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Experimental Therapeutics, Preclinical Pharmacology |
Department of Urology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 [W. A. L.]; Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [Y. H., W. R. F., W. D. W. H.]; and Center for Genetic and Cellular Therapies, Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710 [S. K. N., E. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A new form of immunomodulation using gene transfer techniques is now being actively investigated for a variety of malignancies (12) . This treatment requires the insertion of a plasmid DNA encoding a cytokine gene directly into tumor cells. These "gene-modified" tumor cells then produce this cytokine, resulting in enhanced tumor antigen recognition and a documented increase in an antigen-specific immune response (13, 14, 15) . We have previously investigated this new technique in the MBT-2 murine bladder cancer model (16 , 17) . The MBT-2 tumor was induced in C3H mice by oral administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide, a potent carcinogen that resulted in bladder neoplasms in 80–90% of animals (18) . This poorly differentiated transitional cell carcinoma, which exhibits metastatic progression, resembles, both grossly and histologically, human transitional cell bladder cancer. In addition, treatment regimens that have been successful in this murine model have also shown efficacy in human bladder cancer (19 , 20) . In our previous research using retrovirus as the transfecting agent, cytokine genes including IL-2 and granulocyte macrophage colony-stimulating factor were successfully incorporated and stably expressed in MBT-2 tumor cells in vitro (16 , 17) . In vivo experiments showed that i.p. injection of these irradiated cytokine gene-modified cell preparations in tumor-bearing animals resulted in a significant survival advantage.
Although retroviral vectors provide genomic integration and a permanence of transduction, retroviral vector transfection is a time-consuming process and results in low expression of gene product (21) . Additional limitations, including the safety issues of replication defects in this viral vector system and the fact that only dividing cells can be transduced, inhibit the potential of retroviral vectors as optimal transfecting agents for human application (22) .
This study evaluates an alternative transduction mechanism using cationic liposomes. As reported previously by Vieweg et al. (23) , liposome-mediated transfection has several advantages over viral vector systems in the use of promulgating gene-modified tumor cells. Liposome-mediated transfection has no infectious or little immunogenic potential, characteristics that are critical when considering human application (24 , 25) . Because the transfection is not based on genomic integration, gene product expression is transient (26) . As opposed to the retroviral system, multiple copies of the plasmid can be transduced per cell, leading to higher production of the gene for a shorter period of time. We have hypothesized that if gene expression persists long enough for significant immunomodulation, the transient nature of the transfection becomes less important. Using an adeno-associated virus-based plasmid, we evaluated in vitro liposome-mediated transfection in the MBT-2 model. To establish conditions for optimal transfection, we studied four distinct parameters: (a) toxicity [liposomes have inherent lytic effects on mammalian cells when administered in high concentrations (27) ]; (b) the critical liposome:DNA ratio producing the least toxicity with the highest gene expression; (c) the time of exposure to the liposome-DNA complex, i.e., the least amount of transfection time to produce the greatest gene expression; and (d) the effect of growth factors, such as FBS, on the potential inhibition of liposome-mediated transfection.
Previous investigations studied the MBT-2 bladder cancer model in vivo by using the injection of MBT-2 cells either intradermally or s.c. to establish the tumor (28, 29, 30) . Our laboratory devised an alternative method, i.e., orthotopic implantation. This method involves the direct injection of MBT-2 cells into the bladder wall via a small incision in the suprapubic region and allows us to better observe the growth and metastatic spread of bladder tumor cells growing in their normal in situ environment. Additionally, it provides a more physiological approach to evaluate the immune response to bladder cancer cells. Thus, our in vivo studies evaluated the use of liposome-mediated, irradiated, gene-modified cells as "tumor vaccines" in orthotopically implanted MBT-2 tumor-bearing animals. Parameters including vaccination schedule and the amount of transfected cells/vaccine were investigated with a final end point of survival. All animals that survived the initial tumor implantation were rechallenged with parental MBT-2 cells. Necropsy and splenic CTL analysis were performed on those animals that survived the rechallenge.
Initially, we studied a vaccination schema based on multiple injections of single-agent gene-modified MBT-2 cells. These included plasmids containing IL-2 and the adhesion molecule B7.1 genes. B7.1 was well characterized by Freeman et al. (31) and Chen et al. (32) as a T-cell cofactor that is essential for T-cell activation. As the in vivo evaluation progressed, we revised our vaccination schedule to determine the effects of using different gene products at different vaccination times within the same tumor-bearing animals. Thus, one animal would receive IL-2 or B7.1 gene-modified cells as a single agent throughout or in combination in an alternating vaccination regimen. These "targeted" sequential vaccinations evolved as an opportunity to stimulate different aspects of the immune response in a time-dependent manner, based on our knowledge of the cascade of events that dictate successful immunomodulation.
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MATERIALS AND METHODS |
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Liposome Preparations and Gene Product Analysis.
The liposome used in this study was graciously provided by Vical,
Inc. (San Diego, CA). DMRIE/DOPE was composed of positively charged
lipid DMRIE in combination with neutral lipid DOPE in a 1:1 molar ratio
(27)
. The lipid reagents were stored at 4°C before use.
IL-2 measurement was performed by ELISA (R&D Systems, Minneapolis, MN).
B7.1 quantitative analysis was documented by a fluorescence-activated
cell sorter using FITC-conjugated rat antimouse B7.1 (PharMingen, San
Diego, CA).
Cellular Transfection Procedure.
Twenty-four h before transfection, MBT-2 cells were plated on 60-mm
dishes at a density of 1 x 106 cells/dish.
Various amounts of the liposome (2, 10, 20, 40, 60, 80, 120, and 160
µg) and plasmid DNA (1, 2.5, 5, 7.5, 10, 20, 30, and 40 µg) were
diluted separately in serum-free medium to a total volume of 500 µl
each and then gently mixed together in polystyrene tubes. This solution
was allowed to form complexes at room temperature for 15 min. Adherent
MBT-2 cells were rinsed once with serum-free medium. The liposome-DNA
complex was then diluted with serum-free medium to a final volume of 4
ml and gently added to the cellular monolayers. After a transfection
exposure time ranging from 15 min to 24 h (0.25, 0.5, 0.75, 1, 2,
3, 4, 8, 12, and 24 h), the transfection solution was rinsed off,
and fresh medium with 20% FBS was added. Similar experiments to
determine the effect of the growth factor on the transfection process
required the addition of 10% FBS during the transfection exposure. In
separate studies to determine toxicity, cell counts using trypan blue
exclusion were performed 12 h after the completion of exposure to
the liposome-DNA complex. Twenty-four h after transfection, all cells
were exposed to 70 Gy of irradiation. At the next 24 h time
point, all cells were rinsed with PBS and then
replenished with serum-free medium. Twenty-four h later,
supernatants were collected and frozen at -20°C for future IL-2
ELISA determination. Cell counts were performed at this time to be able
to report IL-2 concentrations as ng/106 cells/24
h. For time course experiments, i.e., evaluation of the gene
product secretion over 30 days, all cells were transfected on day 0 and
irradiated on day 1 and then had complete medium replaced every 3 days.
Cells were rinsed with PBS and replenished with serum-free medium
24 h before supernatant collection. Different sets of cells were
transfected in the same study to evaluate gene product secretion at
days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30. Adherent cells at each
time point were counted for accurate quantification.
Animal Studies.
All animal studies were initiated in 6–8-week-old C3H/HeJ female mice
(Jackson Laboratory, Bar Harbor, ME). Freshly prepared MBT-2 cells were
implanted orthotopically in the animals. Briefly, animals were
anesthetized by an i.p. injection of pentobarbital. Under
magnification, a 0.8-cm incision was made transversely in the abdomen,
just above the pubis. The bladder was delivered into the surgical
field, and the bladder wall was injected with 2 x
104 MBT-2 cells in a total volume of 50 µl of
PBS. The wound was closed with surgical staples that were removed on
postoperative day 5. All animal studies were overseen and approved by
the Research Animal Regulatory Committee advisory groups at both Duke
University Medical Center and Memorial Sloan-Kettering Cancer Center.
The date of implantation was designated as day 0. Multiple tumor vaccination schedules included: (a) days 1, 2, and 3; (b) days 2, 4, and 6; (c) days 3, 6, and 9; (d) days 4, 8, and 12; (e) days 5, 10, and 15; (f) days 6, 12, and 18; and (g) days 7, 14, and 21. Consecutive tumor vaccinations were accomplished with i.p. injection of 5 x 106 MBT-2 cells in a volume of 100 µl of PBS. Transfected MBT-2 cells were used on posttransfection day 2, which was 1 day after irradiation. Adherent cells were removed from plates with 2% trypsin, washed twice with PBS, and then counted on a hemacytometer. A separate aliquot of cells was replated on 60-mm culture dishes for future determination of gene product secretion. The doses of gene-modified cells for vaccination included 1, 2.5, 4, and 5 x 106 MBT-2 cells. Control animals were injected with 100 µl of PBS.
Daily inspection of animals provided general status, tumor size, and symptoms associated with disease (i.e., hematuria and uremic status). Mice with a tumor burden of more than 10% of body weight or mice presenting cahexic status were sacrificed and necropsied with special attention placed on examination of the lungs, kidneys, bladder, and peritoneal cavity. Animals that survived 60 days were rechallenged by orthotopic implantation of 2 x 104 parental MBT-2 cells, and new control animals also received the same inoculum of MBT-2 cells on the same day. These rechallenged animals did not receive any further vaccinations. On day 120 after the original implantation, or 60 days after rechallenge, all animals were sacrificed and necropsied, and CTL analysis was performed on their splenocytes.
CTL Analysis.
Splenocytes were harvested and depleted of RBCs with ammonium chloride
Tris buffer. Splenocytes (1.5 x 107) were
cultured with 7.5 x 105 stimulator cells
(MBT-2/IL-2 cells irradiated at 75 Gy) in 5 ml of Iscove’s modified
Dulbecco’s medium with 10% FBS, 1 mM sodium pyruvate, 100
IU/ml penicillin, 100 mg/ml streptomycin, and 50 µM
ß-mercaptoethanol per well in a 6-well tissue culture plate. Effector
cells were cultured for 5 days at 37°C and 5%
CO2 and harvested for CTL assay.
All 5–10 x 106 target cells were labeled with europium diethylenetriamine pentaacetate for 20 min at 4°C. After several washes, 1 x 104 europium-labeled target cells and serial dilutions of effector cells at varying E:T ratios were incubated in 200 µl of RPMI 1640 with 10% heat-inactivated FBS in 96-well V-bottomed plates. The plates were centrifuged at 500 x g for 3 min and incubated at 37°C and 5% CO2 for 4 h. The supernatant (50 µl) was harvested, and europium release was measured by time resolved fluorescence (Delta fluorometer; Wallace, Inc., Gaithersburg, MD; Ref. 34 ). Spontaneous release was less than 25%. SEs of the means of triplicate cultures were less than 5%.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the extent of IL-2 secretion in this "transient"
liposome-mediated transfection, we studied the gene product expression
over a 30-day period. All cells were transfected on day 0 according to
the optimized conditions as described above. On day 1, cells received
70 Gy of irradiation, and then IL-2 secretion was measured at 3-day
intervals (Fig. 1)
. In three separate
studies, IL-2 levels reached an average of 280
ng/106 cells/24 h by day 3 and maintained 90% of
this peak for up to 9–11 days after transfection. A steady decline in
IL-2 production was then observed, until levels of less than 5
ng/106 cells/24 h were seen beginning on days
15–18. Fourteen to 22% of MBT-2 cells liposomally transfected with
B7.1 plasmid expressed cell surface B7.1 as determined by
fluorescence-activated cell-sorting
analysis.4
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, tumor growth was
prominent 14 days after tumor implantation, and i.p. metastases were
apparent on day 21. These animals used for sacrifice purposes were
found to die at nearly the same time as the animals sacrificed because
of the humane requirements of the animal committees.
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. Of all of the vaccination schedules
studied, the greatest impact on survival was seen with the day 5, 10,
and 15 regimen. Therefore, the group most extensively evaluated was
those receiving i.p. vaccinations of irradiated IL-2 gene-modified
MBT-2 cells on days 5, 10 and 15 after orthotopic implantation. Initial
in vivo experiments showed no survival advantage in those
tumor-bearing mice that received vaccinations of 1 x
106 IL-2 gene-modified MBT-2 cells. Groups that
received vaccinations of 5 x 106 IL-2
gene-modified cells survived for less time than controls, although the
animals were found to have relatively small tumor burdens at the time
of necropsy. Most of these animals developed several signs of toxicity
(i.e., body weight loss, ascites, chattering, and
immobility), and this adverse effect was considered to be IL-2 toxicity
because these animals were injected with cells producing >1000 ng of
total IL-2 secretion per day. An alternative strategy to increase
potential tumor antigen presentation while limiting IL-2 toxicity was
devised. This required vaccinations of nontransfected MBT-2 cells in
addition to IL-2 gene-modified cells. As shown in Table 2
, those
animals receiving vaccinations of 2.5 x 106
liposomally transfected IL-2-secreting cells in combination with
2.5 x 106 nontransfected cells repeatedly
showed a 75% increase in survival as compared with controls. No other
combination of gene-modified and nontransfected cells showed any
consistent increase in survival. Also, none of these groups exhibited
"cure" or survival for 60 days after tumor implantation. Animals
receiving retrovirally gene-modified MBT-2/IL-2 cell preparations had a
median survival of 29 days.
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. MBT-2
cells were transfected using the described optimized
liposome-mediated transfection. The vaccination schedule of
day 5, 10, and 15 was followed. Tumor-bearing animals received a
vaccination with MBT-2 cells containing one transfected plasmid on day
5, followed by MBT-2 cells that were transfected with another plasmid
on days 10 and 15. As seen in the previous experiments, control animals
and those receiving IL-2 gene-modified MBT-2 cell vaccinations followed
the same course (Fig. 2)
. In those
animals receiving B7.1 gene-modified MBT-2 cells alone or B7.1 followed
by IL-2, a survival advantage similar to that seen in the group
treated with IL-2 alone was demonstrated. In repeated studies,
the group of mice given IL-2-secreting cells on day 5 followed by
B7.1-expressing cells on days 10 and 15 showed 75% survival at 60
days. At that time, the survivors were orthotopically rechallenged with
MBT-2 cells, as were a new set of control animals. None of the
survivors received any further vaccinations during the course of the
study. The control mice succumbed as before; however, all of the
IL-2/B7/B7 survivors remained alive for another 60 days, or 120 days
since the original tumor implantation. Interestingly, a set of animals
vaccinated simultaneously with IL-2 and B7.1 cotransfected cells
revealed no survival advantage.
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, splenocytes
harvested from mice vaccinated with IL-2/B7/B7 exhibited very high
levels of CTL activity (approximately 50% at an E:T ratio of 100:1).
Control target L929 demonstrated insignificant lysis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the MBT-2 model, we have demonstrated a clear advantage of liposome-mediated transfection over retroviral system. With optimized liposome-mediated transfection, we were able to observe a 100–150-fold increase in IL-2 production in a fraction of the transfection time (30 min) as compared with the retroviral system. This magnitude of the enhanced gene product secretion persisted for over 10–14 days. Although "transient" in a nonintegrated form as episomes as compared with the stable genomically integrated retroviral transfection, IL-2 expression by liposome-mediated gene-modified cells proved to be persistent enough to stimulate an immune response, the ultimate goal of this form of gene therapy. Indeed, these advantages could have a profound impact when considering a human model for cytokine gene therapy. First, the liposome-mediated transfection does not require dividing cells, as the retroviral system does. Therefore, all cells available for transfection would be potential candidates for gene modification strategies. Second, the issue of safety concerning replication defects in the retroviral system is obviated. The safety of liposomes has been confirmed by their use in human trials for the treatment of melanoma (37 , 38) . In addition, the simplicity and virtual instantaneous transfection significantly reduce the laborious and time-consuming efforts necessary for retroviral transfection, both important improvements when considering human application. The 100-fold increase in cytokine production using the liposome technique also has a profound impact in the human gene therapy model. In human trials, we will not have unlimited amounts of primary tumor cells to transfect and use later as tumor vaccines. Therefore, to be able to produce this logarithmically higher cytokine secretion will afford greater potency to each injection of gene-modified cells. Indeed, in human bladder cancer cell lines and in primary cultures of human bladder cancer obtained from surgical specimens, we have been able to achieve similar enhancement of gene expression using liposome-mediated transfection (39) .
The orthotopic implantation of bladder cancer in an animal model has proved to be beneficial in allowing the observation of a tumor growing directly in bladder tissue, a more physiological approach to evaluate the immune response to this site. The IL-2 gene-modified tumor vaccines appeared to result in a reliable but limited response. Survival was increased, but no cures were observed. IL-2 has been clearly implicated in the attraction of APCs and consequent stimulation of CD4+ and CD8+ cells. However, we did not achieve a lasting immune response or eradication of the pre-established bladder tumor. The explanation of this successful but limited response may lie in the growing belief that multiple signals and stimulation at various sites of the immunological pathway are essential in promoting a potent and persistent cytotoxic tumor effect (32 , 40 , 41) .
This dictum led to the development of the targeted vaccination studies as described in this report. It is also clear that stimulation of CD4+ and CD8+ cells by professional APCs is required to effect the antigen-specific killing of tumor cells. With this in mind, we attempted to understand and modify our approach to liposome-mediated gene-modified cellular vaccines in the MBT-2 tumor-bearing model.
The inclusion of adhesion molecule B7.1, a T-cell cofactor, in
the vaccine regimen was an attempt to investigate alternative modes of
activating an immune response. Thus, multiple combinations of IL-2 and
B7.1 gene-modified tumor vaccines were tested in the in vivo
MBT-2 model. The regimen (IL-2/B7/B7) that began with IL-2-secreting
cells on day 5, followed by the B7.1-transfected cells on days 10 and
15, had a striking effect on survival. The other groups, which included
B7.1 alone (B7/B7/B7) or the alternative schema of B7.1 followed by
IL-2 (B7/IL-2/IL-2), did not prove efficacious. So successful was this
IL-2/B7/B7 schedule that not only did 75% of the animals experience
tumor regression and survive to 60 days after tumor implantation, but
all survivors were resistant to orthotopic rechallenge and remained
tumor free. The evidence that memory CD8+ response against MBT-2 cells
had been achieved in surviving animals is further supported by the CTL
analysis (Fig. 3)
.
Thus far, several previous studies regarding immunogene therapy using B7 and cytokines have shown the controversial results of this strategy in other tumor systems. Gäken et al. (42) have successfully shown that combined expression of murine B7.1 and IL-2 on NC murine adenocarcinoma cells was significantly more effective than either B7.1 or IL-2 alone in inducing the immune-mediated rejection of pre-established NC tumors involving CD4+ lymphocytes. Cayeux et al. (43) have found that immunization with J558-IL2/B7.1 cells followed by challenge with parental murine plasmacytoma J558L cells caused a reduction in systemic protection as compared with J558-B7.1 or J558-IL2 alone. They concluded that "hyperstimulation" of the immune response by genetically modified cancer vaccines could have adverse effects on tumor immunity. It was also true in our current experiment that simultaneously vaccination with IL-2 and B7.1 cotransfected MBT-2 cells resulted in no survival advantage. The differences in immunological mechanisms are not yet completely understood; however, it is suggested that this difference may be due, in part, to the induction of anergy in the potential reactive T cells.
Our results imply that B7.1 is required for successful induction and function of CTLs after IL-2 exposure. B7.1-transfected MBT-2 cells alone were not responsible for tumor regression when used independently; instead, they appeared to be effective only after the initial activation of the immune response by the IL-2 gene-modified MBT-2 vaccinations. One can therefore speculate that the increased attraction of APCs by IL-2 is the initial complementary step in the cascade of immunological events that subsequently permits the function of B7.1 to be realized as a cofactor in the production and stimulation of antigen-specific CTLs. We believe that this concept of timed, programmed, targeted stimulation of the immune response is a logical alternative to continuous single-agent regimens.
Additional studies involving liposome-mediated gene-modified cellular vaccinations in the MBT-2 bladder cancer model are under way. These will focus on the programmed use of multiple cytokines and T-cell factors in targeted vaccination studies. We are currently investigating the different components of the immune response at each activation step to better identify the mechanisms involved. In addition, the use of liposome transfection with cytokine genes in human urological malignancies will be actively pursued. This alternative form of cancer treatment, which uses gene transfer techniques to enhance tumor antigen recognition, has multiple applications and holds great promise.
In summary, liposome-mediated transfection provides a safe, simple, and highly effective mode of gene transfer as compared with retroviral transfection systems. The use of multiple agents in gene-modified tumor cell vaccinations in a time-dependent fashion has elicited a more significant and substantial immune response than single-agent regimens.
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FOOTNOTES |
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1 Supported by an American Foundation for
Urologic Disease Fellowship and the Peter T. Joseph Foundation
(W. A. L.). 
2 To whom requests for reprints should be
addressed, at The Cleveland Clinic Foundation, Department of Urology,
9500 Euclid Avenue, A-100, Cleveland, OH 44195. Phone: (440) 329-7315;
Fax: (440) 329-7316; E-mail: LARCHIW{at}ccf.org
3 The abbreviations used are: IL-2, interleukin 2;
FBS, fetal bovine serum; DMRIE,
1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; DOPE,
dioleolylphosphatidylethanolamine; APC, antigen-presenting cell.
4 W. A. Larchian, unpublished data.
Received 9/16/99; revised 4/ 6/00; accepted 4/ 7/00.
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REFERENCES |
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-interferon genes. Cancer Res., 53: 3561-3568, 1993.-IFN induce effective rejection of established parental tumor. Gene Ther., 6: 253-262, 1999.[CrossRef][Medline]
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