HYBRIDOMA

Volume 9, Number 3, 1990 Mary Ann Liebert, Inc., Publishers

Establishment of Human Monoclonal Antibody Recognizing a New Tumor-Associated Antigen from a Patient with Small Cell Lung Carcinoma TOSHIHIKO IIZASA,1 YUTAKA YAMAGUCHI,1 MASATOSHI TAGAWA,2 HIROAKI SAITO,1 TAKEHIKO FUJISAWA,1 KANEFUSA KATO,3 and MASARU TANIGUCHI2 'Department of Surgery, Institute of Pulmonary Cancer Research, School of Medicine, Chiba University, Chiba 280, Japan 2Division of Molecular Immunology, Center for Immunology and Neurobiology, School of Medicine, Chiba University, Chiba 280, Japan ^Department of Biochemistry, Institute for Developmental Research, Aichi Prefectura! Colony, Kasugai, Aichi 480-03, Japan

ABSTRACT Mouse-human heterohybridoma (3H12) producing human antibody was established by fusing P3/X63-Ag-U1 (P3U1) myeloma cells with lymphocytes from a patient of small cell lung carcinoma (SCLC). This monoclonal antibody reacts to lung cancer cells, especially SCLC, but not to adenocarcinoma or squamous cell carcinoma cells. It does not show any

reactivities to other tumors or normal cells so far examined. An immunoprecipitation experiment with this antibody revealed that the antigen on SCLC was a single chain moiety of 150 kilodaltons (Kd). Judging from the cell type reactivity and molecular size of the antigen, this monoclonal antibody appears to detect a new tumor-associated antigen on human SCLC.

INTRODUCTION SCLC possess unique properties compared with other lung tumors in that they are sensitive to radiotherapy and chemotherapy, although, because of extensive early-stage metastasis, their prognosis is poor. The existence of neuroendocrine granules suggests that they originate from the neural crest and in fact are sometimes associated with a certain syndrome with ectopic hormone production. The finding that they produce peptides such as a gastrin releasing peptide (1) contributed to the establishment of a monitoring system for therapy. With the advent of hybridoma techniques we have been focusing on the establishment of human monoclonal antibody because of several possible advantages for diagnosis and therapeutic trials. Human antibody will also rule out the chance that the antigen recognized by the human immune system might differ from that of the mouse as encountered in the case of mouse hybridomas. We have recently found that stable mouse-human heterohybridoma could be obtained by preferential selection of X chain producers (2). Here we described the human monoclonal antibody (3H12) recognizing a 150 Kd molecule on SCLC, which seems to be classifiable as a new tumor-associated

antigen preferentially expressed

on

SCLC. 211

MATERIALS AND METHODS Cells

Rapidly growing P3/X63-Ag8-U1 (P3U1) was selected for cell fusion by the limiting dilution technique. Neuroblastoma cell lines (Goto, NB-1 and TN-1) were provided by Dr. Kuriyama, Chiba University and lung carcinoma cell lines (HKT 3, HKT6, HKT 7 and HKT 8) were donated by Drs. Baba and Yamakawa, Chiba University (3). The rest of the cell lines tested for reactivity to monoclonal hybridomas were kindly supplied by the Japanese Cancer Research Resource Bank. They were maintained with either RPMI-1640 (GIBCO Laboratories, Chagrin Falls, Ohio), Dulbecco's modified Eagle's medium (Nissui Seiyaku, Tokyo, Japan), or alpha-MEM (GIBCO Laboratories) supplemented with 10% fetal calf serum (FCS) (Boeringer Mannheim, Pentzberg, FRG), 10 mM HEPES buffer, 100 units/ml penicillin and 100 i~g/ml streptomycin. Peripheral blood and bone marrow mononuclear cells were obtained by venipuncture and bone marrow aspiration, respectively. They were collected after FicolPaque selection (Pharmacia Biotechnology, Uppsala, Sweden) and used for cell surface staining. Source of

Lymphocytes

Mediastinal lymphnodes of patients with SCLC were surgically obtained. A singlecell suspension of lymphocytes was prepared by passing the lymphnodes through a sterile mesh (#200), which were used then for cell fusion. Production of Hybridomas

Cell fusions were performed as described (4, 5). In brief, human lymphocytes from patients with SCLC (1-2 x108) were hybridized with murine myeloma cells P3U1 (4 x107) in the presence of 1 ml of 50% (w/v) polyethylene glycol (molecular weight 1,500; BDH Chemicals Ltd., Poole, UK) in RPMI-1640 at 37°C. After removing polyethylene glycol, the cells were resuspended in RPMI-1640/10% FCS and seeded into four 96-well plastic microplates (Coster, Cambridge, MA) at 5 x 105/100|il/well. Hybrid cells were cultured with selection medium (RPMI-1640/10% FCS/10"4M hypoxanthine/4 x 10"7M amlnopterin/1.6 x 10"5M thymidine) for at least 10 days. Proliferating cells

further cultured with RPMI-1640/10% FCS/10"4M hypoxanthine/1.6 x 10"5M thymidine for 5 days and thereafter with RPMI-1640/10% FCS. Supematants from each well with observed growth were tested for screening with enzyme-linked immunosorbent assay (ELISA) (see below). Hybrid cells secreting human monoclonal antibody were then cloned by limiting dilution at 0.3 cell/well with irradiated rat were

thymocytes

as a

feeder

layer.

Cell-Binding Assay for Screening Monoclonal Antibody was used for screening the culture cancer cells (6). Each supernatant (20 i_l) was first reacted to 1 x 105 target cells (previously fixed with 0.25% glutaraldehyde solution) for 1 hr at room temperature, washed with PBS and then reacted to 20 \i\ of alkaline phosphatase-conjugated goat anti-human immunoglobulin. (Kirkegaard & Perry Laboratories, Gaithersburg, ML) (7). A substrate solution (1 OOp.!) containing 15 mM pnitrophenyl phosphate (Sigma Chemical Co., ST. Louis, MO), 1 m diethnolamine, 0.5 mM

cell-binding assay antibodies reactive to lung A

with the ELISA

212

technique

MgCl2 and 2.5 mM levamisole (Sigma Chemical Co.) was further incubated for 30-120 mins at room temperature after washing with PBS. Antibody-binding activity was determined as the absorbance at 405 nm (NJ-2000: Nippon Intermed, Tokyo, Japan). Fluorescence-activated cell sorter (FACS)

analysis

Target cells (1-5 x105) were incubated with 5 \ig of purified antibody for 1 hr at 4 °C in staining buffer (Dulbecco's phosphate buffered saline [PBS]/10% FCS/0.1% NaN3). They were washed three times with the staining buffer and then reacted with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgM. These cells were washed five times and further reacted with propidium iodide (Sigma Chemical Co.) in order to gate out dead cells. They were then analyzed with FACStar (Becton-Dickinson Lab., Sunnyvale, CA) with a logarithmic amplifier. Immunoprecipitations Cells (1

x

107)

were

labeled

metabolically with [35S]-cysteine (37 MBq, ICN

Radiochemicals, Irvine, CA) in 5 ml of cysteine-free RPMI-1640/10% FCS for 8 hrs (8).

Radiolabeled cells were solubllized in 1% Nonidet P-40 (Sigma Chemicals Co.)/PBS at 4°C for 1 hr. The labeled materials were preabsorbed with anti-human IgM coupledProtein A Sepharose (Pharmacia Biotechnology) to remove nonspecific-bound substances before immunoprecipitation. Radiolabeled cell extracts were incubated with either 150 ng of 3H12 or with control human IgM (Cooper Biomédical INC., Malvern, PA) for 2 hrs at 4 °C. They were further reacted with goat anti-human IgM conjugated Protein A Sepharose for 2 hrs at 4° C. After extensive washing with 1% Nonidet P-40/PBS, the précipitants were eluted with Laemmli's sample buffer (1.25% sodium dodecyl sulfate) (9) and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 5% acrylamlde) under reducing and nonreducing conditions (10). The gels were dried and autoradiographied with Kodak XAR 5 film. -

Cell

Type Analysis

of SCLC with

Enzymes

Cytoplasmic enzymes found in SCLC were assayed in order to classify the SCLC cell lines. Cells were suspended in 50 mM Tris-HCI pH 7.5/5 mM MgCl2 and disrupted by sonication. After removing cell debris by centrifugation, the supematants were assayed

for enzyme doses per

protein by enzyme-immunoassay

as

described

(11, 12).

RESULTS Establishment of Heterohvbridomas SCLC

Producing Human Monoclonal Antibody Reacting

Lymphocytes from 5 cases of SCLC were fused with P3U1 individually. Hybrid cells were observed to grow in 89% of the wells, and their culture supematants were tested for binding to the SCLC cell line PC 6. Hybrid cells in positive wells were further examined on PC 6 cells and human fibroblasts by cell-binding assay to select SCLC specific antibody. They were then cloned three times by limiting dilution and finally 3 clones were established, two being IgM k and one IgM X. Since X chain producer is reported to be stable (2) and the present X chain producer reacted to PC6 better than the two k chain producers, we focused on this particular X chain producer 3H12. We examined the reactivity of 3H12 to various types of lung carcinoma with cell surface staining. The results in Table 1 show that this antibody reacts preferentially to 213

Binding Activity

TABLE 1.

Small cell

Cell

lines

lung

cancer

HKT 6 cell carcinoma of PC 13

Antibody To Malignant and Nonmalignant Cells Reactivity

to

3H12

Antibody*

(SCLC)

PC 6 SBC 1 SBC 2 SBC 3 SBC 5 Lu 134 LC-FM LC-MA Squamous cell carcinoma of PC 10 HKT 8 Adenocarcinoma of lung PC 14 HKT 3

Large

of 3H12

+ +

+ + + +

lung

lung +

HKT 7

Neuroblastoma GOTO NB-1 TN-1 Melanoma

SK-Mel-28

Esophagus

tumor

TE-2 T cell leukemia MOLT 4 Burkitt lymphoma

Raji

+

Colon carcinoma COLO 320 COLO 201 WIDr Gastric carcinoma KATO III NUGC-3

Hepatoma Breast

HLE HLF

cancer

YM_>1 '

n„_,¡_-

Ovarian

_

-„-„_, cancer

TYK-nu Liver: Chang liver Flbroblast (3 cases)

Hematopoletlc cells Peripheral

Blood Red Blood Cells Bone Marrow

*

Lymphocytes

was determined by cell surface staining. The mean fluorescence intensity 10 times the control fluorescence is shown as ++ and less than 10 times as +.

Reactivity

over

SCLC cell lines. Four out of eight SCLC cell lines were 3H12 antigen positive. The staining profile of PC 6 by FACStar showed a monophasic pattern (Figure 1), suggesting that the antigen expression is not related to cell cycles. Among the 4 lines, PC 6 and SBC2 were stained brightly, with the mean fluorescence being about 10 fold that of control staining. However, the squamous cell carcinomas and adenocarcinomas tested

214

CD __1

E 0)

o

CD >

_5 CD

CE

10

IO2

10'

Fluorescence

IO3 intensity

FIGURE 1: Cell Surface Staining Profile of PC 6 Cells. Cells were stained with either 3H12 (-) or isotype matched control antibody (--) followed by FITCconjugated goat anti-human IgM.

were

3H12

antigen negative. We examined large cell carcinomas and found that PC 13

Since SCLC is considered to be derived from the neural crest, reactivity to several neuroblastoma cell lines, but none of them expressed the 3H12 antigen on the cell surface. We also extended the screening range to other nonpulmonary tumors and normal cells, and found that none of them (except a cell line of Burkitt lymphoma) showed the reactivity. This indicates, therefore, that this antibody shows preferential reactivity to SCLC. cells

were weakly positive. we examined the

Biochemical

Analysis of

3H12

Antigen

An immunoprecipitation experiment was carried out in order to assess the biochemical properties of the 3H12 antigen. The metabolically labeled PC 6 cells showed that 3H12 antibody precipitated the 150 Kd molecule specifically under both reducing and nonreducing conditions (Figure 2). This suggests that the 3H12 antigen is a single molecule, although we cannot formally rule out the possibility that it is composed of the heterodimer-150 Kd and a small molecule.

FIGURE 2: Autoradiogram of Immunoprecipitation. 35s-cycteine labeled PC 6 cell lysate was immunoprecipitated with either 3H12 (lanes A and C) or isotype matched control antibody (lanes B and D). The précipitants were analyzed by 5% SDS-PAGE under nonreducing (lanes A and B) and reducing (lanes C and D) conditions.

200— 150—

92.5— B

215

Reactivity of 3H12 Antibody

on

Various Cell

Types

of SCLC

investigate 3H12 antibody reactivity against various types of assayed two kinds of enzymes, neuron specific enolase (NSE) and creatine kinase brain ß subunit (CK-BB), which are known to be expressed in SCLC according to their differentiation (13). The results in Table 2 show that, based on the amount of enzymes, there seems to be no strong correlation between the antibody reactivity and the cell types. We also classified the cell lines according to the growth patterns described by Gazdar et al. (typel cell growth in tightly packed floating spheroids; type cell growth in amorphous floating aggregates; type 3 2 large floating cells growing in entwined cords or loose aggregates; type 4 adherent cells lacking the epithelioid features of non-SCLC lines) (14, 15) and found that there was no direct relationship In order to further

SCLC,

we

-

-

-

-

between them.

TABLE 2. Characteristics of SCLC and

Cell line

reactivity of

NSE

Reactivity of 3H12 Antibody CK-BB

Growth

_3H12_(ng/mg)*_(ng/mg)*_pattern" 1711 5422 1 LC-FM + 479 3408 2 SBC1 206 2412 ++ 4 PC 6 SBC 2 ++ 365 2010 3 63 1331 4 SBC3 Lu 134 614 1317 2 LC-MA 151 59 4 SBC 5_+_1_24_5_4;_ -

-

-

*

Dose of enzyme is determined per cellular protein. Morphological classification was determined according to Gazdar et al

(14).

DISCUSSION We have reported here the establishment of human monoclonal antibody against SCLC. More than 100 monoclonal antibodies have been reported (13, 16-23), but as far as we know all of them are of rodent origin, and so this antibody would be the first human antibody for SCLC. Since human antibody would potentially have considerable advantages over rodent antibody, many trials were carried out to establish them efficiently. Three main procedures were employed: human lymphocytes fused with rodent myeloma cells, human-human hybridization, and transformation with EB virus. We selected X chain producing human-mouse heterohybridomas because of their stability (2). In fact, 3H12 hybridoma cells kept in our laboratory have been continuously secreting about 5 ug/ml antibody for the last 2 years. At a recent workshop concerning SCLC antigens, monoclonal antibodies against them were classified according to their reactivity to cell lines and tissues (24). The reactivity of 3H12 antibody to various cell lines is close to those classified as cluster 1 (the cluster 1 antigens are preferentially detected on the cells including SCLC and neuroblastomas). However, 3H12 antibody is distinguished from those of cluster 1 in that this antibody did not show any reactivities to neuroblastoma cells (Table 1). Therefore, this antibody appears to belong to a new cluster. The antigen recognized by this antibody is a putative single chain molecule with a molecular weight of 150 Kd. Hirohashi et al. and Ueda et al. reported that NCC-LU-246 and NE150 antibodies recognized 145 Kd and 150 Kd molecules, respectively (18, 13). However, the molecule recognized by 3H12 antibody seems to be different from those identified by the above antibodies in the following three points: 1) 3H12 antibody does not react to

216

neuroblastoma cells but their antibodies do react, 2) 3H12 antibody binds to PC13 large cell carcinoma, whereas NCC-LU-246 does not (25), 3) 3H12 antibody shows the reactivity to Raji cells, Burkitt lymphoma, which are not recognized by their antibodies (22), 4) the molecules on SCLC recognized 3H12 antibody seems to be scarcely glycosylated because of the distinct band appeared in SDS-PAGE, however, NCC-LU-246 precipitated very broad bands from SCLC (25). Thus, the 3H12 antigen seems to be different from those recognized by the above two antibodies. SCLC cells often produce NSE and CK-BB which are detected in the neuroendocrine system. Ueda et al. have proposed that the differentiation stages of SCLC cells could be possibly determined as neuroendocrine or epithelial type by the amount of these enzymes. They suggested that the amounts of these biomaker enzymes are increased in the neuroendocrine type and decreased in the epithelial type (13). However, the antigen expression of 3H12 does not appear to be correlated with these enzyme doses, and it is suggested that the 3H12 antigen was not the marker for neuroendocrine pathway, as it was not detected on characteristic neuroendocrine type cells such as LC-FM with large amounts of NSE and CK-BB and neuroblastoma cell lines. Taken together, we are led to believe that the 3H12 antigen could be a new marker with considerable potential. We are now in the process of further characterization of the properties of this antigen and are trying to analyze the differentiation stages of SCLC. The applications of this antibody for clinical diagnosis and therapeutic trials are now being considered.

ACKNOWLEGEMENTS This work was supported by Grants-in-Aid for Scientific Research 62440053, Grants-in-Aid for Cancer Research and for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan, the Princess Takamatsu Cancer

Foundation and the Uehara Memorial Foundation, Japan. We appreciate Dr. Ojika, Nagoya University, for measuring the amount of enzymes. We also thank Ms. Memoto and Ms. Saito for technical and secretarial assistance.

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Quantitative distribution of cluster 1 small cell lung cancer antigen in cancerous and non-cancerous tissues, cultured cells and sera. Jpn. J. Cancer Res., 80: 348355. Address reprint requests to : Dr. Masaru Taniguchi Division of Molecular Immunology Center for Neurobiology & Immunology School of Medicine, Chiba University 1-8-1 Inohana, Chiba 280

Japan Received for

Accepted

publication November 20, 1989 after revisions March 8, 1990

219

Establishment of human monoclonal antibody recognizing a new tumor-associated antigen from a patient with small cell lung carcinoma.

Mouse-human heterohybridoma (3H12) producing human antibody was established by fusing P3/X63-Ag-U1 (P3U1) myeloma cells with lymphocytes from a patien...
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