gaithersburg, MD) with a magnetic bar and were then spunon a stir plate at room temperature for 2–3 h, until most of the solid tumor ornormal tissue was dissociated. The cells were filtered through a 70-mm nyloncell strainer (Becton Dickinson, Lincoln Park, NJ) and suspended in RPMI1640 with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT).

Adenovirus. ONYX-015 and wild-type adenovirus were supplied byONYX Pharmaceuticals. ONYX-015 is a chimeric human group C adenovirusthat does not express theMr 55,000 product of the E1B region encoding theMr

55,000 protein (9). The virus contains a deletion between nucleotides 2496 and3323 in the E1B region encoding theMr 55,000 protein. In addition, a C to Ttransition at codon 2022 in E1B generates a stop codon, preventing expressionof a truncated protein from the deleted gene. Wild-type adenovirus is identicalto ONYX-015 except in the E1BMr 55,000 gene region, where the originalwild-type sequence is present.

CPE Assay. In replicate six-well plates, cells (53 105) were grown to50–90% confluence and infected with either ONYX-015 or with wild-typeadenovirus for 90 min at increasing MOIs,i.e., 0, 0.01, 0.1, 1, and 10 pfu/cell(10). Plates were monitored daily for CPE, and the assay was terminated whenessentially total cytolysis was observed at an MOI of 0.01 (MOI of 0.1 forprimary cultures) with wild-type adenovirus. In experiments testing combinedefficacy of chemotherapy and ONYX-015, cisplatin and paclitaxol (10 nM

each) were added to test plates for 24 h immediately after 90-min ONYX-015treatment. The plates were stained with crystal violet and then analyzed.

Cell Viability Assay. Cells were inoculated at densities of 53 105 in60-mm culture dishes 24 h before treatments and treated with ONYX-015 atMOI of 0.1, 0.01, and 0.001 with or without cisplatin and paclitaxol (10 nM foreach). Culture medium alone was used for mock infection. Cell viability wasevaluated at day 8 by trypsinizing the culture plates and staining the cells with

trypan blue, followed by cell counting using a standard hemacytometer (12).All experiments were performed in triplicate.

p53 Mutation Analysis. SSCP of thep53gene was carried out on all lungcancer cell lines and tumor samples.3 A 5.5-ml loading mixture was preparedwith 1 ml of the PCR product, 0.5ml of Genescan 500 size standards(Perkin-Elmer, ABI), a 0.5-ml loading dye (Perkin-Elmer, ABI), 0.5ml of 100mM NaOH, and 3.0ml of deionized formamide. All gels consisted of 0.53Mutation Detection Enhancement gel (FMC, Rockland, ME) with 2.5–10%glycerol (Sigma) and 13TBE (Life Technologies). All gels were run at aconstant 60 W, and the SSCP results were analyzed using Genescan software(ABI) with analysis procedures and settings as described by the manufacturer.The direct DNA sequencing was carried out by University of California at SanFrancisco Cancer Center core facility.

Immunohistochemistry. Immunohistochemistry was done using snap-frozen, OCT (Tissue-Tek, Torrance, CA)-embedded tumors that had been cutinto 6-mm sections, hydrated, and digested with Pronase. The primary antibody(DO-7; DAKO Corp.), which reacts with the wild and mutant types of theintracellular p53 protein (14), was applied at 1:500 dilution, and tissues wereincubated at room temperature for 1 h. Universal DAKO Labeled Streptavidin-Biotin kit (LSAB1) and the secondary antibody was then applied. Diamino-benzidine was used as the chromogen, and the slides were counterstained withhematoxylin.

RESULTS

Primary normal human peripheral lung culture cells and C33A(p53-deficient) cervical carcinoma cells were used as controls for thesensitivity study of ONYX-015 and wild-type adenovirus. Completecytolysis was seen in normal cells treated with wild-type adenovirusat a MOI of 0.01 within 10 days. No significant CPE was observed atthe same time points with a MOI of 1, 0.1, and 0.01 in ONYX-015-treated normal cells. Cytolysis equivalent to that of the wild-type viruswas only evident when a much higher titer was used (MOI.10) inthese cells. C33A, with an inactivating mutation at codon 273 of thep53gene, was used as positive control cell line. When monitored foran extended period of 1–2 days (i.e.,11–12 days), C33A cells werecompletely lysed by the wild-type virus at a MOI of 0.001 and byONYX-015 at a MOI of 0.01.

We tested the cytotoxicity of ONYX-015 against five NSCLC celllines (NCI-H522, NCI-H1703, NCI-H157, NCI-H2347, and NCI-H838) using CPE assays. Both NCI-H1703 and NCI- H522 are lungadenocarcinoma cell lines containing only a mutatedp53gene (Table1). NCI-H157 is a squamous cell lung cancer cell line with a nonsensemutation at p53 codon 298. Both NCI-H2347 and NCI-H838 havewild-type p53gene. In triplicate experiments, ONYX-015 lysed NCI-H522 and NCI-H1703 cells (Fig. 1) in a dose-dependent manner (Fig.2A). Complete cytolysis was evident at a MOI of 1 and 0.1 pfu/cell 10days after infection. In three other lung cancer cell lines NCI-H157,NCI-H838, and NCI-H2347, complete cytolysis was observed at aMOI of 1 (Table 1).

To determine the synergistic effect of ONYX-015 with conven-tional NSCLC chemotherapy, a sensitivity study was carried out onthese NSCLC cell lines. We tested the cytotoxicity of cisplatin and/orpaclitaxol at a concentration of 10mM, 1 mM, 100 nM, 10 nM, 1 nM, and0.1 nM. Cells were killed completely by both agents at 10mM, 1 mM,and 100 nM. At 10 nM cisplatin and paclitaxol, cells were able to growto complete confluence, and minimal cytotoxicity was seen. Theresults are comparable in all these NSCLC cell lines. This chemo-therapy (10 nM cisplatin and 10 nM Taxol) was combined with theONYX-015 treatment for all five NSCLC cell lines (Table 2). Syn-ergistic effects were evident in three NSCLC cell lines with p53mutations,i.e., NCI-H522, NCI-H1703, and NCI-H157. When com-

3 L. Moore, T. Godfrey, C. A. S. Eng, R. Ho, and F. Waldman. Fluorescent SSCPanalysis for fast and sensitive detection of p53 mutations, submitted for publication.

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bined with chemotherapy, the CPE of ONYX-015 was augmented byat least 10-fold (Fig. 3A). Complete cell lysis was shown at a MOI of0.001, 0.01, and 0.1 on NCI-H522, NCI-H1703, and NCI-H157,respectively, in synergistic fashion. These three cell lines were sen-sitized by ONYX-015 to this low-dose chemotherapy, which does notwork effectively on these cells when used alone. No significantsynergistic effect was observed in the two NSCLC cell lines withintact p53 sequence,i.e., NCI-H2347 and NCI-H838.

Seven primary lung cancer cultures, including five NSCLCs andtwo primary lung sarcomas, were studied for the CPE of ONYX-015.In all seven lung cancer primary cultures tested, 90–100% cells werekilled at a MOI of 1. Complete cell lysis in one primary tumor cultureand partial lysis (30–50%) in the other three primary lung cancercultures were evident at a MOI of 0.1. Wild-type adenovirus served asa control for effective infection in all experiments.

In one NSCLC case, we were able to establish primary culturesfrom both normal lung and lung tumor tissues. ONYX-015 lysed mostof the tumor cells at a MOI of 1 and 0.1, whereas no significantcytolytic effect was noticed in normal culture at a MOI of 1. Syner-gistic effects of ONYX-015 with standard chemotherapy were studiedin four of these primary lung cancer primary cultures (Table 2). At aMOI of 0.1, ONYX-015 showed synergistic effect (5–10-fold) withlow-dose standard NSCLC chemotherapy in two of the four tumors,whereas no significant synergistic effect was observed in the othertwo tumors, despite an effect seen with the ONYX-015 virus alone.

A cell viability assay was carried out using NCI-H1703 at 8 daysafter infection; dose-dependent cytolysis is shown (Fig. 2B). A syn-ergistic effect with chemotherapeutic agents (10 nM cisplatin and 10nM paclitaxol) was also evaluated on day 8 (Fig. 3B). The treatmentvariable (with two levels: Mock and Mock1Chemo) was not signif-icant (P 5 0.2471 by theF test). The MOI 0.001versus MOI0.0011Chemo was significantly different, however (P5 0.0024 bythe Studentt test).

The p53 status of these lung cancer cell lines and lung tumors wasdetermined by SSCP and direct sequencing of the 5–8 exon of thep53gene. The p53 status of all five NSCLC cell lines was confirmed byour own SSCP and sequencing analysis. One p53 exon 5 mutation was

detected in the DNA from a lung cancer tumor tissue by SSCP anddirect sequencing (Table 1). In addition, immunohistochemical stain-ing of p53 was performed on all lung cancer tissues (Table 1).

DISCUSSION

These experiments demonstrate that ONYX-015 can infect and lyseeffectively NSCLC cell lines, and more clinically relevant, these dataindicate that the ONYX-015 virus kills fresh human primary lung cancercells and work synergistically with standard NSCLC chemotherapy. Inthis study, no significant cytolytic effect was noticed in primary normallung cultures prepared from freshly resected specimens. ONYX-015 didnot cause significant CPEs in normal human small airway epithelial cells,as shown in an earlier report (10). This virus has been geneticallydesigned to replicate specifically in cells that lack functionalp53 gene,causing tumor cell lysis. Because NSCLC is, in part, a genetic disease,gene-based therapy is both a logical and attractive strategy. More thanhalf of all lung cancers have p53 mutations and LOH. It is likely that amuch higher percentage of lung cancers have some defects in p53-dependent growth regulation pathway (15, 16). Therefore, lung cancer isa very good target for the ONYX-015 virus (3). ONYX-015 has thepotential to offer a new form of therapy based on the molecular geneticsand biology of lung cancer cells. This virus has already been shown to besafe and has entered Phase II clinical trials for the treatment of patientswith head and neck squamous cell carcinoma and adenocarcinomas of thepancreas (11).

We think that primary tumor cultures may provide more clinicallyrelevant information, in addition to other preclinical studies. Origi-nally, nude mice were used to study the antitumor efficacy of ONYX-015 in some cancer cell lines; immunocompetent rodent tumor modelsare not applicable for studying the antitumor efficacy of ONYX-015because adenoviruses can only replicate effectively in human cells.The synergistic effect shown in 50% primary lung cancer culturestested with conventional chemotherapy makes ONYX-015 more at-

Fig. 1. CPE of ONYX-015 on NSCLC cells containing only a mutantp53gene (Table1). A, ONYX-015 lysed NCI-H522 cells completely at a MOI of 0.1 pfu/cell.B, a similareffect was also evident in the NCI-H1703 cell line.

Table 1 CPE of wild-type adenovirus (WT) and ONYX-015 on lung cancer cell linesand lung cancer primary cultures

Cells

CPE

p53 status Ref.WT ONYX-015

Control cellsC33A (cervical carcinoma) 1 0.01 CGT3TGT codon 273

Arg3Cys(25)

Normal lung primary culture 1 NMD,b p53 IHC2c

NSCLC cell linesNCI-H522 1 0.1 CCT3CT codon 191 (26)

1 bp deletion (frameshift)NCI-H1703 1 0.1 GAG3AAG codon 285 (27)

Glu3LysNCI-H157 1 1 CAG3TAG codon 298 (27)

Gln3Stop codonNCI-H2347 1 1 Wild-type (27)NCI-H838 1 1 Wild-type (27)

Primary tumor culturesNSCLC patient 1 1 1 TAC3TGC codon 163

Tyr3Cys, p53 IHC1NSCLC patient 2 1 1 NMD, p53 IHC1NSCLC patient 3 1 1 NMD, p53 IHC1NSCLC patient 4 1 1 NMD, p53 IHC2NSCLC patient 5 1 2 NMD, p53 IHC1LSd patient 6 1 1 NMD, p53 IHC2LS patient 7 1 1 NMD, p53 IHC1a Lowest MOI giving complete cytolysis was presented.b No mutation detected.c Immunohistochemical staining status of p53.d Primary lung sarcoma.

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tractive for treatment of lung cancer. It is possible that in the clinicalsetting ONYX-015 will enhance standard chemotherapy to a greaterextent, because the virus will be administrated at higher titers.

There is increasing evidence that other possible defects in p53signal pathway may be involved in the development of lung cancer.Overexpression of MDM-2 protein was found in 70% of the NSCLCstested (16). Recently, it was reported that the expression of p14-ARFis altered in;40% of NSCLCs (16). The p14-ARF, which links tumorsuppressors Rb and p53 (17), promotes MDM-2 degradation andstabilizes p53 (18). Thus, the p53 protein maybe indirectly inactivatedby mechanisms other than p53 mutations in the rest of lung cancers.This, we believe, may explain in part why ONYX-015 effectivelylyses those tumor cells with intactp53 gene, as described in someother studies (12, 19). In theory, ONYX-015 should kill most, if notall, lung cancers, along with many other types of cancers. Neverthe-less, other unknown mechanisms by which ONYX-015 can replicateindependently of the cell cycle have been suggested (20).

The combination of ONYX-015 and conventional NSCLC chemother-apy had greater antitumor efficacy than ONYX-015 alone in most lungcancer cell lines and primary cultures tested here. The effect appears to besynergistic, because minimal cytotoxicity was observed when either ofthese chemotherapeutic agents was used alone at identical concentrations.The mechanism of this synergistic effect is under study. The possibilitythat each agent works independently on different cell populations cannotbe ruled out, although it is unlikely to be the case here. It has beenreported that the adenovirusE1A gene expression can activate the cellcycle (21) and/or increase the cellular sensitivity to chemotherapy in ap53-independent manner (22). This may be a possible mechanism bywhich the synergistic effect occurs in some NSCLC cells, because allthree p53-mutant NSCLC cell lines showed a synergistic effect whereasno significant effect was noticed in the other two cell lines with thewild-type p53gene. There is a possibility that p53 function may not becompletely lost in these two cell lines with wild-typep53 gene, and itmight explain why these two cell lines were less sensitive to ONYX-015when compared with the two with nonfunctionalp53 genes. Otherunknown functions of p53 in viral replication or cell cycle control mayplay a role in this synergistic effect.

Adenovirus- and retrovirus-mediated wild-type gene transfer tolung cancer patients has entered Phase I clinical trials (7, 8). Wild-type p53 was delivered to tumor cells by direct intratumoral injection.Wild-type p53 can induce apoptosis in tumor cells, therefore causingtumor regression. Like other gene replacement strategies, the prob-lems of gene transfer remain,e.g.,efficient gene transfer requires ahigh-titer recombinant retrovirus; adenovirus can achieve high-effi-ciency transduction but can bind and inactivate wild-type p53. Thereplacement of wild-type p53 might not affect some of those tumorswith a nonfunctional p53 pathway.

One of the major problems for wild-type gene replacement therapyis immune-mediated clearance. Because ONYX-015 is a replicatingvirus, cell-mediated immunity directed toward infected tumor cellsmay actually enhance the efficacy of ONYX-015 in patients withtumor. Antibody-mediated immune response may not affect ONYX-015significantly because of the relatively poor penetration of antibodies into

Fig. 2. Dose-dependent cytolysis of ONYX-015was evident in NSCLC cells.A, dose-dependentcytolysis was seen in NCI-H522 NSCLC cells 6days after infection.B, dose-dependent cytotoxicitywas shown in NCI-H1703 NSCLC cells 8 days afterinfection.Bars,SD.

Table 2 Synergistic effect with chemotherapeutic agents (1028M paclitaxel and

1028M cisplatin)

Cells Chemob

CPEa

p53ONYX-15Synergistic

Effect

NSCLC cell linesNCI-H522 2 0.1 0.01 mutantNCI-H1703 2 0.1 0.01 mutantNCI-H157 2 1 0.1 mutantNCI-H2347 2 1 1 wild-typeNCI-H838 2 1 1 wild-type

NSCLC primary culturesNSCLC patient 2 2 1 1 p53 IHC1c

NSCLC patient 3 2 1 0.1 p53 IHC1NSCLC patient 4 2 1 1 p53 IHC2LSd patient 7 2 1 0.1 p53 IHC1a Lowest MOI giving complete cytolysis was presented.b Drug dose sensitivity was tested with cisplatin alone, paclitaxel alone, and both

together. The control of 1028 M cisplatin plus 1028 M paclitaxel given together had noCPE.

c Immunohistochemical staining status of p53 protein.d Primary lung sarcoma.

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solid tumor masses. ONYX-015 is a tumor-targeting recombinant ade-novirus because it takes advantage of both the normal function of the p53protein and the properties of adenoviral proteins. ONYX-015 replicates,selectively, in any tumor cell with nonfunctional p53. However, efficacyof ONYX-015 therapy may be restricted, in part, by a host immunesystem, which can clear virus before it reaches its tumor target. Topicalor direct intratumoral injection of ONYX-015 to localized tumors, suchas head and neck cancers, focal lung cancers, and primary brain tumors,may be of greatest benefit. Also, it has been shown that ONYX-015 hasthe potential to treat tumors that grow and spread within the body cavity(10). Recently, Phase II clinical trials of ONYX-015 in locally advanced,chemotherapy- and radiotherapy-resistant head and neck squamous cellcancers have shown encouraging preliminary results, with a 60% re-sponse rate (23). On the basis of these data within vitro lung cancertesting and the results of Phase II clinical trials in head and neck cancers,we suggest that the virus should have efficacy in patients with lungcancer.

ACKNOWLEDGMENTS

ONYX-015 and wild-type viruses were kindly provided by ONYX Phar-maceuticals, Inc. (Richmond, CA). We thank Dr. Tony Godfrey for help in thep53 mutational analysis and Jean Y. Chan for help in preparation of themanuscript.

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Fig. 3. Synergistic effect with conventional chemotherapy in NSCLC cells.A, syner-gistic CPE with chemotherapeutic agents paclitaxel (1028 M) and cisplatin (1028 M) inboth NCI-H522 and NCI-H1703 cells.B, synergistic effect was shown in NCI-H1703cells by cell viability assay. The MOI 0.001versusMOI1Chemo 0.001 is significantlydifferent (P5 0.0024, Studentt test).Bars,SD.

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