Intrapulmonary IFN-B Gene Therapy Using an Adenoviral

Vector Is Highly Effective in a Murine Orthotopic Model

of Bronchogenic Adenocarcinoma of the Lung

Michael J. Wilderman, Jing Sun, Arminder S. Jassar, Veena Kapoor,Mohamed Khan, Anil Vachani, Eiji Suzuki, Paul A. Kinniry,Daniel H. Sterman, Larry R. Kaiser, and Steven M. Albelda

Thoracic Oncology Research Laboratory, University of Pennsylvania Medical School, Philadelphia, Pennsylvania

Abstract

Given previous work showing that an adenoviral vectorexpressing IFN-B (Ad.IFNB) was highly effective in eradicatingi.p. mesothelioma tumors, the antitumor efficacy of thisagent was evaluated in an orthotopic model of bronchogenicadenocarcinoma of the lung. These transgenic mice have aconditionally expressed, oncogenic K-rasG12D allele that canbe activated by intratracheal administration of an adenovirusexpressing Cre recombinase (Ad.Cre). K-rasG12D mutant micewere given Ad.Cre intranasally to activate the oncogene. Micewere then given 109 plaque-forming units of a control vector(Ad.LacZ) or Ad.IFNB intranasally 3 and 4 weeks later, a timewhen lung tumors had been established. Cells derived fromK-ras-mutated lung tumors were also grown in the flanks ofmice to study mechanisms of therapeutic responses. In twoseparate experiments, untreated tumor-bearing mice all diedby day 57 (median survival, 49 days). Ad.LacZ-treated mice alldied by day 71 (median survival, 65 days). In contrast, 90% to100% of mice treated with Ad.IFNB were long-term survivors(>120 days; P < 0.001). In addition, immunity to re-challengewith tumor cells was induced. In vitro and flank tumor studiesshowed that Ad.IFNB induced direct tumor cell killing andthat depleting natural killer or CD8+ T cells, but not CD4+

T cells, with antibodies attenuated the effect of Ad.IFNB. Thesestudies, showing remarkable antitumor activity in thisorthotopic lung cancer model, provide strong preclinicalsupport for a trial of Ad.IFNB to treat human non–small celllung cancer. (Cancer Res 2005; 65(18): 8379-87)

Introduction

Lung cancer is the leading cause of cancer-related death in theU.S. and throughout most of the world. It was estimated that in2004, there were 173,770 cases of lung cancer and 160,440 deathsrelated to lung cancer in the U.S. alone, with >80% being non–smallcell lung cancer (NSCLC; ref. 1). Of all patients diagnosed withNSCLC, only 14% survive >5 years. Surgery has been the mainstay oftreatment in early stages of lung cancer. However, most patients(>75%) are not surgical candidates due to their advanced lung cancerstage, and thus medical therapy or radiation are their only options.Unfortunately, medical therapies have been employed with littlesuccess. Clearly, novel treatments for NSCLC are desperately needed.

A potential strategy in the treatment of lung cancer could be theuse of IFNs. Type I IFNs (the IFN-a family and IFN-h) are known toinhibit tumor cell growth and stimulate the immune system (2).IFNs have immunoregulatory effects on antibody production,natural killer (NK) and T cell activation, macrophage function,delayed-type hypersensitivity, and MHC antigen expression (3–6).They have also been shown to have antiproliferative effects (4) andantiangiogenic properties (7).Accordingly, delivery of type I IFN proteins via the i.v. or s.c.

route has been explored in many types of cancers. Unfortunately,success has been limited in solid tumors. Although used at theirmaximally tolerated dose, the short half-life of the protein (<60minutes) makes sustained therapeutic levels difficult to achieve.To counteract these limitations, a number of investigators haveshown that in vivo gene delivery of the IFN-a or IFN-h genesusing gene transfer methods such as plasmids or various viralvectors (e.g., adenovirus), could be effective in tumor models ofmetastatic lung cancer, breast cancer, bladder cancer, cervicalcancer, renal cell carcinoma, glioma, and liver metastases fromcolorectal cancer (8–17). Our group has previously reportedsignificant antitumor activity and improved survival in mousemesothelioma models using an adenovirus vector encoding IFN-h(18–20). We are not aware of studies existing that examine theuse of IFN-h gene therapy in lung cancer.To date, however, there have been few good models of lung

cancer. Most traditional lung cancer models involve injecting lungcancer cells s.c. or i.v. (21, 22). Although useful, these models havethe limitation of generating tumors that grow much more rapidlythan a human tumor would and in nonphysiologic sites (i.e., skin orlung vasculature). Recently, however, an orthotopic mouse modelfor bronchogenic lung cancer involving a conditionally expressedoncogenic K-rasG12D allele has been developed (23). After removalof a lox-flanked stop codon by intrapulmonary administration of anadenovirus expressing the Cre recombinase (Ad.Cre), mutant K-rastranscription occurs leading to the development of lung cancerin situ . The tumors progress in a controlled fashion from smalldiffuse adenomas to widespread sheets of lung cancer. Thepathology is similar to what is seen in humans with bronchogeniclung cancer. Depending on the dose of Ad.Cre, the number lesionsand the extent of tumor can be controlled.Given the previous success using adenoviral vector expressing

IFN-h (Ad.IFNh) in the treatment of other solid tumors, we studiedthis new orthotopic model of advanced bronchogenic lung cancerto evaluate the safety and efficacy of Ad.IFNh delivered via anintratracheal route. Although one potential limitation in this modelwould be that Ad.Cre might generate anti-adenoviral humoralantibodies after the administration of adenovirus (24–27) that

Requests for reprints: Steven Albelda, Thoracic Oncology Research Laboratory,BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160. Phone: 215-573-9969;Fax: 215-573-4469; E-mail: Albelda@mail.med.upenn.edu.

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-0920

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could limit the effectiveness of subsequent Ad.IFNh vectors, wefound that this approach led to remarkable efficacy. We also used acell line derived from K-ras-mutated lung tumors to elucidate someof the mechanisms behind these responses.

Materials and Methods

Animals. Breeding pairs of Lox-Stop-Lox (LSL) K-rasG12D mice ( from a

mixed 129Sv.J and C57BL/6 background) were generously provided by

Dr. David Tuveson of the University of Pennsylvania, Philadelphia, PA. Themice were initially described in a paper by Jackson et al. (23). Mice were

genotyped by PCR amplification of genomic DNA obtained from tail

samples (primer sequences available upon request). LSL-K-rasG12D–positive mice were used for orthotopic lung cancer experiments, whereas

the wild-type littermates were used for flank tumor studies. In addition,

pathogen-free female C57BL/6J F � 129P3/J M F1 hybrids were purchased

from Jackson Labs (Bar Harbor, ME) for flank tumor studies. Animals usedfor all experiments were between 6 and 10 weeks old and were housed in

the animal facility at Wistar Institute (Philadelphia, PA). All protocols were

approved by the Animal Use Committees of the Wistar Institute and

University of Pennsylvania and were in compliance with the guide and careand use of animals.

Cell lines. The murine lung cancer cell line ‘‘LKR’’ was obtained as a gift

from Dr. Joseph Friedberg of the University of Pennsylvania School of

Medicine. These cells were derived from an explant of a pulmonary tumorfrom an activated K-rasG12D mutant mouse grown in Dr. Tyler Jacks’ Lab

at M.I.T. (ref. 28; Boston, MA). Cells were cultured and maintained in high

glucose DMEM (Mediatech, Washington, DC) supplemented with 10% fetalbovine serum (Georgia Biotechnology, Atlanta, GA), 2 mmol/L glutamine,

and 1% penicillin/streptomycin.

To determine levels of MHC class 1 expression, the LKR cells were

analyzed by flow cytometry. LKR cells were left untreated or exposed to50 MOI (multiplicity of infection) of Ad.LacZ, 10 MOI of Ad.IFNh, or 50 MOI

of Ad.IFNh for 48 hours. At this time, the cells were harvested and subjected

to fluorescence-activated cell sorting analysis using a rat anti-mouse

FITC-conjugated monoclonal antibody directed against the H-2Kb molecule(BD PharMingen, San Diego, CA), which is reactive in C57/B6 mice and

expressed on these F1 hybrid mice.

Lung tumor model. To induce tumors, 100 AL of saline containing3 � 1010 particles of Ad.Cre (see below) was given to each LSL-K-rasG12D

mice intranasally. Preliminary studies showed this route to be less

traumatic and equally effective as intratracheal administration. Virus was

suspended in serum-free DMEM medium (which contains 125 mg/L ofNaH2PO4). Fifteen minutes prior to injection of the adenovirus, 0.5 AL of

2 mol/L CaCl2 was added (23). Calcium phosphate coprecipitation has been

shown to improve lung gene transfer (29). Animals were closely observed

daily for signs of distress. When they appeared lethargic, had ruffled fur, orincreased breathing rates, the animals were sacrificed. Massive tumor

infiltration of the lung was confirmed by histology.

Flank tumor models. In pilot studies, we tested 5 � 105, 1 � 106, and

2 � 106 LKR cells. Although all doses formed tumors, the 2 � 106 dose hadkinetics most consistent with our other tumor models and most

consistently formed uniform tumors and was thus selected for further

studies. To create peripheral tumors, mice were thus injected with 2 � 106

LKR cells s.c. in the flanks of C57BL/6J F � 129P3/J M F1 hybrid mice or

transgene-negative transgenic mice. Tumors were measured twice weekly

and volumes were estimated using the formula 3.14 � [largest diameter �(perpendicular diameter)2] / 6.

Immunogenicity tests for murine tumors. The immunogenicity of the

murine lung cancer cell line LKR was tested. Irradiated (50 Gy) tumor cells

(2� 106) were inoculated on one flank of naı̈ve mice followed by challenge by

2� 106 live tumor cells on the opposite flank 14 or 25 days later. As a control,

live tumor cells (2� 106) were inoculated into the flanks of naı̈ve mice at the

same time as the immunized mice received live tumor cell injections.Recombinant adenoviral vectors. The first-generation E1/E3-deleted

type 5 Adenoviral vector encoding the murine IFN-h (Ad.IFNh) gene has

been described (18). A similar E1/E3-deleted Ad.LacZ control virus was

purchased from the University of Pennsylvania Vector Core. The Ad.Cre

virus was provided by Dr. David Tuveson (23). Vector preparations were

shown to be negative for the presence of wild-type adenovirus. We ascribed

the particle to the plaque-forming unit (pfu) ratio of each preparation in

293 cells; this ranged between 50:1 and 100:1.

We evaluated the amount of IFN-h generated by Ad.IFNh after infection

of LKR cells in vitro . LKR cells were seeded in six-well plates and transfected

with Ad.IFNh at various MOI’s. Twenty-four hours after infection, the

supernatants were collected and evaluated using a mouse IFN-h ELISA kit(RDI, Flanders, NJ) following the manufacturer’s protocol. In addition,

flank tumors were collected 3 days after intratumoral injection with 109 pfu

of Ad.IFNh or AdLacZ. Tumors were homogenized, protein levels

determined, and IFN-h detected using the same ELISA kit.

Treatment of tumor-bearing mice with Ad.IFNB. Each LSL-K-

rasG12D mouse was injected intranasally at 21 and 28 days post-Ad.Cretreatment with 1 � 109 pfu Ad.IFNh or Ad.LacZ diluted to 100 AL of DMEM

(125 mg/L NaH2PO4) and mixed with 0.5 AL of 2 mol/L CaCl2. In flank

tumor studies, mice treated with Ad.IFNh received a single intratumoral

dose of 1 � 109 pfu, when flank tumors reached a volume of at least200 mm3.

In vitro cytotoxicity assay. We evaluated the antiproliferative effect of

Ad.IFNh on LKR cells in vitro . LKR cells were seeded in six-well plates andtransfected with Ad.IFNh at various MOI’s. After 6 hours, the cells were

transferred to 96-well plates and evaluated for cell number at 48 and

72 hours after virus exposure. As a positive control for virus effect, another

cell line (AB12, a mouse mesothelioma cell line) known to be sensitive toAd.IFNh (18), was also used. Cancer cell viability was assessed using an MTS

assay, which is a colorimetric test for the quantification of cell viability and

proliferation (CellTiter 96 AQueous nonradioactive MTS cell proliferation

assay, Promega Corp., Madison, WI).To further verify that the cell-killing was due to INF-h, rather than viral

effects, the experiment was repeated, with the addition of 10 Ag/mL of a

neutralizing anti-mouse IFN-h antibody (Associates of Cape Cod, Inc., EastFalmouth, MA) to untreated cells and cells exposed to 50 MOI of Ad.IFNh.Cell proliferation was measured 24 hours after the addition of virus and

antibody using the MTS assay.

Preparation of lymphokine-activated killer cells. To generate

lymphocyte activated killer (LAK) cells with NK cell activity, spleen cells,

at a concentration of 3 � 106/mL, were cultured in RPMI 1640

supplemented with 10% fetal bovine serum, 100 units/mL of penicillin,

100 Ag/mL streptomycin, 10 mmol/L HEPES buffer, 2 mmol/L glutamine,

10 mmol/L pyruvate, and 50 Amol/L h-mercapthoethanol containing

20 ng/mL of recombinant mouse interleukin-2 (R&D Systems Inc., Minnea-

polis, MN) for 5 days. We added 20 ng/mL of interleukin-2 every other day

to the cells.51Cr-release assay. The cytotoxic activity of LAK cells on LKR cells was

determined by the 4-hour 51Cr-release assay. Target cancer cells, LKR andYAC-1 (positive controls for LAK/NK cell cytotoxicity), were labeled with

100 ACi Na2-51CrO4 and washed thrice with PBS.

The 51Cr-labeled target cells were then cultured in triplicate with LAK/

NK (effector cells) cell suspensions in round-bottomed microtiter platesfor 5 hours. The effector to target ratios were 6:1 and 50:1. The percentage

of cytotoxicity was calculated via the following equation (all 51Cr values

in cpm): cytotoxicity (%) = (test 51Cr release � spontaneous release) /(maximum release � spontaneous release) � 100.

Measurement of CTL activity (Winn assay). Winn assays were done

as described previously (30). Splenocytes were isolated and CD8+ T

lymphocytes were purified using the MACs system (Miltenyi Biotec,Auburn, CA). This cell population contained >90% CD8+ cells by flow

cytometry (data not shown). The CD8+ T lymphocyte-enriched populations

were obtained from naı̈ve mice, mice bearing LKR tumors (200-300 mm3 in

size), and mice bearing LKR tumors that were treated 10 days earlier withAd.IFNh. These CD8+ T cells were mixed with naı̈ve viable LKR tumor cells

or tumor cells pretreated with IFN-g ( from BD Biosciences, San Diego, CA;

10 ng/mL for 3 days) at a ratio of three purified CD8+ splenocytes for eachtumor cell, and the mixture was inoculated into the flanks of naı̈ve mice.

Each mixture thus contained 0.5 � 106 tumor cells and 1.5 � 106 CD8+ T

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cells. This ratio has previously been determined to be optimal for detectingpositive and negative effects (21). Tumor growth was measured after 1 week

and expressed as the mean F SE of at least five mice per group.

In vivo depletion of CD4+, CD8+ T cells and natural killer cells. Todeplete specific lymphocyte populations in our model, mice were injectedi.p. with monoclonal antibodies purified from the anti-CD4 hybridoma

GK1.5 or the anti-CD8 hybridoma 53-6.7 (American Type Tissue Culture

Collection, Manassas, VA). Mice were given 300 Ag of purified antibody i.p.

dissolved in 200 AL of PBS for CD4+ and CD8+ antibodies. For NK celldepletion, 100 Ag of polyclonal rabbit anti-asialo GM1 antibody (Wako

Chemicals, Richmond, VA) was used. Antibodies were given 1 day before

and 1 day after treatment injection. Thereafter, a maintenance dose of

300 Ag for CD4+ and CD8+ cell depletion or 100 Ag for NK cell depletion wasdelivered i.p. every sixth day to ensure depletion of targeted lymphocyte

population. Cell depletion was confirmed by flow cytometry of splenic cell

suspensions (data not shown). In addition, we did fluorescence-activatedcell sorting to confirm that the anti-asialo-GM1 antibody did not deplete

CD8+ T cells (data not shown).

Statistical analysis. Unless otherwise noted, data comparing differences

between groups were assessed using one-way ANOVA with appropriate posthoc testing. Survival studies were assessed using Kaplan-Meier survival

curves and analyzed with the Mantel-Cox log rank test. Differences were

considered significant when P value was <0.05.

Results

Establishment of orthotopic model. Using techniques origi-nally described by Jackson et al. (23) and described above, LSL-K-rasG12D–positive mice were injected with Ad.Cre intranasally andtumor progression and survival were followed. Under ourconditions, mice started dying about 1 month after Ad.Creadministration and all of the mice were dead between 47 and 57days (Fig. 1). Mice that were sacrificed to look at tumor progressionat day 21 after Ad.Cre administration (time of the first Ad.IFNhtreatment) showed small adenomas and some small adenocarci-nomas (Fig. 2A). By day 35 after Ad.Cre administration, lungsections showed significant portions of lung replaced by tumor(Fig. 2B). By day 50 after Ad.Cre administration, almost the entiremouse lung was replaced by tumor (Fig. 2C).Survival study of orthotopic lung cancer model treated with

Ad.IFNB. To determine the efficacy of Ad.IFNh on survival in thismodel, 24 LSL-K-rasG12D–positive mice were treated with Ad.Creintranasally at time 0 (control). Ad.LacZ (n = 8) or Ad.IFNh (n = 8)was given intranasally at 21 days, and then again, 28 days after

Ad.Cre administration. One group of mice (n = 8; control) receivedno treatment. The mice were followed until sacrifice (see Materialsand Methods for criteria). Two independent experiments weredone. In the first experiment (Fig. 1A), all mice in the control groupdied by day 57. In the group that received Ad.LacZ, survival wassignificantly prolonged, but all mice were dead by day 77 (P =0.0002 versus control). In the group that received Ad.IFNh, all of themice survived >120 days (P < 0.0001 versus control; P < 0.0001versus Ad.LacZ group). In the second experiment (Fig. 1B), all micein the control group died by day 46. In the group that receivedAd.LacZ, all mice died by day 66 (P = 0.007 versus control). In thegroup that received Ad.IFNh, one mouse died at day 27, theremainder survived >120 days (P < 0.0014 versus control; P < 0.001versus Ad.LacZ group).Characterization of the LRK cell line. To explore the

mechanisms of this striking therapeutic effect, we took advantageof the existence of a murine lung cancer cell line (LKR) that hadbeen derived from an explant of a pulmonary tumor arising in anactivated K-rasG12D-mutated mouse (28).We first analyzed the sensitivity of the line to Ad.IFNh infection.

LKR cells were transduced with different MOIs of Ad.IFNh vectorand the amount of IFN-h generated 24 hours after transductionmeasured using a commercial ELISA kit. Whereas control LKR cellsand cells transduced with control Ad.LacZ produced no detectableIFN-h, we found large and increasing amounts of IFN-h withincreasing MOI of Ad.IFNh. Cells transduced with 10 MOI of IFN-hgenerated 9.6 � 104 pg/mL/24 hours/106 cells of IFN-h and 50 MOIof IFN-h generated 1.4 � 106 pg/mL/24 hours/106 cells of IFN-h; 50MOI of Ad.LacZ generated 0 pg/mL/24 hours of IFN-h.We next evaluated the direct cytotoxic effects of Ad.IFNh on LKR

cells in vitro , using the previously studied AB12 mesothelioma cellline as a sensitive cell line control. LKR and AB12 cells wereinfected with 0, 10, 50, or 100 MOI of Ad.IFNh and assayed after48 hours. Both cell lines were sensitive to the cell-killing effects ofAd.IFNh with the LD50 of both cell lines between 10 and 50 MOI(Fig. 3A). However, LKR cells infected with up to 250 MOI ofAd.LacZ showed minimal cytotoxicity. This experiment wasrepeated with similar results.To confirm that this effect was due to the IFN-h produced (rather

than on nonspecific viral effects), we repeated the experiment, butanalyzed cell numbers 24 hours after vector transduction in the

Figure 1. Ad.IFNh treatment results in significant survival benefit in the orthotopic mouse model of bronchioloalveolar lung cancer. LSL-K-rasG12D–positive micewere given Ad.Cre recombinase intranasally at day 0. Mice were then either left untreated (control; n = 8) or given Ad.IFNh (1 � 109 pfu; n = 8) or Ad.LacZ (1 � 109 pfu;n = 8) intranasally at day 21 and then again at day 28 (arrows ). Mice were then followed for survival. A and B, two separate, independent experiments.

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presence or absence of a neutralizing anti-IFN-h antibody. Asshown in Fig. 3B , we found significant (P < 0.05) cell killing with 50MOI of Ad.IFNh, however, this killing was completely blocked in thepresence of the antibody.

We next evaluated the ‘‘immunogenicity’’ of the cell line usingthe classical prophylactic immunity assay. Two million LKR cellswere irradiated and injected s.c. into the flanks of naı̈ve mice(n = 6). Fourteen and 25 days later, the same number ofnonirradiated LKR cells were injected s.c. into flanks of the micethat received the irradiated cells and into naı̈ve mice (n = 6). Thetumor cells grew equally well in the flanks of the ‘‘vaccinated’’ miceand control mice (data not shown). This lack of inhibition of tumorgrowth is characteristic of ‘‘nonimmunogenic’’ tumors.Given this lack of immunogenicity, we measured MHC I

expression in the LKR cell line before and after treatment withAd.IFNh. As shown in Fig. 3C , we found very low levels ofexpression of H-2Kb at baseline or after exposure to Ad.LacZ. Incontrast, Ad.IFNh at doses of 10 and 50 MOI markedly increasedMHC I expression in the LKR cells.Finally, we evaluated the sensitivity of these cells to NK cell lysis.

LKR cells and the NK-cell sensitive line, YAC-1, were labeled withradioactive chromium, combined with activated NK cells at ratiosof 1:6 and 1:50, and the extent of chromium release determined.As shown in Fig. 3D , marked lysis of YAC-1 and LKR cells was seenat both NK cell/tumor ratios. Consistent with their low levels ofMHC class I expression, LKR cells are extremely sensitive to NK celllysis.Re-challenge of cured animals with LKR tumor cells. To

determine if an immunologic memory response was generated inthe LSL-K-rasG12D–positive mice that were cured by Ad.IFNhtreatment, we re-challenged some of these cured mice (96 daysafter their initial treatment with Ad.IFNh) with 2 million LKRcells injected into their flank. Naı̈ve mice of the same age werealso injected with the same number of LKR cells to serve aspositive controls. Tumor volumes were compared over 25 days.Tumor growth was significantly inhibited (P < 0.001) in the micepreviously treated with Ad.IFNh (Fig. 4). At day 25, the naı̈ve micehad an average tumor volume of 422 F 35 mm3 (n = 5);compared with the cured mice who had an average tumor volumeof 109 F 18 mm3 (n = 5). This experiment was repeated withsimilar results.Efficacy of Ad.IFNB treatment on LKR flank tumors. To

determine if the cell line grown as a flank tumor model wouldbehave similarly to the orthotopic model, mice were injected with2 million LKR cells in each flank. When the tumors reachedf200 mm3, mice were left untreated or given one dose of eitherAd.LacZ or Ad.IFNh (1 � 109 pfu) intratumorally. We confirmedthat the virus produced IFN-h within tumors by making homo-genates from each group and performing ELISA assays. We foundno detectable IFN-h in control tumors or tumors treated withAd.LacZ; however, low, but clearly detectable levels (16 pg/mg oftotal tumor protein) of Ad.IFNh was measured in homogenates oftumors treated with Ad.IFNh.As shown in Fig. 5 at day 36, the untreated mice had an average

tumor volume of 703 F 35 mm3 (n = 5) compared with theAd.LacZ-treated mice who had an average tumor volume of 470 F42 mm3 (n = 5; P < 0.001). However, the mice treated with Ad.IFNhhad complete tumor regressions with an average tumor volume of2 F 2 mm3 (n = 5; P < 0.001). This experiment was repeatedmultiple times with similar results (i.e., see controls for immunecell depletion studies below).Re-challenge of cured flank tumor animals with LKR tumor

cells. To determine if an immunologic memory response wasgenerated using the flank tumor model, as it was in the orthotopicmodel, mice with ‘‘cured’’ LKR flank tumors were re-challenged

Figure 2. Lung sections from mice treated with Ad.Cre recombinase. Mice weregiven Ad.Cre recombinase intranasally on day 0 and were sacrificed at varioustime points. The lungs were harvested, sectioned, and stained with H&E.A, histology (100�) at day 21. Note the scattered areas of adenoma formationwith some satellite areas of carcinoma. B, histology (100�) at day 35. Notethe more diffuse areas of adenoma formation with more areas of carcinoma.Also note that there are still areas of normal lung present in the section.C, histology (100�) day 50. Note the widespread sheets of carcinoma. Most ofthe lung has been replaced by tumor.

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with 2 � 106 LKR cells injected into their contralateral flank38 days after initial treatment with Ad.IFNh. Naı̈ve mice (controls)were also injected with 2 � 106 LKR cells to serve as positivecontrols. At day 24, the naı̈ve mice had an average tumor volume of514.9 F 85 mm3 (n = 5); compared with the cured mice whoshowed no tumor growth at all (n = 5; P = 0.0003). This experimentwas repeated with the same number of mice and similar resultswere obtained.Treated mice generate CTLs. To confirm that this immuno-

logic memory was due to the generation of CTLs, we conductedWinn assays using CD8+ T cells isolated from splenocytes fromcontrol mice, tumor-bearing mice, and tumor-bearing mice treatedwith Ad.IFNh. When these CD8+ T cells were mixed with 5 � 105

LKR cells and injected into the flanks of naı̈ve animals, the tumorssize after 6 days was equal in all groups (146 F 15, 141 F 10, and151 F 10 mm3, respectively) and virtually identical to tumorsgenerated by injection of 5 � 105 LKR cells alone (150 F 11 mm3).Because of the very low MHC class I expression on LKR cells,however, we also mixed the CD8+ T cells with 5 � 105 LKR cells thathad been pretreated with 10 ng/mL of IFN-h (in vitro) to up-regulate MHC class I and injected these mixtures into naı̈ve mice.Pretreatment with IFN-h did not affect tumor growth (154 F10 mm3). The presence of CD8+ T cells from naı̈ve or tumor-bearingmice also did not affect tumor growth (142 F 15 and 134 F15 mm3, respectively). However, addition of the CD8+ T cells fromthe tumor-bearing, Ad.INFh-treated mice significantly (P < 0.01)inhibited the growth of the IFN-h-pretreated LKR cells (5 F3 mm3). This experiment shows that CTL were generated byAd.IFNh treatment, however, they were unable to kill LKR cellsunless MHC class I was up-regulated.Immune cell depletion during Ad.IFNB treatment. To

determine the immunologic mechanisms responsible for the strongAd.IFNh-induced antitumor response, specific immune cell subsetswere depleted using antibodies. Mice were injected with 2 � 106

LKR cells in one flank. When tumors reached a size of f200 mm3,groups of mice were given antibodies to deplete CD4+ T cells, CD8+

T cells or NK cells (see Materials and Methods). Twenty-four hourslater, mice were given one dose of Ad.IFNh (109 pfu) intratumorally.

The appropriate antibody was then given 24 hours after theAd.IFNh, and weekly thereafter. Adequate and specific depletion ofeach subset was confirmed by flow cytometry of spleen cells (datanot shown).

Figure 3. Characterization of the LKR cell line. A, in vitro sensitivity to Ad.IFNh.LKR cells, along with AB12 cells (a mouse mesothelioma cell line serving as apositive control) were exposed to increasing concentrations of Ad.IFNh for48 continuous hours of exposure. LKR cells were also exposed to Ad.LacZ totest the direct toxicity of the adenovirus. An MTS assay was done. LKR andAB12 cells were killed by relatively low concentrations of Ad.IFNh, whereascontrol adenovirus resulted in minimal cytotoxicity. B, use of an antibody to IFN-hto establish specificity of the cytotoxic effect of Ad.IFNh. LKR cells wereexposed to 50 MOI of Ad.IFNh for 24 hours in the presence or absence of aneutralizing mouse antibody to IFN-h (AB). LKR cells were also exposed toAd.LacZ to test the direct toxicity of the adenovirus. An MTS assay was done.About 50% of cells were killed by Ad.IFNh, however, the anti-IFN-h antibodycompletely blocked this response. Control adenovirus resulted in minimalcytotoxicity. C, MHC I expression on LKR cells: LKR cells were exposed toincreasing concentrations of Ad.IFNh to 24 hours. The cells were then harvestedand evaluated using flow cytometry. Baseline levels of MHC I expression werevery low, but increased markedly with increasing concentrations of Ad.IFNh.Control adenovirus resulted in minimal MHC I expression. MFI, mean fluorescentintensity in arbitrary units. The numbers in each tracing represent the percentageof positive cells. D, sensitivity of LKR cells to NK cell lysis. The cytotoxicactivity of activated NK (LAK) cells on LKR cells was determined by the 4-hour51Cr-release assay. Chromium-labeled target LKR cells and YAC-1 cells(positive controls for LAK cell cytotoxicity) were cultured in triplicate with LAK(effector cells) cell suspensions for 5 hours; the effector to target ratio was6:1 and 50:1, respectively. The percentage of cytotoxicity was calculatedvia the following equation (all 51Cr values in cpm): cytotoxicity (%) =(test 51Cr release � spontaneous release) / (maximum release � spontaneousrelease) � 100. LKR cells were extremely sensitive to NK cell–mediated lysis.

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Figure 6A shows that depletion of CD4+ T cells had no effect onthe growth of the tumor. In addition, depletion of CD4+ T cells didnot decrease the efficacy of Ad.IFNh. The Ad.IFNh-treated mice(n = 5) had an average tumor volume of 40 F 7 mm3 (P < 0.001compared with control) whereas the Ad.IFNh + CD4+ antibodygroup (n = 5) had an average tumor volume of 51 F 6 mm3 (P <0.001 compared with control; P = 0.99 compared with Ad.IFNh).This experiment was repeated with the same number of mice andsimilar results were obtained.Figure 6B shows that depletion of CD8+ T cells had no effect on

the growth rate of the tumors, an observation that is consistent withthe previous finding that LKR cells are nonimmunogenic. CD8+ Tcell depletion did diminish the effect of Ad.IFNh, however, theeffects were delayed. At an early time point after Ad.IFNh treatment(i.e., day 16, 4 days after treatment), tumor size was decreasedsignificantly (P < 0.05), but similarly in animals treated withAd.IFNh alone (114 F 22 mm3) and in the Ad.IFNh/CD8+

T cell–depleted animals (145 F 9.2 mm3) when compared withcontrols (275 F 28 mm3). However, after day 20, the tumors in theAd.IFNh/CD8-depleted mice began to grow rapidly, at the same rateas control tumors, whereas the mice treated with Ad.IFNh alonecontinued to decrease in size. By day 28, the Ad.IFNh-treated mice(n = 5) had an average tumor volume of 44 F 4 mm3; whereas theAd.IFNh/CD8+ T cell–depleted group (n = 5) had an average tumorvolume of 323 F 25 mm3 (P = 0.045). The experiment was repeatedwith the same number of mice and similar results were obtained.Figure 6C shows that depletion of NK cells did not affect the

growth of the tumors. However, depletion of NK cells had an effecton the Ad.IFNh-treated mice, but in a different pattern. In contrastto CD8+ T cell depletion that affected the later response toAd.IFNh, NK cell depletion led to loss of the initial response tothe Ad.IFNh. Thus, 4 days after treatment (day 20), the tumors inthe Ad.IFNh/NK cell–depleted group (324 F 27 mm3) were notsignificantly different in size from control tumors (393 F 25 mm3),whereas the tumors in Ad.IFNh group were significantly (P < 0.001)smaller (130 F 20 mm3). After that point, however, the tumorgrowth slowed and actually decreased, with a slope similar to thatof the Ad.IFNh group. By 31 days, the size of tumors in theAd.IFNh/NK cell–depleted group (257 F 41 mm3) was significantlysmaller (P < 0.005) than the control group (885 F 92 mm3).

Discussion

Type I IFN’s stimulate both the innate and adaptive immunesystem and are thus critical mediators in antiviral and antitumoractivity (3, 5, 31). In addition to directly suppressing tumor cellreplication and inducing differentiation or apoptosis (4, 32, 33),type I IFN’s can: (a) stimulate NK cell–mediated tumor lysis (31),(b) induce NK cell proliferation (34), (c) stimulate macrophagesand enhance their tumoricidal properties (8, 35, 36), (d) up-regulateMHC I expression (37), and (e) promote antitumor T cell responsesvia stimulation of memory phenotype subsets and prolongingsurvival of activated populations (38, 39). Type I IFNs havealso been shown to reduce tumor growth via antiangiogenicproperties (40).Despite these impressive potential antitumor activities, admin-

istration of IFN-a and IFN-h proteins have not been very effectivein the treatment of solid tumors, probably because the short half-life requires high doses that result in toxic side effects. In contrast,evidence is mounting that gene therapy approaches for the deliveryof type I IFN’s are both well-tolerated and have much higherefficacy. Successful preclinical experiments transferring the murineIFN-h gene using liposomes or adenovirus vectors have beenreported in immunocompetent animal models of gliomas (14, 41),colorectal liver metastases (17), fibrosarcomas (13), and models ofmalignant mesothelioma (18–20).The data presented here extends these findings by demonstrat-

ing remarkable in vivo antitumor activity of Ad.IFNh in anorthotopic tumor model of bronchogenic lung cancer. In twoindependent studies, intrapulmonary administration of two dosesof Ad.IFNh into animals with well-established tumors led to 90% to100% long-term survivals.An important component of these experiments was the use of an

orthotopic model of lung cancer where tumors arose in the distalairways, much like actual lung carcinomas. This can be contrastedto other lung cancer models where tumor cells are injected i.v.—models that actually more closely mimic distant metastatic diseaserather than primary lung cancer. Although there are aspects of theLSL-K-rasG12D model that are challenging (i.e., generating a

Figure 4. ‘‘Cured’’ mice have immunologic memory towards LRK tumors.LSL-K-rasG12D–positive mice cured by treatment with Ad.IFNh were challengedwith 2 � 106 LKR cells injected in their flank. As controls, naı̈ve mice were alsochallenged with the same number of tumor cells. At 25 days after tumorinjection, the tumor sizes were measured. Columns , mean volume (mm3); bars ,SE. Cured mice had significantly smaller tumors (P < 0.001).

Figure 5. Ad.IFNh has significant antitumor effect in LKR flank tumors. Effect ofAd.IFNh on established flank tumors. Immunocompetent mice (five mice/group)with established LKR flank tumors (f200 mm3 in size, 14 days after tumorinoculation) were treated with either saline (control) or one dose of Ad.IFNh orAd.LacZ intratumorally. Points , mean volume (mm3); bars , SE; , control;

, LacZ; , IFN-h. Ad.IFNh led to complete regression of tumors.

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breeding colony and the difficulty in following tumor growthnoninvasively), the use of an orthotopic lung cancer model such asthis one, in which the pathology and anatomic localization of thetumors closely resembles the human counterpart, has a number ofadvantages for gene therapy studies. First, the immune environ-ment of the lungs is distinctive. For example, the proximity of lungcancers to normal bronchial and alveolar epithelial cells and totumor-associated alveolar macrophages is likely important. Asthe primary site for naturally occurring adenoviral infection, theimmune response generated by the adenoviral vector is verydifferent in the lungs than it would be in a flank tumor. Second, thephysical requirements of treating lung cancer are unique. Lungcancers, especially bronchioloalveolar lung cancers are diffuse andmultifocal, requiring treatment by intratracheal or intrabronchialinstillation of vector. This approach cannot be modeled well usingdirect injection of flank tumors. Third, the molecular trigger of thetumors (mutation in K-ras) in this model is also present in a largepercentage of human lung adenocarcinomas (42). Fourth, the paceof the growth of the tumors is this model is relatively slowcompared with most flank tumor models (and can actually bemodulated), more closely resembling human tumors.One advantage of this particular orthotopic model for thera-

peutic studies, compared with models where tumors arisespontaneously (21, 43, 44), is the ability to initiate tumor formationat a specific time point by using intrapulmonary instillation ofAd.Cre. This allows tumor formation to be controlled and uniform.On the other hand, one potential disadvantage of using Ad.Cre to

initiate tumors is that instillation of an adenoviral vector is knownto sensitize mice to subsequent injections of adenovirus (21, 22,24, 25), thus potentially making subsequent use of therapeuticadenoviral vectors ineffective. Although we did not specificallymeasure humoral or cellular responses to adenovirus, we observedno clinical evidence of toxicity when we sacrificed animals shortlyafter intrapulmonary instillation of Ad.LacZ or Ad.IFNh into theadenovirus-sensitized tumor-bearing mice and examined their lungtissue. However, we did note a brisk inflammatory response withinfiltration of macrophages and neutrophils (data not shown). Webelieve this strong immune response was responsible for thesignificant, but temporary prolongation of survival that weobserved in the Ad.LacZ-treated animals compared with untreatedcontrols (Fig. 1). It is intriguing to note that a previous humanclinical trial for lung cancer using intratumoral injection ofAd.LacZ observed some hint of clinical response (45, 46). Becauseit is known that most humans have been exposed to adenoviruses,the efficacy we see in this model, even though all the animals hadbeen previously exposed to adenovirus, is encouraging fortranslation to clinical trials.Given the strong therapeutic effects of Ad.IFNh in the lung

model and the multiple potential pathways by which IFN-h mightbe acting, it was of interest to dissect some of the mechanismsinvolved. Because of the limited numbers of mice that could begenerated for lung treatment studies, the difficulty in monitoringefficacy of treatment other than by survival studies, and theexperimental limitations imposed by being able to use only

Figure 6. IFN-h’s effects are attenuated by antibodies to CD8+ T cells and NK cells, but not to CD4+ T cells. Immunocompetent mice (five mice/group) withestablished LKR flank tumors (f200 mm3 in size, 12-16 days after tumor inoculation) were treated with either saline (control) or one dose of Ad.IFNh intratumorally.A, CD4+ T cell depletion. Mice were treated with antibodies to CD4+ T cells (arrows ) on day 15 and day 17, 1 day prior and 1 day after treatment with Ad.IFNh(day 16, arrowhead). These antibody-treated mice also were boosted with the antibody at days 23 and 30. Points , mean volume (mm3); bars , SE. Tumor volumeswere significantly smaller in Ad.IFNh-treated groups versus control groups. Anti-CD4 antibodies had no effects. B, CD8+ T cell depletion. Mice were treated withantibodies to CD8+ T cells (arrows ) on days 11 and 13, 1 day prior and 1 day after treatment with Ad.IFNh (day 12, arrowhead). The antibody-treated mice also wereboosted with the antibody at days 19 and 26. CD8+ T cell depletion inhibited the late, but not early, effects of Ad.IFNh. C, NK cell depletion. Mice were treated withan antibodies to NK cells (arrows) on days 15 and 17, 1 day prior and 1 day after treatment with Ad.IFNh (day 16, arrowhead). These antibody-treated mice alsowere boosted with the antibody at days 23 and 30. NK cell depletion inhibited the early, but not late, effects of Ad.IFNh.

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endogenously arising tumors, we took advantage of a cell line(LKR) that was derived from a tumor that had developedspontaneously in a K-rasG12D-positive mouse. This cell line grewwell in the flanks of C57BL/6J F � 129P3/J M F1 mice, the samestrain as the LSL-K-rasG12D–positive mice. We first obtained someinformation about the nature of the tumor cells and were able toshow that: (a) Ad.IFNh could directly inhibit LKR cell growthin vitro (Fig. 3A), (b) these cells were not ‘‘immunogenic’’ becausevaccination of mice with irradiated tumor cells did not prevent thesubsequent growth of injected tumor cells and depletion of CD8+

T cells did not enhance growth (Fig. 6B), (c) the cells expressed lowlevels of MHC class I antigen at baseline that could be up-regulatedby IFN-h (Fig. 3C), and (d) the cells were quite sensitive to NKcell–mediated lysis (Fig. 3D).We used these cells in a number of experiments. Cells were

injected into ‘‘cured’’ mice to determine if an immunologic memoryhad been produced. As shown in Fig. 4, there was, in fact, asignificant slowing of the growth of the cells when injected intopreviously treated lung cancer mice. The ability of the cells to growto a limited degree in these treated mice might be due to the largenumber of cells used to ‘‘challenge’’ the mice. It is also possible thatthe cultured cells had some slight immunologic differences fromthe endogenous tumor and were thus able to escape partially fromimmune surveillance. We also used the cells in a flank tumormodel. Like the lung tumors, the cells growing in the flanks werehighly sensitive to treatment by intratumoral injection of Ad.IFNh(Fig. 5A). By depleting specific lymphocyte populations, we wereable to show that efficacy was not dependent on CD4+ T cells(Fig. 6A). Instead, we saw that early inhibition of tumor growth wasmediated by NK cells (Fig. 6C), whereas late tumor growthinhibition required CD8+ T cells (Fig. 6B). We also directly showedgeneration of cytotoxic CD8+ T lymphocytes, although interestingly,these CTL required up-regulation of MHC class I on the LKR tumorcells to induce cell lysis. In vivo , it is likely that such MHC class Iup-regulation was induced directly by IFN-h from the vector, orindirectly by IFN-g release by NK cells.Although it is well-established that Ad.IFNh can have effects on

both NK cells and CD8+ T cells, this is the first example, to ourknowledge, of a tumor where both activities were required for fullantitumor activity. In our previous studies of Ad.IFNh inmesothelioma, we found that efficacy was almost exclusively dueto CD8+ T cells (18, 19). In a fibrosarcoma model, immunologicmemory was observed and it was found that the antitumor effects

of Ad.IFNh were completely lost in severe combined immunode-ficiency mice, suggesting complete dependence on T cells (13).Natsume et al. also saw immunity to reinjected tumor cells andfound that administration of anti-CD8 monoclonal antibodiesblocked the antitumor cytolytic activity of Ad.IFNh in theirglioma model (41). However, NK cell–mediated cell killing hasalso been observed to be dominant in other models. For example,Tada et al. (17) showed a strong NK cell–mediated component intheir studies with colorectal tumors. Some of these differencesmay be due to the sensitivity of the tumors to NK cell lysis. Forexample, although we did see a marked influx of NK cells in theperitoneal fluid of tumor-bearing mice treated with Ad.IFNh, themesothelioma cell lines used in our study were almost completelyresistant to NK cell–mediated lysis, unlike the LKR cells used here(18, 19).In conclusion, this study shows that adenovirus-mediated IFN-h

gene therapy can induce a potent antitumor effect in a poorlyimmunogenic orthotopic model of bronchogenic NSCLC thatresembles the human condition. We found that this profoundantitumor effect was initially due to natural killer cells, but wasthen followed by the generation of cytotoxic CD8+ T cells. Blockingeither of these pathways with monoclonal antibodies attenuatedthe therapeutic effects. Our group is currently conducting a phase Itrial for malignant mesothelioma and metastatic pleural diseaseusing intrapleural administration of Ad.human IFNh. Early resultshave shown that the approach is safe and both clinical andimmunologic responses have been seen. Given the results from thisstudy and the lack of effective therapy for lung cancer, we proposethat Ad.IFNh should be further investigated in the treatment ofhuman lung cancer. Given the lack of suitable therapeutic optionsand the especially favorable anatomy for intrapulmonary instilla-tion, bronchioalveolar cell carcinoma might be an especiallyattractive target.

Acknowledgments

Received 3/21/2005; revised 6/16/2005; accepted 7/11/2005.Grant support: National Cancer Institute PO1 CA 66726.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank David Tuveson, M.D., Ph.D. and Tyler Jacks, Ph.D. for generouslyproviding us with the initial breeding pair of Lox-Stop-Lox K-rasG12D–positive mice,Joseph Friedberg, M.D. for providing us with the LKR cell line, and Jennifer Brown ofBiogenIdec for assistance in measuring IFN-h levels.

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IFN-b Gene Therapy for an Orthotopic Lung Cancer Model

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2005;65:8379-8387. Cancer Res   Michael J. Wilderman, Jing Sun, Arminder S. Jassar, et al.   Bronchogenic Adenocarcinoma of the LungVector Is Highly Effective in a Murine Orthotopic Model of

Gene Therapy Using an AdenoviralβIntrapulmonary IFN-

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