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*
Department of Neurosurgery and Georgetown Brain Tumor Center and
Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC 20007, and
Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussion References |
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production. There was no
significant tumor growth inhibition after intratumoral inoculation of
s.c. CT26 tumors in athymic mice. We conclude that this defective HSV
vector system is an effective method for cytokine gene delivery to
tumors in situ and IL-12 expression in tumors synergizes the antitumor
activity mediated by the replication-competent HSV helper virus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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We have developed a multigene mutant of herpes simplex virus type-1
(HSV-1),3 G207 with deletions
in the 34.5 gene and a lacZ insertion in the ICP6 gene,
that has proved efficacious in tumor therapy and is nonpathogenic (2).
G207 replicates in dividing cells with resultant cell death, whereas
its growth in nondividing cells is highly attenuated. Inoculation of
established tumors in athymic mice with G207 leads to inhibition of
tumor growth and prolonged animal survival due to tumor selective
replication (2, 3). Additionally, in immunocompetent mice, intratumoral
G207 inoculation induces a tumor-specific immune response that is able
to inhibit the growth of noninoculated tumors (4). In this case, G207
is behaving as an "in situ cancer vaccine."
We have used a defective HSV vector to deliver IL-12 in combination
with G207 as a helper virus. Defective HSV vectors consist of defective
particles containing tandem repeats of an amplicon plasmid (encoding
IL-12 in this case) and helper HSV (5). Any conditional-lethal or
replication-competent HSV mutant can be used as helper virus. Because a
viral genome length of DNA (
153 kb) is packaged, each defective
particle contains 15 copies of the IL-12 gene that can transduce
both dividing and nondividing cells at high efficiency. The viral DNA
does not integrate into the infected cell genome (6) and with the CMV
promoter driving IL-12, expression is strong but transient (7).
Integrating vectors run the risk of long term transgene expression with
chronic cytokine stimulation of the immune system.
IL-12 is a heterodimeric cytokine, composed of 35 kDa (p35) and 40 kDa
(p40) subunits, that binds to receptors present on NK and T cells (8).
The high affinity receptor is composed of two ß-type cytokine
receptor subunits that individually behave as low affinity receptors
(9). IL-12 plays a multifunctional role in the immune system,
augmenting the proliferation and cytotoxic activity of T cells and NK
cells, regulating IFN- production, and promoting the development of
CD4+ Th1 cells (10, 11). The antitumor activity of IL-12
has been demonstrated in a number of different murine tumor models,
both solid and metastatic, with systemic administration of recombinant
IL-12 (12, 13, 14, 15), fibroblasts or tumor cells engineered to secrete IL-12
(16, 17, 18, 19), viral vectors expressing IL-12 (20, 21). However, IL-12
immunotherapy is less effective with other tumor cell lines such as
CT26 (22), C26 (18), MCH-1-A1 (23), and TS/A (24).
We have chosen to use the murine colorectal carcinoma cell line CT26, which is poorly immunogenic and does not induce detectable tumor-specific CTLs (25, 26). CT26 tumors are somewhat refractory to IL-12 treatment (22, 27), although with sufficient levels of IL-12, tumor regression can be induced (14, 18). The immunodominant MHC class I-restricted Ag for CT26 has been identified as a nonamer peptide derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus (26). Adoptive transfer studies have established the correlation between induction of tumor-specific CTL and an antitumor effect on established s.c. CT26 tumors (26). In these studies, we demonstrate that local expression of IL-12 can enhance the antitumor activity of replication-competent attenuated HSV in both inoculated and noninoculated established CT26 tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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African green monkey kidney (Vero) cells (kindly provided by Dr. D. Knipe, Harvard Medical School) were cultured in DMEM containing 10% calf serum. CT26 is a colon epithelial tumor cell line (H-2d) derived by intrarectal injections of N-nitroso-N-methylurethan from a female BALB/c mouse (28, 29) (kindly provided by Dr. N. P. Restifo, National Cancer Institute, National Institutes of Health, Bethesda, MD). MC-38 mouse colon adenocarcinoma (obtained from Dr. N. P. Restifo) (15), Harding-Passey mouse melanoma (kindly provided by Dr. N. W. Fraser, Wistar Institute) (30, 31), MDA-MB-435 human breast adenocarcinoma (obtained from the Lombardi Cancer Center, Georgetown University Medical Center) (32), and CT26 cells were grown in DMEM containing 10% heat-inactivated FCS (HyClone, Logan, UT) and penicillin-streptomycin (Sigma Chemical, St. Louis, MO). A20, a B cell lymphoma cell line (Ig+, Ia+, H-2d) derived from a spontaneous reticulum cell neoplasm in BALB/c mice (33) (American Type Culture Collection, Rockville, MD, ATCC TIB 208), was grown in RPMI 1640 containing 10% heat-inactivated FCS, 50 µM 2-ME, 2 mM glutamine, 20 mM HEPES buffer, and penicillin-streptomycin.
Generation of dvs
The double-cassette amplicon plasmid pHCL-tk was constructed by
inserting the HSV-1 thymidine kinase (TK) gene, blunt-ended
BamHI fragment from pHSV-106 (Life Technologies, Rockville,
MD) into the blunt-ended SpeI site of pHCL (7) (Fig. 1
A). The cDNA for
murine IL-12 p40 (BL-pSV40) was kindly provided by Dr. U. Gubler
(Hoffmann-La Roche) (34). The cDNA for murine IL-12 p35 and the
internal ribosome entry site (IRES) of equine encephalomyocarditis
virus from DFG-mIL-12 (19) was kindly provided by Dr. H. Tahara
(University of Pittsburgh, Pittsburgh, PA). The coding regions of p40,
BamHI fragment from BL-pSV40, and the IRES-p35,
BamHI fragment from DFG-mIL12, were subcloned into
LITMUS 28 (New England Biolabs, MA) at the
BglII/BamHI site to generate p40-IRES-p35. The
IL-12-encoding double-cassette amplicon plasmid pHCIL12-tk
was constructed by insertion of the p40-IRES-p35 cassette,
SnaB1/AflII fragment, into the blunt-ended
SalI site of pSR-ori (7) and then inserting the HSV TK
blunt-ended BamHI fragment into the blunt-ended
SphI site to produce pHCIL12-tk (Fig. 1B).
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34.5 gene and
an Escherichia coli lacZ insertion inactivating the
ICP6 gene (2), was used as helper virus for the generation
of defective vector (dv) stocks. Vero cells were cotransfected with
purified amplicon plasmid DNA (pHCIL12-tk and pHCL-tk) and G207 viral
DNA using lipofectAMINE (Life Technologies), as described by the
manufacturer, and then cultured at 34.5°C until cells exhibited
complete cytopathic effect. Virus was then harvested and passaged at a
1:4 dilution in Vero cells until inhibition of helper virus replication
was observed (7). For ease, the IL-12-containing dv will be termed
dvIL12/G207 and the lacZ-containing dv will be termed
dvlacZ/G207. Titering of dv stocks
Defective vector stocks were titered after a freeze-thaw/sonication regimen and removal of cell debris by low speed centrifugation (2000 x g for 10 min at 4°C). G207 helper virus titer was expressed as the number of plaque-forming units (PFUs) after plaque assay on Vero cells at 34.5°C. For dvIL12/G207, IL-12 expression was determined and the passage with the highest level was used (passage 4), with a G207 helper virus titer of 5 x 107 PFU/ml. The titer of dvlacZ/G207, determined by counting 5-bromo-4-chloro-3-indolyl-ß-D-glactopyranoside histochemistry-positive single cells (defective particle units (DPU)) after formation of plaques by G207, was 5 x 106 DPU/ml and 5 x 107 PFU/ml of helper virus.
Detection of IL-12
Cells were infected with dvIL12/G207 at a multiplicity of infection (MOI) of 1 PFU per cell, and 24 h postinfection aliquots of infected cell supernatant were removed, quick frozen in a dry ice/ethanol bath, and stored at -80°C for detection of IL-12. Tumors and blood were collected from dv-treated mice and snap-frozen in a dry ice/ethanol bath. Frozen tissue was homogenized in ice-cold PBS containing 500 µM PMSF, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin. The homogenate was then sonicated twice for 10 s and cleared by centrifugation in a microfuge for 5 min at 4°C. Immunoreactive IL-12 levels were determined by sandwich ELISA, using Ab pairs and rIL-12 kindly provided by Dr. D. H. Presky (Hoffmann-La Roche, Nutley, NJ) (35). The rIL-12 standards were diluted in the same media or buffer as the samples (i.e., mouse serum for the serum samples). Briefly, 96-well plates coated with an anti-mouse IL-12 mAb (9A5) were incubated overnight at room temperature with the test samples. After washes, the plates were incubated with peroxidase-labeled anti-mouse IL-12 p40 Ab (5C3) for 2 h and developed. Absorbance was measured at 450 nm.
s.c. tumor model
BALB/c and BALB/c-nu/nu mice were obtained from the
National Cancer Institute or Charles River (Wilmington, MA). All animal
procedures were approved by the Georgetown University Animal Care and
Use Committee. For surgical procedures, each mouse was anesthetized
with an i.p. injection of a 0.25- to 0.30-ml solution consisting of
84% bacteriostatic saline, 10% sodium pentobarbital (1 mg/ml; Abbott
Laboratories, Chicago, IL), and 6% ethyl alcohol. CT26 tumor cells
(1 x 105) were injected s.c. in the bilateral flanks
of mice. When s.c. tumors were palpably growing (5 mm in maximal
diameter), mice were unilaterally inoculated into the right side tumor
with either 50 µl of defective HSV vector (7 x 105
PFU of helper virus) in virus buffer (150 mM NaCl, 20 mM Tris, pH 7.5)
or 50 µl of virus buffer, followed by a second injection 7 days
later. For some studies, where indicated, mock extract (2) was used in
place of virus buffer. Tumor size was measured by external caliper, and
tumor volume was calculated (V =
h x w x d). If
animals appeared moribund or the diameter of their s.c. tumors reached
18 mm, they were killed and this was recorded as the date of death for
survival studies. Statistical differences were calculated using
StatView 4.5 (Abacus Concepts, Berkeley, CA) where mean tumor volume
was assessed by unpaired t test, survival means by ANOVA
(Fisher’s post hoc comparisons), and differences in survival
by log rank (Mantel-Cox) test.
To generate lymphocytes for CTL assays, FACS analysis, and IFN-
production assays, mice were killed 12 days after virus inoculation,
and their spleens were isolated. Single-cell suspensions of splenocytes
from individual inoculated mice were prepared in ACK lysing buffer
(BioWhittaker, Walkersville, MD) followed by washing in RPMI 1640
medium containing 10% heat-inactivated FCS.
IFN- assays
Single-cell suspensions of splenocytes were washed and
resuspended in RPMI 1640 medium containing 10% inactivated FCS. Cells
(3 x 106/ml) were cultured in 24-well plates for
24 h. Supernatants were collected and assayed by a sandwich ELISA
using anti-IFN- Ab pairs obtained from Endogen (Woburn,
MA).
Flow cytometry analysis
Single-cell suspensions of splenocytes were washed and resuspended in PBS containing 0.3% BSA and 0.1% sodium azide. Briefly, aliquots of 3 x 106 cells were stained with FITC-conjugated anti-mouse CD4 mAb and R-phycoerythrin (PE)-conjugated anti-mouse CD8a mAb (PharMingen, San Diego, CA) for 30 min at 4°C. After staining, samples were fixed with 0.5% paraformaldehyde and analyzed on a FACScan flow cytometer focusing on the lymphocyte cluster (Becton Dickinson, Mountain View, CA).
Peptides
Peptide AH1 (nonamer SPSYVYHQF) is an immunodominant Ag identified in CT26 cells (26), derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia virus and presented by the MHC class I Ld molecule (26). The peptide was synthesized by Peptide Technologies (Washington, DC) to a purity of >99% as determined by HPLC and amino acid analysis. H-2Ld-restricted peptide P815AB, LPYLGWLVF, is an immunodominant Ag derived from murine mastocytoma P815 cells (36) (kindly provided by Dr. N. P. Restifo).
CTL Assays
Single-cell suspensions of splenocytes were cultured in RPMI 1640 medium with 10% inactivated FCS, 50 µM 2-ME, 2 mM glutamine, 20 mM HEPES, and penicillin-streptomycin in 24-well plates at a concentration of 3 x 106 cells/ml. In addition, either 1 x 106 inactivated CT26 cells or 1 µg/ml peptide AH1 was added to the medium. For inactivation, CT26 tumor cells were incubated for 1 h in culture medium containing 100 µg/ml of mitomycin C and then washed twice. Effector cells were harvested after 6 days of in vitro culture.
Four-hour 51Cr release assays were performed as previously described (37). In brief, target cells were incubated with 50 µCi of Na51CrO4 (51Cr) for 60 min. A20 cells were pulsed with 1 µg/ml of the Ld-restricted peptides AH1 or P815AB for 1 h before labeling. Target cells were then mixed with effector cells for 4 h at the E:T ratios indicated. The amount of 51Cr release was determined by gamma counting, and the percentage of specific lysis was calculated from triplicate samples as: [(experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm)] x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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Amplicon plasmids, pHCIL12-tk and pHCL-tk, of similar size
encoding the two subunits of murine IL-12, p40 and p35, or
lacZ, respectively, under control of the CMVIE
promoter (Fig. 1
) were constructed. IL-12 is functional as a
heterodimer, and therefore both subunits were expressed from a single
dv as a bicistronic message using an IRES. dv stocks were generated
from these amplicon plasmids using HSV-1 G207 as helper virus.
Infection of CT26 cells with dvIL12/G207 or G207 alone results in cell
death with >99% cytotoxicity by 4 days postinfection at a MOI of 1
(data not shown). The expression and secretion of IL-12 heterodimer
were determined by ELISA assay after infection of tumor cells in
culture. Infection of CT26 (murine colon carcinoma),
Harding-Passey (murine melanoma), MCA38 (murine colon
adenocarcinoma), and MDA-MB-435 (human breast adenocarcinoma) cells
with dvIL12/G207 resulted in secretion of up to 1.5 ng of murine
IL-12/105 tumor cells/24 h (Fig. 2). No IL-12 was detected in the
supernatants of uninfected tumor cell cultures or those infected with
dvlacZ/G207. Levels of IL-12 synthesis and secretion peaked 1 day after
dvIL12/G207 infection of CT26 cells and decreased to undetectable by 3
days postinfection, likely due to cell death.
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The antitumor efficacy of the combined dvIL12/G207 therapy was
evaluated in the CT26 s.c. tumor model in syngeneic BALB/c mice.
Bilateral tumors were established in the flanks of BALB/c mice by the
implantation of 105 CT26 cells per side. Treatment was
initiated when s.c. tumors reached 5 mm in maximal diameter. Each
animal then underwent a unilateral intratumoral inoculation of dv stock
(7 x 105 PFU of G207 helper virus) in the right flank
tumor, followed by a second inoculation 7 days later. dvlacZ/G207
rather than helper virus G207 alone was used as a control for
dvIL12/G207 inoculation so that differences in viral factors (i.e.,
particle:PFU ratio) present in dv stocks vs G207 stocks would be
accounted for. Both G207 and dvlacZ contain E. coli
lacZ, and therefore no additional foreign Ags were expressed
by the control dv. G207 infection of s.c. CT26 tumors results in
anti-HSV immunity, as detected by production of neutralizing serum
Ab (data not shown).
Inoculation with dvIL12/G207 elicited a very prominent antitumor
effect, with both the inoculated tumors and their noninoculated
contralateral counterparts demonstrating a significant reduction in
tumor growth (Fig. 3,
top). Two of six of the dvIL12/G207-inoculated tumors
regressed to an undetectable size. Inoculation with dvlacZ/G207 also
resulted in a significant reduction in tumor growth of both inoculated
and noninoculated tumors compared with controls, although to a much
lesser extent than dvIL12/G207 (Fig. 3, top). A
similar degree of s.c. CT26 tumor growth inhibition (as seen with
dvlacZ/G207) was detected after intratumoral inoculation of G207 alone
(4). Mice were also followed for survival (Fig. 4), where sacrifice occurred when either
of the bilateral tumors became larger than 18 mm in diameter. Survival
of the dv-treated animals is therefore reflective of the growth of the
noninoculated tumors and is significantly longer than control
animals. Mice treated unilaterally with dvIL12/G207 survived longer
than dvlacZ/G207-treated mice (Fig. 4). Heterodimeric IL-12 was
detected in the dvIL12/G207-inoculated tumors 1 and 5 days
postinoculation (50–100 pg/tumor), with no IL-12 detected in
the serum.
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5 mm in maximal diameter)
followed by a second inoculation 7 days later. Although there was a
slight delay in growth of right side tumors injected with dvIL12/G207,
no significant tumor growth inhibition was observed in either the
inoculated or contralateral noninoculated tumors (Fig. 3, bottom). CT26 tumors grew somewhat more rapidly in
the athymic mice than in the immunocompetent mice (Fig. 3). Intratumoral inoculation with defective HSV vectors elicits a CT26-specific CTL response
To test whether inhibition of tumor growth was associated with
increased CTL activity, we examined the capacity of intratumoral
inoculation with defective HSV vectors to elicit CT26-specific CTL
activity in vitro using a 51Cr release assay. BALB/c
mice were inoculated with dvIL12/G207 or dvlacZ/G207 intratumorally
when s.c. tumors reached 5 mm in maximal diameter, followed by a
second inoculation 7 days later. Effector cells were generated in vitro
from splenocytes obtained 5 days after the second inoculation, by
culture with mitomycin C-treated CT26 cells or 1 µg/ml of the CT26
immunodominant MHC class I-restricted peptide, AH1 (26). Effector
cells, from dvIL12/G207-treated mice, restimulated with mitomycin
C-treated CT26 cells exhibited specific lysis of CT26 target cells, as
well as A20 cells pulsed with peptide AH1 (Fig. 5). No apparent lysis of A20 cells (data
not shown) or A20 cells pulsed with Ld-restricted peptide
P815AB, the immunodominant Ag derived from murine mastocytoma P815
cells (36), was observed (Fig. 5). Effector cells restimulated with
peptide AH1, from mice treated with dvIL12/G207 or dvlacZ/G207,
exhibited specific lysis of target A20 cells pulsed with peptide AH1 as
well as CT26 cells but not unpulsed A20 cells (Fig. 6). The level of CTL activity generated
by dvIL12/G207 was significantly greater than that generated by
dvlacZ/G207. Effector cells from dvIL12/G207 inoculated animals, not
restimulated, were able to specifically lyse CT26 but not A20 cells
(data not shown).
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production. Splenocytes were isolated 5 days after the second
inoculation of dvIL12/G207 or dvlacZ/G207 and tested for IFN-
production by ELISA and splenic T lymphocyte subsets by FACS analysis.
Similar percentages of Th cells (CD4) and cytotoxic T cells (CD8a) were
found in dvIL12/G207 and dvlacZ/G207-treated mice (Fig. 7). Splenocytes from mice treated with
dvIL12/G207 produced significantly greater amounts of IFN- than
those treated with dvlacZ/G207 (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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The defective IL-12 vector infected a number of different tumor cells, which then produced and secreted IL-12 in vitro. B16 murine melanoma, which has been used in a large number of immunotherapy studies, is highly resistant to HSV infection (38) and did not produce detectable IL-12 after dvIL12/G207 infection (data not shown). The variation in IL-12 production by different cell lines could be due to different susceptibilities of the cells to defective HSV infection or lysis by the helper virus G207. Cells that are highly susceptible to HSV infection, but where G207 replicates poorly and are therefore not rapidly destroyed, may be the highest producers of IL-12 in vitro. The actual levels and timing of delivery or synthesis of IL-12 in vivo can have large effects on the outcome of therapy. For example, Noguchi et al. (39) found that a low dose of IL-12 induced the largest tumor-specific CTL response, whereas higher doses actually suppressed the CTL response, yet the highest doses of IL-12 had the most potent antitumor effect.
Systemic delivery of rIL-12 has been shown to have potent antitumor effects in various animal models (12, 13, 14, 15, 22, 27); however, prolonged exposure to IL-12 can have deleterious side effects like many cytokines (40, 41). Transfer of cytokine genes directly to the tumor cells is advantageous because cytokines are expressed within the tumor at the site of putative tumor Ags. Although localized gene therapy is very attractive, current retroviral-based vectors for gene transfer require ex vivo manipulation and therefore are not practical for routine clinical use.
The approach that we have chosen involves direct genetic modification of the tumors in situ, using defective HSV vectors to deliver cytokine genes, thereby making the tumor cells themselves a source of cytokine production. As a control for these studies, we used dvlacZ/G207. Both G207 and dvlacZ express ß-galactosidase and lacZ is under the same transcriptional control in dvlacZ as IL-12 is in dvIL12. In addition, it has been shown that CT26 cells stably transfected with lacZ are just as tumorigenic (42) and as poorly susceptible to systemic IL-12 therapy as CT26 cells (22). Mice vaccinated with a recombinant poxvirus expressing ß-galactosidase were not protected from challenge with CT26 cells (42). Therefore, even though ß-galactosidase is a MHC class I-processed Ag of foreign origin, it failed to illicit a measurable antitumor immune response and does not seem to have any independent effect on antitumor therapy.
Other viral vectors (adenovirus and vaccinia virus) have been
constructed to deliver both subunits of IL-12 in vivo by direct
inoculation (21, 43). In this study, IL-12 production was detected in
the inoculated tumor but not in the serum. The distribution of T cell
subtypes was not significantly altered in the spleen by IL-12
expression at the tumor site; however, tumor-specific CTL activity and
production of IFN- by splenocytes were significantly enhanced. The
local expression of IL-12, as an adjuvant, improved the antitumor
efficacy of vaccination with the attenuated HSV helper virus in
reducing growth of inoculated and noninoculated tumors. This
improvement was correlated with heightened induction of tumor-specific
CTL activity and IFN- production by splenocytes. In contrast, use of
rIL-12 as adjuvant with IL-2-transduced tumor cells as vaccine improved
antitumor efficacy with enhanced IFN- production but no effect on
CTL activity (44).
It is unclear what mechanism(s) is involved in the generation of specific antitumor immunity after HSV infection of tumor cells. It has recently been shown that tumor Ags can be transferred to host bone marrow-derived APCs before MHC class I presentation to CD8+ T cells (26). Local HSV infection of the tumor might induce circulating precursors to differentiate into APCs. It has been suggested that GM-CSF plays an important role in the maturation and/or function of specialized APCs (1, 45). We found that intratumoral inoculation of GM-CSF-expressing defective HSV vectors had only limited antitumor efficacy compared with dvlacZ/G207 in the same s.c. CT26 tumor model (M. Toda, R. L. Martuza, and S. D. Rabkin, unpublished results). A subset of macrophages can process exogenous Ags by the same class I pathway as endogenous Ags and present them to CD8+ T cell clones (46, 47). Viral destruction of tumor cells might release tumor Ags that are then picked up by APCs, carried to the draining lymph nodes and presented to T cells. Histologically, the dvIL12-treated tumors had much larger areas of necrosis than the dvlacZ tumors, whereas the mock-treated tumors only had small areas of necrosis. Both dvIL12- and dvlacZ-treated tumors had a large influx of neutrophils, with none apparent in the mock-treated tumors (data not shown)
Although HSV-1 replicates in a wide variety of tumor cell types, G207,
which is highly attenuated, is much more restricted and unable to grow
in most rodent tumor cell lines. CT26 cells have only limited
susceptibility to G207, so that at a MOI of 0.1 only 60% of the
cells are killed (data not shown), and G207 inoculation did not inhibit
tumor growth in athymic mice. Therefore, in this system, G207 the
helper virus is functioning much more as a tumor vaccine than by direct
cytocidal action. The local production of IL-12 is acting as an
adjuvant to the antitumor immunity induced by the helper virus. We did
not find evidence of elevated IL-12 in the serum, but we did find
elevated IL-12 in the contralateral, noninoculated tumor that is
probably being produced endogenously. IL-12 is secreted by
"professional" APCs, monocytes/macrophages, dendritic cells, and B
cells (48). HSV infection of mice can lead to up-regulation of IL-12
expression (49), and this may be the cause of the elevated IL-12 in the
noninoculated tumors or the inoculated tumors at later time points.
G207 has been found to be nonpathogenic in a number of animal models (2) and is progressing toward clinical trials for the treatment of primary human brain tumors. This vector combination of viral cancer vaccine and immune enhancement with cytokine expression can be administered directly into the tumor in situ without any ex vivo manipulations of the tumor cells or knowledge of tumor Ags. The presence of IL-12 in particles separate from the helper virus and the large number of copies of IL-12 delivered in each defective particle may account for the significant antitumor effect seen both in established inoculated tumors and distal noninoculated tumors.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Samuel D. Rabkin, Department of Neurosurgery, Georgetown University Medical Center, 3970 Reservoir Road, NW., Washington, DC 20007.
3 Abbreviations used in this paper: HSV, herpes simplex virus; PFU, plaque-forming unit; DPU, defective particle unit; MOI, multiplicity of infection; dv, defective vector; IRES, internal ribosome entry site; TK, thymidine kinase; PE, R-phycoerythrin.
Received for publication July 11, 1997. Accepted for publication December 29, 1997.
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References |
|---|
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TopAbstractIntroductionMaterials and MethodsResultsDiscussion References |
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production. J. Immunol. 153:1697.[Abstract]
-mediated tumor growth inhibition induced during tumor immunotherapy with rIL-12. Int. Immunol. 8:855. in vivo. Int. Immunol. 6:157.This article has been cited by other articles:
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 |
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  Y. Ino, Y. Saeki, H. Fukuhara, and T. Todo Triple Combination of Oncolytic Herpes Simplex Virus-1 Vectors Armed with Interleukin-12, Interleukin-18, or Soluble B7-1 Results in Enhanced Antitumor Efficacy Clin. Cancer Res., January 15, 2006; 12(2): 643 - 652. [Abstract] [Full Text] [PDF]
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|
 |
|
 R. Liu, S. Varghese, and S. D. Rabkin Oncolytic Herpes Simplex Virus Vector Therapy of Breast Cancer in C3(1)/SV40 T-antigen Transgenic Mice Cancer Res., February 15, 2005; 65(4): 1532 - 1540. [Abstract] [Full Text] [PDF]
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W. Ren, R. Strube, X. Zhang, S.-Y. Chen, and X. F. Huang Potent Tumor-Specific Immunity Induced by an In vivo Heat Shock Protein-Suicide Gene-Based Tumor Vaccine Cancer Res., September 15, 2004; 64(18): 6645 - 6651. [Abstract] [Full Text] [PDF]
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X. F. Huang, W. Ren, L. Rollins, P. Pittman, M. Shah, L. Shen, Q. Gu, R. Strube, F. Hu, and S.-Y. Chen A Broadly Applicable, Personalized Heat Shock Protein-Mediated Oncolytic Tumor Vaccine Cancer Res., November 1, 2003; 63(21): 7321 - 7329. [Abstract] [Full Text] [PDF]
|
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K. A. Tolba, W. J. Bowers, J. Muller, V. Housekneckt, R. E. Giuliano, H. J. Federoff, and J. D. Rosenblatt Herpes Simplex Virus (HSV) Amplicon-mediated Codelivery of Secondary Lymphoid Tissue Chemokine and CD40L Results in Augmented Antitumor Activity Cancer Res., November 15, 2002; 62(22): 6545 - 6551. [Abstract] [Full Text] [PDF]
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 |
|
 M. Katae, Y. Miyahira, K. Takeda, H. Matsuda, H. Yagita, K. Okumura, T. Takeuchi, T. Kamiyama, A. Ohwada, Y. Fukuchi, et al. Coadministration of an Interleukin-12 Gene and a Trypanosoma cruzi Gene Improves Vaccine Efficacy Infect. Immun., September 1, 2002; 70(9): 4833 - 4840. [Abstract] [Full Text] [PDF]
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A. Wu, A. Mazumder, R. L. Martuza, X. Liu, M. Thein, K. R. Meehan, and S. D. Rabkin Biological Purging of Breast Cancer Cells Using an Attenuated Replication-competent Herpes Simplex Virus in Human Hematopoietic Stem Cell Transplantation Cancer Res., April 1, 2001; 61(7): 3009 - 3015. [Abstract] [Full Text]
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 |
|
 J. Nemunaitis, F. Khuri, I. Ganly, J. Arseneau, M. Posner, E. Vokes, J. Kuhn, T. McCarty, S. Landers, A. Blackburn, et al. Phase II Trial of Intratumoral Administration of ONYX-015, a Replication-Selective Adenovirus, in Patients With Refractory Head and Neck Cancer J. Clin. Oncol., January 15, 2001; 19(2): 289 - 298. [Abstract] [Full Text] [PDF]
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T. Todo, R. L. Martuza, M. J. Dallman, and S. D. Rabkin In Situ Expression of Soluble B7-1 in the Context of Oncolytic Herpes Simplex Virus Induces Potent Antitumor Immunity Cancer Res., January 1, 2001; 61(1): 153 - 161. [Abstract] [Full Text]
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 H. Kojima, M. Toda, and M. V. Sitkovsky Comparison of Fas- versus perforin-mediated pathways of cytotoxicity in TCR- and Thy-1-activated murine T cells Int. Immunol., March 1, 2000; 12(3): 365 - 374. [Abstract] [Full Text] [PDF]
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S. A. Ali, C. S. McLean, M. E. G. Boursnell, G. Martin, C. L. Holmes, S. Reeder, C. Entwisle, D. M. Blakeley, J. G. Shields, S. Todryk, et al. Preclinical Evaluation of "Whole" Cell Vaccines for Prophylaxis and Therapy Using a Disabled Infectious Single Cycle-Herpes Simplex Virus Vector to Transduce Cytokine Genes Cancer Res., March 1, 2000; 60(6): 1663 - 1670. [Abstract] [Full Text]
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 S. S. YOON, H. NAKAMURA, N. M. CARROLL, B. P. BODE, E. A. CHIOCCA, and K. K. TANABE An oncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma FASEB J, February 1, 2000; 14(2): 301 - 311. [Abstract] [Full Text]
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 T. A. Steele Recent Developments in the Virus Therapy of Cancer Experimental Biology and Medicine, February 1, 2000; 223(2): 118 - 127. [Abstract] [Full Text]
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 E. S. Lambright, D. J. Caparrelli, A. E. Abbas, T. Toyoizumi, G. Coukos, K. L. Molnar-Kimber, and L. R. Kaiser Oncolytic therapy using a mutant type-1 herpes simplex virus and the role of the immune system Ann. Thorac. Surg., November 1, 1999; 68(5): 1756 - 1760. [Abstract] [Full Text] [PDF]
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 C. S. Schmidt and M. F. Mescher Adjuvant Effect of IL-12: Conversion of Peptide Antigen Administration from Tolerizing to Immunizing for CD8+ T Cells In Vivo J. Immunol., September 1, 1999; 163(5): 2561 - 2567. [Abstract] [Full Text] [PDF]
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D. A. Kooby, J. F. Carew, M. W. Halterman, J. E. Mack, J. R. Bertino, L. H. Blumgart, H. J. Federoff, and Y. Fong Oncolytic viral therapy for human colorectal cancer and liver metastases using a multi-mutated herpes simplex virus type-1 (G207) FASEB J, August 1, 1999; 13(11): 1325 - 1334. [Abstract] [Full Text]
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G. Coukos, A. Makrigiannakis, E. H. Kang, D. Caparelli, I. Benjamin, L. R. Kaiser, S. C. Rubin, S. M. Albelda, and K. L. Molnar-Kimber Use of Carrier Cells to Deliver a Replication-selective Herpes Simplex Virus-1 Mutant for the Intraperitoneal Therapy of Epithelial Ovarian Cancer Clin. Cancer Res., June 1, 1999; 5(6): 1523 - 1537. [Abstract] [Full Text] [PDF]
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 J. H. Sampson THE PREUSS FOUNDATION SEMINAR ON VACCINE THERAPY FOR MALIGNANT PRIMARY BRAIN TUMORS February 15-17, 199 8, La Jolla, Calif. Neuro-oncol, January 1, 1999; 1(1): 33 - 42. [PDF]
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N. C. Fernandez, J.-P. Levraud, H. Haddada, M. Perricaudet, and P. Kourilsky High Frequency of Specific CD8+ T Cells in the Tumor and Blood Is Associated with Efficient Local IL-12 Gene Therapy of Cancer J. Immunol., January 1, 1999; 162(1): 609 - 617. [Abstract] [Full Text] [PDF]
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 J. N. Parker, G. Y. Gillespie, C. E. Love, S. Randall, R. J. Whitley, and J. M. Markert From the Cover: Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors PNAS, February 29, 2000; 97(5): 2208 - 2213. [Abstract] [Full Text] [PDF]
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