Activation of Human Effector Cells by a Tumor Reactive ...

Vol. 2, 1951-1959. December 1996 Clinical Cancer Research 1951
Activation of Human Effector Cells by a Tumor Reactive
Recombinant Anti-Ganglioside GD2 Interleukin-2
Fusion Protein (chl4.18-1L2)’
Jacquelyn A. Hank,2 Jean E. Surfus, Jacek Gan,
Peter Jaeger, Stephen D. Gillies,
Ralph A. Reisfeld, and Paul M. Sondel
Department of Human Oncology. University of Wisconsin-Madison,
Madison. Wisconsin 53792 Ii. A. H.. J. E. S., J. G.. P. J., P. M. 5.1:
Fuji ImmunoPharmaceuticals Corporation. Lexington. Massachusetts
02173 15. D. G.l: The Scripps Research Institute. La Jolla. Califrrnia
92037 iR. A. RI: and Departments of Pediatrics and Medical
Genetics, University of Wisconsin-Madison, Madison, Wisconsin
53792 P. M. 5.1
ABSTRACT
Cytotoxic effector cells interact with target cells
through various mechanisms. CTLs use the antigen-specific
T cell receptor, whereas Fe receptor-positive natural killer
cells use this receptor to interact with antibody-coated target
cells. We evaluated the tumor-binding and lymphocyte-activating
capability of a recombinant fusion protein consisting
of a tumor-selective human/mouse chimeric anti-ganglioside
GD2 antibody (chl4.18) and recombinant human
interleukin-2 (1L2) (chl4.18-IL2). This fusion protein bound
specifically to GD2-positive melanoma and neuroblastoma
tumor cell lines, and its 1L2 component stimulated in vitro
proliferation of an IL2-dependent cell line, as well as peripheral
blood mononuclear cells, in healthy control individuals
and in cancer patients receiving continuous infusion of 1L2.
The 1L2 presented by the fusion protein, when bound to
tumor cells, induced proliferation of IL2-responsive cells as
well as a comparable amount of soluble 1L2 did. This suggests
that localization of 1L2 at the site of contact between
tumor and effector cells is an effective way of presenting this
cytokine to 1L2-responsive cells. The chl4.18-1L2 fusion
protein also mediated antibody-dependent cellular cytotoxicity
with Fc receptor-positive effector cells to an extent
similar to chl4.18. These results, together with those of
previous studies documenting antitumor efficacy against human
tumor xenografts in SCID mice and GD2-positive murine
tumors in immunocompetent syngeneic mice, suggest
that the chl4.18-1L2 fusion protein should be tested in Phase
I and II trials in patients with GD2-positive tumors.
INTRODUCTION
Immunotherapy with IL23 is of benefit to some patients
with renal cell cancer and melanoma ( I ). Although multiple
immune mechanisms are activated in patients receiving IL2, the
immune components induced by lL2 necessary for antitumor
activity have not been identified. Continuous infusion of IL2. at
doses tolerated in the outpatient setting. induces systemic
lymphoid activation in virtually all treated patients: however,
only a minority of such patients achieve antitumor responses
( 1-4). Included in this lymphoid cell activation is an expansion
and activation of the CDl6, FcR NK cells (5. 6). Currently.
attempts are being made clinically to target these cells to tumor
through the use of tumor-specific mAbs (7. 8). These include
clinical evaluation of regimens combining effector cell activation
through 1L2 infusions with infusions of munine or chimeric
tumor-selective mAb, such as the l4.G2a or chl4. 18 antibodies,
which recognize the ganglioside GD2 expressed on neuroblastoma.
melanoma, and certain other tumors (7).
In an effort to augment the stimulation of the FcR antibody-directed”
effector cells and activate FcR effector cells
that express IL2 receptors (2, 9), a chl4.l8-IL2 fusion protein
has been constructed by fusion of a synthetic sequence coding
for human IL2 to the carboxyl end of the C-yl gene of the mAb
ch I 4. 1 8 ( 1 0). When the antitumor variable region of this fusion
protein binds to tumor, IL2 should be concentrated in the tumor
microenvironment and provide activation of FcR effectors,
such as NK cells, that did bind to the immunoglobulin Fe
domain of the tumor-bound fusion protein. Furthermore, the IL2
component of this tumor-bound fusion protein may activate
IL2-responsive cells. such as cytotoxic T cells and helper T
cells, that do not necessarily have FcRs and the subpopulation of
NK cells that express IL2 receptors but lack the FcR (9), thereby
recruiting additional effector cells into the tumor microenvironment.
The chI4.l8-1L2 fusion protein was previously shown to
maintain antigen-binding activity and 1L2 activity ( 10) and has
provided antitumor effects in SCID mice bearing human tumor
xenografts of neuroblastoma ( I I ) and melanoma ( I 2), as well as
in a syngeneic murine melanoma model ( 13). We have extended
these findings by demonstrating that chl4. l8-IL2 bound to
GD2 tumor cells can be visualized by flow cytornetry, detecting
either the chl4.l8 or the 1L2 portion of this construct. The
IL2 component of the fusion protein is able to stimulate lL2-
dependent cells. The fusion protein bound to GD2 tumors
Received 10/24/95; revised 8/27/96: accepted 9/18/96.
‘ This research was supported by NIH grants and contracts CA-5344 I.
CA-05436. CA-32685, CM-87290, CA- I 4520, CA- I 3539. CM-47669,
HL-02I43. RR-03186, and American Cancer Society Grant CH-237.
2 To whom requests for reprints should be addressed, at K4/454 CSC.
6()0 Highland Avenue, Madison. WI 53792. Phone: (608) 263-7262:
Fax: (608) 263-4226.
3 The abbreviations used are: lL2. interleukin 2: NK. natural killer: FcR,
Fe receptor: mAb. monoclonal antibody: ADCC. antibody-dependent
cellular cytotoxicity: HLR, Hoffmann LaRoche; PBMC, peripheral
blood mononuclear cell: LAK. lymphokine-activated killer.

1952 Anti-GD2-IL2 Fusion Protein
stimulates proliferation to the same extent as soluble fusion
protein or soluble 1L2. The chl4.l8-1L2 fusion protein also
stimulates cytolytic activity in vitro by cells able to mediate
ADCC. FcR NK cells obtained from melanoma patients in
vivo following therapy with continuous infusion 1L2 are targeted
to GD2 tumors that have bound chl4.l8-IL2 in vitro, and the
lytic activity was comparable to that induced by the combination
of free antibody and soluble 1L2.
MATERIALS AND METHODS
Recombinant IL2. HLR IL2 was provided through the
Cancer Treatment and Evaluation Program of the National Cancer
Institute. The National Cancer Institute-Biological Response
Modifiers program standard for unit dosage was used, and the
specific activity of the IL2 was 15 X 106 units/mg. This unit
corresponds closely with the international standard for IL2
unitage (14).
Chimenc Antibody and Fusion Protein. The mouse/
human chimeric 14.18 antibody was constructed by combining
the variable regions of the murine anti-GD2 14.18 antibody with
the constant regions of human IgG1 heavy chain and K light
chain (15, 16). The 14.l8-IL2 fusion protein was constructed by
fusion of a synthetic sequence coding for human IL2 to the
carboxyl end of the human C-yl gene of the mAb chl4. 18 (10).
The fused gene was inserted into the vector pdHL2-l4. 18 as
described previously (15). Transfection of the expression plasmids
in Sp2/0-Agl4 cells and propagation of the clones secreting
chl4. 18-1L2, as well as its purification, have been described
previously (10). To compare the 1L2 activity in the soluble IL2
preparation and in the fusion protein, concentrations were based
on weight/volume calculations of 1L2 in the two preparations.
Because the fusion protein molecule consists of 80% chimeric
14.18 immunoglobulin and 20% 1L2 by molecular weight, the
fusion protein 1L2 concentration was based on IL2 comprising
20% of the weight of the fusion protein. Thus, 50 ng/rnl of
fusion protein would correspond to 10 ng/ml of IL2 in the fusion
protein. Because 10 ng/ml of soluble IL2 corresponds to 150
units/mi of soluble HLR 1L2, we have used this conversion to
describe the units of 1L2 anticipated for the fusion protein
preparation based on the molecular weight of IL2 and using the
specific activity of 15 X 106 units/mg for the HLR IL2.
Tumor Cell Lines. The GD2-positive LA-N-S neuroblastoma
target cell line, kindly provided by R. Seeger (Children’s
Hospital of Los Angeles, Los Angeles, CA), was maintamed
as an adherent monolayer in Leibovitz’ s medium
supplemented with 15% heat-inactivated fetal bovine serum. A
trypsin-EDTA solution was used to harvest the cell monolayer.
The M2l human melanoma line (GD2’) was described previously
(15), and the BT-20 human breast carcinoma cell line
(GD2) was obtained from American Type Culture Collection.
Both cell lines were maintained as adherent monolayers in
RPM! 1640 supplemented with penicillin and streptomycin
(P/S), L-glutamine, HEPES buffer, and 10% fetal bovine serum.
Flow Cytometry. Cell-bound fusion protein and antibody
were detected by standard indirect immunofluorescence
methods (Becton Dickinson, San Jose, CA). Antibodies ineluded
a goat antihuman IgG (Caltag, San Francisco, CA) and a
rabbit antihuman IL2 (Genzyme, Cambridge, MA).
Proliferation Assays. Fresh PBMCs from healthy volunteer
human donors or from patients who were treated with a
96-h continuous infusion of IL2 (3. 4, 17) were cultured in
0.2-ml round-bottom microplates at a concentration of 1 X l0
cells/well in RPMI 1640 supplemented with 10% human serum
(Pel-Freez, Rogers, AR), 25 msi HEPES, 100 units/mI penicillin,
and 100 p.g/ml streptomycin sulfate (RPMI-HS). Recombinant
IL2 and fusion protein were added at concentrations as
indicated in the “Results”. Concentrations of recombinant soluble
1L2 were also tested, which corresponded to the concentration
of 1L2 in the fusion protein preparation based on the
molecular weight and concentration of fusion protein: I i.g of
the fusion protein contains approximately 3000 units of IL2
(I 1). In experiments in which chl4. I 8 or fusion protein-coated
M21 and LA-N-S cells were used as a proliferative stimulus, the
antibody or fusion protein at S ig/ml was incubated with the
tumor cells for 1 h on ice. Irradiation of these cells took place
during this incubation, with LA-N-S and M2l receiving 10,000
and 40,000 rads, respectively. Cultures were incubated at 37#{176}C
in 5% CO2 for 48-72 h, pulsed with I pCi [3H]thymidine for
18 h, and harvested with a Filterrnate 196 Packard harvester, and
[3H]thymidine incorporation was quantitated with a Matrix
9600 direct 13 counter with a 5-mm counting period. Informed
consent forms, approved by the University of Wisconsin Human
Subjects Committee, were obtained prior to collection of all
human blood specimens.
For some proliferative studies, the 1L2-responsive cells
used were the Tf-l myeloid leukemia cell line transfected with
the gene for the 1L2 receptor 3 chain. This transfected line was
designated Tf-l , and the mock-transfected control line contaming
the LXSN vector but no IL2R3 gene was designated
Tf-IL (18). This Tf-l3 cell line responded to 1L2 using intermediate
affinity receptor complexes ( 1 7. I 8) and thus is
analogous to the majority of NK cells in 1L2-treated patients,
which also use intermediate affinity IL2 receptors (2). The Mik
11 monoclonal antibody directed against the p70 IL-2 receptor
1 chain was used in the blocking studies ( 19. 20).
ADCC and Fusion Protein-mediated Cellular Cytotoxicity.
All ADCC assays were performed in RPMI-HS. Effector
cells in a total volume of SO pi were plated in quadruplicate
into 96-well U-bottomed microtiter plates at the indicated effector/target
ratios. Just prior to the addition of target cells, SO
il of antibody, antibody plus 1L2, or fusion protein were added
to the effectors. While the effectors were being prepared, target
cells were labeled for 2 h with 250 pCi of 5tCr in 0.2 ml of
RPMI-HS. Target cells were mixed every 15-30 mm during
labeling to keep the cells in suspension. After being washed
twice with RPMI, S x l0 target cells in 50 il of RPMI-HS
were added to effector cells and centrifuged at 200 X g for S
mm. In the experiments using fusion protein-coated target cells.
chl4.l8-1L2 was added to the targets following one wash and
incubated on ice for 1 h. Two subsequent washes removed
excess fusion protein and 51Cr. The effector cells were also
plated in medium and in IL2 to determine their ability to
mediate lysis of target cells in the absence of antibody or fusion
protein. The plates were incubated at 37#{176}Cat 5% CO2 for 4 h,
and the supernatants were harvested using the Skatron Harvesting
System (Skatron, McLean, VA). Maximum 5tCr release was
measured by lysing target cells with the detergent cetrimide

Clinical Cancer Research 1953
M21
LAN-5
BT-20
chl4.18
chl4.18-1L2
JL
Rabbit
IgG
Rabbit
Anti-1L2
Rabbit
lgG
Rabbit
Anti-1L2
. . . .-
1u
Fig. I Detection of antibody component and 1L2 component in tumor-bound fusion protein. The GD2 M2 1 melanoma and LA-N-S neuroblastoma
and GD2 BT-20 breast carcinoma cell lines were each coated with I p.g of either chl4.18-1L2 fusion protein or chl4.18 antibody. To detect the
antibody component (top panels). a fluorescein-conjugated goat antihuman IgG secondary antibody was used. To detect the 1L2 component (bottom
panels). a rabbit anti-IL2 antibody was used, followed by a phycoerythrin-conjugated goat antirabbit antibody.
(Sigma). Spontaneous 5tCr release was measured by incubating
target cells in RPMI-HS alone. Percent cytotoxicity values were
calculated for each effector/target ratio as follows:
C/
cytotoxicity
experimental release - spontaneous release
=l00X-- .
maximum release - spontaneous release
Results are expressed as percent cytotoxicity or as lytic
units, where 1 lytic unit is the number of effector cells necessary
to achieve 20% lysis of S X I0 targets.
RESULTS
Fusion Protein chl4.18-1L2 Binds to GD2 Tumor
Cells. The data shown in Fig. I demonstrate the binding of
chl4.l8-IL2 to the GD2 M2l human melanoma and LA-N-S
human neuroblastoma cell lines. The fusion protein binds to the
tumor cells with a fluorescence intensity similar to that of the
parental ch I 4. 18 antibody (Fig. 1, top panels). The specificity of
ch I 4. 18-IL2 and ch I 4. 18 antibody is demonstrated by their lack
of binding to the GD2 BT-20 human breast carcinoma cell
line. The IL2 component of ch 14. 1 8-1L2 can be detected by
rabbit anti-IL2 antibody when the fusion protein is bound to
M-2l and LA-N-S cells, respectively (Fig. I. bottom pane/c).
These data document that the IL2 component of the fusion
protein remains associated with the tumor cells that bind
chl4.l8-1L2 on their surfaces and is detectable by anti-IL2
antibody. Furthermore, separate flow cytometric analyses have
shown that M-2l and LA-N-S cells do not express either the a
or 3 chains of the 1L2 receptor (data not shown). Thus, binding
of the fusion protein to these cells is mediated through the
chl4.18 rather than the IL2 component of the fusion protein.
Soluble chl4.18-1L2 Stimulates IL2-induced Proliferative
Responses. Fig. 2 documents proliferative responses to
1L2 attained with the Tf-I3 cell line and PBMCs from a melanoma
patient obtained 24 h after a 96 h continuous iv. infusion
of IL2. Tf-l is a GM-CSF-dependent myeloid leukemia-derived
cell line that constitutively expresses the common cytokine
receptor -y chain. A variant cell line bearing functional intermediate
affinity IL2 receptors (3-y dimers), designated Tf- I , was
obtained by stable transfection of Tf- I with eDNA encoding the
1 chain of the IL2 receptor ( 1 8). The Tf- 1 3 cell line that
retained responsiveness to GM-CSF also responded to IL2 in a
dose-dependent fashion. The mock-transfected Tf-lL cell line
did not proliferate in response to 1L2, and the Tf-l3 cell line did
not respond to chl4.l8 antibody (data not shown). The results of
proliferative assays shown in Fig. 2 were obtained by using
dilutions of IL2 and chl4.l8-1L2 to achieve equivalent molar

1954 Anti-GD2-IL2 Fusion Protein
Cl)
I-
z
0
C)
1200
1000
10000
600
400
200
8000
6000
4000
2000
0
0 -
400
L2 UImI 1.6 3.0 00 12 25 50 100 200
FP JmI.B2O 1.20 2.0 5.0 10 20 40 80 ISO
Fig. 2 Proliferation induced by soluble IL2 and IL2 within the soluble
fusion protein. The Tf-l3 cell line (A) and PBMCs obtained from a
patient following continuous infusion lL2 (B) were stimulated with
increasing concentrations of soluble IL2 (A) and soluble chl4.l8-1L2
fusion protein (s). Dilutions of IL2 and fusion protein were made to
achieve equivalent molar concentrations of IL2. The counts were determined
for [3H]TdR incorporation by proliferating cells.
concentrations of IL2. Assuming the same specific activity of
1L2 within the fusion protein as the recombinant HLR product,
there are approximately 400 units/ml of 1L2 contained within
the chl4.18-IL2 when it is applied at a concentration of 160
ng/ml. When these data are expressed based on the concentration
of 1L2, dose-response curves indicate that at concentrations of
1L2 less than or equal to SO units/rn], the fusion protein is slightly
more efficient than free 1L2 at stimulating the Tf-l(3 cell line to
proliferate. This finding was reproduced in two additional proliferative
assays using the Tf- 13 cell line as the responding cell.
PBMCs obtained from six patients with melanoma who
had just completed a continuous infusion of IL2 were also tested
for their responsiveness to IL2 and chl4.l8-IL2. Results from
one representative patient are shown at the bottom of Fig. 2.
Previous studies have shown that NK cells obtained following in
vivo IL2 treatment show rapid proliferative responses to restimulation
with IL2 in vitro, using primarily the intermediate
affinity 13’y receptor complex (2, 21). Similar proliferation by
these cells was observed at most concentrations of IL2 and
chl4.18-IL2 tested. These data indicate that the antibody component
in the fusion protein did not adversely affect the ability
of IL2 to interact with the IL2R complexes either on the Tf-l 3
cells or on the PBMCs of a melanoma patients treated with IL2,
nor did it affect the ability of the 1L2 component of the fusion
protein to stimulate proliferation. chl4. I 8 antibody alone, at
comparable concentrations, did not stimulate patient PBMCs to
proliferate (data not shown). Table 1 presents data from two
separate experiments indicating the in vitro proliferative response
induced by 100 units of soluble IL2 and by a similar
amount of IL2 contained within 40 ng of chl4.18-IL2. The
responding cells were PBMCs obtained from five melanoma
patients and PBMCs from one healthy control individual. The
patient PBMCs were obtained 24 h after completion of a 96-h
continuous infusion of IL2 in vivo. In these experiments, the
fusion protein stimulated proliferation that was similar to that
induced by the soluble 1L2 for patient I and for the control
donor in experiment 1 and for the three patients in experiment 2.
The chimeric chl4.l8 antibody itself was not stimulatory.
Fusion Protein-induced Proliferation Mediated through
an Intermediate Affinity #{176}y 1L2 Receptor Is Specifically
Blocked by Antibody to the Chain of the IL2 Receptor.
Fig. 3 demonstrates the proliferation induced by IL2 and fusion
protein on cells with intermediate affinity receptors for 1L2,
Tf-l 3, and PBMCs obtained from cancer patients following a
4-day continuous infusion of 1L2. Cells with high-affinity aj
IL2 receptors (Kit 225 cells) were also assayed. Tf- 1 3 and
patient PBMCs responded to 1L2 and the fusion protein in a
Table I Proliferative response to soluble IL2 and to chl4.l8-1L2 fusion protein
PBMCs from a healthy volunteer individual (control) and from two patients were assayed in experiment 1. and PBMCs from three patients were
assayed in experiment 2. The patient PBMCs were obtained 24 h after a 96-h continuous infusion of IL2. They were stimulated with IL2 or
chl4.l8-IL2 fusion protein at concentrations containing equivalent amounts of IL2 or with an excess of chl4.I8 antibody. The cells were cultured
for 4 days at 37#{176}C in 5% CO,. pulsed with 3H-thymidine for 16 h, and harvested, and counts per 5 mm were obtained with a Packard 9600 Matrix
counter.
3H-Thymidine incorporati on (count per 5 mm)
Experiment
I
Control
Patient 1
Patient 2
II Patient 16
Patient 17
Patient 20
Medium 100 units/mI IL2 40 ng/ml chl4.18-1L2 O.5g/ml chl4.18
197
160
184
165
49
31
14,194
9,741
24,381
9,959
2,321
4,135
13.387
7,916
7,413
11,201
2,700
6,317
190
161
207
59
46
43

Clinical Cancer Research 1955
120000 120000
90000 90000
L1
I-
60000 60000
30000 30000
0 - 0
200 50 12.5 66.5 16.6 4.1
16000 16000
C-)
0.
I-
a-
12000
8000
4000
0
200
50
I 2000
8000
Fig. 3 Antibody to the 3 chain of the IL2 receptor
blocks fusion protein induced proliferation by cells
expressing intermediate affinity, but not cells expressing
high-affinity lL2 receptors. Tf-l cells.
PBMCs, and KIT 225 cells were cultured with lL2
(leftpa,iels) or the chI4.18-1L2 fusion protein (right
panels). The humanized Mik 3l antibody at a final
concentration of 3 p.g/ml (A) or human serum diluted
66 5 16 6 4 1 1 1/50 as the control () was added at the initiation of
12.5 . . a 3-day I3HITdR incorporation assay. #{149}, medium.
100000
100000
I0
C.”
75000
75000
I-
50000
50000
25000
25000
0-
200
‘ . 0 ‘
50 12.5 66.5 16.6 4.1 1
IL-2 CONCENTRATION ( U/mi) 14.18-IL-2 CONCENTRATiON (ug/mi)
dose-dependent manner, and this proliferation was abrogated by
inclusion of the Mik 3I antibody, which recognizes the 3 chain Coated
of the IL2 receptor (20). Over the dose range examined, there Tumor Cells
was a strong
. . #{149}500
proliferative response to both soluble IL2 and 60000
#{149}
fusion protein by the Kit 225 cell line, which expresses the U)
50
high-affinity afty IL2 receptor. The Mik 3 1 antibody did not I-
block this proliferative response, as shown previously for IL2 z
40000
(19).
chl4.18-1L2 Bound to GD2 Cells Stimulates Proliferation.
The flow cytometric studies (Fig. 1 ) document that the
chl4.18-IL2 binds to GD2 tumor cells via the antibody vanable
region and that its IL2 component is recognized by rabbit
antihuman 1L2 antibody. Subsequent experiments tested the
functional activity of the IL2 component of the fusion protein
when bound to tumor cells. In this case, the M21 and LA-N-S
cell lines were coated with either chl4.18-IL2 or chl4.l8 and
then washed free of any excess and cultured with the Tf-l
responding cell line or the Tf-lL control cell line. The data
shown in Fig. 4 demonstrate that the ch 14. 18-IL2 fusion protein.
when bound to a GD2 tumor cell line, effectively presents IL2
to the Tf- 1 3 cell line. When ch I 4. 18-IL2 is bound to S X 102
M21 or LA-N-S cells it is able to efficiently present the bound
0
C)
80000
20000
chl4.18
M21
FP
LIt
chl4.18
FP
LA-N-5
Fig. 4 Proliferation induced by the lL2 component of the tumor-bound
fusion protein. The M2l melanoma and LA-N-S neuroblastoma cell
lines were coated for I h at 4#{176}Cwith either chI4.18 antibody or the
chl4.18-1L2 fusion protein (FP). Irradiation took place during this
incubation. These coated tumor target cells were washed twice and
diluted to 500, 100, or 50 cells per well and used to stimulate l0 Tf-l
cells per well. Proliferation was quantitated after 72 h by I3HITdR
incorporation.

1956 Anti-GD2-lL2 Fusion Protein
Table 2 Stimulatory activity of IL2 in tumor-bound fusion protein
The Tf-l3 and control Tf-lL cell lines (experiment 1) or Tf-l3 cells and PBMCs from IL2-treated patients (experiment 2) were stimulated in
a 3-day proliferative assay with soluble IL2 or tumor cells coated with chI4.18 antibody or the chl4.18-IL2 fusion protein. Tumor cells (106) were
incubated with S i.g of chl4.18, washed twice, and diluted. Sixty % of the fusion protein used to coat the M2l cell remained bound to the cells:
therefore, 100 cells have 0.3 ng of fusion protein, corresponding to approximately 1 unit of bound lL2. The total volume of the microwell is 0.2 ml,
resulting in a total of 5 units of IL2/ml in the wells with chl4.18-IL2-coated tumor cells. The cells were cultured for 2.5 days at 37#{176}Cin 5% CO2.
pulsed with 3H-thymidine for 16 h, and then harvested, and counts per 5 mm were obtained with a Packard 9600 Matrix counter. The proliferation
of Tf- 113 cells induced by 5 units/mI 1L2 as tumor-bound fusion protein was similar to the proliferation induced by 50 units/ml of soluble IL2. and
the proliferation of patient PBMCs induced by 25 units/mI 1L2 as tumor-bound fusion protein was similar to the proliferation induced by SO units/mI
of soluble IL2.
3H-TdR incorporation (coun
Is X I0), responding cell
Experiment
I
Experiment 2
Stimulus
Medium
GM-CSF (5 ng/ml)
1L2 (100 units/mI)
IL2 (50 units/mI)
1L2 (25 units/mI)
100 M21 cells labeled with 0.5 ng of chl4.18-1L2 fusion protein (5.0 units of 1L2/ml)
500 M2l cells labeled with 0.5 ng of chl4.l8-1L2 fusion protein (25 units of IL2/ml)
‘a NT, not tested.
Tf-l 3 Tf- 1L
6.7 10.8
67.4 91.1
64.2 13.9
50.7 12.1
41.9 12.8
56.6 14.4
70.8 13.4
Tf- I 3
Patient
0.8 .03
NT’
NT
18.6 4.1
9.3 1.7
3.2 0.6
1.2 0.4
4.2 2.1
IL2. As few as SO such cells coated with the fusion protein can
still produce a significant 1L2-specific proliferative response.
The control Tf-lL cell line did not respond to tumor-bound
fusion protein in these experiments (data not shown).
Proliferative Response to “Equivalent” Doses of Tumor-bound
Fusion Protein. In an attempt to determine
whether there is a dose effect of the cell-bound fusion protein on
the proliferative response, we compared the proliferation induced
by soluble 1L2 to that induced by an equivalent amount of
ch I 4. 18-1L2 coated on tumor cells. To make dose comparisons,
we determined the amount of fusion protein that remained
bound to the coated GD2 tumor cells, based on the measurement
of free fusion protein concentration in the supernatant
following incubation. This indicated that approximately 60% of
the chl4.l8-IL2 used to coat the cells actually remained bound
to the cells. Data shown in Table 2 are from an experiment in
which 106 M2l cells were coated with S ag ofchl4.18-1L2 and
washed twice. These cells were then diluted, and 100 or 500
fusion protein-coated M2 1 cells per well were used to stimulate
Tf- I 13 cells. With 60% of fusion protein bound to the M2 I cells,
this corresponds to a total of 0.3 ng of fusion protein or approximately
1 unit of IL2 per 100 cells in the 0.2-ml microtiter well.
This corresponds to 5.0 and 25 units of IL2/ml in the proliferative
assay with 100 and 500 fusion protein-coated M21 cells,
respectively. Two experiments are presented in Table 2.
chl4.18-IL2 fusion protein-coated M2l melanoma cells stimulated
proliferative responses by the Tf-l 3 cell line (experiments
1 and 2) and patient PBMCs obtained following an in vivo
infusion of IL2 (experiment 2). These results indicate that the
IL2 component of the fusion protein that remains coated to M2l
cells is presented in a conformation able to stimulate the IL-2
receptor and show that the tumor-bound IL-2 is as stimulatory as
soluble 1L2.
chl4.18-1L2 Fusion Protein Is Capable of Eliciting
ADCC by PBMCs on GD2 Tumor Targets. PBMCs from
two melanoma patients receiving IL2 were assayed for lytic
L
Y
I
C
U
N
T
S
400.
300.
200.
oo
200.
150.
100.
50.
LAN 5m4 18
LAN-5
+112 FP
c81418 +11.2
cI,14.1O
12
chI4.18
IL.2 (units) 2.5 25 ‘ 100
FP (ng) I 10 40
Fr
Patient 1
Patient 2
Control
M2i
M24.18+1U
±14 10 +112
414.18
18
Fp
11.2
112 Fr
2.5 25 100
I 10 40
Fig. 5 Soluble fusion protein facilitated ADCC on GD2 tumor cells.
PBMCs obtained from two patients following a 96-h continuous infusion of
1L2 and from a volunteer control donor were the effector cells in a 4-h 51Cr
relea.se assay. The GD2 LA-N-S and M21 cell lines were used as targets.
The assay was performed in medium supplemented with 1L2 alone at 2.5.
25, or 100 units/mI; with chl4.18-IL2 fusion protein (FP) alone at 1, 10, or
40 ng/ml (corresponding to 2.5, 25, and 100 units of 1L2/ml): with the
chI4.l8 antibody alone at 1, 10, or 40 ng/ml: or with a combination of 1L2
and chl4.18 antibody at the same concentrations.

Clinical Cancer Research 1957
300
250
M21
A
600
S
800
600
8 igG
200
150
0 IgG+iL2
iichl4.18
Oichl4.18+1L2
U FP
U)
400
z
2000 200
:
, 400
100
. 0
(I)
50
0
#{163}
,Ju1,J
chl4.18 FP chl4.18 FP
M21
LA-N-5
600
_I
5oo
400
LA-N-5
B Fig. 7 ADCC ofGD2 M21 and LA-N-S cells was facilitated by either
chl4.l8 antibody or chl4.l8-IL2 fusion protein. Six separate 51Cr
release cytotoxic assays using PBMCs obtained from nine patients
receiving continuous infusion IL2 are included. Three ofthe assays used
target cells coated with the chl4.18 antibody and fusion protein (-)
and 3 assays used the antibody and fusion protein in soluble form
( ). For each experiment, the paired comparisons of ADCC by the
chl4.18 or the fusion protein are connected by the line.
300
200
100
0
Patient 6 Patient 7 PatIent 20 Patient 17
Fig. 6 Tumor-bound fusion protein facilitated ADCC. Patient PBMCs
obtained from four patients following a 96-h continuous infusion of 1L2
were cryopreserved and thawed on the morning of the 4 h 51Cr release
assay. The GD2 M2l (A) and LA-N-S (B) targets were coated with the
chl4.18, control antibody (IgG), or the chl4.l8-IL2 fusion protein (FP)
during the 51Cr labeling. For the chl4.18 antibody-coated targets. soluble
1L2 was added at l()O units/mI during the 4-h assay. Targets coated
with the chl4.l8-IL2 fusion protein received no additional soluble 1L2.
activity against the GD2 LA-N-S and M2l cell lines (Fig. 5).
The fusion protein added in soluble form during the 4-h 51Cr
release assay enhanced lysis mediated by both patients PBMCs.
The ch 14. 18-IL2 fusion protein mediated levels of ADCC similar
to those mediated by ch I 4. 18 alone or antibody combined
with soluble IL2 when tested on the NK-resistant LA-N-S target
cells. Against the M2 I targets, the fusion protein also enhanced
lysis over that achieved with 1L2 alone; however, the fusion
protein was not as effective as antibody combined with soluble
IL2. Similar effects were observed with PBMCs from a healthy
volunteer donor that were not previously primed in vito with
IL2.
ADCC with Tumor Target Cells Coated with chl4.18-
1L2 Fusion Protein. PBMCs obtained from four melanoma
patients 24 h after a 96-h continuous infusion of IL2 were
cryopreserved and thawed the day of the assay for use as
effector cells. The GD2 tumor cell targets were coated with
either chl4.l8-IL2, chI4.l8, or control lgG immediately following
the 5tCr labeling. Soluble IL2 (100 units/ml) was added
to the medium during the 4-h assay with control IgG or mAb
chl4.l8. Tumor-bound fusion protein elicited ADCC with both
M21 and LA-N-S targets (Fig. 6, A and B). The ADCC against
M2 1 target cells was comparable to that induced by mAb
chl4.l8 alone. The level of cytotoxicity against the LA-N-S
target cell was comparable to the augmented cytotoxicity observed
when soluble IL2 was combined with chl4.l8. This
similar pattern of lysis was also noted with the fusion protein
added in soluble form (Fig. 4). These results suggest that for the
LA-N-S neuroblastoma target, fusion protein is capable of augmenting
the level of ADCC observed with chl4.l8 alone.
Fig. 7 shows the results from six separate experiments
using PBMC samples from nine different cancer patients comparing
the cytotoxicity on both M2l melanoma and LA-N-S
neuroblastoma targets. In three of the experiments, ch 14.18
antibody or fusion protein was added in soluble form (as in Fig.
5). and in three experiments the target cells were coated with the
ch 14. 18 antibody or the fusion protein and washed free of any
excess prior to the cytotoxic assay (as in Fig. 6). No reproducible
difference in ADCC was noted between coated targets or
targets to which soluble fusion protein was added. Overall, the
data presented in Fig. 7 show that the ADCC mediated by the
ch I 4. 1 8 fusion protein was comparable to or better than that
seen with an equivalent concentration of chl4.l8 antibody
against both targets.
DISCUSSION
The fusion protein chl4. 18-1L2 was designed to create a
molecule that would achieve enhanced in vivo effects over the
combined use of the antibody and IL2 as separate molecules ( 10,
1 1 ). The function of the antibody component of this fusion
protein is to facilitate ADCC and to target the cytokine IL2 to
the area of GD2 tumors. Thus, chl4.l8-IL2 is a single molecule
containing both antitumor specificity and immunopotenti-

1958 Anti-GD2-lL2 Fusion Protein
ating capabilities. The results of in vitro experiments presented
here confirm and extend previous reports demonstrating that the
anti-GD2 cytokine fusion protein chl4.l8-IL2 retains the functional
activity of both the antibody and IL2 (10). The fusion
protein bound specifically to GD2 tumor cell lines, resulting in
the same fluorescence intensity as the parent chl4. 1 8 antibody
(Fig. 1 and Ref. 10). In addition, we demonstrated that tumor
cell-bound fusion protein could be detected with antibody specific
for human IgG or IL2.
A previous study using murine CTLL-2 cells as responders
(10) demonstrated that the IL2 component of the chl4.l 8-1L2
fusion protein was as active as native soluble 1L2 in inducing
proliferative responses. In contrast, Fell et al. (22), using a
fusion protein consisting of the Fab’ region of the human
carcinoma-specific L6 antibody linked to IL2, found the specific
activity of the IL2 component of that fusion protein to be
200-fold less than native rIL2 when measured in a proliferative
assay with the CTLL-2 cell line. We examined the ability of
chl4.18-IL2 to stimulate proliferation of a human myeloid Ieukemia
line, Tf-l3, previously shown to respond to IL2 in a
dose-dependent manner (18) and to stimulate PBMCs obtained
from melanoma patients following 96-h continuous infusions of
IL2. Both of these responding cell populations proliferate to IL2
via interaction with the intermediate affinity (3y) IL2 receptor
complex (2, 17, 21, 23). Both of these responding cell populations
demonstrated that the 1L2 component of the chl4. l8-IL2
fusion protein was as stimulatory as soluble recombinant human
1L2. In addition, the Mik 3l antibody specific for the 3 chain of
the IL-2 receptor blocked these fusion protein-induced proliferative
responses, demonstrating that the activation was due to the
IL-2 component. More importantly, we noted that tumor-bound
fusion protein was capable of stimulating 1L2-induced proliferative
responses. Quantitative comparisons indicated that the 1L2
component of the tumor cell-bound fusion protein was as stimulatory
as soluble 1L2.
Inclusion of 1L2 in vitro in the 51Cr release assay has been
noted to augment 1L2-dependent LAK and ADCC activity mediated
by PBMCs obtained following in vivo therapy with IL2
(24, 25). We have noted that the IL2 within the chl4.18-IL2
fusion protein also facilitates 1L2-dependent LAK killing of the
Daudi target by patient PBMCs (data not shown). The current
experiments demonstrate that the 1L2 component of the tumorbound
fusion protein also augments ADCC. When the fusion
protein was used to coat tumor target cells prior to washing and
inclusion in the cytotoxic assay, enhanced ADCC (over that
induced by the chl4.l8 antibody) was noted with patient PB-
MCs as effectors on the LAN-S target in over 50% of the assays.
On the M2 I melanoma target, the level of ADCC was comparable
to that seen with antibody alone. This ADCC activity
extends the prior observation that chl4. l8-IL2 fusion protein
can enhance T cell-mediated killing of an autologous tumor cell
line (10). In the previously published case, using the human 660
TIL line as a tumor-specific effector population, increased lysis
of the autologous GD2 tumor was obtained by addition of the
chl4. 18-IL2 fusion protein as compared to that mediated by
addition of chl4.l8 antibody or IL2 alone (10).
There is reason to hypothesize that in vivo localization of
IL2 to the tumor via the tumor-selective chl4.18-1L2 fusion
protein may induce more effective antitumor destruction than an
equivalent amount of free soluble ]L2. Other studies have demonstrated
that 1L2 produced in viva by tumor cells (following
gene transfer) can result in enhanced rejection of the IL2-
producing tumor cells (26). The fact that this rejection was
immunologically mediated suggests that a higher local concentration
of 1L2 may improve immune recognition of tumor.
Previous findings with the 14.l8-IL2 fusion protein demonstrated
suppression of human neuroblastoma tumor growth in an
experimental hepatic metastases model in SCID mice (I I). In
this model, human LAK cells and relatively low doses of
chl4.18-IL2 fusion protein induced prolonged survival of animals
bearing micrometastases, comparable to the survival noted
only with very high doses of recombinant human 1L2 (1 1).
Mixtures of low doses of mAb chl4.18 plus IL2 do not effectively
prolong the life span nor eradicate established metastases
of neuroblastoma or melanoma in SCID mice, whereas the
fusion protein chl4.l8-IL2 is able to accomplish both of these
tasks (11, 12). Becker et a/. (13) also demonstrated that this
chl4.l8-IL2 fusion protein is effective against pulmonary and
hepatic melanoma metastases in a syngeneic murine tumor
model and that T cells are essential for achieving this antitumor
effect. We note in the present in vitro study that in some
instances, such as the lytic activity against M2l and LA-N-S
targets, the fusion protein did not function any better than the
combination of 1L2 and chl4.l8 as 2 independent reagents, yet
four separate murine studies have noted a clear advantage of the
fusion protein in vivo over the combination of soluble IL2 and
chl4.l8 antibody (1 1-13, 27). This could be due to a number of
factors. The human immunoglobulin component of the fusion
protein actually lengthens the serum half life of IL2 (27) from a
li/2f3 of 6 mm for recombinant human IL2 to a ‘1/2(3 of 30 h for
the chl4.l8-IL2 fusion protein. In addition, in vivo localization
studies have shown targeting of anti-GD2 mAb to GD2 tumor
in viva (28). Thus, it is likely that the ch 14. 1 8-IL2 fusion protein
can specifically localize to tumor sites in vivo and deliver IL2
directly to tumor sites. This may result in augmented stimulation
of IL2-responsive effector cells, including both NK and T cells.
These preclinical in vitro data presented here and the
previously obtained in vivo data from the munine xenograft and
syngeneic models suggest that the chl4. 18-1L2 fusion protein
prolongs the serum half-life of IL2, localizes to sites of GD2
tumor metastases, activates ADCC through FeR-bearing effectors,
and activates IL2-responsive NK and T cells at tumor sites,
potentially mediating a protective antitumor response. The current
in vitro study documents that the 1L2 component of the
tumor-bound fusion protein is as effective as soluble 1L2 in
stimulating proliferation and cytotoxicity by effector cells obtamed
from patients following in vivo therapy with IL2. The IL2
component of the fusion protein facilitates ADCC of neuroblastoma
and melanoma targets by in vivo activated human effector
cells. Toxicity testing of this fusion protein in experimental
animals is now underway to determine how best to test ch 14. 18-
IL2 in Phase I clinical trials for patients with GD2 tumors,
including melanoma (29) and neuroblastoma.
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