Vol. 1. 425-434, April 1995 Clinical Cancer Research 425
An Immunoconjugate of Lys3-Bombesin and Monoclonal Antibody
22 Can Specifically Induce FcyRI (CD64)-dependent Monocyte-
and Neutrophil-mediated Lysis of Small Cell Carcinoma
of the Lung Cells1
Jian Chen, Michael Mokotoff, Jie-Hua Zhou,
Michael W. Fanger, and Edward D. Ball2
Division of Hematology/Bone Marrow Transplantation, University ofPittsburgh Medical Center and Pittsburgh Cancer Institute, Pittsburgh,Pennsylvania 15213 [J. C., J-H. Z., E. D. B.]; Department ofPharmaceutical Sciences, University of Pittsburgh, Pittsburgh,
Pennsylvania 15213 [M. M.]; and Department of Microbiology,
Dartmouth Medical School, Lebanon, New Hampshire 03756[M. W. F.]
ABSTRACT
Small cell carcinoma of the lung (SCCL) accounts for
25% of all lung cancers and has a very poor prognosis. It is
known that SCCL cells produce gastrin-releasing peptide, a
peptide which has similar biological actions to that of bom-
besin, an amphibian counterpart of gastrin-releasing pep-
tide, and express high affinity cell surface bombesin/gastrin-
releasing peptide receptors. These receptors can serve as
targets for specific immunotherapy. Cell surface receptors
for the Fc portion of IgG (FcyR) are a family of molecules
that can mediate a variety of immune reactions, including
tumor cell cytotoxicity. We hypothesized that an immuno-
conjugate of bombesin and a mAb directed to the high-
affinity FcyRI (mAb 22) should be able to trigger specific
cytotoxicity against SCCL cells. In this article, we report the
construction of this immunoconjugate and demonstrate its
capacity to redirect immune effector cells toward SCCL
cells and elicit lysis of these target cells. The immunoconju-
gate stained the majority of cells from four SCCL cell lines
and reacted with Fc�yRI on activated monocytes and neu-trophils. After preincubating monocytes and neutrophils
with recombinant ‘y interferon to enhance the expression of
FcyRI on the cell surface, we demonstrated that 60-98% ofSCCL cells could be lysed in the presence of the immuno-conjugate in a chromium release assay. Tumor cell lysis was
observed over a wide range of immunoconjugate concentra-
tions, was dependent on the ratio of E:T cells, and could be
blocked by the addition of either parental molecule of the
immunoconjugate. Bispecific molecules redirecting immune
efTector cells to target SCCL cells may have clinical appli-
cation in the therapy of SCCL.
INTRODUCTION
The prognosis for SCCL3 remains poor despite recent
advances in the management of certain other cancers. An esti-
mated 42,500 cases of SCCL were diagnosed in the United
States in 1993, accounting for 25% of all lung cancers (1, 2).
Although the majority of SCCL patients respond to chemother-
apy, the response is of short duration (3, 4). Median survival for
patients with limited-stage disease is 14-18 months, and for
patients with extensive disease is 9-1 1 months. Only 15-25%
of limited-stage patients survive for more than 2 years (1-4).
Many combinations of chemotherapeutic agents have been used
clinically without much improvement in the long-term survival
rate. Recent efforts have been focused on the development of
other therapeutic strategies for the treatment of SCCL, including
immunological and hormonal therapy (5, 6) and intensive che-
motherapy with autologous bone marrow or peripheral stem ceil
support (7-9).
BN is a 14-amino acid peptide which was initially isolated
from the skin of the frog Bombina bombina (10). The mamma-
han analogue of BN, GRP, contains a COOH-terminal hep-
tapeptide sequence identical to that of BN (1 1). The majority of
human SCCL cell lines produce GRP and express a single class
of high-affinity receptors for BN/GRP (12-16). It has also been
demonstrated that GRP functions as an autocrine growth factor
for human SCCL cells. Blockage of this autocrine growth stim-
ulatory activity of GRP in SCCL has been the focus of several
studies. A mAb against GRP and a number of BN/GRP receptor
antagonists have been shown to inhibit the growth of SCCL
cells both in vitro and in vivo (5, 17-19). Since BN/GRP
receptors have limited distribution in the body, they can serve as
targets for specific immune reactions. A novel approach of
immunotherapy targeting the BN/GRP receptors expressed on
SCCL cell surface has been developed in our laboratory. Wehave made an immunoconjugate between a BN-like peptide and
a mAb against human high-affinity FcyR (Fc-yRI, CD64), which
is expressed on the surface of human monocytes and activated
neutrophils. We hypothesized that this immunoconjugate should
Received 8/26/94; accepted 12/12/94.C This work was supported by Grant CA37868 from the National Cancer
Institute, NIH, Bethesda, MD.2 To whom requests for reprints should be addressed, at Division of
Hematology/Bone Marrow Transplantation, Montefiore University Hos-
pital, 200 Lothrop Street, Pittsburgh, PA 15213.
3 The abbreviations used are: SCCL, small cell carcinoma of the lung;BN, bombesin; GRP, gastrin-releasing peptide; Fc-yR, Fc -y receptor;ADCC, antibody-dependent cell-mediated cytotoxicity; SATA, N-sue-
cinimidyl S-acetylthioacetate; Sulfo-SMCC, sulfosuccinimidyl 4-(N-male-imidomethyl) cydlohexane-1-carboxylate; R-HPLC, reversed phaseHPLC; SH, free sulfhydryl group; rIFN--y, human recombinant IFN--y;MFI, mean fluorescence intensity; G-CSF, granulocyte-colony-stimu-lating factor; PBA, phosphate-buffered saline-bovine serum albumin-
sodium aside.
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0
Lys3-BN
�H -C0-CH2-S-Ac
IN H 2 0 H
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stAb22-Lys-BN iaaunocon)ugate
Fig. I Schema of the chemical conjugation between Lys3-BN and mAb 22 or its F(ab’), fragments.
426 Immunoconjugate of Lys3-BN and mAb 22 Can Induce Lysis of SCCL
Lys3-BN + Ac�S�CH2COO_NO
NH2 SATA .1/0
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be able to redirect these immune effector cells toward SCCL
cells and elicit specific ADCC against these cells.
MATERIALS AND METHODS
Cell Lines. SCCL cell lines, NCI-H69, NCI-H345, and
SHP-77, were maintained in RPMI 1640 medium (GIBCO-
BRL, Grand Island, NY) supplemented with 10% FCS, 2 mr�t
L-glutamine, 100 units/ml penicillin, and 100 p.g/ml streptomy-
din (GIBCO-BRL) at 37#{176}Cin a humidified atmosphere with 5%
CO2. Another SCCL cell line, DMS273 (20), was maintained in
Waymouth’s MB 752/1 medium (GIBCO-BRL) supplemented
with 10% FCS.
Antibodies and Reagents. Anti-Fc-yRI (mAb 22; Ref.
21), F(ab’)2 fragments of mAb 22 (F(ab’)2), and FITC-labeled
mAb 22 were obtained from Medarex, Inc. (Annandale, NJ).
SCCL-1, an IgG2a mAb reactive with transferrin receptor (22),
was produced in this laboratory. Lys3-BN, a BN analogue with
similar binding affinity to the BN/GRP receptor (1 1, 13), and
hydroxylamine were purchased from Sigma Chemical Company
(St. Louis, MO). Conjugation chemicals, SATA and Sulfo-
SMCC, were obtained from Pierce Chemical Co. (Rockford,
IL).
Protein Conjugation. Lys3-BN was freshly dissolved in
0.1 M sodium phosphate buffer (pH 7.4) containing 2.5 m�i
EDTA and the SATA was freshly dissolved in 100% dimeth-
ylformamide. The SATA was mixed with L-BN in a final molar
ratio of 10:1. After 30 mm of reaction at room temperature, the
Lys3-BN-SATA conjugate was separated from nonreacted
Lys3-BN and SATA by R-HPLC on a Vydac C18 analytical
column. The R-HPLC eluent containing Lys3-BN-SATA was
adjusted to pH 7.0 by adding 1 M sodium phosphate (pH 8.0).
The free sulthydryl group was generated by deacetylation with
hydroxylamine at 4#{176}Cfor 2 h. A second R-HPLC was per-
formed to separate Lys3-BN-SH. The fraction containing Lys3-
BN-SH was collected and neutralized to pH 7.0. The presence of
free sulthydryl groups could be determined via reaction with
Ellman’s reagent. At the same time, mAb 22 or F(ab’)2 frag-
ments of mAb 22 was reacted with Sulfo-SMCC to produce a
maleimide-activated antibody. The activated antibody was sep-
arated from unreacted Sulfo-SMCC by centrifugation through a
Centricon 30 apparatus (Amicon, Beverly, MA). The final con-
jugation between Lys3-BN-SH and the activated antibody was
carried out by mixing an equal molar amount at room temper-
ature overnight. The unreacted Lys3-BN and other by-products
were removed by centrifugation through a Centricon 30 appa-
ratus. The concentration of the immunoconjugate was quantified
using a Bio-Rad DC protein assay (Bio-Rad Laboratories, Rich-
mond, CA) and its purity was checked by SDS-PAGE. The
scheme for the peptide conjugation is shown in Fig. 1.
Immunofluorescence. SCCL cells were washed twice
with ice-cold PBS containing 0.1% BSA and 0.1% sodium azide
(PBA solution) and incubated with different amounts of the
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Clinical Cancer Research 427
immunoconjugate at 4#{176}Cfor 1 h in the presence of 100 p.g/ml
human IgG. The final concentration of immunoconjugate added
was 5, 25, and 50 jig/mI per 5 X 106 cells. After washing three
times with PBA solution, the cells were resuspended and incu-
bated with FITC-labeled goat F(ab’)2 anti-mouse immunoglob-
ulin (Caltag Lab., South San Francisco, CA) for 30 mm at 4#{176}C.
After washing, the cells were fixed in 1% paraformaldehyde.
Monocytes and neutrophils, before and after IFN-y stimulation,
were stained directly with FITC-Iabeled mAb 22 to evaluate the
expression of FcyRI. All of the samples were analyzed by
FACScan flow cytometry (Becton Dickinson, San Jose, CA).
The number of antibody sites bound per cell was deter-
mined by using a standard bead mixture containing beads with
six different concentrations of FITC labeling (23). The standard
beads were purchased from Flow Cytometry Standards Corpo-
ration (San Juan, Puerto Rico; Quantum 24 and 25). The median
fluorescent intensities of the six bead peaks were used to set a
standard curve for determination of the amount of FITC/cell.
Antibody/cell was calculated by dividing the FITC/cell by the
flourescein:protein ratio for the standard bead mixture. Appro-
priate isotype-matched negative controls were performed to
subtract nonspecific binding of antibody.
Stability of Lys3-BN x 22 Immunoconjugate Binding to
SCCL Cells. It has been reported that BN/GRP receptors are
rapidly internalized upon binding BN or GRP (24). To deter-
mine the stability of immunoconjugate binding, SCCL cells
were incubated with 20 p.g/ml immunoconjugate for 30 mm and
then washed to remove unbound immunoconjugate. The immu-
noconjugate-bound cells were incubated at 37#{176}Cin fresh media
for various times before the addition of FITC-Iabeled antimouse
antibody at 4#{176}C.The binding of the immunoconjugate to SCCL
cells at the initiation of incubation at 37#{176}Cwas defined as 100%.
Isolation of EfTector Cells from Peripheral Blood.
Monocytes were separated from Leuko-Packs obtained from the
Pittsburgh Central Blood Bank. After Ficoll-Hypaque gradient
centrifugation, mononuclear cells were washed twice with
HBSS (GIBCO-BRL) containing 1 mr�t EDTA and then cultured
in a flask with RPMI 1640 medium containing 10% FCS for 2 h
at 37#{176}C.The nonadherent cells were removed. The adherent
cells were detached, collected, stained with anti-CD14, anti-
CD45, anti-CD3, anti-CD13, and anti-CD56 (Becton Dickin-
son), and analyzed by FACScan flow cytometry to determine
the purity. The neutrophils were isolated from normal donors. A
20-ml blood sample was centrifuged on Ficoll-Hypaque. The
cell pellet was suspended in an equal volume of RPMI 1640
medium. Four hundred p.1 of 5% dextran were added to each ml
of blood sample. After sedimentation at 1 X g for 1 h at 4#{176}C,the
neutrophil-rich supernatant was collected. The purity of neutro-
phils thus isolated was usually higher than 95%.
Activation of Immune Effector Cells. rIFN--y was
kindly provided by Genentech (San Francisco, CA). The con-
centration of rIFN-’y used in this study (200 units/ml) has been
shown to saturate its receptors and to induce a maximum in-
crease in the expression of Fc�yRI on the surface of monocytes
and neutrophils (25). Isolated monocytes and neutrophils were
incubated with rIFN--y in RPMI 1640 medium containing 10%
FCS for 18 h at 37#{176}Cbefore the ADCC assay. The expression
of Fc-yRI before and after rIFN--y incubation was determined by
staining with FITC-labeled mAb 22 and analyzed by FACScan
flow cytometry.
ADCC Assay. The assay was performed in 96-well
round-bottomed microtiter plates (Rainin Instrument Co., Woburn,
MA). The target SCCL cells were washed once with RPMI 1640
medium and incubated with 100 jiCi sodium [5tCr]chromate
(New England Nuclear, Boston, MA) for 1 h at 37#{176}C.After
washing several times, cells were resuspended in RPM! 1640
medium containing 10% FCS to a concentration of 1 X i05
cells/ml. Activated monocytes or neutrophils serving as effector
cells were suspended in RPMI 1640 medium in a final concen-
tration of 2 x i0� cells/ml. Effector cells (100 p.1) were added
to the first row of wells and serial dilution was performed with
an equal volume of RPMI 1640 medium. Then, 100 p.1 of target
cells were added in the wells to yield a final E:T ratio of 100:1,
50:1, 25:1, and 12:1. In a standard assay, 5 jig immunocongu-
gate were added to yield a final concentration of 25 jig/mI. The
mAb SCCL-1 was included in each assay as a positive control
to measure the activity of the monocytes and neutrophils. 5ev-
eral other controls with E:T ratio of 100:1 were also incorpo-
rated in each assay, including incubation of target cells with
immunoconjugate alone, incubation of target and effector cells
without any antibody, and incubation of target and effector cells
with unconjugated mAb 22. In each assay, a 10-fold excess of
Lys3-BN and unconjugated mAb 22 along with the immuno-
conjugate were incubated along with the target and effector cells
to determine whether the tumor cell lysis could be blocked by
either of the parental molecule. To determine whether the pres-
ence of human immunoglobulin could interfere with the redi-
rected tumor cell killing, human IgG at a final concentration of
1 mg/ml was added in some assays. After incubation at 37#{176}Cfor
4 h or 18 h, the microplates were centrifuged and the supernatant
was collected for the determination of 51Cr release. Maximum
lysis was achieved by the addition of 100 p.1 5% NP4O to 100 p.1
target cells. The percentage of cell lysis was calculated as:
(experimental cpm - spontaneous release mean cpm)
x 100/(maximum release mean cpm
- spontaneous mean cpm)
Spontaneous release of 5tCr from the target cells was less than
10% of maximum release in a 4-h assay and less than 20% in an
18-h assay. For dose-response assays, the immunoconjugate was
serially diluted and added. The E:T ratio in those assays was
100:1. Since the amount of immunoconjugate added in a stan-
dard assay was 25 p.g/ml, we defined the percentage of tumor
cell lysis with that amount of immunoconjugate as 100% activ-
ity. The tumor cell lysis achieved with diluted immunoconjugate
was calculated accordingly.
Statistical Analysis. Each experimental result was ob-
tamed from triplicates and reported as the mean ± SD. Signif-
icance level was determined by paired Student’s t test when
applicable.
RESULTS
Binding of the Lys3-BN x 22 Immunoconjugate to
SCCL Cells. Unconjugated mAb 22 and its F(ab’)2 fragments
did not react with SCCL cells. After conjugation with Lys3-BN,
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DMS273 cells DMS273 cells300
200
100
0
H345 cells H345 cells300
I.’.’. 0�200CC. 1001 0 20 3 0 40 � 5’O D � 10 � 20 � � � 5�0
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Concintratlon (Ng/ml)
10 20 30 40 50
Concsntratlon (�gIml)
428 lmmunoconjugate of Lys3-BN and mAb 22 Can Induce Lysis of SCCL
10 20 30 40 50 0 10 20 30 40 50
Fig. 2 Flow cytometric analysis of four SCCL lines stained with the
immunoconjugate made from Lys3-BN and mAb 22 (#{149})or F(ab’), (0).Each point represents the mean of three individual analyses and the SD
is less than 10% for positivity and 20% for MFI. Isotypic control of
mouse IgG was included in all of the analyses. Both the percentage of
positive cells and the MFI increase with the amount of immunoconju-
gate added. Compared with the immunoconjugate made between
Lys3-BN and mAb 22, the immunoconjugate made between Lys3-BN
and F(ab’)2 has a similar percentage of positivity, but consistently lower
MFI.
the binding of the immunoconjugate to SCCL cells could be
detected by the addition of FITC-labeled antimouse antibody.
The results of flow cytometric analysis of the binding of two
immunoconjugates to four SCCL cell lines are illustrated in
Fig. 2. The binding was directly proportional to the amount of
immunoconjugate used to stain the cells. This was manifested
both by an increase in the absolute percentage of cells stained
positively and by an augmentation of the MFI of the entire cell
population. As the amount of immunoconjugate was increased
from 5-50 p.g/ml, the percentage of positive cells generally
increased from 50-85%, and the MFI generally increased from
< 100 to >200. In general, the immunoconjugate prepared be-
tween the whole antibody of mAb 22 and Lys3-BN had a higher
MFI than the one prepared between the F(ab’), fragments of
mAb 22 and Lys3-BN. The number of BN/GRP receptors has
been estimated to be 1500 and 1000, respectively, for H345 and
H69 cell lines (16, 24). We have not quantified the BN/GRP
receptor numbers on DMS273 and SHP-77 cells. By flow cy-
tometric analysis, it appears that DMS273 cells express approx-
imately the same number of BN/GRP receptors, while the
SHP-77 cells have slightly fewer BN/GRP receptors when com-
pared to H345 cells.
The binding of the Lys3-BN X 22 immunoconjugate to
normal peripheral lymphocytes and to two leukemia cell lines
was also tested. The immunoconjugates did not bind to normal
peripheral lymphocytes. The mAb 22 and its F(ab’)2 fragments
stained 20-50% of cells from the HL-60 and NB4 leukemia cell
lines with very dim fluorescence, consistent with the known
expression of Fc-yRI on these cells. There was no change in
fluorescence intensity when these cells were stained with the
immunoconjugate, although the percentage of positive cells
increased slightly.
The number of immunoglobulin molecules per SCCL cell
was calculated using a bead calibration method. H345 cells
bound an average of 61 16 immunoconjugate molecules, H69
cells bound 5036, DMS 273 cells bound 9473, and SHP-77 cells
bound 2399. The number of bound immunoconjugates is greater
than the published number of BN/GRP receptors per cell (14-
16). Since there are three types of BN/GRP receptors present, it
is possible that the immunoconjugate may bind to low-affinity
BN/GRP receptors or cross-bind to other family members of
GRP receptors.
It has been reported that the BN/GRP receptors are quickly
internalized upon binding BN/GRP. To determine the stability
of Lys3-BN x 22 immunoconjugate binding, cells from four
SCCL lines were incubated with a saturation amount (25 jig/mI)
of immunoconjugate for 30 mm and then washed to remove
unbound immunoconjugate. The cells were incubated at 37#{176}Cin
fresh media for various times before the addition of FITC-
labeled antimouse antibody. The results from three experiments
are shown in Fig. 3. The percentage of positive cells did not
change significantly during the first 4-h period. After 24-h
incubation, the majority of immunoconjugate binding was lost.
Binding of the Lys3-BN x 22 Immunoconjugate to
Monocytes and Neutrophils. We also tested the binding of
the Lys3-BN X 22 immunoconjugate to peripheral monocytes
before and after incubation with 200 units/ml rIFN--y for 18 h.
This is shown in Table 1 . rIFN--y dramatically increased the
expression of Fc-yRI on human monocytes as defined by the
increase of MFI from <30 to > 120. In contrast, there was no
change in the expression of FcyRI on human peripheral lym-
phocytes. The conjugation of Lys3-BN to mAb 22 did not
interfere with its binding to Fc-yRI. Neutrophils without rIFN--y
treatment did not express cell surface FcyRl or bind to the
immunoconjugate. After incubation with rIFN-�y for 18 h,
>90% of neutrophils bound to the Lys3-BN X 22 immunocon-
jugate with a median MFI of 50.
Lys3-BN x 22 Immunoconjugate Redirected TumorCell Lysis. The ability of the Lys3-BN X 22 immunoconju-
gate to elicit monocyte-mediated and neutrophil-mediated tumor
cell lysis was tested in a series of 5tCr release assays. The
optimal concentration of the immunoconjugate-induced cyto-
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4 of 11 Celltrion, Inc., Exhibit 1016
120 -
1 00 � � DMS273
� H345
80 DH69B SHP-77
60
40- T2:.
Fig. 3 Stability of the immunoconjugate binding to four SCCL cell lines was analyzed by flow cytometry. The percentage of binding is defined as
100% at the initiation of incubation at 37#{176}C(time 0). Results from three individual experiments are presented as mean ± SD. There is no significant
change in immunoconjugate binding to SCCL cells in the first 4 h.
24 h
150
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050
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Clinical Cancer Research 429
a’C
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Tab! e I Binding of the immunoconjugate to monocytes befor e and after rIFN-y incubation”
Before rIFN--y incubation After rIFN-�y incubation
% Positive ± SD MFI ± SD % Positive ± SD MFI ± SD
mAb 22F(ab’)2mAb 22 x Lys3-BNF(ab’)2 X Lys3-BN
83.5 ± 2.2 52.0 ± 26.0 (2)
70.7 ± 15.6 29.9 ± 14.6 (2)
86.7 ± 3.4 25.7 ± 0.10 (2)
72.3 ± 10.7 26.7 ± 8.57 (2)
85.2 ± 16.7
84.7 ± 17.2
92.1 ± 7.60
85.8 ± 16.3
210.1 ± 46.7 (2)
124.6 ± 25.5 (2)
188.0 ± 85.1 (2)
1 19.6 ± 20.9 (2)
a Flow cytometric analysis of the binding of mAb 22, F(ab’)2 of mAb 22, and two immunoconjugates to human monocytes before and after 18-h
rIFN--y incubation. MFI increased significantly after rIFN--y incubation.
1 10 100 1000 10000 100000
Immunoconjugate concentration (ng/mI)
Fig. 4 Effect of different concentrations of immunoconjugate Lys3-BN
x 22 on cytotoxicity of 5tCr-labeled SHP-77 cells by human monocytes
at an E:T ratio of 100:1. Results of two experiments are shown. Tumor
cell lysis by monocytes achieved with 25 p.g/mI immunoconjugate was
defined as 100%.
toxicity was determined by a dose-response assay using acti-
vated monocytes as effector cells and SHP-77 cells as target
cells. Results of two such assays are presented in Fig. 4. Max-
imum tumor cell lysis was observed over a wide range of
concentrations. Since the flow cytometry analysis indicated that
the maximum binding of the immunoconjugate to SCCL cells
occurred at concentrations above 25 jig/mI, we used 25 p.g/ml
immunoconjugate in most cytotoxicity assays.
The results of monocyte-mediated cytotoxicity of SCCL
cells are presented in Fig. 5. Since the effector cells came from
different donors in each experiment, the cytotoxic potency var-
ied greatly among donors. For each SCCL cell line, >80% of
cells could be lysed by monocytes from some donors. Tumor
cell lysis was primarily dependent on E:T ratio in each individ-
ual donor. The greatest lysis was consistently achieved at an E:T
ratio of 100:1. Pilot experiments using 4-h assays showed mod-
erate cytotoxicity. Increasing the incubation time from 4 h to
18 h resulted in a higher percentage of tumor cell lysis. We also
observed that monocytes from the same donor could have dif-
ferent cytotoxic potency against different SCCL cell lines. One
SCCL cell line could be better killed by monocytes from one
donor, but resistent to monocytes from another donor.
Monocyte-mediated cytotoxicity was studied under differ-
ent assay conditions in each experiment. The result of a typical
experiment is shown in Fig. 6. Incubating target cells with
activated monocytes in the absence of immunoconjugate re-
suited in 15-60% of tumor cell lysis, depending on the donor.
The addition of parental antibody, mAb 22, did not further
increase the lysis. In the presence of immunoconjugate, tumor
cell lysis was increased to 50-95%. This increase is significant
for each individual donor (P < 0.05) and is consistent in each
Research. on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
5 of 11 Celltrion, Inc., Exhibit 1016
DMS273 cells H69 cells
S Donorl
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#{163} Donor3#
100
80�
60�
40�
20�
12:1 25:1 50:1 100:1
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Fig. 5 Cytotoxicity of 5tCr-Iabeled SCCL cells by human monocytes. Assays were performed at E:T ratios ranging from 12:1 to 100:1. Monocytes
were separated from different donors in each individual assay. A 4-h assay was performed in pilot experiments. Since the tumor cell lysis was greatly
enhanced by the monocytes of the same donor after 18-h incubation, subsequent assays were done using an 18-h incubation. There is no differencein cytotoxicity between the immunoconjugate made from Lys3-BN and mAb 22 or its F(ab’)2 fragments. �, 4-h assay; #, immunoconjugate made
between L-BN and F(ab’), of mAb 22.
430 Immunoconjugate of Lys3-BN and mAb 22 Can Induce Lysis of SCCL
100
800
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individual assay. The immunoconjugate-induced SCCL cell ly-
sis could be significantly (P < 0.05) blocked by adding exces-
sive amounts of either unconjugated mAb 22 or Lys3-BN in
each assay. The presence of 1 mg/mI human IgG did not
interfere with immunoconjugate-induced tumor cell lysis (data
not shown). We also compared the potency of tumor cell lysis
between rIFN-y stimulated and nonstimulated monocytes from
the same donor. The result is shown in Fig. 7. Monocytes after
rIFN-�y stimulation had a significantly higher cytotoxic potency
(P < 0.05) in different E:T ratios. There was no difference in the
observed cytotoxicity from immunoconjugate made with whole
mAb 22 versus its F(ab’), fragments.
The results of neutrophil-mediated SCCL cell lysis are
shown in Fig. 8. Cytotoxic potency varied greatly among do-
nors. More than 75% of SHP-77 cells were lysed by neutrophils
from two different donors. The percentage of tumor cell lysis
was greatly increased by neutrophils from one donor when the
incubation time of the assay was prolonged from 4 h to 18 h. It
appears that H69 cells are more resistent to cytotoxicity from
neutrophils. The percentage of cell lysis ranged from 17-45% at
an E:T ratio of 100:1. Since neutrophils do not express Fc-yRI on
their cell surface without rIFN--y stimulation, nonstimulated
neutrophils had much less cytotoxicity when they were incu-
bated with target cells. Again, the immunoconjugate-induced
tumor cell lysis could be significantly blocked by adding excess
amounts of either mAb 22 or Lys3-BN in each individual assay
(data not shown).
DISCUSSION
Recently, a variety of bispecific antibodies and immuno-
toxins have been shown to confer antitumor effects in vitro as
well as in vivo (26-29). The major advantage of these bispecific
molecules lies in their ability to target therapy selectively to
tumor cells while simultaneously triggering cytotoxicity by en-
dogenous effector cells. In this report we have demonstrated that
immunoconjugates made of Lys3-BN and mAb 22, or F(ab’),
fragments of mAb22, can effectively induce lysis of SCCL cells
in the presence of rIFN--y activated monocytes and neutrophils
in vitro. The Lys3-BN X 22 immunoconjugate binds to more
than 85% of SCCL cells at concentrations above 25 p.g/ml,
while cytotoxicity is noted at 1000-fold lower concentrations.
This suggests that tumor cell lysis does not require saturation of
BN/GRP receptors. The cytotoxicity observed at low immuno-
conjugate concentrations is particularly important since the BN/
GRP receptors are occupied to a certain extent by endogenous
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DMS273 cells
TflnH69 cslls
100
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. 50.
60
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0
.- 100#{149}0
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excessive amounts of L-BN.
Clinical Cancer Research 431
Fig. 6 Comparison of cytotoxic effects by monocytes under different conditions. They were incorporated in each cytotoxicity assay with 25 p.g/ml
immunoconjugate and an E:T ratio of 100:1. One typical experiment is presented here. CI, incubation oftarget cells with immunoconjugate, without
monocytes. The immunoconjugate itself has no cytotoxic effects. C2, incubation of target cells with rIFN-�y activated monocytes, without
immunoconjugate. T, incubation of target cells with rIFN-y activated monocytes in the presence of the immunoconjugate. The cytotoxicity of
monocytes was significantly higher (P < 0.05) in the presence of immunoconjugate. C3, incubation of target cells with rIFN-y activated monocytes
and mAb 22, but no immunoconjugate. C4, incubation of target cells with rIFN--y activated monocytes, immunoconjugate, and 10-fold excess of mAb
22. The cytotoxicity was significantly blocked (P < 0.05) by the addition of excessive amounts of mAb 22. CS, incubation of target cells with rIFN-�y
activated monocytes, immunoconjugate, and 10-fold excess of Lys3-BN. The cytotoxicity was significantly blocked (P < 0.05) by the addition of
BN/GRP, and antibody delivery to tumor sites in vivo may be
limited. Compared with the approach of using BN/GRP antag-
onists, the immunotherapy described in this article offers several
advantages. First, some antagonists against BN/GRP receptors
have been shown to partially inhibit the growth of SCCL cells in
vitro and less effectively in vivo (30). They could be quickly
degraded by serum proteases before they can reach tumor sites
(16). The conjugation of these small peptides to a large immu-
noglobulin should greatly retard their degradation. Although the
Lys3-BN used in our study is an agonist of BN/GRP receptors,
the immunoconjugate thus made did not show any stimulatory
effect on the growth of SCCL cells (data not shown). An
immunoconjugate made between an antagonist of BN/GRP re-
ceptor and mAb 22 will have additional therapeutic benefits by
inhibiting the growth of SCCL cells and redirecting immune
effector cells to SCCL targets. Second, it has been shown that
BN/GRP receptors on the SCCL cell surface are internalized
quickly upon binding BN/GRP (24). This property of the recep-
tor may be disadvantageous for the administration of an antag-
onist alone because the loss of active binding site may lead to
the loss of inhibitory effect on SCCL cells. The immunoconju-
gate we constructed can greatly retard the internalization of
BN/GRP receptors and is capable of inducing tumor cell lysis in
a relatively short time. Third, we showed that the immunocon-
jugate made between Lys3-BN and F(ab’)2 fragments of mAb
22 had a similar binding profile to both target and effector cells,
and equal capacity to induce ADCC against SCCL cells. Since
the F(ab’)2 fragments are smaller than the whole molecule of
mAb, they should more readily reach tumor sites in vito. Fi-
nally, it has been shown that multiple growth factors are in-
volved in the growth stimulation of SCCL cells (16). The
blockage of one pathway, such as BN/GRP receptors by their
antagonists, is not likely to inhibit the growth of SCCL cells in
vivo given the redundancy of growth factor receptors. However,
the use of BN/GRP receptors as targets for immunotherapy will
allow elimination of SCCL cells without this limitation.
Cell surface receptors for the Fe portion of IgG (Fc-yR)
play a central role in various immune and inflammatory re-
sponses. Three distinct classes of Fe-yR have been identified.
Fc-yRI is a potent cytotoxic trigger molecule on monocytes,
macrophages, and activated neutrophils. FcyRII is a cytotoxic
trigger molecule present on monocytes, macrophages, neutro-
phils, eosinophils, platelets, and other cells which are not cyto-
toxic to tumor cells. Fc-yRIII is also a cytotoxic trigger molecule
on macrophages and natural killer cells. The expression of
Fc�yRI on neutrophils occurs in certain clinical situations (31),
after rIFN-�y stimulation (32), and after G-CSF stimulation (33,
34). Activation of FcyRI on monocytes and neutrophils is as-
sociated with increased function of these cells, including phago-
cytosis, the triggering of cytotoxicity, degranulation, the gener-
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7 of 11 Celltrion, Inc., Exhibit 1016
H345 cellsDMS273 cells
100
4 112:1 25:1 50:1 100:1
Effeclor to target cell nIb
H69 cells
100
kJ)4ILTIL#{149}80�2o�0 �
12.1 25:1 50:1 100:1
Eflecbon to I.ng.I cell nelbo
12:1 25:1 50:1 100:1
Effeclor 10 lenget cell relbo
SHP.77 cells
12:1 25:1 50:1 100:1
Elfeclon to lergel cell ratio
U PrebnctAaied wiI� gsmma.IFN
� Wtttout preinctEaibon with
Fig. 7 Comparison of cytotoxic effects of monocytes with and without rIFN-y preincubation. The monocytes in these experiments were isolated from
the same donor. Preincubation of monocytes with rIFN-y resulted in significantly (P < 0.05) higher cytotoxicity at each data point. Results of one
experiment of three are presented here.
432 Immunoconjugate of Lys3-BN and mAb 22 Can Induce Lysis of SCCL
ation of superoxide, and the release of various cytokines. The
cytotoxic activity of monocytes and neutrophils against SCCL
cells in our experiments is significantly increased by the en-
hanced expression of Fc-yRI, and can be blocked by an excessive
amount of parental mAb 22. Both indicate that Fc-yRI is the key
molecule in redirecting immune effector cells to target cells and
in triggering cytotoxicity. An additional advantage of choosing
mAb 22 in the immunoconjugate is that mAb 22 binds to Fc-yRI
outside of the normal Fc-yRI ligand binding site (21). Thus,
endogenous immunoglobulin will not be able to compete with
the immunoconjugate for Fc-yRI binding sites, and will not
interfere with the functional efficiency of the immunoconjugate
to trigger cytotoxicity. Therapy with other mAbs may have been
hampered by this limitation in previous clinical trials.
The capacity of the Lys3-BN X 22 immunoconjugate to
trigger cytotoxicity against SCCL cells by neutrophils is impor-
tant for several reasons: (a) they are potent mediators of anti-
body-dependent cytotoxicity and the most abundant leukocytes
in circulation. Since the cytotoxicity against SCCL cells is
dependent on the E:T ratio in our studies, the number of effector
cells that can reach the tumor site is crucial in the clinical setting
of cancer; (b) they can be increased quantitatively and qualita-
tively by growth factors such as G-CSF and granulocyte-mac-
rophage-colony-stimulating factor. It has been shown that neu-
trophils isolated from patients treated with G-CSF had enhanced
Fc-yRI expression and abilities to mediate ADCC (34). It has
been shown that another bispecific antibody comprised of mAb
St
08
C-
22 and an anti-HER-2/NEU mAb induced the release of endo-
genous G-CSF in a clinical trial involving patients with HER-
2INEU-positive breast and ovarian cancer (35); (c) neutrophils
are capable of infiltrating into tumor sites in animal studies (36);
and (d) neutrophil function is probably not altered in the ma-
jority of patients with cancer.
We envisage a therapeutic application for the Lys3-BN X
22 immunoconjugate as an adjunct in the therapy of SCCL,
focusing on the elimination of residual disease. Since the ma-
jority of SCCL patients respond initially to chemotherapy but
eventually relapse, the elimination of residual tumor cells by
adjuvant immunotherapy may be useful. Notably, G-CSF has
already been used for SCCL patients after chemotherapy to
reduce the period of neutropenia and fever and augment de-
fenses against infections (33). The administration of G-CSF can
significantly increase the number of neutrophils and enhance the
expression of Fc-yRI on these cells and cytotoxic potency in vivo
(34). A future clinical trial examining the hypothesis that the
administration of the Lys3-BN X 22 immunoconjugate will
trigger cytotoxicity by neutrophils and monocytes against resid-
ual SCCL tumor cells and decrease relapse rates seems war-
ranted. To achieve the goal of launching clinical trials of the
Lys3-BN X 22 immunoconjugate, we will first test the ability of
the immunoconjugate to target SCCL tumors in xenografts of
human SCCL in immunodeficient mice. In addition, we will
examine the ability of human effector cells to migrate to SCCL
xenografts and mediate antitumor effects. These studies will
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at
aat
SU
0
E
I-.
H69 cells
100�
80�
60�
40�
20�
0�
100
a Donorl
-0--- Donor 2
#{163} Donor 3
- a
50:1 100:112:1 25:1
SHP-77 cells
at
a 80a
.?‘ 60
atU 40
I 20
1 2:1 25:1 50:1 100:1
Clinical Cancer Research 433
S Donor 1 (4h assay)
-0--- Donor 2
#{163} Donor 3-��--�y� -----Donor 4
-c:t---- Donor 1 (18h assay)
Effector to target cell ratio
Fig. 8 Cytotoxicity of StCr�labeled SCCL cells by human neutrophils in the presence of immunoconjugate Lys3-BN X 22. Assays were performed
at E:T ratios ranging from 12:1 and 100:1. Neutrophils were separated from different donors in each individual assay. �, 4-h assay.
allow us to determine the stability of the immunoconjugate in
vivo, its ability to bind to SCCL in vivo and to direct leukocyte
effector cells, and to assess its possible therapeutic effects.
Depending on the results of these studies, we would begin Phase
I studies to determine the maximally tolerated dose and the
optimal biological dose in vivo in patients with SCCL. Finally,
Phase II studies to determine the efficacy of the immunoconju-
gate would be performed in patients with minimal disease
following chemotherapy. The need for cytokine administration
and adoptively transferred in vitro activated effector cells would
be estimated from the Phase I studies. Ultimately, as in many
areas of new agents for the treatment of cancer, randomized
Phase III studies will most likely be needed to prove efficacy.
ACKNOWLEDGMENTS
We thank Karol Rosengarth for her assistance in the preparation of
this manuscript.
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1995;1:425-434. Clin Cancer Res J Chen, M Mokotoff, JH Zhou, et al. small cell carcinoma of the lung cells(CD64)-dependent monocyte- and neutrophil-mediated lysis ofantibody 22 can specifically induce FcgammaRI An immunoconjugate of Lys3-bombesin and monoclonal
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