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Vol. 3, 409-417, March 1997 Clinical Cancer Research 409
Phase I Evaluation of Intravenous Recombinant Human Interleukin
12 in Patients with Advanced Malignancies1
Michael B. Atkins,2 Michael J Robertson,
Michael Gordon, Michael T. Lotze,
Michelle DeCoste, Jon S. DuBois, Jerome Ritz,
Alan B. Sandler, Howard D. Edington,
Pamela D. Garzone, James W. Mier,
Christine M. Canning, Linda Battiato,
Hideaki Tahara, and Matthew L. Sherman
Tupper Research Institute and the Division of Hematology/Oncology,Tufts-New England Medical Center, Boston, Massachusetts 021 1 1[M. B. A., J. S. D., J. W. M.]; Division of Hematologic Malignancies,Dana-Farber Cancer Institute, Boston, Massachusetts 021 15 [M. J. R.,J. R., C. M. C.]; Division of Hematology/Oncology, IndianaUniversity School of Medicine, Indianapolis, Indiana 46202 [M. 0.,A. B. S., L. B.]; Department of Surgical Oncology, Pittsburgh CancerInstitute, Pittsburgh, Pennsylvania 15261 [M. T. L., H. D. E., H. I.];
and Genetics Institute, Inc., Cambridge, Massachusetts 02140 [M. D.,P. D. 0., M. L. S.]
ABSTRACT
A Phase I dose escalation trial of i.v. administered recom-
binant human interleuldn 12 (rhIL-12) was performed to de-termine its toxicity, maximum tolerated dose (MTD), pharma-
cokinetics, and biological and potential antineoplastic effects.
Cohorts of four to six patients with advanced cancer, Karnof-
sky performance �70%, and normal organ function received
escalating doses (3-1009 ng/kg/day) of rhIL-12 (Genetics Insti.
tute, Inc.) by bolus i.v. injection once as an inpatient and then,
after a 2-week rest period, once daily for five days every 3weeks as an outpatient Therapy was withheld for grade 3toxicity (grade 4 hyperbilirubinemia or neutropenia), and dose
escalation was halted if three of six patients experienced adose-limiting toxicity (DLI). After establishment of the MTD,eight more patients were enrolled to further assess the safety,
pharmacokinefics, and immunobiology of this dose.Forty patients were enrolled, including 20 with renal can-
cer, 12 with melanoma, and 5 with colon cancer; 25 patients
had received prior systemic therapy. Common toxicities in-
cluded fever/chills, fatigue, nausea, vomiting, and headache.
Fever was first observed at the 3 ng/kg dose level, typically
Received 8/20/96; revised 1 1/21/96; accepted 12/2/96.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 Grants MOl-RR-00054 and MOl-RR7SO from the Division of Research Resources, NIH, and a stipendfrom Genetics Institute, Inc.2 To whom requests for reprints should be addressed, at Division ofAdult Hematology/Oncology, Department of Medicine, New EnglandMedical Center, 750 Washington Street, Box 542, Boston, MA 021 1 1.Phone: (617) 636-6527; Fax: (617) 636-0342.
occurred 8-12 h after rhIL-12 administration, and was incom-pletely suppressed with nonsteroidal anti-inflammatory drugs.
Routine laboratory changes included anemia, neutropenia,
lymphopenia, hyperglycemia, thrombocytopenia, and hy-poalbuminemiL DLTs included oral stomatitis and liver func-lion test abnormalities, predominantly elevated transaminases,
which occurred in three offour patients at the 1000 ng/kg dose
leveL The 500 ngflg dose level was determined to be the MTh.This dose, administered by this schedule, was associated withasymptomatic hepatic function test abnormalities in three pa-tients and an onstudy death due to Closb*Iia perfringens sep-
ticemia but was otherwise well tolerated by the 14 patients
treated in the dose escalation and safety phases. The T,,� elim-
ination of rhIL-12 was CalCUlated to be 5.3-9.6 h. Biologicaleffects included dose-dependent increases in circulating IFN-�y,
which exhibited attenuation with subsequent cycles. Serum
neopterin rose in a reproducible fashion regardless of dose orcycle. Tumor necrosis factor a was not detected by ELISA.
One of 40 patients developed a low titer antibody to rhIL-12.
Lymphopenia was observed at all dose levels, with recovery
occurring within several days of completing treatment without
rebound lymphocytosis. There was one partial response (renalcell cancer) and one transient complete response (melanoma),both in previously untreated patients. Four additional patients
received all proposed treatment without disease progression.rhIL-12 administered according to this schedule is biologically
and clinically active at doses tolerable by most patients in anoutpatient setting. Nonetheless, additional Phase I studies ex-aniining different schedules and the mechanisms of the specificDLTs are indicated before proceeding to Phase II testing.
INTRODUCTION
IL-l23 is a heterodirnenc cytokine with potent immunoregu-
latory activity. It was initially given the names natural killer cell
stimulatory factor and cytotoxic lymphocyte maturation factor (1,
2) based on its stimulatory effects on these cytolytic lymphokine
populations. The cDNAs encoding the two IL-l2 subunits are
unrelated and encode for two distinct proteins having molecular
masses of approximately 35 and 40 kDa (3, 4). The p35 subunit is
distantly related to IL-6, granulocyte colony-stimulating factor, and
chicken myelomonocytic growth factor, and the p40 subunit is
related to the extracellular domain of the IL-6 receptor, as well as
to the ciliaiy neurotrophic growth factor receptor (5, 6).
In vitro studies have shown that monocytes appear to be the
major source of IL-l2 in activated PBMC suspensions (7). Al-
though EBV-transforrned B cells appear to produce IL-l2, signif-
3 The abbreviations used are: IL, interleukin; PBMC, peripheral bloodmononuclear cell; NK, natural killer; TNF, tumor necrosis factor; AST,aspartate aminotransferase; DLT, dose-limiting toxicity; MID, maxi-
mum tolerated dose; ANC, absolute neutrophil count.
Research. on January 3, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
410 Phase I Trial of iv. rhIL-12 in Advanced Malignancies
icant production by T cells or NK cells has not been demonstrated.
Two low-affinity rhIL-l2 receptor subunits have been cloned,
which together constitute a high-affinity IL-12 receptor (8). High-
affinity IL-12 receptors have been identified on phytohernaggluti-
nm-activated T cells and IL-2-activated NK cells (9, 10). In addi-
tion, specific binding sites (possibly low-affinity) have recently
been detected on the Bl subset of B lymphocytes (1 1).
IL- I 2 has been shown to regulate a number of activities
central to both cellular and humoral immune responses. For
example, in vitro studies have demonstrated that IL- 1 2 can
enhance the non-MHC-restricted cytolytic activity of NK cells
( I 2, 1 3) and facilitate specific allogeneic human cytolytic T
lymphocyte responses against allogeneic irradiated melanoma
cells (14). IL-12 has been shown to be mitogenic, allowing the
continued proliferation of both activated T lymphocytes and NK
cells (15-17). IL-12 has also been shown to be a potent inducer
of IFN-�y release from both T lymphocytes and NK cells (1, 18)
while inducing much lower amounts of inflammatory cytokines,
such as TNF-a, especially when compared with IL-2 (13, 19).
Furthermore, IL-l2 promotes T helper 1 cell development while
inhibiting T helper 2 cell development and characteristic cyto-
kine expression (20-22), favoring the development of cellular
immunity and immunological memory over hurnoral immunity.
In this regard, exposure to IL-12 enhanced survival in murine
models of toxoplasmosis and leishmaniasis, two intracellular
infections that require host T helper 1 cell responses (23-25).
IL- 12 has also shown potent antitumor activity in a number
of in vivo murine tumor models, including the Bl6FlO mela-
norna, RENCA renal cell adenocarcinoma, and M5076 reticu-
lum cell sarcoma (26, 27). i.p. administration of IL-l2 sup-
pressed the growth of both pulmonary and s.c. BI6F1O
melanoma deposits and induced regression of RENCA. In the
RENCA model, long-term survival and resistance to rechallenge
were observed in mice injected peniturnorally with IL-l2. An-
titumor effects were demonstrated both in microscopic disease
models and in animals bearing large established tumors in which
IL-l2 was initiated 7, 14, or even 28 days after tumor inocula-
tion (26, 27). The antiturnor activity observed in these model
systems was more potent than that observed with IL-2 (26).
Antitumor activity was slightly reduced in cytotoxic granule-
deficient beige mice, but was nearly eliminated in T cell-defi-
cient nude mice (26). Depletion of the CD8+ (but not CD4+)
T-cell subset by systemic administration of monoclonal antibod-
ies diminished the efficacy of IL- 1 2 in the RENCA system (26),
whereas depletion of either CD8+ or CD4+ T cells reduced
antitumor activity in other models (27). In addition, most studies
have shown that administration of antibodies to IFN-’-y signifi-
cantly reduces IL-12 antitumor activity (4). These experiments
suggest that the antitumor effect of IL-l2 is largely mediated by
CD4+ and CD8+ T cells through the release of IFN--y. How-
ever, the antitumor effect of IL- I 2 in most tumor systems is
superior to that of IFN-�y, and furthermore, efficacy is absent in
nude mice despite dramatically enhanced IFN-’y production
(26), suggesting that other factors besides IFN-� must contribute
to the antitumor effects of IL-l2. These preclinical data suggest
that IL- 1 2 may have potent irnmunoregulatory and antiturnor
activities that might be useful in the treatment of human infec-
tions and malignancies and encouraged us to proceed with this
Phase I clinical trial of rhIL-12 in cancer patients.
PATIENTS AND METHODS
Patient Selection. All patients were adults with histologi-
cally proven advanced malignancy that was measurable, clearly
progressive, and refractory to conventional therapy. Patients were
required to have a Kamofsky performance status of �70% and
adequate organ function defined by white blood count > 4000/p.L,
hemoglobin > 10 g/dl, platelet count > 100,000/pA, creatinine <2
mg/dl, creatinine clearance > 60 mI/mm, bilirubin < 2.0 mg/dl,
AST < 2 times the upper limit of normal, electrocardiogram and
chest X-ray without clinically significant nonmalignant abnormal-
ities, and an FEV1 > 2.0 liters or 75% of predicted. Patients with
history of peripheral neuropathy, brain metastases, or other central
nervous system disease, prior exposure to nephrotoxic or neuro-
toxic chemotherapy agents, more than two prior systemic therapies,
history of autoimmune disease, concurrent requirement for corti-
costeroids, seropositivity for human immunodeficiency virus or
hepatitis BSAg, serious concurrent infection or other major active
illness, or who had received other chemotherapy, biological ther-
apy, or any investigational drug within the preceding 3 weeks were
ineligible.
Study Design. The study was an open-label, nonrandom-
ized, multicenten Phase 1 dose escalation trial. The treatment
protocol was approved by the Human Investigation Review
Board at the participating institutions, and written informed
consent was obtained from each patient. rhIL- 12 was supplied
by Genetics Institute, Inc. (Cambridge, MA) and administered
by rapid iv. injection into a rapidly flowing iv. line.
The treatment schedule is displayed in Fig. I. A test dose
was administered on day 1 , and patients were observed over-
night as inpatients and were seen in follow-up on days 5 and 8.
Patients who tolerated the test dose without DLT received
rhIL-l2 by rapid iv. injection daily for 5 days beginning on day
15 in an outpatient setting. Treatment cycles were repeated
every 21 days. Vital signs, including supine blood pressure,
pulse, respiratory rate, and temperature, were measured at fre-
quent regular intervals for up to 6 h after rhIL-l2 administration
on each day of the first 5-day course and for I h after IL-l2
administration on all subsequent cycles. Weight was measured
before each rhIL-l2 administration, and rhIL-l2 dosages were
recalculated before each treatment course. Patients were evalu-
ated for tumor response at the end of every second 21-day
treatment cycle, and patients with stable or regressing disease
could receive a maximum of six S-day treatment courses.
rhJL-l 2 doses were increased from 3 to 1000 ng/kg in suc-
cessive cohorts of patients. There was no intrapatient dose escala-
tion. A minimum of four patients were enrolled at each dose level,
and all patients had to have completed day 35 (end of first 21-day
cycle) before initiating enrollment to the next dose level. If any
patient developed grade 3 or greater toxicity at a particular dose
level, then an additional two patients were enrolled at that dose
level. Dose escalation was halted when three patients at a particular
dose level experienced a grade 3 or greater toxicity and the pre-
ceding dose level was designated the MTD.
Patients experiencing a fever after the test dose could
receive prophylactic acetaminophen (up to 2600 mg/day) for
subsequent cycles. If fever recurred then indomethacin (25 rng,
p.o., every 8 h) was provided. No other concomitant rnedica-
tions were routinely administered.
Research. on January 3, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
test dose
rhIL.12
cycle I cycle 2
++t++TumorMeasurements
Table 1 Patient characteristics
Day I 15 19 36 40 50
. Dose levels: 3, 10, 30, 100, 250, 500, 1000 ng/kg
. 4 6 patients per dose cohort
. Maximum: test dose and six 21 day cycles
Fig. 1 Schema for this study.
No. of patients
40
52 years (25-76)24/16
13
21
6
20
12
5
2514
12
38
2
Clinical Cancer Research 411
Toxicity was assessed using the National Cancer Institute
Common Toxicity Criteria with slight modifications to address
issues of fever, neutropenia, lymphopenia, elevated bilirubin, and
capillary leak syndrome. Grade 3 elevations in bilirubin or de-
creases in neutrophil count and grade 4 lyrnphopenia were not
considered DLTs. Grade 3 capillary leak syndrome was defined as
fluid retention associated with hypotension, or symptomatic or
laboratory evidence of organ dysfunction. Patients experiencing
grade 3 or grade 4 toxicity were taken off the study, except for
those patients treated at the highest dose level administered (be-
yond the MTD), who were allowed to continue therapy at the
preceding dose level. Otherwise, no intrapatient dose modifications
were permined.
Pharmacokinetics. Serial blood specimens were col-lected for determination of circulating rhIL-l2 levels before the
administration of rhIL-l2 on day 1 and 10, 20, and 30 mm, and
1, 2, 4, 6, 10, 12, and 24 h post-drug administration. Additional
blood specimens were also collected 10 mm after rhIL-l2 ad-
ministration in subsequent treatment cycles. Serum was isolated
and analyzed by an ELISA (28) with a lower limit of detection
of 40 pg!ml.
Immunobiology. Serum for neopterin, IFN--y, and TNF-a
determinations was obtained before each rhIL-12 administration, at
all interim visits, and at the final study visit. Sera for quantitative
immunoglobulin measurements and PBMC for FACS analysis
were obtained before the test dose and immediately before each
rhIL-12 5-day treatment course, and at the final visit. PBMCs were
shipped overnight to Nichols Laboratory (San Juan Capistrano,
CA) and analyzed for the CD3, CD4, CD8, CD14, CD16, CD2O,and CD56 expression using standard techniques.
rhIL-12 Antibodies. Serum for anti-rhIL-12 antibody
determinations was obtained pretreatment, at the beginning of
each treatment cycle and at the final visit.
Assessment of Tumor Response. Tumor measurements
were obtained after the second 21-day cycle and then every
other cycle. Responses were determined according to previously
developed and published criteria (29).
RESULTS
Patient Population. Thirty-two patients participated in
the dose escalation phase of this study at seven different dose
levels. After determination of the MTD, an additional eight
patients were enrolled at that level to further assess its safety.
Table 1 depicts the relevant demographic and previous treat-
ment information for all 40 study patients. As can be seen, the
majority of patients had either renal cell cancer or melanoma
and had received prior therapy. In particular, 9 of the 20 patients
Total patientsMedian age (range)Gender (Male/Female)Performance status
(Karnofsky)
100%90%80%
Tumor typesRenal cell cancerMelanoma
Colon cancerParotid cancer
Cervical cancerAdenoid cystic cancer
Prior therapyChemo/immunotherapy(IL-2)
RadiotherapySurgeryNone
with renal cell carcinoma and S of the 12 patients with meta-
static melanoma had received prior IL-2 therapy.
Treatment. Three patients received only the test dose of
rhIL-l2 due to either the clinical appearance of previously unsus-
pected brain metastases (two patients with melanoma treated at the
250 and 500 ng/kg dose levels) or transient grade 3 hepatic trans-
aminase elevations (one patient at the 500 ng/kg dose level).
Six additional patients were not treated beyond the first
5-day treatment course due to disease progression (3 patients),
asymptornatic premature ventricular contractions ( 1 patient),
and transient liver function test abnormalities (2 patients). Five
of the remaining 3 1 patients received the test dose and all six of
the intended 21-day treatment cycles.
Clinical and Laboratory Toxicity. Common side effects
associated with rhIL-l2 included fever, chills, fatigue, headache,
and, less commonly, nausea and vomiting. The fever was first
observed at the 3 ng/kg dose level and occurred in the majority of
patients receiving � 10 ng/kg rhIL-l2. Fever was delayed at onset,
typically occurring 8-12 h after the rhIL-12 injection, and was
incompletely suppressed with nonsteroidal anti-inflammatory
drugs. Significant laboratory toxicities according to dose level are
displayed in Table 2. Frequent laboratory changes included anemia,
neutropenia, lymphocytopenia, hyperglycemia, thrombocytopenia,
and hypoalbuminemia. Most of these abnormalities appeared mdc-
pendent of dose, peaked at day 5 of a treatment cycle, and resolved
quickly. Grade 4 lymphocytopenia occurred in the majority of
patients at all dose levels and resolved without rebound lympho-
cytosis. No consistent changes in serum creatinine or neurological
toxicity were observed.
Dose-dependent abnormalities included stomatitis, which
first became apparent during the sixth 5-day treatment cycle in
one patient at the 100 ng/kg dose level and was evident in three
of four patients at the 1000 ng/kg dose level, and liver function
test abnormalities, which first became evident in one patient at
the 250 nglkg dose level and were universal by the 1000 ng/kg
dose level. Liver function test abnormalities consisted predom-
inantly of elevations in hepatic transaminases and lactate dehy-
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412 Phase I Trial of iv. rhIL-l2 in Advanced Malignancies
Table 2 Number of pati ents experiencin g selected Ia boratory toxicity in all cycles (grad es 2, 3)
Dose level (ngfkg)(n)
3 10 30 100 250 500 1000Toxicity (4) (4) (4) (4) (6) (14) (4)
Hepatic
Alkaline phosphatase ( 1 , 0) ( 1, 0) (7, 2) (1, 1)AST (1, 0) (0, 1) (2, 2) (6, 3) (2, 2)
Bilirubin (1, 1) (1, 0) (1, 6) (0, 1)
Hyperglycemia (1, 0) (1, 0) (2, 0) (2, 0) (2, 1) (3, 1) (1, 0)Creatinine (2, 0)Hematologic
ANC (1, 0) (0, 3) (3, 1) (3, 5) (3, 1)
Hemoglobin (3, 0) (1, 0) (2, 0) (0, 1) (2, 0) (3, 1) (1, 1)
Platelets (1, 0) (1, 0)
drogenase, but alkaline phosphatase and bilirubin were occa-
sionally elevated as well. Stomatitis usually resolved over 3
days, whereas liver function test abnormalities resolved over
7-10 days. Mucosal biopsies were not performed, but herpes
simplex virus cultures from the oral lesions were negative on
multiple occasions in several patients.
Determination of MTD. During the dose escalation
phase, one patient each at the 250 and 500 ng/kg dose levels had
DLT (grade 3 nausea and vomiting secondary to previously
undiagnosed brain metastases and grade 3 elevations of AST,
respectively). Consequently, two additional patients were added
to each dose level and tolerated therapy without difficulty.
Three of four patients treated at the 1000 ng/kg dose level
experienced DLT. One patient developed grade 3 elevation of
hepatic transaminases, grade 3 stomatitis, and a mild capillary
leak syndrome after three doses. Another patient developed
grade 3 elevation of hepatic transaminases and grade 2-3 sto-
matitis after four doses, and the third patient developed grade 3
elevation of alkaline phosphatase after completing all five doses.
The fourth patient at this dose level experienced both grade 2
stomatitis and grade 2 elevations in hepatic transaminases. On
the basis of this experience, the DLTs were defined as liver
function test abnormalities and stomatitis, and the MTD for this
schedule was determined to be 500 nglkg.
Safety Experience. After determination of the MTD, an
additional eight patients were treated at this dose level to verify
its safety. The results of all 14 patients treated at the 500 ngfkg
dose level are displayed in Table 3. One patient went off study
after only receiving the test dose for early disease progression
and was considered inevaluable for toxicity. Nine patients re-
ceived all of the first 5-day treatment course without significant
toxicity. Two of these nine patients experienced potential DLT
during later cycles: an upper gastrointestinal bleed in one patient
after three cycles and an admission for dehydration and ortho-
stasis in another patient after the second 5-day treatment course.
Both of these toxicities were believed not to be directly related
to rhIL-12. Three of the remaining four patients had doses held
for hepatotoxicity: grade 3 elevations in transaminases or alka-
line phosphatase that were largely transient and asymptomatic.
One of these patients was able to continue to receive therapy
without additional grade 3 hepatic toxicity. The final patient
developed grade 3 elevation of alkaline phosphatase after four
doses in the first 5-day treatment course and had his treatment
stopped. Subsequently, he developed abdominal pain and ane-
mia and was admitted for evaluation of potential gastrointestinal
bleeding. No mucositis was documented. Within 24 h he devel-
oped fever, followed by rapid onset of irreversible shock and
death. Blood cultures grew Clostridia perfringens from an un-
known source. No autopsy was performed. The death was felt to
be secondary to sepsis, presumably from necrotic intra-abdom-
inal tumor, and was classified as “possibly related to rhIL-l2.”
Toxicity was fairly consistent from one cycle to the next
within individual patients without evidence of either cumulative
effect or attenuation corresponding to the changes observed in
some biological parameters (see below). Fig. 2, A and B, dis-
plays hepatic transaminase levels and neutrophil counts follow-
ing the test dose and subsequent cycles for four patients who
received at least two 5-day courses of therapy at the 500 ng/kg
dose level. AST levels were maximal at day 5 of the first 5-day
treatment course, with possible attenuation with subsequent
cycles. ANC nadirs occurred around day 5 of each cycle without
apparent cumulative toxicity. ANC recovery occurred by day 15
of each cycle without rebound neutrophilia.
Tumor Response. Objective tumor responses were ob-
served in two patients. One patient with metastatic renal cell
carcinoma with small pulmonary nodules and hilar adenopathy
experienced a partial response that is still ongoing at 22+ months.
The second patient, a 57-year-old woman with melanoma, experi-
enced a complete regression of small pleural-based nodules and
cervical adenopathy lasting approximately 4 weeks. Four additional
patients completed six 21-day treatment cycles with stable disease.
Of the 14 patients treated at the MTD (6 renal, 4 melanoma; 3
without previous therapy), no responses were seen.
Biological Parameters. Mean circulating IFN-�y concen-
trations for the top three cohorts are displayed in Fig. 3. IFN--y
appeared to be induced in a dose-dependent manner with peak
concentrations exceeding 1000 pg/mi observed 24 h after a test
dose of 1000 nglkg rhIL-12, with return to baseline by day 8.
During the 5-day treatment courses, IFN--y concentrations were
highest at 48-72 h after the first rhIL-12 injection and returned
to baseline by 72 h after the last dose. Peak IFN-�y concentra-
tions in general were higher during the first 5-day treatment
course than after the test dose. Peak IFN--y concentrations dur-
ing subsequent cycles tended to progressively decline.
Mean serum neopterin concentrations are displayed in Fig.
4. Neopterin tended to rise in a non-dose-dependent manner,
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Clinical Cancer Research 413
Table 3 Results of patients treated at the 500 ng/kg dose level ofrhIL-12
Patientno.
No. of cyclescompleted Results
Dose escalation 023 2 No DLTphase
Safety phase
024025
026027
028
033
034035
036
037
038
039
040
22
Test doseTest dose
2
1”
2�2”
3
4’
2
2
1”
No DLTNo DLT
Grade 3 AST, discontinuedBrain metastases,
discontinued
No DLI
Grade 3 alkalinephosphatase.sepsis/deathe
No DLIGrade 3 orthostasis,1
discontinued
Gastrointestinal bleed!discontinued
Grade 3 liver function test,dose held in cycle 2,continued on therapywithout subsequent DLT
No DLI
No DLT
Grade 3 alkalinephosphatase,discontinued
a Received four doses in cycle 1.
/) Received two doses in cycle 2.C Received four doses in cycle I.d Received three doses in cycle 1.e Possibly related to rhIL-l 2.
1Not directly related to rhIL-12.
reaching a maximum at day 5 and declining slowly thereafter. In
contrast to IFN--y, no attenuation in peak concentrations was
observed with successive rhIL-12 cycles. TNF-a was not de-
tected in any of the plasma specimens at any dose level.
Immunophenotyping. Mean lymphocyte and monocyte
counts fell promptly during therapy and recovered quickly after
treatment without significant rebound or dose dependence. Phe-
notypic analyses showed reduction in all lymphocyte subsets
(data not shown).
IL-12 Antibodies. One patient treated at 500 ng/kg de-
veloped low titer antibodies to rhIL-12 after 2 weeks of therapy.
No other patient developed antibodies to rhIL-l2.
Pharmacokinetics. rhIL- 12 concentrations in patients
receiving either 3 or 10 ng/kg were below the limit of assay
quantitation. The pharmacokinetic parameters for all other pa-
tients are shown in Table 4. Both maximum concentration
(Cmax) and area-under-concentration versus time curve extrap-
olated to infinity (AUC0...,�) were proportional to the dose
administered, indicating that over the dose range of 30-1000
ng/kg, the pharmacokinetic characteristics of rhIL-12 were un-
ear and not dependent on dose. The elimination half-life was
calculated to be between 5.3 and 10.3 h. Fig. 5 displays the
concentration versus time profiles of patients receiving 500
ng/kg, the MTh in this study. All patient profiles fell well
within the range around the mean expected for this limited
number of samples, and there was no correlation observed
between rhIL- 1 2 elimination half-life and toxicity.
DISCUSSION
IL-12 has potent immunomodulatory and antitumor effects
in preclinical studies. This report describes the results ofthe first
clinical trial of this promising cytokine. In this study of rhIL- I 2
administered by bolus iv. injection initially as a single test dose,
then daily for 5 days every 3 weeks, we defined the MTD for
rhIL-12 to be 500 ng/kg. DLT, transient reversible stomatitis
and/or hepatic dysfunction, occurred in three of four patients
treated at the 1000 ng/kg dose level. One patient treated at the
500 ng/kg dose level died of C. perfringens sepsis. It is possible
that some effect of rhIL-12 on intestinal mucosa or bacterial
surveillance may have contributed to this event; however, be-
cause mucositis was not a prominent toxicity at this dose level,
and because this dose and schedule were well tolerated by most
other patients in an outpatient setting, we felt this dose did not
exceed the MTD. Other prominent clinical abnormalities in-
cluded fever and chills, fatigue, and, less commonly, nausea,
vomiting, and headache. Commonly observed laboratory abnor-
malities included anemia, neutropenia, lymphopenia, thrombo-
cytopenia, hyperglycemia, and hypoalbuminemia. The fever
was unusually delayed, typically occurring 8-12 h after an in-
jection rather than at 2-4 h as is typically seen with other py-
rogenic cytokines such as IL-i, IL-2, IL-4, IL-6, and the inter-
ferons (30-34). The half-life of rhIL-l2 was also unusually
prolonged relative to these other cytokines (5-10 h versus 20
mm) perhaps explaining the delay in onset of the fever.
Similar to preclinical observations, rhIL- 12 was found to
be a potent inducer of IFN--1’. IFN--y was observed to be induced
in a dose-dependent manner, but to a progressively lesser degree
with each successive 5-day treatment course. Many of the side
effects observed with rhIL-1 2 administration, in particular the
liver function test abnormalities, fever, flu-like symptoms, fa-
tigue, hernatological changes, and hyperglycemia, could be di-
rectly or indirectly attributable to IFN-�y production because
they have been observed in clinical trials with this cytokine (35).
The etiology of the stomatitis is less clear. It was a sur-
prising DLT because it had not been observed in preclinical
toxicology studies. Potential mechanisms include: decrease in
mucosal IgA immunity, an autoirnmune phenomenon, viral ac-
tivation, or inhibition of constitutive mucosal epithelial prolif-
eration. Although in the absence of histological confirmation it
is difficult to distinguish between these possibilities, no unusual
bacterial, fungal, or viral organisms were obtained from any
cultures, and no other evidence of autoimmune phenomena was
observed, making the first three possibilities unlikely. IL-12 has
been observed to enhance the mucosal toxicity associated with
radiation therapy, presumably through an IFN--y-mediated
mechanism (36), and IFN-�y has been shown to inhibit TGF-�3
production (37), a factor purported to be important in maintain-
ing mucosal integrity, perhaps implicating IFN-’-y in this toxicity
as well. However, IFN-y did not cause mucositis in Phase I
trials (35), arguing against it having a sole causative role in the
stomatitis associated with rhIL-l2. Perhaps the more sustained
release of IFN-y in response to prolonged presence of IL- I 2 or
the concomitant effect of other factors may have enabled this
new toxic effect to become manifest. This side effect limited
further escalation of rhIL-12 on this schedule. Whether a better
understanding of the underlying mechanism for this toxicity or
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414 Phase I Trial of iv. rhIL-l2 in Advanced Malignancies
x xxxxx
AST (SGOT)
xxxx x
A
-I
I-0C,Cl)
I-Cl)4
xxxxx400�
350�
300�
250�
200�
150�
100�
50#{149}
B
0 10 20 30 40 50 60 70 80
DAY
xxxxx
ANC
xxxxx xxxxx
10 20 30 40 50 60 70 80
13
12
11
10
DAY
Fig. 2 Serial AST (SGOT; A) and neutrophil (ANC; B) levels for four individual patients over the first 70 days of therapy at the 500 ng/kg dose level.x,rhIL-l2 administration (500 ng/kg).
aggressive preventative treatment will enable higher doses of
rhIL-l2 to be safely administered remains to be determined.
Other cytokines have been shown to be potent inducers of
TNF-a (19), but despite the ability of IL-12 to induce some TNF
in vitro and the appearance of some side effects potentially
attributable to TNF, no TNF was detectable in the plasma of
patients receiving rhIL- 12. Therefore, in contrast to mechanisms
for toxicity proposed for other cytokines, toxicities observed in
response to rhIL-l2 administration are unlikely to be related to
secondary TNF production.
IL- 12 was shown in preclinical studies to allow the con-
tinued proliferation of preactivated NK and T cells, to enhance
Research. on January 3, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
MEAN INTERFERON GAMMA
�
L
17000 x
16000
15000
14000
�. 13000
E.�l 12000a.
.� 11000
�� 9000
C, 8000
z 7000 �\� 6000 �wU- 5000 :� � 1%.� 4000
z 3000 .
- � �0 � 40 5� 60 70 80 90 100
DAY
Fig. 3 Serial mean serum IFN--y levels for patients treated at the top three rhIL-l2 dose levels. �, 250 ng/kg; #{149}, 500 ng/kg; 0, 1000 ngfkg. x,
rhIL-12 administration.
MEAN NEOPTERIN
300
250
-J� 2000EC
z 150
wI-0.
0wz
50
0
100
DAY
Fig. 4 Serial mean serum neopterin levels for patients treated at the top three rhIL- 12 dose levels. E, 250 ng/kg; #{149}, 500 ng/kg; 0, 1000 ng/kg. x,rhIL-l2 administration.
x�0tx �
0 10 20 30 40 50 60 70 80 90
Clinical Cancer Research 415
cytotoxic activity of T cells and NK cells, and to induce T helper
1 cell and inhibit T helper 2 cell differentiation (20-22). Special
studies to determine potential effects of rhIL-l2 on TH cell
distribution were not performed and, therefore, must await fu-
ture rhIL-12 studies. PBMC phenotypic analyses showed reduc-
tion in all lymphocyte subsets.
Tumor responses were observed in one patient each with
melanoma and renal cell carcinoma treated near the MTD.
Because this study involved few patients per dose level and
many of these patients had previously received other immuno-
therapy agents (most commonly IL-2), it is impossible to deter-
mine the cancer immunotherapeutic potential of rhIL-12 or the
most appropriate dose for Phase II testing. However, based on
this study, we feel that Phase II testing eventually will be
Research. on January 3, 2021. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Parameter
c,,,a� (pg/mI)
30 100
744.3 ± 313.1 2877.8 ± 904.7 6640.0 ± 1385.3 1 1 136.8 ± 3529.8 19575.0 ± 5269.0
v� (mI/kg) 48.4 ± 29.7 43.4 ± 13.3 47.8 ± 23.2 56.7 ± 8.6 62.5 ± 14.3
T112�.1,�, (hours) 5.3 ± 0.69 5.6 ± 1.1 7.2 ± 2.5 9.6 ± 2.6 9.1 ± 2.1
CL (mI/hr/kg) 6.8 ± 5.3 5.6 ± 2.0 5.8 ± 5.5 4.4 ± 1.5 5.2 ± 2.5
AUC��. (pg X hr/mI) 61 19.2 ± 3232.9 19821.0 ± 7162.9 64276.5 ± 30675.8 123864.4 � 37559.8 220096.9± 75237.5
250 500 l00()
20
416 Phase I Trial of iv. rhIL-l2 in Advanced Malignancies
Table 4 Mean pharmacokinetic parameter estimates in patients receiving rhIL-12 as single iv. dose on day I
All values are mean ± SD. Cm�x, maximum concentration; V�, the volume of distribution at steady state: T112�11,,, the terminal or elimination
half-life: CL, clearance: and AUC0_,�, area-under-concentration versus time curve extrapolated to infinity.
Dose (ng/kg)
.�1
E0)
0.
c.,J
0C0
(0
C
C0
C)
100000
10000
I 000
I 00
0 5 10 15
Time After Administration (hrs)
Fig. 5 Concentration versus time profiles of all patients receiving 500ng/kg IL-l2 as a single iv. dose on day I. . individual patients:-, mean of all patients.
warranted, and if this schedule is to be used, that doses in the
range of 500 ng/kg should be tolerated by most patients.
Of note, a Phase II trial of rhIL-l2 in metastatic renal cell
carcinoma using this schedule (without the test dose) at the 500
ng/kg dose level was initiated, but had to be closed because of
unexpected severe toxicity (38). Although the reason for the
severe toxicity in this Phase II trial is still being explored, initial
results indicate that the omission of the test dose was a major
contributing factor.4 The observation from this study that IFN-�y
levels progressively declined with each successive cycle of
rhIL-l 2 supports the contention that prior rhIL-l2 administra-
tion may in some way exert a protective effect against subse-
quent toxicity and that this effect may be sustained over at least
4 J_ P. Leonard, M. L. Sherman, 0. L. Fisher. L. J. Buchanan, 0. Larsen, M.B. Atkins, J. A. Sosman, J. P. Dutcher, N. J. Vogelzang, and J. L. Ryan,Effects of single-dose interleukin 12 (IL-12) on IL-l2-associated toxicityand interferon--y production. manuscript in preparation.
a 10-17-day interval. Ifthis hypothesis is confirmed in ongoing
preclinical and subsequent clinical testing, then this would have
a major impact on the design of future rhIL- I 2 studies. Because
of the severe toxicity associated with minor schedule changes,
additional Phase I studies are necessary to examine alternative
schedules and further evaluate the significance and mechanism
underlying this “desensitization effect.”
ACKNOWLEDGMENTS
We thank Kerry Kappler, R. N., M. S., N. P., Sandy Dunstan, R. N.,
Lynn Colicchio, R. N., Lisa O’Connor, R. N., and Donna Kinzler, R. N.,for nursing support; Deborah Rovner, Steven Chartier, and Dr. Beth
Harrison-Mann for data management; Christine Cameron, Dr. Zhiling
Yu, and Kyle McCarthy for laboratory assistance: and Jacqueline L.
Myers for assistance in preparing the manuscript.
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