phase lb trial of chimeric antidisialoganglioside...
TRANSCRIPT
Vol. 3, 1277-1288, August 1997 Clinical Cancer Research 1277
Phase lB Trial of Chimeric Antidisialoganglioside Antibody Plus
Interleukin 2 for Melanoma Patients’
Mark R. Albertini,2 Jacquelyn A. Hank,
Joan H. Schiller, Masoud Khorsand,
Agnes A. Borchert, Jacek Gan, Robin Bechhofer,
Barry Storer, Ralph A. Reisfeld, and
Paul M. SondelUniversity of Wisconsin Comprehensive Cancer Center, University of
Wisconsin, Madison, Wisconsin 53792 [M. R. A., I. A. H., I. H. S.,
M.K., A.A.B.,J.G., RB., B.S., P.M.S.l: and Scripps ResearchInstitute, La Jolla, California 92037 [R. A. R.l
ABSTRACTWe conducted a Phase lB trial of antidisialoganglioside
chimeric 14.18 (chl4.18) antibody and interleukin 2 (IL-2) to
determine the maximal tolerated dose (MTD), immunolog-
ical effects, antitumor effects, and toxicity of this treatment
combination. Twenty-four melanoma patients received im-
munotherapy with chl4.18 antibody and a continuous infu-
sion of Roche IL-2 (1.5 x 106 units/m2/day) given 4 days/
week for 3 weeks. The chl4.18 antibody (dose level, 2-10
mg/m2/day) was scheduled to be given for 5 days, before,
during, or following initial systemic IL-2 treatment. The
chl4.18 MTD was 7.5 mg/m2/day, and 15 patients were
treated with the chl4.18 MTD. Immunological effects in-
cluded the induction of lymphokine-activated killer activityand antibody-dependent cellular cytotoxicity by peripheralblood mononuclear cells. In addition, serum samples ob-
tamed following chl4.18 infusions were able to facilitate in
vitro antibody-dependent cellular cytotoxicity. Antitumor
activity included one complete response, one partial re-sponse, eight patients with stable disease, and one patient
with >50% decrease of hepatic metastases in the face of
recurrence of a s.c. lesion. Dose-limiting toxicities were a
severe allergic reaction and weakness, pencardial effusion,
and decreased performance status. Most patients treated atthe MTD had abdominal, chest, or extremity pain requiring
i.v. morphine. One patient had an objective peripheral neu-
ropathy. This IL-2 and chl4.18 treatment combination in-
duces immune activation in all patients and antitumor ac-
tivity in some melanoma patients. We are attempting to
Received 3/4/97; revised 4/28/97; accepted 5/1/97.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
I This work was supported by NIH Grants CA614498-Ol , NO1 -
CM87290, and 3-MOI-RR03186-07S2.
2 To whom requests for reprints should be addressed, at K4/4l4 Clinical
Science Center, University of Wisconsin Hospital and Clinics, 600
Highland Avenue, Madison, WI 53792. Phone: (608)263-0117: Fax:
(608) 263-4226.
enhance this treatment approach by addition of the anti-
GD3 R24 antibody to this IL-2 and chl4.18 regimen.
INTRODUCTIONOver the past several years, a vast amount of clinical data
has been obtained with a variety of human recombinant IL-23
regimens ( I ). The rationale for the use of IL-2 to treat human
cancers is at least twofold (2-5). First, in some murine tumors,
antigen-specific T cells are able to specifically recognize and
destroy syngeneic neoplastic tissue. Such cells appear to have
their destructive capacities augmented by treatment with IL-2.
Some in vitro data suggest that certain patients with metastatic
neoplasms (for example, melanoma) have antigen-specific T
cells that may be enhanced in their antitumor effects by IL-2 (4,
5). Second, a separate population of IL-2-responsive cells, mi-tially designated LAK cells, reflect predominantly activated NK
cells that do not have mature T-cell markers or antigen-specific
receptor molecules. These IL-2-activated NK cells are able to
preferentially destroy a variety of neoplastic tissues in vitro and
possibly in vivo. Such cells can destroy tumors that are “non-
immunogenic,” demonstrating that treatment with IL-2 does not
require the existence of documented antigen-specific tumor-
reactive T cells (3-5). A variety of regimens using various doses
of IL-2 have induced measurable antitumor responses in patients
with cancer, particularly melanoma and renal cell carcinoma (6,
7). Although the mechanism of action remains uncertain, in vivo
IL-2 treatment does induce cells with LAK activity able to
destroy tumor in vitro (8) and possibly in vivo. Unfortunately,
the toxicity associated with these regimens is significant, and the
responses are generally of brief duration.
Although LAK cells can destroy tumor cells, they also can
destroy, to a lesser extent, normal tissue in vitro (9, 10). In
murine studies, this appears to account for the severe toxicity of
IL-2, particularly the capillary leak phenomenon (I I). Although
even larger doses of IL-2 may potentially augment in vivo LAK
activity to a greater degree, thereby inducing more tumor de-
struction, augmented destruction of normal cells would likely
result. This may account for the life-threatening toxicity of very
high doses of IL-2. Further improvements in clinical results with
IL-2 require more selective tumor destruction by the immune
responses induced. More selective induction of tumor-specific T
cells rather than LAK cells may provide better specificity (4, 5).
However, in vivo tumor-selective T-cell reactivity has been
difficult to document for most human tumors ( I 2). Furthermore,
3 The abbreviations used are: IL-2, interleukin 2; LAK. lymphokine-
activated killer: NK, natural killer: mAb, monoclonal antibody: ADCC,
antibody-dependent cellular cytotoxicity: GD2, disialoganglioside:
chl4.18, chimeric 14.18; Id, idiotypic: MTD, maximal tolerated dose:
ECOG, Eastern Cooperative Oncology Group: CNS, central nervoussystem: PBMC, peripheral blood mononuclear cell.
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1278 IL-2 and chl4.18 Antibody for Melanoma
4 M. Sznol, personal communication.
some data suggest that IL-2 treatment may actually cause a
decrease in specific T-cell function by endogenous T cells (13,
14). One other approach involves focusing the cytotoxic activity
of LAK cells to mediate more tumor-specific destruction. In
vitro, this can be accomplished with mAbs able to facilitate
ADCC, a process mediated primarily by Fc receptor-bearing NK
cells (15-17).
Murine mAb 14.18, an IgG3 directed against murine GD2,
recognizes GD2 expressed on human neuroblastoma, mela-
noma, and certain other tumors (16, 18). The l4.G2A antibody
is an IgG2A class switch variant of the 14.18 antibody, designed
to mediate enhanced ADCC (19). To improve the potential
clinical utility of this antibody, a chimeric construct was formed
using the murine variable genes and human genes for the con-
stant regions of IgGl and K chains (20, 21). This chl4.18 is
50-100 times more potent in vitro at ADCC than the murine
mAb (22). The purpose of this study was to combine IL-2
treatment with administration ofthis chl4.18 antibody. The IL-2
regimen is one that has been well tolerated on an outpatient
basis yet is able to effectively induce LAK activation and other
IL-2-dependent immune changes in vivo (23).
Clinical trials with mAbs have shown some benefit in
occasional patients (24). For those antibodies able to mediate
ADCC, it is possible that inadequate effector cell function was
the limiting factor. The rationale for concurrent treatment with
IL-2 and chl4.I8 is to provide in vivo activation of effectors
with IL-2 in an attempt to enhance in vivo ADCC (25). We have
described previously the anti-Id response to the chl4.18 anti-
body in patients receiving immunotherapy with IL-2 and the
chl4.18 antibody (26). This report describes the toxicity, im-
munological effects, MTh, and antitumor effects of IL-2 ad-
ministered with the anti-GD2, melanoma-reactive chl4. 18 anti-
body.
MATERIALS AND METHODS
Clinical Protocol
Patients. From June, 1993, through December, 1994, 24
melanoma patients were enrolled in this clinical trial (National
Cancer Institute-Biological Response Modifiers Program Proto-
col B90-00l4). All patients signed informed consent forms.
These patients had biopsy-proven refractory melanoma that was
surgically or medically incurable by standard clinical ap-
proaches. Patients could have either measurable or evaluable
disease using standard ECOG criteria or no evidence of disease,
if the patient had prior surgical resection of distant or multiple
regional recurrences. All patients had an ECOG performance
status of 0 or 1 and a life expectancy of at least 4 months.
Eligibility criteria included normal hematological parameters
(leukocyte count, �3500/ml; hemoglobin, � 10.0 gm/dl; and
platelet count, > lOO,000/ml), adequate liver function (total se-
rum bilirubin, <2.0 mg/dI; and transaminases, <3X normal),
and adequate renal function (serum creatinine, <2.0 mg/dl; or
creatinine clearance, >60 ml/min). Criteria for patient exclusion
included treatment with cytotoxic chemotherapy within 3
weeks, radiation therapy within 2 weeks, or treatment with
glucocorticoids within 2 weeks prior to entry into study. Patients
with CNS disease, including intracerebral CNS metastases or a
history of CNS metastases, were eligible for treatment if the
CNS disease was previously treated and clinically stable for at
least 4 weeks following radiotherapy. Patients who required
continued therapy with corticosteroids, aspirin, or nonsteroidal
anti-inflammatory agents were ineligible. Patients with signifi-
cant cardiac disease or symptomatic respiratory disease were
also not eligible for this study. Patients who had previously
received biological therapy with murine mAb or with humanl
mouse chimeric antibody and patients who had received prior
murine mAb for tumor imaging or for any other reason were
ineligible for this study.
Recombinant IL-2 and chl4.18. Recombinant IL-2 was
provided through the National Cancer Institute-Biological Re-
sponse Modifiers Program by Hoffmann-LaRoche, Inc. (Nutley,
NJ). The drug was lyophilized and reconstituted with sterile
saline. Unitage corresponds to the initial Hoffmann-LaRoche
IL-2 unit, which also corresponds to that of the National Cancer
Institute-Biological Response Modifiers Program standard IL-2
unit, as used previously in our published IL-2 trials (27). The
current dosing conversion from Hoffmann-LaRoche IL-2 units
to commercially available Chiron (Chiron Therapeutics, Em-
eryville, CA) IL-2 units is: 1 Roche unit = 3 international units
of Chiron IL-2.4
The chl4.l8 antibody is a chimeric construct containing
the same murine variable region as murine mAb l4.G2a that is
specific for the GD2 antigen, which is expressed on human
neuroblastoma, melanoma, glioblastoma, sarcoma, and small
cell lung carcinoma (20, 21). The chl4.18 antibody was con-
structed by joining the cDNA for the variable region of the
murine antibody with the constant regions of ‘yl heavy chain
and the K light chain (21). This antibody was developed by
Stephen D. Gillies (Fuji Immunopharmaceutical Corporation,
Lexington, MA) and was provided through the National Cancer
Institute-Biological Response Modifiers Program (Frederick,
MD).
Study Design. The patients were entered into one of four
treatment groups (Table 1). Treatment group 1 consisted of 15
patients who received treatment with IL-2 and with chl4.18
antibody at four separate dose levels of chl4.18 antibody (an-
tibody dose levels between 2 and 10 mg/m2/day). These patients
were scheduled to receive IL-2 at a dose of 1 .5 X 106 units/m2/
day, which was given 4 days per week for 3 weeks. The IL-2
was administered as a continuous infusion throughout each 96-h
period, except in its 2nd week of administration, when each
scheduled 24-h infusion of IL-2 was accelerated to 20 h to
prevent the cytokine being infused during antibody infusions.
The chl4.18 antibody was given as a daily 4-h iv. infusion for
5 days in a row during week 2 of the treatment. Following a
2-week observation, patients who still met all eligibility criteria
and had stable disease or a clinical antitumor response could
receive an additional course of treatment. Patients without an
ECOG grade 3 IL-2 toxicity during their first 3 weeks of IL-2
treatment were eligible for an IL-2 dose escalation to 2 X 106
units/m2/day. Patients who received a second course of treat-
ment were scheduled to receive an additional 5 days of chl4.18
antibody at the same dose as given initially. This was also
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1279
Table I Treatmen t schedule for melanoma patients receiving combina tion immunotherapy
Treatment groups”
Treatment”chl4.l8 dose
(mg/m2/day)Week ch 14. 1 8� lL-2”
Treatment group 1 ( 15 patients) 2-10Course I I
2
3
4-5, observe
+
+
+
+
Course 2 6
7
8
9-10. observe
+
+
+
+
Treatment group 2 (3 patients) 7.5Course I 1
2, observe
+
Course 2 3
45
6-7, observe
+
+
+
+
Treatment group 3 (3 patients) 7.5
Coursel
Course2
I2, observe
3
4
5
6-7, observe
+
+
+
+
+
+
Treatment group 4 (3 patients) 7.5
Course 1 12
3, observe
+
+
+
Course 2 4
5
67-8, observe
+
+
+
+
‘, The 24 melanoma patients were enrolled into treatment group I (15 patients): treatment group 2 (3 patients): treatment group 3 (3 patients):
and treatment group 4 (3 patients).
b The + symbol indicates treatment weeks during which chl4.l8 antibody and/or IL-2 was scheduled to be administered. Treatment course 3,
when given, was the same as the prior treatment course 2.C The chl4.18 antibody was scheduled to be administered as five daily 4-h infusions during each antibody treatment week.
d The IL-2 dose of I .5 X I 0” units/m2/day was scheduled to be given as a continuous infusion over four consecutive days during each IL-2 week
except for not being given during the 4-h antibody infusions.
administered during the 2nd week of this second course of
treatment.
Three additional treatment groups were then evaluated
to determine whether the ch 14. 1 8 MTD from treatment group
I could be administered with different schedules of systemic
IL-2 infusion, as well as to determine the influence of sys-
temic IL-2 on the anti-Id response to chl4.18. Treatment
group 2 was composed of three patients who received an
initial 5-day treatment course of chl4.18 antibody alone,
without any IL-2. The dose of chl4. 18 antibody used for this
group (7.5 mg/m2/day) was the highest dose of chl4.l8
antibody that was well tolerated by the patients in treatment
group 1, and it was given as a 4-h infusion daily for 5 days.
These three patients were observed for 1 week without treat-
ment. Then, in weeks 3, 4, and 5, they were scheduled to
receive the same treatment regimen as used during weeks I,
2, and 3 for treatment group 1, with IL-2 given at 1.5 X 106
units/m2/day, 4 days per week, for 3 weeks. The chl4.l8
antibody was scheduled to be given during the 2nd week of
IL-2 administration (week 4 of the protocol regimen), 5 days
in a row, at 7.5 mg/m2/day. Following a 2-week observation
period, these three patients were again eligible for an addi-
tional 3-week treatment consisting of the identical IL-2 and
chl4.l8 antibody treatment administered during weeks 3, 4,
and 5. Treatment group 3 consisted of three patients who
received treatment as outlined for treatment group 2, with the
addition of IL-2 at a dose of 1.5 X 106 units/m2/day given
for 4 days during the initial week of repetitive ch I 4. 18
infusions. Treatment group 4 was composed of three patients
who received treatment as outlined for treatment group 2,
with the addition of IL-2 at a dose of 1 .5 X I 06 units/m2/day
given for 4 days during the initial week of repetitive chl4. I 8
infusions, as well as during the week before the chl4.l8
infusions.
All patients were treated in the inpatient or outpatient
facility of the University of Wisconsin General Clinical Re-
search Center. The IL-2 alone treatments were administered on
an outpatient basis. All treatments involving chl4.l8 antibody
were administered as inpatient therapy. On each day of antibody
treatment, each patient received an initial test dose of 1/20 of
their scheduled daily antibody dose over 10 mm, followed by 20
mm of observation for evidence of allergic reaction. The re-
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1280 IL-2 and chl4.18 Antibody for Melanoma
maining antibody administration was then completed as a 3.5-h
continuous infusion. Premedications for the chl4.l8 infusions
included: 0.3-ml s.c. injection of Sus-phrine (a 1 :200 aqueous
suspension of a brand of epinephrine; Steris Laboratories, Inc.,
Phoenix, AZ); 2-mg iv. injection of morphine; and 650 mg of
acetaminophen and 50 mg of diphenhydramine hydrochloride,
each administered p.o.
Toxicity Grading and Dose Modifications. The
ECOG common toxicity criteria were used for grading of tox-
icities. The University of Wisconsin Comprehensive Cancer
Center clinical toxicity grading scale for IL-2 studies was used
for weight gain, systolic blood pressure, temperature, and de-
dine in performance status (7). Mild (grade I) toxicity corre-
sponded to a �20 mmHg decrease in systolic blood pressure, a
5-10% body mass weight gain, fever of �38#{176}C, or a decline in
performance status of one grade. Moderate toxicity corre-
sponded to grade 2 toxicity of a 20-40 mm/Hg decrease in
systolic blood pressure, I 1-14% body mass weight gain, fever
of 38. 1-39.9#{176}C, or a decline of two grades in performance
status. Severe toxicity corresponded to grade 3 with a �40
mmHg decrease in systolic blood pressure, � I 5% body mass
weight gain, fever of �40#{176}C, or a decrease of three grades in
performance status. When grade 3 toxicity occurred, treatment
was withheld until the toxic reaction(s) improved to a grade of
� I or symptoms returned to baseline. Treatment was then
resumed at a 50% dose.
Response Criteria. A complete response was defined as
complete disappearance of all evident tumor and return of
tumor-related abnormal test values to normal levels. A partial
response was defined as a decrease by at least 50% of the sum
of the areas of all known lesions in the absence of progression
of any lesion or the appearance of any new lesions. Progressive
disease was defined as any of the following: development of any
new area of malignant disease; increase by at least 25% in any
pretreatment area of measurable malignant disease; or signifi-
cant clinical deterioration related to malignant disease. Stable
disease was defined as a change in measurable disease too small
to meet the requirements for partial response or progression and
no new lesions appearing.
Immunological Monitoring
As previously reported, serum samples were obtained from
all patients to determine the anti-Id response to chl4.l8 anti-
body (26). Additional immunological monitoring was per-
formed on patients in treatment groups I and 2. The patients in
treatment groups 3 and 4 were evaluated at the chl 4. 18 MTD to
determine the influence of the schedule of IL-2 administration
on the anti-Id response to chl4. 18, and additional immunolog-
ical monitoring was not planned for those patients. For the
immunological monitoring of treatment group 1 and 2 patients,
two baseline blood samples were obtained pretreatment. Blood
samples were also obtained on treatment protocol days 6, 13,
and 20 for treatment group 1 patients and on protocol day 6 for
treatment group 2 patients. Serum samples from selected pa-
tients were obtained immediately prior to and 1 h following a
4-h in vito chl4. 18 infusion for the in vitro serum ADCC
assays.
Cell Lines. The Daudi Burkitts lymphoma cell line and
the BT2O human breast carcinoma cell line were obtained from
the American Type Culture Collection (Rockville, MD). The
LA-N-S human neuroblastoma cell line was kindly provided by
R. Seeger (Children’s Hospital of Los Angeles, Los Angeles,
CA).
mAb. The chl4.18 antibody was described above. ING-l
is a mouse-human chimeric antibody of the human IgG I sub-
class with mouse-encoded variable regions (28, 29). It reacts
with a surface antigen on adenocarcinoma cells, including
breast, lung, and colon, and was kindly provided by R. Robinson
(XOMA, Santa Monica, CA).
Surface Marker Analysis. The expression of T-cell
marker (CD3), Fc receptor (CDI6), a chain of the IL-2
receptor (CD25), and NK marker (CDS6) was examined on
PBMCs gated for lymphocytes. Double marker analysis was
used to determine the percentage of cells expressing both
CD56 and CDI6. All fluorescence labeling was performed at
4#{176}Cin the dark for 30 mm. Patient PBMC populations were
characterized by incubating 2 X 10� Ficoll-Hypaque-isolated
PBMCs in 100 p.1 of PBS with FITC- or phycoerythrin-
conjugated antibodies (Becton Dickinson, San Jose, CA) at
the concentrations recommended by the manufacturer’s pro-
cedure. Propidium iodide, at a final concentration of I p.g/ml,
was added just prior to analysis to separate live from dead
cell populations.
ADCC. All ADCC assays were performed in RPMI I 640
medium supplemented with 10% human serum (Pel-Freez, Rog-
ers, AR), 25 mM HEPES, 100 units/mI penicillin, and 100 p.g/ml
streptomycin sulfate (RPMI-HS) and in RPMI-HS supple-
mented with IL-2 at a final concentration of 100 units/ml.
Effector cells in RPMI-HS, in a total volume of 50 p.1, were
plated in quadruplicate into 96-well U-bottomed microtiter
plates at E:T ratios of 50:1, 16.7:1, and 5.6:1. Fifty �il of
medium or IL-2 at a final concentration of 100 units/mI were
added to the effector cells and incubated for 30-45 mm at 37#{176}C
in an incubator with 5% CO,. Immediately prior to the addition
of target cells, SO p.1 of medium containing human mouse
chimeric antibody chl4.18, ING-l, or 50 pJ of the indicated
patient serum sample was added to the effectors. The final
concentration per well was 0.25 p.g/ml for both ING-l and
chl4.l8. Effector cells in medium and IL-2 were also plated to
determine their ability to mediate lysis of target cells in the
absence of antibody. Target cells were tumor cells labeled with
250 �i.Ci of 51Cr in 0.2 ml of RPMI-HS. These cells were mixed
every 15-30 mm during labeling to keep the cells in suspension.
After washing twice with RPMI 1640, 5 X l0� target cells were
plated with effector cells and centrifuged at 200 X g for S mm.
The plates were incubated at 37#{176}Cwith 5% CO2 for 4 h, and the
supernatants were harvested using the Skatron Harvesting Sys-
tern (Skatron, McLean, VA). Maximum 51Cr release was meas-
ured by lysing target cells with the detergent cetrimide (Sigma
Chemical Co.). Spontaneous 51Cr release was measured by
incubating target cells in RPMI-HS medium alone. Percentage
of cytotoxicity values were calculated for each E:T ratio as
follows:
% cytotoxicity
experimental release - spontaneous release= .
maximum release - spontaneous release
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1281
Table 2 Characteristics of patients receiving imm unotherapy with
chl4.18 antibody plus IL-2
Characteristic No of patients”
Total 24
SexMale 16
Female 8
Prior therapySurgery 21
Radiation therapy 8
Chemotherapy 8Hormonal therapy 2
Immunotherapy 4Hyperthermia I
Sites of disease
Nodal, skin, s.c. 17
Lung 10
Liver 10
Central nervous system 3Spleen 3
Bone 2
Other (adrenal, mesentery, bowel, pelvic mass) 5No evidence of disease 3
Performance status (ECOG)
0 III 13
(1 Median age, 46: range, 29-75 years.
Results are expressed as lytic units, in which I lytic unit is the
number of effector cells necessary to achieve 20% lysis of
5 x l0� targets (30).
LAK Cell Functional Assays. PBMCs were assayed for
their ability to kill the NK-resistant, LAK-sensitive, Burkitt’s
lymphoma line Daudi. PBMCs in 50 p.1 of RPMI-HS were
plated in quadruplicate U-bottomed microtiter wells at E:T
ratios of 50: 1 , 16.7: 1, and 5.6: 1 . RPMI-HS or RPMI-HS sup-
plemented with IL-2 at a final concentration of 100 units/mI
were added to quadruplicate wells. Target and effector cells
were handled as described above.
Evaluation of Anti-Idiotype Levels. Detailed methods
have recently been reported (26). Briefly, a 96-well, flat-bot-
tomed, polystyrene plate (Maxmsorp; NUNC, Roskilde, Den-
mark) was coated with 150 �il of chl4. 18 antibody per well in
a concentration of 2 pg/ml in 0.05 M sodium carbonate buffer
(pH 9.6) overnight at 4#{176}C.The plate was washed three times
with PBS, pH 7.2, containing 0.05% Tween 20 (Sigma). Serum
test samples and controls (100 ji.l) were diluted 1 :5 in sample
buffer (PBS + Tween 20 + gelatin + milk) and incubated
overnight at 4#{176}C.After being washed five times with PBS/
Tween 20, the plate was incubated with 100 p.1 of biotin-labeled
chl4. 18 per well in a I :500 dilution in sample buffer for 3 h at
room temperature before washing five times with 0. 1 M Tris/
Tween 20 (pH 7.4). Bound biotinylated antibody was detected
by adding ExtrAvidin conjugated to alkaline phosphatase (Sig-
ma) diluted to 1 :5000 in 0. 1 M Tris-HC1, pH 7.4, plus 0.05%
Tween 20. After a 1 -h incubation at room temperature and
washing as above, staining was performed with p-nitrophenyl-
phosphate (Sigma) in a concentration of 2 mg/ml in diethanol-
amine buffer. After I -h incubation at room temperature in the
dark, substrate conversion to colored product was determined at
Table 3 Dose escal ation of chl4.l8 antibod y in treatment group I
chl4.l8 dose
Dose level” No. of patients (mg/m2/day)
a 3 2
b 3 5
c 3 10
d” 6 7.5a Patients in treatment group I were sequentially entered into dose
levels a-d.
I, Dose level d was established after dose-limiting toxicity was seen
in two of three patients in dose level c.
405 nm with an ELISA reader (model EAR 400 AT: SLT Lab
Instruments, Groeding/Salzburg, Austria).
Statistical Analysis
Treatment effects were assessed as the change in parameter
values from baseline to various times during treatment, and
means are reported with SEs. Paired t tests were used to test for
treatment effects within groups of patients. The immunological
monitoring was performed for patients in treatment group 1 at
baseline and on protocol days 6, 13, and 20, as well as for
patients in treatment group 2 at baseline and on protocol day 6.
Linear regression analysis was used to test for a dose effect
within group I . In general, because of the small sample size and
variability of measures of immunological function, dose de-
pendency was not apparent. Consequently, the data from treat-
ment group 1 are pooled and shown in the appropriate table or
figure. The data from the three patients in treatment group 2 are
discussed in the appropriate section.
RESULTS
Patient Characteristics
Twenty-four patients were entered into this study, and their
pretreatment characteristics are outlined in Table 2. Sixteen of
the 24 patients were men, the median age was 46 years, and all
patients were ECOG performance status 0 or 1 . Eight patients
had received prior radiation therapy, eight patients were treated
with prior chemotherapy, and four patients had received prior
immunotherapy. The most common sites of metastatic disease
included nodal, skin, or s.c. disease (17 patients), pulmonary
metastatic disease (10 patients), and metastatic disease to the
liver (10 patients). Three patients had prior metastatic disease to
the brain. Three patients had no clinical evidence of disease at
the time of entry into this study.
Treatment Summary
Fifteen patients were evaluated in treatment group I to
determine the MTD of chl4.l8 when given with IL-2 as corn-
bined immunotherapy. The dose escalation scheme is shown in
Table 3. The chl4.l8 antibody dose levels of 2 mg/m2/day and
S mg/m2/day were tolerated without dose-limiting toxicity.
Three patients were then entered into the study at the chl4. 18
antibody dose level of 10 mg/m2/day. Dose-limiting toxicity
was seen in two of these three patients and included a severe
allergic reaction in one patient, as well as weakness, pericardial
effusion, and decreased performance status in the other patient.
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Patientno.”
Treatmentgroup”
ch 14. 18(mg/m2/day) No. of antibody courses Anti-Id antibody� Clinical response”
Duration of response orstable disease (months)
I (5) V 2.0 1 0 Progression2 (6) 1’ 2.0 2 0 Stable 3
3 (7) 1 2.0 1 0 Progression
4 (8) 1 5.0 1 0 Progression
5 (9) 1 5.0 2 0 Progression
6 ( 10) 1 5.0 2 0 Stable 2
7 ( 17) l� 10.0 1 + Progression
8 ( 18) 1 10.0 1 + Progression
9 ( I 9) 1 10.0 1 + Stable 6
10 ( I 1 ) le 7.5 2 0 Progression
11 (12) I 7.5 1 0 Progression
12 (13) 1 7.5 1 0 Progression
13 (14) 1 7.5 1 0 Progression
14 (15) 1 7.5 1 0 Progression
15 (16) 1 7.5 1 0 Progression
16 (20) 2 7.5 1 + Stable 2
17 (21) 2 7.5 3 + Stable 6
18 (22) 2” 7.5 3 + Stable 5
19 (23) 3 7.5 3 + Stable 120 (24) 3 7.5 2 0 Progression
21 (25) 3 7.5 3 + Stable 322 (26) 4’� 7.5 3 + Complete response >2423 (27) 4(1 7.5 2 0 Progression
24 (28) 4 7.5 3 + Partial response I
,, Each patient was given a sequential patient number, and the number in parentheses refers to that same patient’s designation in our publication
describing the pattern of anti-Id responses in this trial (26).
I, Modifications in planned chl4.18 infusions were required for patient 7 (2 days given), patient 9 (3 days given), patient 10 (3 days given during
course I and 4.2 days given during course 2), patient 14 (4.7 days given), and patient 16 (no second course given).
( Patients who had anti-Id antibody detected at any time during protocol treatment are identified with a + , and patients who did not have anti-Id
antibody detected at any time during protocol therapy are identified with a 0.
‘/ Patients 16, 17, and 18 had no measurable disease at the time of starting protocol therapy.
e IL-2 dose reductions were required for this patient.I Patient 22 received an inadvertent 10-fold excess of planned IL-2 administration on treatment day 35.
1282 IL-2 and chl4.18 Antibody for Melanoma
Table 4 Treatment summary
The l0-mg/m2/day chl4. 18 dose level thus exceeded the MTD
of chl4.l8 when combined with IL-2, as defined for this study.
Six patients were then evaluated at the intermediate chl4.l8
dose level of 7.5 mg/m2/day, and this dose level was tolerated
without dose-limiting toxicity. Thus, 7.5 mg/m2/day was deter-
mined to be the MTD of chl4. 18 when given with this dose and
schedule of IL-2. Patients were then sequentially entered into
treatment groups 2-4 to determine the influence of the timing of
systemic IL-2 administration on the clinical and immunological
effects of chl4.l 8 administration.
A treatment summary describing the treatment group, an-
tibody dose level, number of antibody courses, presence of
anti-Id antibodies, clinical response, and duration of response
for each patient treated on this study is provided in Table 4. The
detailed pattern of anti-Id antibody seen in each of these patients
was recently published (26). The patients who required IL-2
dose reductions and modifications in planned ch 14. 18 infusions
are identified. Seven patients required a 50% dose modification
of their IL-2 infusion (patients 1, 2, 7, 10, 18, 22, and 23). The
reasons for this required IL-2 dose modification included IL-2-
related hepatotoxicity (three patients), hypotension (two pa-
tients), decreased performance status (one patient), and throm-
bocytopenia (one patient). In addition, patient 10 did not receive
IL-2 in the 3rd week of protocol treatment due to IL-2-related
thrombocytopenia. Patient 22 received an inadvertent 10-fold
excess of planned IL-2 administration due to a pharmacy error
on treatment day 35. Modifications in the planned chl4.l8
infusion occurred in five patients. Two of these patients were
treated at the chl4.l8 antibody dose level of 10 mg/m2/day and
required treatment modifications because of weakness, pericar-
dial effusion, and decreased performance status (patient 7), or
due to a severe allergic reaction (patient 9). Patient 10 only
received three of the five planned initial infusions of chl4.18
antibody due to IL-2-related thrombocytopenia. In addition, a
dose modification was required in course 2 because of neuro-
pathic pain. Patient 14 had a chl4. I 8 antibody dose modifica-
tion due to neuropathic pain, and patient 16 did not receive any
of his second course of ch 14. 1 8 antibody infusion because of an
objective peripheral neuropathy that occurred following his first
course of treatment with chl4. 18 antibody. Treatment delays
between courses 1 and 2 included a I -week delay due to throm-
bus (patient 5), a 2-week delay as a consequence of infection
(patient 21), and a 3-week delay because of either infection
(patient 19) or a pulmonary embolus (patient 10). There was a
2-week delay between treatment courses 2 and 3 due to infection
for patient 18.
Toxicity
Most patients treated on this clinical study had the well-
described IL-2 constitutional symptoms, including fever, chills,
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1283
Table 5 Significant clinical toxicities d uring IL-2 and chl4.18 therapy
No. of patients No. of treat ment courses
Clinical toxicity” (total = 24) (total = 43)
Fever �40#{176}C 5 5Hypotension (>40 mmHg reduction in systolic blood pressure) 14 18
Brief 12 16
Extended 2 2
Requiring pressors 0 0
Decline in ECOG performance status of 3 (0 -� 3) 2 2
Weight gain �l5% of total body weight 1 1
Central venous catheter thrombosis 5 5
Infection requiring iv. antibiotics 8 8
Transient dyspnea or hypoxemia requiring oxygen 5 5
Pulmonary embolus I I
Severe allergic reaction I IObjective peripheral neuropathy 1 1
Pain
Requiring iv. morphine 10 12Uncontrolled with iv. morphine 2 2
‘C The clinical toxicities that were � grade 3 by either the common toxicity criteria or the University of Wisconsin Comprehensive Cancer Center
clinical toxicity grading scale for IL-2 studies are indicated. Most patients had some constitutional toxicities including fever, malaise, anorexia, and
a decline in performance status.
myalgias, anorexia, and decrease in performance status. Addi-
tional clinical toxicities that were greater than or equal to grade
3, by either the common toxicity criteria or the University of
Wisconsin Comprehensive Cancer Center clinical toxicity grad-
ing scale for IL-2 studies, are shown in Table 5. Patients on this
study did not receive prophylactic antibiotics, and all patients
were treated with I mg of daily coumadin as prophylaxis for
catheter-related thromboses. One patient had a pulmonary em-
bolus that occurred after being off all protocol treatment for 6
days and was believed to be most likely attributable to under-
lying disease status. A severe allergic reaction occurred in one
patient at the 10-mg/m2/day dose level, and this was one of the
dose-limiting toxicities of this treatment. This patient experi-
enced swelling of the neck, face, and tongue in association with
receiving an infusion of chl4.18 antibody. These symptoms
rapidly resolved following stopping the chl4.l8 infusion and
s.c. treatment with epinephrine and diphenhydramine hydro-
chloride. Severe allergic reactions did not occur in additional
patients. Note that patients treated on this study were premed-
icated for their chl4.l8 infusions with diphenhydramine hydro-
chloride, acetaminophen, morphine, and s.c. epinephrine.
Neuropathic pain in association with infusion of the
chl4. 18 antibody occurred in the majority of patients treated at
the chl4.18 MTD of 7.5 mg/m2/day. This pain was most typi-
cally described as a sharp pain in the back or upper abdomen
and usually began within I h of starting the chl4.18 antibody
infusion. Although this pain could be sufficiently severe as to
require parenteral morphine to control the pain, it could be
satisfactorily controlled with i.v. morphine in all but two pa-
tients. The pain in association with the chl4.18 antibody infu-
sion typically resolved within I h following completion of the
chl4.18 antibody infusion. Some patients also experienced a
self-limited distal extremity pain, particularly in the legs, fol-
lowing the chl4.18 antibody infusion. This pain would typically
resolve within 4-6 hours of completing the chl4.18 infusion.
One patient (patient 16), who received treatment with
chl4. I 8 antibody at 7.5 mg/m2/day in treatment group 2, had an
Table 6 Significant laboratory changes during IL-2 and chl4.18
therapy
No. of
No. of patients treatment courses
Laboratory value (total - 24)(total = 43)
Hematological
Hemoglobin, < 10 g/l00 ml I 3 18
Neutrophil count
500-900/p.l 5 5
<500/�d 0 0
Eosinophil count. >lO.000/p.l 14 17
Platelet count, <50,0004i.I 1 1
Hepatic
Aspartate aminotransferase
2.5-5 times normal 8 8
>5 times normal 3 3
Total bilirubinI .5-3 times normal 3 3
Renal (creatinine)2.0-2.9mg/dI 3 4
objective peripheral neuropathy. The initial symptom was a
patchy decreased temperature sensation to cold and primarily
involved the distal legs and arms. Neurological evaluation in-
cluded nerve conduction velocities that were abnormal and
indicated the presence of a sensory demyelinating polyneurop-
athy. He did not receive any further chl4.18 antibody infusions
due to the objective nature of his polyneuropathy. These symp-
toms improved following his treatment, but confirmatory elec-
trodiagnostic evaluation was not possible because the patient
developed intracranial metastatic disease and a rapid clinical
decline shortly following his protocol treatment. No additional
patient on this study developed objective evidence of peripheral
neuropathy. One patient treated at the lO-mg/m2/day dose level
(patient 7) experienced transient diffuse, objective weakness
with subsequent complete clinical resolution of this symptom
within a few days. This generalized weakness occurred in as-
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
12OOO�
��)oLymphocyte
count
(cells/vt) � � �#{176}-:�uoo
i
i� �i�� Pi...-i� �
1284 IL-2 and chl4.I8 Antibody for Melanoma
Baseline Day 6 Day 13 Day 20
Sample timepoint
Fig. I Lymphocyte counts were determined for patients in treatment
group I at baseline before beginning protocol therapy (a 15) and on
protocol days 6 (following 1 week of IL-2: n = 15). 13 (following theIL-2 plus chl4.18 administration: ,z = 15), and 20 (following the last
week of IL-2: n 14). C’olu,nns, mean counts: bars, SE. The changefrom baseline was determined by paired t test and was significant for the
lymphocyte counts on days 6, 13, and 20 (P < 0.005).
sociation with pleural and pericardial effusions, and these symp-
toms were dose-limiting for this patient.
Significant laboratory changes noted during this IL-2 and
chl4. 18 antibody treatment are indicated in Table 6. Toxicities
were related to the IL-2 and improved following protocol treat-
ment. These laboratory changes were similar to those seen in our
prior IL-2-containing protocols (3 1), and they were felt to rep-
resent toxicities attributable to IL-2.
Antitumor Effects
The best clinical response and the duration of that response
is shown for all patients in Table 4. One of the patients identified
as having progressive disease experienced a >50% decrease in
cross-sectional area of bulky hepatic metastases but developed a
recurrent s.c. nodule as the only area of disease progression. The
one patient who achieved a complete response (patient 22) had
measurable disease at the start of protocol therapy, which con-
sisted of a pathologically enlarged right axillary lymph node.
This patient was treated in treatment group 4 and required a 50%
decrease in the IL-2 dose during the first course of IL-2 treat-
ment. The patient then received an inadvertent 10-fold excess of
a planned IL-2 dose during course 2 (treatment day 35). The
patient achieved a complete clinical response, and this has
remained a durable complete response for now more than 24
months. Because there was no disease status reevaluation until
completion of treatment course 2, it is not possible to determine
the effect of the treatment day 35 IL-2 dose on the subsequent
clinical response.
Immunological Results
Lymphocyte Number and Phenotype. Peripheral blood
samples were obtained from patients in treatment group 1 to
determine the treatment-associated changes in lymphocyte
count and phenotype. As shown in Fig. I , there was a progres-
sive increase in lymphocyte counts after each 96-h IL-2 infu-
sion. Lymphocyte counts obtained from patients on protocol
treatment days 6, 1 3, and 20 were significantly increased from
baseline values.
Lymphocyte cell surface phenotype was also evaluated for
patients in treatment group 1. As shown in Table 7, the lym-
phocyte cell surface phenotype had a therapy-induced drop in
the percentage of CD3 + T lymphocytes, with a corresponding
increase in the percentage of both CD I 6 + Fc receptor-express-
ing cells and CDS6 + NK cells. These changes were initially
seen following the 1st week of treatment with IL-2 alone and
were maintained during each of the subsequent 2 weeks of
protocol treatment. There was an increase in the percentage of
PBMCs positive for CD25 on day 13, but this was not main-
tamed on day 20 of protocol treatment.
Patients in treatment group 2 showed no significant change
in lymphocyte number or phenotype on day 6 compared to
baseline (data not shown). Note that only three patients were
entered into this group, making any detailed analysis of this
group difficult.
LAK and ADCC Activity. Fresh PBMCs from patients
in treatment group I were evaluated for LAK activity at baseline
and treatment days 6, 13, and 20. As shown in Fig. 2, significant
LAK activity was induced in vivo with this combined IL-2 plus
chl4.l8 therapy protocol. This LAK activity was seen following
the 1st week of treatment with IL-2 alone and was maintained
during the next 2 weeks of therapy. In addition to the LAK
activity against the Daudi target, LAK activity was induced
against the BT2O human breast carcinoma cell line. As shown in
Fig. 3, fresh PBMCs were able to kill the BT2O target on
protocol days 6, 13, and 20. This lytic activity was significantly
increased from baseline and was initially present on protocol
day 6, following treatment with IL-2 alone. This enhanced lytic
activity was then maintained during the next 2 weeks of therapy.
In addition to this LAK activity, ADCC against the BT2O human
breast carcinoma cell line, measured in vitro, was also enhanced
by this in vivo treatment. Fresh PBMCs were evaluated at
baseline and on protocol days 6, 13, and 20 either in the
presence or absence of 0.25 p.g/ml of the ING-l antibody. This
ING-l antibody is known to mediate ADCC against the BT2O
breast carcinoma line. As shown in Fig. 3, enhanced ADCC with
ING-l antibody against the BT2O cell line was seen on protocol
days 6, 13, and 20.
The three patients in treatment group 2 had no significant
change in LAK activity for the Daudi or BT2O target cells on
day 6 compared to baseline (data not shown). However, there
was a decrease in ADCC by fresh PBMCs tested with ING-l
against the BT2O cell line seen on protocol day 6 compared to
baseline (P = 0.05). The mean lytic unit value at baseline for
these 3 patients (265 ± 24) decreased on treatment day 6 (122 ±
18) when the assay was done in HS-RPMI, supplemented with
a final IL-2 concentration of 100 lunits/mI. Note that only three
patients were entered into this group.
Serum samples from patients receiving in vivo therapy with
chl4. 18 antibody were also evaluated to determine whether the
administered ch 14. 1 8 antibody results in serum concentrations
sufficient to mediate ADCC. The tumor cell line evaluated for
these assays was the LA-N-S neuroblastoma cell line known to
express GD2 and to bind the chl4.18 antibody. As shown in Fig.
4, PBMCs from normal donors can mediate ADCC against the
LA-N-S neuroblastoma cell line in the presence of0.25 �i.g/ml of
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
300
Lytic units/1 0’ ettectors
Lytic units/10� effectors
Baseline Day 6 Day 1 3 Day 20
Sample timepoint
Baseline Day 6 Day 13 Day 20
Sample timepoint
Clinical Cancer Research 1285
Ta ble 7 Cell surface phenotype
Sample time”
% of PBMCs positive”
CD3 CDI6 CD25 CD56
Baseline 64 ± 4 19 ± 3 19 ± 2 22 ± 2
Day6Day 13
44±4C
45 ± 5C
30±4’
27 ± 4”
22±2
26 ± 3d
42±3’
38 ± 5’
Day 20 36 ± 4C 38 ± 4” 21 ± 2 52 ± 4’
a The cell surface phenotype of lymphocytes from patients in treatment group 1 was determined at baseline before beginning protocol therapy
(n = 14) and on protocol day 6 (ti = 14), day 13 (n = 13), and day 20 (n = 13). PBMCs were isolated by density gradient centrifugation and directly
labeled with FITC- or phycoerythrin-conjugated antibodies.1� The mean percentage of cells positive (±SE) is shown and is based on PBMCs gated for lymphocytes.
C The change from baseline was determined by paired r test (P < 0.005).d The change from baseline was determined by paired I test (P < 0.05).
Fig. 2 Lytic activity against Daudi cells by lymphocytes from patients
in treatment group I obtained baseline before beginning protocol ther-
apy (n = 15) and on protocol days 6 (n = 14). 13 (pi = 13), and 20 (n =
13). C’olumns, mean lytic unit values: bars, SE. The assays were run in
HS-RPMI supplemented with IL-2 at a final concentration of 100units/mi. The change from baseline was determined by paired t test and
was significant for values on days 6, 13, and 20 (P < 0.05).
antibody chl4. 18. Serum was then obtained from patient 8 both
on protocol day 8, immediately prior to initial in vivo therapy
with the 10 mg/m2 dose ofchl4.l8, and on protocol day 13, 1 h
following the 5th daily administration of chl4.l8 at 10 mg/rn2!
day. Although patient serum obtained prior to in vivo chl4. I 8
antibody therapy could not mediate ADCC against this cell line,
So �iJ of patient serum obtained 1 h following in vivo chl4.18
infusion was able to mediate ADCC against the neuroblastoma
cell line. The magnitude of ADCC obtained using patient serum
following in vivo chl4.l8 infusion was similar to that seen with
the addition of 0.25 p.g/mI chl4.l8 antibody to normal donor
PBMCs. Similar assays were performed with serum samples
from patients receiving 7.5 mg/m2!day chl4. 18 antibody and
confirmed that their serum could also mediate ADCC following
infusions of the chl4. 18 antibody (data not shown).
DISCUSSION
This study evaluates combined immunotherapy with IL-2
and chl4. 18 antibody for patients with melanoma. The MTD for
chl4.l8 antibody, when given on each of five consecutive days
Fig. 3 Lytic activity against the BT2O human breast carcinoma cell
line by lymphocytes from patients in treatment group 1 obtained base-
line before beginning protocol therapy (n = 15) and on protocol days 6(n 14), 13 (n 13), and 20 (n = 13). The BT2O target cells were
evaluated either alone (BT2O) or following addition of ING- 1 at a final
concentration of 0.25 �i.WmI to the effector cells immediately prior to thecytotoxicity assay (BT2O + ING-l). C’olumns, mean lytic unit values:bars, SE. The assays were run in HS-RPMI supplemented with IL-2 at
a final concentration of 100 units/ml. The change from baseline was
determined by paired t test and was significant for increased lysis of the
BT2O cell line on days 6, 13, and 20 both in the absence (P < 0.05) andpresence (P < 0.05) of the 1NG-l antibody.
as concurrent therapy with this IL-2 regimen, is 7.5 mg/m2/day.
Dose-limiting toxicities were a severe allergic reaction and
weakness, pericardial effusion, and decreased performance sta-
tus. Most patients treated at the ch I 4. 18 MTD had abdominal,
chest, or extremity pain requiring iv. morphine. A similar pain
syndrome has been described in other patients receiving immu-
notherapy with the chl4.l8 antibody (32). Although the mech-
anism for this pain was not directly tested in this study, the
timing of the pain was clearly related to the chl4.18 antibody
infusions. This pain could be adequately controlled to the sat-
isfaction of most patients and was not dose-limiting. As neural
tissue also expresses GD2, one concern with the combination of
an anti-GD2 antibody with IL-2 was the possibility for signifi-
cant peripheral neuropathy. One patient treated on this study did
develop objective evidence of a demyelinating process. Al-
though this symptom was clinically improving following treat-
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
. + Media
��Q +Serum2
2 3 4 � 1 2 3 4
Normal donor A Normal donor B
1286 IL-2 and chl4.I8 Antibody for Melanoma
Fig. 4 Lytic activity against the LA-N-S neuroblastoma cell line bynormal donor lymphocytes (donors A and B) in the presence of: column
1. HS-RPMI (media): column 2, 0.25 �i.g/ml chI4.18 antibody(chl4. 18): cOllflflfl 3, 50 p1 of serum from patient 8 on protocol day 8,immediately prior to in vito chl4. 18 therapy (serum 1): or column 4, 50
p.1 of serum from patient 8 on protocol day 13, 1 h following in vivo
chl4.18 infusion (serum 2). C’olumns. lytic unit values.
ment, the patient developed CNS metastatic disease and expired
before this toxicity resolved. Careful assessment for potential
neurotoxicity remains an important component of clinical man-
agement for patients receiving anti-GD2 antibodies. No patient
treated on this study developed prolonged symptoms of motor
weakness. The remaining toxicities were similar to those seen
during earlier studies of this IL-2 regimen given alone. There
was no significant hyponatremia associated with this chl4.18
treatment. This is consistent with other studies of the chl4.l8
antibody (20, 32), but different from the hyponatremia associ-
ated with murine l4.G2a antibody administration (33). Thus, the
chl4.I8 antibody can be safely administered with this IL-2
regimen. In separate studies of the chI4. 18 antibody, either as a
single agent (20) or combined with granulocyte-macrophage
colony-stimulating factor (32), higher doses of chl4.18 were
administered. The single chl4.18 dose of 100 mg was admin-
istered without dose-limiting toxicity by Saleh et a!. (20). This
suggests that IL-2 may have potentiated the toxicity of chl4.l8.
However, differences in the administration schedules of chl4.l8
prevent a direct comparison of chl4.l8 MTD in these studies.
The repetitive administration ofchl4.l8 in this study resulted in
a total weekly chl4. 18 dose of 37.5 mg/rn2 at the MTD. Six
patients were eligible for three antibody-containing courses of
therapy at the 7.5 mg/m2/day dose level ofchl4.l8, and none of
these patients had dose-limiting toxicity due to chl4.l8. Thus, it
is difficult to directly compare the chI4.18 MTD in this study
with the chl4.18 MTD established in other studies which were
administered as a single infusion of chl4.l8.
We have recently reported that administration of systemic
IL-2 before, during, and after chl4.l8 antibody administration
appears to inhibit the anti-Id response to this antibody (26). In
addition, administration of systemic IL-2 1 week after chl4.18
administration may boost the anti-Id response to chl4.l8 pre-
viously given without IL-2 (26). The formation of an anti-Id
response could result in antibodies that bind the therapeutically
administered antibody and prevent binding of the therapeutic
antibody with its target tissue (34). This interaction would be
expected to decrease the potential clinical benefit of the admin-
istered antibody, and treatment strategies to decrease the im-
mune response to the administered antibody would be expected
to result in an improved antitumor effect. It is of interest that an
anti-Id response was absent in 8 of 15 patients at the chl4.18
MTD dose of 7.5 mg/m3/day, and all 8 of these patients had
progressive disease. The other seven patients had an anti-Id
response detected during protocol therapy, and five patients had
stable disease, one had a partial response, and one had a com-
plete clinical response. Although the stable disease and partial
response durations were brief, the complete clinical response
has now been durable for more than 24 months. Thus, a patient
can achieve a durable antitumor response in the presence of
anti-Id antibodies. The relevance of induction of anti-Id anti-
bodies for an antitumor response is unknown, but the Id network
has been hypothesized to be important for antitumor immunity
(35). It is possible that the anti-Id response may reflect an
activation of the Id network that could potentially influence the
antitumor response. In addition, avoiding an anti-Id response
was clearly insufficient, by itself, as a means to achieve a greater
antitumor effect.
There were several immunological changes associated with
this combined IL-2 plus chl4.18 therapy. There was a treat-
ment-related increase in lymphocyte count, and there was an
increase in LAK activity. These changes are similar to those
described previously with in vivo administration of IL-2 alone
(36). There was an increase in the percentage of CD16 + and of
CD56 + lymphocytes, and there was a corresponding decrease
in the percentage ofCD3 + lymphocytes. These activated CDI6
+ effectors can mediate enhanced ADCC when compared with
pretreatment lymphocytes. Thus, this treatment is associated
with the in vivo activation of effector cells to mediate ADCC.
Treatment with chl4. 18 alone appeared not to be sufficient to
cause this effect and may even decrease the amount of ADCC
that these effectors can mediate in vitro with a different anti-
body. However, serum obtained from patients following
chl4.l8 infusion demonstrate its ability to achieve serum con-
centrations that can facilitate ADCC.
Although immune activation was demonstrated in all pa-
tients evaluated, antitumor activity was more limited. A durable
complete response was seen in one patient, and this persists over
24 months after the completion of all protocol therapy. Although
this response is of considerable importance, the complete re-
sponse rate of 4% is clearly in need of further improvement. In
addition, the patient with the complete response had several
alterations in the planned IL-2 administration, including an
inadvertent 10-fold excess of planned IL-2 dose on treatment
day 35. Any relationship between this single increased IL-2 dose
and the observed antitumor response cannot be determined, as
the initial disease status reevaluation occurred following that
treatment course. Although another patient achieved a partial
response, this response was only maintained for 1 month. Eight
of the patients maintained stable disease following treatment,
but this lasted not more than 6 months.
In conclusion, the combined immunotherapy with IL-2 and
chl4.l8 antibody produces immune activation in all patients and
can be administered with acceptable toxicity. Further improve-
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Clinical Cancer Research 1287
ments are needed to enhance the antitumor activity of this
approach. Although most melanomas express the GD2 antigen
and would be expected to bind with chl4.18 antibody (37), the
actual level of expression is variable (38). Ganglioside GD3 is
an antigen on melanomas that binds the anti-GD3 antibody R24
(39, 40), and the R24 antibody can mediate ADCC with IL-2-
activated effector cells (3, 41, 42). We are currently evaluating
IL-2 in combination with both chl4.18 and murine R24 anti-
body as immunotherapy for patients with GD2 and GD3-posi-
tive tumors. In addition, a chl4.l8-IL-2 recombinant fusion
protein can bind to GD2-positive melanoma and neuroblastoma
(43) and has been shown to induce cellular antitumor responses
(44). Preclinical murine testing has demonstrated effective an-
titumor responses against established melanoma following treat-
ment with the chl4.18-IL-2 fusion protein (45, 46). These
approaches require clinical testing and represent promising
strategies to improve our immunotherapy of human melanoma.
ACKNOWLEDGMENTSWe thank Karen Huseby-Moore and the nurses of the University of
Wisconsin Comprehensive Cancer Center for meticulous clinical care,
D. Meltzer for administrative coordination, K. Schell for assistance with
figure preparation, and K. Purcell for manuscript preparation.
REFERENCES1. Oppenheim, M. H., and Loize, M. T. Interleukin-2: solid-tumor
therapy. Oncology (Basel), 51: 154-169, 1994.
2. Parkinson, D. H. Enhancement of the antineoplastic activity of in-
terleukin-2. In: M. B. Atkins and J. W. Mier (eds.), Therapeutic Appli-cations of Interleukin-2, pp. 439-454. New York: Marcel Dekker, Inc.,
1993.
3. Soiffer, R. J., Chapman, P. B., Murray, C., Williams, L., Unger, P.,Collins, H., Houghton, A. N., and Ritz, J. Administration of R24
monoclonal antibody and low-dose interleukin 2 for malignant mela-noma. Clin. Cancer Res., 3: 17-24, 1997.
4. Itoh, K., Platsoucas, D., and Balch, C. M. Autologous tumor-specific
cytotoxic T lymphocytes in the infiltrate of human metastatic melano-mas, activation by interleukin-2 and autologous tumor cells and involve-
ment of the T-cell receptor. J. Exp. Med., 168: 1419-1441, 1988.
5. Rosenberg. S. A., Packard, B. S., Aebersold, P. M., Solomon, D.,
Topalian, S. L., Toy, S. T., Simon, P., Lotze, M. T., Yang, J. C., Seipp,
C. A., Simpson, C., Carter, C., Bock, S., Schwartzentruber, D., Wei, J.P., and White, D. E. Use of tumor infiltrating lymphocytes and inter-leukin-2 in the immunotherapy of patients with metastatic melanoma.
N. Engl. J. Med., 319: 1676-1680, 1988.
6. Rosenberg, S. A., Yang, J. C., Topalian, S. L., Schwartzentruber,D. J., Weber, J. S., Parkinson, D. R., Seipp, C. A., Einhorn, J. H., andWhite, D. E. Treatment of 283 consecutive patients with metastatic
melanoma or renal cell cancer using high-dose bolus interleukin 2.J. Am. Med. Assoc., 271: 907-913, 1994.
7. Sosman, J. A., Kohler, P. C., Hank, J. A., Moore, K. H., Bechhofer,
R., Storer, B., and Sondel, P. M. Repetitive weekly cycles of IL-2. II.Clinical and immunologic effects of dose, schedule, and addition ofindomethacin. J. Nail. Cancer Inst. (Bethesda), 80: 1451-1460, 1988.
8. Hank, J. A., Kohler, P. C., WeiI-Hillman, G., Rosenthal, N., Moore,
K. H., Storer, B., Minkoff, D., Bradshaw, J., Bechhofer, R., and Sondel,P. M. In vivo induction of the lymphokine-activated killer phenomenon:
interleukin 2-dependent human non-major histocompatibility complex-
restricted cytotoxicity generated in vivo during administration of humanrecombinant interleukin 2. Cancer Res., 48: 1965-1971, 1988.
9. Sondel, P. M., Hank, J. A., Kohler, P. C., Chen, B. P., Minkoff, D. Z.,and Molenda, J. A. Destruction of autologous human lymphocytes by
IL-2 activated cytotoxic cells. J. Immunol., 137: 502-51 1, 1986.
10. Damle, N. K., Doyle, L. V., Bender, I. R., and Bradley, E. C. IL-2
activated human lymphocytes exhibit enhanced adhesion to normal
vascular endothelial cells and cause their lysis. J. Immunol., 138:
1779-1785, 1987.
11. Ettinghausen, S. E., Puri, R. K., and Rosenberg, S. A. Increased
vascular permeability in organs mediated by the systemic administration
of lymphokine-activated killer cells and recombinant lL-2 in mice.
J. NatI. Cancer Inst. (Bethesda), 80: 177-188, 1988.
12. Ferradini, L., Mackensen, A., Genevee, C., Bosq, J.. Duvillard, P.,
Avril, M. F., and Hercend, T. Analysis of T cell receptor variability in
tumor-infiltrating lymphocytes from a human regressive melanoma.
J. Clin. Invest., 91: 1183-1190, 1993.
13. Wiebke, E. A., Rosenberg, S. A., and Lotze, M. T. Acute immu-
nologic effects of interleukin-2 therapy in cancer patients: decreased
delayed type hypersensitivity response and decreased proliferative re-
sponse to soluble antigens. J. Clin. Oncol., 9: 1440-1449, 1988.
14. Hank, J. A., Sosman, J. A., Kohler, P. C., Bechhofer, R., Storer, B.,
and Sondel, P. M. Depressed T cell responses concomitant with in-creased IL-2 induced responses following in vivo treatment with IL-2.J. Biol. Response Modif., 9: 5-14, 1990.
15. Munn, D. H., and Cheung, N. K. V. Interleukin-2 enhancement of
monoclonal antibody-mediated cellular cytotoxicity against human mel-
anoma. Cancer Res., 47. 6600-6605, 1987.
16. Honsik, C. J., Jung, G., and Reisfeld, R. A. LAK cells targeted bymonoclonal antibody to the disialogangliosides GD2 and GD3 specifi-
cally lyse human tumor cells of neuroectodermal origin. Proc. NatI.
Acad. Sci. USA, 83: 7893-7897, 1986.
17. Shiloni, E., Eisenthal, A., Sachs, D., and Rosenberg, S. A. Anti-
body-dependent cellular cytotoxicity mediated by murine lymphocytesactivated in recombinant interleukin-2. J. Immunol., 138: 1992-1998,1987.
18. Mujoo, K., Cheresh, D. A., Yang, H. M., and Reisfeld, R. A.
Disialoganglioside GD2 on human neuroblastoma cells: target antigen
for monoclonal antibody-mediated cytolysis and suppression of tumorgrowth. Cancer Res., 47: 1098-1 104, 1987.
19. Mujoo, K., Kipps, T. J., Yang, H. M., Cheresh, D. A., Wargalla, U..Sander, D. J., and Reisfeld, R. A. Functional properties and effect ongrowth suppression of human neuroblastoma tumors by isotype switchvariants of monoclonal antiganglioside GD2 antibody 14.18. Cancer
Res., 49: 2857-2861, 1989.
20. Saleh, M. N., Khazaeli, M. B., Wheeler, R. H., Allen, L., Tilden,
A. B., Grizzle, W., Reisfeld, R. A., Yu, A. L., Gillies, S. D., andLoBuglio, A. F. Phase I trial of the chimeric anti-GD2 monoclonal
antibody chl4.18 in patients with malignant melanoma. Hum. Antib.
Hybrid., 3: 19-24, 1992.
21. Barker, E., Mueller, B. M., Handgretinger. R., Herter, M.. Yu,
A. L., and Reisfeld, R. A. Effect of a chimeric anti-ganglioside GD,antibody on cell-mediated lysis of human neuroblastoma cells. CancerRes., 54: 144-149, 1991.
22. Mueller, B. M., Romerdahl, C. A., Gillies, S. D., and Reisfeld, R. A.
Enhancement of antibody-dependent cytotoxicity with a chimeric anti-
GD2 antibody. J. Immunol., 144: 1382-1386, 1990.
23. Goldstein, D., Sosman, J. A., Hank, I. A., Weil-Hillman, G., Moore,
K. H.. Borchert, R., Storer, B., Kohler, P. C., Levitt, D., and Sondel.
P. M. Repetitive weekly cycles of interleukin-2 (IL-2): outpatient treat-ment with a lower dose of IL-2 maintains heightened non-MHC re-stricted killer activity. Cancer Res., 49: 6832-6839, 1989.
24. Schlom, J. Monoclonal antibodies in cancer therapy. In: V. T.DeVita, Jr., S. Hellman, and S. A. Rosenberg (eds.), Biologic Therapy
of Cancer, pp. 507-521. Philadelphia: I. B. Lippincott Co., 1995.
25. Hank, J. A., Surfus, J., Gan, J., Chew, T. L., Hong, R., Tans, K.,Reisfeld, R., Seeger, R. C., Reynolds, C. P., Bauer, M., Wiersma, S.,
Hammond, D., and Sondel, P. M. Treatment of neuroblastoma patients
with antiganglioside GD2 antibody plus interleukin-2 induces antibody-
dependent cellular cytotoxicity against neuroblastoma detected in vitro.
J. Immunother., 15: 29-37, 1994.
26. Albertini, M. R., Gan, J., Jaeger, P., Hank, J. A., Storer, B.. Schell,
K., Rivest, T., Surfus, J., Reisfeld, R. A.. Schiller, J. H., and Sondel.
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1288 IL-2 and chl4. 18 Antibody for Melanoma
P. M. Systemic interleukin-2 modulates the anti-idiotypic response to
chimeric anti-GD2 antibody in patients with melanoma. J. Immunother.,
19: 278-295, 1996.
27. Albertini, M. R., Sosman, J. A., Hank, J. A., Moore, K. H.,
Borchert, A., Schell, K., Kohler, P. C., Bechhofer, R., Storer, B., andSondel, P. M. The influence of autologous lymphokine-activated killercell infusions on the toxicity and antitumor effect of repetitive cycles of
interleukin-2. Cancer (Phila.), 66: 2457-2464, 1990.
28. Liao, S. K., Horton, L., Flahart, R. E., O’Rear, L., Crumpacker, D.,
Imbaratto, J. W., Yannelli, J. R., Robinson, R. R., and Oldham, R. K.Binding and functional properties of a mouse-human chimeric mono-
clonal antibody of the human IgGl subclass with specificity for human
carcinomas. Hum. Antib. Hybrid., 1: 66-76, 1990.
29. Robinson, R. R., Chattier, J., Jr., Chang, C. P., Horwitz, A. H., and
Better, M. Chimeric mouse-human anti-carcinoma antibodies that me-diate different anti-tumor cell biological activities. Hum. Antib. Hybrid.
2: 84-93, 1991.
30. Pross, H. F., and Maroun, J. A. The standardization of NK cell
assays for use in studies of biological response modifiers. J. Immunol.
Methods, 68: 235-249, 1984.
31. Hank, J. A., Albertini, M., Wesly, 0. H., Schiller, J. H., Borchert,
A., Moore, K., Bechhofer, R., Storer, B., Gan, I., Gambacorti, C.,Sosman, J., and Sondel, P. M. Clinical and immunological effects oftreatment with murine anti-CD3 monoclonal antibody along with inter-leukin 2 in patients with cancer. Clin. Cancer Res., 1: 481-491, 1995.
32. Murray, J. L., Kleinerman, E. S., Jia, S. F., Rosenblum, M. G., Eton,
0., Buzaid, A., Legha, S., Ross, M. I., Thompson, L., Mujoo, K., Tieger,P. T., Saleh. M., Khazaeli, M. B., and Vadhan-Raj, S. Phase Ia/lb trialof anti-GD2 chimeric monoclonal antibody 14.18 (chl4.18) and recom-binant human granulocyte-macrophage colony-stimulating factor
(rhGM-CSF) in metastatic melanoma. J. Immunother., 19: 206-217,1996.
33. Saleh, M. N., Khazaeli, M. B., Wheeler, R. H., Dropcho, E., Liu, T.,
Urist, M., Miller, D. M., Lawson, S., Dixon, P., Russell, C. H., and
LoBuglio, A. F. Phase I trial of the murine monoclonal anti-GD2 anti-
body l4G2a in metastatic melanoma. Cancer Res., 52: 4342-4347,
1992.
34. Khazaeli, M. B., Conry, R. M., and LoBuglio, A. F. Human immune
response to monoclonal antibodies. J. Immunother., 15: 42-52, 1994.
35. Kennedy, R. C., Zhou, E. M., Lanford, R. E., Chanh, T. C., andBona, C. A. Possible role of anti-idiotypic antibodies in the induction oftumor immunity. J. Clin. Invest., 80: 1217-1224, 1987.
36. Albertini, M. R., Hank, J. A., and Sondel, P. M. Strategies for
improving antitumor activity utilizing IL-2: preclinical models and
analysis of antitumor activity of lymphocytes from patients receiving
IL-2. Biotherapy (Dordrecht), 4: 189-198, 1992.
37. Hamilton, W. B., Helling, F., Lloyd, K. 0., and Livingston, P. 0.
Ganglioside expression on human malignant melanoma assessed by
quantitative immune thin-layer chromatography. mt. J. Cancer, 53:
566-573, 1993.
38. Tsuchida, T., Saxton, R. E., Morton, D. L., and Irie, R. F. Ganglio-
sides of human melanoma. Cancer (Phila.), 63: 1 166-1 174, 1989.
39. Urmacher, C., Cordon-Cardo, C., and Houghton, A. N. Tissuedistribution of GD3 ganglioside detected by mouse monoclonal anti-
body R24. Am. J. Dermatopathol., 16: 577-581, 1989.
40. Vadhan-Raj, S., Cordon-Cardo, C., Carswell, E., Mintzer, D.,
Dantis, L., Duteau, C., Templeton, M. A., Oettgen, H. F., Old, L. J.,
and Houghton, A. N. Phase I trial of a mouse monoclonal antibodyagainst GD) ganglioside in patients with melanoma: induction of
inflammatory responses at tumor sites. I. Clin. Oncol., 6: 1636-1648, 1988.
41. Hard, W., Shau, H., Hadley, C. G., Morgan, A. C., Jr., Reisfeld,
R. A., Cheresh, D. A., and Mitchell, M. S. Increased lysis of melanomaby in vivo-elicited human lymphokine-activated killer cells after addi-
tion of antiganglioside antibodies in vitro. Cancer Res., 50: 631 1-63 15,1990.
42. Bajorin, D. F., Chapman, P. B., Wong, G., Coit, D. G., Kunicka,
J., Dimaggio, J., Cordon-Cardo, C., Urmacher, C., Dantes, L.,Templeton, M. A., Liu, J., Oettgen, H. F., and Houghton, A. N. PhaseI evaluation of a combination of monoclonal antibody R24 and
interleukin 2 in patients with metastatic melanoma. Cancer Res.. 50:
7490-7495, 1990.
43. Hank, J. A., Surfus, J. E., Gan, J., Jaeger, P., Gillies, S. D., Reisfeld,
R. A., and Sondel, P. M. Activation of human effector cells by a tumorreactive recombinant anti-ganglioside GD2 interleukin-2 fusion protein
(chl4.l8-1L2). Clin. Cancer Res., 2: 1951-1959, 1996.
44. Becker, J. C., Varki, N., Gillies, S. D., Furukawa, K., and Reisfeld,
R. A. An antibody-interleukin 2 fusion protein overcomes tumor heter-
ogeneity by induction of a cellular immune response. Proc. Nail. Acad.Sci. USA, 93: 7826-7831, 1996.
45. Becker, J. C., Pancook, J. D., Gillies, S. D., Furukawa, K., andReisfeld, R. A. T cell-mediated eradication of murine metastatic mela-
noma induced by targeted interleukin 2 therapy. J. Exp. Med., 183:
2361-2366, 1996.
46. Becker, J. C., Varki, N., Gillies, S. D., Furukawa, K., and Reisfeld,
R. A. Long-lived and transferable tumor immunity in mice after targetedinterleukin-2 therapy. J. Clin. Invest., 98: 2801-2804, 1996.
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
1997;3:1277-1288. Clin Cancer Res M R Albertini, J A Hank, J H Schiller, et al. interleukin 2 for melanoma patients.Phase IB trial of chimeric antidisialoganglioside antibody plus
Updated version
http://clincancerres.aacrjournals.org/content/3/8/1277
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/3/8/1277To request permission to re-use all or part of this article, use this link
Research. on May 19, 2018. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from