Phase lB Trial of Chimeric Antidisialoganglioside...

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.

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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



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-




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




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.



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



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.


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,




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-



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



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


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-



. + 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-




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.



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.



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

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