Download A Phase I Study of Recombinant Interleukin 2 plus Recombinant

[CANCER RESEARCH 48. 3875-3881. July 1. 1988]
A Phase I Study of Recombinant Interleukin 2 plus Recombinant ^-Interferon
Robert L. Krigel,1 Kristin A. Padavic-Shaller,
Alfred R. Rudolph, Samuel Litwin, Michael Konrad,
Edward C. Bradley, and Robert L. (omis
Departments of Medicine and Biostatistics, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [R. L. K., K. A. P-S., S. L., K. L. C.], and Cetus Corporation,
Emeryville, California 94608 [A. R. R., M. K., E. C. B.J
ABSTRACT
Interleukin 2 (IL-2) therapies have antitumor activities against several
neoplasms. In vitro these activities are enhanced by 0-interferon (IF V
tf ). Therefore, we initiated a Phase I trial with a combination of IL-2 and
IFN-/3 three times weekly. The IFN-/3 was administered i.v. Initially, the
IL-2 was administered s.c. However, neutralizing antibody to the IL-2
developed in five patients, and the route of administration of the IL-2
was changed to i.v.
Forty-seven patients were entered on the study. The maximum toler
ated doses for the combination given i.v. were 5x10"
units/m2 of IL-2
and 10 x IO6 units/m2 of IFN-/3. Dose-limiting toxicities were profound
fatigue/decreased performance status, anorexia/weight loss, depression,
and arthralgias. Hypotension, exfoliative skin rash, thrombocytopenia,
diarrhea, temperature >40.6°C, and peripheral edema were rarely dose
limiting.
Thirty-two
activity to levels higher than those seen with either substance
alone (19). Interestingly, IFN-/3 but not a-interferon was syn
ergistic with IL-2 in the cytotoxic T-cell mediated destruction
of autologous transformed cells (20). In addition, IFN-/3 is also
synergistic with 7-interferon, a lymphokine which is generated
by cytotoxic T-lymphocytes stimulated by IL-2 (21-23).
We now report the results of a Phase I study combining IL2 with IFN-0 which demonstrate the following: (a) that IL-2
and recombinant IFN-ßcan be given together at doses that are
tolerable; (b) that IL-2 and recombinant IFN-/3 can generate
LAK activity in vivo; and (c) that IFN-7 can be generated with
IL-2 with this therapy.
MATERIALS
patients were évaluablefor response.
AND METHODS
After 4 weeks of
treatment, 21 patients had stable disease, three patients had a minor
response, and one patient had a partial response. Significant lymphokineactivated killer cell (LAK) activity was seen in seven patients (22%) and
required S x 10* units/m2 of IL-2. Those who had progressive disease
had significantly less LAK activity than those with either stable disease
or a response. This therapy also induced more than 60 units/ml of
endogenous -v-interferon 4 h after the i.v. IL-2 administration.
This study demonstrates that (a) intermittent i.v. bolus IL-2 therapy
can generate LAK activity, (b) LAK activity may be associated with an
antitumor response, (c) significant levels of -y-interferon are induced by
this therapy, and (</) IL-2 and IFN-/J given three times weekly i.v. is
both tolerable and biologically active. The recommended Phase II dose
is 5 x 10' units/m2 of IL-2 plus 6 x 10' units/m2 of IFN-/8.
INTRODUCTION
Interleukin 2-based treatments have activity against a variety
of cancers in both experimental animals and humans (1-6).
However, IL-22 therapy has been associated with significant
toxicity which has been shown to be directly related to the dose
of IL-2 and the duration of its administration. These trials have
often included the coadministration of ex vivo IL-2-activated
autologous peripheral blood lymphocytes (lymphokine-activated killer cells). It is unclear whether the use of LAK adds to
the efficacy of IL-2 used alone and how much LAK contributes
to toxicity. Therefore, many investigators are examining alter
native dose regimens, schedules, and routes of administration
in order to minimize toxicity and improve efficacy without the
coadministration of ex vivo activated LAK (5-9).
^-Interferon is a type I interferon, a class of interferons which
has shown activity in a wide spectrum of diseases (10-12). IFNßhas also been shown to be synergistic with IL-2 both in
stimulating NK activity and in increasing expression of IL-2
receptors (13-18). In patients with AIDS the combination of
type I interferons and IL-2 significantly increases the NK
Received 11/24/87; revised 3/16/88; accepted 3/31/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Department of
Medicine, Fox Chase Cancer Center, Philadelphia, PA 19111.
3The abbreviations used are: IL-2, human recombinant interleukin 2; II-N.
interferon; LAK, lymphokine-activated killer cell(s); NK, natural killer cell; PD,
progressive disease; SD, stable disease; ELISA, enzyme-linked immunosorbent
assay; LU, lytic units,,, .
Patient Population and Treatment Plan
The patients selected for this study had histologically proven solid
tumor malignancies which were refractory to standard therapy or for
which there was no generally accepted therapy. All patients had meas
urable or évaluabledisease; Karnofsky performance status >70; ade
quate bone marrow (granulocytes, >l,000/mm3, platelets, >50,000/
mm1); renal (creatinine, <2.0 mg/dl) and hepatic (bilirubin, <1.5 mg/
dl) function; and a minimum life expectancy of at least 3 months.
Patients with clinically significant cardiac disease, active infection
requiring systemic antibiotic therapy, or central nervous system métas
tases were excluded from entry. Throughout the duration of the trial,
patients did not receive other anticancer treatments, aspirin, nonsteroidal antiinflammatory drugs, corticosteroids, cimetidine, ranitidine, bar
biturates, or other investigational agents.
Initially, patients were treated with II \ ,< as a 1 min i.v. bolus
immediately followed by IL-2 given s.c. During the course of the study,
changes were made in the route of administration due to the develop
ment of neutralizing antibody in several patients to the s.c. administered
IL-2 and after dose-limiting formulation problems which prevented
further IL-2 dose escalations. Subsequently, IL-2 was given i.v. as a 1min bolus injection. The first 35 patients received IL-2 s.c. and the last
12 received it i.v. Treatments were administered three times weekly for
4 weeks in the absence of progressive disease or unacceptable toxicity.
Patients who exhibited either stable disease or tumor regression were
continued on therapy until there was evidence of tumor progression or
unacceptable toxicity. The protocol schema is shown in Table 1.
Toxicity Criteria
The toxicity scale was a modification of the World Health Organi
zation criteria. Patients exhibiting Grade IV toxicity were removed
from the study. Grade IV toxicity included the following: granulocyte
decrease to <250/mm3; platelet decrease to <20,000/mm3; bilirubin,
>3.0 mg/dl: serum glutamic and oxaloacetic transaminases, >10x
baseline; creatinine. >3.5 mg/dl; intractable vomiting necessitating i.v.
fluids; diarrhea requiring i.v. fluids: >10% weight loss; >30-point
decrease from baseline Karnofsky performance status; profound motor
weakness or autonomie dysfunction; coma or seizures; temperature
>40.6°C;or symptomatic fluid accumulation with weight gain >20%.
If Grade III toxicity was evident, doses of both agents were reduced
by 50%. Grade III toxicity included the following: granulocyte decrease
to >250/mm:1 and <500/mm3; platelet decrease to >20,000/mm3 and
<40,000/mm3: bilirubin. >2.5 mg/dl but <3.0 mg/dl; serum glutamic
and oxaloacetic transaminases, >6 but <10x the baseline; creatinine,
>3 mg/dl but <3.5 mg/dl; 6-10 episodes of emesis/day; >7 episodes
of diarrhea/day despite antidiarrhea medications; a >20-point decrease
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PHASE I STUDY OF IL-2 AND IFN-0
Table 1 Protocol schema
(s.c.)
(units/in2)10"10'10'5X
Dose
levelIL-2
S.C.IIIIIIIVVVIVIIIL-2
i.v.VVIVIIIL-2
(i.v.)
(units/in2)2x
10'5
10'5
x
10'5x
x
10'5
10'2x
10'2x
10'2x
IO66x
10'10
10'20
x
10'6x
x
10«5
x
10'5x
x
10'IFN-/3
10'10
10'20
x
x 10'
from baseline Karnofsky performance status; marked decrease of deep
tendon reflexes or marked motor weakness or sensory deficits; confu
sion or disorientation; temperatures >39.4°Cbut <40.6°Clasting 3 h
or more; weight gain >15% but <20% associated with peripheral
edema; ascites or pleural effusion while asymptomatic. If Grade III
toxicity continued at the reduced dose, the dose of IFN-/3 was again
reduced by 50%. If toxicity continued or progressed to Grade IV, the
patient was removed from study.
Four patients were entered at each dose level. If any 2 of 4 patients
at a dose level experienced Grade III or IV toxicity, an additional 4
patients were entered at that dose level. The maximum tolerated dose
was defined as that dose at which 3 of 8 treated patients experienced
Grade III or IV toxicity.
Response criteria were as follows: partial response required a mini
mum of 50% reduction in the sum of the products of the perpendicular
diameters of all measurable lesions; minor response was defined as a
25-50% decrease in the sum of the products of the perpendicular
diameters of all measurable lesions; stable disease constituted a less
than 25% change in tumor measurements; and progressive disease was
defined as a 25% or greater increase in the measurable disease.
ELISA was used to quantitate levels of antibodies specific to IL-2 or
IFN in patient sera (25, 26). Separate assays were done to measure IgG
and IgM specifically. An initial screen was done on duplicate 10-fold
dilutions of sera, and if the net absorbance was greater than 0.5, a
second assay on additional 3-fold serial dilutions was performed. The
ELISA titer was defined as the product of absorbance and dilution
factor, when the absorbance is between 0.1 and 0.5. The limit of
sensitivity of the IgG assay is 1 unit/ml, with 10 units/ml being a
definite positive. The background and variability of the IgM assay is
about 3-fold higher. Measurements of IL-2-binding capacity of patients'
sera containing IL-2-specifïcIgG indicate that 1 ELISA unit corre
sponds to 3 ng/ml of IL-2, or 30 ng/ml of IgG. Since the detection of
IgG has no quantitative relation to the identity of the antigen, as long
as it is in excess, 1 ELISA unit/ml will correspond to about 30 ng/ml
of IgG for any antigen.
Sera from patients on trial for 60 days or longer and having an IgG
titer of 10 or greater were assayed for ability to neutralize IL-2 activity.
IL-2 was added to 3 aliquots of sera to obtain titers of 4000, 400, and
40 units/ml. After incubation at 4°Covernight, the mixture was assayed
for IL-2 activity. The neutralizing titer is defined as the fold reduction
of IL-2 activity (over the value observed in control sera) times the final
serum dilution in the assay.
The activity of IFN was measured using an antiviral assay with a
visual cytopathic end point (27). The IFN titer was determined by
comparison to cells treated with serial dilutions of an NIH standard
IFN-/8 preparation of 100 units/ml. Values are reported as the median
of triplicate assays.
Cell-mediated Cytotoxicity Assays
Heparinized whole blood (20 ml) was collected from all patients
entered into the study prior to dosing (i.e., 72 h after the last IL-2 dose)
at Weeks 0, 3, and 5 and then monthly until time off study. No IL-2
was present in these specimens, which were kept at room temperature
and shipped by overnight delivery to the Cetus Corp.
Effector Cell Preparation. Patient and normal peripheral blood
mononuclear cells were isolated from heparinized blood samples by
Product Descriptions
Ficoll-Hypaque density gradient centrifugation. The samples were
IL-2 and recombinant IFN-/3 were provided by the Cetus Corp. The
mixed (approximately 20 ml) with an equal volume of RPMI 1640,
IL-2 had a nominal specific activity of 3 x IO6Cetus units/mg (8 x IO6 carefully layered over 20-25 ml of Ficoll-Hypaque, and centrifuged for
Biological Response Modifier Program units/mg) equivalent to 1 mg
30 min at 400 x g. The interface of the plasma and Ficoll-Hypaque
of product. The IL-2 was supplied as a lyophilized powder which when
was removed, the peripheral blood mononuclear cells were washed
reconstituted had a concentration of 3 x IO6 Cetus units/ml. The ß- twice in RPMI 1640 and then resuspended in 20-30 ml of RPMI plus
interferon had a nominal specific activity of 1.8 x 10s lU/mg. The
5% human AB+ serum, and the concentration was adjusted to 1 x IO7
IFN-/3 was supplied as a lyophilized cake which when reconstituted
peripheral blood mononuclear cells/ml.
yielded 45 x IO6 lU/ml.
Target Cell Preparation. A subclone of the natural killer cell-resistant
Daudi
human lymphoblastoid cell line was used as the target to evaluate
IL-2 Pharmacokinetics
LAK activity. Target cells were labeled by a 2-h incubation of 2-4 x
IO6cells in 0.5 ml of culture medium supplemented with 200-400 pCi
Serum samples were collected in 10-ml serum separator tubes (Becof 51Cr (250-500 ¿iCi/mg;Amersham Corp., Arlington Heights, IL).
ton-Dickinson) from all patients entered into the study at 1, 5, 10, 30,
Cells were washed three times, with gentle resuspension after pelleting,
60, 120, 240, and 360 min following their initial IL-2 and IFN-0
and then adjusted to 2 x IO5 cells/ml. One hundred /¿I
(2 x IO4 cells)
dosing. The specimens were processed by centrifugation and pipeted
off into cryotubes. The serum samples were then frozen at —40°C
until
of each cell type were counted in a Beckman gamma counter with a
51Cr isoset to determine the level of 5lCr uptake; 10,000-20,000 cpm/
they were shipped overnight on dry ice to the Cetus Corp. for assay.
IO4cells was a standard value.
Serum levels were determined by a biological assay which has been
Four-H "Cr Release Assay. Labeled target cells (2 x 10" cells in 100
described (24). Essentially, IL-2 levels were determined by measuring
/¿I)
were added to 96-well, round bottomed microtiter plates. Effector
the incorporation of tritiated thymidine by an IL-2-dependent cell line
(HT2). The serum was heated at 50°Cfor 30 min before the assay to cells were added in a volume of 100 pi, at ratios of effector to target
cells of 50:1, 17:1, and 6:1. Each assay plate also included target cells
inactivate serum components which are toxic to the HT2 cells. The
incubated in medium alone and target cells incubated in medium
sensitivity of the assay is usually 0.3 unit/ml (in the serum sample).
containing 0.5% Nonidet P-40 detergent. All assay points were done in
One unit/ml is defined relative to an IL-2 standard and produces
triplicate. After a 4-h incubation at 37°C,the microtiter plates were
approximately 50% of maximum incorporation in the well containing
the test cells. One Cetus unit has been determined to be equal to 2.3 centrifuged at 500 x g for 3 min. The supernatant fluids were removed
with a Skatron harvester (Skatron, Inc., Sterling, VA) and counted in
units as defined by the IL-2 standard supplied by the NIH Biological
a gamma counter. The results were quantitated as
Response Modifier Program.
Assay for Anti-IL-2 Antibodies
% of specific lysis =
Serum samples were collected in 10-ml serum separator tubes (Becton-Dickinson) from all patients entered into the study prior to dosing
at Weeks 0, 3, and 5 and then monthly until they went off study. The
serum was separated and then frozen at —40°C.
A standard indirect
test cpm - spontaneous cpm
— x 100%
total cpm —spontaneous cpm
Spontaneous cpm were defined by the target cells incubated alone; total
cpm were defined by target cells in medium plus detergent. Results
were calculated in lytic units30%/107effector cells (28).
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PHASE I STUDY OF IL-2 AND IFN-0
•y-Interferon
Assay
Serum samples were collected with the pharmacokinetic samples in
10-ml serum separator tubes (Becton-Dickinson) from all patients
entered into the study at 2, 60, 120, 240, and 360 min following the
initial IL-2 and IFN-/3 dosing. The serum was separated, then frozen
at -70°Cuntil assayed. IFN-7 levels were determined using the IMRX
7-interferon radioimmunoassay test kit (Centocor, Inc.). One anti-IFN7 mouse monoclonal antibody-coated polystyrene bead was placed into
each well of a 25-well reaction tray. Pipeted into the wells was 0.2 ml
of the following: (a) normal human serum standards; (/)) positive
control; and (c) specimen sample. The product was then incubated for
2 h at room temperature. Following incubation the liquid was aspirated
and the beads were washed three times with a total of 3 ml of distilled
water, '"¡-labeled anti-IFN-7 antibody (0.2 ml) was then pipeted onto
all of the beads and incubated 2 h at room temperature. The liquid was
again aspirated and the beads were washed as before. The beads were
then transferred to counting tubes placed in a gamma scintillation
counter.
RESULTS
Patient Characteristics. After obtaining informed consent, 47
patients were entered on the clinical trial. All 47 patients were
évaluablefor toxicity analysis. Fifteen patients were withdrawn
from the study program prior to completing the initial 4-week
treatment period. Eight patients developed early evidence of
progressive disease, three patients were removed because of
development of infection (one with influenza and two with
postobstructive pneumonia), and four patients had unusual,
early, non-Grade III or IV toxicities (see below).
The remaining 32 clinically évaluablepatients (Table 2)
ranged in age from 24 to 70 years with a mean age of 53 years
and include IS males and 17 females. The patients had the
following diagnoses: colorectal carcinoma, 12; non-small cell
lung carcinoma, 4; renal cell carcinoma, 4; malignant mela
noma, 3; metastatic breast carcinoma, 3; carcinoid, 1; esophageal carcinoma, 1; oat cell lung carcinoma, 1; ovarian carci
noma, 1; synovial sarcoma, 1; and adenocarcinoma of unknown
primary, 1. Of this population, 26 patients had received prior
chemotherapy (including 1 with prior IFN-/3), 10 had received
prior radiotherapy, and 1 had received monoclonal antibody
therapy. Three patients had received radiotherapy as their only
prior treatment modality.
Clinical Effects (Dose Escalation). IL-2 was administered s.c.
to the first 35 patients entered on the trial. The dose was limited
to 5 x IO6 units/m2 because of the small volume which could
be administered by this route. In addition, neutralizing antibod
ies to the IL-2 developed in five patients (see below). For these
Table 2 Characteristics of patients évaluable
for both toxicity and response
Total
Age (years)
Sex
Diagnoses
Colorectal
Non-small cell lung cancer
Renal
Melanoma
Breast
Other
No.
32°
24-70
15 M, 17 F
12
4
4
3
3
6
Prior treatments
Chemotherapy
26
Radiotherapy
10
Monoclonal antibody
1
"An additional 15 patients were available for pharmacological
toxicity analysis only.
and acute
reasons IL-2 was given i.v. to subsequent patients entering the
study. This is shown in Table 1. With 5 x IO6 units/m2 of IL2 s.c., the maximum tolerated dose for IFN-/3 was 6 x IO6
units/m2. However, with this same dose of IL-2 administered
i.v., the maximum tolerated dose for IFN-/3 was 10 x IO6units/
m2. This was based upon the observed cumulative toxicities of
fatigue, anorexia with weight loss, depression, and decrease in
overall performance status.
Four patients were removed or asked to be removed from the
study program because of unacceptable side effects. One patient
at Level IV developed a progressive, generalized urticaria! rash
during Week 1 which may have been treatment related. The
second patient was at Dose Level IV; he was 70 years of age
with a primary diagnosis of adenocarcinoma of the lung. After
each of three treatments he experienced pronounced dyspnea
secondary to rigors and complicated by anxiety. The third
patient in this subgroup was at Dose Level IV; he was 56 years
of age with a primary diagnosis of melanoma. Each of three
doses produced a transient fever of 40.6°Cpreceded by rigors
of approximately 1-h duration. During the episode of rigors,
the patient had 3-4 episodes of vomiting despite standard
antiemetic therapy. On Day 8 of treatment the patient was
given 50% doses, due to a decrease in performance status, with
similar side effects again recorded, and he asked to be with
drawn from study. The fourth of these patients was at Dose
Level VI; she was 31 years of age with a primary diagnosis of
malignant melanoma. Each of 2 doses produced a transient
fever of <39.8°Cpreceded by rigors for approximately 40 min.
The patient also experienced nausea and a single episode of
vomiting during her rigors. The patient deemed the side effects
unacceptable and withdrew from the study. The toxicity data
on all 47 patients are summarized in Table 3.
Pharmacokinetics. The pharmacokinetic curves of IL-2 (Fig.
1) were generated from data compiled on those patients who
received 5 x 106/m2 by either s.c. or i.v. administration. One
min after i.v. bolus injection detectable serum levels ranged
from 2000 to 6100 units/ml with a mean value of 4060 units/
ml. IL-2 given i.v. exhibited biphasic serum clearance kinetics
with an initial clearance of approximately 1-3 min followed by
a slower phase of approximately 50 min. Administration of IL2 by the s.c. route resulted in peak serum levels of approximately
1-3% of those seen with i.v. administration. These peak levels
occurred at 240-360 min following s.c. administration. At 6 h,
comparable IL-2 levels for i.v. and s.c. administration ranged
from 36 to 110 units/ml and 8.2 to 190 units/ml, respectively.
Response Data. Of the 32 évaluablepatients, 27 had meas
urable disease and 5 had évaluabledisease, to assess for re
sponse. All but one of the patients had progressive disease at
time of entry on study. One patient with renal carcinoma had
a stable renal mass.
Seven patients had progressive disease upon completing 4
weeks of therapy and were removed from study [3 at Dose Level
I, 1 at Level II, 1 at Level V (s.c.), 1 at Level VI (i.v.), and 1 at
Level VII (s.c.)].
Twenty-one patients had stable disease after Week 4. One
patient was removed from study due to Grade IV fever toxicity
after 5 weeks of therapy and remains alive. The mean on-study
time for patients with stable disease was 101.2 days and ranges
from 28 to 252 days. A total of 10 patients remain alive in the
SD group (50%).
Three patients had a minor response while on therapy. The
first of these patients (at Level III) had a carcinoid tumor
metastatic to bone and soft tissue. After Week 4 of therapy, his
bone scan and soft tissue disease remained stable. After Week
7 of therapy, his soft tissue disease had decreased by 20% and
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PHASE I STUDY OF IL-2 AND IFN-)3
Table 3 Toxicities ofll.-2 and IFN-ßin combination
toxicity*Fatigue/performancestatus1133243Anorexia/wt
with Grade III or IV
ofpatientsentered/evaluable"4/44/45/37/37/35/33/35/44/43/3No.requireddosereduction0012332243No.
DoselevelIIIIIIIVV
loss1121332Depression2222Arthritis122OtherIe1"1*''A1*1'y
(S.C.)VI
(s.c.)VII
(s.c.)V
(i.V.)VI
(i.V.)VII
(i.V.)No.
°After 4 weeks of treatment.
* Note that individual patients are included under more than one toxicity.
' Hypotension.
d Exfoliative skin rash.
' Thrombocytopenia.
r Diarrhea.
* Fever.
* Peripheral edema.
sH
to
H
1500
200.0
250.0
4000
TIMK (miniiU-s)
JÕL
Fig. 1. Pharmacokinetics of IL-2 with i.v. (IV; A) and s.c. (SQ; O) adminis
tration at Dose Level V. Dose Level V uses 5x10" units/m2 of IL-2 and 6x10'
units/m! of IFN-/3. The area of the IV curve is 25,898 unit-min/ml, and the area
of the SQ curve is 17,498 unit-min/ml (P < 0.05).
10,000
100.000
1.000.000
10.000.000
DOSE IL-2 (I'NITS/M2)
bone disease continued as stable. After 9 weeks of therapy, the
soft tissue response was at 50% reduction, but there was no
change in his bone disease. However, at Day 133 he was
removed from study due to tumor progression. The second
patient [at Level V (s.c.)] had multiple, bilateral lung métastases
from her synovial sarcoma. After 4 weeks of therapy, her
measurable disease had decreased by 25% and remained stable
H
until disease progression at on-study Day 63. The third patient
Z
[at Level V (s.c.)] with malignant melanoma metastatic to the
orbit had a stable computed tomography scan after Week 4 of
therapy. However, her physical examination demonstrated a
marked decrease (to near normal) in the degree of orbital
protuberance and complete resolution of pain and inflammation
of the surrounding tissues and structures. She was removed
from study at Day 31 due to a decrease in performance status.
Two of these patients remain alive.
One patient with melanoma had a partial response after Week
1,000.000
10,000,000
100.000.000
4 of therapy at Dose Level IV. A measurable, biopsy-proved,
DOSE IFN-BETA (UNITS/M1)
right neck mass decreased by >50%. At Week 11 of therapy,
Fig. 2. A, maximum LAK activity by treatment dose level of s.c. IL-2; constant
the neck examination was negative; however, a nonmeasurable
dose IFN-/3, 2 x 10' units/m2. B, maximum LAK activity by IFN-/3 dose level;
hilar mass remained unchanged. The patient was removed from constant dose IL-2, 5 x 10' units/m2. Bars, SEM.
study at Week 38 (Day 270) because of progressive disease.
LAK Activity. The mean LAK activity steadily increased as
the dose of IL-2 increased. Significant LAK activity required 5 the difference between mean LU for all dose levels using this
x IO6 units/m2 (Fig. 2A). With this dose of IL-2,6 x IO6units/
dose of IL-2 was not statistically significant (Fig. 2B).
m2 of IFN-/3 generated the highest mean value for LU. However,
No patient had baseline LAK activity >8 LU. However, seven
Bo
SO"
rii
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PHASE I STUDY OF IL-2 AND IFN-0
évaluablepatients had treatment-stimulated peak LAK activity
>15 LU (7 of 32 or 21.9%). These patients had a variety of
tumors: colon carcinoma, 2 of 12; melanoma, 2 of 3; breast
carcinoma, 1 of 3; renal carcinoma, 1 of 4; and ovarian carci
noma, 1 of 1. Of the 12 évaluablecolorectal cancer patients, 3
had PD at Week 4, and 9 had SD. Of these 9 patients, 7 did
not exhibit significant LAK activity and had a mean duration
of disease stabilization of 87.8 days. Four of these patients are
alive. One of the 11 had significant LAK activity at Dose Level
V (i.v.), with an on-study time of 58 days. One patient, at Dose
Level VII (s.c.), had PD at Week 4 and exhibited significant
LAK activity. Both of these patients remain alive to date. Of
the 3 évaluablepatients with melanoma, 1 had SD at Week 4
with PD at Week 5. Two patients exhibited significant LAK
activity at Levels IV and V (s.c.). One had a marginal response
of 31+ days and the second patient had a partial response
lasting for 270 days. One patient is alive to date. Of the 3
évaluablepatients with breast cancer, the 1 patient with SD at
Week 4 also had significant LAK activity at Level VI (i.v.).
This patient had a disease stabilization duration of 71 + days.
One patient with metastatic ovarian cancer and SD at Week 4
exhibited significant LAK activity at Dose Level VII (i.v.). This
patient elected no further therapy due to severe fatigue at Week
10. Of the 4 évaluablepatients with renal cell carcinoma, 2 had
SD including the 1 patient with significant LAK activity with
an on-study time of 81 days. Three of these patients remain
alive to date.
The overall rate of LAK activity >15 LU for the évaluable
patients receiving s.c. doses of IL-2 was 18% (4 of 22). The rate
of LAK activity >15 LU in the group of évaluablepatients
receiving i.v. IL-2 was 30% (3 of 10). Seven of the 21 (33%)
évaluablepatients receiving 5 x 10* units/m2 of IL-2 had LAK
activity >15 LU, including 4 of 11 (36%) receiving s.c. IL-2.
Peak LAK activity occurred at the end of the second week of
treatment in 3 patients, at the end of the fourth week of
treatment in 2 patients, and at the end of 8 weeks of treatment
in 2 patients. There appeared to be a correlation between degree
of response and LAK activity (Fig. 3). There was a significant
difference in LAK activity between patients having progressive
disease (mean, 5.0) and those having either stable disease (mean.
!!;/>=
0.047) or a response (mean, 53; P = 0.019). The
difference in LAK activity between patients with stable disease
and a response was not statistically significant (P = 0.10).
Antibody Studies. In order to follow the possible development
of antibodies to IL-2 and IFN, an ELISA specific for IgG and
for IgM to each of these proteins was performed on serum
taken before treatment and at approximately 2-week intervals
during their administration. Two or more serum samples were
available from 38 of the 47 patients enrolled in this trial. The
antibody levels are expressed as titers, defined as the product
of the absorbance in the ELISA and the serum dilution factor.
A titer of 1 corresponds to approximately 30 ng/ni I of IgG and
is the lower limit of the sensitivity of this assay.
IgM specific to IL-2 was seen in 12 patients, although the
level was only marginally detectable in 7 of these. IgG specific
to IL-2 was seen in 24 patients, with the level being marginal
in 5 (Fig. 4). In only 2 patients was a n<mmarginal level of IgM
not associated with IgG. The maximum IgM titer was 80, while
the maximum IgG titer seen was 1600. The four highest IgM
titers were found in the group of 12 patients receiving IL-2 by
the i.v. route. However, the highest IgG titers were found in
patients receiving s.c. injections. There appears to be a signifi
cant correlation between maximum IgG titers and number of
days on trial only for the group of 26 patients in the s.c. group,
as shown in Fig. 4. The sera from all patients with IL-2 antibody
titers greater than 10 who received IL-2 for 50 days or longer
were assayed for IL-2-neu tral i/ing activity. Among these 9
patients, the sera from 5 had neutralizing activity, with the
highest titer being 9000.
The immunogenicity of IFN, given by the i.v. route to the
entire group, was modest compared to that of IL-2. One patient
developed an IFN-specific titer of 660, 3 had titers of 1 or 2,
and the remaining patients were negative. Four had non-zero
IFN-specific IgM titers, but only in the range of 2-4. Interest
ingly, the patient with the IFN antibody titer of 660 also had
the highest IL-2 antibody ELISA titer and IL-2-neutralizing
activity.
7-Interferon Levels. Endogenous IFN-7 was induced as
shown in Fig. 5 following the administration of IL-2 and IFNß.At equivalent doses of IL-2 (5 x 10* units/m2) i.v. adminis
tration induced significantly more IFN-7 than did s.c. admin
istration (P = 0.047; see legend to Fig. 5). In addition, the
maximum induced IFN-7 level occurred 4 h after the IL-2
û= SQ DOSING
G = IV DOSING
MR/PR
n=4
mean-53.10
stm =35 61
>1000-
•Z <1000-
•f.
<
2
H
>
D D
Q D
K
< 100-1
D G
û û
CO
a
=
°
t.
O
01
0-
SD
n-21
D
A û
in =fi 02
n=2.96
<20
<80
DAYS
Fig. 3. Mean peak LAK activity according to response. There is a significant
difference in LAK activity between patients with progressive disease (PD) and
those with either stable disease (SD) (P = 0.047) or a response (P = 0.019). MR.
minor response; PR. partial response. The difference in LAK activity between
patients with stable disease and a response is not statistically significant (/' =
0.10). Bars, SEM.
Fig. 4. Maximum IgG titers in 26 patients receiving i.v. (/r. D) or s.c. (SQ.
A) IL-2. The number of days is divided into 4 groups and the IgG titers into 5
groups. There is a significant correlation between maximum IgG titers and
number of days on trial for the SQ group (Spearman's p rank correlation, O.S4).
There is no correlation between titer and number of days on study for the IV
group (Spearman's /) rank correlation. —0.43).
3879
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PHASE I STUDY OF IL-2 AND IFN-0
Table 4 Toxicities
Acute
Fever
Rigors
Vomiting
Local inflammation'
Diarrhea
Rash
Chronic
Fatigue/performance
Anorexia/wt loss"
Arthritis/arthralgias"
Depression"
Peripheral edema"
status"
Hypotension
Insomnia
Anemia
Thrombocytopenia
" The maximum tolerated dose was determined by these chronic toxicities
(Grade III-IV).
' After S.C.IL-2.
o.o
r>o.o loo.o 150.0 200.0 ','fjo.o 300.0 350.0 400.0
TIMK (iniiiuli's)
Fig. 5. Endogenous IFN-i following 5x10' units/m2 of IL-2 either i.v. (D)
or s.c. (A). Time points, means of 12 patient values. Linear regression was used
to test if the s.c. and i.v. routes were equivalent. Having rejected this (P = 0.047),
the data were fit by functions of the form a(/)(f"/>)exp(—b*t), i —1, 2 for this
graph. The ratio of the two curves is thus constant over time since they differ
only in the coefficients a(i). The i.v. curve is uniformly about 3.3 times higher
than the s.c. curve. With these fitted curves, the P value is much lower (P =
0.001).
administration, with a sharp rise between 2 and 4 h and a
gradual decline thereafter.
DISCUSSION
Initial enthusiasm followed the first reports on the use of
interleukin 2 in humans (1, 2). However, significant toxicity
was associated with this therapy, and subsequent reports mini
mized the potential benefit of this new treatment (29-31). Phase
II studies of IL-2 have shown efficacy in renal cell carcinoma
and malignant melanoma (3, 4, 8, 32). These two diseases are
also responsive to the type I interferons, a- and /3-interferons
have reported response rates between 10 and 30% in renal cell
carcinoma and between 10 and 20% in malignant melanoma
(33, 34).
The combination of IL-2 and IFN-/3 was prompted not only
by these clinical data but also by preclinical in vitro data
demonstrating synergism between these two agents both in
stimulating NK activity and in increasing expression of IL-2
receptors (13-18). Interleukin 2 also generates IFN--y in vitro,
a substance that is synergistic both with type I interferons and
with interleukin 2 (35). IFN--y has been shown to generate IL2 receptors and thereby presumably enhance IL-2 binding (36,
37). In addition, IFN-7 binds to a different receptor than do
the type I interferons, subsequently enhancing the antiproliferative effects of each other (21-23).
In the current study, IL-2 and IFN-/3 were given by i.v. bolus,
three times weekly. The doses of interleukin 2 were comparable
to those given with interleukin 2 alone in a similar schedule.3
Over a 4-week period of time, 60 million units/m2 of interleukin
2 are administered using this regimen. The IL-2 plus IFN-0
regimen has been shown to be well tolerated and with somewhat
different toxicities than are seen with the high dose therapy
used by Rosenberg (3). Specifically, the dose-limiting toxicities
were the chronic toxicities of profound fatigue, lethargy, depres
sion, weight loss, and a decrease in performance status (Table
4). Hypotension, peripheral edema, and pulmonary edema, the
major dose-limiting toxicities reported in high dose IL-2 or IL2 plus LAK studies, were uncommon in this moderate dose,
intermittent IL-2 plus IFN-/3 trial.
This combination was able to generate LAK activity with the
3 E. Hersh, personal communication,
1987.
interleukin 2 administered both s.c. and i.v. However, the s.c.
dosing induced neutralizing antibodies. There was a 90% inci
dence of antibodies when patients were treated s.c. for more
than 50 days and 5 of 9 of these were neutralizing. No neutral
izing antibodies were seen with the i.v. dosing of IL-2. More
over, there was no change in the generation of IL-2 antibodies
formed by the addition of IFN-0. Of particular note is that the
one patient with a partial response had significant levels of
LAK activity. This LAK activity disappeared when she devel
oped neutralizing antibodies to the IL-2. As typified by this
patient, however, the ability to generate LAK was correlated
with response to treatment (Fig. 3). As the degree of response
improved from progression to stable disease to minor or partial
response, the mean level of LAK activity increased as well.
While there was not a statistical difference between the LAK
activity in patients with stable disease as compared to those
with a response, the trend toward improvement is apparent.
The use of i.v. IL-2 resulted in much higher serum levels of
interleukin 2 for approximately 3 h as compared to the use of
s.c. IL-2. This may account for the difference in the ability to
generate 7-interferon between these two routes of administra
tion.
Attempts are now ongoing to minimize the toxicity seen with
high doses of IL-2 and to assess the contribution to efficacy of
the administration of LAK. Recent reports have challenged the
need for the ex vivo activation of LAK (32, 38, 39). However,
most data support the importance of LAK as the mechanism
of activity for interleukin 2. Attempts to minimize toxicity and
maintain efficacy have included administering the interleukin 2
by continuous infusion, using a lower dose of interleukin 2, and
using the same dose of interleukin 2 without the coadministration of LAK. Others have reported on the use of interleukin 2
i.p., s.c., and intermittently i.v. (38-40). The current report
presents a novel approach to potentially enhancing the efficacy
of moderate dose IL-2 by combining IL-2 with low dose IFN0.
The recommended dose for Phase II studies is 5 x 10' units/
m2 IL-2 plus 6 x IO6 units/m2 IFN-/3, both administered i.v.
This dose is one level below the maximum tolerated dose and
would appear to be an adequate dose for generating LAK
activity by this schedule. The use of IL-2 i.v. also avoids the
problem of neutralizing antibodies seen with IL-2 s.c. One
patient with malignant melanoma had a partial tumor regres
sion and one patient had a minor tumor regression. Thus
melanoma would appear to be a likely tumor type for Phase II
testing. Both IL-2 and IFN-/3 have independent activity in renal
cell carcinoma, and therefore this tumor type might also be an
appropriate candidate for Phase II studies. There is also the
potential for this combination to have synergy with the induced
IFN-y.
In summary, this Phase I study demonstrates that: (a) high
doses of IL-2 are not necessary for LAK activation in vivo; (b)
activation of LAK may be associated with an antitumor re-
3880
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PHASE I STUDY OF IL-2 AND IFN-0
sponse; (c) significant levels of IFN-i can be generated with
this dose and schedule of IL-2; and (d) 5 x IO6 units/m2 IL-2
plus 6 x IO6 units/m2 IFN-/3 given i.v. three times weekly is
both a tolerable dose for Phase II studies and a biologically
active dose.
17.
18.
ACKNOWLEDGMENTS
19.
The authors wish to thank Dr. Louis Weiner for his careful review
of the manuscript and for performing the IFN-7 assay, to Donna
Dodaro for typing the manuscript, and to Perry Watts for programming
and graphics.
20.
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3881
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A Phase I Study of Recombinant Interleukin 2 plus Recombinant
β-Interferon
Robert L. Krigel, Kristin A. Padavic-Shaller, Alfred R. Rudolph, et al.
Cancer Res 1988;48:3875-3881.
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