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Endocrinology Vol. 140, No. 5 2152-2158
Copyright © 1999 by The Endocrine Society

ARTICLES

Effective Genetic Therapy of Established Medullary Thyroid Carcinomas with Murine Interleukin-2: Dissemination and Cytotoxicity Studies in a Rat Tumor Model1

Rusheng Zhang, Francis H. Straus and Leslie J. DeGroot

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Replication-defective adenovirus (AdCMVmIL2) expressing murine interleukin-2 was directly injected into rat medullary thyroid carcinomas to examine antitumor activity. AdCMVmIL2 cured 42.9% of all treated tumor bearing animals. Most cured rats were protected against tumor growth after subsequent rechallenge with wild-type tumor cells, reflecting the immunity obtained from the original treatment. Studies of viral dissemination showed that the intratumoral inoculated viruses can enter the circulation, infect peripheral tissues, and express genes driven by the CMV promoter. Liver is the main target organ. In a toxicity study, AdCMVmIL2 was administered iv at a dose five times higher than that given directly into tumor. No detectable side effect was found. Histological studies showed variable degrees of lymphocyte infiltration in the livers of studied animals, and no functional change indicated by the normal serum level of glutamic oxalacetic transaminase and glutamic pyruvic transaminase was found in all animals studied. These data demonstrate that AdCMVmIL2 is an effective antitumor agent in this animal model, and that virus treatment can be given without significant toxicity to other organs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS CLEAR THAT tumor cells can express antigens that induce immune responses by the host (1, 2). Unfortunately the host antitumor immune response, or immunosurveillance, does not always prevent the occurrence of lethal cancers. This failure may be due to a defect in immune stimulation, or regulation, rather than the absence of tumor specific antigens. Tumor cells evade immune recognition in several ways (1). Class I MHC expression can be down-regulated on tumor cells so that they cannot present complexes of processed peptides. Lack of MHC II and costimulatory molecules on tumor cells may impair T cell activation. Tumor products such as transforming growth factor-ß may suppress antitumor immune response. The host may be tolerant to tumor antigens, as demonstrated for tumors caused by the murine mammary tumor virus. The loss of surface antigen expression on tumor cells by antibody binding (antigenic modulation) or the masking of antigens by glycocalyx can prevent an efficient immune response. Selection of mutant tumor cells that no longer express immunogenic peptides can also allow tumors to evade an immune response. For effective immunotherapy, it is necessary to design ways to increase the immune response to tumor antigens.

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-{alpha} (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?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell lines
Wag/Rij rats were bred and maintained in the Carlson Biocontainment Suite under standard conditions, according to the guidelines of the Animal Research Center. Four-week-old rats were used in our studies.

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 {chi} square tests were used to analyze the data. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tumorigenicity of rMTC cells transduced by AdCMVmIL2
Tumorigenicity of rMTC cells infected with AdCMVmIL2 was evaluated in both sygeneic rats and SCID mice. Groups of 10 WAG/Rij rats were injected sc in the right flank with either wild-type rMTC cells, or rMTC cells infected with AdCMVmIL2 or AdCMVLacZ vectors. Both wild-type and AdCMVLacZ infected rMTC cells induced tumor development in all injected rats, whereas only one out of 10 rats injected with AdCMVmIL2 infected rMTC cells developed a tumor (Fig. 1Go).



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  		Figure 1. Tumorigenicity of rMTC cells after in vitro infected with AdCMCVmIL2. Rat MTC cells were infected at 100 moi with either AdCMVmIL2, or AdCMVLacZ, or were left uninfected. Cells were then injected sc into one flank of WAG/Rij rats (10 rats per group). Tumor development was inspected every 2 days. All tumor-bearing rats were killed when the tumor size reached more than 25 mm.

 
To examine the tumorigenicity of AdCMVmIL2 infected rMTC cells in SCID mice, 1 x 106 rMTC cells infected with either AdCMVmIL2, AdCMVLacZ, or uninfected cells, were inoculated into three groups of five SCID mice. No tumors developed in mice injected with AdCMVmIL2 infected rMTC cells, whereas all mice injected with either AdCMVLacZ or wild-type rMTC cells developed tumors within 10–20 days after inoculation (Fig. 2Go). 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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  		Figure 2. Tumorigenicity of infected rMTC cells in BALB/c-SCID mice. Rat MTC cells were infected at 100 moi with AdCMVmIL2, or AdCMVLacZ, or were left uninfected. Cells were then injected sc into the abdomen of BALB/c-SCID mice (five mice per group). Tumor development was checked every 2 days. All tumor-bearing mice were killed when the tumor size reached more than 25 mm.

 
Effect of intratumoral administration of AdCMVmIL2
We found that the antitumor efficiency of AdCMVmIL2 was related to the tumor size. The smaller the tumor, the better the therapeutic efficacy. Table 1Go 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. 3AGo). 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 1Go, Fig. 3BGo). 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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  		Table 1. Summary of studies on preestablished tumors treated in vivo with AdCMVmIL2

 


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  		Figure 3. Average tumor sizes after treatment by direct intratumoral administration. Tumor-bearing rats were treated either with AdCMVmIL2, or AdCMVLacZ, at a dose of 2 x 109 p.f.u., or with medium alone, in a volume of 100 µl. The treatment was performed when the tumor volume was equal or less than 100 mm3 (Fig. 3AGo), or larger than 100 mm3 (Fig. 3BGo).

 
The presence of neutralizing antibody was examined in treated animals. All adenoviral vector treated animals developed high titer antiandenovirus neutralizing antibodies as early as 3 days after the treatment (data not shown), which may reduce the expression of injected vector and inhibit the usefulness of repeat administration.

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 2Go), indicating that most animals were immunized against the tumor cells.


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  		Table 2. Challenge of cured rats with wild-type of rMTC cells

 
In vitro evaluation of the antitumor immune response
Lysis of tumor target cells and YAC-1 cells by splenocytes harvested from rats after adenoviral vector treatment was measured in CTL assay and NK assay for each rat in the treatment groups. Whether or not the treated animal was cured, spleen cells from animals treated with adenoviral vector did not result in any clear CTL and NK activity. CTL and NK activity was highly variable within the animals in these groups (data not shown). Overall the in vitro CTL and NK activity were weak and did not correlate with in vivo tumor rejection.

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. 4Go). 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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  		Figure 4. Time-course of AdCMVLacZ dissemination following intratumor administration. Tumor-bearing rats received an intratumoral injection of 1 x 109 p.f.u. of AdCMCLacZ on day 0. On days 1, 3, 5, and 7, every two animals were killed and various tissues were removed, homogenized, and assayed for LacZ activity as an indicator of virus dissemination.

 
Cytotoxicity of adenoviral vectors
To detect possible damage to liver and other tissues after administration of AdCMVmIL2, this vector was administrated iv at doses of 2 x 109 or 1 x 1010 p.f.u. per rat. This provides a viral load greater than that given directly into the tumor but allowed direct iv access to all tissues. There was no change in behavior in all of the treated animals compared with that of untreated animals. Rats were killed on day 3 or day 7 after virus administration. Although liver is the major target organ after administration, no evidence of functional change was found in any treated animal (Table 3Go). 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. 5Go). 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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  		Table 3. GPT and GOT assays after administration iv of AdCMVmIL2

 


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  		Figure 5. Pathological change of liver in AdCMVmIL2 treated animals. AdCMVmIL2 was administered iv at doses of 2 x 109 or 1 x 1010 p.f.u. Tissues of treated animals were harvested 3 or 7 days after treatment. Possible pathological changes of various tissues were examined after H&E staining. All livers of treated animals showed mild to extensive lymphocyte infiltration. A, Liver from 1 x 1010 p.f.u. treated animal, 3 days after treatment, showing minimal pericentral lymphocyte infiltration with slight increase in sinusoidal lymphocyte infiltration. B, Liver from 1 x 1010 p.f.u. treated animal, 7 days after treatment, showing marked portal triad and sinusoidal infiltration of lymphocytes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many studies have found that the interleukin-2 expressed by tumor cells generates a systemic immune response specific for parental tumor cells and protects the animals from a tumor challenge. There are limited reports on dissemination and expression of the viral vector after intratumoral administration.

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.


   Acknowledgments
 
We are grateful to our colleagues Cyprian Gardine, Minemura Kesami, Yoshikuni Sawai, and Tsuyoshi Kouki for helpful discussion and careful reading of the manuscript, and to Miss Myrna Zimberg for her excellent secretarial work.


   Footnotes
 
1 Supported by the David Wiener Fund, the Pardee Foundation, and a Center of Excellence award from Knoll Pharmaceutical Co. Back

Received August 8, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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