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Thyroid Study Unit/MC 3090 (R.Z., L.J.D.), Department of Medicine, Department of Pathology (F.H.S.), The University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One useful therapy is to introduce genes that alter the local
immunological microenvironment and activate immune effector cells. A
variety of genes have been used for this purpose, including
costimulator molecules such as B7–1 (3, 4, 5), B7–2 (5, 6, 7), MHC
molecules (5, 8, 9), cytokine genes such as interleukin (IL)-1 (10, 11), IL-2 (10, 12), IL-4 (10, 13, 14), IL-6 (10, 15), IL-7 (10, 16),
IL-10 (10, 17), IL-12 (10, 18), tumor necrosis factor-
(10, 11),
interferon (10), granulocyte colony-stimulating factor (G-CSF) (10, 19), and granulocyte-mononucleocyte colony-stimulating factor (GM-CSF)
(10, 20, 21, 22). Among these molecules, IL-2 has been extensively studied
and always showed reliable antitumor activity.
IL-2 is an important T cell growth factor that stimulates effector cells such as NK, CD4+, and CTL cells. IL-2 promotes proliferation and differentiation of these immune effector cells, thus activating both nonspecific and specific responses to tumor cells. A high concentration of administered IL-2 is needed to induce a therapeutic immune response. This systemic dose of IL-2 always results in severe side effects, which have limited the clinical usefulness of IL-2 in treatment of cancer patient (23). Development of gene transfer technology circumvents this limitation because high concentrations of IL-2 can be generated locally at the tumor site by direct intratumoral administration. This technique enhances antitumor immunity as evidenced in several experimental animal models (24, 25, 26, 27, 28, 29).
Gene transfer by recombinant replication-defective adenoviral vectors has many advantages and is currently widely applied in both animal and clinical trials. This family of viruses possesses a broad host range, can accept large DNA inserts (up to 8 kb), and infects cells independent of cell division. Rapidly dividing tumor cells are selectively targeted over the surrounding normal tissue (30). The adenovirus DNA induces transient gene expression but does not integrate into the genome. It is easy to get very high titers of virus, and the virus has a very good safety record (31).
We previously reported the construction of a replication defective adenovirus vector harboring the mouse IL-2 gene (AdCMVIL2). In vitro infection of murine medullary thyroid carcinoma (MTC) cells with AdCMVIL2 abrogated their tumorigenicity and induced a long lasting state of immunity in syngeneic BALB/C mice (32). We also confirmed that intratumoral injection of AdCMVIL2 results in the rejection and/or stabilization of preestablished tumors in treated mice (33). In this study, we aimed to answer the following questions: 1) Does AdCMVmIL2 induce antitumor activity after intratumoral administration in our rat MTC model? 2) Does dissemination of adenoviral vector occur after intratumoral injection? 3) Does AdCMVmIL2 cause significant toxicity in peripheral tissues?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rat MTC cell line was purchased from American Type Culture Collection (ATCC) (Rockville, MD), and maintained in DMEM (Gibco, Life Technologies, Inc.) supplemented with 10% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. YAC-1 cells were kindly provided by Dr. H. Schreiber (Department of Pathology, University of Chicago, Chicago, IL) and maintained in complete DMEM (supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin). 293 cells, a transformed human cell line that has the E1 region of the adenovirus type 5 integrated in its genome (34, 35), were purchased from Microbix Biosystems Inc. (MBI) (Ontario, Canada), and maintained in complete MEM (containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin). CTLL-2 cells were kindly provided by Dr. Edward Cohen (University of Illinois at Chicago). CTLL-2 cells were maintained in complete RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol and 20 U/ml recombinant human IL-2.
Recombinant defective adenoviral vectors
Construction of the replication-defective adenoviral vectors
containing mIL-2 complementary DNA, or the Lac Z gene, under the
transcriptional control of the human cytomegalovirus immediate early
(HCMV i.e.) promoter/enhancer system (AdCMVmIL2, AdCMVLacZ)
has been described (32). Viral stocks were prepared by infection of 293
cells. The viruses were harvested 48 h after infection and
purified by double cesium chloride gradient ultracentrifugations (34).
Viral titers (p.f.u./ml) were determined by plaque assay using 293
cells. Viral stocks were stored in 10% glycerol at -80 C.
Analysis of gene expression
Murine IL-2 bioactivity was measured by a standard bioassay
protocol using an IL-2 dependent murine T cell line, CTLL-2, as
described previously (36). The transduced rMTC cells were cultured for
24 h, and supernatant was saved for mIL-2 assay. CTLL-2 cells
(5 x 103) were incubated with a mIL-2 containing
sample in a volume of 180 µl for 24 h at 37 C in 96-well
microtiter plates. 3H-thymidine (0.5 µCi/well in 20 µl)
was added, and the incorporation of radiolabeled thymidine into DNA was
determined after overnight incubation (36).
Tumorigenicity of AdCMVmIL2 transduced rMTC cells
Rat MTC cells were infected with 100 multiplicity of infection
(moi) of AdCMVmIL2 for 2 h in 500 µl infection solution (DMEM
supplemented with 2% FBS). Infected cells were then washed with
serum-free medium. A total of 1 x 106 infected cells
in 100 µl serum-free medium were injected sc into one flank of
WAG/Rij rats or the abdomen of SCID mice. The AdCMVLacZ vector served
as the control. Injected animals were inspected every 2 days for
development of tumors.
Tumor production and calculation of the volume of sc tumors
Rat MTC cells were washed in serum-free DMEM, counted, and
injected sc into syngeneic rats (1 x 106 cells per
rat). The injected rats always developed palpable tumors within 7–10
days, and tumors grew progressively.
Tumor volumes were determined from the formula v = a2b/2, where a is the shortest diameter, and b is the longest diameter of the tumor. Tumor growth was measured every 2 or 3 days.
Direct intratumoral delivery of the vectors
AdCMVmIL2 or AdCMVLacZ was diluted in serum-free DMEM to 2
x 1010 p.f.u./ml. One-hundred microliters of diluted
vector (2 x 109 p.f.u.), or serum-free DMEM (as
control) were injected into preestablished MTC tumors.
Distant site challenge with parental rMTC cells in tumor-free
rats
Tumor-free rats previously treated with different adenoviral
vectors were challenged sc with wild-type rMTC cells in the opposite
flank after 60 days. The tumorigenic dose of cells (5 x
105 cells/rat) was used in this study.
Histology
At the time of necropsy, tumor and organs were harvested, and
placed into Zamboni’s fixative solution (Newcomer Supply, Middleton,
WI). The specimens were then embedded in paraffin, sectioned, and
stained with hematoxylin and eosin (H&E).
Neutralization antibody assay
Serum samples were tested for neutralizing antibodies
using the method described before with modification (37). Serial
dilutions of serum were prepared in serum-free DMEM. An aliquot of 200
µl of diluted serum was mixed with a 200 µl aliquot of AdCMVLacZ
diluted to 5 x 106 p.f.u./ml in serum-free DMEM and
incubated at 37 C for 1 h. At the same time, serum-free medium
mixed with AdCMVLacZ served as an antibody negative control, and a
neutralizing antibody containing rat serum mixture served as an
antibody positive control. An aliquot of 100 µl of this mixture was
added to one well of a 24-well plate seeded 6 h earlier with
2 x 105 Hela cells. After incubation for 1 h at
37 C, nonadsorbed virus solution was washed out of the wells and the
plates were incubated for a further 24 h with new complete DMEM.
The cells in each well were washed with 0.1 M PBS, and then
lysed by addition of 100 µl of lysis buffer (25 mM
Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM
1,2-diaminocyclohexane-N,N,N',N'-tetraacetic
acid, 10% glycerol, 1% Triton X-100) for 15 min at room temperature
(RT). A 900-µl aliquot of substrate solution composed of 150 µg/ml
Chlorophenol red-ß-D-galactopyranoside (CPRG,
Boehringer Mannheim Corp., Indianapolis, IN), 1
mM MgCl2, 45 mM ß-mercaptoethanol, in 0.1
M PBS, pH 7.5 was added to each well and incubated at 37 C
for 1 h. The reaction was terminated by the addition of 500 µl
of 1 M Na2CO3. The optical density
of the solution in each well was measured at 570 nm. The titer of
neutralization antibody was expressed as the inverse of the dilution
required to produce 50% reduction in LacZ expression as measured by
absorbance at 570 nm.
Cell mediated cytotoxicity assays
Nonradioactive Cytotoxicity Assay kit (Promega Corp., Madison, WI) was used to evaluate cell-mediated
cytotoxicity as previously described (32). Splenocytes from animals
free of tumor after treatment were used to assay tumor specific
cytotoxic T lymphocytes (CTL). Splenocytes were collected and
stimulated in vitro by incubating 3 x 106
of effector cells/ml with 2 x 105 of mitomycin-C
treated tumor cells/ml in 75-ml flasks for 5 days at 37 C in the
presence of 20 U/ml rIL2. As a control, splenocytes from untreated rats
were also collected and used in the assays. Target cells (1 x
104) were mixed with stimulated effector cells at final
effector/target (E:T) ratios between 100:1 and 12.5:1 in 96-well
U-bottom plates. The plates were lightly centrifuged at 500 rpm for 4
min and incubated for 4 h at 37 C, 5% CO2. Forty-five minutes
before harvesting supernatants, 20 µl of 10x lysis solution was
added to the target maximum release wells and the volume correction
control wells. The plates were centrifuged at 500 rpm for 4 min and 50
µl aliquots of supernatants were transferred from all wells to a
fresh 96-well flat bottom plate. Fifty microliters of substrate were
added to each well and incubated for 20 min at RT. Fifty microliter of
stop solution was added and the absorbance was recorded at 490 nm.
The percentage of specific lysis was calculated using the formula:
% cytotoxicity = (experimental LDH release-effector cell spontaneous LDH release-target cell spontaneous LDH release)/(target cell maximum LDH release-target cell spontaneous LDH release).
NK assays were performed using the NK-sensitive cell line YAC-1 as the target cell.
Dissemination of adenovirus following intratumoral
administration
AdCMVLacZ was directly injected into tumors at the dose of
2 x 109 p.f.u. in 100 µl serum-free medium. After
1, 3, 5, and 7 days, rats were killed. Samples of tumor, liver, lung,
kidney, and spleen were homogenized. Supernatants from a 10,000 x
g centrifugation were saved and kept at -80 C. LacZ
activity was examined as described below. Serum for each rat was also
saved for liver function tests and neutralization antibody assays.
LacZ activity assay
Five microliters sample and 295 µl substrate solution (150
µg/ml CPRG, 1 mM MgCl2, 45 mM
ß-mercaptoethnol in 0.1 M PBS pH 7.5) were mixed and
incubated at RT for 30 min. Five hundred microliters stop solution (1
M Na2CO3) was added to each
reaction. OD570 was measured in an ELISA reader.
Toxicity of AdCMVmIL2 after iv administration
AdCMVmIL2 was administrated iv at doses of 2 x
109 and 1 x 1010 p.f.u. in 200 µl PBS
buffer. Treated animals were inspected every day for their behavior,
and killed 3 or 7 days after treatment. Serum was save from each animal
to test liver function, and organs and tissues were harvested for
pathological examination.
Transaminase assay
A commercial kit was used for quantitative colormetric
determination of glutamic oxalacetic transaminase (GOT) and glutamic
pyruvic transaminase (GPT) (Sigma Chemical Co., St. Louis,
MO). The procedure from the company was followed.
Statistical calculations
Student’s t and 
square tests were used to analyze the data.
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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). In
another similar trial with transduced rMTC cells in SCID mice, one
mouse in the AdCMVmIL2 infected group developed a tumor, and all mice
in the AdCMVLacZ infected or wild-type rMTC control groups developed
tumors.
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summarizes the results of in vivo studies on tumor
bearing rats using AdCMVmIL2 vector. When the initial treatment tumor
size was equal to or less than 100 mm3, 9 out of 21
AdCMVmIL2 treated rats (42.9%) became tumor free, compared with 2
out of 14 (14.3%) among AdCMVLacZ treated animals. All control rat
tumors kept growing. The average tumor size in AdCMVmIL2-treated
animals was much smaller than AdCMVLacZ-treated or control rats,
indicating that effective antitumor activity was induced in
AdCMVmIL2 treated animals (Fig. 3A).
In animals with tumor size larger than 100 mm3, a
significant antitumor effect of AdCMVmIL2 was found, but less effective
than in small tumors (Table 1, Fig. 3B). It should be noted that the
average tumor size in AdCMVLacZ treated group was always smaller
than in the control group in small size tumors, indicating a
"nonspecific" antitumor effect from the viral vector, consistent
with our previous observation (32, 33).
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Challenge with wild-type rMTC cells in cured rats
Rat MTC cells (5 x 105 cells/rat) were used to
rechallenge rats cured of tumor by prior injection of AdCMVmIL2. More
than 78% of cured rats did not develop tumor (Table 2), indicating that most animals were
immunized against the tumor cells.
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Dissemination of adenovirus following intratumoral
administration
To examine virus dissemination following intratumoral injection,
replication defective vector AdCMVLacZ was used as a marker to detect
infected cells in various tissues. Sixteen tumor-bearing animals
received an intratumoral injection of AdCMVLacZ at a dose of 2 x
109 p.f.u. in 100 µl serum free medium. Tissues were
harvested on days 1, 3, 5, or 7 after injection. LacZ activity was
examined in homogenized supernatants for each sample. One day after
injection, a high level of LacZ activity was detected in the tumor, and
no LacZ activity was found in other tissues (Fig. 4). Three days after injection, LacZ
activity was detected in liver, indicating dissemination from the
injected tumor and expression in this organ. A high level of LacZ
expression was seen in the liver, but not in other tissues, suggesting
that the liver is the major site of dissemination following
intratumoral administration of adenovirus vector.
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). GPT and GOT
levels in treated animals were not significantly different from
untreated control animals. All animals given virus iv had mild to
extensive lymphocyte infiltration in the sinusoids and triads of the
liver, but this was not clearly related to dose (Fig. 5). Spleens of some virus treated animals
showed decreased white pulp, apparently increased hematopoiesis, and
2/12 animals showed areas of cell necrosis in the spleen. No specific
changes were found in lungs and kidneys.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initiated the present study to explore the feasibility and safety of new approaches for the treatment of medullary thyroid carcinomas. Our results confirmed and extended the antitumor activity of mIL2. In our rMTC animal model, treatment with AdCMVmIL2 vector cured about 43% of the rats and most cured animals developed systemic immunity to parental tumor cells. This antitumor activity came from both specific and nonspecific immune effector cells. The challenge study confirms the specific immunity, because after treatment with AdCMVmIL2 most cured rats did not develop tumors when injected with wild-type rMTC cells. Loss of the tumorigenicity of AdCMVmIL2 infected rMTC cells in SCID mice indicates the presence of NK activity because these mice have no T cells. That the average tumor sizes in AdCMCLacZ vector treated animals are always smaller than in medium-treated control group also suggest nonspecific antitumor activity.
We could not find a consistent antitumor immune response by assay of CTL and NK activity in vitro. Numerous factors may interfere with the in vitro studies. Other studies also reported that in vitro results do not always correlate with or reflect in vivo function (37).
Previous reports have demonstrated that an adenoviral vector can disseminate from the injection site of tumor and infect peripheral tissues (38, 39, 40). Our results confirmed this phenomenon and verified that liver is the main target organ of dissemination after intratumoral administration. When viral vectors are employed for gene therapy of solid tumors, it is always hoped that the virus will primarily transduce cells in the vicinity of inoculation. However, it is quite clear that the virus can enter the circulation following intratumoral administration and infect the peripheral tissues. Previous studies confirmed the dissemination of viruses in multiple tissues when virus was given intratumorly using a luciferase expression virus as a marker (38, 39). At least 5 orders of magnitude greater sensitivity can be obtained using this marker. We believe this is the reason that it has been possible to detect the very low levels of leaking expression in the tissues.
One danger of viral dissemination is that it will give rise to inflammation and dysfunction of the infected tissues. To explore the possible side effect of the AdCMVmIL2 vector, a high dose of virus (up to 1 x 1010 p.f.u.) was given iv to rats. We inspected the behavior and examined the pathologic and functional change of liver and other tissues. We found only a variable degree of lymphocyte infiltration in the livers of treated animals, and no evidence of altered GOT or GPT levels in serum. This result suggests that adenoviral vector transducing IL-2 is safe in gene therapy.
The present studies support the approach of using an adenoviral vector transducing IL-2 for medullary thyroid cancer gene therapy. It is clear that intratumoral administration of AdCMVmIL2 is safe. A clinical trial will be necessary to evaluate AdCMVIL2-based cancer gene therapy in practice.
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Acknowledgments |
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Footnotes |
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Received August 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1/2 domain polymorphism
and in vivo expression pattern in tumor resistance: studies
with transgenic mice and lymphoma cell transfectants. J Immunother 14:175–181
and interferon-. J
Immunother 15:223–241
,
IFN-
and TNF-. Lab Invest 74:146–157
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