[CANCER RESEARCH 61, 6201– 6212, August 15, 2001]
Eradication of Intraperitoneal and Distant Tumor by Adenovirus-mediated
Interferon-␤ Gene Therapy Is Attributable to Induction of
Systemic Immunity1
Makoto Odaka, Daniel H. Sterman, Rainer Wiewrodt, Yi Zhang, Michael Kiefer, Kunjlata M. Amin, Guan-Ping Gao,
James M. Wilson, James Barsoum, Larry R. Kaiser, and Steven M. Albelda2
Thoracic Oncology Research Laboratory, Division of Pulmonary, Allergy, and Critical Care Medicine [D. H. S., R. W., M. K., S. M. A.], Department of Surgery [M. O., K. M. A.,
L. R. K.], Institute for Human Gene Therapy [Y. Z., G-P. G., J. M. W.], University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, and Biogen, Inc.,
Cambridge, Massachusetts 02142 [J. B.]
ABSTRACT
Malignant mesothelioma remains an incurable disease for which im-
mune-modulatory therapies, such as exogenous cytokines, have shown
some promise. One such cytokine, IFN-␤, has potent antiproliferative and
immunostimulatory activity in vitro, but its in vivo use has been limited by
toxicity. We thus conducted studies evaluating intracavitary delivery of a
replication-deficient adenoviral (Ad) vector encoding for the murine
IFN-␤ gene (Ad.muIFN-␤) in mouse models of malignant mesothelioma.
In contrast to multiple injections of recombinant protein, a single i.p.
injection of Ad.muIFN-␤ into animals with established tumors elicited
remarkable antitumor activity leading to long-term survival in >90% of
animals bearing either AB12 or AC29 i.p. mesotheliomas. A control
adenovirus vector had minimal antitumor effect in vivo. Significant ther-
apeutic effects were also seen in animals treated with large tumor burdens.
Importantly, treatment of i.p. tumor also led to reduction of growth in
tumors established at a distant site (flank). A number of experiments
suggested that these effects were attributable to an acquired CD8ⴙ T-cell-
mediated response including: (a) the induction of long-lasting antitumor
immunity; (b) loss of efficacy of Ad.muIFN-␤ in tumor-bearing, immune-
deficient (SCID, SCID/beige) mice; (c) detection of high levels of specific
antitumor cytolytic activity from unstimulated splenocytes harvested from
Ad.muIFN-␤-treated animals that was abolished by CD8ⴙ T-cell deple-
tion; and (d) abrogation of antitumor effects of Ad.muIFN-␤ in tumor-
bearing CD8ⴙ T-cell-depleted animals. These data show that intracavitary
IFN-␤ gene therapy using an adenoviral vector provides strong CD8ⴙ
T-cell-mediated antitumor effects in murine models of mesothelioma and
suggest that this may be a promising strategy for the treatment of local-
ized tumors such as mesothelioma or ovarian cancer in humans.
INTRODUCTION
Malignant mesothelioma, an asbestos-related neoplasm of the pleu-
ral and peritoneal space, occurs in about 2000 –2500 patients/year in
the United States and in ⬃10,000 patients worldwide (1, 2). The
incidence, especially in Europe, is rising (3). Malignant pleural me-
sothelioma is unresponsive to chemotherapy and radiotherapy regi-
mens and typically recurs even after the most aggressive attempts at
surgical resection (4). Multimodality approaches have been of some
benefit in prolonging survival in very highly selected subgroups of
patients but have had a relatively small impact upon the majority of
the patients diagnosed with this disease (5). As the incidence of
pleural mesothelioma peaks over the next 10 –20 years, new thera-
peutic measures will be necessary.
Received 3/14/01; accepted 6/8/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by Grant NCI PO1 66726 and Specialized Program of
Research Excellence Grant P50-CA-83638 from the National Cancer Institute. Support
was also provided by the Benjamin Shein Foundation for Humanity. R. W. is a postdoc-
toral fellow of the Mildred Scheel Stiftung für Krebsforschung der Deutschen Krebshilfe
e.V. (D/98/02288). J. M. W. holds equity in Targeted Genetics.
2
To whom requests for reprints should be addressed, at University of Pennsylvania
Medical Center, Room 856, BRBII/III, 421 Curie Boulevard, Philadelphia, PA 19104.
Fax: (215) 573-4469; E-mail: Albelda@mail.med.upenn.edu.
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Despite repeated observations that mesothelioma is well known to
inhibit host cellular and humoral antitumor immune responses, there
may be a role for intrapleural immunotherapy in the treatment of this
refractory neoplasm (6, 7). Using animals models that closely mimic
the human disease (8), positive therapeutic results have been seen with
IFN-␣ (9), interleukin 2 (10), interleukin 12 (11), and antisense
TGF3-␤ treatment (12). Some of these findings in animal models have
been translated into clinical studies. In several Phase I/II clinical trials
conducted over the past decade, some antitumor efficacy has been
demonstrated with systemic and local administration of recombinant
human interleukin 2 (13, 14) and granulocyte/macrophage-colony
stimulating factor (15).
An area of especially active interest and promise in the treatment of
mesothelioma has been the administration of IFNs. IFNs have immu-
noregulatory effects upon antibody production, NK and T-cell acti-
vation, macrophage function, delayed-type hypersensitivity, and
MHC antigen expression (16 –19). They also have antiangiogenic
properties (20, 21), as well as direct antiproliferative effects (19, 22).
IFNs have been demonstrated to directly inhibit the cellular prolifer-
ation of several mesothelioma cell lines in vitro (23, 24), and antitu-
mor activity has been shown in animal models (9). Accordingly, both
type I IFNs (␣ and ␤) and type II IFN (IFN-␥) have been studied in
clinical trials, although the results have not proved as promising as
hoped. For example, s.c. administration of IFN-␣2a to patients with
mesothelioma yielded overall response rates of ⬍20% (25, 26). The
Southwest Oncology Study Group reported no clinical responses after
6 weeks of systemic IFN-␤ treatment in 14 patients with pleural
mesothelioma and but observed significant toxic side effects (27).
Improved responses were observed when intrapleural natural IFN-␤
was studied in 29 patients with malignant pleural effusions, 11 of
whom had “complete remission of pleural effusion,” including one
patient with mesothelioma (28). IFN-␥ has also been studied in the
treatment of mesothelioma, especially in the context of intrapleural
immunotherapy for mesothelioma. Some positive responses have been
seen, especially in patients with very early disease (29, 30).
One hypothesis for the relatively low response rates of IFNs in
antitumor therapy is that systemic delivery does not engender the
sustained intratumoral cytokine levels necessary for inducing antitu-
mor responses. For example, pharmacokinetic studies after i.v. injec-
tion have shown that the half-life of recombinant IFN-␤ is only ⬃5
min (31). A potential method for increasing local tumor cell exposure
to IFNs with minimal systemic side effects could be tumor cell
transduction with IFN genes. A number of investigators have tested
this hypothesis for IFN-␤ by stably transducing tumor cells with
IFN-␤ and showing marked decreases in tumorigenicity (32–34). This
idea has been extended recently to show that in vivo delivery of the
3
The abbreviations used are: TGF, transforming growth factor; Ad, adenoviral; tk,
thymidine kinase; RSV, Rous sarcoma virus; pfu, plaque-forming unit; MOI, multiplicity
of infection; NK, natural killer; CMV, cytomegalovirus; MTS, 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium; SCID, severe combined
immunodeficient.
 Ad.IFN-␤ FOR MESOTHELIOMA

IFN-␤ gene to flank or brain tumors using various vector approaches 160), which has low levels of MHC class I and is sensitive to NK cell-mediated
resulted in good therapeutic efficacy (35–37). lysis, has been described elsewhere (44). YAC-1 cells were cultured in RPMI
A number of characteristics make pleural malignant mesothelioma 1640 with 10% FBS, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, and
an especially attractive target for gene therapy (38). The location in 2 mM glutamine.
the potential space of the thoracic cavity makes the tumor highly REN cells are a human mesothelioma cell line isolated in our laboratory
from a patient’s tumor specimen (38). I-45 is a human mesothelioma cell line
accessible, facilitating directed administration of gene therapy vectors
obtained as a gift from Dr. Joseph Testa (Fox Chase Cancer Institute, Phila-
and subsequent analysis of treatment effects. In addition, local exten- delphia, PA). These cells were cultured and maintained in RPMI 1640 with
sion of disease, rather than the development of widespread distant 10% FBS, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, and 2 mM
metastases, is responsible for the majority of the morbidity and glutamine.
mortality associated with this neoplasm. Thus, small increments of Recombinant Ad Vectors. Ad vectors encoding the murine and human
improvement in local control could lead to significant improvement in IFN-␤ genes were provided by the Vector Core of the Institute for Human
survival or palliation. Accordingly, our group has successfully com- Gene Therapy (University of Pennsylvania Medical Center). An adenovirus
pleted several Phase I clinical trials of intrapleural gene therapy for transfer vector encoding the murine IFN-␤ gene driven by the CMV early
mesothelioma involving intrapleural instillation of recombinant ad- promoter, pAdCMV-muIFN-␤, was constructed by ligating a cDNA insert
enoviral vectors containing the herpes simplex virus tk (HSVtk) gene encoding murine IFN-␤1a into the plasmid pAd.CMVlink1 (45). pAd.CMV-
muIFN-␤ was cotransfected with the ClaI-restricted H5.010CMVEGP viral
in combination with systemic ganciclovir (39 – 42).
backbone into 293 cells for homologous recombination. The white recombi-
Because of the lack of effective therapy, the potential for response
nant plaques were isolated by a green/white selection process (46). The white
to IFN immunotherapy, and the proven feasibility of local Ad gene plaques were expanded for confirmation in restriction enzyme analysis of the
therapy, malignant mesothelioma represents an ideal tumor to test IFN virus genome and muIFN-␤ expression. A similar virus was prepared by
gene therapy. On the basis of recent successes using an adenovirus ligating a cDNA insert containing the human IFN-␤1a (35) into the same
expressing the human and murine IFN-␤ gene (35, 36), the present shuttle vector.
study was designed to assess the potential of using a recombinant, As a control vector, replication-deficient Ad vector encoding the herpes
replication-deficient adenovirus carrying the murine IFN-␤ gene (Ad- simplex type I tk gene driven by the RSV promoter (Ad.RSVtk) was used as
.muIFN-␤) to treat established i.p. tumors and to explore the mecha- described previously (47). Virus preparations were produced in 293 cells and
nisms of the observed therapeutic effects. purified on CsCl gradients after three rounds of plaque isolation. Vector
preparations were shown to be negative for the presence of wild-type adeno-
We show that Ad.muIFN-␤ efficiently transfects murine mesothe-
virus. The particle:pfu ratio of each preparation was determined in 293 cells
lioma cells both in vitro and in vivo, and that a single i.p. adminis-
and ranged between 50:1 and 150:1.
tration of Ad.muIFN-␤ can effectively treat established malignant Recombinant muIFN-␤. Recombinant muIFN-␤ (41 units/ng) was pro-
mesothelioma tumors in syngeneic, immune-competent mice leading vided by Biogen Inc. The protein was kept at ⫺70°C and did not undergo more
to significantly prolonged survival. The effects were attributable to than one freeze-thaw.
immunological mechanisms because: (a) a single i.p. administration Quantitation of muIFN-␤ Protein Levels by ELISA. One million AB12
of Ad.muIFN-␤ conferred long-lasting, systemic antitumor immunity cells in six-well plates were infected with Ad.muIFN-␤ at a MOI of 50. After
associated with tumor-specific CTL reactivity; and (b) minimal anti- 24 h, the wells were washed and refed with 1 ml of medium containing 1%
tumor efficacy was noted with i.p administration of Ad.muIFN-␤ in FBS. Twenty-four h later, supernatants were collected, and IFN-␤ concentra-
tumor-bearing immune-deficient mice. This systemic immune re- tion was quantified by ELISA. Cell counts per well at the end of the collection
sponse was adequate to inhibit growth of a distant flank tumor upon period were ⬃4 million.
To determine the levels and duration of expression of muIFN-␤ after
treatment of i.p. disease. Thus, intracavitary delivery of the IFN-␤
administration of Ad.muIFN-␤, naı̈ve or tumor-bearing mice received injec-
gene via an Ad vector provides a remarkably effective treatment for tions i.p. with 10 9 pfu of Ad.muIFN-␤. At various time points, animals
locally established murine mesothelioma, as well as generating distant (n ⫽ 3/group) were sacrificed, bled, and subjected to peritoneal lavage with 3
antitumor effects. The mechanism of action in this model appears to ml of PBS. muIFN-␤ levels in blood and peritoneal lavage fluid were then
be primarily dependent on CD8⫹ T lymphocytes rather than on innate determined by ELISA (see below).
immune mechanisms, antiangiogenesis or direct antiproliferative ef- Murine IFN-␤ Assay. Ninety-six-well plates were coated overnight at 4°C
fects. with a monoclonal antibody to mouse IFN-␤ (muIFN-␤ MB-7; Yamasa Shoyu
Co., Ltd., Tokyo, Japan). The antibody was used at a concentration of 10
␮g/ml in the coating buffer containing 50 mM sodium bicarbonate/carbonate,
MATERIALS AND METHODS 0.2 mM MgCl2, and 0.2 mM CaCl2 (pH 9.6). After the plates were blocked with
0.5% nonfat dry milk in PBS for 1 h at room temperature, medium from
Cell Lines. The murine mesothelioma cell lines AB12 and AC29 were infected cells or muIFN-␤ protein standards (Biogen, Inc.) diluted in 0.5%
provided by Dr. Jay K. Kolls of the Louisiana State University School of nonfat dry milk and 0.05% Tween 20 in PBS were added. The plates were then
Medicine (New Orleans, LA). These cell lines were originally generated by Dr. successively incubated at room temperature for 1 h with an anti-muIFN-␤
Bruce Robinson at the Queen Elizabeth II Medical Center (Nedlands, Perth, rabbit sera (produced at Biogen; 1:2000 dilution), 1 h with horseradish per-
Western Australia) by i.p. implantation of asbestos fibers in BALB/c and oxidase-conjugated donkey antirabbit antibody (Jackson ImmunoResearch;
CBA/J mice, respectively, and have been well characterized (8, 43). AB12 and 1:5000 dilution), and the substrate solution (4.2 mM tetramethylbenzidine and
AC29 cells were cultured and maintained in high glucose DMEM (Mediatech, 0.1 M sodium acetate-citric acid, pH 4.9). After the reaction was stopped with
Washington, DC) supplemented with 10% FBS (Georgia Biotechnology, At- 2N H2SO4, absorbance was measured at 450 nm.
lanta, GA), 100 units/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM In Vitro Cytotoxicity Assay. To evaluate the direct antiproliferative effect
glutamine. L1C2 is a murine (BALB/c) bronchoalveolar carcinoma cell line of Ad.muIFN-␤, AB12 and AC29 cells were infected with Ad.muIFN-␤ or
obtained as a gift from Dr. Steven Dubinett (University of California–Los Ad.RSVtk at various concentrations (MOIs of 0.01, 0.1, 1, 10, and 50) and
Angeles, Los Angeles, CA). L1C2 cells were cultured in RPMI 1640 with 10% seeded in 96-well plates at a density of 2 ⫻ 10 3 cells/well. Similar experiments
FBS, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, and 2 mM glutamine. were conducted with Ad.huIFN-␤ using REN or I-45 cells. Survival of infected
The EJ62 line (EJ-6-2-Bam-6a) is a 3T3 mouse fibroblast cell line transformed cells compared with uninfected cells was measured 3 days after infection using
with the H-ras oncogene that forms tumors in BALB/c mice. It was purchased the MTS assay, which is a colorimetric test for the quantification of cell
from the American Type Culture Collection (Manassas, VA) and maintained in viability and proliferation (Cell Titer 96 Aqueous Non-Radioactive MTS Cell
DMEM with 10% FBS, 100 units/ml penicillin G, 100 ␮g/ml streptomycin, Proliferation Assay; Promega Corp., Madison, WI). The percentage of inhibi-
and 2 mM glutamine. The murine lymphoma cell line YAC-1 (ATCC TIB- tion of cell growth was calculated according to the following formula:
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 Ad.IFN-␤ FOR MESOTHELIOMA
1 ⫺ A490 of treated group
Inhibition 共 % 兲 ⫽ ⫻ 100
A490 of control group
Experimental Animals. Female BALB/c, female CB17-SCID, and female
CB17-SCID/beige mice (6 – 8 weeks of age; weight, approximately 20 –25 g)
were obtained from Taconic Laboratory (Germantown, NY). Female CBA/J
mice (6 – 8 weeks of age; weight, ⬃20 g) were obtained from The Jackson
Laboratory (Bar Harbor, ME). Animals were housed in the animal facility at
the Wistar Institute (Philadelphia, PA). All mice were maintained in a patho-
gen-free animal facility for at least 1 week before each experiment. The
Animal Use Committees of the Wistar Institute and University of Pennsylvania
approved all protocols in compliance with the Guide for the Care and Use of
Laboratory Animals.
Treatment of Tumor-bearing Mice with Ad Vectors. Two independent
intracavitary tumor models of murine mesothelioma (AB12 and AC29) were
established to examine the treatment efficacy of locally administered Ad-
.muIFN-␤. Murine mesothelioma (AB12 and AC29) cells were grown to 80%
confluence in culture flasks, the medium was aspirated, and cells were har-
vested using 0.05% trypsin-versene. Cells were then pelleted (1000 rpm for 3
min) and resuspended at 1 ⫻ 105 cells/ml in serum-free DMEM for i.p.
administration. AB12 and AC29 cells (5 ⫻ 105 in 0.5 ml) were injected i.p.
into BALB/c and CBA/J mice, respectively, using 25-gauge needles. Four to
7 days after tumor cell injection, macroscopic (3– 4 mm) tumor nodules could
be identified on the small bowel mesentery. Later, tumor masses could be
observed on the diaphragm, peritoneal surface, porta hepatis, lesser sac, and
retroperitoneum. Survival studies were performed in lieu of tumor burden
assessment because of the difficulty in harvesting tumor densely adherent to
the abdominal viscera. Animals were sacrificed when they met predetermined
criteria established for minimizing pain and suffering. Treatment was initiated
when approximately 3– 4-mm tumor nodules were identified after tumor cell
inoculation. This was usually 4 days after injection of AB12 cells and 7 days
after injection of AC29 cells.
For protein experiments, three groups (n ⫽ 9) of AB12 tumor-bearing mice
were treated i.p. with saline or with recombinant murine IFN-␤ at a dose of
3000 units/dose three times weekly (“low dose”) or 30,000 units/dose (“high
dose”) three times weekly for as long as the mice survived.
In gene therapy experiments, unless otherwise noted, mice received a single
i.p. administration of Ad.muIFN-␤ at a dose of 1 ⫻ 109 pfu. In control mice,
Ad.RSVtk was injected at an identical dose of 1 ⫻ 109 pfu without adminis-
tration of ganciclovir to control for nonspecific effects of Ad transduction.
To study the effects of Ad.muIFN-␤ in tumor-bearing immunodeficient
mice, we used both SCID mice and SCID/beige mice, which underwent i.p
injection of murine mesothelioma cell lines (AB12 and AC29) as described
above.
For the in vivo dose-escalation experiments, BALB/c mice received an i.p.
injection of 5 ⫻ 10 5 tumor cells diluted in 500 ␮l of serum-free DMEM. Four
days after tumor implantation, mice received a single i.p. injection of 500 ␮l
of DMEM containing varying doses of Ad.muIFN-␤ (1 ⫻ 10 6, 1 ⫻ 107,
1 ⫻ 10 8, and 1 ⫻ 10 9 pfu). Animals were then observed closely, and survival
studies were performed.
“Bulky” Tumor Model. To examine the effect of Ad.muIFN-␤ instillation
against bulkier i.p. tumors, we staggered the time of vector instillation (4, 7,
and 10 days after tumor inoculation) to allow some of the animals to develop
larger (5–7 mm) i.p. nodules, as well as allow more tumor involvement in the
porta hepatis and small bowel mesentery. BALB/c mice received i.p injections
of 5 ⫻ 10 5 tumor cells in 500 ␮l of DMEM on day 0; subsequently on day 4,
7, or 10, test animals underwent single i.p. instillation of 500 ␮l of DMEM
containing 1 ⫻ 10 9 pfu of Ad.muIFN-␤. Survival studies were then per-
formed.
Tumor Rechallenge of Long-Term Survivors. Animals who were long-
term survivors from the above experiments (survival ⬎2 months after primary
tumor instillation and single Ad.muIFN-␤ treatment) were rechallenged in one
flank with s.c. flank injections of 5 ⫻ 10 6 cells of the parental tumor cell line
(AB12 and AC29) in 100 ␮l of serum-free DMEM. Each group contained 10
animals. Long-term survivors in the AB12 model were also challenged s.c.
with 5 ⫻ 10 6 cells in the contralateral flank with an additional murine
bronchoalveolar lung cancer cell line, L1C2, syngeneic in BALB/c to evaluate
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the specificity of the antitumor response. Approximately 11 days after the s.c.
challenge, flank tumors were excised and weighed.
To determine the role of T-cell subsets in rechallenge immunity, long-term
survivors from AB12/Ad.muIFN-␤ experiments were rechallenged with AB12
cells as above. Three groups (each group with 5 mice and 10 flank tumors)
were studied. One group received i.p. injections of saline, one group was
depleted of CD4⫹ T cells before and during tumor growth using the schedule
described below, and one group was depleted of CD8⫹ T cells before and
during tumor growth. After 11 days, animals were sacrificed, and tumor
weights were determined.
In Vivo Depletion of CD4ⴙ and CD8ⴙ T Cells. To deplete specific
immune effector cells subsets prior to and during treatment with Ad.muIFN-␤
in the AB12 model, BALB/c mice received i.p. injections of 200 ␮g of purified
monoclonal antibodies purified from the anti-CD4⫹ hybridoma GK1.5 and the
anti-CD8⫹ hybridoma 53-6.7 (obtained from the American Type Culture
Collection). Injections were administered 3 days and 1 day prior to inoculation
with AB12 cells. Thereafter, a maintenance dose of antibody was injected i.p.
every 7 days throughout the entire experimental period to ensure depletion of
the targeted cell type. CD4⫹ and CD8⫹ T-cell depletion was confirmed by
flow cytometry of splenic suspensions at the time of tumor injection and
weekly afterward. BALB/c mice received i.p. injections of 5 ⫻ 10 5 AB12 cells
in 500 ␮l of DMEM on day 0. Four days after tumor inoculation, mice received
a single i.p. injection of 500 ␮l of DMEM containing 1 ⫻ 10 9 pfu Ad-
.muIFN-␤. Survival studies were then performed as described above.
Effects of Treatment of i.p. Tumor on Tumors at Distant Sites. To
examine the effect of treatment of i.p. treatment of tumor with Ad.muIFN-␤ on
the growth of tumor at distant sites, four tumor models of s.c. and/or i.p. AB12
tumors were established. In group 1, 10 mice were injected with 1 ⫻ 106 AB12
cells in 100 ␮l of serum-free DMEM in each flank. In Group 2, 10 mice were
injected with 1 ⫻ 106 AB12 cells in each flank plus 5 ⫻ 105 AB12 cells and
500 ␮l of DMEM i.p. In group 3, 10 mice were injected with 1 ⫻ 106 AB12
cells in each flank and 4 days later received a single i.p. injection of 500 ␮l of
DMEM containing 1 ⫻ 109 pfu of Ad.muIFN-␤. This group served as a control
to see whether systemic circulation of IFN-␤ would affect tumor growth. In
Group 4, 10 mice were injected with 1 ⫻ 106 AB12 cells in each flank plus
5 ⫻ 105 AB12 cells i.p. Four days later, the animals were treated with 1 ⫻ 109
pfu Ad.muIFN-␤ i.p. This group served as the experimental group to test
whether treatment of i.p. tumor would affect the growth of the flank tumors.
On day 19 after tumor injection (15 days after treatment with Ad.muIFN-␤),
the flank tumors were harvested and weighed.
To determine the importance of the B- and T-lymphocytes in distant
response, a similar experiment was performed in three groups of SCID mice
(n ⫽ 10) using the conditions described above for groups 2, 3, and 4.
CTL Assay. We performed standard chromium-release assays on murine
splenocytes to evaluate for the development of tumor-specific CTLs resulting
from Ad.muIFN-␤ treatment of mice bearing i.p. tumors. Splenocytes (two
mice/group) were harvested 10 days after i.p. treatment with Ad.muIFN-␤ and
used as effector cells. AB12 and EJ62 cells were used as target cells. Impor-
tantly, the effectors (murine splenocytes from animals post-Ad.muIFN-␤ treat-
ment) were not stimulated in vitro with target cells prior to testing in the
chromium-release assay. To evaluate NK cell activity, effector cells were
reacted with YAC-1 cells. Target cells (AB12, EJ62, and YAC-1 cells) were
labeled with 51Cr by incubating 1 ⫻ 104 cells in a 96-well plate with 3.7 MBq
of 51Cr (New England Nuclear Life Science Products, Boston, MA) in normal
growth medium for 1 h and then washed five times with PBS. Varying
numbers of effector cells were mixed with target cells to yield E:T ratios of
100:1 to 6.25:1 and were incubated for 5 h in a V-bottomed, 96-well microtiter
plate. 51Cr release was measured in wells containing effector cells and target
cells (cpm test), wells containing target cells in medium alone (cpm sponta-
neous), and wells containing target cells plus 10% SDS (cpm total). The
percentage of lysis was calculated using the following formula:
cpm test ⫺ cpm spontaneous
% lysis ⫽ ⫻ 100
cpm total ⫺ cpm spontaneous
To deplete CD8⫹ T-cells from the splenocyte population, aliquots of effec-
tor cells were incubated for 30 min at 4°C with magnetic MicroBeads conju-
gated to antimouse CD8⫹ T-cell antibodies (Ly-2; Miltenyi Biotec, Inc.,
Auburn, CA). Beads and bound CD8⫹ T-cells were magnetically removed
 Ad.IFN-␤ FOR MESOTHELIOMA

from the incubation medium. The CD8⫹ T-cell-depleted splenocytes were then
tested for CTL activity as described above.
Statistics. The significance of the in vitro inhibition results were deter-
mined by Student’s t test (two-tailed) comparing growth inhibition after
administration of Ad.IFN versus Ad.RSVtk. Differences in flank tumor
weights in the animal experiments were determined by one-way ANOVA
adapted for multiple comparisons (Tukey-Kramer test; JMP; SAS Institute
Inc., Cary, NC). Statistical significance was set at P ⬍ 0.05. Kaplan-Meier
survival curves were analyzed with the Mantel-Cox log-rank test. Results are
expressed as mean ⫾ SEM.
RESULTS
Effects of Ad.IFN-␤ on in Vitro Mesothelioma Cell Growth
To determine the sensitivity of mesothelioma cells to the direct
antiproliferative effects of Ad.IFN-␤, we first tested the effect of
IFN-␤ gene transfer on human mesothelioma lines in vitro. Because
there is limited cross-species reactivity, Ad.huIFN-␤ was used. Two
mesothelioma cell lines (REN and I-45) were infected with various
MOIs (MOI ⫽ number of pfu of adenovirus/cell) of Ad.huIFN-␤ or a
control virus, Ad.RSVtk (used without addition of ganciclovir as a
control for adenoviral toxicity). After three days, cell number was
determined using an MTS assay. The degree of cell growth inhibition
was calculated by comparison to uninfected mesothelioma cells. As
shown in Fig. 1, A and B, doses of Ad.huIFN-␤ as small as 0.01 MOI
induced significant (30 – 40%) growth-inhibitory effects. At MOIs of
10, 60 –70% growth inhibition was seen. There was minimal cytotox-
icity with the control adenovirus. At every dose, the percentage of
inhibition induced by Ad.huIFN-␤ was significantly greater than the
corresponding dose of Ad.RSVtk control virus (P ⬍ 0.001).
To study murine mesothelioma cell lines, an Ad.muIFN-␤ virus
was constructed as described in “Materials and Methods.” The vector
was then used to infect murine mesothelioma AB12 cells at an MOI
of 50. One day after infection, fresh medium was added and collected
for 24 h. Medium supernatants were analyzed by ELISA and found to
contain 786 ng/ml ⫾ 71 ng/ml of IFN-␤ per 106 cells. This corre-
sponds to ⬃32,000 units of IFN-␤ per 106 cells/24 h.
The effects of Ad.muIFN-␤ on the growth of two murine mesothe-
lioma cells (AB12 and AC29) were studied as above. As shown in
Fig. 1, C and D, growth was also inhibited by Ad.muIFN-␤. At every
dose, the percentage of inhibition of Ad.muIFN-␤ was significantly
greater than the corresponding dose of Ad.RSVtk control virus
(P ⬍ 0.001). However, these cells were less sensitive to the antipro-
liferative effects of the virus, with maximum inhibition being only
50 – 60% of control virus at the highest dose of 50 MOI. This could
represent the fact that these murine lines are less infectable by ade-
novirus than are the human lines, or that murine protein has an
inherently lower antiproliferative activity than human protein.
These studies show that Ad.IFN-␤ has direct antiproliferative ef-
fects on human and murine mesothelioma cells in vitro. Although the
human mesothelioma cells are quite sensitive to the direct toxic
effects of IFN-␤, mouse studies using these lines would have to be
conducted in immunodeficient animals. Because many of the antitu-
mor effects of IFN-␤ are thought to be attributable to its effects on the
immune system (36), we reasoned that such studies would not model
the clinical situation in which we would actually use this vector.
Accordingly, we conducted the animal studies using the two murine
mesothelioma cell lines.
Treatment of Established i.p. Mesothelioma with Recombinant
Murine IFN-␤ or a Single Administration of Ad.muIFN-␤
To study a clinically relevant model of mesothelioma, we used two
models of murine mesothelioma, both based on injection of mouse
mesothelioma cells injected into the peritoneal cavity of the appro-
priate strain of mice. Cell line AB12 grows in BALB/C mice. Cell line
AC29 grows in CBA/J mice. Both lines can be injected i.p., where
they form diffuse tumors throughout the peritoneal cavity (43), similar
to the presentation of peritoneal human mesothelioma.
As a point of comparison, we first tested the efficacy of injection of
IFN-␤ protein in the AB12 model. After visible tumor nodules were
confirmed, three groups (n ⫽ 9) of tumor-bearing mice were treated
i.p. with saline or with recombinant murine IFN-␤ at a dose of 3000
units/dose three times weekly (“low dose”) or 30,000 units/dose
(“high dose”) three times weekly for as long as the mice survived. The
low dose of 3000 units of IFN-␤ corresponds approximately to the
maximally tolerated doses used in a humans (10 million units or
⬃143,000 units/kg body weight). As shown in Fig. 2, administration
of recombinant IFN-␤ at either dose led to significant (P ⫽ 0.02 by
log-rank test) increases in median survival from 25 days (control) to

Fig. 1. Effects of Ad.IFN-␤ on in vitro mesothe-

lioma cell growth. Human mesothelioma cells,
REN (A) and I-45 (B) cells were infected with
Ad.huIFN-␤ or Ad.RSVtk at various doses (MOIs
of 0.01, 0.1, 1, 10, and 50) and seeded in 96-well
plates at a density of 2 ⫻ 103 cells/well. After 3
days, cell number was determined using a colori-
metric MTS assay. The percentage of inhibition of
cell growth of Ad.IFN-infected or Ad.RSVtk-in-
fected cells compared with noninfected cells is
plotted. Thus, growth was inhibited by ⬃75% in
REN cells infected with Ad.huIFN-␤ at an MOI of
50. Similar experiments were conducted with Ad-
.muIFN-␤ using the murine mesothelioma AB12
(C) or AC29 (D) cell lines. A–D: bars, SEM.

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 Ad.IFN-␤ FOR MESOTHELIOMA

were not able to detect activity in the serum at any time point.
Peritoneal lavage levels were 43.2 ⫾ 14 ng/ml at 5 min after injection,
15.5 ⫾ 2.4 ng/ml at 30 min after injection, and undetectable after 4 h.
After one injection of Ad.muIFN-␤, 1 day after i.p. injection, serum
levels averaged 2.7 ⫾ 0.7 ng/ml. At the same time, peritoneal lavage
levels averaged 12.5 ⫾ 4.5 ng/ml. By days 4 and 14, however,
muIFN-␤ levels were below the level of detection in both serum and
lavage fluid. Similar results were seen when 10 9 pfu of Ad.muIFN-␤
were injected into tumor-bearing animals; clearly detectable levels
were present in peritoneal lavage fluid 1 and 2 days after injection but
were undetectable by 4 days. Thus, as expected, peritoneal levels were
very short lived after injection of recombinant protein (⬍4 h), whereas
Fig. 2. Effects of recombinant IFN-␤ on survival in a malignant mesothelioma tumor detectable levels of IFN-␤ were present up to 2 days after the single
model. Three groups (n ⫽ 9) of mice bearing i.p. AB12 tumors were treated i.p. with
saline (✳) or with recombinant murine IFN-␤ at a dose of 3000 units/dose three times
injection of Ad.muIFN-␤.
weekly (“low dose”; F) or 30,000 units/dose (“high dose”; E) three times weekly for as
long as the mice survived. Administration of recombinant IFN-␤ at either dose led to Dose-Response Experiments
significant (P ⫽ 0.02 by log-rank test) increases in median survival from 25 days (control)
to 37 days; however, there was only one long-term (⬎60 days) survivor. There was no To examine the dose-response characteristics of Ad.muIFN-␤, two
difference between the low- and high-dose groups.
sets of experiments were performed using the AB12 model. In the first
experiment, groups of 10 mice were injected with 5 ⫻ 105 AB12
37 days; however, there was only one long-term (⬎60 days) survivor. tumor cells on day 0 and, after confirmation of visible tumor nodules
There was no difference between the low- and high-dose groups. on day 4, were injected with one dose of virus or vehicle. Doses of
Fig. 3 shows the Kaplan-Meier survival curves of groups of tumor- Ad.muIFN-␤ were 109, 108, 107, or 106 pfu. Animals were then
bearing mice (10/group) treated with one dose of Ad.muIFN-␤ (109 followed for survival. As shown in the Kaplan-Meier survival curve
pfu), a control adenoviral vector Ad.RSVtk (109 pfu) given without (Fig. 4A), all control animals died by day 27. Compared with controls,
ganciclovir, or vehicle alone. Vector was injected after confirmation a clear dose-response effect of vector was seen with long-term sur-
of the presence of visible tumor nodules (3 mm in size) in the vival rates of 90% in the animals injected with 109 pfu (P ⬍ 0.0001)
peritoneum. This occurred either 4 days (AB12) or 7 days (AC29) and 30% in the animals injected with 108 pfu (P ⬍ 0.0001). Survival
after injection of tumor cells. was slightly but significantly increased in the 107 pfu animals
All animals injected with adenoviral vectors showed slight ruffling (P ⬍ 0.03). There was no increase on the survival rate in animals
of their fur for 2–3 days after injection; however, no differences were treated with 106 pfu of Ad.muIFN-␤.
noted between control and muAd.IFN-␤-injected animals. Similarly,
no side effects were observed in non-tumor-bearing animals injected
with the vector. In the AB12-injected animals (Fig. 3A), all control
animals (saline and Ad.RSVtk) died by day 31 with a median survival
of 22 days. In contrast, animals injected with one dose of Ad-
.muIFN-␤ had a 100% survival rate (P ⬍ 0.0001). In the AC29-
injected animals (Fig. 3B), the vehicle-injected control animals were
all dead by 60 days with a median survival of 40 days. Animals
injected with the control adenovirus had a slight prolongation of
median survival (40 days versus 54 days), and there were two long-
term survivors. This difference did not quite reach significance
(P ⫽ 0.065 via log-rank test) compared with controls. However,
animals injected with one dose of Ad.muIFN-␤ had a 100% survival
rate (P ⬍ 0.0001), similar to the results with the AB12 cell line. These
experiments have been repeated multiple times with virtually identical
results (see below).
These studies demonstrate that although multiple doses of recom-
binant muIFN-␤ result in a small survival advantage, a single dose of
Ad.muIFN-␤ is remarkably effective in treating two separate models
of i.p. established murine mesothelioma. In both experiments, all of
the animals survived after Ad.muIFN-␤ treatment.

Evaluation of Serum and i.p. Levels of IFN-␤ after i.p.

Injection of muIFN-␤ and Ad.muIFN-␤

To determine the local and systemic levels of IFN-␤ after i.p.

injection of muIFN-␤ or Ad.muIFN-␤, animals were given a single
dose of 30,000 units of recombinant protein or one dose of 10 9 pfu
of Ad.muIFN-␤ via an i.p. injection. At various time points after
injection, animals were sacrificed, and the levels of IFN-␤ in serum or
peritoneal lavage fluid were determined by ELISA. Levels in serum
and after a 3-ml peritoneal lavage at baseline were below detection
levels (⬍0.4 ng/ml). After one injection of recombinant muIFN-␤, we
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Fig. 3. Effects of Ad.muIFN-␤ on survival in two malignant mesothelioma tumor
models. A single i.p. dose (arrows) of 1 ⫻ 109 pfu of Ad.muIFN-␤ (E) or Ad.RSVtk (Œ)
was administered to BALB/c mice bearing AB12 tumors 4 days after tumor injection (A)
or to CBA/J mice bearing AC29 tumors 7 days after injection (B). Control mice received
500 ␮l of saline i.p. (✳). Animals (n ⫽ 10/group) were then followed for survival.
Significant long-term survival (100%) was observed in treated mice receiving Ad-
.muIFN-␤ in both models (P ⬍ 0.0001).

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 Ad.IFN-␤ FOR MESOTHELIOMA
Fig. 4. Dose-response experiments. A, BALB/c mice bearing AB12 tumors were
treated with a single dose of Ad.muIFN- ␤ via the i.p. route at varying doses, i.e., 109 (E),
10 (F), 107 (䡺), 106 (f) pfu at day 4 after tumor inoculation. Control mice received 500
8 extend to a second tumor type.
␮l saline i.p. (✳). Animals were then followed for survival. Significant increases in
survival were seen in animals given 109, 108, and 107 pfu of virus. B, BALB/c mice
(n ⫽ 9/group) received i.p injections of AB12 tumor cells on day 0. Subsequently, on day
4 (E), day 7 (F), or day 10 (䡺), test animals were administered a single i.p. dose of
1 ⫻ 109 pfu of Ad.muIFN-␤ and were followed for survival. Control animals received a
dose of saline on day 4 (✳). Significant increases in survival were seen in all treated
groups.
In the second experiment, we varied the time of vector injection
after tumor inoculation to determine whether Ad.muIFN-␤ would be
effective against larger tumors. Groups of 11 mice were injected with
5 ⫻ 105 AB12 tumor cells on day 0. On days 4, 7, and 10 after tumor
injection, two mice were sacrificed to confirm the size of visible
tumor nodules. At day 4, multiple 3– 4-mm tumor nodules were noted
on the small bowel and mesentery. One group of animals received an
i.p. injection of medium alone (control), and a second group of
animals (group 1) were injected i.p. with one dose of Ad.muIFN-␤
(109 pfu). On day 7, the nodules had increased to 5– 6 mm in size. At
this time, group 2 animals were injected i.p. with one dose of Ad-
.muIFN-␤ (109 pfu). By day 10, there was extensive bulky disease
with large irregularly shaped 7– 8-mm nodules throughout the perito-
neal cavity, as well as smaller nodules on the bowels, mesentery, and
diaphragm. At this time, group 3 animals were injected i.p. with one
dose of Ad.muIFN-␤ (109 pfu).
As shown in Fig. 4B, control animals died rapidly with a median
survival of 17 days and with all animals dead by 28 days. All treated
groups had significant increases in survival compared with control
animals (P ⬍ 0.0001) with a clear dose-response effect noted. Ani-
mals injected on day 7 had a 33% long-term survival rate with an
increase in median survival to 44 days (P ⬍ 0.0001). Even those
animals with large tumors treated on day 10 had a significant
(P ⫽ 0.0003) increase in median survival (31 days versus 17 days)
with one long-term survivor. Consistent with our previous study (Fig.
3), animals injected at day 4 had 100% survival.
Thus, Ad.muIFN-␤ successfully treats tumors in a dose-responsive
fashion. The dose inducing complete survival was 109 pfu. Significant
therapeutic effects were also seen in animals with large tumor bur-
dens.
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Induction of Long-Term Immunity after Ad.muIFN-␤ Therapy
To determine whether treatment with Ad.muIFN-␤ therapy induced
an acquired immune response against the tumor cells, treated animals
“cured” of their tumors were rechallenged with a s.c. injection of
tumor. Groups of 10 mice “cured” of AB12 tumors (⬎60 days after
tumor injection and treatment with Ad.muIFN-␤) or naı̈ve, control
BALB/c mice were injected in both flanks with either 5 ⫻ 106 AB12
cells or L1C2 cells. L1C2 cells are a mouse lung adenocarcinoma cell
line that also grows in BALB/c mice.
After 11 days, the flank tumors were removed and weighed. As
shown in Fig. 5, there were statistically significant differences
(P ⬍ 0.0001) in the size of the tumors induced by injection of AB12
cells into “cured” mice versus previously untreated mice. Specifically,
tumors in previously treated mice did not grow, in contrast to the
marked growth of the same cells injected into naı̈ve BALB/C mice.
Interestingly, there appeared to be some “cross-sensitization” to the
L1C2 cell line, because these cells did not grow well in the “cured”
mice compared with control mice (P ⬍ 0.0001). Similar results were
seen with the AC29 model, where none (0 of 10) of the “cured” mice
injected with AC29 cells developed flank tumors, whereas all of the
control CBA/J mice (10 of 10 tumors) developed large flank tumors
(data not shown).
These data indicate that animals successfully treated with Ad-
.muIFN-␤ develop long-lasting protective immunity upon rechallenge
with the same tumor. In one case, this antitumor response appeared to
Effects of Ad.muIFN-␤ in Immunodeficient Animals
Although our rechallenge experiments indicated development of
long-term immunity to tumor cells, these studies did not define how
tumor cells were eliminated after i.p. injection. IFN-␤ has been shown
to work by a number of mechanisms, including direct cytotoxicity,
inhibition of angiogenesis, NK and macrophage activation, as well as
induction of an acquired (T-cell-mediated) immune response (see
“Discussion”). To determine which of these mechanisms were oper-
ative in our model, we tested the efficacy of Ad.muIFN-␤ after
injection into tumor-bearing immunodeficient mice.
Groups of 10 SCID/beige mice were injected with 5 ⫻ 105 AB12
Fig. 5. Existence of long-term antitumor immunity. Long-term survivors in the AB12
model (survival ⬎2 months; see Fig. 3A) were rechallenged with the s.c. parental AB12
tumor cell line (䡺) or with the syngeneic L1C2 tumor cell line (f). Previously untreated
control animals were injected with the same number of tumor cells. Eleven days after the
s.c. challenge, flank tumors were excised and weighed. In the control mice, both tumor
cell lines produced tumors that grew to large size. In the previously “cured” animals,
AB12 tumor growth was completely inhibited. Interestingly, growth of L1C2 cells was
also markedly diminished. These data demonstrate the induction of long-term antitumor
immunity. Bars, SE.
 Ad.IFN-␤ FOR MESOTHELIOMA
tumor cells on day 0. On day 4, two mice were sacrificed to confirm
the presence of visible tumor nodules. After confirmation, animals
were injected with one dose of Ad.muIFN-␤ (109 pfu) or vehicle.
Animals were then followed for survival. As shown in Fig. 6A, in
contrast to the immunocompetent BALB/c mice injected with AB12
cells (see Fig. 3), the therapeutic efficacy of Ad.muIFN-␤ was almost
completely lost, with no increase in median survival and no survival
advantage. Because SCID/beige mice are deficient in B cells, T cells,
and NK cells, we conducted a similar experiment in SCID mice that
lack B and T cells but have intact NK function. As shown in Fig. 6B,
virtually identical results were obtained; the therapeutic efficacy of
Ad.muIFN-␤ was almost completely lost, with virtually no increase in
median survival and no survival advantage.
The SCID mouse experiment was repeated using the AC29 cells.
Seven days after tumor inoculation and confirmation of tumor growth,
animals were injected with one dose of Ad.muIFN-␤ (109 pfu) or
vehicle. As shown in Fig. 6C, in this model, there was a slight increase
in median survival from 26 to 32 days (P ⬍ 0.0001); however, all of
the treated animals were dead by day 39.
These data show that in the AB12 model, almost all of the thera-
peutic effects of Ad.muIFN-␤ are attributable to factors related to
acquired immunity. In the AC29 model, there appears to be a small
increase in survival (which may be attributable to Ad or IFN-␤
effects), but these effects do not lead to long-term survival.
Fig. 6. Effect of Ad.muIFN-␤ treatment in immunodeficient mouse strains. Four days
after the injection of AB12 cells, a single i.p. dose of 1 ⫻ 109 pfu of Ad.muIFN-␤ (E;
arrows) was administered to SCID/beige (A) or SCID (B) mice. The same dose of virus
was administered to SCID mice 7 days after i.p. injection of AC29 cells (C). Control mice
received 500 ␮l saline i.p. (✳). Animals (n ⫽ 10/group) were then followed for survival.
The therapeutic efficacy of Ad.muIFN-␤ was markedly diminished in the immunodefi-
cient mice.
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Fig. 7. Effect of in Ad.muIFN-␤ on BALB/c mice bearing AB12 tumor cells after
depletion of CD4⫹ or CD8⫹ T cells. A single i.p. dose of 1 ⫻ 109 pfu of was administered
to BALB/c mice who had been injected i.p. with AB12 cells 4 days earlier. Antibodies
specific for CD8⫹ and CD4⫹ T cells were used to deplete these cell types before and
during treatment with Ad.muIFN-␤. There were no differences in survival between the
untreated animals (✳) and the CD8⫹ T-cell-depleted animals treated with Ad.muIFN-␤
(f). The animals depleted of CD4⫹ T cells (Œ) showed a significant increase in survival
but with no long-term survivors. Fully immunocompetent mice treated with Ad.muIFN-␤
(E) had an 80% survival rate in this experiment.
Determination of the Immune Mechanism of Tumor Killing
Depletion of CD4 and CD8 Cells. The experiments described
above indicate that acquired immune responses are the primary mech-
anism of the effects of Ad.muIFN-␤ in our models. To more precisely
define this mechanism, groups of mice were treated with anti-CD4 or
anti-CD8 antibodies to deplete these populations of cells. After ade-
quacy of depletion was confirmed by performing fluorescence-acti-
vated cell sorter analysis of spleen cells (data not shown), groups of
10 BALB/c mice received injections of 5 ⫻ 105 AB12 tumor cells on
day 0. On day 4, after confirmation of the presence of visible tumor
nodules, animals were injected with one dose of Ad.muIFN-␤ (109
pfu) or vehicle and followed for survival. Depletion of the appropriate
T-cell population was confirmed weekly by analysis of spleen cells by
flow cytometry (data not shown).
As shown in Fig. 7, there were no significant differences in survival
between the untreated animals and the CD8⫹ T-cell-depleted animals
treated with Ad.muIFN-␤. In contrast, the animals depleted of CD4⫹
T cells showed a marked increase in survival, although not to the level
of intact animals (P ⬍ 0.0004 versus the saline group). This experi-
ment indicates that, in this model, CD8⫹ T cells are central to the
therapeutic effects of Ad.muIFN-␤. CD4⫹ T cells also play a role in
the generation of antitumor immunity, but even in their absence,
significant antitumor effects are still observed.
To determine the role of CD4⫹ and CD8⫹ T cells in established
tumor immunity, groups (n ⫽ 5) of naı̈ve mice or mice cured of AB12
tumors by previous injection of Ad.IFN-␤ were reinjected with AB12
cells in both flanks (Fig. 8). In one group, CD4⫹ T cells were depleted
using antibody injections at the time of tumor rechallenge of “cured”
animals, and in a second group, CD8⫹ T cells were depleted by
antibody injections. Tumor growth was then assessed at 11 days.
Consistent with previous data (Fig. 5), AB12 cells injected into the
flanks of naı̈ve animals grew to large size, whereas tumor growth was
completely inhibited in previously treated, “cured” mice (Fig. 8).
Depletion of CD8⫹ T cells completely blocked this antitumor effect,
allowing tumors to grow to near control size in these animals. How-
ever, depletion of CD4⫹ T cells had very little effect on tumor growth
(tumor size was not significantly different from that seen in nonde-
pleted “cured” mice). This experiment demonstrates that CD8⫹ T
cells are absolutely required for antitumor immunity. CD4⫹ T cells
not directly involved in the recall antitumor effects but seem to be
more important in helping to generate CD8⫹ T cells.
 Ad.IFN-␤ FOR MESOTHELIOMA

2, 10 mice were injected with 1 ⫻ 106 AB12 cells in each flank plus
5 ⫻ 105 AB12 cells i.p. This group served as a control group to rule
out inhibitory effects on flank tumor growth by the concurrent growth
of i.p. tumor. In group 3, 10 mice were injected with 1 ⫻ 106 AB12
cells in each flank and 4 days later were treated i.p. with 1 ⫻ 109 pfu
of Ad.muIFN-␤. This group served as a control to see whether
systemic circulation of IFN-␤ after i.p. injection would affect tumor
growth. In group 4, 10 mice were injected with 1 ⫻ 106 AB12 cells
in each flank plus 5 ⫻ 105 AB12 cells i.p. Four days later, the animals
were treated with 1 ⫻ 109 pfu of Ad.muIFN-␤ i.p. This group served
as the experimental group to test whether treatment of i.p. tumor
would affect the growth of the flank tumors.
Fig. 8. Importance of CD4⫹ or CD8⫹ T cells for long-term antitumor immunity. On day 19 after tumor injection (15 days after Ad.muIFN-␤), the
Long-term (cured) survivors in the AB12 model (survival ⬎2 months) were rechallenged
with bilateral flank injections of parental AB12 tumor cell line. Previously untreated flank tumors were harvested and weighed. As shown in Fig. 10A,
control (naı̈ve) animals (n ⫽ 5) were injected with the same number of tumor cells. In one AB12 flank tumors grew to large size in the control animals (group 1),
cured group (n ⫽ 5), no T-cell depletion was performed. In one cured group (n ⫽ 5),
CD4⫹ T cells were depleted using antibody injections at the time of tumor rechallenge of
“cured” animals. In the third cured group (n ⫽ 5), CD8⫹ T cells were depleted by
antibody injections. Tumor growth was then assessed by weight at 11 days after tumor
injection. AB12 cells injected into the flanks of naı̈ve animals grew to a large size,
whereas tumor growth was completely inhibited in previously treated, “cured” mice.
Depletion of CD8⫹ T cells completely blocked this antitumor effect (tumors grew to a
large size and were not significantly different in size than naı̈ve controls). Depletion of
CD4⫹ T cells did not prevent the inhibition of tumor growth [tumor size was not
significantly different from that seen in nondepleted “cured” mice; both groups were
significantly different from naı̈ve mice (P ⬍ 0.05)]. This experiment demonstrates that
CD8⫹ T cells, but not CD4⫹ T cells, are required for antitumor immunity. Bars, SE.

Evaluation of Cytotoxic T Cells. To confirm the importance of

cytotoxic CD8⫹ T cells, control BALB/c mice, AB12 tumor-bearing
mice treated with a control Ad.RSVtk adenovirus, and AB12 tumor-
bearing mice treated with Ad.muIFN-␤ were sacrificed 10 days after
vector treatment (14 days after tumor instillation). Splenocytes were
isolated and assessed for the presence of CTLs using chromium-
labeled AB12 or EJ62 (a murine fibrosarcoma that grows in BALB/c
mice) cell targets. Of note, the splenocytes were not stimulated in
vitro. Fig. 9A shows that there was significant cytolytic activity of
splenocytes from the Ad.muIFN-␤-treated animals against AB12 tar-
gets. In contrast, there was no cytolytic activity seen in control
splenocytes or splenocytes from the animals treated with the control
Ad.RSVtk vector against the AB12 targets. No cytolytic activity was
seen against EJ62 targets using splenocytes from the Ad.muIFN-␤-
treated animals or any treatment group (Fig. 9B).
To confirm that the cytolytic activity was attributable to CD8⫹ T
cells, splenocytes from control or Ad.muIFN-␤-treated animals were
depleted of CD8⫹ T cells using anti-CD8-coated beads prior to use in
a CTL assay. This treatment completely eliminated cytolytic activity
of the cells against the chromium-labeled AB12 targets (Fig. 9C).
Splenocytes from control or Ad.muIFN-␤-treated animals were also
tested for cytolytic effects against labeled YAC-1 target cells. No
significant lysis was seen at E:T ratios of up to 100 (data not shown),
indicating minimal NK cell activity.
These experiments demonstrate that treatment of i.p. AB12 tumor
with Ad.muIFN-␤ generates tumor-specific CD8⫹ cytotoxic T cells
and that these cells account for almost all of the in vitro antitumor
activity. No evidence for active NK cell lysis was seen. Fig. 9. In vitro cytotoxic activity of splenocytes from tumor-bearing BALB/c mice
treated with i.p. Ad.muIFN-␤. A single i.p. dose of 1 ⫻ 109 pfu of Ad.muIFN-␤ or
Ad.RSVtk was administered to BALB/c mice bearing AB12 tumors 4 days after tumor
Effects of Treatment of i.p. Tumor on Tumors at Distant Sites injection. Ten days after i.p. treatment, splenocytes were harvested and used as effector
cells. AB12 (A and C) or syngeneic EJ62 (B) cells were chromium labeled and used as
The demonstration of systemic cytotoxic CD8⫹ T lymphocytes target cells. The effector cells were not stimulated in vitro with target cells prior to testing
suggested that antitumor effects might not be limited to the local (i.p.) in the chromium-release assay. As shown in A, there was significant cytolytic activity of
splenocytes from the Ad.muIFN-␤ treated animals (E) against AB12 targets. In contrast,
tumor site. To determine whether successful treatment of local (i.p.) there was no cytolytic activity against the AB12 targets using control splenocytes (✳) or
disease could lead to antitumor effects at distant (flank) sites, the splenocytes from the animals treated with the control Ad.RSVtk vector (Œ). As shown in
following four groups of animals were established. In group 1, 10 B, no cytolytic activity was seen against EJ62 targets using splenocytes from any
treatment group. In C, splenocytes depleted of CD8⫹ T cells (⽧) by the use of magnetic
mice were injected with 1 ⫻ 106 AB12 cells in each flank. This group beads completely lost their cytotoxic activity against the parental AB12 cells when
served as the control group for the growth of flank tumors. In group compared with nondepleted splenocytes (E).
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 Ad.IFN-␤ FOR MESOTHELIOMA
Fig. 10. Effects of treatment of i.p. tumor on tumors at distant sites. A, to examine the
antitumor effects on distant sites after i.p. treatment, four treatment groups were estab-
lished. In group 1, 10 mice were injected with 1 ⫻ 106 AB12 cells in each flank. In group
2, 10 mice were injected with 1 ⫻ 106 AB12 cells in each flank plus 5 ⫻ 105 AB12 cells
i.p. In group 3, 10 mice were injected with 1 ⫻ 106 AB12 cells in each flank and 4 days
later were treated i.p. with 1 ⫻ 109 pfu of Ad.muIFN-␤. In group 4, 10 mice were injected
with 1 ⫻ 106 AB12 cells in each flank plus 5 ⫻ 105 AB12 cells i.p., and 4 days later, the
animals were treated with 1 ⫻ 109 pfu of Ad.muIFN-␤ i.p. On day 19 after tumor
injection, the flank tumors were harvested and weighed. Flank tumors grew to large size
in groups 1–3. In contrast, flank tumor growth was markedly inhibited in group 4
(P ⬍ 0.05, all comparisons). Bars, SE. B, to determine the importance of T and B cells
in this distant tumor effect, groups 2, 3, and 4 were established as described above using
immunodeficient SCID mice. Flank tumors grew to a large size in all groups. In contrast
with the experiment above, group 4 tumors were not inhibited. Bars, SE.
and this growth was not affected by the presence of tumor growing i.p.
(group 2) or by the injection of Ad.muIFN-␤ into the peritoneal cavity
(group 3; P ⬎ 0.05 for all comparisons). However, flank tumor
growth was markedly inhibited in the group 4 animals (P ⬍ 0.05, all
comparisons).
To confirm that this antitumor response was dependent on acquired
immune responses, the experiment was repeated using immunodefi-
cient SCID mice instead of BALB/c animals (in this experiment,
group 1 animals were not studied). In contrast to the results in
immunocompetent animals, the growth of flank tumors in the group 4
SCID animals was not inhibited (Fig. 10B). These experiments show
that treatment of i.p. tumor also results in “distant” antitumor effects
that require an intact immune system.
DISCUSSION
The data presented here demonstrate remarkable in vivo antitumor
activity of murine IFN-␤ gene delivery using an adenoviral vector
(Ad.muIFN-␤) in two murine models of mesothelioma. More than
90% of animals bearing either AB12 (Fig. 3A) or AC29 (Fig. 3B) i.p.
mesotheliomas tumors showed complete tumor regression. A control
adenovirus vector had minimal antitumor effect in vivo. In contrast,
multiple i.p. injections of high doses of recombinant mu.IFN-␤ had
only minimal therapeutic effects (Fig. 2). Significant therapeutic
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effects were seen in animals treated with various Ad.muIFN-␤ doses
(Fig. 4A) and with very large tumor burdens (Fig. 4B). A dose of
1 ⫻ 10 9 pfu of Ad.muIFN-␤ led to almost 100% survival. Taking into
account size differences (⬃3000-fold), this dose would be the human
“equivalent” of 3 ⫻ 10 12 pfu of virus, a dose similar to the highest
dose of Ad.HSVtk (1 ⫻ 10 12 pfu) that we administered in our
previous mesothelioma clinical trial (40). Even a single dose of vector
one log lower, 1 ⫻ 10 8 pfu, significantly prolonged survival com-
pared with control vector and resulted in complete tumor regression in
⬃30% of the treated animals. A single i.p injection of Ad.muIFN-␤ in
mice bearing bulky intra-abdominal mesothelioma tumors (7 or 10
days after instillation) also resulted in prolongation of survival com-
pared with controls, although with less antitumor efficacy than mice
treated at earlier tumor stages (day 4). These results suggest that
treatment of mesothelioma at an early stage or in association with a
surgical “debulking” procedure to remove gross disease may be
successful strategies for Ad.muIFN-␤ gene therapy in future human
clinical trials. In addition to effective treatment of local i.p. tumors,
we were also able to demonstrate that treatment of i.p. tumor led to
inhibition of growth of tumor cells at a distant (flank) site (Fig. 10).
This finding raises the exciting potential of using i.p. (or intrapleural)
therapy as a way to treat systemic disease.
The type I IFNs, IFN-␣ and IFN-␤, are a family of closely related
cytokines that possess potent antiviral and immunoregulatory activi-
ties (17, 18, 48). IFN-␣ and IFN-␤ are produced by both immune and
nonimmune cell types and can be induced to high systemic levels in
response to viral infections. Type I IFNs can inhibit tumor growth
directly by suppressing cell replication and inducing differentiation or
apoptosis (19, 22, 49) and indirectly by activating NK-cell mediated
lysis (48) and tumoricidal properties of macrophages (32, 33, 50).
Type I IFNs can also promote tumor regression by suppression of
angiogenesis (21) and stimulation of specific cellular immune re-
sponses. Additionally, IFN-␤ also has the potential to induce NK cell
proliferation (51) and to promote antitumor T-cell responses by in-
ducing proliferation of memory phenotype subsets and prolonging
survival of activated populations (52, 53). Finally, type I IFNs can
up-regulate MHC class I expression (54).
Taking advantage of these multiple potential mechanisms for anti-
tumor effects, a number of groups have shown that expression of
IFN-␤ by tumor cells (through ex vivo or in vivo transfection) results
in impressive antitumor effects in animal models. In 1994, Yagi et al.
(55) showed antitumor effects of cationic liposome encapsulated
huIFN-␤ in human gliomas that had been transplanted into nude mice.
More recent work by the same group has shown antitumor effects and
the generation of cellular immunity by liposomal-mediated transfer of
murine IFN-␤ into mouse gliomas (37, 56). Studies performed by Xie
et al. (32) and Xu et al. (33) of injecting human and murine cancer
cells transfected with murine IFN-␤ into nude mice demonstrated that
cell growth of transduced tumor cells was markedly inhibited, with the
antitumor effects being attributed to up-regulation of inducible nitric
oxide synthase in murine macrophages. Qin et al. (35) showed that an
adenovirus expressing the human IFN-␤ gene (Ad.huIFN-␤) could be
injected into established human tumors (breast, colon, cervical, and
hepatoma) growing as flank tumors in SCID or nude mice and induce
significant inhibition of tumor growth. Because these latter studies
were done in immunodeficient animals, effects were likely attributa-
ble primarily to innate immune responses, direct antiproliferative
effects, or antiangiogenic activity.
In experiments with perhaps more relevance to an actual clinical
scenario, Lu et al. (36) showed that an Ad.muIFN-␤ vector multiply
injected into a murine sarcoma line growing in the flanks of immu-
nocompetent C3H mice led to tumor growth inhibition. In these
studies, an antitumor immune response was postulated because: (a)
 Ad.IFN-␤ FOR MESOTHELIOMA
injection of Ad.muIFN-␤ induced a prominent T-cell infiltration into
the tumors; (b) reinjected tumors did not grow in previously treated
animals; and (c) the vector was ineffective when injected into tumors
growing in immunodeficient mice.
Given this multiplicity of effects, it was thus of interest to define the
mechanisms by which i.p. injection of Ad.muIFN-␤ led to its antitu-
mor effects in our mesothelioma models. Our studies show that
acquired, CD8⫹ T-cell-mediated immune responses are primarily
responsible for the therapeutic efficacy observed. There appear to be
relatively small contributions from other mechanisms, such as macro-
phage or NK cell activation, direct antiproliferative effects, or anti-
angiogenesis. This conclusion is based on the following data:
(a) Successful treatment led to antitumor immunity, evidenced by
the failure of AB12 cells to grow in the flanks of previously treated
animals (Fig. 5). This immunity was completely abolished by deple-
tion of CD8⫹ T cells at the time of rechallenge (Fig. 8). Interestingly
(see below), this antitumor immunity extended to a second, unrelated
lung cancer tumor.
(b) The therapeutic effects of Ad.muIFN-␤ were almost completely
attenuated in SCID/beige and SCID mice (Fig. 6). The complete lack
of efficacy in SCID/beige mice, who lack B, T, and NK cells, shows
that the antiproliferative activity of IFN-␤ on the tumor cells and the
antiangiogenic properties were minimal. This is in marked contrast to
the effects of IFN-␤ injected into human tumors growing on SCID/
beige mice reported by Qin et al. (35). The experiments in SCID mice,
which lack B and T cells but have functional macrophages, neutro-
phils, and NK cells, were particularly useful in showing that the
IFN-␤-dependent macrophage and NK cell activation that appear
prominent in other models were of only minor significance important
in our system. It is interesting that in at least one other syngeneic
murine tumor model (the growth of liver metastases after injection of
the CT26 colon carcinoma line), NK cell activation appears to be very
important in the antitumor effects.4
(c) We were able to demonstrate that splenocytes isolated from
Ad.muIFN-␤-treated animals (but not control animals or animals
treated with control adenoviral vector) had high specific cytolytic
activity against AB12 tumor cells (Fig. 9). This lytic activity was
completely abolished by depletion of CD8⫹ T cells (Fig. 9C) showing
that CD8⫹ T cells, rather than NK cells, were of primary importance.
Of note is the fact that CTL activity was elicited in cells without the
need for in vitro stimulation, suggesting very strong CTL responses.
(d) We were able to abrogate completely the antitumor effects of
Ad.muIFN-␤ by depleting animals of CD8⫹ T cells using injection of
anti-CD8 antibodies (Fig. 7). A significant, but lesser, inhibitory
effect was seen after depletion of CD4⫹ T cells, suggesting that both
arms of the acquired immune system play important roles in gener-
ating antitumor immunity. However, depletion of CD4⫹ T cells did
not block the ability of “cured” mice to reject a rechallenge of tumor
cells (Fig. 8).
These findings share similarities and differences with the previous
studies of Natusme et al. (56) and Lu et al. (36). Similar to our
findings, Natusme et al. (56), who injected liposomally encapsulated
IFN-␤ into murine brain tumors, found immunity to re-injected tumor
cells and detected splenic CTLs, the specific antitumor cytolytic
activity of which could be abrogated completely by CD8⫹ T-cell
depletion. They were also able to block antitumor effects in animals
by administration of anti-CD8 monoclonal antibodies. In contrast to
our results, however, detection of CTL activity required two to three
rounds of in vitro stimulation, and no cross-sensitization responses
were noted. Although a more limited immunological analysis was
4
F. Spitz, University of Pennsylvania, personal communication.
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Research.
performed, the study of Lu et al. (36) was similar to ours in that
immunity to re-injection of tumor cells was observed and that the
therapeutic effects of Ad.muIFN-␤ were largely lost in SCID mice,
thus suggesting minimal effects of NK cells. Important differences
between the studies included: (a) that multiple doses of vector were
administered to flank tumors by Lu et al. (Ref. 36; compared with a
single i.p. dose in our study); (b) no cross sensitization was observed;
and (c) an “immunogenic” tumor (UV-2237m) versus “non-immuno-
genic” tumor (AC29; Ref. 57) was used. Experiments showing that
local treatment could lead to distant therapeutic effects were not
performed in either of the other studies.
It is interesting to speculate on why administration of Ad.muIFN-␤
was so effective in our models of mesothelioma. An exceptionally
powerful T-cell response was generated because treatment with only
a single dose of Ad.muIFN-␤ not only produced a high percentage of
long-term tumor-free survivors but also resulted in inhibitory effects
on tumors cells growing at a distant site. In addition, we saw CTL
activity in splenic cells without the need for in vitro stimulation. In
previous immunotherapy experiments using liposomes containing
DNA and using the same cell lines, but with multiple treatments, we
found that at least one round of in vitro stimulation was needed to
detect CTL activity (43). Treatment in the current study led to the
generation of systemic immunity to challenge with parental tumor
cells; however, somewhat surprisingly, we also observed immunity to
another syngeneic, nonmesothelioma tumor, the L1C2 murine lung
adenocarcinoma. The appearance of such cross-sensitization has been
reported (58) but is unusual.
There are a number of possible explanations for the strength of
these effects. For example, the route of administration may be impor-
tant. The peritoneal cavity is an anatomical site where immune re-
sponses are vigorous, and thus the i.p. route has been used often for
in vivo immunization (59, 60). One reason for this is the large surface
area available. Another advantage could be the presence of large
numbers of free floating macrophages and dendritic cells in this cavity
(61) that have free access to the tumors growing at the surface of the
peritoneum. In addition, it has been recognized recently that normal
mesothelial cells also have antigen-presenting capability and may be
participating in the generation of antitumor immune responses (60, 62,
63). Experiments are ongoing to test the effects of Ad.muIFN-␤
injected into flank tumors compared with i.p. tumors. The extent of
gene transfer after vector administration may also be important. We
have shown previously that large numbers of tumor cells and normal
mesothelial cells are transduced after i.p. injection of adenovirus (38).
There may also be gene transfer into leukocytes within the peritoneal
cavity, although we have not specifically examined this issue. Future
studies are aimed at exploring some of these questions.
There are also some factors specific to mesothelioma that may also
be important. Unlike many tumors, mesotheliomas express high levels
of MHC class I antigens (64) that may make them more resistant to
NK cell activity and more susceptible to CD8⫹ T-cell lysis. Both
AB12 and AC29 cells express high levels of MHC class I molecules
(data not shown). Mesothelioma cells make large amounts of TGF-␤
that may induce an immunosuppressive environment (12). Bielefeldt-
Ohmann et al. (9) showed that IFN-␣ suppressed TGF-␤ mRNA
expression in mesothelioma tumors. Although not examined in our
study, it is possible that IFN-␤ inhibited the production of TGF-␤ in
a similar fashion, thus improving immune responses. Finally, the fact
that mesothelioma cells are sensitive to the antiproliferative and
apoptotic effects of Ad.muIFN-␤ (Fig. 1) could play a key role in
generating appropriate “danger signals” needed to drive effective
antitumor responses (65, 66). The importance of having the tumor
cells transduced is suggested in preliminary experiments, where i.p.
injection of Ad.muIFN-␤ followed by injection of tumor cells is
 Ad.IFN-␤ FOR MESOTHELIOMA
ineffective whereas ex vivo transduction of tumor cells prior to i.p.
injection leads to complete cure in immunocompetent mice but not in
SCID mice.
In summary, we report that adenovirus-mediated muIFN-␤ gene
therapy can exert potent antitumor activities in in vivo models of
murine malignant mesothelioma. A single i.p. administration of Ad-
.muIFN-␤ was sufficient to cause complete regression of i.p. tumors
in immunocompetent animals and induce long-lasting immune re-
sponses that protected them from s.c. challenge with parental tumor
cells and at least one other syngeneic tumor. Effects on distant tumor
were also seen. This potent antitumor effect, likely resulting from the
local immune stimulatory effects of muIFN-␤, could be a critical
factor in cancer gene therapy trials in which the degree of gene
delivery to tumor cells is likely to be limited and a significant
bystander effect will be required. Although not prominent in our
animal models, it is possible that in clinical trials, the increased
sensitivity of human mesothelioma cells to the antiproliferative effects
of human IFN-␤, plus the potent antiangiogenic and immunostimula-
tory effects on macrophages and NK cells, could further contribute to
antitumor activity. Therefore, intracavitary IFN-␤ gene therapy pro-
vides a promising strategy for the treatment of some solid tumors such
as mesothelioma or ovarian cancer in humans.
ACKNOWLEDGMENTS
We thank Patina Zarcone for performing the IFN-␤ ELISAs and Veena
Kapoor for technical assistance. We also recognize Dr. Stephen Eck and the
other members of the Thoracic Oncology Laboratory for input and useful
discussion.
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Eradication of Intraperitoneal and Distant Tumor by
Adenovirus-mediated Interferon- β Gene Therapy Is Attributable
to Induction of Systemic Immunity
Makoto Odaka, Daniel H. Sterman, Rainer Wiewrodt, et al.

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