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

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-ac-

tivating capability of a recombinant fusion protein consist-

ing of a tumor-selective human/mouse chimeric anti-gangli-

oside 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 periph-

eral 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 sug-

gests 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 cytotox-

icity 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 hu-

man tumor xenografts in SCID mice and GD2-positive mu-

rine 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.

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.

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 activa-

tion 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 neuroblas-

toma. melanoma, and certain other tumors (7).

In an effort to augment the stimulation of the FcR� anti-

body-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 ( 10). 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 microenviron-

ment.

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, detect-

ing 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

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.

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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 Can-

cer 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 plas-

mids in Sp2/0-Agl4 cells and propagation of the clones secret-

ing 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 neuro-blastoma target cell line, kindly provided by R. Seeger (Chil-

dren’s Hospital of Los Angeles, Los Angeles, CA), was main-

tamed 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 previ-

ously (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 anti-body were detected by standard indirect immunofluorescence

methods (Becton Dickinson, San Jose, CA). Antibodies in-

eluded a goat antihuman IgG (Caltag, San Francisco, CA) and a

rabbit antihuman IL2 (Genzyme, Cambridge, MA).

Proliferation Assays. Fresh PBMCs from healthy vol-

unteer 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 penicil-

lin, and 100 p.g/ml streptomycin sulfate (RPMI-HS). Recombi-

nant IL2 and fusion protein were added at concentrations as

indicated in the “Results”. Concentrations of recombinant sol-

uble 1L2 were also tested, which corresponded to the concen-

tration 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 con-

taming the LXSN vector but no IL2R�3 gene was designated

Tf-IL (18). This Tf-l�3 cell line responded to 1L2 using inter-

mediate affinity � receptor complexes ( 1 7. I 8) and thus isanalogous to the majority of NK cells in 1L2-treated patients,

which also use intermediate affinity IL2 receptors (2). The Mik

1�1 monoclonal antibody directed against the p70 IL-2 receptor1� chain was used in the blocking studies ( 19. 20).

ADCC and Fusion Protein-mediated Cellular Cytotox-icity. All ADCC assays were performed in RPMI-HS. Effec-

tor cells in a total volume of SO pi were plated in quadruplicate

into 96-well U-bottomed microtiter plates at the indicated ef-

fector/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-HSwere 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 Harvest-

ing System (Skatron, McLean, VA). Maximum 5tCr release was

measured by lysing target cells with the detergent cetrimide



M21

chl4.18-1L2chl4.18

BT-20

JLRabbit Anti-1L2

Rabbit IgG

Rabbit lgGRabbit Anti-1L2

. . . .�-

Clinical Cancer Research 1953

LAN-5

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 theantibody 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 ofch 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 Prolifera-

tive Responses. Fig. 2 documents proliferative responses to

1L2 attained with the Tf-I�3 cell line and PBMCs from a mel-

anoma 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 interme-

diate affinity IL2 receptors (�3-y dimers), designated Tf- I �, wasobtained by stable transfection of Tf- I with eDNA encoding the

1� chain of the IL2 receptor ( 1 8). The Tf- 1 �3 cell line thatretained 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-l�3 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



1200

1000

600

400

200

0

10000

8000

6000

4000

2000

1954 Anti-GD2-IL2 Fusion Protein

Cl)

I-z

0C�)

0 -�L2 UImI 1.6 3.0 00 12 25 50 100 200 400FP ��JmI.B2O 1.20 2.0 5.0 10 20 40 80 ISO

Fig. 2 Proliferation induced by soluble IL2 and IL2 within the solublefusion protein. The Tf-l�3 cell line (A) and PBMCs obtained from apatient following continuous infusion lL2 (B) were stimulated withincreasing concentrations of soluble IL2 (A) and soluble chl4.l8-1L2

fusion protein (s). Dilutions of IL2 and fusion protein were made toachieve equivalent molar concentrations of IL2. The counts were deter-mined 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 concentra-

tion 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 prolif-

erative assays using the Tf- 1 �3 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 re-

stimulation 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 com-

ponent 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 re-

sponse 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 throughan Intermediate Affinity �#{176}y1L2 Receptor Is SpecificallyBlocked 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 a�j

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 wereassayed 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 culturedfor 4 days at 37#{176}Cin 5% CO,. pulsed with 3H-thymidine for 16 h, and harvested, and counts per 5 mm were obtained with a Packard 9600 Matrixcounter.

Experiment

3H-Thymidine incorporati on (count per 5 mm)

Medium 100 units/mI IL2 40 ng/ml chl4.18-1L2 O.5�g/ml chl4.18

I ControlPatient 1Patient 2

197160184

14,1949,741

24,381

13.3877,916

7,413

190161

207

II Patient 16Patient 17

Patient 20

16549

31

9,9592,321

4,135

11,2012,7006,317

594643



120000 120000

90000 90000

60000 60000

30000 30000

0 � �- 0

200 50 12.5 66.5 16.6 4.1

16000 16000

L1�I-

C-)

0.I-a-

I0

C.”

I-

I 2000

8000

200

12000

8000

4000

0

100000

75000

50000

25000

50

100000

75000

50000

25000

0-200

IL-2 CONCENTRATION ( U/mi) 14.18-IL-2 CONCENTRATiON (ug/mi)

80000

60000

40000

20000

U)I-z

0C)

chl4.18

LIt�chl4.18 FP

LA-N-5FP

M21Fig. 4 Proliferation induced by the lL2 component of the tumor-bound

fusion protein. The M2l melanoma and LA-N-S neuroblastoma celllines 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 thisincubation. 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 I3HITdRincorporation.


Fig. 3 Antibody to the �3 chain of the IL2 receptor

blocks fusion protein induced proliferation by cells

expressing intermediate affinity, but not cells ex-

pressing 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 (rightpanels). The humanized Mik �3l antibody at a finalconcentration 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.

‘ . 0 � ‘50 12.5 66.5 16.6 4.1 1

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. . #{149}500

was a strong proliferative response to both soluble IL2 and #{149}�fusion protein by the Kit 225 cell line, which expresses the � 50

high-affinity afty IL2 receptor. The Mik �3 1 antibody did not

block this proliferative response, as shown previously for IL2

(19).

chl4.18-1L2 Bound to GD2� Cells Stimulates Prolifer-ation. The flow cytometric studies (Fig. 1 ) document that the

chl4.18-IL2 binds to GD2� tumor cells via the antibody van-

able 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



Patient 1

400.

300.

200.

L �oo

LAN 5m4 18

+112 FP

cI,14.1O

18

Fp

11.2

Y

Patient 2I

C

U

N

T

S

M2i

±14 10 +112

ControlLAN-5

200.

150.

100.

50.

c81418 +11.2� chI4.18Fr

12

M2�4.18+1U

414.18

112 Fr

IL.2 (units) 2.5 25 ‘ 100FP (ng) I 10 40

2.5 25 100I 10 40

Fig. 5 Soluble fusion protein facilitated ADCC on GD2� tumor cells.

PBMCs obtained from two patients following a 96-h continuous infusion of1L2 and from a volunteer control donor were the effector cells in a 4-h 51Crrelea.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, or40 ng/ml (corresponding to 2.5, 25, and 100 units of 1L2/ml): with thechI4.l8 antibody alone at 1, 10, or 40 ng/ml: or with a combination of 1L2

and chl4.18 antibody at the same concentrations.

1956 Anti-GD2-lL2 Fusion Protein

two melanoma patients receiving IL2 were assayed for lytic

Table 2 Stimulatory activity of IL2 in tumor-bound fusion protein

The Tf-l�3 and control Tf-lL cell lines (experiment 1) or Tf-l�3 cells and PBMCs from IL2-treated patients (experiment 2) were stimulated ina 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 proliferationof 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. andthe 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.

Stimulus

3H-TdR incorporation (coun

Experiment I

Is X I0�), responding cell

Experiment 2

Tf- I �3 PatientTf-l �3 Tf- 1L

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)

6.7 10.8

67.4 91.164.2 13.950.7 12.1

41.9 12.8

56.6 14.4

70.8 13.4

0.8 .03

NT’ NT18.6 4.1

9.3 1.7

3.2 0.6

1.2 0.4

4.2 2.1

‘a NT, not tested.

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 Tu-

mor-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 in-

duced 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 measure-

ment 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 approx-

imately 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 prolifer-

ative 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 stimu-

lated 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



M21A300

250

200

150

100

50

0

8 igG

0 IgG+iL2

iichl4.18

Oichl4.18+1L2

U FP

600 �

U)

� 400z

� 200

800

� 600

#{163}(I)

S

:

LA-N-5�600

_I 5oo

400

300

200

100


0

,Ju1�,J

Patient 6 Patient 7 PatIent 20 Patient 17

Fig. 6 Tumor-bound fusion protein facilitated ADCC. Patient PBMCsobtained 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. sol-

uble 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 sim-

ilar 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 fol-

lowing the 5tCr labeling. Soluble IL2 (100 units/ml) was added

,� 400

0 � 200

chl4.18 FP chl4.18 FP

M21 LA-N-5

�. 0

B Fig. 7 ADCC ofGD2� M21 and LA-N-S cells was facilitated by eitherchl4.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 thechl4.18 or the fusion protein are connected by the line.

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 ob-

served 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 aug-

menting 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 com-

paring 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 reproduc-

ible 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 mole-

cule 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 func-

tional 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 spe-

cific 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 Ieu-

kemia line, Tf-l�3, 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 popula-

tions 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 prolifer-

ative 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 prolifer-

ative responses. Quantitative comparisons indicated that the 1L2

component of the tumor cell-bound fusion protein was as stim-

ulatory as soluble 1L2.

Inclusion of 1L2 in vitro in the 51Cr release assay has been

noted to augment 1L2-dependent LAK and ADCC activity me-

diated 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 tumor-

bound 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 compa-

rable 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 dem-

onstrated 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 concen-

tration of 1L2 may improve immune recognition of tumor.

Previous findings with the 14.l8-IL2 fusion protein demon-

strated 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 ani-

mals 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 effec-

tively 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 effec-

tors, and activates IL2-responsive NK and T cells at tumor sites,

potentially mediating a protective antitumor response. The cur-

rent 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 ob-

tamed from patients following in vivo therapy with IL2. The IL2

component of the fusion protein facilitates ADCC of neuroblas-

toma 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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1996;2:1951-1959. Clin Cancer Res J A Hank, J E Surfus, J Gan, et al. (ch14.18-IL2).recombinant anti-ganglioside GD2 interleukin-2 fusion protein Activation of human effector cells by a tumor reactive

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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