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

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