HYBRIDOMA Volume 11, Number 2, 1992 Mary Ann Liebert, Inc., Publishers

Characterization of a Human-Human Hybridoma Antibody, C-OUl, Directed Against a Colon Tumor-Associated Antigen KARIN

BORUP-CHRISTENSEN,2 HENRIK DITZEL,1 JOHN CHEMNITZ,1 HELMUTH HAAS,3 and

ERB,1

PER

JENS CHRISTIAN JENSENIUS4

'Department of Medical Microbiology, University of Odense, DK-5000, Denmark 2Department of Surgery, University Hospital, Odense, DK-5000, Denmark ~'Forschungs-institut Borste!, D-2061, Germany "Institute for Medical Microbiology, University of Aarhus, DK-8000, Denmark

ABSTRACT The human hybridoma cell line, B9165, was obtained after fusion of lymphocytes from lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon with the human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2). B9165 secretes the human monoclonal antibody, C-OUl (IgM, kappa). Immunocytochemical and immunohistochemical analysis showed that the antibody bound to a differentiation antigen. Electron microscopy of colonie adenocarcinoma cells, intact tumor and colonie epithelium by the immunogold technique demonstrated that the C-OUl antibody reacted with a molecule associated with areas of disruption of the intermediate filaments in the cytoplasm of the tumor cells. No reaction was seen with interme¬ diate filaments in normal colonie epithelium. The molecular weight of the antigen was shown to be 43 Kda by SDS-PAGE and Western blotting of tumor extracts, and isoelectric focusing of sonicated extracts demonstrated reaction with molecular species of pi 5.4-6.2. These findings suggest that the C-OUl antigen is a modified cytokeratin 18. The B9165 cell line has proved to be quite stable, and the antibody is of potential clinical value. Its usefulness for localizing tumors in patients is

being investigated.

INTRODUCTION

is one of the most frequent malignant diseases and one of the of cancer deaths. The prognosis has not improved in recent decades methods of detection and adjuvant therapy for this type of cancer are

Colorectal

leading

cancer

causes

and new needed. Monoclonal antibodies might play an important role in this respect. So far most monoclonal antibodies raised against cancer-associated antigens have been of murine origin (1). The antigens recognized by these reagents have, therefore, generally been determined by their immunogenicity in the mouse, and they do not necessarily correspond to antigens that are important in man. Murine monoclonal antibodies have been of limited value for human therapy, because they are recognized as foreign proteins and are often neutralized by the resulting immune response (2,3). Human hybridoma technology may provide less immunogenic and more relevant

diagnostic technology in

cancer

therapeutic agents for administration to patients with cancer. The provides possibilities for investigating the humoral immune response patients. and also

121

We have isolated lymphocytes from regional draining lymph nodes of a patient with adenocarcinoma of the colon and fused them with a human B-lymphoblastoid cell line, WI-L2-729-HF2 (4). In the present study we describe and characterize the human monoclonal antibody C-OUl produced by the hybridoma cell line B9165.

MATERIAL AND METHODS

Fusion Cell Line The human B-lymphoblastoid cell line WI-L2-729-HF2 (729-HF2) was obtained from Techniclone International, California, USA (4). Although the cell line is charac¬ terized as a non-secretor, we found a production of about 40 ng/ml of IgM (kappa

light chain)

at

overgrowth.

fusion, the cell line was grown in RPMI-1640 medium (GIBCO, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (FBS) (GIBCO), penicillin 100 U/ml, streptomycin 100 |ig/ml, and 0.1 mM 6-thioguanine (Sigma). Before

Human Tissues

Lymphocytes were obtained from mesenteric lymph nodes draining the tumor region in a patient with adenocarcinoma of the colon. The lymph nodes were minced under sterile conditions and debris was removed by filtration through cotton wool. The lymphocytes were then used for fusion with 729-HF2 without in vitro stimulation. Fusion Procedure Fusion was performed as described by Köhler (5). The ratio between 729-HF2 and lymphocytes (2.3x10 ) was 1:2, and samples of 2xl05 cells in 200 µ were plated in 96-well microplates (Costar, Cambridge, MA, USA). Feeder cells were not required for maintenance. The cells

weeks in HAT medium (RPMI-1640 with 10% FBS, M thymidine). M aminopterin and 3.2x10 4x10 Further in the same medium without aminopterin. Growing was performed hybrids appeared between 10 days and 4 weeks after fusion. Cloning by limiting dilution was carried out without feeder cells.

maintained for

were

containing

2x10 culture

M

two

hypoxanthine,

Cancer Cell Lines

The colonie adenocarcinoma cell line, Colon 137, was established by Dr. Peter Ebbesen, The Cancer Research Inst., Aarhus, Denmark. The malignant melanoma cell line, HU373 was established by Dr. Ingolf Nielsen, The Finsen Inst., Copenhagen,

The following cell lines were obtained from the American Type Culture Collection, Rockville, MA, USA (ATCC): the colonie adenocarcinoma cell line, COLO 201, the hepatocellular carcinoma cell line, HepG2, the mammary carcinoma cell line, MCF7, the duodenal carcinoma cell line, HuTu80, and Burkitt's lymphoma, EB2. Denmark.

Control Antibodies The human-human hybridoma antibodies, F11348 and G4146, both of the IgM isotype, have been described (6). Human myeloma IgM and human polyclonal IgM were purchased from Cappel Laboratories, Cochranville, P.A. Murine monoclonal anti-human cytokeratin 18 (CK2) was purchased from Boehringer

Mannheim and normal

chromatography.

polyclonal

mouse

IgG

was

122

purified

from

mouse serum

by Protein-A

Enzyme-linked Immunosorbent Assays

Enzyme-linked immunosorbent assay (ELISA) was performed for both the quantitat¬ ive and qualitative analysis of the C-OUl antibody. The immunoglobulin concentration determined by solid-phase ELISA as previously described (6). For the was identification of the light-chain class of C-OUl immunoglobulin, the antibody was added to an immunoplate (Nunc A/S, Kamstrup, Denmark) precoated with Fab'-rabbit anti-human kappa or Fab'-rabbit anti-human lambda at a concentration of 1 pg/ml. The Fab'-fragments of the antibodies (from Dakopatts) were obtained by pepsin digestion and reduction (7), followed by chromatography on Sephacryl S-300 (Pharmacia). The ELISA was terminated after successive incubation with secondary alkaline phosphatase (AP)-labeled rabbit antibody to human µ chain and with chromogenic substrate as described (6). A modified ELISA was used to determine antibody specificity. The method will be published in detail elsewhere (8). Briefly, immunoplates were coated with ultrasonic lysates of the various cancer cell lines. The hybridoma supernatants and control antibodies were added. Development was performed by adding enzyme-labeled secondary antibody

and

chromogenic

substrate.

Immunohistochemical Analyses

antibody was concentrated by precipitation with 2M (NH^)2S04 and applied sections of various cancers and normal tissues. Frozen blocks of cancerous and normal tissues were cut into 5 µ sections and air dried overnight at room temperature. After fixation in acetone for 10 min and washing in PBS-Tween, the sections were preincubated with Fab'-rabbit anti-human IgM for 4 hours at 4°C in a humidified chamber to block endogenous immunoglobulin (7). The sections were then washed for 10 min in 0.05% Tween 20 in phosphate-buffered saline (PBS-Tween), and incubated overnight at 4°C with C-OUl antibody (0.5pg IgM/ml PBS-Tween), followed by incubation for 30 min at 4°C with rabbit anti-human IgM labeled with horse-radish peroxidase (HRP, Boehringer-Mannheim, FRG) as the second antibody. This was prepared from the rabbit antibodies also used for the preparation of Fab' fragments (above), C-OUl

to frozen

the enzyme being coupled to the antibody by the two-step glutaraldehyde method (9). After additional washing, the bound enzyme was visualized by development with chromogenic substrate (0.01% H2O2 in a solution of diaminobenzidine at 0.6 ^g/ml PBS (DAB)). The slides were washed, counterstained with hematoxylin, and mounted in

(Histo Lab., Bethlehem Trading Ltd., Goteborg, Sweden). Previous results have shown that all human IgM preparations (including myeloma IgM) bind to frozen, acetone-fixed colon epithelium independently of antigen. This antigen-independent binding of IgM was not seen on formalin-fixed epithelium (10). Because of this, C-OUl was also analyzed on sections of tissue fixed with 4% for¬ maldehyde in 0.075 M sodium phosphate, pH 7.0, for 24 hours and embedded in paraffin. The sections were deparaffinized in xylol, rehydrated through graded alcohols, and stained as described above. Pertex

Immunocytochemical

Analysis

Cell smears were prepared from human tumor cell lines and peripheral human blood and fixed in buffered formol-acetone (60 ml acetone, 33 ml 40% formalin, 38 ml H20, 8 ml 1.5 M phosphate buffer, pH 7.4). Approximately 50 µ of C-OUl antibody (1 pg/ml) was placed on the smears and incubated at 4°C in a humidified chamber overnight. The smears were then rinsed and incubated at room temperature for 1 hour with HRP-rabbit anti-IgM (the same second antibody as used for the immunohistochemical staining) diluted 1/80 in PBS-Tween. They were washed and developed with DAB, counterstained and mounted as described above. Human myeloma IgM (Cappel) was used as control in a concentration at 10 /Jg/ml. During the course of these investigations, Cappel switched from supplying IgM purified from myeloma serum to IgM purified from normal human serum. No difference in reaction or lack of

leucocytes,

-

reaction

was -

observed between these

preparations.

123

SDS-PAGE and Western

Blotting

Cultured cancer cells (10' cells/ml) resuspended in PBS containing enzyme inhibitors (1 mM iodoacetamide, 2 mM tranexamic acid (Cyclocapron, KABI), 1 mM ethylenediaminetetraacetic acid (EDTA), 0. 5 mM phenylmethylsulfonyl-fluoride (PMSF) and 4 U/ml aprotinin (Trasylol, Boehringer Mannheim)). The cell suspension was mixed with an equal volume of sample buffer (2% sodium dodecyl-sulphate (SDS), 4 M urea, 10 mM iodoacetamide) and incubated at room temperature for 15 min before PAGE

(polyacrylamide gel electrophoresis) on 5 to 20% gradient gels. The load was 5x10 cells/cm gel. The protein bands were transferred onto nitrocellulose sheets as described (11), ethanol being substituted for methanol in the transfer buffer. Experiments proved ethanol to be as efficient as methanol in promoting the blotting process.

A section of the blot was stained for proteins with colloidal gold (12). The remainder of the nitrocellulose sheet was cut into 3 mm strips and placed in disposable 8-channel polypropylene trays (Octa-trays, PATEOF, Tranegaardsvej 4, Hellerup, Denmark), and immunostained as previously described (13). In brief; after quenching in TRIS-buffered saline (TBS) containing 0,1% Tween-20, the blots were incubated with C-OUl antibody, followed by secondary alkaline-phosphatase labeled rabbit anti-human µ chain antibody and chromogenic substrate.

Subcellular Localization and Distribution of the

1:

Labeling

Antigen

of live colon adenocarcinoma cells

Approximately 2x10 COLO 201 cells were resuspended in 200 µ B9165 supernatant containing about 2 pg/ml C-OUl antibody and incubated at 4°C for 2 hours. Human myeloma IgM at 10 pg/ml in RPMI-1640 containing 10% FBS was used as control. Human monoclonal antibody with known reactivity to cell surface antigens was not available, and a mouse monoclonal antibody, NS 19-9 (14), with known cell surface reactivity, was used as positive control. After incubation the cells were washed

with PBS and further incubated at 4°C for 1 hour in 200 µ HRP-rabbit anti-human IgM (Dakopatts) diluted 1/80 in PBS containing 0.1% BSA (for C-OUl and human myeloma IgM), or HRP-rabbit anti-mouse Ig (Dakopatts) diluted 1/50 in the same medium (for NS 19-9). After washing in PBS, cell smears were prepared and fixed in 0.17% glutaraldehyde in PBS for 15 min. The slides were washed in PBS, developed in DAB, counterstained with hematoxylin and mounted. 2: Immune electron

microscopy

of cells labeled in

suspension

Incubation of cells with C-OUl, human myeloma IgM and NS 19-9 was carried out above. The cells were then incubated with rabbit anti-human IgM (for C-OUl and human myeloma IgM), or rabbit anti-mouse Ig (for NS 19-9) followed by goat antirabbit IgG-coated gold probes of 15 nm (Jansen Inc., Brussels, Belgium). The labeled cells were fixed with 0.1% (w/v) glutaraldehyde in 0.1 M PBS, pH 7.2, for 30-60 min at 4°C and washed in PBS supplemented with 0.1% BSA and lysineHC1 overnight at 4°C. The cells were dehydrated with ethanol at -20°C, impregnated with Lowicryl K4M at -35°C and polymerized overnight under ultraviolet light at 35°C followed by 2 days at room temperature (15). Ultrathin sections were examined in a JEOL 100-CX electron microscope operating at 80 kV. as

-

3: Immune electron

Lowicryl

K4M

microscopy

embedding

was

of labeled fixed cells and tissues

carried

out as above

on

colonie adenocarcinoma cells

(COLO 201), duodenal adenocarcinoma cells (HuTu80), fragments of biopsy from human colonie adenocarcinoma implanted in a nude mouse, fresh adenocarcinoma of the colon,

adjacent normal epithelium. Ultrathin sections were cut and mounted on nickel-coated grids. The grids were placed on drops of 1% (w/v) NaBH4 in PBS for 10 min, washed in 0.1% BSA in Tris buffer for 2x5 min and 1x15 min. The grids were incubated with C-OUl supernatant or control human IgM for 1 hour at room temperature, washed twice with 0.1% BSA-Tris and the

124

for 10 min. and incubated with rabbit anti-human IgM diluted 1/100 in 3% BSA-Tris for another hour at room temperature. After washing in 0.1% BSA-Tris, the grids were incubated with goat anti-rabbit IgG-coated gold probes diluted 1/100 in 3% BSA-Tris for 30 min at room temperature. The grids were washed twice with 0.1% BSA-Tris followed by redistilled water and dried. The sections were then stained with 1% uranyl acetate for 10 min and 0.4% lead citrate for 2 min at room temperature and examined in an electron microscope. Molecular Characterization of the

Antigen

bv Isoelectric

Focusing

Extract of cells and tissues (Colon 137 cells, human colonie adenocarcinoma tissue and normal colonie epithelium) was prepared according to Dr. J.v.d. Bosch, Research Institute Borstel (unpublished procedure). Packed Colon 137 cells (10' cells) or 0.2 g minced tissue was suspended in 0.3 ml of extraction buffer (75 mM NaCl, 75 mM KC1, 10 mM HEPES, 5 mM EDTA, 5% 2-mercapto-ethanol, 5 ^g/ml aprotinin, 0.1 µ Leupeptin and 10 µ Pepstatin) and ultrasonicated for 4x30 seconds on ice. 0.5 g/ml urea and 80 mg/ml sucrose were then added. After incubation for 30 min at 37°C, insoluble material was removed by centrifugation (5 min at 15000 g). Isoelectric focusing was performed in a 1% thin-layer agarose gel (Agarose IEF, Pharmacia, Sweden) containing 6 M urea, 3% (w/v) Servalytes 3-10 and 1% Servalytes 4-6 (Serva). Gelbond film (LKB) was used as support. The amount of protein applied was 10-20 µg/cm as assessed by the Pierce Protein Assay. Current was limited to 15 mA, voltage to 2000 V, and power to 30 W. The temperature of the coolant was set to 9°C. The duration of a run was about 120 min. Proteins were detected by staining with Coomassie Brilliant Blue. The pH of the gel was determined by means of a surface electrode. Immunoblotting was performed as described elsewhere (16). In brief, after transfer of the separated extracts to nitrocellulose and blocking with buffer containing 0.05 % Tween-20, the paper was incubated with human monoclonal antibody (diluted to about 200 ng/ml). After washing with PBS-Tween, the blots were incubated either with AP-rabbit anti-human IgM (Dakopatts) or, to increase the sensitivity, with unlabeled rabbit anti-human IgM followed by AP-goat anti-rabbit IgG (Jackson Immunoresearch). This was followed by development in substrate-chromogen mixture as described for Western Blotting. Proteins bound to nitrocellulose were stained nonspecifically with Indian ink (17).

RESULTS

The human hybridoma cell line, B9165, was obtained after fusion between regional node lymphocytes and the human B-lymphoblastoid cell line 729-HF2. C-OUl was selected from Ig-positive primary supernatants because of its binding to sections of colonie adenocarcinoma but not to sections of normal tonsillar tissues on immunohistochemical analysis. B9165 has been cloned and recloned 4 times, and has been in culture for about 5 years. The culture supernatants at overgrowth were estimated by ELISA to contain about 2 µg/ml IgM. In the light-chain ELISA the antibody reacted with anti-kappa but not with anti-lambda coated plates.

lymph

C-OUl

Analyzed

Cancer Cell Extracts bv ELISA

on

coated with sonicated cancer cell lines (Table I) showed and the murine monoclonal anti-human cytokeratin 18 antibody, colonie adenocarcinoma cell lines (COLO 201, Colon 137) and the mammary carcinoma cell line (MCF7); a slight reaction was observed with the hepatoma cell line (HepG2) while neither antibody showed binding to the duodenal adenocar¬ ELISA

in wells

reactivity of C-OUl CK2, against the two ,

or the melanoma cell line (HU373). Polyclonal IgM (Cappel) control showed no reaction and F11348 used as positive control with all cell lines tested.

cinoma cell line (HuTu80)

used as reacted

a

negative

strongly

125

TABLE I.

ELISA of C-OUl Reactivity Against Various Sonicated Cancer Cell Lines. Values Are Means Of Optical Densities At 405 run Of Duplicate Wells. All Ig Concentrations Were ^g/ml.

Antibody Cell Lines

Preparations

And Control

C-OUl

CK2

IgM

mlgG

PBS-T

Mab

Colon 137

1.872

2.267

0.194

0.147

0.148

1.455

COLO 201

0.894

1.667

0.198

0.145

0.147

1.992

MCF7

0.927

2.086

0.201

0.148

0.144

2.108

HepG2

0.263

0.695

0.196

0.151

0.144

2.108

HuTu80

0.189

0.140

0.225

0.154

0.149

1.730

HU373

0.185

0.133

0.200

0.154

0.145

2.342

CK2: Murine monoclonal anti-human cytokeratin 18 antibody; IgM: human IgM; mlgG: murine polyclonal IgG; PBS-T: PBS-Tween; Mab: Human-human hybridoma IgM antibody, F11348.

Polyclonal

Immunohistochemical Analyses The reaction

pattern of C-OUl antibody with different tumors and normal tissues of frozen, acetone-fixed tissue sections is shown

analyzed by immunohistochemistry in Tables II and III.

TABLE II.

of Tumor Tested) of C-OUl with Frozen, Acetone-Fixed Tissue Sections of Human Cancer Tissues Analyzed

Reactivity (Number Positive/Number

by Immunohistochemistry.

C-OUl

TISSUES

(0.5 µ

)

Myeloma IgM (10 µ / 1)

Colon adenocarcinoma

21/21

Ovarian adenocarcinoma

2/2

6/21J 1/2J

Renal adenocarcinoma

1/2

0/2

Mammary carcinoma

7/9

0/9

Lung adenocarcinoma

7/7

0/7

0/6

0/6

Non-seminoma testicular carcinoma

1/1

0/1

Sarcoma

0/4

0/4

0/7

0/7

B-Lymphoma

0/3

0/3

Thymoma

0/2

0/2

contrast to the

diffuse

Squamous

cell

Malignant

lung carcinoma

melanoma

1

Myeloma IgM showed only focal reactivity in staining obtained with C-OUl

126

antibody (0.5 µg/ml) reacted with all of the frozen, acetone-fixed colonie specimens examined and stained 82% of the formalin-fixed colonie specimens. Staining of frozen, acetone-fixed sections of other tumors The antibody showed binding to frozen sections of ovarian was also found (Table II) cancer, renal cancer and non-seminoma testicular cancer. Furthermore, binding was shown in 7 highly differentiated breast infiltrating ductal carcinomas, while there was no binding to 2 poorly differentiated breast carcinomas. The antibody also reacted with all lung adenocarcinomas tested, but not with squamous cell carcinoma's of the lung. At the same concentration the C-OUl antibody showed no binding to a variety of normal tissues (Table III). However, binding was observed in normal mammary tissue, hepatocytes and prostatic epithelium. A property common to all IgM antibodies examined is that they bind to normal colonie epithelium with a diffuse staining pattern when analyzed on frozen, acetone-fixed tissues. Because of this antigen-independent binding of IgM, the staining pattern of C0U1 in colonie epithelium was also investigated on formalin-fixed tissues and compared to autologous colonie adenocarcinoma. In 5 cases of 17 positive tumors C-OUl

adenocarcinoma adenocarcinoma

.

TABLE III. of C-OUl with Frozen, Acetone-Fixed Tissue Sections of Human Normal Tissues Analyzed by Immunohistochemistry.

Reactivity

C-OUl TISSUES

Normal colon

(0.5pg/ml)

Myeloma IgM (10 µ / 1)

+/-

+/-

epithelia

Ovarian stroma

epithelia glomeruli

Ovarian Renal

Renal tubules

Mammary tubules Mammary ductuli

Lung

alveolus

Bronchial

epithelia

Testes

Epidermis Tonsillar

lymphatic

Tonsillar

epithelia

Smooth muscles

Pancreatic duct epithelia

Hepatocytes

Biliary

duct

epithelia

Blood vessels

Prostatic

epithelia

Strong reaction: No reaction:

;

+++

; Moderate

Binding

of

reaction:

Ig only

found in

-

127

++

; Weak

some

reaction:

areas:

+/-

+

binding to the corresponding normal colonie epithelia, while in the 12 cases there was some staining, largely confined to the superficial layer of the epithelium. Human myeloma IgM reacted neither with colonie adenocar¬ cinoma nor with normal colonie epithelium after formalin fixation of the tissues. there

was

no

remaining

Immunocytochemical Analyses The reactivity pattern of C-OUl against a range of cancer cell lines and normal human leucocytes was tested by immunocytochemical staining of cell smears. The antibody bound to colonie adenocarcinoma, mammary carcinoma and hepatoma cells. The binding was most pronounced at the periphery of cells which were larger and binding was morphologically different from the negative cells in the smear. No normal human observed in melanoma, duodenal carcinoma, Burkitt's lymphoma or leucocytes. Control human immunoglobulin showed no binding to any of the cells tested.

SDS-PAGE and Western

Blotting

C-OUl bound to a protein of Mr of 43 kDa present in two colonie adenocarcinoma cell lines (Colon 137 and COLO 201), a hepatoma cell line (HepG2) and a mammary adenocarcinoma cell line (MCF7) (Figure 1). This component was not stained in blots of a duodenal carcinoma cell line (HuTu80) or the melanoma cell line (HU373). Incubation of the blots with a murine monoclonal anti-human cytokeratin 18 antibody, CK2, stained a protein of the same molecular weight. This protein was only present in the cell lines to which C-OUl showed binding.

abed

abed

abed

abed

abed

abed

Blotting of Different Cancer Cell Lines Stained with a) C-OUl, b) Cappel IgM, c) CK2 and d) Mouse Ig. The Cancer Cell Lines are A) COLO 201; B) Colon 137; C) HepG2; D) MCF7; E) HuTu80; F; HU373. The Numbers indicate molecular weight 1) 100 kDa and 2) 43 kDa. FIGURE 1. SDS-PAGE

Isoelectric

Focusing

antigen

for C-OUl was visualized by isoelectric focusing followed by of extracts of the colonie adenocarcinoma cell line (Colon 137), colonie adenocarcinoma tissues, and morphologically normal colonie epithelium from cancer patients. C-OUl reacted with the cancer tissues and the cancer cell line, detecting a molecule with a pi ranging from 5.4-6.2. As a control, the human-human The target

immunoblotting

128

8.5

-

123451234512 34 51

2 345

abed FIGURE 2.

Isoelectric

Focusing

of Extracts

from two Different Colonie

Epithelia

(a and b), Colon 137 (c) and Colonie Adenocarcinoma Tissue (d) Transferred to Nitrocellulose Sheets and Stained as Follows: 1) Indian Ink for Total Protein; 4) C-OUl; 5) G4146; 2) and 3) Controls only Incubated with Buffer and

Adjacent

to Cancer

Secondary Antibody. was found to detect molecules with a pi ranging from 5.36.0. In some but not all of the preparations of normal colonie epithelium, C-OUl bound to a molecule with a pi of 5.2. G4146 stained bands of identical mobility on blots of normal colonie epithelium and adenocarcinoma of the colon.

hybridoma antibody, G4146,

Subcellular Localization and Distribution of the

Antigen

Live COLO 201 cells were incubated with C-OUl antibody to test for reaction antigen on the cell surface. A mouse monoclonal antibody (NS 19-9), reactive a cell-surface antigen, was used as control. Although C-OUl stained some of the COLO 201 cells, electron microscopical examination showed that the cell membranes of stained cells were perforated, and the gold probes were localized to molecules in the cytoplasm and not on the cell surface. By contrast, NS 19-9 bound to the cell

with with

surface of all cells analyzed. No reactivity was observed when control human myeloma IgM was used. Immunoelectron microscopy of fixed cells and tissues was performed to localize the elements in the cytoplasm with which C-OUl reacts, and to examine the distribu¬ tion of the antigen in different cancer cells, colon cancer tissues and colonie

epithelium. C-OUl

found to react with COLO 201, human colonie adenocarcinoma from a in a nude mouse, and on adenocarcinoma of the colon from a patient. By contrast, C-OUl did not stain colonie epithelium adjacent to the cancer; nor did it react with the duodenal tumor cells (HuTu80). The antibody was found to bind to sites associated with the intermediate filaments in colonie cancer cells. The gold tumor

was

implanted

129

FIGURE

3.

Electron

Microscopy

of

Glutaraldehyde Fixed, Ultrathin Sections of

Different Cell Lines and Tissues Incubated with C-OUl Antibody, a) COLO 201 cells (x52000); b) COLO 201 Tumor from Nude Mouse (x42000); c) Human Colon Cancer Tissue (x86000); d) the Corresponding Normal Colonie Epithelium (x52000)

probes seemed rupted.

to

be localized to

areas

where the intermediate filaments

were

dis¬

DISCUSSION

colorectal cancerwith colonie cancer have been used as source of antigen-primed lymphocytes (18,19,20,21). We have similarly fused such lymphocytes with a human lymphoblastoid cell line, 729-HF2 (4), and obtained a number of antibody-producing hybridomas. One of these, coded B9165, secretes the human monoclonal antibody C-OUl, which because of its reactivity with adenocarcinoma of the colon and only to a limited extent with normal tissues seemed potentially useful for in vivo diagnostics. Evaluation of human monoclonal anti-cancer antibodies for imaging requires large quantities of antibody. Unfortunately, human cell lines producing monoclonal antibodies usually secrete only small amounts of Ig, and this has seriously hampered In attempts to produce human monoclonal antibodies binding associated antigens, lymphocytes from regional lymph nodes of

130

to

patients

hybridoma cell line which produces C-OUl has been compared with other human hybridoma cell lines secretes relatively large amounts of antibody (1 µg Ig/10° cells/day). Staining of frozen tissue sections by an immunohistochemical technique showed that the C-OUl antigen is found in nearly all adenocarcinomas tested including colonie, ovarian, renal, lung and mammary carcinomas, and the antibody also reacted with a non-seminoma testicular cancer. When analyzed for reactivity to normal tissues and tissue components, at a concentration of 0.5 µg/ml C-OUl showed only slight binding to colonie, mammary and prostatic epithelia. clinical evaluation. The B9165 stable for about 5 years and

both C-OUl and all other human IgM preparations tested As shown earlier (6) (including normal human IgM and myeloma IgM) bind to normal colonie epithelium. Controls omitting IgM showed no binding of the enzyme-conjugated anti-IgM antibody. Kan-Mitchell et al. (22) suggested that such binding of IgM to epithelial colonie tissue is non-specific. We have recently demonstrated that this binding is mediated by the epithelial cell secretory component (SC) which binds both IgA and IgM. We also observed that the binding of IgM to SC does not occur when formalin-fixed ,

tissue is used (10). Formalin-fixed tissue may thus in some cases be superior to cryo-sections with regard to non-specific binding, even though it is known that formalin fixation is destructive to many determinants recognized by monoclonal antibodies (23). On formalin-fixed tissues C-OUl reacted with 17/21 colonie cancers but only with 12/21 adjacent colonie epithelia. The binding in the colonie epithelium was largely localized to the superficial layer of the epithelia. Human myeloma IgM did not bind to formalin-fixed colonie epithelium. Isoelectric focusing of sonicated colonie adenocarcinoma cells and colonie tumor extracts showed that the C-OUl antibody reacted with molecules of pi 5.4-6.2, and the control antibody G4146 reacted with molecules of pi 5.3-6.0. On extracts of normal colonie epithelia G4146 stained molecules of identical pi, while C-OUl either did not react or reacted with a molecule of lower pi than those stained in extracts of cancer. These results are in accordance with the immunohistochemical findings, where C-OUl shows a variable reaction with colonie epithelium adjacent to the cancer and does not react with colonie epithelium from non-cancer patients (24). The binding of C-OUl antibody to live colonie cancer cells appeared, by light microscopy to be localized on the cell surface of some of the cells. It is difficult to make safe conclusions concerning the antigen localization from such studies. Haspel et al. (25) reported that two human IgM monoclonal antibodies (16-88 and 28A32) against antigens associated with colorectal cancer reacted with molecules localized on the cell surface. Later it was found that the 28A32 antibody reacted with a cytoplasmatic, and possibly also a cell surface antigen, while 16-88 only showed binding to a cytoplasmatic molecule (26). Hansmann et al. (27) have concluded that ultra-structural localization of the target antigen detected by human monoclonal antibodies may be important before embarking on their therapeutic

application.

Studies by electron microscopy of preparations of live colon cancer cells incubated with C-OUl revealed that the cells to which the antibody had bound were in fact probably dead cells, since their cell membranes were ruptured. Binding could only be observed in the cytoplasm and not on the cell surface. Further electron microscopical studies showed that C-OUl reacts with a molecule associated with the intermediate filaments in the cytoplasm of colonie cancer cells and intact tumor, while it did not bind to the intermediate filaments in normal colonie epithelium from

a

patient. specificity of

cancer

the monoclonal antibody was further analyzed by incubation with nitrocellulose blots of SDS-PAGE separated tumor cell extract. The staining clearly showed that C-OUl was not merely a broadly cross-reacting IgM antibody, but identified a distinct molecule of Mr about 43kDa. A protein with the same mobility was identified by the anti-cytokeratin antibody (CK2) in the same cell lines. The epitopes recognized by the two antibodies are, however, not the same as shown by their different stability to boiling (13). The molecular weight of the C-OUl antigen (43 kDa) and its localization to intermediate filaments in endoderm-derived epithelial tumors suggest that the antigen may be a cytokeratin 18 like molecule. Other human cell lines established from lymphocytes of tumor-draining lymph nodes have been reported to secrete human monoclonal IgM anti-cytokeratin 18 antibodies (28,29). Those antibodies, in contrast to C-OUl, reacted strongly with both frozen, acetone-fixed and formalin-fixed, The

131

paraffin-embedded normal colonie epithelium. Furthermore, Veale et al. (30) reported that patients with squamous cell carcinoma of the esophagus, in contrast to normal individuals, have circulating antibodies reacting with modified cytokeratin 18. The differential reactivity of C-OUl antibody with normal and malignant tissues might be due to the claimed differences in the amount of cytokeratin 18 in benign and malignant epithelia (31). Alternatively, C-OUl may be directed against a modified cytokeratin 18 molecule. The 16.88 antibody (32), which reacts with an antigen of nearly the same tissue distribution and molecular weight as C-OUl antigen, was claimed to react with a cancer-associated cytokeratin showing less than 65% homology with the corresponding normal cytokeratin. It is obvious to suppose that antibodies reacting with cancer-specific surface antigens are necessary for the imaging or treatment of cancer. It has nevertheless been shown that antibodies reacting only with cytoplasmatic antigens can localize tumors in vivo (33,34). In this respect it should be mentioned that it now appears that all cells present on their surface peptide fragments of endogenous proteins in association with class I MHC molecules. On the basis of its observed reactivity with what is probably a cancerassociated differentiation antigen, C-OUl appears to be of potential clinical value.

ACKNOWLEDGMENTS The authors thank Mrs. Karin Kejling and Mrs. Anette Kliem for technical assistance and Professor Otto Uttenthal for the critical reading of the manuscript. The work was supported by grants from the Danish Cancer Society and Carla Cornelia Storch Mullers Foundation.

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of

Address

colorectal

reprint requests

to:

Karin Erb

Department

Microbiology University of Odense J.B. Winslowsvej 19

of Medical

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