an immunoconjugate of lys3-bombesin and monoclonal ... · stab22-lys-bn iaaunocon)ugate fig. i...

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.

Research. on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

1 of 11 Celltrion, Inc., Exhibit 1016

http://clincancerres.aacrjournals.org/

0

Lys3-BN

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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

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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




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,




DMS273 cells DMS273 cells300

200

100

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H345 cells H345 cells300

I.’.’. 0�200CC. 1001 0 20 3 0 40 � 5’O D � 10 � 20 � � � 5�0

H69 cells H69 cells

100

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SHP-77 cells SHP-77 cells

300

200

100

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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-




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

� 100

0

050

0

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-0-- Experiment 2


a’C

�0C

.0

0

Sa’S

CSU1�S

a.

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




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

H345 cells

0

0

SU

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0

0

SU

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E

I-

12:1 25:1 50:1 100:1

SHP-77 cells

80

60

40

100 100

#{149}80

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:; -0--- Donor2U 4� S Donorl #{163} Donor3

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0 #{149} . i I 0 � #{149} �

12:1 25:1 50:1 100:1 12:1 25:1 50:1 100:1

Effector to target cell ratIo

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.


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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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-




H345 cellsDMS273 cells

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12:1 25:1 50:1 100:1

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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.


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




at

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at

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S Donor 1 (4h assay)

-0--- Donor 2

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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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Research. on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from 10 of 11 Celltrion, Inc., Exhibit 1016


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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