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Vol. 4, 75-85, January /998 Clinical Cancer Research 75

Pilot Study of Subcutaneous Recombinant Human Interleukin 12 in

Metastatic Melanoma’

Emilio Bajetta,2 Michele Del Vecchio,

Roberta Mortarini, Rosemary Nadeau,Ashok Rakhit, Lorenza Rimassa, Camilla Fowst,

Alessandra Born, Andrea Anichini, and

Giorgio Parmiani

Divisions of Medical Oncology B [E. B., M. D. V., L. R.] and

Experimental Oncology D [R. M., A. B., A. A., G. P.], IstitutoNazionale per lo Studio e la Cura dci Tumori, Milan, Italy, and from

Hoffmann-La Roche, Milan, Italy [C. F.] and Nutley, New Jersey[RN., AR.]

ABSTRACT

The aim of this study was to evaluate the safety profileof s.c. administered recombinant human interleukin 12(rHuIL-12). Pharmacokinetics and pharmacodynamics ofrHuIL-12 and any evidence of antitumor effect were alsoconsidered. Ten pretreated patients with progressive meta-static melanoma were enrolled in this pilot study. Patientsreceived a fixed dose of rHuIL-12 (0.5 pig/kg) for two iden-tical 28-day cycles, with injections given on days 1, 8, and 15

of each cycle. In case of any evidence of response or diseasestabilization, the treatment was continued for two further28-day cycles. Toxicity mainly consisted of a flu-like syn.drome. Transient increases in transaminasemia (6 of 10patients) and triglyceridemia (8 of 10 patients) were oh-served. Peak serum IL-12 levels were reached 8-12 h afterthe first injection in all patients; no serum LL-12 was detect-able in 6 of 9 evaluable patients after the last injection of thesecond cycle. No antibody response to rHuIL-12 could be

detected in any of the patients. A marked, transient reduc-tion in circulating CD8� and CD16� lymphocytes and neu-trophils was observed after the first administration and highlevels of serum IFN-y and IL-lO were detected in all patients

within 24-48 h. Tumor shrinkage, not reaching partial or

complete remission, involved the regression of s.c. nodules (2

of 3 patients), superficial adenopathies (1 of 3 patients), andhepatic metastases (1 of 3 patients); regressions were de-tected after the first cycle of treatment and were maintained

in spite of progression at different sites. s.c. rHuIL-12 treat-

Received 6/4/97; revised 10/15/97; accepted 10/21/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported in part by Hoffmann-La Roche Inc., Nutley,NJ.2 To whom requests for reprints should be addressed, at Division ofMedical Oncology B. Istituto Nazionale per lo Studio e Ia Cura deiTumori, Via Venezian I, 20133 Milan, Italy. Phone: 39-2-2390500;Fax: 39-2-2367219.

ment was well tolerated and had marked effects on immuneparameters and potential antitumor activity.

INTRODUCTION

The incidence of melanoma is rapidly increasing in various

countries ( 1 , 2). When the disease recurs after the surgical

excision of a primary lesion, a proportion of patients can benefit

from adjuvant therapy with IFN-a2b (3), whereas chemotherapy

or radiotherapy have little impact on most advanced metastatic

patients. Dacarbazine [5-(3,3-dimethyl-b-triazeno)-imidazole-4-

carboxamide] is the most effective monotherapy, with an overall

response rate of -�-20%, but durable responses are uncommon

and cure is very rare (4). Available results suggest that no more

than 25% of patients may transiently benefit from current bio-

logical therapies (Refs. 5-7; rHuIFN-a,3 IL-2 with or without

bymphokine-activated killer cells), and although a 34% response

rate has been reported in the case of tumor-infiltrating lympho-

cytes + IL-2, this treatment is highly toxic, costly, and difficult

to use routinely (8).

These results highlight the need for new therapeutic ap-

proaches. There is evidence that human melanoma may be

responsive to immunological therapies aimed at boosting the T

cell-mediated response to tumor-associated antigens. In fact, the

results of various studies indicate that human melanoma cells

express a wide range of genes coding for tumor antigens rec-

ognized by T cells (9). Furthermore, analysis of the T-cell

receptor repertoire in melanoma lesions has revealed selective

expansions of T cell clonotypes with specificity for autologous

tumor (10). These findings support the notion that at least some

human mebanomas may be immunogenic in vivo in the autolo-

gous host, which provides the rationale for attempting new

therapeutic approaches that may have an impact on the activa-

tion, proliferation, and function of tumor-specific T cells and

thus improve the host response to autologous tumors.

IL-I 2 is a relatively new cytokine with a number of bio-

logical functions that justify its potential use as a modulator of

the immune response to neoplastic cells, including the stimula-

tion of proliferation and activation of both NK cells and CTLs

and the promotion of ThI-dependent responses (1 1). Further-

more, experimental models of immunotherapy have indicated

that IL-12 has significant antineoplastic activity based on van-

ous mechanisms. IL-12 treatment in mice induces the infiltration

of neoplastic lesions by NK cells, as well as by CD4 � and

CD8� T lymphocytes (12, 13). Leukocyte subset depletion

studies have revealed that the NK and T cells infiltrating the

3 The abbreviations used are: rHu, recombinant human; IL, interleukin;bFGF, basic fibroblast growth factor; mAb, monoclonal antibody; NK,natural killer; VEGF, vascular endothelial growth factor; TNF, tumor

necrosis factor; GM-CSF, granulocyte-macrophage colony-stimulating

factor; TGF, transforming growth factor.

Research. on July 27, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

Table 1 Patient characteristics

a p5�, performance status.

76 Pilot Study of rHuIL-l2 in Metastatic Melanoma

4 A. Rakhit, personal communication.

lesions are responsible for the antitumor activity and that IL- 12

treatment can induce a long-lasting antitumor immunity (12-15)

that is at beast partially mediated by the promotion of Thl-

dependent responses (16, 17). Furthermore, although the anti-

neoplastic activity of IL-12 is not entirely dependent on IFN-y

( 12), the therapeutic efficacy of IL- 12 is abrogated by systemic

treatment with anti-IFN-’y mAb (18) in some animal models,

thus underlining the relevance of this cytokine in T cell and NK

cell-mediated responses.

In addition to boosting T cell-mediated immunity to tumor

antigens, new therapeutic approaches to melanoma aim at block-

ing tumor-dependent angiogenesis, a process that is affected by

IL- I 2. Human melanomas produce angiogenetic factors such as

VEGF/vascular permeability factor that can increase tumor

growth and metastasis (19), and it has recently been shown that

the inhibition of melanoma angiogenesis beads to the impair-

ment of growth and metastasis formation (20). Tumor-induced

angiogenesis can be inhibited by IL-12 (21, 22). This occurs by

means of an IFN--y-dependent mechanism that is mediated by

the chemokine IP-lO, a potent antiangiogenetic factor (23).

Finally, a further antitumor effect induced by IL-12 (once again

mediated by IFN-�y) is dependent on the synthesis of an induc-

ible type of nitric oxide synthase, which in turn drives the

synthesis of nitric oxide, a potential inhibitor of tumor growth

(24).

On the basis of these experimental results, 10 patients with

advanced melanoma were enrolled in a pilot study aimed at

establishing the safety of a fixed s.c. rHuIL-12 dose of 0.5

p.g/kg. Pharmacokinetics and pharmacodynamics of rHuIL-I2

and any evidence of antitumor effect were also considered.

PATIENTS AND METHODS

Patient Selection. Italian Ministry of Health regulations

allow a maximum of 10 patients to be treated with a drug that

has never been used before. The eligibility criteria were as

follows: measurable and/or evaluable, histologically confirmed,

advanced and/or metastatic melanoma; Karnofsky performance

status �80%; age, 18 -75 years; a life expectancy of at least 4

months; no clinical or computerized tomography scan evidence

of brain metastases; no clinical history of significant cardiovas-

cular diseases or liver or metabolic/endocrine disorders; no

significant extravascular fluid accumulations, such as ascites or

pleural effusion; no systemic bacterial/fungal infections or Thl-

mediated autoimmune diseases (such as rheumatoid arthritis and

systemic bupus erythematosus); prior therapy limited to one

chemotherapy regimen and/or one immunotherapy regimen for

metastatic disease; creatinine, � 1.5 mg/dl; bilirubin, �1.2 mg/

dl; WBC count, �3,000/pJ; platelet count, �75,000/p.l; granu-

locytes, �2,000/p.l; hemoglobin, � 10 g/db; aspartate amin-

otransferase/abanine aminotransferase, �2.5 X normal; alkalin

phosphatase, �2.5 x normal; and normal CO diffusion capac-

ity. Patients were considered ineligible for entry in the presence

of any one of the following criteria: major surgery during the

previous 4 weeks; chemotherapy, immunotherapy, hormonal

treatment, or radiotherapy within the previous 3 weeks; depot

systemic corticosteroid treatment within the previous 6 weeks;

seropositivity for hepatitis B or C virus or HIV; or blood

transfusions or growth factors administered within the previous

No. of patients 10

Median age (range) 50 (26-61)Sex

Male 5

Female 5P.S.” (Eastern Cooperative Oncology Group)

0 8

1 2

Previous treatment

Surgery 10(100%)Chemotherapy 9 (90%)Radiotherapy 3 (30%)Hormonotherapy 3 (30%)Immunotherapy 8 (80%)

Dose0.5 pg/kg 10

0.3 pg/kg INo. of cycles

I 12 94 2

Site of metastatic diseaseSkin or soft tissue 8

Superficial adenopathies 2Abdominopelvic nodes/organs 3

Lung 4

Liver 2

2 weeks. All of the enrolled patients had received only one line

of biological treatment and/or chemotherapy for metastatic dis-

ease (Table 1). The treatment protocol was approved by the

Ethical and Scientific Committees of our Institute, and written

informed consent was obtained from each patient. rHuIL-12 was

supplied by Hoffmann-La Roche.

Study Design. This study was an open-label, nonran-

domized, single-center pilot study designed to evaluate the

safety, tolerability, pharmacodynamics, and pharmacokinetics

of s.c. administered rHuIL-i2 in patients with metastatic mela-

noma. All of the patients received two identical 28-day cycles,

with the s.c. injections given on days 1, 8, and 15 ofeach cycle.

The injection sites were rotated (e.g., day + 1 in the right upper

arm, day +8 in the left upper arm, day + 15 in the right

shoulder, and so forth). A fixed dose of 0.5 pg/kg was used

throughout the study, but if a patient experienced grade III or

recurrent grade II toxicity, this was reduced to 0.3 �ig&g. The

0.5 p.g/kg dosage was chosen on the basis of preliminary results

of the United States Trial SO 14547.� In that study, the maxi-

mum tolerated dose was reached at 1 .0 jig/kg, given once

weekly with two grade III toxicities (increase in transaminases

and leukopenia). Therefore, 0.5 p,g/kg was the only reasonable

and acceptable maximum dosing scheme available. Tumor re-

sponse was assessed at the end of the second cycle, after which,

the responding patients and those with stable disease could

receive a maximum of two additional cycles. After completing

the last cycle, the patients were followed up for a further 28 days

to check for any adverse event. Vital signs, including blood



Clinical Cancer Research 77

pressure, pulse, and temperature, were measured at frequent and

regular intervals on days 1, 2, 5, 8, 12, 15, and 19 during the first

cycle and on days 1, 8, and 15 during subsequent cycles. On

days 1, 8, and 15 of each cycle, the measurements were made

before the injection and 8 and 16 h after the injection. Weight

was measured prior to each rHuIL- 12 administration. Electro-

cardiogram and CO diffusion capacity were recorded at baseline

and on day 19 ± 3 of each cycle. In the case of grade II fever,

500 mg paracetamol p.o. every 8-12 h was provided; no other

concomitant medications were routinely administered. Toxicity

was assessed using the National Cancer Institute Common Tox-

icity Criteria (25). The patients experiencing grade I pulmonary

toxicity (except cough), grade II hypotension, weight gain, or an

increase in transaminases levels plus a decrease in the number of

leukocytes were to be hospitalized, and the treatment was dis-

continued until recovery. For any patients experiencing grade III

toxicity, drug administration was withheld and then, when the

toxicity had recovered to grade 0-I, the therapy was continued

at a dose of 0.3 p.g/kg. If grade III toxicity occurred, the

treatment was discontinued unless the investigator positively

decided otherwise.

Patient Characteristics. Ten patients entered the study:

five males and five females, with a median age of 50 years

(range, 26-61). The Karnofsky performance status was 100% in

eight patients and 80% in the remaining two patients. The initial

dose given to all patients was 0.5 p.g/kg; it was reduced to 0.3

�i.g/kg in one patient because of grade III neutropenia after the

first and second administration of the first cycle, possibly as a

result of a previous heavy treatment with dacarbazine and Fote-

mustine. This patient received only one cycle of treatment. The

remaining nine patients all received at beast two cycles, and two

follow-up cycles were given to two responsive patients. The

main sites of metastatic disease were soft tissue (80%) and lung

(40%), followed by superficial adenopathies (20%), abdomi-

nopelvic nodes or organs, and liver (Table 1). All of the patients

had undergone surgery of primary lesion. Patient 1 had received

immunotherapy with IFN-a, initially in adjuvant setting and

then for metastatic disease for 4 months. All of the other patients

had received chemotherapy, and three had also received radio-

therapy for superficial adenopathies; three had received hormo-

nal therapy with tamoxifen; and seven had received immuno-

therapy with IL-2 and/or IFN-a.

rHuIL-12 Pharmacokinetics. Serial blood samples (2

ml) were collected immediately before the rHuIL-l2 injection

and 4, 8, 12, 24, 30, and 48 h after the first injection (day +1)

of the first cycle. During the second cycle, the samples were

taken immediately before the last injection (day + 15) and then

4, 8, 12, 24, 30, and 48 h afterward. The serum bevels of

rHuIL- 12 were measured using a two-step assay involving an-

tibody capture followed by a functional bioassay; this method is

specific for rHuIL- I 2 and has a sensitivity limit of about 50

pg/mb.

Pharmacodynamics. Blood (10 ml) and urine samples

(10 ml of a 12-h collection) were taken at baseline and 24, 48,

and 96 h after the first injection of the first cycle. During the

second cycle, the samples were taken 24 h after the first injec-

tion, 24 h after the second injection, and before and 24, 48, and

96 h after the third injection. During the fourth week of the

second cycle, one end-of-study sample was taken. Serum levels

were determined using commercially available ELISA kits for

the following factors: IL-2, IL-4, IL-6, IFN-’y, TNF-a, GM-

CSF, TGF-� 1 , IL- 10, (Genzyme Corporation, Cambridge, MA),

IL-8 (Bender MedSystems, Vienna, Austria), and bFGF (Onco-

gene Science, Cambridge, MA). ELISA kits were also used to

determine the bevels of bFGF and VEGF (Genzyme Corpora-

tion) in urine samples. The beukocyte subsets were evaluated by

means of direct immunofluorescence using phycoherythrin-con-

jugated antibodies (Becton Dickinson, Sunnyvale, CA) on

whole blood, followed by flow cytometry analysis using a

FACScan instrument (Becton Dickinson). For each sample, the

proportion of lymphocytes, monocytes, and granulocytes was

established by gating on forward versus side scatter plots. The

following markers were analyzed: CD3, CD4, CD8, CD 16,

CD3O, and CD45RO. The expression of each marker was eval-

uated on the whole leukocyte population or on lymphocytes

only, after appropriate gating on forward versus side scatter

plots.

rHuIL-12 Antibodies. The serum for anti-rHuIL-l2 an-

tibody determinations (2-ml blood samples) was obtained prior

to treatment and at the beginning of each administration (with

the exception of day 8 of the second cycle). A further sample

was drawn during the off-study observation period (19-28 days

after the last injection). The sensitivity limit of the antibody

detection assay was 29.3 ng/ml.

RESULTS

rHuIL-12 Pharmacokinetics

IL- 12 was detected in the serum of 9 of the 10 patients as

early as four h after the first s.c. injection (Fig. 1); in the

remaining patient (patient 8), it was found 8 h after the injection.

Circulating IL-l2 peaked 8-12 h after the first injection in all of

the patients; the mean peak value was 250 ± 122 pg/mb (Fig. 1).

The levels dropped below detectable values 24-30 h after drug

administration in most patients. The same monitoring of IL-l2

bevels was repeated after the last injection (day + 15 of the

second cycle). At this time, there was no detectable IL-l2 in the

serum of 6 of 9 evaluable patients up to 48 h after the injection

(patient 6 withdrew from trial at the end of the first cycle; Fig.

1). In the remaining three cases (patients 4, 5, and 10), the peak

IL- 12 values were much lower than after the first injection of the

first cycle: the mean (± SD) peak concentration in these patients

was 94 ± 35 pg/mb between 8 and 12 h after the injection. The

estimated serum half-life of rHuIL- 1 2 (Table 2) was 12 h after

the first dose. The mean apparent systemic clearance was 1.22

mL/min-kg after single s.c. dose of 0.5 p.g/kg. The pharmacoki-

netic parameters could not be evaluated at the end of the second

cycle because of the nonmeasurable levels of IL- I 2 in serum.

These findings indicate that the initial s.c. rHuIL- 12 ad-

ministration led to significant serum levels of the cytokine;

however, between the first and the last injection, an adaptive

response occurred in all of the patients that progressively inhib-

ited the diffusion of rHuIL-l2 into the bloodstream or led to

more rapid clearance of rHuIL- 1 2 from the blood. The evalua-

tion of the patients’ sera failed to reveal the presence of anti-

rHuIL-12 antibodies in any of the patients up to 28 days after the

completion of the two treatment cycles (data not shown), thus

indicating that the progressive inhibition of IL- I 2 diffusion into



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Day: +1 � +43 +44 Day: +1 +2 +43 +44 Day: +1 +2 +43 +44

Fig. 1 Serum IL-b2 levels in melanoma patients receiving s.c. rHuIL-l2. Circulating IL-12 was measured by ELISA in serum samples obtained fromeach patient immediately before the first lL-l2 injection (hour 0) and then at 4, 8, 12, and 24 h (day 1), as well as at 30 and 48 h (day +2) after thefirst IL-12 administration. The same schedule of serum sampling was repeated on days +43 and +44 after the last IL-12 administration (day +43).Patient 6 (0) withdrew from the trial at the end of the first cycle.

78 Pilot Study of rHuIL-12 in Metastatic Melanoma

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Table 2 Main pharmacokinetic parameters after the first

IL-l2 injection

Parameter Value, mean (SD)

Tmax (h)” 10 (2.8)

Cmax (pg/ml) 277 (139)

ti,2 dim (h) 12 (4.1)

CL/F (ml/minkg) 1.22 (0.31)AUC 0-*� (pg�h/mb) 7357 (2314)

a T�,,j,, time at which maximum plasma concentration was reached;

Cmax, maximum concentration at Tm� t1,� dim’ elimination half-life;CL/F. clearance; AUC 0-*oo, area under the concentration versus timecurve extrapolated to infinity.

the bloodstream could not be explained on the basis of an

antibody response to the injected cytokine.

Treatment-related Toxicity

A detailed list of the observed treatment-related toxic re-

actions is shown in Table 3. A flu-like syndrome affected 100%

of patients; grade II fever was easily treated with paracetamol.

The fever began within 12-24 h of administration and waned in

a median of 3 days. The common side effects associated with

fever included chills, fatigue, and arthromyalgias. No nausea or

vomiting was observed. Transient increases in serum transami-

nases occurred in six patients, but the values returned to within

the normal range at the end of the treatment. Additional abnor-

mabities in liver function consisted of an increase in lactate

dehydrogenase, but no change in alkaline phosphatase or bili-

rubin was observed; eight patients experienced a transient in-

crease in triglyceridemia, with an inversion of the cholesterol-

emia/triglycenidemia ratio that returned to normal in a median of

12 days in most cases. Evidence of pulmonary toxicity was

observed in only two patients, as indicated by a grade I reduc-

tion in CO diffusion capacity; this side effect was not dose-

related, and lung function subsequently improved even when

rHuIL- 12 treatment was continued at the same dosage. One case

of hyperfibrinogenemia and two cases of anemia not requiring

transfusion were also observed. Grade Ill neutropenia and grade

I thrombocytopenia occurred in the same patient. There were no

significant changes in serum creatinine levels, nor any signs of

neurological toxicity or autoimmune phenomena. Only one pa-

tient experienced grade I stomatitis, which spontaneously re-

solved within a few days. No patient died because of treatment-

related toxicity, nor was any case of infection observed.

Pharmacodynamics

Effects on Leukocyte Subsets. One day after the first

administration (day +2 of the cycle), a marked leukopenia was

evident in all patients. The mean total number of circulating

lymphocytes dropped from a pretreatment value of 1.49 ±

0.45 X l03/mm3 on day -4 to 0.53 ± 0.22 X i03/mm3; 7 days

after the first injection, the number of lymphocytes returned to

pretreatment values (1.45 ± 0.59 X i03/mm3). Monocytes

showed a similar pattern of decline and recovery but the initial



Table 3 Toxicity profile

% ofCategory of toxicity Toxicity course Grade”

Systemic Flu-like syndrome 100% IIFever 90% IISkin rash 20% I

Local (site of injection) Subcutaneous induration 10% I

Pulmonary CO diffusion capacity 20% IGastrointestinal Stomatitis 10% IHepatic Transitory 60% I

hypertransaminasemia

Increased levels of 20% Ilactate dehydrogenase

Hematobogicab Anemia 20% IINeutropenia 10% IIIThrombocytopenia 10% I

Coagulation Hyperfibrinogenemia 10% IMetabolic Hypertriglyceridemia 80%

a National Cancer Institute Common Toxicity Criteria.I

80

60

40

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Lymphocytes

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Fig. 2 Leukocyte subset analysis in patients treated with IL-12. Flowcytometry analysis was performed on whole blood samples from each

patient after red cell depletion. The relative proportion of lymphocytes,monocytes, and granulocytes was established by gating on forwardversus side scatter plots after collecting 10,000 events for each sample.

Note that the flow cytometry results are relative proportions and notabsolute counts; thus, the increase in lymphocytes on day +5 in com-

parison to day -4 is only apparent and actually reflects the drop in theabsolute neutrophil count on day +5. The same consideration applies tothe apparent increase in granulocytes on day +2, which is actually dueto the decline in absolute lymphocyte counts on the same day.


reduction was less pronounced (day-4: 0.41 ± 0.13 X l03/mm3;

day +2: 0.28 ± 0.11 X l03/mm3; day +8: 0.46 ± 0.16 X

103/mm3). On the contrary, neutrophil kinetic pattern was dif-

ferent, with a marked reduction becoming evident 4 days after

the first injection (from 3.55 ± 1.33 X 103/mm3 on day -4 to

1.74 ± 1.22 x 103/mm3 on day +5), followed by an almost

complete recovery on day +7 (2.83 ± 1.37 X 103/mm3).

Interestingly, all these effects were either absent (in the case of

lymphocytes and neutrophils) or much weaker (in the case of

monocytes) between 1 and 5 days after the last injection of the

second cycle (data not shown).

The effect of rHuIL- 12 treatment on the leukocyte subsets

was also evaluated by means of flow cytometry on whole blood

and confirmed a marked alteration in the relative proportions of

circulating leukocytes in all of the patients within 24 h of the

first injection of the first treatment cycle (Fig. 2). By gating on

forward versus side scatter plots, a marked reduction in the

proportion of lymphocytes associated with an apparent increase

in granubocytes was observed. On day +5 of the cycle, flow

cytometry analysis confirmed the reduction in granubocytes al-

ready indicated by the conventional neutrophil count: at the

same time there was a transient increase in the proportion of

monocytes that reflected the decrease in the number of granu-

locytes. A similar analysis made on day +44 (24 h after the last

injection of the second cycle) revealed a much more limited

change in the proportions of leukocytes than on day +2 (Fig. 2).

Staining with anti-CD3, CD4, CD8, CD16, CD3O and CD45RO

mAbs revealed a major shift in the CD4:CD8 ratio on day +2 of

the first cycle (24 h after the first injection on day + 1); whereas

the proportion of CD3� T cells remained constant (data not

shown), there was a marked reduction in CD8� cells and an

increase in CD4� cells leading to a transient inversion of the

CD4:CD8 ratio in all patients on day +2 (Fig. 3). There was

also a clear reduction in CD16” cells (Fig. 3). None of these

changes were observed on day +44, with the exception of a

decrease in CDl6� cells in some of the patients. No significant

changes were observed in the proportion of cells expressing the

other markers CD3O and CD4SRO.

U)aU0a

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Pharmacodynamics

Effects on Serum Cytokine Levels. The serum/urine

levels of a number of cytokines/growth factors were measured

by means of ELISA during the planned two cycles of treatment.

The serum samples from each patient were tested for the pres-

ence of IL-2, IL-4, IL-6, IL-8, IL-b, IFN-’y, TNF-a, GM-CSF,

TGF-�31, and bFGF. The serum IFN--y levels in all of the

patients markedly increased within 24 h of the first rHuIL-12

injection (Fig. 4), reaching a peak concentration in the ng/ml

range in six patients; in most cases, these levels returned to their

baseline values within 3-6 days. The increase in serum IFN--y

was also observed during the second cycle of treatment, but with

the exception of patients 8 and 9, the peak levels were much

bower than those observed after the first rHuIL-l2 injection (Fig.

4). IL-lO was the only other cytokine the serum bevels of which

considerably increased in all of the patients. As shown in Fig. 5,

the IL-lO modulation pattern was more complex than in the case



CD4

Fig. 3 Lymphocyte subset analysis in patients re-ceiving s.c. rHuIL-l2. The proportion of circulat-

ing CD4�. CD8�. and CDl6� lymphocytes andthe CD4:CD8 ratio was established by direct im-munofluorescence with PE-conjugated mAbs onred cell-depleted whole blood samples taken be-

tween day -4 and day +50 of the treatment cycle.The results represent the proportion of positivecells evaluated after gating for lymphocytes on

forward versus side scatter plots. By these criteria,

the proportion of CD3� cells (not shown) did notchange as a result of rHuIL-12 therapy.

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80 Pilot Study of rHulL-l 2 in Metastatic Melanoma

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of IFN--y. In S of 9 evaluable cases (patients 4, 5, 8, 9, and 10),

the highest IL-lO levels were observed during the second cycle

of treatment rather than immediately after the first rHuIL-l2

injection. However, as in the case of IFN--y, the increase and

decline in IL-b bevels appeared to be completed within 3-6

days after rHuIL-l2 administration.

All of the other factors showed a less consistent pattern of

change. Only 1-2 patients displayed moderate increases or

decreases in serum IL-2, IL-4. TNF-a, and GM-CSF and urine

VEGF levels in comparison to baseline (data not shown). IL-6,

IL-8, TGF-�3l, and bFGF levels were up- or down-regulated in

30-60% of the patients (data not shown), but no correlation

could be established between these changes and the clinical

response to rHuIL-12 treatment. In two of the three responding

patients, we observed a reduction in urinary bFGF bevels (a drop

in patient 1 and a minor reduction in patient 5; data not shown);

this could support a possible antiangiogenic activity of IL-l2.

Clinical Responses

The details of the responding patients are shown in Table 4.

Three of the 10 patients achieved regressions of some tumor

lesions. One patient (patient 1), who had been pretreated with

surgery and IFN--y (first in an adjuvant setting and then for

metastatic disease) and had three s.c. nodules (diameters, 1 , 1,

and 0.5 cm) and an abdominopelvic mass (global diameters, 8 X

3 cm), received a total of four cycles of rHuIL-l2. After only

two administrations (first cycle), the s.c. lesions had disap-

peared; at the end of the second cycle, the deep adenopathies

showed no change, and the complete response of the s.c. lesions

continued. This patient therefore underwent two additional cy-

des, during which two s.c. nodules reappeared in the same

location, one of which subsequently disappeared, possibly as a

result of a late response. After four cycles, computerized tomog-

raphy revealed a progression in the abdominopelvic nodes.

The second responding patient (patient 5) had been pre-

treated with chemo/immuno/hormonal therapy consisting of a

combination of dacarbazine, carboplatin, mytomycin-C, tamox-

ifen, and IFN-a. This patient had soft tissue and abdominopelvic

node disease. The first cycle led to the complete remission of the

soft tissue lesions (a superficial adenopathy with a diameter of

2 X 3 cm and a s.c. lesion with a diameter of 0.5 cm); this

response continued, but at the end of the fourth cycle, ultra-

sonography revealed marked progression in the deep nodes.

The third patient (patient 10), who had been pretreated with

hyperthermic L-phenylalanine mustard perfusion, had s.c. tissue

and hepatic parenchyma disease. After the first two rHuIL-12

injections, a complete remission of hepatic lesions occurred

(documented by ultrasonography and then by nuclear magnetic

resonance), but progressive s.c. disease, with the appearance of

a number of confluent and echo-documented nodules, was also

observed (Table 4).

Another patient (patient 8) had s.c. disease (at least seven

mammographically confirmed right breast nodules with a max-

imum diameter of 1 cm and a left arm nodule with a diameter of

2 cm) and a right adrenal lesion. Given that disease stabilization

was observed at the end of the first two cycles, the treatment was

continued; however, a new s.c. nodule appeared under the right

eye after the first administration of the first follow-up cycle, and

so the treatment was stopped.

DISCUSSION

IL-l2 is a cytokine of great immunological interest that has

been shown to have significant antitumor activity in a number of

animal models. The present trial of rHuIL-l2 in metastatic

melanoma was designed to evaluate its side effects, pharmaco-

kinetics, and immunological parameters and to document any

evidence of clinically relevant activity. The results indicated that

the once-weekly, fixed-dose s.c. administration of rHuIL-l2 for

two consecutive 28-day cycles is a well-tolerated regimen that

has significant pharmacodynamic effects on circulating leuko-

cytes and on serum levels of various cytokines. Furthermore,

although there were no partial or complete remissions, tumor



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Fig. 4 Serum IFN-�y levels determined by ELISA in patients receiving s.c. rHuIL-l2. Arrowheads, time ofIL-12 injection (days +1, +8, +15, +29,+36, and + 43). The serum samples for WN--y evaluation were obtained on days -4, +2, +3, +5, +8, + 12, +30, +37, +43, +44, +45, +47,

and + 50. Serum samples of days + 8 and +43 were taken immediately before IL-l2 injection. Patient 6 (0) was removed from the trial at the endof the first cycle.


regression in the form of mixed responses was induced in 3 of

10 previously treated patients with metastatic melanoma. The

treatment was well tolerated by most patients, and no pulmonary

toxicity was observed in spite of the evidence obtained in

experimental models (26). Many of the side effects observed

after rHuIL-l2 administration (in particular, liver function ab-

normalities, fever, fatigue, hematobogical changes, and hyper-

triglyceridemia) may reflect the induction of IFN--1s because

similar effects have been observed in clinical trials using WN-y

(27, 28). Fever (the most frequent side effect) was unusually

delayed: it typically occurred 12 h after injection, rather than the

2-4 h observed with the use of other pyrogenic cytokines, such

as IL-], IL-2, and the IFNs. Taken together, these results mdi-

cate that s.c. administered rHuIL-l2 has greater immunomodu-

latory effects and suggest that it may have therapeutic potential

in advanced melanoma.

The pharmacokinetics of the fixed-dose regimen were as-

sociated with a striking difference between the first and last

rHuIL-l2 injection. IL-l2 could be readily detected in the serum

of all of the patients after the first injection, but not in 6 of 9

evaluable patients 48 h after the last injection of the second

cycle; in the remaining three evaluable patients, peak serum

levels after the last injection were much lower than after the first

administration. This reduction in serum IL-12 levels at the end

of the two cycles of treatment suggests that an adaptive response

occurred in all of the patients and led either to greater removal

of free IL-l2 from peripheral blood or the inhibition of the

access of the injected rHuIL-l2 to the bloodstream. This adapt-

ive response could not be explained on the basis of an antibody

response to the injected cytokine because no anti-IL-l2 antibod-

ies could be detected in any of the patients. One possibility is

that the initial rHuIL-12 injections led to an up-regulation of the

high-affinity receptors for this cytokine: it is known that high-

affinity IL- 12 receptors are expressed on activated T and NK

cells (29) and that such receptors are composed of two distinct

�3-type subunits (30). The initial rHuIL-l2 injections may

“prime” the host by leading to an enhanced expression of

high-affinity IL-l2 receptors, and this process may, subse-

quently, provide a mechanism for the enhanced suppression of

serum IL-12 levels after later injections. In mice, the adminis-

tration of IL-12 up-regulates the expression of the mRNA for

both the 1� 1 and �32 IL- 12 receptor subunits, and an inverse



V V V V V

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82 Pilot Study of rHuIL-l2 in Metastatic Melanoma

5 Maurice Gately (Hoffmann-La Roche Inc., Nutley, NJ) personal com-

munication.

.�

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Fig. 5 Serum IL-b levels in patients receiving s.c. rHuIL-12. Arrowheads, time oflL-l2 injection (days + 1, +8, + 15, +29, +36, and +43). The

serum samples for IL-b evaluation were obtained on days -4, +2, +3, +5, +8, + 12, +30, +37, +43, +44, +45, +47, and +50. Serum samplesof days +8 and +43 were taken immediately before IL-l2 injection. Patient 6 (0) was removed from the trial at the end of the first cycle.

correlation has been found between the levels of IL- 1 2-receptor

mRNA and serum IL-l2 levels,5 which supports the hypothesis

that there is a positive feedback loop.

The contrast between the high initial serum bevels of IL-l2

after the first injection and the bow or zero levels detected at the

end of the two-cycle treatment was reflected by the pattern of

changes in leukocyte subsets and IFN--y bevels. Interestingly, the

progressive attenuation of serum IFN--y levels at subsequent

IL-l2 injection has been observed even when IL-12 is iv.

administered (3 1). More importantly, all local clinical responses

of superficial adenopathies and soft tissues appeared after the

first two injections of the first cycle: only one late response,

involving a s.c. lesion, occurred, during the follow-up cycles

administered to patient 1 . No responses occurred during the

second cycle. The available evidence, therefore, suggests that a

high initial serum level of IL-12 has both immunomodulatory

and antitumor effects. Continued antitumor activity during or

after the second cycle of treatment may be inhibited by the same

adaptive mechanism(s) that blocks the presence of significant

bevels of IL-12 in peripheral blood. On the other hand, the

kinetics of IL-lO serum levels in 5 of 9 patients indicate that

peak levels were reached during the second cycle of rHuIL-12

injections, thus suggesting that the cytokine still had a signifi-

cant, although greatly reduced, biological activity.

rHuIL- 12 treatment led to a marked but transient reduction

in circulating leukocyte subsets bevels. The lowest lymphocyte

and neutrophil counts were observed 24 h and 4 days after

rHuIL-l2 injection, respectively, which suggests that they were

partially removed from the circulation by different mechanisms.

The drop in the proportion of CDl6� cells 24 h after rHuIL-l2

injection was not unexpected because of the known chemotactic

activity of IL-l2 in relation to NK cells and its ability to

stimulate the adhesion of NK cells to the vascular endothelium

(32). Furthermore, the induction of high IFN-�)’ levels after

rHuIL-l2 injection may lead to the synthesis of IP-lO, a known

chemoauractant and stimulator of adhesion of monocytes and T

cells to endothebial cells (33, 34). Interestingly, IP-lO mRNA

has recently been detected in the lymphocytes of a series of

patients with renal cell carcinoma receiving s.c. IL-12 (35).

Additional indirect mechanisms triggered by IL-12 may con-

tribute to explaining the drop in neutrophib counts and may

involve the induction of adhesion molecules, such as E-sebectin

(induced by IL-lO and IFN--y; Refs. 36-38) or ICAM-l (39), on

endothelial cells.

In line with the known biological functions of IL-12 (40),

IFN--y and IL-b were transiently induced in all treated patients.

IL-l2 can induce concomitant IL-lO and IFN--y secretion by

CD4� and CD8� T cells (41, 42), and therefore, it was not



SurgeryIFN-cs

Patientno. Previous therapy Age/sexP.S.” Site of tumor

58/M 0 Subcutaneous

Table 4 Characteristics of responding patients

Response”/duration(months)

CR11

Liver

NC/2

CR/4

CR/4

NC/4

PD

CR110

‘I � performance status: CR, complete remission; NC. no change; L-PAM, L-phenylalanine mustard; PD. progression of disease.b Response category refers to the individual lesions reported in this table.


S SurgeryChemo/immuno/hormonal therapy

10 Surgery

Perfusion with L-PAM

Abdominopelvic nodes

26fF 0 Superficial node

Subcutaneous

Abdominopelvic nodes

57/F 0 Subcutaneous

Size of tumor(cm X cm)

lXl

lXl

0.5 X 0.58X3

3X2

0.5 x 0.5

2.5 x 2.53 X 4.5

1 X 1.5IXI

0.5 X 0.51.9 X 1.91.0 X 1.0

unexpected to find both cytokines in the serum of the patients.

However, the kinetics of the two cytokines were different:

unlike IFN-y, in 6 of 9 patients, peak serum IL-b levels were

observed during the second rather than the first cycle of treat-

ment. IL-lO is a major inhibitor of IL-l2 production by antigen

presenting cells, and its production in response to IL-12 is

regarded as a negative feedback mechanism (43). Therefore, it is

possible that the increased IL-lO levels detected in the sera of

many patients during the second cycle of treatment may reflect

an ongoing negative feedback response aimed at contrasting the

presence of IL- 12.

The original hypothesis that IL-bO may be considered a

down-regulator of cell-mediated immunity (44) has been par-

tiabby contradicted by more recent results. In fact, the systemic

administration of high IL-lO doses to mice bearing established

tumors (including melanoma) led to tumor rejection, an effect

that was abrogated by sublethal irradiation (45). Furthermore,

the injection of IL-lO can suppress both experimental and spon-

taneous metastases in mice bearing murine or human melano-

mas, by means of a process dependent on NK cells (46). On the

basis of this evidence, the induction of IL-lO in all patients of

this study may be considered as a contribution to the antitumor

effects of IL-12, rather than simply as a negative feedback

mechanism induced by rHuIL- 12 administration. However, fur-

ther studies are needed to clarify the precise role of IL-lO in this

setting because recent results have indicated that it can induce

long-lasting and antigen-specific anergic state in CD4� cells

(47).

The limited antitumor activity of rHuIL-b2 observed in 3 of

10 patients did not correlate with any of the measured immu-

nobogicab parameters. In mouse models, the antineoplastic ac-

tivity of systemic rHuIL-l2 has been found to be dose depend-

ent and can be observed on s.c. tumors, as well as on pulmonary

and hepatic metastases (12-14, 48, 49). The regressions of s.c.

lesions and hepatic metastases observed in this study confirm

the antitumor activity of IL-12 previously obtained in murine

models. A seemingly higher proportion of tumor regressions

were observed in our study (3 of 10 treated patients) in com-

parison to the results recently described by Atkins el a!. (31)

using iv. IL-12 (1 transient complete response out of 12 mel-

anoma patients). This may depend on individual patient char-

acteristics, but it is also possible that differences in the route (iv.

versus s.c.) and in the schedule of cytokine administration

between the two studies may play a role in the antitumor

efficacy of IL-12.

Finally, experimental models indicate that the therapeutic

effects of IL-12 are dose dependent (14, 15); thus, it is possible

that an increased antitumor activity in patients may be obtained

by changing the dose and/or the administration schedule. In the

present study, a dose higher than 0.5 p.g/kg was not attempted.

A possibility to be investigated in future trials is the safety and

tolerability of a 0.75 p.g/kg dose, which may improve the

clinical efficacy of rHuIL-l2, possibly without reaching the

limiting liver toxicity and leukopenia observed at 1 .0 �i.g/kg in

the United States Trial SO 14547 (as mentioned in “Patients and

Methods”). The problem of dose-finding is highly relevant

because the possibility of increasing dosages without reaching

limiting toxicity could lead to an improvement in the therapeutic

index. A further and perhaps more significant challenge facing

any improvement in the therapeutic efficacy of this cytokine is

to devise protocols that can reverse or contrast the adaptive

response that leads to the progressive inhibition of IL- 12 serum

levels. It is of note that the absence of a measurable antibody

response to the injected cytokine suggests that the recombinant

form of human IL-12 backs immunogenicity in human hosts,

thus providing a significant advantage for further clinical trials.

ACKNOWLEDGMENTS

We thank Dr. M. Gately (Hoffmann-La Roche Inc.) and Dr. H.Parmar (Roche Products Ltd., Welwyn Garden City. United Kingdom)for critically reading the manuscript. We also thank G. Pezzaglia. G.

Mangiacorti. E. Di Dedda, A. Tamburo. and L. Cennamo for their

nursing support.

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1998;4:75-85. Clin Cancer Res E Bajetta, M Del Vecchio, R Mortarini, et al. in metastatic melanoma.Pilot study of subcutaneous recombinant human interleukin 12

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