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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
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
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
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
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
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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