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-



U,>.U,

�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-




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



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)


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



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.


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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