Optimal Scheduling of Interleukin 12 and Chemotherapy...
Transcript of Optimal Scheduling of Interleukin 12 and Chemotherapy...
Vol. 3, 1661-1667, September 1997 Clinical Cancer Research 1661
Optimal Scheduling of Interleukin 12 and Chemotherapy in the
Murine MB-49 Bladder Carcinoma and B16 Melanoma1
Beverly A. Teicher,2 Guishan Ara, David Buxton,
John Leonard, and Robert G. Schaub
Dana-Farber Cancer Institute and Joint Center for Radiation Therapy,Boston, MA 021 15 [B. A. T., G. A., D. B.]; and Genetics Institute,
Inc., Andover, MA 01810 [J. L., R. G. S.]
ABSTRACT
The antitumor activity of interleukin (IL)-!2, a nat-
urally occurring cytokine, has been demonstrated in
several murine solid tumors. Animals bearing established
B16 melanoma or MB-49 bladder carcinoma were used to
study the most effective scheduling of recombinant mu-
rine IL-!2 (rmIL-!2), along with systemic chemotherapy.
rmIL-!2 (0.45, 4.5, or 45 rig/kg) was more effective as a
single agent when administered to mice bearing the
MB-49 bladder carcinoma at the highest dose for ! ! doses
rather than for 5 doses. In combination with chemother-
apy (Adriamycin, cyclophosphamide, or 5-fluorouracil),
rmIL-12 administration did not increase the toxicity of
the chemotherapy, and there was increased antitumor
activity with each rmIL-12-drug combination. Adminis-
tering rmIL-!2 (45 pig/kg) on days 4-14, along with Ad-
riamycin, cyclophosphamide, or 5-fluorouracil on days
7-! !, resulted in 2.2-2.7-fold increases in tumor growth
delay, compared with the chemotherapy alone against the
primary tumor, and a marked decrease in the number of
lung metastases on day 20. Because the B!6 melanoma
grows more slowly than the MB-49 bladder carcinoma,
allowing multiple courses of chemotherapy, cyclophosph-
amide could be administered. The rmIL-!2 (45 �.agIkg)-
cyclophosphamide combination regimen that was most
effective overlapped 2 days with the terminal portion of
the chemotherapy treatment. There was a parallel in-
crease in the response of the primary tumor and meta-
static disease to the lungs. Administration of rmIL-!2 to
animals bearing the MB-49 bladder carcinoma or the B!6
melanoma was compatible with coadministration of
chemotherapy at full dose without additional toxicity.
Received 2/28/97; revised 5/27/97; accepted 5/30/97.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
� This work was supported by a grant from Genetics Institute, Inc.
(Cambridge, MA).2 To whom requests for reprints should be addressed, at Dana-Farber
Cancer Institute, 44 Binney Street, Boston, MA 021 15. Phone:
(617) 632-3122; Fax: (617)632-2411.
INTRODUCTION
IL-123 is a naturally occurring cytokine that serves as a link
between the innate and the cognate cellular immune systems
( 1-4). IL- 12 has the ability to act as a NK-cehl and a T-cell
growth factor (5-7) to enhance NK-Ilymphokine-activated kill-
er-cell cytolytic activity (7-9), to augment cytolytic T-cell re-
sponses (8) and to induce secretion of cytokines, particularly
IFN--y from T and NK cells (10).
IL- 1 2 has been shown to induce tumor regression and
rejection in a variety of murine tumor models when adminis-
tered as a single agent (1 1-15). This tumor regression results
from activation of immune mechanisms that involve IFN--y,
CD4�, and CD8� cells (12, 13). IL-12 has also been described
as an antiangiogenic agent through the induction of IFN--y (16).
Both T and NK cells have been implicated as antitumor
effector cells ( 17), and IFN--y has been shown to have antitumor
activity in animals ( 1 8, 19). IL- 12 has the potential to be used as
an immunomodulatory cytokine in the therapy of malignancies
(18, 20, 21), as well as in gene therapy (22, 23). Brunda et a!.
(12) have shown that systemic administration of murine IL-12
can slow and, in some cases, inhibit the growth of both estab-
hished s.c. tumors in mice and experimental pulmonary or he-
patic metastases of B16F1O murine melanoma, M5076 reticu-
hum cell sarcoma, or RenCa renal cell adenocarcinoma and that
local peritumoral injections of IL- 1 2 can result in regression of
established s.c. tumors. On the basis of results obtained using
mice deficient in lymphocyte subsets and antibody depletion
experiments, Brunda and colleagues (12, 24) concluded that the
antitumor efficacy of IL-12 is mediated primarily through
CD8� T cells.
Most anticancer therapeutic regimens involve systemic
treatment with chemotherapy and/or local treatment with radi-
ation therapy. In previous studies, the treatment of animals
bearing Lewis lung carcinoma with IL-12 in addition to
fractionated radiation therapy was markedly dose modifying,
indicating that IL- 1 2 was acting synergistically with radiation
(25). The current study was undertaken to understand the most
effective scheduling of IL-12 administration with systemic
chemotherapy in two murine tumors known to be metastatic and
responsive to IL-12.
MATERIALS AND METHODS
Drugs
rmIL-12 was supplied by Genetics Institute (Cambridge,
MA). Cyclophosphamide was purchased from Sigma Chemical
Co. (St. Louis, MO). Adriamycin and 5-fluorouracil were pur-
chased from the Dana-Farber Cancer Institute pharmacy.
3 The abbreviations used are: IL, interleukin; NK, natural killer; rmIL-12, recombinant murine IL-12; M-CSF, macrophage colony-stimulating
factor.
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1662 Scheduling IL-12 with Chemotherapy
Tumor
The MB-49 bladder carcinoma and B16 melanoma, grow-
ing in CS7BL mice, were chosen for tumor growth delay studies
because these tumors are relatively resistant to many cancer
therapies and are highly metastatic to the lungs from s.c. im-
plants. The B16 melanoma was carried in male C57BL mice
(Taconic Farms, Germantown, NY). For experiments, I X 106
tumors cells prepared from a brei of several stock tumors were
implanted s.c. into the legs of male mice, ages 8-10 weeks. The
MB-49 bladder carcinoma was carried in male C57BL mice
(Taconic Farms). For experiments, 2 X 106 MB-49 bladder
tumors cells prepared from a brei of several stock tumors were
implanted s.c. into the legs of male mice, ages 8-10 weeks.
Tumor Growth Delay Experiments
Experiment !. Animals bearing MB-49 bladder carci-
noma were treated with rmIL-12 (0.45, 4.5, or 45 p.g/kg),
administered i.p. daily on days 4-8, days 4-14, days 10-14, or
days 7-1 1 and 14-18 post-tumor cell implantation, alone or in
combination with anticancer chemotherapy. The doses of
rmIL- 12 were chosen to span the range from a low effective
dose to a maximum tolerated dose to assess potential toxicities
from the combination regimens. The four schedules of rmIL- 12
were designed to assess the efficacy of the combination regi-
mens when rmIL- 12 was administered: prior to chemotherapy
(days 4-8); throughout the main growth period of the tumor that
is prior to, during, and after chemotherapy (days 4-14); after
chemotherapy (days 10-14); and simultaneously with chemo-
therapy and after chemotherapy on a 2-week, Monday-through-
Friday schedule (days 7-1 1 and 14-18).
When the tumors were approximately 100 mm3 in volume,
on day 7 post-tumor cell implantation, chemotherapy was initi-
ated. Animals were treated with Adriamycin (I .25 mg/kg) i.p.
daily on days 7-1 1, cyclophosphamide (100 mg/kg) i.p. on days
7, 9, and I 1, or 5-fluorouracil (30 mg/kg) i.p. daily on days
7-11.
Experiment 2. Animals bearing Bl6 melanoma were
treated with rmIL-12 (4.5 or 45 jig/kg), administered i.p. daily
on days 10-14, days 14-18, days 10-14 and 18-22, days
14-18 and 21-25, or days 14-18 and 28-32 post-tumor cell
implantation, alone or along with cyclophosphamide. The doses
of rm!L-12 were chosen to be effective, with the high dose being
the maximally tolerated dose. The schedules for rmIL- 1 2 were
designed to assess potential regimens integrating treatment with
rmIL-l2 and chemotherapy, such that rmIL-12 was adminis-
tered: overlapping and after the chemotherapy (days 10-14); the
week following the chemotherapy (days 14-1 8); alternating
with the chemotherapy (days 10-14 and 18-22); for 2 weeks
after the chemotherapy (days 14-18 and 21-25); or for 3 weeks
after the chemotherapy (days 14-18, 21-25, and 28-32).
When the tumors were approximately 100 mm3 in volume,
on day 7 post-tumor cell implantation, cytotoxic chemotherapy
was initiated. Cyclophosphamide (125 mg/kg) was administered
i.p. on days 7, 9, and 11 or on days 7, 9, 11, 28, 30, and 32, or
cyclophosphamide (62 mg/kg) was administered i.p. on days 7,
9, 11, 15, 17, and 19.
The progress of each tumor was measured thrice weekly
until it reached a volume of 500 mm3. Tumor growth delay was
calculated as the days taken by each individual tumor to reach
500 mm3 compared with the untreated controls. Each treatment
group had six animals, and each experiment was repeated three
times (n = 18). Days of tumor growth delay are the mean ± SE
for the treatment group compared to the control group. Tumor
growth delay is the difference in days for treated versus control
tumors to reach 500 mm3 (25). The time after s.c. tumor cell
implantation for control tumors to reach 500 mm3 were as
follows: for MB-49 bladder carcinoma, 14. 1 ± 1 . 1 days; and for
B16 melanoma, 17.6 ± 1.0 days.
Lung metastases were examined on day 30 in B 16 mela-
noma-bearing animals and on day 20 in MB-49 bladder carci-
noma-bearing animals. Untreated control animals died from
lung metastases on days 32-35 with B 16 melanoma and on days
21-25 with MB-49 bladder carcinoma. The numbers of external
lung metastases were counted from two animals per group and
scored as �3 mm or <3 mm in diameter. Metastases that were
�3 mm in diameter were counted as large (vascularized;
Ref. 25).
RESULTS
rmIL-l2 was found to be an active antitumor agent in the
MB-49 bladder carcinoma. The antitumor activity was depend-
ent upon rmIL- 1 2 dose, the duration of treatment, and the tumor
burden at the initiation of treatment (Table 1 and Fig. 1). When
rmIL-12 treatment was initiated on day 4 (tumor volume, ap-
proximately 30 mm3), there was no statistically significant dif-
ference between the tumor growth delay produced by rmIL-12
(at 0.45 or 4.5 p.g/kg) on 5- and 1 1-dose regimens. However,
when rmIL-12 was administered to the animals at 45 p.glkg, the
tumor response was significantly greater with the 1 1-dose reg-
imen than with the 5-dose regimen. Delaying rmIL-12 treatment
until day 10, when the tumors were approximately 200 mm3 in
volume, resulted in decreased tumor growth delay, so that only
the highest dose of rmIL-12 (45 jig/kg) produced a significant
tumor response. The number of lung metastases in these animals
on day 20 was significantly decreased only at the highest dose
of rmIL-12 (45 p.glkg), and the percent of large (vascularized)
lung metastases was not different from that seen in the controls.
In the design of treatment regimens including systemic
administration of rmIL- 12 and chemotherapy, two major issues
were: possible damage of the rmIL-12-targeted T cells by the
chemotherapy, resulting in ablation of the rmIL-12 effect; and
increased toxicity of the combination therapy. Therefore,
rmIL-12 was studied over a dosage range, consisting of 0.45,
4.5, and 45 �i.gIkg, with the chemotherapy and on schedules
prior to, after, and overlapping with the chemotherapy. Each of
the chemotherapeutic agents studied, Adriamycin, cyclophosph-
amide, and 5-fluorouracil, were active antitumor agents against
the MB-49 bladder carcinoma (Table 2). rmIL-12 treatment did
not increase the toxicity of the chemotherapy. There was in-
creased anticancer activity when rmIL- I 2 administration was
added to treatment with each chemotherapeutic agent. The in-
creased tumor response was dependent on rmIL- 12 dose and
schedule, with overlapping therapy producing the greatest effect
(Table 2). Adriamycin (1.75 mg/kg) on days 7-1 1 produced
10.8 days of tumor growth delay. However, the greatest tumor
growth delay was obtained with extended rmTL-l2 treatment, on
days 4-14, combined with Adriamycin treatment, which re-
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�0
4-JLu0
Fig. 1 Growth delay of the murine MB-49 bladder carcinoma aftertreatment of the tumor-bearing animals with IL-l2 (0.45, 4.5, or 45
iig/kg) by daily i.p. injection, on days 4-8 (#{149}),on days 4-14 (0), or ondays 10-14 (). Data points, means of three experiments; bars, SE.a Tumor growth delay is the difference between the number of days
for treated tumors to reach 500 mm3 and the number for untreatedcontrol tumors to reach that size. Untreated control tumors reach 500mm3 in about 14 days. Mean ± SE of 18 animals.
0.1 1.0 11 100
rmIL-12 TOTAL DOSE, ug
Clinical Cancer Research 1663
Table 1 Tumor growth delay and number and size of lungmetastases in animals bearing the MB-49 bladder carcinoma treated
with rmIL-12 on different schedules
Cumulative Tumor growth No. of lungdose delay” metastases
Treatment group (p.g) (days) (% large)
Control 22 (44)Daily, days 4-8 post-tumor
implantation
rmIL-12 (45 p.g/kg) 5 7.3 ± 1.2 12 (38)
rmIL-12 (4.5 p.g/kg) 0.5 5.9 ± 0.9 17 (35)rmIL-l2 (0.45 �Lg/kg) 0.05 3.8 ± 0.6 18.5 (46)
Daily, days 4-14 post-tumorimplantation
rmIL-12 (45 p.g/kg) I I 10.9 ± 1.5 8 (42)
rmIL-l2 (4.5 p.g/kg) 1.1 5.7 ± 0.9 14 (35)rmIL-12 (0.45 p.g/kg) 0.1 1 4.3 ± 0.6 18 (42)
Daily,days7-ll and 14-18post-tumor implantation
rmlL-12 (4.5 �i.g/kg) 1 4.5 ± 0.7
Daily, days 10-14 post-tumorimplantation
rmIL-12 (45 rig/kg) S 3.2 ± 0.6 9 (43)rmIL-l2 (4.5 p.g/kg) 0.5 1.6 ± 0.3 14 (45)rmIL-l2 (0.45 p.g/kg) 0.05 1.5 ± 0.3 16 (47)
sulted in 23.4 days of tumor growth delay. Cyclophosphamide
(100 mg/kg), administered on days 7, 9, and 1 1, produced 8.0
days of tumor growth delay. When rmIL-12 was administered
along with cyclophosphamide on the longer schedule, days
4-14, a tumor growth delay of 21.7 days was produced. 5-Flu-
orouracil (30 mg/kg), administered on days 7-1 1, produced 6.2
days of tumor growth delay. rmIL-l2 treatment, extended to
days 4-14, along with 5-fluorouracil treatment resulted in 16.5
days of tumor growth delay. Shorter-duration treatment with
rmlL- 12 before or after chemotherapy regimens was less effec-
tive in all treatment groups (Table 2). Interestingly, administer-
ing rmIL-12 (45 jig/kg) simultaneously with the chemotherapy
and then again on days 14-1 8 was not a very effective combi-
nation therapy (Table 2).
Including rmIL-l2 in the therapeutic regimen markedly
increased its efficacy against metastatic disease (Table 2). The
highest dose of rmIL-12, 45 �i.gfkg, was most effective against
metastasis to the lungs. Unlike tumor growth delay with
rmIL-12 alone, the combinations with Adriamycin, cyclophos-
phamide, or 5-fluorouracil had similar efficacies at all schedules
evaluated. In combination with Adriamycin or 5-fluorouracil,
there was little impact of rmIL-l2 administration on the percent
of lung metastases that were �3 mm in diameter, indicating that
these treatments were not altering the growth pattern of the
metastases. However, rmIL-12 in combination with cyclophos-
phamide did decrease the percent of large lung metastases,
indicating that the growth rate of the metastases was slowed.
The B16 melanoma is a highly metastatic murine solid
tumor that grows more slowly than the MB-49 bladder carci-
noma, thus providing a convenient model in which to address
the question of cycling rmIL-l2 administration with cytotoxic
therapy. Several schedules of rmIL-12 and cyclophosphamide
were tested in which rmIL-12 administration was initiated after
cyclophosphamide therapy in a manner that overlapped the
terminal portion of the chemotherapy regimen or that started 2
days after the completion of the chemotherapy regimen and
extended for 1, 2, or 3 weeks (Table 3). Cyclophosphamide (125
mg/kg) was administered for one course (days 7, 9, and 1 1) or
for two courses (days 7, 9, 1 1, 28, 30, and 32). Both rmIL-12
and cyclophosphamide were active antitumor agents against the
B16 melanoma. The tumor growth delay produced by rmIL-l2
was dependent on the dose and duration of treatment. Admin-
istration of 45 p.g/kg of rmIL-!2 was more effective than
administration of 4.5 p.g/kg of rmIL-12. Administration of
rmIL- 12 for 2 weeks was more effective than administration of
rmIL-l2 for 1 week. However, administration of rmIL-12 for 3
weeks did not increase the tumor response further. Greater
tumor growth delay resulted when the 5-day rmIL-12 regimen
was administered simultaneously with the terminal portion of
the chemotherapy treatment than if a 2-day break was allowed
from completion of the cyclophosphamide treatment to initia-
tion of the rmIL-l2 administration (Fig. 2). Extending the
rmlL-l2 administration to 2 weeks (10 injections) resulted in a
highly effective therapeutic regimen, with a tumor growth delay
of about 31 days. Adding a 3rd week of rmIL-l2 administration
to that regimen increased the tumor growth delay by only 2.4
days. When the dose of rmIL-l2 was decreased to 4.5 �i.g/kg, the
tumor growth delays observed with the combination regimens
were decreased to 23 or 24 days, which was significantly greater
than the delay with cyclophosphamide alone. Administration of
two courses of cyclophosphamide on days 7, 9, and 1 1 and again
on days 28, 30, and 32 produced a tumor growth delay of about
28.5 days. When rmIL-l2 (45 igfkg) was administered between
and after completion of the cyclophosphamide courses, a tumor
growth delay of 40 days resulted, which was greater than cx-
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1664 Scheduling IL-12 with Chemotherapy
Table 2 Tumor growth delay and number and size of lung metastases in animals bearing the MB-49 bladder carcinoma treated with rmIL-l2and anticancer agents
Treatment group Tumor growth delay” (days) No. of lung metastases” (% large)
Control 22 (44)Adriamycin (1.25 mg/kg), days 7-I 1
Alone 10.8 ± 1.2 18 (42)
+ rmIL-l2 (45 p.glkg), days 4-8 17.1 ± 2.0 7.5 (33)
+ rmIL-12 (4.5 p.g/kg), days 4-8 15.0 ± 1.7 15 (37)+ rmIL-l2 (0.45 p.g/ltg), days 4-8 14.6 ± 1.6 17 (38)+ rmIL-l2 (45 p.glkg), days 4-14 23.4 ± 3.7 8 (38)+ rmlL-l2 (4.5 rig/kg), days 4-14 15.1 ± 1.8 1 1.5 (38)+ rmIL-l2 (0.45 p.g/kg), days 4-14 13.8 ± 1.7 12 (38)+ rmIL-12 (45 p.g/kg), days 7-1 1, and 14-18 12.8 ± 1.7
+ rmIL-l2 (45 p.g/kg), days 10-14 14.8 ± 1.4 5 (42)
+ rmIL-l2 (4.5 lLgIkg), days 10-14 13.6 ± 1.4 12 (46)+ rmIL-12 (0.45 p.g/kg), days 10-14 13.0 ± 1.3 14 (41)
Cyclophosphamide (100 mg/kg), days 7, 9, and 11Alone 8.0 ± 0.8 12 (38)+ rmIL-l2 (45 p.g/kg), days 4-8 17.0 ± 1.7 3 (17)+ rmIL-12 (4.5 p.gfkg), days 4-8 15.6 ± 1.6 4 (38)+ rmIL-l2 (0.45 p.g/kg), days 4-8 15.0 ± 1.3 3.5 (28)+ rmIL-12 (45 p.g/kg), days 4-14 21.7 ± 3.3 4 (21)
+ rmIL-l2 (4.5 p.g/kg), days 4-14 16.5 ± 1.8 5 (23)+ rmIL-12 (0.45 sag/kg), days 4-14 14.3 ± 1.5 6 (28)+ rmIL-12 (45 �LgIkg), days 7-1 1, and 14-18 12.8 ± 1.6+ rmIL-12 (45 �Lg/kg), days 10-14 17.6 ± 1.9 1.5 (50)+ rmIL-l2 (4.5 jig/kg), days 10-14 14.6 ± 1.7 5 (33)
+ rmIL-12 (0.45 p�g/kg), days 10-14 12.7 ± 1.2 8 (27)5-Fluorouracil (30 mg/kg), days 7-11
Alone 6.2 ± 0.7 17 (42)
+ rmIL-12 (45 p.g/kg), days 4-8 12.3 ± 1.1 10 (25)+ rmIL-12 (4.5 �agIkg), days 4-8 1 1.9 ± 1.0 1 1 .5 (39)+ rmIL-12 (0.45 l.Lg/kg), days 4-8 10.2 ± 0.9 16 (38)+ rmIL-l2 (45 p.g/kg), days 4-14 16.5 ± 1.8 10 (27)+ rmIL-12 (4.5 �LgIkg), days 4-14 1 1.0 ± 1.0 13.5 (35)+ rmIL-12 (0.45 p.glkg), days 4-14 8.5 ± 0.7 19 (39)+ rmIL-12 (45 p.g/kg), days 7-1 1 and 14-18 7.1 ± 0.6+ rmIL-12 (45 p.g/kg), days 10-14 1 1.2 ± 1.1 1 1 (43)+ rmIL-l2 (4.5 p.gfkg), days 10-14 9.2 ± 0.9 21 (44)+ rmIL-12 (0.45 p.g/kg), days 10-14 9.1 ± 0.9 22 (41)
a Tumor growth delay is the difference between the number of days for treated tumors to reach 500 mm3 and the number of days for untreatedcontrol tumors to reach the same size. Untreated control tumors reach 500 mm� in about 14 days. Mean ± SE of 18 animals.
b The number of external lung metastases on day 20 post-tumor implant was counted manually and scored as �3 or <3 mm in diameter. Data
are the means from 6-12 pairs of lungs. Numbers in parentheses, number of large (vascularized) metastases (� 3 mm in diameter).
pected for the additivity of the two therapies. When the same
treatment regimen was carried out with the lower dose of
rmIL-12 (4.5 p.g/kg), the tumor growth delay observed was
about 28 days. To explore the effect of cyclophosphamide dose
and schedule, a total dose of 375 mg/kg of cyclophosphamide,
administered as three injections of 125 mg/kg alone, was di-
vided into six injections of 62 mg/kg administered over two
courses (Table 3). Decreasing the dose intensity of the cyclo-
phosphamide resulted in a decrease in the tumor growth delay
from 16.8 days for 125 mg/kg (three doses) cyclophosphamide
to 6.8 days for 62 mg/kg (six doses) cyclophosphamide. Ad-
ministering the rmIL-12 (4.5 or 45 p.g/kg) between and after the
chemotherapy treatment resulted in the additivity of the two
therapies.
DISCUSSION
Curative anticancer regimens will likely include several
treatment modalities. The current studies were conducted be-
cause the optimal scheduling of a cytokine such as IL- 12 with
cytotoxic chemotherapy was unclear. The results of these stud-
ies indicate that daily prolonged treatment with IL-!2 at a high
dose through the cytotoxic chemotherapy or overlapping with
the cytotoxic therapy and extending past the chemotherapy
resulted in the best therapeutic regimens in both the MB-49
bladder carcinoma and the B16 melanoma. Daily administration
of IL-l2 was more effective than administration on alternate
days or once weekly administration, leading to the same total
dose (26). In both the MB49 bladder carcinoma and the B 16
melanoma, optimal scheduling of rmIL-12 and chemotherapy
resulted in additive to greater-than-additive tumor growth delays
for the two therapies. Recently, Brunda et a!. (1 1) reported that
IL-!2 (45 �igIkg), administered by i.p. injection on days 14-18,
21-25, 28-32, 35-39, 42, and 43, along with Adriamycin (5
mg/kg), administered once per week, was a more effective
therapy than either treatment administered alone. One drawback
of this study was that the Adriamycin regimen alone had no
antitumor activity in this tumor. The same IL-12 regimen was
combined with etoposide (10 mg/kg) administered once per
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Treatment groupTumor growthdelay” (days)
4.6 ± 0.45.1 ± 0.46.4 ± 0.5
6.3 ± 0.5
6.8 ± 0.5
8 (21)
10 (23)3 (25)
10 (23)8 (44)
Clinical Cancer Research 1665
Table 3 Tumor growth delay of the B 16 melanoma and number and size of lung metastases on day 30 produced by treatment with IL- I 2 and/or
cyclophosphamide
No. of lung metastases”
- - (% large)
Control 21 (49)L-12 (45 p4/kg), i.p.
Days 10-14
Days 14-18
Days 10-14 and 18-22
Days 14-18 and 2 1-25
Days 14-18, 21-25, and 28-32L-12 (4.5 p.g/kg), i.p.
Days 10-14 2.1 ±0.3 11 (24)Days 10-14 and 18-22 3.2 ± 0.3 16 (27)
CTXC (125 mg/kg), i.p.
Days7,9, 11 16.8± 1.4 3.5(31)Days 7, 9, 1 1; 28, 30, 32 28.5 ± 2. 1 3.5 (29)
CTX + IL- 12 (45 p.g/kg)CTX, days 7, 9, and 1 1; + IL-12, days 10-14 25.8 ± 2.7 2.5 (27)CTX,days7,9,and 11; + IL-12,days 14-18 19.0± 1.6 3.5(43)CTX, days 7, 9, and 11; + IL-12, days 14-18 and 21-25 30.9 ± 2.5 2 (0)CTX, days 7, 9, and 11; + IL-12, days 14-18, 21-25, and 28-32 33.4 ± 2.1 1 (0)
CTX, days 7, 9, and 11; + IL-12, days 14-18 and 21-25; + 40.0 ± 2.2 1 (0)CTX, days 28, 30, and 32; + IL-12, days 35-39
CTX + IL-l2 (4.5 p.g/kg)CTX, days 7, 9, and 11; + IL-l2, days 14-18 and 21-25 23.2 ± 1.3 6 (17)
CTX, days 7, 9, and 1 1; + IL-l2, days 14-18, 21-25, and 28-32 23.7 ± 1.7 2.5 (0)
CTX, days 7, 9, and 11; + IL-12, days 14-18 and 21-25; + 28.2 ± 1.9 2 (25)CTX days 28, 30, and 32; + IL-12, days 35-39
CTX (62 mg/kg) i.p. Days 7, 9, 1 1, 15, 17, and 19 6.8 ± 0.5 6 (50)
CTX, days 7, 9, and 1 1; + IL-12 (45 p.g/kg) days 10-14; + 1 1.6 ± 1.0 0.5 (8)CTX, days 15, 17, and 19; + IL-l2 (45 p.g/kg), days 18-22
CTX, days 7, 9, and 1 1; + IL-12 (4.5 p.g/kg) days 10-14; + 9.1 ± 0.8 4.5 (22)CIX, days 15, 17, and 19; + !L-12 (4.5 pg/kg) days 18-22
a Tumor growth delay is the difference between the number of days for treated tumors to reach 500 mm3 and the number of days for untreatedcontrol tumors to reach the same size. Untreated control tumors reach 500 mm3 in about 14 days. Mean ± SE of 18 animals.
b The number of external lung metastases on day 20 post-tumor implant was counted manually and scored as �3 or <3 mm in diameter. Dataare the means from 6-12 pairs of lungs. Numbers in parentheses, number of large (vascularized) metastases (�3 mm in diameter).
C CTX, cyclophosphamide.
week to animals bearing s.c. Bl6 melanoma, and no benefit of
the chemotherapy was observed. In this tumor system, more
frequent administration of a higher dose of etoposide may have
provided a better result. Several generalizations of combined
modality therapeutic regimens may pertain to optimizing the
scheduling of IL-12 and chemotherapy. First, both the chemo-
therapy and IL-12 should have activity against the tumor. 5cc-
ond, the therapies should be scheduled so that the more cyto-
cidal agent is administered as early as possible in the therapeutic
regimen when the greatest tumor burden is present in an effort
to decrease the bulk of the disease in the host, thus allowing the
IL-!2-induced immunotherapy to attack the residual disease.
Third, both the chemotherapy and IL-]2 should be administered
at maximally tolerated doses because there was a clear dose-
response effect for the IL-12 with the highest dose tested,
resulting in the greatest antitumor activity.
In all of the combination treatment regimens of IL-l2 with
chemotherapy, there was a marked effect on disease metastatic
to the lungs with each of the tumors studied. IL-12 has been
described as an antiangiogenic agent (16). The antiangiogenic
activity of IL-12 appears to be due to the induction of IFN-y by
the cytokine (16). Although the mechanism by which IFN--y
exerts antiangiogenic effects remains unelucidated, several stud-
ies have shown that the IFNs inhibit production of matrix
metalloproteinases (27-30). Gohji et a!. (27) found that incuba-
tion of human KG-2 renal cell carcinoma cells with IFN43 or -�y
suppressed transcription of the Mr 72,000 gelatinase gene and,
hence, production of gelatinase activity. These inhibitory effects
of IFNs were independent of their antiproliferative effects.
Treatment of KG-2 cells with IFN43 or --y significantly inhibited
cell invasion through reconstituted basement membrane toward
chemoattractants produced by kidney fibroblasts. The inhibitory
activity of IFNs was specific to the KG-2 cells because gelatin-
ase activity by various fibroblasts was unaffected. In human
A2058 melanoma cells, Hujanen et a!. (28) found that IFN43
and --y were potent regulators of both Mr 72,000 and Mr 92,000
type-IV collagenase/gelatinase A and B genes, showing biphasic
and parallel effects on mRNA levels of both enzymes, depend-
ing on the treatment time, and that the Mr 72,000 metallopro-
teinase/gelatinase A was the predominant basement membrane-
degrading type-IV collagenase in the A2058 human melanoma
cell line. Norioka et a!. (29) found that IFN--y alone and in
combination with IL-i inhibited the proliferation of human
umbilical vein endothelial cells stimulated with basic fibroblast
growth factor in culture. Local administration of IFN-y induced
basic fibroblast growth factor and stimulated angiogenesis in
Research. on June 4, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Fig. 2 Growth delay of the murine B16 mela-noma after treatment of the tumor-bearing animalswith cyclophosphamide (125 mg/kg) by i.p. injec-
tion, on days 7, 9, and 11 (Cyclophosphamide), orwith cyclophosphamide (125 mg/kg) by i.p. injec-tion on days 7, 9, 11, 28, 30, and 32 (C7X X 2),
alone or along with rmIL-l2 (45 p.g/kg) by i.p.injection on the schedule shown. Columns, meansof three experiments; bars, SE.
non. 10-14 14-18 1418; 14-18; 1418;21-25 21.25; 21-25;
28-32 35-39
SCHEDULE IL-12 (45 ug/kg)ip, DAYS
non.
1666 Scheduling IL-12 with Chemotherapy
Cl)>-40
>-
4-JLu
0
mouse skin. IFN--y, especially in combination with IL-!, down-
regulated expression of basic fibroblast growth factor receptor
on the endothehial cells. On the other hand, Hiscox et a!. (30)
found that IL-l2 directly inhibited the auachment of the human
colon cancer cell lines HRT18, HT29, and HT115 to Matrigel.
IL-12 did not affect the growth of these colon carcinoma cell
lines. Flow cytometry, Western analysis, and immunohisto-
chemistry showed an up-regulation of E-cadherin cell surface
adhesion molecules. These direct effects of IL-12 on colon
cancer cells suggest a potentially important role for IL-12 in
metastasis. Therefore, administration of IL-12 may act as an
antiangiogenic agent, directly and/or indirectly, by preventing
invasion and extravasation of tumor cells through vasculature
and by preventing angiogenic activity in implanted metastatic
tumor cells.
The immune basis of IL-12 activity would suggest that
combination of IL-12 with other therapies that enhance immune
response could potentiate the antitumor activity of IL-12. The
combination of IL-12 with IL-2, a cytokine with a similar
pharmacological profile, was found to be no more effective than
the optimal dose of IL-12 alone (24, 31). It was hypothesized
that this outcome may have resulted from the substantially
increased toxicity associated with IL-!2-IL-2 combination ther-
apy (24); however, pulse IL-2 along with IL-12 was less toxic
and more efficacious (31). The combination of IL-!2 with
M-CSF, a macrophage activator and growth factor, was syner-
gistic, especially with local fractionated radiation therapy (25).
These results concur and extend those of Lu et a!. (32), who
showed that M-CSF is effective in enhancing the response of the
Lewis lung carcinoma to radiation therapy. Macrophages are
present in tumors (33), have a significant role in antigen pres-
entation and lymphocyte activation, and have been identified as
a primary source of endogenous IL-12 (24, 34). They produce a
variety of other inflammatory cytokines, such as tumor necrosis
factor a, IL-h, and IFN-a, �3 as well as oxygen radicals and other
cytostatic and cytolytic factors. M-CSF augments many of these
antitumor functions (35).
Administration of IL-12 to animals bearing the MB-49
bladder carcinoma or the B 16 melanoma was compatible with
coadministration of chemotherapy at full dose, without addi-
tional toxicity and with, in general, additive antitumor activity
of the two therapies. Previous studies have shown IL-12 admin-
istration to be compatible with fractionated radiation therapy
(26), as well as with administration of other cytokines (24-26,
3!). Thus, a clinical trial of IL-!2 as a component of combina-
tion therapy protocols is warranted.
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1997;3:1661-1667. Clin Cancer Res B A Teicher, G Ara, D Buxton, et al. murine MB-49 bladder carcinoma and B16 melanoma.Optimal scheduling of interleukin 12 and chemotherapy in the
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