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Experimental Therapeutics, Preclinical Pharmacology |
Scott Department of Urology [G. W. H., M. A. M, Y. N., C. H. B., G. Y., S. S., H-M. L., J. W., J. A., S. E., T. S., T. L. T., T. C. T.] and Departments of Cell Biology [T. C. T.] and Radiology [T. C. T.], Baylor College of Medicine, Houston, Texas 77030, and Veterans Affairs Medical Center, Houston, Texas 77030 [T. L. T., T. C. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To develop new therapeutic approaches for prostate cancer, we have conducted extensive preclinical studies of various adenovirus-mediated gene therapy protocols. We have evaluated "suicide gene therapy" extensively in preclinical studies using adenovirus-mediated expression of the HSV-tk gene, followed by the systemic administration of the prodrug GCV studies (4, 5, 6, 7, 8) . In our orthotopic models of prostate cancer, tumors are established by intraprostatic injection of well-characterized prostate cancer cell lines. Approximately 1 week later, the tumor is injected directly with the therapeutic adenoviral vector, and the animals are followed for 2–3 weeks. HSV-tk + GCV gene therapy led to marked growth inhibition of the local prostate cancer with suppression of spontaneous and induced metastases using two-cell mouse prostate cancer cell lines, RM-1 and RM-2 (5 , 6 , 8) . In these studies, antimetastatic activity consistent with the induction of an antitumor immune response was also associated with HSV-tk + GCV therapy. These preclinical studies led to the first clinical trial of in situ gene therapy for prostate cancer, which was shown to be safe and suggested that in situ gene therapy with HSV-tk + GCV could suppress serum PSA levels in men who have local recurrence of prostate cancer after radiotherapy (9) . We have subsequently accrued additional patients and documented alterations of the PSA doubling times and the presence of activated CD8 immunocyte populations in the peripheral blood of patients who received gene therapy.5
Human (10) and mouse (11 , 12) prostate cancer cells are generally considered to be poorly immunogenic despite the presence of antigens that may be tumor specific (13) . This lack of immune stimulation may be overcome by enhancing the presentation of the tumor antigens to T cells and by delivering immunostimulatory cytokines. Modification of the immune response against cancer using specific cytokines may prove effective against cancers such as prostate cancer. IL-12, a heterodimeric cytokine, was found to be elevated after HSV-tk + GCV therapy in melanoma models (14) and has potent antitumoral properties in several preclinical models of cancer (15, 16, 17) . However, administration of cytokines systemically to treat cancers has resulted in significant toxicity in both preclinical studies and early human trials. Using gene therapy to deliver immunomodulatory genes directly into tumors is one technique to limit the toxicity associated with systemic administration of cytokines. We reported the efficacy of a single injection of an adenovirus expressing IL-12 to treat a poorly immunogenic orthotopic murine model of prostate cancer. Treatment of the primary tumor resulted in a reduction of primary tumor weight by 58%, a significant survival advantage, and a reduction in the number of pre-established lung metastases with minimal toxicity (11) . The mechanism of efficacy in our system appeared to involve NK-mediated cytolysis, increased activation of nitric oxide synthase in macrophages and, potentially, T-cell activities.
Antigen presentation requires binding of processed peptides complexed with MHC molecules to the T-cell receptor. Professional antigen-presenting cells express costimulatory molecules such as B7-1 (CD80) that interacts with its specific receptor, CD28, expressed on T cells to generate long-lasting antitumor responses (18) . B7-1 is poorly expressed on most tumor cells; thus, genetically engineered tumor cells that express B7-1 have been studied as whole-cell vaccines in several preclinical models (19 , 20) . In addition to its costimulatory role in activation of T cells, B7-1 has also been shown to enhance nonspecific NK-mediated cytolysis of a poorly immunogenic murine lung carcinoma (21) .
In this study, we compared the efficacy of adenovirus-mediated expression of IL-12 and B7-1 (AdmIL-12/B7) to an adenovirus expressing IL-12 alone (AdmIL-12) and a control adenovirus (Ad/CMV/ßgal) in the treatment of a poorly immunogenic prostate cancer cell line, RM-9. We document IL-12 secretion and enhanced surface expression of B7-1 after in vitro infection with AdmIL-12/B7. In our orthotopic model, we show a dose-dependent reduction of tumor weight that is significantly better than AdmIL-12 alone. This vector also offered a significant survival advantage that involved long-term survivors/apparent cures and reduced the number of pre-established lung metastases. We also developed a model for cell-based vaccine therapy with irradiated RM-9 cells infected with either AdmIL-12 or AdmIL-12/B7 and demonstrated therapeutic activity of both vectors using this protocol.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of in Vitro Infected Cells.
Adenoviral infection of the mouse prostate cell line, RM-9, with
AdmIL-12 and AdmIL-12/B7 was tested in vitro to evaluate for
direct cytotoxicity, IL-12 secretion, and expression of cell surface
markers. The RM-9 prostate cancer cell line was derived from the mouse
prostate reconstitution model system using C57BL/6 mice as described
previously (24
, 25)
. Triplicate aliquots of 10,000 RM-9
cells were plated on 24-well plates in DMEM with 10% fetal bovine
serum, 10 mM HEPES, 100 units/ml penicillin, and
100 µg/ml streptomycin and allowed to adhere for 24 h. The
medium was then aspirated, and the adherent cells were washed gently
with PBS. The cells were then infected with AdmIL-12, AdmIL-12/B7, or
Ad/CMV/ßgal at a MOI of 100 using serum-free media (DMEM, 10
mM HEPES, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.1% BSA). The medium was gently aspirated after
2 h and replaced with 1 ml of serum containing DMEM as described
above. Cell counts were measured in triplicate 24, 48, 72, and 96 h after transduction to evaluate for direct cytotoxicity of the vectors
on the RM-9 cell line. Medium was collected from each of the wells at
24, 48, 72, and 96 h and assayed for murine IL-12 by ELISA
(BioSource International, Camarillo, CA). Cell suspensions of 5 x
105
infected and parental RM-9 prostate cancer
cells were incubated for 30 min on ice with biotin-conjugated antimouse
MHC class I, II, B7-1, and B7-2 antibodies (PharMingen, San Diego, CA)
and then with fluorescein-conjugated avidin (Pierce Corp., Rockford,
IL). The cells were washed, and 10,000 cells were analyzed on EPICS
XL-MCL (Coulter Electronics, Westbrook, ME) for expression of MHC class
I, II, B7-1, and B7-2 antigen by mean fluorescence intensity as
described previously (11)
.
Orthotopic Tumor Induction.
After trypsinization, RM-9 cells were counted and resuspended in HBSS.
For orthotopic tumor inoculation, syngeneic C57/BL6 mice were
anesthetized with sodium pentobarbital. A low transverse abdominal
incision was made, and the dorsolateral prostate was exposed. Injection
of 5000 cells in 10 µl directly into the right lobe of the
dorsolateral prostate resulted in efficient and reproducible orthotopic
tumor formation. The tumors were allowed to grow for 7–8 days prior to
treatment. At this time point, most mice had tumors ranging between 5
and 15 mm3
, as measured with calipers.
In Vivo IL-12/B7-1 Gene Therapy.
Previously, Nasu et al. (11)
had
determined the optimal therapeutic dose of AdmIL-12 to be 1 x
108 using this orthotopic model of prostate
cancer. To determine an optimal therapeutic viral dose of
AdmIL-12/B7, escalating viral doses from 2.5 x
107 to 3 x 108 PFUs
of AdmIL-12/B7 were directly injected into established prostate tumors
7–8 days after tumor cell inoculation. Ad/CMV/ßgal and PBS were used
as controls. A 27-gauge needle was placed through an intracapsular,
transprostatic tract prior to tumor penetration to minimize leakage of
virus at the time of needle withdrawal. The needle point was then moved
around within the tumor during injection to maximize the area exposed
to the vectors. A volume of <25 µl was used for all virus
injections.
Animals were euthanized on the 14th day after virus injection or at seven different time points in a kinetic analysis (1, 3, 5, 7, 10, and 14 days after viral injection). For survival analysis studies, animals were evaluated at death, or they were euthanized when appearing in distress, as evidenced by lethargy, ruffled fur, or weight loss. All animals underwent a careful necropsy for gross metastasis. The primary tumor and spleen were removed and weighed. Blood was collected, allowed to clot, and serum frozen at -80°C for analysis of IL-12 as described above. The spleen was placed in sterile medium until the splenocytes could be harvested to perform flow cytometry, NK cell activity assays. In addition, the pelvic and retroperitoneal lymph nodes and samples of lung were excised, regardless of gross appearance, and along with a portion of the primary tumor processed for histological analysis. Samples of tissue were placed in formalin, paraffin embedded, cut into 4–5-µm sections, and stained with H&E for histological examination or immersed in Tissue-Tec OCT medium and quick frozen in liquid N2.
All mice were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care, and all animal studies were conducted in accordance with the principles and procedures outlined in the NIH’s Guide for the Care and Use of Laboratory Animals.
Pre-established Metastases and Treatment.
In our model of preexisting lung metastases, mice receive a tail vein
injection of RM-9 cells (100,000 cells in 100 µl of HBSS) to induce
lung metastases at the same time as orthotopic tumor inoculation with
5000 cells. Six days after inoculation, animals were randomized to
receive a single injection of either AdmIL-12, AdmIL-12/B7, PBS, or a
control vector, Ad/CMV/ßgal, directly into the orthotopic prostate
tumor. In previous studies, the presence of lung metastases has been
confirmed at the time of virus injection (11)
. Animals
with preexisting metastases were sacrificed on the eighth day after
virus injection, at which time the lungs were weighed and lung
metastases were counted with the aid of a dissecting microscope by two
blinded observers.
Vaccination Protocol.
Vaccination consisted of three s.c. injections at weekly intervals of
one million irradiated RM-9 cells either uninfected or infected with
adenovirus. Control vaccination consisted of injection of HBSS. The
RM-9 cells were infected in vitro at an MOI of 100 1 day
prior to irradiation. The next day cells were trypsinized, centrifuged,
and resuspended in HBSS at 1 x 106 cells/ml
and then irradiated with 300 Gy with a gamma cell irradiator. The cells
were then centrifuged and resuspended in HBSS at a concentration of
1 x 106 cells/100 µl and injected s.c. in
the right upper abdominal quadrant. This dose of irradiation was
determined to prevent further cell growth either in vitro or
in vivo but did not affect the ability of the
adenovirus-infected, irradiated cells to continue to express IL-12
in vitro (not shown). Three weeks after the last
vaccination, the mice were challenged with orthotopic injection of 5000
RM-9 cells as described above for orthotopic tumor induction. The mice
were sacrificed 22 days later and evaluated for prostate tumor
formation, and the wet weights of tumors were recorded.
Quantitative Immunohistochemistry.
Frozen tissues were sectioned with a cryostat, and sections reacted
with CD4, CD8, and F4/80 antibodies and visualized as described
previously (11)
. Computer-assisted image analysis was used
to determine the number of immunopositive
cells/mm2
(11)
.
In Vitro Cytolytic Assays.
NK cell lytic activity was measured in the splenocytes harvested from
the different treatment groups. NK activity was determined at several
time points after treatment with 51Cr-labeled YAC
cells as the target cells. A standard assay was used as described
previously (11)
. Various effector (splenocytes):target
cell (YAC) ratios were used. The YAC cell line was obtained from
American Type Culture Collection.
Statistical Analysis.
ANOVA was used to compare tumor weights. Survival analysis was with
the log-rank test (Mantel-Cox test). All statistical analyses were
performed with Statview 4.02 (Abacus Concepts, Berkeley, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a). In vitro IL-12
production was higher in the AdmIL-12/B7-infected cells compared with
AdmIL-12-infected cells (at 24, 48, and 72 h after infection) and
controls (Fig. 1
b), which is consistent with previous
reports in a breast cancer cell line (23)
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, we show that RM-9 cells have low
expression of B7-1 and B7-2. However, infection with AdmIL-12/B7 led to
an up-regulation of B7-1 surface expression by 3.1 times compared with
parental RM-9 cells (mean fluorescence increased from 2.9 to 9.0; Fig. 2
). AdmIL-12/B7 infection did not affect
MHC I, MHC II, or B7-2 surface expression. Infection of cells with
AdmIL-12 alone did not alter surface expression of MHC I, II, B7-1, or
B7-2.
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).
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). More than 20% of the
animals survived over an extended time period. All animals had
measurable tumors at the time of adenoviral vector injection. Six of 19
animals were examined at day 50 for the presence of tumor, and there
was no evidence of malignancy within the prostate gland, suggesting
that these animals had been cured of their disease. In contrast, only 1
of 38 animals injected with AdmIL-12 demonstrated long-term survival
without evidence of disease.
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). Both doses of AdmIL-12/B7 (5 x
107 and 1 x 108 PFU)
demonstrated a trend toward enhancement of antimetastatic effect
compared with AdmIL-12, but this did not reach statistical significance
(P = 0.18 and P = 0.52).
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A, we show that IL-12/B7
suppressed tumor growth compared with control or AdmIL-12-injected
tumors. The spleen from each animal was weighed at the time of
sacrifice, and as seen in Fig. 6
B, the AdmIL-12/B7-treated
animals had the largest increase in spleen weight, with a maximum
increase somewhat delayed compared with the AdmIL-12-treated animals.
Consistent with our previous study (11)
, serum IL-12
levels peaked 1 day after adenoviral vector injection with either
AdmIL-12 or AdmIL-12/B7 (Fig. 6
C). With equivalent vector
doses (1 x 108), a somewhat larger but more
variable peak serum level of IL-12 was obtained with the AdmIL-12
vector. The serum levels at subsequent time points were similar for the
two vectors at this dose. The optimal therapeutic dose of AdmIL-12/B7
(5 x 107) yielded lower serum IL-12 levels.
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. There was
a 2–3-fold increase in the number of CD4-reactive T cells on day 7
after either vector (Fig. 7
A). The number was slightly
larger in the AdmIL-12 group compared with the AdmIL-12/B7 group. An
increase in CD8-reactive T cells was even more pronounced at day 7
after both vectors (Fig. 7
B). The AdmIL-12/B7 induced a
somewhat greater increase in CD8+ cell numbers at day 7, and this
increase was sustained through day 14 in contrast to the AdmIL-12 group
in which there was a substantial decrease in CD8 counts by day 14. The
levels of cells reactive with the macrophage-selective antibody (F4/80)
were elevated for both treatment groups on day 2 relative to the
control Ad/CMV/ßgal (on day 14) and to the control levels observed
previously at the time of injection (11)
. The number of
F4/80-positive cells in AdmIL-12-treated tumors initially decreased
more rapidly than the AdmIL-12/B7 group after the day-2 peak (Fig. 7
C).
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. This NK activity returned to control
levels by day 5 (data not shown).
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A). Vaccination with
AdmIL-12-infected RM-9 cells protected 20% (8 of 10) of the mice. No
mice vaccinated with HBSS alone or irradiated, uninfected cells were
tumor free. The weight of the orthotopic tumors in the animals with
tumors also suggested that vaccination with adenovirus-infected cells
generated a systemic immune response that suppressed the growth of the
orthotopic challenge tumors (Fig. 9
B). There was no
significant difference in tumor weight between the two vaccine
treatments, but both were significantly smaller than controls
(P < 0.02).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or pre-established lung metastases (Fig. 5)
. Although
in vitro production of IL-12 appeared to be increased in the
AdmIL-12/B7 vector (Fig. 1
B), this did not translate into
increased serum levels in vivo after orthotopic injection
into RM-9 tumors (Fig. 6
C). Therefore, the therapeutic
benefits of AdmIL-12/B7 are not likely to be explained as simply
increased production of IL-12. A possible mechanism of increased
potency was revealed by the sustained intratumoral CD8 response after
AdmIL-12/B7 injection (Fig. 7
B). Although a lower titer of
AdmIL-12/B7 may achieve a similar therapeutic response as AdmIL-12,
careful monitoring of the safety of this vector is warranted because
high doses tended to produce increased spleen weight compared with
AdmIL-12 (Fig. 6
B) at a lower dose (5 x
107 PFU orthotopically). Although there are numerous studies that have used IL-12 and/or B7-1 under various conditions, there are very few that have directly compared IL-12 and IL-12 + B7.1 using adenoviral vector-mediated in situ transduction of these therapeutic, immunostimulatory genes. One study to which our current study can be compared is that in which AdmIL-12 and AdmIL-12/B7 were compared directly in an orthotopic mouse model of breast cancer. In this study, AdmIL-12/B7 was significantly more effective in delaying orthotopic tumor growth and inducing partial or complete regression. Furthermore, AdmIL-12/B7 in situ gene therapy also protected against a second inoculation of tumor cells when rechallenged 2–3 months later in those animals that had experienced complete primary tumor regression (23) . However, in these studies, the breast cancer cell line was derived from transgenic mice in which breast cancer was induced by the potent immunogenic polyoma middle T antigen (23) . In our studies, we used RM-9 metastatic mouse prostate cancer cells that have been shown previously to be of low immunogenicity (11) and therefore similar in that regard to human prostate cancer (10) .
An additional study that compared IL-12 with IL-12 + B7 using a stable transfection protocol in the CMT93 mouse colorectal tumor was also able to demonstrate that the combination of IL-12 + B7 induced significantly higher levels of systemic protection against inoculation with live CMT93 cells relative to IL-12 or B7-1 alone (26) . However, in this study, the CMT93 alone cells alone were also able to induce systemic antitumor immunity, and thus, the pronounced superior effect of IL-12 + B7 compared with IL-12 alone may also be related to intrinsic immunogenicity of these cells. In a liver tumor cell line that expressed low MHC class I and no MHC class II, ex vivo retroviral transduction of IL-12 + B7 was more effective than either IL-12 or B7 alone, but treating established s.c. tumors with retrovirus producing lines did not reveal a difference between IL-12 and IL-12 + B7 (27) . Depletion studies suggested that CD4+ T cells appeared to be the major contributor to the antitumor effect of B7 and both CD4+ and CD8+ T cells were implicated in the effect of IL-12 alone. However, the combination of IL-12 and B7 depended mainly on CD4+ T cells with non-T cells also contributing. The increased efficacy and biological activities associated with IL-12 + B7 compared with IL-12 alone documented in this study of prostate cancer could reflect a difference in the mechanisms of antitumor effects resulting from the tumor type or possibly the tumor site, i.e., s.c. versus prostatic. Although the results of these earlier studies are highly informative, in studies of prostate cancer it is important to compare the results of IL-12 with IL-12 + B7 in situ gene therapy in a model of documented low immunogenicity as we have done. Indeed, this is the first study to make these comparisons in such a prostate cancer model system.
The AdmIL-12/B7 vector may also be useful in cell-based vaccine
approaches for prostate cancer. Although our study used only one
vaccination schedule, we were able to induce an immune response that
led to rejection of an orthotopic tumor in one-third of the animals
that received the IL-12/B7 vaccine. The IL-12 vaccine was also
effective with 20% of the animals protected. These studies are
encouraging and suggest that both viruses may prove useful in
developing further optimized cell-based vaccines for prostate cancer.
Both vectors are capable of inducing NK cells after orthotopic
injection into tumors (Fig. 8)
. Additional studies will be required to
determine whether specific levels of NK or CTLs are induced by the
cell-based vaccination protocols.
In prostate cancer, a clinical trial with a vaccine derived from
autologous tumor cells infected with a retrovirus expressing
granulocyte/macrophage-colony stimulating factor has demonstrated that
this approach is safe and capable of eliciting systemic immune
responses as detected by development of a delayed-type hypersensitivity
response (28)
. Three of eight patients also generated
antibodies that recognized specific proteins that were present in their
prostate cancer as well as in prostate cancer cell lines and normal
prostate epithelium. A limitation of this strategy is the inability to
grow sufficient numbers of prostate tumor cells in vitro to
provide the cancer cells for genetic modification via a retroviral
vector and use as a vaccine. Other vaccine type approaches for prostate
cancer have relied on delivery of autologous dendritic cells pulsed
with prostate membrane-specific antigen (29)
. Reductions
in serum PSA have been reported in 
30% of the treated patients,
even those with hormone-refractory metastatic disease (29
, 30)
. The vaccine approach that we describe here uses recombinant
adenoviral vectors to transduce the cell-based vaccines. For prostate
cancer, this approach is novel and might have unique value clinically
for prostate cancer cell-based vaccines that have proven exceedingly
difficult because of the difficulty in culturing prostate cancer cells
in vitro (28)
.
Overall, AdmIL-12/B7 is the most effective single-vector combination in situ gene therapy strategy that we have tested thus far in our preclinical model systems. When used as an in situ approach, this therapy was more effective in comparison to IL-12 alone in suppressing localized tumor growth, as well as in models of pre-established lung metastases. It is remarkable that 6 of 19 animals treated with AdmIL12/B7 survived long term with apparent eradication of their disease in contrast to 1 of 38 animals treated with AdmIL12. In additional protocols, we also demonstrated that AdmIL-12/B7 (as well as AdmIL-12) was effective in generating systemic antitumor immunity using a cell-based vaccine strategy. On the basis of these studies, IL-12 + B7 gene therapy approaches should now be considered in both in situ and vaccine strategies as a single-vector gene therapy approach or potentially in combination with cytotoxic therapies that could potentially contribute to an immune response (8) . Further development of these approaches hopefully will lead to more rational and effective therapies for this devastating disease.
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FOOTNOTES |
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1 Supported by NIH Specialized Program of Research
Excellence Grant P50-58204. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Scott Department of Urology, Baylor College of Medicine,
6560 Fannin, Suite 2100, Houston, TX 77030. Phone: (713) 799-8718;
Fax:(713) 799-8712; E-mail: timothyt{at}.bcm.tmc.edu
4 The abbreviations used are: PSA,
prostate-specific antigen; HSV-tk, herpes simplex virus-thymidine
kinase; GCV, ganciclovir; IL, interleukin; NK, natural killer; Ad,
adenovirus; CMV, cytomegalovirus; ßgal, ß-galactosidase; PFU,
plaque-forming unit; MOI, multiplicity of infection.
5 B. J. Miles, M. Shalev, E. Aguilar-Cordova,
H.-M. Lee, G. Yang, H. L. Adler, K. Kernen, T. L. Timme, C. K.
Pramudji, A. Frolov, G. Ayala, T. M. Wheeler, E. B. Butler, D. Kadmon,
and T. C. Thompson, submitted for publication.
Received 4/ 5/00; revised 7/24/00; accepted 7/25/00.
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) or 3 days (
) after virus injection.