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Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain by plasmid-mediated
gene transfer in head and neck, as well as prostate tumors to provide a
potential target. This was followed by successfully treating xenograft
tumor-bearing nude mice with IL-13R-directed cytotoxin (IL13-PE38QQR).
Although we did not observe an indirect cytotoxic bystander effect
conveyed to nontransduced tumor cells in vitro, our approach in vivo
led to a complete regression of established tumors transfected with
IL-13R2 chain in most animals. We found that the tumor eradication
was achieved in part by infiltration of macrophages and NK cells,
assessed by immunohistochemistry. Moreover, head and neck tumors
xenografted in macrophage-depleted nude mice were less sensitive to the
antitumor effect of IL-13 cytotoxin. Because we did not observe
vector-related toxicity in any vital organs, our novel combination
strategy of gene transfer of IL-13R2 chain and receptor-directed
cytotoxin therapy may be a useful approach for the treatment of
localized cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Over the last 7 years, we have identified the IL-13R as a specific
tumor cell surface target for receptor-directed cytotoxin therapy. To
target IL-13Rs on human solid cancer cells, we produced a recombinant
agent that binds to IL-13R on tumor cells (7). This
molecule is a chimeric protein composed of IL-13 and a truncated form
of a powerful bacterial toxin called Pseudomonas exotoxin
(fusion protein termed IL13-PE38QQR or IL-13 cytotoxin). We have shown
that this toxin is highly cytotoxic to IL-13R-positive tumor cells in
vitro and in vivo in animal models of human tumors
(8, 9, 10, 11, 12, 13, 14, 15, 16). Although tumor cells derived
from glioblastoma, AIDS-associated Kaposi’s sarcoma, renal cell
carcinoma, and head and neck cancer express high levels of receptor for
IL-13, tumor cells derived from breast, colon, ovarian, prostate, and
lung cancer express a modest or low level of IL-13R, which may not be
sufficient for the receptor-targeted cytotoxin therapy
(8, 9, 10, 11, 12, 13, 14, 15, 16). To overcome this limitation, we hypothesized that
tumor cells coaxed to express more IL-13R might be more susceptible to
the effects of IL-13R-targeted cytotoxin. To achieve this goal, we used
the IL-13R subunit IL-13R2 chain as a possible target for this
strategy, as the IL-13R2 chain is known to bind IL-13 with highest
binding affinity and is internalized after binding to its ligand
(17, 18, 19, 20). Using cDNA transfection of several tumor cell
lines, we have demonstrated that gene transfer of IL-13R2 chain can
sensitize tumor cells to the cytotoxic effect of IL-13 cytotoxin
(21). More recently, we demonstrated that treatment
with IL-13 cytotoxin mediates complete regression of IL-13R2
chain-transfected tumors established in immunodeficient mouse models of
human cancer (12, 15).
In the current study, to explore this approach
further as well as more closely mimic an actual clinical situation, we
intratumorally injected IL-13R2 chain-encoding plasmid into tumors
established from human head and neck (A253) and prostate (DU145) cancer
cell lines by s.c. implantation in nude mice. These mice were treated
systemically or intratumorally with IL-13 cytotoxin. We evaluated
gene transfer efficiency of IL-13R2 chain in vivo, vector migration
by determining expression in vital organs, and the mechanism
responsible for antitumor activity resulting from the combination of
gene transfer of IL-13R2 chain, followed by IL-13 cytotoxin therapy.
Based on our observation of a remarkable antitumor activity, we propose
that this approach could be applied to a variety of solid tumors in
vivo and perhaps in the clinic for the treatment of localized tumors
that either do not constitutively express, express low levels, or
express heterogeneous levels of IL-13R2 chain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human A253 and DU145 cell lines were purchased from the
American Type Culture Collection (Manassas, VA) and cultured, as
previously described (12, 15). rIL13-PE38QQR was produced
and purified in our laboratory (7, 16). cDNA encoding
human IL-13R2 chain (17, 22) was cloned into the VR1020
mammalian expression vector (a kind gift from Vical, San Diego, CA)
using PstI and BglII sites, and the sequences of
the flanking regions of the junctions were verified by direct
sequencing (ABI Prism 310; PerkinElmer, Wellesley, MA). Gene expression
of IL-13R2 cDNA in the VR1020 vector is driven by the CMV promotor
(23). The resulting construct was expanded in
Escherichia coli and purified using endotoxin-free EndoFree
Mega kit (Qiagen, Valencia, CA).
Protein synthesis inhibition assay
The in vitro cytotoxic activity of IL13-PE38QQR was measured by the inhibition of protein synthesis (11). All assays were performed in quadruplicate, and the concentration of IL-13 cytotoxin at which 50% inhibition of protein synthesis occurred was calculated (IC50).
Animal studies
Athymic nude mice 4 wk old (
20 g in body weight) were
purchased from Frederick Cancer Center Animal Facilities (Frederick,
MD). Animal care was in accordance with the guidelines of National
Institutes of Health Animal Research Advisory Committee. Human head and
neck and prostate tumor models were established in the nude mice by
s.c. injection of 5 x 106 A253 or DU145
cells in 150 µl PBS into the flank. These mice with established
tumors were injected intratumorally with 25 µg of IL-13R2
cDNA-encoding vector mixed with 20 mM
N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium
chloride:cholesterol (1:1 molar ratio) liposome (Sigma-Aldrich, St.
Louis, MO) (24, 25). For antitumor activity, mice were
injected with IL13-PE38QQR or excipient either i.p. (500 µl/mouse) or
intratumorally (30 µl/tumor), and tumors were carefully measured by
Vernier calipers. Tumor size was calculated by multiplying length and
width of the tumor on a given day. The statistical significance of
tumor regression was calculated by Student’s t test. All
statistical tests were two sided.
Reverse-transcriptase PCR
Total RNA was isolated using TRIzol reagent (Life Technologies, Grand Island, NY), and RT-PCR was performed as described (26).
Immunohistochemistry
Immunohistochemistry was performed using the Vector ABC peroxidase kit (Vector Laboratories, Burlingame, CA), according to the manufacturer’s instructions. Subcutaneous tumor samples were harvested 3 days after the IL13-PE38QQR treatment (day 12) and fixed with 10% Formalin (paraffin-embedded sections) or snap frozen with OCT compound (frozen sections). Paraffin-embedded sections were deparaffinized by xylene treatment and washed with alcohol (100–50%) and PBS. Slides were incubated with Abs against murine macrophage (F4/80; Caltag Laboratories, Burlingame, CA), NK cells (NK1.1; Caltag Laboratories), or inducible NO synthase (iNOS,4 M19; Santa Cruz Biotechnology, Santa Cruz, CA) (0.4–1 µg/ml) or isotype control for 18 h at 4°C. Slides were then developed using diaminobenzidine substrate-biotinylated peroxidase reagent (Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich). Immunohistochemical assays were performed two to three times independently with similar results, and slides were assessed by two investigators (K. Kawakami and M. Kawakami).
For immunofluorescent assays, frozen sections were stained with
anti-human mAb for IL-13R2 (Diaclone, Besancon, France)
or costained with Abs for macrophages and iNOS. Slides were fixed in
acetone at -20°C for 5 min and air dried. Nonspecific binding was
blocked by treatment with 10% serum for 1 h, followed by
incubation with Abs or isotype control. Sections were subsequently
incubated for 1 h with secondary Abs that had either
tetramethylrhodamine isothiocyanate or FITC tags. After three washes
with PBS, slides were dried and layered with Vectashield
antifluorescence fading mounting medium (Vector Laboratories) and a
covership. The slides were viewed in an Olympus IX70 fluorescence
microscope using appropriate filters (Olympus Optical, Melville, NY).
Images were compiled from sets of three consecutive single optical
sections using SPOT INSIGHT V 3.2 software (Diagnostic Instruments,
Sterling Heights, MI).
Macrophage depletion
Macrophage depletion in animals was performed, as described previously (27, 28). Carrageenan (type II; Sigma-Aldrich) was dissolved in sterile PBS at 5 mg/ml. The solution was heated to 56°C to ensure complete solubilization. Mice were treated by i.p. injection of 200 µl (1 mg) of carrageenan on days 3, 7, and 14 after the tumor implantation. Control mice received 200 µl of sterile PBS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain in vitro
We stably transfected head and neck (A253) and prostate cancer
(DU145) cell lines with IL-13R2 chain. Expression level was
confirmed by RT-PCR and radiolabeled IL-13-binding studies (12, 15). Using cloned tumor cells, we determined the sensitivity of
IL-13 cytotoxin in vitro and also determined whether cells in the
process of dying as a consequence of exposure to IL-13 cytotoxin can
mediate bystander cytotoxic effect to non-IL-13R2-transfected tumor
cells. Tumor cells transfected with IL-13R2 chain were mixed with
vector-only-transfected (mock control) cells in various ratios, and the
cytotoxic activity of IL-13 cytotoxin was evaluated by protein
synthesis inhibition assays (Fig. 1
A). As the concentration of
IL-13R2 chain-positive cells increased, the cytotoxic effect of
IL-13 cytotoxin increased. The IC50 (IL-13
cytotoxin concentration causing 50% inhibition of protein synthesis)
correlated positively with the level of IL-13R2 expression. Highest
cytotoxicity of IL-13 cytotoxin was observed when 100% cells expressed
IL-13R2 chain.
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2 chain-positive and
IL-13R2 chain-negative cell mixtures in different ratios, incubated
with IL-13 cytotoxin for 5 days, and then counted viable cells by
trypan blue staining (Fig. 1
B). At a concentration of 10
ng/ml, IL-13 cytotoxin was enough to kill >75% of both A253 and DU145
tumor cells when 100% of tumor cells were positive for IL-13R2
chain. Cytotoxicity of both tumor cell lines decreased proportionally
when IL-13R2-transfected cells were mixed with nontransfected tumor
cells. These results indicate that the cytotoxicity of IL-13 cytotoxin
depends on IL-13R2 chain expression and that IL-13R2
chain-positive dying tumor cells do not mediate bystander cytotoxic
effect to nontransduced tumor cells.
Antitumor effect of IL-13 cytotoxin on head and neck tumors with
heterogeneous expression of IL-13R2 chain in vivo
Because cytotoxic activity of IL-13 cytotoxin in vitro correlated
with IL-13R2 chain expression in tumor cells, we investigated to
what degree of heterogeneity of IL-13R2 expression is required in
tumors to reveal optimal antitumor activity of systemic IL-13 cytotoxin
treatment in animals. For this assessment, A253 tumor cells transfected
with IL-13R2 chain were mixed with vector-only-transfected (mock
control) cells in various ratios, and 5 x
106 cells were injected s.c. in nude mice. Mice
were then i.p. injected with IL-13 cytotoxin (50 µg/kg, twice per day
(b.i.d.) for 5 days) from day 4 through 8. As shown in Fig. 2, in all the animal groups except for
0%, IL-13R2-expressing tumor (only mock control cells
injected)-bearing mice A253 tumor growth was inhibited during IL-13
cytotoxin administration. After the treatment period, tumors gradually
grew again; however, mean size of 25% IL-13R2-expressing tumors (74
mm2) was significantly smaller than 0%
IL-13R2 control tumors (182 mm2)
(p < 0.0005) by day 35. IL-13 cytotoxin showed
antitumor activity against A253 tumors in IL-13R2
expression level-dependent manner, and complete disappearance
of tumor was observed in one of six 75% IL-13R2-expressing
tumor-bearing mice and four of six 100% IL-13R2-expressing
tumor-bearing mice by day 35. These results suggest that the
correlation between IL-13R2 expression and sensitivity to IL-13
cytotoxin is not linear, and that heterogeneous expression of
IL-13R2 chain (at low levels) on tumor cells can still successfully
sensitize them to the cytotoxic effect of IL-13 cytotoxin in
vivo.
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2 chain expression in tumors in vivo after
intratumoral vector administration
To achieve gene expression of IL-13R2 chain in vivo, A253 and
DU145 tumors rapidly growing in the flank of the nude mice were
intratumorally injected with plasmid vector-encoding IL-13R2 chain
cDNA. Plasmids were mixed with liposomes for optimal gene transfer in
vivo and injected on days 4, 5, and 6 after tumor implantation. Tumors
were subsequently resected at various time points after plasmid
injections and subjected to RT-PCR and immunofluorescence microscopy
analyses for IL-13R2 gene expression. IL-13R2 chain mRNA was
found to be expressed continuously at high levels until day 18 in both
tumor models (Fig. 3A). The
level of expression was decreased on day 21 posttumor implantation, and
by day 27 no detectable signal could be observed. These results were
confirmed by immunofluorescence microscopy in A253 tumor sites using
mAb to IL-13R2 chain (Fig. 3B). After three injections of
IL-13R2 cDNA on days 4, 5, and 6, intense protein expression was
observed on day 12, decreasing by day 21, and finally disappearing on
day 27 (data not shown). Tumor slides from several animal experiments
demonstrated that mean IL-13R2 expression level in tumors was
20–30% on day 12 and 5–10% on day 21. These results indicate that
the expression of IL-13R2 in the gene-transferred tumor site was
maintained for 12–15 days after three injections of plasmid
vector.
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2 chain-targeted antitumor activity of IL-13 cytotoxin
Once we were able to express consistent level of IL-13R2 chain
in s.c. growing solid tumors, we tested the antitumor activity of IL-13
cytotoxin in an A253 human head and neck tumor xenograft model. First,
we developed tumors in both the right and left flanks of nude mice.
Then animals received either a vector-only injection (right flank) or
an injection of IL-13R2 chain plasmid (left flank) intratumorally on
days 4, 5, and 6. Subsequently, these animals were treated from days 5
through 9 with IL-13 cytotoxin using either an i.p. (25 or 50 µg/kg
b.i.d. for 5 days) or intratumoral (100 or 250 µg/kg once per day
(q.d.) for 5 days) route of administration (Fig. 4A). Tumors injected with
vector only (right flank) were insensitive to IL-13 cytotoxin treatment
and grew rapidly. By day 36, tumor growth in control
(excipient-injected) and IL-13 cytotoxin-treated animals did not show
any significant difference. In contrast, tumors injected with
IL-13R2 chain plasmid (left flank) started to regress during the
treatment period in both i.p. and intratumoral treated animals.
Although in mice treated i.p. complete regression of tumors could not
be achieved, the mean size of tumors measured on day 36 was
significantly reduced (72 mm2 in 25
µg/kg, and 54 mm2 in 50 µg/kg groups,
respectively) compared with the control (174 mm2)
(p < 0.0005). In contrast, intratumoral
administration of IL-13 cytotoxin resulted in dose-dependent antitumor
activity associated with complete regression of tumors. In the 250
µg/kg dose group of mice, tumors were completely not palpable by day
16. On day 28, tumors recurred in two of the six mice; however, their
size remained significantly smaller (p <
0.0005) than control mice, and four of the six (250 µg/kg) mice
remained completely tumor free in their left flanks out to the end of
the experiment on day 36. Results for a representative mouse from each
group are depicted (Fig. 4B).
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2 chain, followed by IL-13 cytotoxin
treatment using a protocol similar to the one for the A253 tumor model
(Fig. 4C). Again, tumors targeted with vector only (right
flank) did not respond to the IL-13 cytotoxin treatment and grew as
aggressively as tumors implanted in mice treated with excipient. For as
long as we followed these animals (day 50), mean tumor size in control
(excipient only) and treated groups failed to exhibit any difference.
In contrast, tumors modified to express IL-13R2 chain by plasmid
injection (left flank) responded extremely well to the antitumor effect
of IL-13 cytotoxin. After i.p. administration (50 µg/kg b.i.d. x 5
days), the rapid growth of IL-13R2-targeted tumors was arrested in
all mice, and by the last day of the experiment (day 50), mean size of
tumors was significantly smaller compared with control (163
mm2 vs 38 mm2,
p < 0.0005). In addition, one of five test animals
showed complete disappearance of an established tumor. In mice treated
intratumorally, a much better tumor response was observed. Tumors in
three of six (100 µg/kg) and five of six (250 µg/kg) mice
completely regressed by day 12, and had not recurred by day 50.
Overall, the mean tumor size in IL-13 cytotoxin-treated mice remained
significantly smaller (p < 0.0005) compared
with excipient-treated animal throughout the experiment.
IL-13 cytotoxin shows extremely potent antitumor activity to large
tumors targeted with IL-13R2 chain
To further determine whether our combination approach using
IL-13R2 chain gene transfer followed by IL-13 cytotoxin therapy is
effective in much larger tumors, we implanted A253 tumor cells in the
right and left flanks of nude mice, letting them grow until reaching 80
mm2 in either side by day 18. Tumors were then
injected with vector only (right flank) or IL-13R2 chain-encoding
vector (left flank) on days 18, 19, and 20, followed by either i.p. (50
µg/kg b.i.d. x 7 days) or intratumoral (250 µg/kg q.d. x 7 days)
administration of IL-13 cytotoxin (Fig. 5). Regardless of the route of
administration, only IL-13R2-targeted tumors (left flank) regressed
dramatically during the treatment period. In the i.p. injected group,
only IL-13R2-targeted tumors showed significant decrease in size
compared with excipient-injected control (179 mm2
vs 52 mm2, p < 0.0005) on day
36. In animals injected intratumorally with IL-13 cytotoxin, five of
the six showed complete regression of tumors by the end of the IL-13
cytotoxin injection (day 25). Although one tumor recurred later, four
of the six mice remained tumor free in their left flanks in a long-term
(day 90) followup.
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2
plasmid and IL-13 cytotoxin treatment
To investigate the mechanism of antitumor response by approach of
in vivo gene transfer, followed by a targeted cytotoxin therapy, we
examined treated tumors for cellular infiltration. Three days after the
completion of combination therapy, A253 and DU145 tumors were resected
and examined histologically. In IL-13R2-targeted A253 tumors treated
with 100 µg/kg (q.d. x 5 days) of intratumoral IL-13 cytotoxin
administration, several necrotic areas were observed in sections of
dying tumor. These areas were surrounded by fibrotic elements (Fig. 6, A and B).
Interestingly, a number of phagocytes, most resembling monocytes
morphologically, were observed in necrotic areas and in remaining
viable tumor cell area. In contrast, in A253 tumors injected with
vector only, no cell infiltration was observed (data not shown). We
also observed similar phenomenon in IL-13R2-targeted DU145 tumors
after 50 µg/kg (b.i.d. x 5 days) of IL-13 cytotoxin administered
i.p. (data not shown).
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, A and B (A253 tumors). We found moderate numbers
of macrophage marker (F4/80)-positive cells (Fig. 6C), NK
marker (NK1.1)-positive cells (Fig. 6D), and iNOS marker
(M19)-positive cells (Fig. 6E). Although some cancer cells
were positive for iNOS, other infiltrating cells also appeared to be
positive for iNOS. To determine whether some of these iNOS-positive
cells were macrophages, we costained tumor sections with F4/80 (green)
and M19 (red) Abs and examined the sections by fluorescence microscopy.
As shown in Fig. 6F, A253 tumors that received the
IL-13R2 gene followed by IL-13 cytotoxin treatment demonstrated
colocalization of macrophage and iNOS stains. However, staining for
either macrophages or iNOS could not be detected in untreated A253
tumors (data not shown).
In vivo IL-13R2 gene-transferred tumors showed less sensitivity
to IL-13 cytotoxin in macrophage-depleted animals
We observed that some F4/80-positive phagocytes infiltrated within
the dying tumors. To assess whether these infiltrated phagocytes mount
an impact on the tumor regression mechanism during the cancer therapy
approach or were recruited as a result of cell death, we evaluated the
antitumor activity of IL-13 cytotoxin after IL-13R2 gene transfer in
macrophage-depleted nude mice. Mice injected with A253 tumor cells s.c.
(day 0) were then injected with carrageenan (type II) on days 3, 7, and
14. IL-13R2 cDNA injection and IL-13 cytotoxin treatment (50 µg/kg
dose, i.p. or intratumorally) was performed following the schedule
mentioned in Fig. 4. As shown in Fig. 7, during the IL-13 cytotoxin treatment period, tumor regression in both
macrophage-depleted and not-depleted groups were observed. However, the
extent of tumor regression in macrophage-depleted mice was less
pronounced compared with not-depleted mice. By the end of the
experiment (day 38), mean tumor size in macrophage-depleted mice was
significantly larger (87 mm2) compared with mice
without macrophage depleted (61 mm2) in i.p.
IL-13 cytotoxin treatment groups (p < 0.002).
Intratumoral route was superior in inhibiting tumor growth compared
with i.p. route even though doses administered were the same. By day
38, mean tumor size in macrophage-depleted mice was again significantly
larger (68 mm2) compared with mice without
macrophage depletion (29 mm2) in
intratumoral treatment groups (p < 0.0005).
These results confirm that macrophages infiltrating into regressing
tumors as a result of IL-13R2 gene transfer, followed by IL-13
cytotoxin therapy, play at least some role in antitumor mechanism of
this approach.
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2 chain was not detected in any vital organs after
intratumoral plasmid administration
To examine whether intratumorally administrated IL-13R2 plasmid
migrated to distant, nontarget vital organs resulting in IL-13
cytotoxin-mediated organ toxicity, we collected vital organs including
liver, lung, kidney, spleen, heart, and blood as well as A253 tumors on
day 12 (3 days after the completion of IL-13R2 gene transfer and
i.p. or intratumoral IL-13 cytotoxin therapy). All tissue specimens
were snap frozen. Total RNA and tissue sections were analyzed for
IL-13R2 expression or any evidence of cellular damage. IL-13R2
chain mRNA was not detected by sensitive RT-PCR in any vital organs or
blood cells, except in IL-13R2 plasmid-injected tumor tissues (Fig. 8A). Similarly, coupled with
immunofluorescence using mAb to IL-13R2 chain, no detectable protein
expression was observed in any organs examined. Using this approach,
IL-13R2 chain protein was only detected in A253 tumors that were
administrated with IL-13R2 plasmid (Fig. 8B). In
addition, we did not observe any organ toxicity by histological
examination (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 chain followed by
IL-13R-targeted cytotoxin administration represents promising new
approach for locoregional cancer therapy. In our preliminary studies,
we failed to show desirable and pronounced antitumor activity of IL-13
cytotoxin after one injection of IL-13R2-encoding plasmid.
Consequently, we decided to perform three injections: three
intratumoral injections of IL-13R2 chain-encoding vector mixed with
liposome were sufficient for target expression lasting for 12–15 days.
These observations suggest that one can delay IL-13 cytotoxin
administration for a longer period of time, allowing maximum expression
of transgene. Furthermore, multiple injections of IL-13 cytotoxin could
be performed for optimal antitumor response. The lack of IL-13R2
expression or toxicity in any nontarget vital organs suggests IL-13
cytotoxin may eliminate IL-13R2-expressing tumors exclusively. To
our knowledge, this is the first report involving tumor cells enforced
to express an artificial receptor target, followed by therapy comprised
of a unique receptor-directed recombinant antitumor agent.
Based on published reports, we assumed that plasmid-mediated gene
transfer in vivo may not result in the transgene transduction of every
tumor cell. Because the cytotoxic effect of IL-13 cytotoxin is specific
to IL-13R2 chain expression in tumor cells transfected in vitro, we
hypothesized that this combination therapy may not cause a bystander
effect or other apoptotic pathway-mediated tumor cell death in vivo
(29, 30, 31). It seemed that this approach in animal models of
human cancer would not result in a desired, robust antitumor effect.
However, to our surprise, we found that tumors manipulated to express
IL-13R2 chain gene responded to IL-13 cytotoxin therapy. Moreover,
complete regression of tumors was achieved in many animals. By
histological examination, we found that these successful results were
obtained because of cellular infiltration of phagocytes and NK cells,
in addition to a direct IL-13 cytotoxin effect. Some of these
infiltrating phagocytes secreted iNOS. However, whether iNOS-positive
macrophages participated in IL-13 cytotoxin-induced tumor regression or
these cells were infiltrating as a result of tumor necrosis induced by
IL-13 cytotoxin was not clear. To address this important issue, we
performed macrophage depletion experiment. Mice injected with A253
tumor cells s.c. were injected with carrageenan (type II) on days 3, 7,
and 14. IL-13R2 cDNA was then intratumorally injected, followed by
IL-13 cytotoxin treatment by two routes. IL-13 cytotoxin by both i.p.
and intratumoral routes caused lower antitumor activity when
macrophages were depleted before treatment compared with undepleted
groups. The difference in mean tumor size between control and
macrophage-depleted animals was statistically significant by both
routes of IL-13 cytotoxin administration. These results confirmed a
role for phagocytes in the tumor regression observed; however, other
mechanisms must also be involved. Thus, combination of IL-13R2 chain
gene transfer and IL-13 cytotoxin treatment seems to enhance host
immune response at the tumor site, thereby eliminating residual tumor
cells that escaped the cytotoxic effect of IL-13 cytotoxin. In
addition, although nude mice do not have a functionally competent
immune system, they have B cells, monocytes, and NK cells that may be
sufficient to eliminate remaining tumor cells following IL-13R2
chain gene injection and IL-13 cytotoxin therapy. Further delineation
of the role of the immune response in the mechanism of tumor regression
is under investigation in our laboratory.
We also considered a possibility that other mechanism(s) may be
involved in the pronounced antitumor effect of IL-13 cytotoxin in less
than 100% IL-13R2-positive tumors. It is possible that IL-13R2
transfection of host endothelial cells or fibroblasts results in an
antiangiogenesis/antistromal effect, which by itself may be basis of
tumor rejection. To address these issues, we performed a mixing
experiment, in which in vitro IL-13R2 stable transfected A253 tumor
cells were mixed in various ratios with mock vector-transfected control
tumor cells (Fig. 2). These mixtures of cells were s.c. injected into
nude mice, and then mice were treated with IL-13 cytotoxin by two
routes of injection. Tumor-expressing 0% IL-13R2 chain continued to
grow, forming large nodules; however, tumors expressing various
percentages of IL-13R2 chain showed statistically significant
antitumor response as a result of IL-13 cytotoxin treatment. These
results suggest that tumor rejection proceeds when host nontumor cells,
e.g., endothelial cells or fibroblasts, are not directly exposed to
intralesional plasmid, and that the antitumor effect is not mediated
through antiangiogenesis or antistromal effect of plasmid
injection.
IL-13R2 chain has been shown to be highly expressed on cell lines
derived from certain types of solid tumors, including malignant glioma
and primary cell cultures derived from glioma tumors. The extent of
receptor expression in vivo remains unknown (8, 10, 11, 32, 33). In addition, localized neoplasias such as pancreatic, head
and neck, ovarian, and prostate cancer do not express IL-13R2 chain,
or only limited percentage of tumor cells express IL-13R2 chain
(12, 15, 16, 21, 26, 34, 35). Therefore, these tumors will
not be sensitive to the cytotoxic effect of IL-13 cytotoxin. Moreover,
in some situations, IL-13 cytotoxin may not be able to home to tumor
target in sufficient concentrations to mediate an antitumor effect even
if these tumors express sufficient numbers of IL-13Rs. In these
situations, direct gene transfer of IL-13R2 chain may sensitize
these tumor cells to the antitumor effect of IL-13 cytotoxin at low
doses. Our current results support these hypotheses and demonstrate a
potent antitumor effect of IL-13 cytotoxin in two tumor models.
In conclusion, we demonstrate that combined forced expression of
IL-13R2 chain in tumor cells, followed by target-direct cytotoxin
therapy yields excellent antitumor activity enlisting the aid of the
host’s immune system. Because this novel combinational approach
elicits complete regression of extremely large tumors in animal models
of human head and neck, and prostate cancer without any induction of
vital organ toxicity, we believe this strategy could be applied in the
clinic against many types of cancer.
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Acknowledgments |
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Footnotes |
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2 K.K. and M.K. contributed equally to this paper.
3 Address correspondence and reprint requests to Dr. Raj K. Puri, Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, National Institutes of Health Building 29B, Room 2NN10, 29 Lincoln Drive MSC 4555, Bethesda, MD 20892. E-mail address: puri{at}cber.fda.gov
4 Abbreviations used in this paper: iNOS, inducible NO synthase; b.i.d., twice per day; q.d., once per day.
Received for publication December 11, 2001. Accepted for publication October 16, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain: a novel tumor associated transmembrane protein in primary explants of human malignant gliomas. Cancer Res. 60:1168.2 chain dramatically enhances the antitumor effect of IL-13 receptor-targeted cytotoxin in human prostate cancer xenografts. Cancer Gene Ther. 8:861.[Medline]
chain. J. Biol. Chem. 271:16921.2: molecular cloning, characterization, and comparison with murine IL-13 receptor 1. J. Immunol. 161:2317.2 chain: an essential component for binding and internalization but not for IL-13 induced signal transduction through the STAT6 pathway. Blood 97:2673.2 chain. J. Biol. Chem. 276:25114. chain. Hum. Gene Ther. 11:1829.[Medline]
2 chain inhibits tumorigenicity of human breast and pancreatic tumors in immunodeficient mice. J. Exp. Med. 194:1743.This article has been cited by other articles:
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); IP, 25 (
) or 50
µg/kg (
); and IT, 100 (
) or 250 µg/kg (
).
B, Pictures shown are of A253 tumor-bearing mice on day
36.
