Affiliations of authors: M. F. X. Gnant, Surgery Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD, and Department of Surgery, University of Vienna, Austria; L. A. Noll, R. E. Terrill, Laboratory Animal Medicine Section, Animal Sciences Branch, Division of Basic Sciences, National Cancer Institute; K. R. Irvine, M. Puhlmann, H. R. Alexander, Jr., D. L. Bartlett, Surgery Branch, Division of Clinical Sciences, National Cancer Institute.
Correspondence to: David L. Bartlett, M.D., National Institutes of Health, Bldg. 10, Rm. 2B07, Bethesda, MD 20892 (e-mail: dbart{at}nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Virtually all experimental in vivo data of tumor-directed gene therapy have been reported in rodent models involving mice or rats, mostly because of the lack of a large-animal model. The developmental gap between rodents and humans limits the applicability of these experiments.
We now report on the development of a large-animal (rabbit) model of experimental liver metastases. We used this tumor model for investigating the pattern of transgene expression following intravenous vector administration in a gene therapy setting. The vector system that we used is a recombinant vaccinia virus attenuated by the disruption of its thymidine kinase gene (3) and carrying the reporter gene-firefly luciferase. We determined the pattern of transgene activity in tumors and other tissues over time. In addition, we monitored the time course of elimination of the circulating vector by the host and the development of specific anti-vector antibodies.
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MATERIALS AND METHODS |
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Human ovarian cancer cells HeLa S3 and monkey kidney cells CV-1 were obtained from the American Type Culture Collection (Manassas, VA) and were used for the generation, amplification, and titration of recombinant vaccinia viruses. They were cultured in complete medium, i.e., Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1% penicillin-streptomycin, 1% gentamicin, and 0.2% fungizone (all from Biofluids, Rockville, MD) at 37 °C in a 5% CO2 incubator.
VX-2 is a transplantable and highly malignant neoplasm of rabbits (5). VX-2 tumor cells do not grow in vitro and are maintained by serial transplantation in carrier animals (6), as described below.
Plasmids and Recombinant Vaccinia Viruses
The recombinant vaccinia viruses used are based on a novel vaccinia shuttle plasmid, pCB023II, which is described in detail elsewhere (2). Briefly, the multiple cloning site of pBluescript KS II (Stratagene Cloning Systems, La Jolla, CA) was inserted in between the thymidine kinase-flanking regions of vaccinia. The Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt) gene under the control of the p7.5 vaccinia early/late promoter was inserted for selection purposes. The gpt gene has been used for positive selection in a purine salvage pathway because it catalyzes the conversion of xanthine, hypoxanthine, and guanine to their respective monophosphates. Cells that express the gpt gene can utilize xanthine as a purine analogue and can convert it to hypoxanthine in the presence of mycophenolic acid, which inhibits the mammalian pathway. To prevent antisense transcription from native vaccinia promoters, an early termination signal (TTTTTNT) was designed for all three possible open reading frames and was inserted just downstream of the left of vaccinia virus thymidine kinase-flanking region. Any gene within the multiple cloning site is driven by the synthetic early/late promoter (7,8). The firefly luciferase gene was cut out of pGEM-luc (Promega Corp., Madison, WI) and was inserted into the shuttle plasmid (pCB023II). The luciferase gene allows for quantitative analysis of gene expression in a rapid, convenient assay for luminescence. The correct gene sequence was confirmed by automated sequencing (ABI Prism; Perkin-Elmer Applied Biosystems, Norwalk, CT).
Recombination of vaccinia viruses (strain Western Reserve) was performed by infection of CV-1 cells with vaccinia virus, followed by transfection of the shuttle plasmid (pCB023II) by CaCl2 precipitation (9). Double recombination occurs between homologous regions of the thymidine kinase gene in the shuttle plasmid and the virus, resulting in the insertion of the gene cassette into the thymidine kinase gene in the virus (thus inactivating the viral thymidine kinase gene). Recombinant vaccinia viruses are isolated by mycophenolic acid selection and multiple purification steps. The final thymidine kinase-negative viral construct was designated vvLuc. Recombinant viruses were amplified in HeLa S3 cells by use of spinner flasks and standardized methods (9).
Animal Care
Female New Zealand White (NZW) rabbits (body weight, 3-4 kg) were obtained from Covance Research Products (Denver, PA) and housed in groups of four or five animals per run. Runs were 118 inches x 46 inches x 92 inches in size with epoxy flooring. Animals had access to food (high-fiber rabbit pellets; Zeigler Brothers, Gardners, PA) and water ad libitum. Temperature was maintained at 18 °C ± 1 °C; relative humidity was maintained at 50% ± 10%. Environmental enrichment included a Colorbust Dumbbell (7 inches; AJ Buck & Son, Owing Mills, MD), Goofy Links (large; AJ Buck & Son), and Prima-Hedrons (18 inches; Primate Products, Redwood City, CA). After inoculation with vaccinia virus, the animals were housed individually in a barrier room; Biosafety Level 2 standard procedures were followed. All rabbits tested negative for anti-vaccinia antibodies before starting the experiments.
All animal protocols were approved by the institutional Animal Care and Use Committee and were conducted in strict compliance to the guidelines established by the Animal Research Advisory Committee of the National Institutes of Health.
Tumor ModelGeneration of Disseminated Liver Metastases
Disseminated liver metastases were established by direct injection of tumor cells into the portal vein. Rabbits were anesthetized by intramuscular injections of 40 mg/kg ketamine (Fort Dodge Laboratories, Fort Dodge, IA), 10 mg/kg xylazine (Miles, Shawnee Mission, KS), and 0.2 mL of glycopyrrolate (AH Robbins, Richmond, VA). Animals were intubated with a #3 endotracheal, cuffed, Murphy eye tube (Kendall Sheridan, Mansfield, MA) with the use of a Flagg-0 laryngoscope (Welch-Allyn, MacGaw Park, IL), and anesthesia was maintained with 2%-3% isoflurane USP (Abbott Laboratories, North Chicago, IL) with 1.5 L of oxygen per minute. Ringer's lactate (Abbott Laboratories) was infused via a lateral ear vein at a rate of 10 mL/kg per hour.
After clipping and surgical prepping of the abdomen, we performed a median epigastric laparotomy. The peritoneal cavity was opened, and the liver was briefly mobilized. The portal vein was identified, and VX-2 cells were injected slowly in 5 mL of Hanks' balanced salt solution (HBSS) with the use of a 30-gauge needle (Becton-Dickinson, Franklin Lakes, NJ). Hemostasis was achieved by application of moderate pressure on the injection site for 3 minutes. The abdominal cavity was briefly irrigated and closed in anatomic layers with resorbable sutures (Vicryl®; Ethicon, Somerville, NJ). Skin closure was performed with an intracutaneous suture technique in which 3-0 Vycril® (Ethicon) was used. Animals were closely monitored thereafter. For analgesia, 0.02 mg/kg buprenorphine HCl (Rickett & Colman, Richmond, VA) was administered intramuscularly.
For maintenance of the tumor cell line, carrier animals received an injection of VX-2 cells in the thigh muscles. When the tumor reached approximately 3 cm in diameter, carrier animals were killed, and their tumor was immediately harvested under sterile conditions. Tumors were either transplanted directly into the next carrier animal or digested for 6-8 hours with enzymes (collagenase type IV, hyaluronidase type IV, and deoxyribonuclease type IV; all from Sigma Chemical Co., St. Louis, MO), processed through a 112-µm Nitex® nylon mesh (Lawshe Instruments, Rockville, MD), washed three times with HBSS, and frozen at -80 °C in 90% FBS-10% dimethyl sulfoxide (Fluka Chemical Corp., Milwaukee, WI) (10). If frozen cells were used for tumor inoculation, they were thawed quickly, washed three times in HBSS, and, after viability assessment with 0.4% trypan blue dye (Sigma Chemical Co.), adjusted to the desired concentration.
Non-tumor-bearing animals were used as controls in all experiments.
Assessment of Reporter Gene Expression In Vivo
In the experiments designed to assess the reporter gene expression, animals (tumor-bearing or non-tumor-bearing) received an injection of the vaccinia virus vector encoding luciferase gene (vvLuc) at different doses 14 days after tumor inoculation. With the use of light anesthesia and analgesia as described, virus (in 50 mL of HBSS) was injected slowly (duration of >15 minutes) via a lateral ear vein. Groups of animals were then killed at various time points. Tumors and normal tissues (liver, spleen, kidneys, pancreas, uterus, ovary, bowel, heart, lung, muscle, skin, and brain) were harvested, immediately frozen, and stored at -80 °C until assayed for luciferase activity.
Also, serum samples were taken at various time points. While the animal was under light sedation, 5 mL of blood was drawn from the central ear artery. To minimize irritation of individual animals, we rotated the rabbits throughout the serum-sampling schedule and drew blood from them during scheduled euthanasia whenever possible. Serum was frozen at -80 °C until assays were performed.
Titration of Virus Amounts in Viral Stocks and in Serum Samples
Viral stock and animal serum samples were assayed for quantity of virus by use of a plaque assay. Briefly, CV-1 cells were plated into six-well plates (Corning Costar, Cambridge, MA) and grown until confluent. Virus-containing samples were serially diluted in HBSS containing 0.1% bovine serum albumin (BSA) (Calbiochem Corp., La Jolla, CA) and added to the wells. After incubation for 2 hours with gentle movement, wells were overlaid with complete medium and Bacto-Agar (1 : 1; Difco Laboratories, Detroit, MI). After further incubation for 48 hours, the gel was cautiously removed from the well, and cell monolayers were briefly exposed to 500 µL of 0.1% Crystal Violet, rinsed, and dried. Dry wells were then enumerated for viral plaques, and the number of plaques (or plaque-forming units [pfu]) was calculated per milliliter. Approximately 30 viral particles per pfu were present based on calculations from DNA measurements.
Enzyme-Linked Immunosorbent Assay (ELISA) for Anti-Vaccinia Antibodies
Serum was collected from rabbits at time points as outlined earlier after administration of viral vector. Microtiter plates were coated overnight at 4 °C with 5 x 105 pfu of wild-type vaccinia virus in 50 µL of phosphate-buffered saline (PBS) per well or 50 µL of 1 µg/mL BSA in PBS per well as control antigen. After the unattached antigen was rinsed off, the plates were incubated with 5% BSA in PBS in each well for 1 hour to prevent nonspecific antibody binding, which was followed by a 1-hour incubation with 50 µL of fivefold dilutions (starting at 1 : 25) of test serum samples or anti-vaccinia polyclonal mouse antisera (obtained from vaccinated mice) as positive controls. After the plates were washed with 1% BSA in PBS, 50 µL of horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin G F(ab')2 fragments (at 1 : 2000 dilution in 1% BSA in PBS) (Amersham International, Amersham, U.K.) was added for 1 hour to detect antibodies immobilized on the wells. The resulting complex was detected by the chromogen o-phenylenediamine (Sigma Chemical Co.). Absorbance was read on a Multiskan Plus Reader (Titertek, Huntsville, AL) with the use of a 490-nm filter. Results are presented as serum dilutions observed at an optical density of 0.25.
Firefly Luciferase Assay
Luciferase activity was assayed by use of a commercially available assay system (Promega Corp.). Frozen tissue samples were briefly thawed, homogenized, and lysed in 750 µL of 1x strength reporter gene lysis buffer provided by the manufacturer. Samples were centrifuged (Eppendorf Centrifuge #5415C; Brinkman Instruments, Westbury, NY) for 5 minutes at 13 000 rpm (10 000g) at room temperature to pelletize the cellular debris. Supernatant (10 µL) was added to 100 µL of Luciferase Assay Reagent in an 8-mm x 50-mm disposable cuvette and immediately placed into a luminometer (TD-20/20; Turner, Sunnyvale, CA). After a 2-second delay, light emission was measured for 10 seconds. Each sample was measured in duplicate, and mean values were recorded. The concentration of total protein in each sample was then determined with a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL) with the use of a BSA standard. All measurements were done in triplicate. The final firefly luciferase activity is expressed in relative light units per milligrams of protein.
Histology
Tumor and tissue specimens were harvested from animals killed at the indicated time points and were immediately fixed with 10% paraformaldehyde in 0.1 M phosphate buffer. Paraffin embedding, sectioning, and hematoxylin-eosin staining of the tissues were performed at Paragon Biotech (Baltimore, MD).
Statistical Analysis
Results are presented as means ± standard deviation. Two-tailed Student's paired t test was used for making comparisons.
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RESULTS |
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Groups of four rabbits each received an injection of 1 x
105, 1 x 106, or 5 x 106 VX-2 tumor
cells directly into the portal vein, and their survival pattern was
monitored. Tumor take in the group given the lowest dose was
inconsistent (two of four animals), and survival times varied (26 days
and 53 days) in this group. The group given the highest dose died
quickly from progressive liver metastases; four of four animals in this
group died between day 18 and day 21 after inoculation. Injection of 1
x 106 cells resulted in 100% tumor take, with four of
four animals dying of disseminated liver metastases between day 29 and
day 35. Tumor nodules (seen as white spots in Fig. 1,
A) became visible around 5-7 days after inoculation. Fig. 1,
A, shows
the macroscopic appearance of these nodules on day 14 after
inoculation. The livers of the rabbits were progressively invaded by
metastatic nodules originating from the portal venous system (Fig. 1,
B
[original magnification x10]; Fig. 1,
C [original
magnification
x40]), eventually leading to fatal liver failure as indicated by
serum chemistry (data not shown). Necropsy revealed the cause of death
to be from complete replacement of hepatic tissue by tumor, and 20%
(two of 10 examined) had small, clinically insignificant lung
metastases. In all subsequent experiments, 1 x 106 VX-2
tumor cells were used.
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We performed a dose-escalation experiment to determine the optimal dose range of the vaccinia virus. We used 1 x 108 pfu of vvLuc as the lowest dose level (n = 4). Additional experimental groups received 1 x 109 pfu (n = 4), 5 x 109 pfu (n = 4), or 3.75 x 1010 pfu (n = 2) of vvLuc. Clinical symptoms of viremia included lethargy, conjunctivitis, rhinorrhea, abstinence from food, and oliguria. A sampling of liver chemistry and complete blood cell counts were within the normal range, even at toxic viral levels (data not shown). Rabbits receiving 1 x 108 pfu of vvLuc did not show any clinical symptoms. Rabbits receiving 1 x 109 pfu of vvLuc showed only mild signs of viral infection on days 3 and 4 after injection. Animals receiving 5 x 109 pfu of vvLuc had notable viral disease beginning on day 2, displayed maximum number of symptoms by day 4, but were fully recovered by day 7 after virus administration. Dose-limiting toxicity was clearly reached at 3.75 x 1010 pfu, with one animal dying within 2 hours after virus inoculation and the other developing severe sickness necessitating parenteral infusion and eventual euthanasia 12 hours after infection. Thus, 5 x 109 pfu of vvLuc was considered to be the maximum tolerated dose in this system.
Pattern of Transgene Expression After Systemic Vector Administration
The established rabbit model of liver metastases was used to determine the in vivo expression of the reporter gene. After intravenous administration of 5 x 109 pfu of vvLuc in the tumor-bearing rabbits, the reporter gene activity in different tissues was determined over time. On days 2, 4, 6, 8, 10, and 12 after virus administration, groups of animals (n = 4 per time point) were killed and tumor and tissue samples were harvested and assayed as described in the "Materials and Methods" section.
As shown in Table 1, substantial reporter gene activity was
measurable for about 1 week. The luciferase expression in tumors was markedly higher than that
in any other tissue, and it peaked on day 4. Tumor tissue demonstrated a 16-fold increase in
luciferase activity compared with that in the ovary (P<.001), a 62-fold increase
compared with that in the spleen (P<.001), a 180-fold increase compared with that in
the kidneys (P<.001), and a 285-fold increase compared with that in the lungs (P<.001). On day 6, luciferase activity in tumors was still about 37-fold higher compared
with that in the ovaries (P<.001). In general, the ovaries showed the highest gene
expression among the organs not carrying tumor metastases. Luciferase activity in other organs,
such as kidneys or lungs, was usually about two logs lower than that in tumor over the entire
observation period. There was no difference in gene expression in these organs between
tumor-bearing and non-tumor-bearing animals (data not shown).
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To assess the impact of viral dosage after systemic delivery on
reporter gene expression in tumors and other tissues, an experiment was
performed with the use of 1 x 108 pfu of vvLuc as compared
with 5 x 109 pfu of vvLuc. As shown in Fig.
2, intratumoral reporter gene activity was
significantly decreased in the lower dose group (days 2 and 4:
P<.001; day 6: P<.02). While tumor specificity
was preserved, differences in transgene expression between tumor and
other tissues were less dramatic than in the high-dose group (e.g.,
about 3.5-fold compared with the ovaries). In addition, the duration of
tumor-specific gene expression showed a trend toward being shortened
(approximately 5 days in the group receiving 1 x 108 pfu
of vvLuc compared with 7 days in the group receiving 5 x
109 pfu of vvLuc).
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Tumor-bearing rabbits received an intravenous injection of 5 x
109 pfu of vvLuc, and serum samples were taken on days 2,
4, 6, 8, 10, and 21 and thereafter. Sera of at least three animals per
time point were assayed for the presence of circulating virus. As shown
in Fig. 3, A, maximum levels of circulating viral
particles occurred on day 4 after systemic injection. It is interesting
that viral titers on day 2 were consistently low, indicating an initial
uptake of virus into tissues shortly after systemic application and
subsequent viremia after initial intracellular amplification. The
pattern of virus presence in serum coincides with the clinical symptoms
observed in these animals.
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Tumor-bearing animals were tested by ELISA for antibody reactivity
on days 0, 2, 4, 6, 8, 12, 14, 17, and 21 after virus inoculation. For
long-term studies, serum from naive animals given an injection of vvLuc
was collected at identical time points as well as on days 42, 64, and
85 after virus challenge. As Fig. 2 demonstrates, anti-vaccinia
antibodies could be detected as early as 6 days after virus
administration, with rapidly increasing titers thereafter. Maximum
titers were observed on day 21 after vector administration (Fig. 3,
B).
High antibody levels were maintained for at least 3 months. There was no difference
in antibody titers between tumor-bearing and non-tumor-bearing animals.
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DISCUSSION |
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Large-animal tumor models are rarely described in experimental cancer gene therapy, mostly because of the lack of adequate tumor lines. As of November 25, 1998, MEDLINE® has recorded 1166 published reports on experimental cancer gene therapy involving animal studies; however, not one of them was performed in other than rodent experimental systems (11-13), except those assessing toxicology in non-tumor-bearing primates (14). We believe that a large-animal model may be useful to narrow the gap between in vivo research in rodents and early clinical application of novel therapeutic concepts in humans. To the best of our knowledge, this study represents the first report on in vivo application of tumor-directed gene delivery by systemic vector administration in tumor-bearing, large animals.
VX-2 rabbit tumors originated from a papillomatous reaction to Shope virus infection (15), and these tumors have been maintained by serial transplantation. Their histopathologic and morphologic features have been described extensively (16). VX-2 tumors have been used in previous studies to induce tumors in several organs and locations in rabbits, including liver tumors (6,17-20). Most investigators (19,21-24) induced liver tumors by direct implantation of a small tumor piece or by injection of a cell suspension into the liver parenchyma. In our opinion, direct tumor implantation does not accurately mimic the properties of liver metastases that reach the liver hematogenously. There is a single report on injection of tumor cells via the short gastric vein (25) as well as another report describing tumor inoculation via the superior mesenteric vein (26). We prefer injection directly into the portal vein because hemostasis can be achieved easily and tumor deposits outside the liver (such as in the pancreas or duodenal wall) do not occur.
The most important basis for the success of the gene delivery system that we used is the unique ability of recombinant vaccinia viruses to specifically infect tumors (2)a crucial feature for a vector used in tumor-directed gene therapy (27). Other gene therapy vectors do not show this tumor-tracking ability. Adenoviruses, for example, have extensive hepatotropic characteristics in some models, leading to hepatotoxicity (13,28). We have observed some transgene expression in other organs, but the reported increased activity of the genes within tumors as compared with the activity within other tissues provides a therapeutic window for gene therapy approaches. Our observations could be strengthened with data on viral recovery or DNA levels within the organs, but transgene expression provides therapeutically relevant data.
Several possible explanations for the tumor selectivity of thymidine kinase-negative vaccinia virus have been reported (29,30). We have suggested previously that the disruption of the vaccinia virus thymidine kinase by the inserted gene may be the main reason for tumor-specific gene delivery (2). The lack of functional viral thymidine kinase inhibits viral amplification in nondividing cells, whereas vaccinia virus can utilize the abundant nucleotides present in tumor cells and can replicate normally in the absence of viral thymidine kinase (31). The 16-fold difference in maximum transgene expression between tumors and other tissues in the rabbit model is lower than in murine tumor models using a similar vector (32). The permissiveness of different species for vaccinia virus may vary (33).
While circulating viral particles disappear from the circulation shortly after intravenous injection, we observed viremia on day 2 to day 4 thereafter. The viral particles responsible for this secondary viremia are most likely derived from the second and subsequent viral generations, amplified intracellularly predominantly in tumor cells. Despite this amplification capability (3), the animal's intact immune system inhibits long-lasting systemic viral infection. Not only is circulating vector cleared from the circulation within a week, but also long-term immunity is initiated as indicated by the presence of high antibody titers. The lack of transgene persistence in our experiment is most likely secondary to immune elimination of cells expressing the transgene and other vaccinia genes. We have shown elsewhere (32) that the absence of T-cell response leads to prolonged transgene expression in tumors and improved treatment results in an in vivo suicide gene therapy setting.
The systemic dosage of virus seems to be associated with the extent and duration of intratumoral transgene expression. Currently, it is unknown, however, whether the duration and level of transgene expression are sufficient to exhibit therapeutic benefit in the model described. It is also unclear whether the percentage of tumor cells expressing the gene of interest is high enough to be therapeutically significant. In situ immunohistochemistry or marker gene staining would help define this percentage. Using the purine nucleoside phosphorylase/6-methylpurine deoxyriboside system (4), we have demonstrated in murine models that prodrug delivery over a period of only 3 days is sufficient for a 50% cure rate. Therefore, even a brief duration of gene expression can be therapeutically significant.
Poxviruses are highly immunogenic because of their large size and the large number of viral proteins (33). In a potential clinical application, the immunogenicity will most likely preclude repeated applications of the same vector and also be an issue for preimmunized patients (34). However, the protective immunity also provides a safety feature for the systemic application of these viruses in the clinical setting (35). The smallpox eradication experience (36) and the several clinical phase I immunotherapy trials of recombinant vaccinia viruses in immunocompetent cancer patients have shown no vector-associated morbidity (35,37), encouraging the future use of the vector system in a tumor-directed gene therapy setting (38). However, experience with intravenous injection of these vectors is still lacking. If in a gene therapy approach multiple viral applications are intended, either non-cross-reactive heterologous vectors could be used (39) or the host immune response could be temporarily manipulated.
In summary, our investigation demonstrates that tumor specificity and high transgene expression can be achieved with minor side effects in a large-animal system. The results enhance knowledge about tumor-directed gene therapy using thymidine kinase-negative vaccinia viruses and contribute to the translation of these promising experimental systems into clinical practice.
M. F. X. Gnant is a recipient of a research grant by the Max Kade Foundation, New York, NY.
We thank Dr. Steven Rosenberg (National Cancer Institute, Bethesda, MD) for helpful comments and general advice. We gratefully acknowledge the support of Dr. Scott Gazelle (Massachusetts General Hospital, Boston) and Dr. Steven Curley (The University of Texas M. D. Anderson Cancer Center, Houston) for assistance with VX-2 cells. We are also grateful to Liz Scanlon for expert animal care and to Barbara Owen for assistance with the manuscript.
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Manuscript received December 17, 1999; revised August 9, 1999; accepted August 23, 1999.
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