Phase I Trial of Twice-Weekly Intravenous Interleukin 12 ... · Jared A. Gollob,2 James W. Mier, Korina Veenstra, David F. McDermott, Daniel Clancy, Marguerite Clancy, and Michael

Phase I Trial of Twice-Weekly Intravenous Interleukin 12 inPatients with Metastatic Renal Cell Cancer or MalignantMelanoma: Ability to Maintain IFN-g Induction IsAssociated with Clinical Response1

Jared A. Gollob,2 James W. Mier,Korina Veenstra, David F. McDermott,Daniel Clancy, Marguerite Clancy, andMichael B. AtkinsBeth Israel Deaconess Medical Center, Division ofHematology/Oncology, Boston, Massachusetts 02215

ABSTRACTThe aim of this study was to examine the tolerability,

antitumor activity, and biological effects of a new scheduleof i.v. recombinant human interleukin 12 (rhIL-12). Twenty-eight patients were enrolled in a Phase I trial in whichrhIL-12 was administered twice weekly as an i.v. bolus for 6weeks. Stable or responding patients were eligible to receiveadditional 6-week cycles until there was no evidence ofdisease or until tumor progression. Patient cohorts weretreated with escalating doses of rhIL-12 (30–700 ng/kg). Themaximum tolerated dose (MTD) was 500 ng/kg, with dose-limiting toxicities consisting of elevated hepatic transami-nases and cytopenias. At the MTD (n 5 14), there was onepartial response occurring after 6 cycles of rhIL-12 in apatient with renal cell cancer. Two additional renal cellcancer patients treated at the MTD had prolonged diseasestabilization, with one of these exhibiting tumor regressionafter 8 cycles of rhIL-12. IFN-g, IL-15, and IL-18 wereinduced in patients treated with rhIL-12. Whereas IFN-gand IL-15 induction were attenuated midway through thefirst cycle in patients with disease progression, those patientswith tumor regression or prolonged disease stabilizationwere able to maintain IFN-g, IL-15, and IL-18 induction.The down-modulation of IFN-g induction during rhIL-12treatment did not relate to IL-10 production or alterationsin rhIL-12 bioavailability but was associated with an ac-quired defect in lymphocyte IFN-g production in responseto IL-12, IL-2, or IL-15. This defect could be partially

overcomein vitro through combined stimulation with IL-12plus IL-2. These findings show that the chronic administra-tion of twice-weekly i.v. rhIL-12 is well-tolerated, stimulatesthe production of IL-12 costimulatory cytokines and IFN-g,and can induce delayed tumor regression. Strategies aimedat maintaining IFN-g induction, such as the addition of IL-2,may further augment the response rate to this schedule ofrhIL-12.

INTRODUCTIONIL3-12 is a cytokine with considerable promise for the

treatment of human malignancies because of its pleiotropicimmunostimulatory effects on lymphocytes (1–5), dendriticcells (6), and neutrophils (7–8), as well as its potent antitumoractivity in murine tumor models (9–10). Whereas immune ac-tivation by IL-12 in mice has resulted in both tumor necrosisfactor and NO production, the antitumor effect of IL-12 hasbeen more dependent on IFN-gproduction (10) and the activa-tion of either CD81T cells (9–11) or NKT cells (12). Thereseem to be a number of mechanisms through which IL-12 caninduce tumor regression, including the direct killing of tumorcells by activated lymphocytes, the antiangiogenic effects ofIL-12-induced IFN-g(13), and injury both to the tumor micro-circulation and to the tumor itself by activated neutrophils (11).

The immunomodulatory activity of IL-12 is considerablydependent on costimulatory cytokines. When the ability ofIL-12 to activate unmanipulated peripheral blood NK cells andCD81 T cells in humans was examined, it was found that theselymphocyte subsets responded to IL-12 only when stimulatedtogether with IL-2 (14). Both IL-15 and IL-18 are also keycostimulatory cytokines, which, when combined with IL-12,induce strong IFN-gproduction by T and NK cells (15, 16). Inmice treated with IL-12, the neutralization of endogenous IL-18significantly blunts IFN-gproduction (17), further emphasizingthe fact that the biological activity of IL-12in vivo is likelydependent on the presence and/or induction of endogenouscostimulatory cytokines.

The promising preclinical data showing IL-12 to be highlyeffective against murine melanoma, renal cell cancer, and sar-coma led to its testing in clinical trials in cancer patients startingin 1994. In the first published trial, rhIL-12 was administeredi.v. daily for 5 days, with a 2-week break between cycles. In

Received 12/20/99; revised 2/18/00; accepted 2/18/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 Supported in part by NIH Grants CA78055 and CA74401 as well as bya stipend from Genetics Institute, Inc.2 To whom requests for reprints should be addressed, at Division ofHematology/Oncology, Beth Israel Deaconess Medical Center, 330Brookline Avenue, East Campus/Room KS-158, Boston, MA 02215.Phone: (617) 667-1930; Fax: (617) 975-8030.

3 The abbreviations used are: IL, interleukin; rh, recombinant human;NO, nitric oxide; DLT, dose-limiting toxicity; MTD, maximal tolerateddose; PR, partial response; PBMC, peripheral blood mononuclear cell;CT, computed tomography; NK, natural killer; ppb, parts per billion.

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addition, a single test dose was given 2 weeks before the firstcycle. With that dosing schedule, the MTD was 500 ng/kg, withDLTs consisting of liver function test abnormalities and stoma-titis (18). Although signs of immune activation were observed,including dose-dependent IFN-gproduction and reversible de-creases in CD81T cell and NK cell numbers (19), only tworesponses were seen among 40 patients (one PR in a patient withrenal cell cancer and one transient complete response (CR) in apatient with melanoma). Similarly low response rates wereobserved in two subsequent trials of weekly s.c. rhIL-12 inmelanoma (20) and renal cell cancer (21), as well as in a trialtesting a thrice weekly schedule of s.c. rhIL-12 (22).

In patients treated with either i.v. or s.c. rhIL-12, IFN-gproduction inducedin vivo by rhIL-12 has attenuated rapidlywith consecutive cycles (18, 20–22), which indicates that thebiological response to rhIL-12 is down-modulated during ther-apy. Even a single test dose administered 2 weeks before thefirst cycle of rhIL-12 seemed to attenuate IL-12-induced IFN-gproduction (23). This down-modulation of IFN-g production hasbeen shown to result in diminished IL-12-induced tumor regres-sion in mice (24). In addition, multiple doses of IL-12 have alsobeen shown in animals to induce a temporary state of immuno-suppression (25–26), perhaps analogous to the down-modula-tion of IFN-g production in patients receiving multiple doses ofrhIL-12. This paradoxical immunosuppression after a relativelybrief period of immune activation by rhIL-12 may explain thelimited antitumor activity observed to date in rhIL-12 clinicaltrials. Although the mechanism of this IL-12-induced down-modulation of subsequent IFN-ginduction remains undefined,data from animal models have suggested that IL-12-induced NOmay be operative (26), whereas observations from clinical trialshave also implicated changes in rhIL-12 pharmacokinetics(20, 22).

In June of 1998, we initiated a Phase I dose escalation trialof i.v. rhIL-12 in patients with renal cell cancer and melanoma,using a new dosing schedule. To try to prevent or delay thedampening of IFN-ginduction, we eliminated the test dose. Inaddition, we implemented a twice-weekly dosing schedule todetermine whether moderate and sustained IFN-g productioncould be stimulated without prohibitive toxicity. Although im-portant aims of this trial included determining the safety andtolerability as well as the antitumor activity of this regimen, thisstudy was also undertaken to further explore the mechanismthrough which rhIL-12 activates the immune systemin vivo andto examine how IFN-ginduction by rhIL-12 is modulated withchronic dosing.

PATIENTS AND METHODSPatient Selection. All of the patients were adults with

histologically proven advanced malignancy that was metastaticor unresectable and for which standard curative or palliativemeasures did not exist or were no longer effective. All of thepatients had measurable or evaluable disease that was clearlyprogressive. Patients were required to have an Eastern Cooper-ative Oncology Group (ECOG) performance status of 0 or 1 andadequate organ function defined by WBC.4000/ml, plateletcount.100,000/ml, creatinine,1.5 mg/dl, bilirubin,1.5 mg/dl, aspartate aminotransferase,2 times the upper limit of nor-

mal, and electrocardiogram and chest X-ray without clinicallysignificant nonmalignant abnormalities. Patients with brain me-tastases, seizure disorders, organ allografts, concurrent require-ment for corticosteroids, more than two prior chemotherapyregimens, more than two prior immunotherapy regimens, orprior IL-12 therapy were ineligible.

Study Design. The study was an open-label, nonrandom-ized, single-center Phase I dose escalation trial. The treatmentprotocol was approved by the Cancer Therapy Evaluation Pro-gram (CTEP) of the National Cancer Institute (protocol T97-0053) and by the Human Institutional Review Board at the BethIsrael Deaconess Medical Center (protocol 97-1083), and writ-ten informed consent was obtained from each patient. rhIL-12,produced by Genetics Institute, Inc. (Cambridge, MA), wassupplied by the National Cancer Institute (IND 6798). TherhIL-12 was administered by i.v. bolus injection.

The treatment schedule is shown in Fig. 1. Patients weretreated in the General Clinical Research Center at the Beth IsraelDeaconess Medical Center, and received i.v. bolus injections ofrhIL-12 twice weekly, with doses given 3–4 days apart. A cycleof therapy lasted 6 weeks, with patients receiving a total of 12doses during that period. During the first cycle only, patientswere admitted overnight after the first, second, and seventhdoses of rhIL-12 for observation and serial blood draws. All ofthe remaining doses were administered on an outpatient basis,with patients observed for 1 h after each dose. Patients wereevaluated for tumor response at the end of each 6-week cycle,and patients with stable or regressing disease could continuereceiving additional cycles until there was no evidence of dis-ease or until there was disease progression. Patients were al-lowed up to a 2-week break between cycles for the resolution ofany significant rhIL-12-induced toxicity.

The rhIL-12 dose was increased from 30 to 700 ng/kg insuccessive cohorts of patients. No intrapatient dose escalationwas permitted. A minimum of three patients were enrolled ateach dose level, and all of the patients had to have completed thefirst 3 weeks of cycle 1 before initiating enrollment to the nextdose level. Toxicity was assessed using the National CancerInstitute Common Toxicity Criteria. In general, grade 3 orgreater toxicities were considered dose-limiting. However, liverfunction test abnormalities were not classified as dose-limitinguntil the total bilirubin was.3 times normal or the hepatictransaminases or alkaline phosphatase were.10 times normal.In addition, the WBC count and neutrophil count were notconsidered dose-limiting until criteria for grade 4 toxicity weremet, and no degree of lymphopenia was dose-limiting. Grade 2cardiovascular toxicity (except for hypotension) and neurolog-ical toxicity were considered dose-limiting. The IL-12 dose wasescalated when 0 of 3 patients at a dose level had a DLT. If 1 of3 experienced a DLT, three more patients were enrolled at thatdose level, and the dose was escalated if no more than 1 of 6patients had a DLT. Patients experiencing a DLT could resumethe IL-12 at the next lowest dose level if the toxicity resolvedwithin 2 weeks. When two or more DLTs were experienced ata dose level, the MTD was determined to be the next previousdose level.

All of the patients received ranitidine for the duration oftheir IL-12 treatment. Acetaminophen was administered prophy-lactically for 24 h after each IL-12 dose and could be taken as

1679Clinical Cancer Research



needed thereafter. Indomethacin was used to control fever thatwas not responsive to acetaminophen, and demerol was used totreat rigors.

Assessment of Tumor Response.Tumor measurementswere obtained by CT scan at the end of each 6-week cycleof IL-12.

Measurement of IL-12- and rhIL-12-Induced Cyto-kines. Serial blood specimens were collected in heparinizedtubes immediately before and 4, 8, 12, 16, 20, and 24 h after thefirst, second, and seventh rhIL-12 doses during the first cycle.The tubes were centrifuged immediately after collection, and theplasma was then removed and stored at220°C. Plasma IL-12levels were measured using an ELISA that detects only the p70IL-12 heterodimer (Endogen, Cambridge, MA, sensitivity,3pg/ml). ELISA kits were also used to measure plasma IFN-g(Endogen, sensitivity,2 pg/ml), IL-10 (Endogen, sensitivity,3 pg/ml), IL-15 (R&D, Minneapolis, MN, sensitivity,1pg/ml), and IL-18 (R&D, sensitivity,15 pg/ml).

In Vitro Assays of Lymphocyte Cytokine Responsive-ness. Blood specimens were collected in heparinized tubesimmediately before the first and seventh doses of rhIL-12during cycle 1. PBMCs were isolated from blood samplesthrough density gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, MO). PBMCs were incubated in96-well U-bottomed plates at 53 104 cells/well with mediumalone (RPMI 1640 plus 15% FCS, 2%L-glutamine, 1%sodium pyruvate, 1% gentamicin, and 1% penicillin-strepto-mycin) or with medium plus one of the following: (a)50 ng/ml IL-2 (Chiron Corporation, Emeryville, CA, specificactivity 18 3 106 units/mg); (b) 1 nM IL-12 (Genetics Insti-tute, Cambridge, MA, specific activity 1.73 107 units/mg);(c) 10 ng/ml IL-15 (Endogen, specific activity$2 3 106

units/mg); (d) IL-2 1 IL-12; or (e) IL-15 1 IL-12. Condi-tions were plated in triplicate, and after a 72-h incubation at37°C, aliquots of supernatants from each well were harvested

immediately before pulsing each well with 1mCi [3H]thymi-dine (DuPont-New England Nuclear, Boston, MA). TheIFN-g concentration in the harvested supernatants was as-sayed using an IFN-gELISA (Endogen). Cell proliferationwas determined by measuring [3H]thymidine incorporation8 h after pulsing, as described previously (27).

Measurement of NO in Expired Air. The concentrationof NO in expired air was measured in five patients receivingIL-12 at either the 500-ng/kg or the 700-ng/kg dose levels.Measurements were made immediately before and 24 h after thefirst and second IL-12 doses. Expired air was collected inself-sealing balloons after first clearing the upper airway ofambient air NO by having patients take four deep inspirationsthrough a tube fitted with a charcoal filter (Omega EngineeringCo.). The NO concentration in the air expired after the fourthbreath was measured using a high-sensitivity NO detector basedon a gas-phase chemiluminescent reaction between NO andozone (Model 280 Nitric Oxide Analyzer, Sievers Instruments,Inc., Boulder, CO). Patients receiving high-dose IL-2 (600,000IU/kg i.v. every 8 h) were used as positive controls. IL-2patients had NO samples obtained before the start of the 1stweek of IL-2 and then daily for the 1st 3 days of IL-2 treatment.

RESULTSPatient Characteristics

Between June 1998 and June 1999, 28 patients were en-rolled in this study. Patient characteristics are shown in Table 1.The majority of patients had metastases to two or more sites(including 15 of 28 with liver, adrenal, and/or kidney involve-ment and 10 of 28 with bone metastases), and 23 of 28 hadreceived one or more prior immunotherapy regimens (primarilyIL-2-based regimens). Only 3 of 23 patients had responded totheir prior immunotherapy.

Fig. 1 Schema for clinical trial oftwice-weekly i.v. rhIL-12 without atest dose.

1680Phase I Trial of Twice-Weekly i.v. rhIL-12



Treatment Administered and ToxicityDose Escalation Phase. Three patients were treated at

each of the first three rhIL-12 dose levels (30, 100, and 300ng/kg), with no DLTs. Common side effects included self-limited fever and chills, occurring 6–10 h after the dose, andmild malaise. These side effects were observed even at the30-ng/kg dose level, and were greatly attenuated by the start ofthe 3rd week of therapy. One patient, treated at 100 ng/kg,developed a supraventricular tachycardia (Table 2) in the settingof fever after the second rhIL-12 dose, which resolved sponta-neously. Small, minimally symptomatic, transient oral aphthousulcers developed during the first cycle of therapy in one patientat the 300-ng/kg dose level. No significant (Grade 2 or greater)cytopenias or liver-function test abnormalities were noted at thefirst three dose levels.

At the 500-ng/kg dose level, the fever and chills were moresevere with previously untreated patients or with patients forwhom more than 1 year had passed since prior therapy. Feverswere highest after the second dose and were minimal-to-absentby the 3rd week of therapy in the majority of patients. Indo-methacin was added to acetaminophen for the control of feversand chills in only 3 of 14 patients and was never requiredbeyond the first week of therapy. Minimal nausea and anorexia

were observed, but no diarrhea or gastrointestinal bleeding.Stomatitis was uncommon and was never greater than grade 2.Grade 1–2 elevations of serum transaminases were common,usually peaking after the second dose and normalizing by thestart of week 3 (Table 2). Orthostatic hypotension 24 h afterthe second rhIL-12 dose occurred in one patient and constitutedthe one DLT among the six patients treated at the 500-ng/kgdose level during the escalation phase. No fluid retention orevidence of capillary leak syndrome was observed at either the500-ng/kg dose level or any other rhIL-12 dose level, nor wasthere any renal or pulmonary toxicity.

A total of five patients were treated at the 700-ng/kg doselevel. Two patients (one with melanoma and one with renal cellcancer) who had received high-dose IL-2,6 months before therhIL-12 had either no fever or low-grade fevers and minimal-to-no liver function test abnormalities during rhIL-12 treatment.In contrast, the other three patients who received either high-dose IL-2 therapy.1 year previously or low-dose IL-2.6months previously experienced higher and more sustained fe-vers (requiring both acetaminophen and indomethacin duringthe first 2 weeks of therapy) as well as more protracted consti-tutional symptoms (including malaise and anorexia). Two DLTswere observed among these three patients, including grade-3hemolytic anemia (occurring during week 5 of cycle 1) in onepatient and a grade-3 elevation of serum hepatic transaminases(occurring after the second dose of rhIL-12) in another (Table2). The hemolytic anemia was Coombs negative and requiredboth the discontinuation of the rhIL-12 and a 1-week course ofprednisone to resolve. IL-12-induced hypersplenism leading toextravascular hemolysis was suspected because CT scansshowed the development of splenomegaly after the first cycle ofrhIL-12 (not shown). The grade 3 transaminase elevation re-solved within 1 week of stopping the rhIL-12.

Safety Phase. On the basis of the two DLTs observed atthe 700-ng/kg dose level, the MTD for the twice-weekly sched-ule of i.v. rhIL-12 administered without a test dose was deter-mined to be 500 ng/kg. To better assess the safety of the MTD,an additional eight patients were treated at 500 ng/kg. As shownin Table 3, 7 of 8 patients tolerated the rhIL-12 well without anyDLTs. One patient tolerated cycle 1 without difficulty but thendeveloped grade-4 neutropenia after the first 2 weeks of cycle 2.Bone marrow biopsy revealed agranulocytosis, which resolvedafter discontinuation of the IL-12 and treatment with prednisoneplus low-dose oral cyclophosphamide.

With the exception of the case of agranulocytosis, nounusual or severe toxicities occurred among patients receivingtwo or more uninterrupted 6-week cycles of rhIL-12, includingtwo patients who had been on rhIL-12 for 36 and 48 weeks,respectively (Table 3). Several patients, including the one onrhIL-12 for 36 weeks, experienced grade 1–2 arthralgias (Table2), involving primarily the shoulders and fingers, beginningwith the second cycle of therapy. The arthralgias were episodic,unaccompanied by joint swelling or tenderness, and responsiveto therapy with nonsteroidal anti-inflammatory drugs.

Biological Effects of Twice-Weekly i.v. rhIL-12In Vivo IFN-g Induction. IFN-g levels were obtained in

eight patients treated at the 500-ng/kg dose level as well asin two patients enrolled at the 700-ng/kg dose level. As shown

Table 1 Patient characteristics

No. of patients

Total patients 28Median age (range) 56 yr (32–72 yr)Gender (male/female) 20/8Performance status

ECOGa 0 15ECOG 1 13

Tumor typesRenal cell cancer 20Melanoma 8

Prior therapyChemo/Immunotherapy 24

High-dose IL-2 14Low-dose IL-2 6IFN a-2b 12Biochemotherapy 3Chemotherapy 1

Surgery 20Radiotherapy 13None 1

Prior systemic treatment regimens0 41 122 103 2

Prior response to chemo/immunotherapy 3Disease sites

Lung 21Lymph nodes 13Skin or soft tissue 5Liver 9Bone 10Adrenal/kidney 6

Number of disease sites1 62 9

$3 13a ECOG, Eastern Cooperative Oncology Group.




in Fig. 2 and Table 4, we were able to discern three patterns ofIFN-g induction among these 10 patients. In all of the patterns,the first significant rise in plasma IFN-goccurred between 4 and8 h after the rhIL-12 dose, corresponding to the onset of fevers/chills. In the type-I pattern (Table 4 and Fig. 2A, top), the IFN-glevel peaked at a modest 450-1600 pg/ml (with peaks occurringbetween 8 and 24 h for individual patients) after the first rhIL-12dose (week 1/day 1). After the second dose (week 1/day 4), peaklevels were 2–3-fold higher than those induced by the first dose.However, after the seventh dose (week 4/day 1), peak IFN-glevels were comparable with those after the first dose. Patientswith this type-I pattern tended to have modest fever/chills aftereach rhIL-12 dose during cycle 1, with the most prominentsymptoms occurring after the second dose. However, whereasIFN-g could be detected in the plasma 24 h after an IL-12 dose,it always dropped to undetectable levels by the time of the nextdose 2–3 days later (Fig. 2,A-C, top). Patients exhibiting the

type-I pattern of IFN-ginduction had all been treated previouslywith an IL-2-based regimen and were either.6 months past alow-dose IL-2 regimen or.1 year past a high-dose IL-2regimen.

In the type-II pattern, peak IFN-glevels after the first dosewere, on the average, 2-fold higher than those measured inpatients with the type-I pattern (Table 4 and Fig. 2B, top). Theaugmentation in peak IFN-glevels after the second dose wasalso higher in the type-II pattern compared with the type-Ipattern, increasing 2- to 4-fold over the peak levels after dose 1.This difference in the magnitude of IFN-g production wasassociated with higher fevers and more pronounced chills/rigorsin these patients after the first two doses of rhIL-12 comparedwith patients exhibiting the type-I pattern of IFN-g induction.However, despite this larger surge of IFN-gproduction after thesecond dose, IFN-ginduction after the seventh dose of rhIL-12was markedly curtailed compared with IFN-glevels after the

Table 2 Number of patients experiencing select toxicities during treatment with IL-12 (grades 2, 3, 4)

Toxicity

Dose level, ng/kg (no. of patients)

30 (3) 100 (3) 300 (3) 500 (14) 700 (5)

HepaticASTa (4, 0, 0) (0, 1, 0)Bilirubin (1, 0, 0) (1, 0, 0)Alk phosphatase (1, 0, 0) (1, 0, 0)

HematologicNeutropenia (2, 1, 1) (2, 0, 0)Anemia (2, 0, 0)Hemolytic anemia (0, 1, 0)Thrombocytopenia (1, 0, 0) (1, 0, 0)

Oral mucositis (2, 0, 0) (1, 0, 0)Cardiovascular (hypotension) (1, 0, 0)Cardiovascular (arrhythmia) (1, 0, 0)Fever (1, 0, 0) (2, 1, 0) (2, 0, 0)Arthralgia (2, 0, 0)

a AST, aspartate aminotransferase; Alk, alkaline.

Table 3 Summary of tolerability of IL-12 among patients treated at the MTD of 500 ng/kg

Patientno.

No. ofcycles

completed Results

Dose escalation phase 10 8 No DLT11 3 No DLT12 1 Grade 2 orthostatic hypotension; IL-12

dose reduced to 300 ng/kg13 6 No DLT14 1 No DLT15 1 No DLT

Safety phase 21 1 No DLT22 2a Grade 4 neutropenia (agranulocytosis);

IL-12 discontinued23 1 No DLT24 2 No DLT25 1b No DLT26 1 No DLT27 3 No DLT28 2 No DLT

a Received four doses in cycle 2.b Received only two doses in cycle 1 because of rapid disease progression.




first dose. This was associated with a greatly diminished-to-absent febrile response to rhIL-12 by the third week of cycle 1.All of the three patients with this pattern of IFN-ginduction hadnot received any prior immunotherapy for their metastatic dis-ease.

The type-III pattern was characterized by modest peakIFN-g levels in response to the first dose of rhIL-12, followedby the rapid attenuation of IFN-gproduction (Table 4 and Fig.2C, top). Whereas peak IFN-glevels reached their maximumafter the second rhIL-12 dose in the type-I and type-II patterns,in the type-III pattern, peak IFN-glevels were lower after dose2 compared with those after dose 1 (Table 4), and they contin-ued to decline when measured again after the seventh dose. Thistype-III pattern was associated with a weak-to-absent febrileresponse to IL-12, and, of the three patients exhibiting thispattern, all had received one course or multiple courses ofhigh-dose IL-2,6 months before starting the rhIL-12.

In Vivo Induction of IL-15 and IL-18. To determinewhether IL-15 and IL-18 were induced by rhIL-12 in cancerpatients and to examine whether there was an association be-tween IL-15/IL-18 induction and IFN-ginduction by rhIL-12,we measured the plasma levels of IL-15 and IL-18 at the sametime points used to measure IFN-glevels. As shown in Fig. 2,A-C (middle), IL-15 was not detectable in the plasma beforestarting rhIL-12 but was detectable at low levels 4 h after thefirst injection. With each pattern of IFN-ginduction, plasmaIL-15 levels reached their maximum either before, or at thesame time as, peak IFN-glevels. However, the magnitude ofIL-15 induction did not correlate with the magnitude of IFN-ginduction, because peak IFN-g levels after the second rhIL-12dose in the type-I and type-II patterns were not associated withsimilar increases in peak IL-15 induction. Nonetheless, therewas an association between the ability to sustain comparablelevels of IL-15 induction during a cycle of rhIL-12 and the

Fig. 2 Patterns ofin vivo cytokine induction in patients during rhIL-12 therapy. Plasma concentrations of IFN-g, IL-15, and IL-18 were measuredin 10 patients treated with rhIL-12 at the 500-ng/kg and 700-ng/kg dose levels. Cytokine concentrations were determined before and after (every 4 hover a 24-h period) the first (Wk1D1), second (Wk1D4), and seventh (Wk4D1) rhIL-12 injections during cycle 1. The values for IFN-g, IL-15, andIL-18 at each time point inA represent the mean and SD derived from four patients with the type-I pattern of IFN-ginduction. InB andC, representingthe type-II and type-III patterns of IFN-ginduction, respectively, the values at each time point for IFN-gand IL-15 represent the mean and SD derivedfrom three patients for each pattern, whereas the data for IL-18 were from one patient (similar results seen with two patients examined) for eachpattern.




ability to sustain IFN-ginduction. As shown in Fig. 2A(middle),only small differences in the peak and plateau levels of IL-15after the first, second, and seventh rhIL-12 doses were evident inpatients with the type-I IFN-gpattern. In contrast, there was a50–70% drop in the peak and plateau IL-15 levels by week 4 inpatients with the type-II and type-III IFN-g patterns (Fig. 2,B-C, middle), and no IL-15 could be detected in the plasmabefore the seventh rhIL-12 dose in these patients.

In all of the patients tested, small amounts of IL-18 weredetected in the plasma before starting rhIL-12 (Fig. 2,A-C,bottom), with higher levels present before the second and sev-enth rhIL-12 doses. However, plasma IL-18 levels after anrhIL-12 injection usually peaked later than IFN-g. In addition,the loss of IFN-gproduction after the seventh rhIL-12 doseamong patients with the type-II and type-III IFN-g patternsoccurred despite continued IL-18 production at levels compara-ble with those measured immediately before and after the firstrhIL-12 dose.

IL-10 Induction and rhIL-12 Pharmacokinetics. Asshown in Fig. 3A, IL-10 was induced after the first dose ofrhIL-12 in a similar manner in all of the patients, returning toundetectable levels before the next rhIL-12 dose. A strongerincrease in IL-10 production was observed after the second dosein all of the patients, with the highest peak levels detected inpatients with the type-II IFN-gpattern (Fig. 3A,middle). How-ever, IL-10 induction continued after the seventh dose in all ofthe patients, including those with the type-I IFN-g pattern (Fig.3A, top), as well as those with the type-III pattern exhibiting asignificant suppression of IFN-g (Fig. 3A,bottom).

When the pharmacokinetics of rhIL-12 was examined, wefound that the levels of IL-12 in the plasma fell more quicklyafter the second and subsequent rhIL-12 doses compared withafter the first dose (Fig. 3B). However, these changes were notnecessarily associated with changes in IFN-ginduction. Forexample, for patients with the type-I IFN-g induction pattern,plasma IFN-glevels were comparable after the first and seventhIL-12 doses despite the more rapid drop in IL-12 levels after theseventh dose. Furthermore, as shown in Fig. 3B, after the secondand seventh doses of IL-12, the molar concentrations of IL-12

measured in the plasma 4–24 h after the IL-12 injection werelower for a patient with the type-I IFN-gpattern (Fig. 3B,top)compared with a patient with the type-III IFN-g pattern (Fig.3B, bottom).

Clinical Response to rhIL-12: Association with IFN-gInduction Pattern

Among nine patients treated at rhIL-12 dose levels thatwere below the MTD of 500 ng/kg, there were no responses, nordid any of these patients achieve disease stabilization for morethan two cycles. At the MTD (n5 14), one patient with renalcell cancer had a PR, and two others with renal cell cancer havehad long-term disease stabilization for 61 and 121months(Table 4). Among the 7 of 14 patients who received more thanone cycle of IL-12 (Table 3), none required a break betweencycles. The patient with the PR had multiple, large, pleural-based, and parenchymal lung metastases (Fig. 4A) as well as asolitary liver metastasis, and had disease progression after acourse of low-dose IL-2 before starting the IL-12. After the firstthree cycles of rhIL-12, the patient had stable disease but, afterthe fourth cycle, showed the first signs of tumor regression inthe lung. After six cycles, he had achieved a PR, with completeresolution of most of the parenchymal and pleural-based tumorsand a 70% reduction in the size of the largest pleural-based mass(Fig. 4B). The single liver lesion remained unchanged. TherhIL-12 was stopped during the seventh cycle because of thedevelopment of a small cerebellar metastasis, which was ex-cised. Follow-up CT scans 4 months after stopping the rhIL-12revealed a continued regression of the remaining lung tumors(Fig. 4C).

One of the patients (Table 4, patient 10) with long-termdisease stabilization had multiple lung, liver, bone, and lymphnode metastases, and a history of disease progression while onhigh-dose IL-2. She remains on rhIL-12 therapy after havingcompleted 8 cycles. CT scans after cycle 8 showed the first signsof tumor regression, with shrinkage of lesions in the liver andabdominal lymph nodes (not shown). The other patient withlong-term disease stabilization (Table 4, patient 22) had lymphnode, bone, and lung metastases and also had progressive dis-

Table 4 Relation between pattern of IFN-ginduction by IL-12 during cycle 1, clinical response, and cytopenias

IFN-ginductionpattern Disease

Cycle 1 Peak IFN-glevel (pg/ml) IL-12 doselevel

(ng/kg) Response CytopeniaWk1D1a Wk1D4 Wk4D1

Type IPatient 10 RCC 1600 2560 1200 500 SD @48 wk None

13 RCC 1100 2235 1340 500 PR @36 wk None18 RCC 450 1290 400 700 SD @20 wk Hemolytic anemia22 RCC 1000 1840 850 500 SD @24 wk Agranulocytosis

Type IIPatient 24 RCC 2365 4960 220 500 PD @12 wk None

26 RCC 2040 9060 248 500 PD @6 wk None28 Mel 1560 5918 77 500 PD @12 wk None

Type IIIPatient 21 Mel 2229 1476 159 500 PD @6 wk None

23 Mel 1256 951 90 500 PD @6 wk None19 RCC 1800 890 125 700 PD @6 wk None

a Wk1D1, week 1 day 1; Wk1D4, week 1 day 4; Wk4D1, week 4 day 1; RCC, renal cell cancer; Mel, melanoma; SD, stable disease; PD,progressive disease.




ease before beginning rhIL-12 despite having received high-dose IL-2 1 year earlier. The rhIL-12 was discontinued in thispatient because of the development of agranulocytosis, but hecontinues to have stable disease 4 months after the cessation ofthe rhIL-12.

At the 700-ng/kg dose level, one renal cell cancer patientwith extensive lung, bone, and lymph node metastases, who hadfailed prior treatment with low-dose IL-2, received 5 weeks ofrhIL-12 before it was stopped because of the development of aCoombs’ negative hemolytic anemia (Table 4, patient 18). Thispatient continues to have stable disease 4 months after stopping

the rhIL-12. Among the remaining 15 patients treated at the500-ng/kg and 700-ng/kg dose levels, all had disease progres-sion during the first 1–3 cycles of rhIL-12, with the majorityprogressing during the first cycle.

When we examined the responses among the 10 patientsselected for IFN-g/IL-15/IL-18 analysis (Table 4), we foundthat, of the patients who were unable to sustain IFN-ginductionthrough the first cycle (type-II and type-III patterns), all haddisease progression. In contrast, of the four patients who hadeither a PR or prolonged disease stabilization, all exhibited thetype-I pattern of sustained IFN-g induction. In addition, the two

Fig. 3 A, IL-10 induction in patients treated with rhIL-12. Plasma IL-10 levels were measured before and during a 24-h period after the first, second,and seventh rhIL-12 doses in patients with the type-I (top), type-II (middle), and type-III (bottom) IFN-ginduction patterns. Each panel shows thedata from one patient in each group (similar results were obtained from two patients in each group).B, plasma IL-12 levels in patients after injectionsof i.v. rhIL-12. The p70 IL-12 heterodimer was measured in the plasma at regular intervals over a 20-h period starting 4 h after a rhIL-12 injection.This analysis was performed after the first (Wk1D1), second (Wk1D4), and seventh (Wk4D1) IL-12 injections during cycle 1. The results from twopatients treated at the 500-ng/kg dose level are shown, including one patient with the type-I IFN-g induction pattern (top) and the other with thetype-III pattern (bottom). These are representative of what was observed in four patients treated with 500 ng/kg rhIL-12. The concentration of IL-12represented on the Y axis is converted from pg/ml to molar concentration at several points, as indicated next to the horizontal dotted lines on eachgraph.




Fig. 4 Antitumor response in a patient treated withtwice-weekly i.v. rhIL-12. CT scans of the chest in apatient with renal cell cancer metastatic to the lung,performed before starting rhIL-12 (A), after six cyclesof rhIL-12 (B), and 4 months after the completion ofsix cycles of rhIL-12 (C).




episodes of cytopenias, including the Coombs negative hemo-lytic anemia that was responsive to steroids and the agranulo-cytosis that was responsive to steroids plus low-dose cyclophos-phamide, occurred among patients with the type-I IFN-ginduction pattern.

Although patients with the type-II or type-III IFN-ginduction pattern all had progressive disease on rhIL-12, twopatients with metastatic melanoma developed ecchymosesthat appeared only over their s.c. metastases (Fig. 5). Theseecchymoses developed during the first 2 weeks of rhIL-12therapy but were not associated with any change in the size

or consistency of the metastases. Neither patient had anymeasurable coagulopathy or significant thrombocytopeniaduring IL-12 therapy (data not shown), and there were noother cutaneous or mucosal changes. By the 6th week oftherapy, the ecchymoses over the s.c. metastases had fadedconsiderably in both patients, and there was no measurabletumor regression. Excisional biopsy of one of these s.c.lesions at the end of cycle 1 (at which time the overlyingbruising had resolved) revealed viable tumor with no lym-phocytic infiltration and no ischemic necrosis (data notshown).

Fig. 5 Skin changes in melanoma pa-tients treated with rhIL-12. Two patients(A andB) with melanoma developed ex-tensive ecchymoses limited to the sitesof s.c. metastases during the first severalweeks of rhIL-12 therapy.




Effect of rhIL-12 Therapy on Lymphocyte CytokineResponsiveness

Among patients exhibiting the type-II and type-III IFN-ginduction patterns, we found that the proliferative response toIL-12, IL-2, and IL-15 was preserved at the midway point ofcycle 1 (Fig. 6A,top). In contrast, the ability of IL-2, IL-12, andIL-15 to induce IFN-gproduction was completely abrogated bythe start of week 4 (Fig. 6A,bottom). The induction of IFN-gbythe combination of IL-12 plus IL-15 was also greatly diminishedat the start of week 4 compared with the start of week 1. Thischange in IFN-gproduction was not associated with any changein the number of circulating CD41and CD81T cells or NKcells, nor was it associated with any change in lymphocyte IL-12or IL-2 receptor expression (data not shown). Although IFN-gproduction in response to IL-12 plus IL-2 decreased as well

during rhIL-12 therapy, the combination of IL-12 plus IL-2 stillinduced higher levels of IFN-g in vitro at week 4 than thoseinduced by IL-12 plus IL-15 at week 1 (Fig. 6A, bottom). Whenwe examined thein vitro cytokine response of PBMC isolatedfrom patients exhibiting the type-I pattern of IFN-g induction,we found that proliferation and, to a large extent, IFN-g pro-duction were preserved from weeks 1 to 4 (Fig. 6B).

NO Production in Patients Receiving rhIL-12 or IL-2For patients treated with i.v. rhIL-12 at the 500-ng/kg and

700-ng/kg dose levels, expired air was collected before startingrhIL-12 and on the morning after the first two doses. BeforerhIL-12, the mean NO concentration was 216 9.5 ppb. Themean peak NO level after the first two doses of rhIL-12 wasonly 27.5 6 7.8 ppb, with patients, on average, exhibiting a

Fig. 6 In vitro response of PBMCs to cytokines before and midway through the first cycle of rhIL-12. PBMCs were isolated from the blood beforethe start of rhIL-12 therapy (Wk1D1) and before the start of the 4th week of cycle 1 (Wk4D1). Cells were then cultured in medium alone (neg), IL-2,IL-12, IL-15, IL-2 1 IL-12, or IL-15 1 IL-12 for 72 h, after which both proliferation (A andB, top) and IFN-gproduction (AandB, bottom) weremeasured.A shows the data from a patient with the type-II IFN-gpattern (representative of three type-II patients examined), whereasB shows thedata from a patient with the type-I IFN-gpattern (representative of three type-I patients examined).




1.5-fold increase (range, 1- to 2-fold) over the baseline value(Fig. 7). The one patient with the modest 2-fold increase in NOproduction was treated at the 700-ng/kg dose level and had apeak plasma IFN-glevel of 40,000 pg/ml after the second doseof rhIL-12. Among patients with renal cell cancer and mela-noma who were receiving the standard i.v. bolus high-dose IL-2regimen (600,000 IU/kg every 8 h3 14 doses; Ref. 28), themean NO concentration was 14.76 5.6 ppb before starting IL-2and 536 33 ppb during IL-2 therapy. Patients, on average,exhibited a 3.5-fold increase (range, 2- to 6-fold) in exhaled airNO concentration compared with the baseline value (Fig. 7).

DISCUSSIONIn this report, we have shown that a new schedule of i.v.

rhIL-12, consisting of twice-weekly injections administeredwithout a test dose for 6 weeks, has acceptable toxicity at theMTD of 500 ng/kg and is capable of inducing antitumor re-sponses. We observed one response (ongoing PR) among 14patients treated at the MTD, with no responses at doses,500ng/kg. The kinetics of this patient’s response is of particularinterest, because regression did not begin until he had beencontinuously on the rhIL-12 for 6 months. This pattern ofprolonged disease stabilization followed by tumor regressioncould be indicative of an antiangiogenic effect of rhIL-12 (29).A similar pattern of delayed response to therapy in renal cellcancer patients has been reported with thalidomide (30), anotheragent shown to have antiangiogenic effects (31). Among theother three patients with renal cell cancer who were treated at,or above, the MTD and who have had long-term disease stabi-lization, only one has remained on IL-12 for more than twocycles. After 8 cycles, this patient is starting to exhibit the firstsigns of regression of multiple liver and abdominal lymph nodemetastases. Therefore, for patients treated with twice-weeklyi.v. rhIL-12, disease stabilization that is maintained for a pro-longed period of time may have a significant likelihood oftranslating into tumor regression. This finding provides a com-pelling reason for dosing rhIL-12 chronically, a strategy that

may also prove to be necessary for antitumor responses toagents that are more “pure” antiangiogenic drugs.

Although the majority of patients enrolled in the trial oftwice-weekly i.v. rhIL-12 had received prior immunotherapy,their response to prior therapy was not predictive of a responseto rhIL-12. In fact, the patients with renal cell cancer whoexhibited disease regression or prolonged disease stabilizationin response to rhIL-12 had all failed prior therapy with IL-2.This finding suggests that IL-12 and IL-2 may mediate tumorregression through distinct mechanisms and is an indication thatrhIL-12 may be a viable treatment option in renal cell cancerpatients who have not responded to IL-2.

One of the goals of measuringin vivocytokine induction inour patients who were receiving rhIL-12 was to determinewhether there was any association between patterns of IFN-ginduction and antitumor responses. Importantly, it was not themagnitude of IFN-gproduction but rather the ability to sustainIFN-g induction by rhIL-12 during the first cycle that wasassociated with outcome. Plasma IFN-g levels that were inducedby the twice-weekly schedule at the MTD of 500 ng/kg werecomparable with the levels induced by the daily-for- 5-daysschedule with a test dose at 500 ng/kg (18, 19). However,consistent with what has been demonstrated in previous trials ofrhIL-12, the majority of patients treated with twice-weekly i.v.rhIL-12 lost the ability to produce IFN-grelatively soon afterthe start of dosing. Importantly, those few patients with thetype-I IFN-g induction pattern, who produced only modestamounts of IFN-gbut could sustain that induction over thecourse of the first cycle, exhibited signs of antitumor immunitysuch as tumor regression or disease stabilization. This is the firstdemonstration in cancer patients that sustained IFN-g inductionby rhIL-12 may be necessary for antitumor responses, and it isconsistent with the murine data which have shown that rhIL-12-induced tumor regression is IFN-g-dependent (10). The as-sociation of high plasma IFN-glevels (5,000–10,000 pg/ml)after the second rhIL-12 dose with subsequent down-modulationof IFN-g induction (type-II IFN-g induction pattern) suggeststhat overactive immune stimulation may lead to the early cur-tailment of IL-12 responsiveness. If antiangiogenic and cytolyticantitumor responses to rhIL-12 require chronic immune stimu-lation, then strong levels of immune activation that can bemaintained for only brief periods of time are likely to becounterproductive in patients treated with rhIL-12.

Hemolytic anemia or agranulocytosis, requiring cessationof the rhIL-12 and treatment with a brief course of low-doseCytoxan and/or prednisone, occurred in 2 of 19 patients treatedat the 500-ng/kg and 700-ng/kg dose levels on the twice-weeklyschedule. These toxicities have never been reported amongpatients treated with i.v. or s.c. rhIL-12 on other dosing sched-ules (18, 20–22, 32), nor have they been observed in patientstreated with IL-2 (33). The finding that these rhIL-12-inducedcytopenias persisted for 1 week after stopping the rhIL-12 andwere resolved only after treatment with immunosuppressivedrugs such as prednisone and Cytoxan suggests they may havebeen autoimmune phenomena and raises the possibility that thismay be a unique toxicity of chronic stimulation with rhIL-12 incertain susceptible patients. However, it is also notable thatthese cytopenias were restricted to two of the four patients whowere able to maintain IFN-ginduction during cycle 1 (Table 4).

Fig. 7 NO production in patients treated with either high-dose i.v. IL-2or twice-weekly i.v. rhIL-12.f, the mean baseline concentration of NOin the expired air of patients before starting either IL-2 (n5 11) orrhIL-12 (n 5 5); u, the mean peak NO concentration in expired airmeasured during IL-2 or rhIL-12 therapy.




In mice, IL-12 has been shown to induce bone marrow suppres-sion as well as splenomegaly, both of which are dependent onIL-12-induced IFN-g(34). Therefore, the agranulocytosis andCoombs negative hemolytic anemia may not have been theresult of specific immune responses directed against erythroid ormyeloid antigens. Instead, direct toxic effects of rhIL-12-induced IFN-gon myeloid precursors, as well as stimulatoryeffects of IFN-gon NK cells (34), monocytes, and other reticu-loendothelial elements in the spleen, may have led to the ob-served cytopenias.

The bruising that developed over the s.c. metastases in twopatients with melanoma was provocative, for it appeared duringthe first several weeks of cycle 1 when IFN-g was being inducedby rhIL-12 and then resolved when IFN-g induction was shutdown. Whereas it is possible that these ecchymoses representedthe early phase of an antiangiogenic process that was abortedwhen IFN-ginduction ceased, there was no tumor regression orbiopsy evidence of tumor necrosis/microvessel damage to sup-port this hypothesis.

IFN-g production in vivo indicates that the elements re-quired for the elicitation of a T helper type-1-like immuneresponse by IL-12 are present and intact. As IL-12 by itself isonly a weak inducer of IFN-gproduction by PBMCsin vitro,the induction of endogenous costimulatory cytokines such asIL-15 and IL-18 may be necessary to induce IFN-g productionin vivo. Our results show for the first time that rhIL-12 doesinduce both IL-15 and IL-18 productionin vivo in cancerpatients. Because IL-15 and IL-18 are synthesized by activatedmonocytes and dendritic cells (35, 36), it is possible that thedirect activation of these antigen-presenting cells by rhIL-12 (6)in vivo was responsible for the induction of these costimulatorycytokines. However, as IFN-g was often induced concurrentlywith IL-15 and IL-18, it is not possible to deduce whether theIL-15 and IL-18 augmented IFN-gproduction by IL-12 (36, 37),or whether rhIL-12-derived IFN-g may have augmented anti-gen-presenting cell production of IL-15 and IL-18. It is possiblethat both mechanisms were operative. It is notable that IL-18could be detected in the plasma even before the first dose ofrhIL-12, which suggests that low-level constitutive productionof IL-18 may allow for the immediate synergistic activation oflymphocytes early during an immune response when IL-12 isfirst being synthesized. Whereas IL-15 was not detectable in theplasma before starting IL-12, small amounts (5–10 pg/ml) ofIL-15 were induced by rhIL-12 within 4 h after an injection.However, it is not clear whether the small amount of IL-15detected in plasma with an ELISA represents the true amountavailablein vivo for lymphocyte activation, or whether such alow effective concentration would be capable of influencingIFN-g production together with IL-12 and IL-18. It is notablethat monocytes have been shown to express a membrane-boundform of IL-15 that is capable of stimulating lymphocytes (38),because this suggests that the amount of IL-15 induced byrhIL-12 in vivo and available to activate lymphocytes in con-junction with rhIL-12 may be greater than the amount detectedin the plasma by ELISA.

Because IL-15 and IL-18 are induced by rhIL-12 and maybe involved in rhIL-12-induced IFN-ginduction, it is reasonableto speculate that changes in the production of these costimula-tory cytokines could underlie the attenuation of IFN-gproduc-

tion during rhIL-12 therapy. In patients with sustained IFN-ginduction during cycle 1, the magnitude of IL-15 inductionremained constant, whereas peak and plateau levels of IL-15diminished 50–70% by mid-cycle in patients unable to sustainIFN-g induction at week 4. This may be an indication that IL-15production is operative in IFN-g production by rhIL-12in vivo,or it may simply reflect the fact that the production of smalleramounts of IFN-gleads to weaker IFN-g-induced IL-15 pro-duction. Unlike IL-15, IL-18 induction remained fairly intact bymid-cycle, even in patients whose IFN-g induction had greatlyattenuated. If IL-18 is necessary for IFN-g induction by rhIL-12,this observation may be an indication that lymphocyte respon-siveness to IL-18 was diminished midway through the first cycleof rhIL-12. Alternatively, because an IL-18 binding protein(IL-18BP) that neutralizes the activity of IL-18 has recentlybeen identified (39), it is possible that the down-modulation ofIFN-g production may have involved the induction of IL-18BPby rhIL-12.

The attenuation of IFN-gproduction at week 4 of cycle 1in patients with the type-II and type-III IFN-g induction patternswas associated with an acquired defect inin vitro IFN-g pro-duction stimulated by IL-12, IL-2, or IL-15. This defect wasselective, inasmuch as proliferation induced by these cytokineswas unaffected. Furthermore, the defect was not observed inpatients with the type-I IFN-ginduction pattern. Because IL-12did not diminish the number of circulating T or NK cells and didnot down-regulate IL-12 or IL-2/IL-15 receptor expression,4 it islikely that the diminished IFN-g response to these cytokinesrepresents either an acquired defect in lymphocyte cytokinesignaling and/or a defect in monocyte/dendritic cell function. Itremains to be determined whether this change in IL-2/IL-15-and IL-12-induced IFN-gproduction is attributable to alter-ations in the Jak/Stat (40), mitogen-activated protein kinase(41), or NF-kB (6) signaling pathways used by these cytokines,or whether it might involve changes in theIFN-g gene itself,such as methylation of the promoter (42). Although IL-10 wasinducedin vivo in patients with the type-II and type-III IFN-gpatterns, it was also strongly induced in patients with the type-Ipattern. It seems unlikely, therefore, that the observed defect inIFN-g productionin vitro was simply the result of an inhibitoryeffect of IL-10 on lymphocytes or monocytes/dendritic cells. Inaddition to its potential inhibitory effects on IFN-g production,IL-10 can also stimulate IFN-g production by NK cells incombination with IL-18 (43). This dual effect of IL-10 may havecontributed to our inability to detect an association betweeninvivo IL-10 and IFN-gproduction in patients receiving rhIL-12.

Our analysis of plasma IL-12 levels after bolus i.v. injec-tions of rhIL-12 showed that rhIL-12 was cleared more rapidlyfrom the blood after the second and seventh doses comparedwith after the first dose. However, this accelerated clearance byitself did not seem to be responsible for the attenuation of IFN-gproduction midway through the first cycle, because the samepharmacokinetics were observed in patients with the type-I andtype-II/type-III IFN-g induction patterns. Although diminishedIFN-g production has correlated with the down-regulation of

4 J. A. Gollob, K. Veenstra, and J. W. Mier, unpublished observations.




serum IL-12 levels (44) in patients and mice receiving multipledoses of s.c. IL-12, the mechanisms underlying the decrease inIFN-g and IL-12 levels have not yet been elucidated. As peaklevels of rhIL-12 in the plasma are considerably higher after i.v.bolus injection than after s.c. injection (18, 20), acceleratedclearance occurring with repeated injections may have a moredetrimental impact on the effectivein vivo concentration ofrhIL-12 in patients treated via the s.c. route.

Whereas NO has been implicated in mice as a cause ofIL-12-induced immune suppression (26), we detected little NOproduction in patients treated with twice-weekly i.v. rhIL-12.This was in contrast to patients treated with high-dose IL-2, inwhom we observed 3- to 5-fold increases in NO productionduring therapy. Although it is possible that expired air NO didnot reflect systemic NO production in our patients, this isunlikely because, in animals treated withlipopolysaccharide(LPS), the augmentation of systemic NO production correlateswell with changes in the NO concentration in expired air (45,46). Furthermore, becausein vitro cytokine-induced IFN-gpro-duction was largely unaffected in patients treated with high-doseIL-24 (whereas it was inhibited in many patients treated withrhIL-12), it seems unlikely that NO production is an importantcause of diminished IFN-gproduction in patients receivingrhIL-12.

If, as our results suggest, chronic T helper type-1-likeimmune activation involving IFN-g production is necessary forrhIL-12-induced antitumor effects, is it possible to prevent ordelay the down-modulation of IFN-ginduction in patientstreated with rhIL-12? Whereas genetic factors, tumor burden, orprior treatment history may be determinants of the type of IFN-ginduction pattern that a patient will exhibit during rhIL-12therapy, it is clear that the majority of patients will fail to sustainIFN-g induction and will not respond to rhIL-12. However, ourfindings indicate that the down-modulation of IFN-g inductionby i.v. rhIL-12 may not be an insurmountable problem, for it isnot attributable to irreversible factors such as the loss of keylymphocyte subsets, the down-regulation of cytokine receptorexpression, or greatly diminished bioavailability of the admin-istered rhIL-12. Whereas our data indicate that changes ininvivo costimulatory cytokine production and lymphocyte respon-siveness to these cytokines probably contribute to the attenua-tion of IFN-g induction, we have also shown that lymphocyteIFN-g production by IL-12 can be revivedin vitro if IL-2 isadded. It is possible, therefore, that strategies involving theaddition of IL-2 to i.v. rhIL-12 may be able to lengthen theduration of immune stimulation by rhIL-12in vivo, therebyaugmenting its antitumor activity.

ACKNOWLEDGMENTSWe thank Donna Martin, Nancy Brown, Christine Moan, JoAnne

Swenson, Julie Carpenter, Nancy Salonpuro, Debora Weed, and the restof the staff in the Clinical Research Center of the Beth Israel DeaconessHospital for their nursing support.

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2000;6:1678-1692. Clin Cancer Res Jared A. Gollob, James W. Mier, Korina Veenstra, et al. with Clinical Response

Induction Is AssociatedγMelanoma: Ability to Maintain IFN-Patients with Metastatic Renal Cell Cancer or Malignant Phase I Trial of Twice-Weekly Intravenous Interleukin 12 in

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Phase I Trial of Twice-Weekly Intravenous Interleukin 12 ... · Jared A. Gollob,2 James W. Mier, Korina Veenstra, David F. McDermott, Daniel Clancy, Marguerite Clancy, and Michael