HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Liebert, Inc., Publishers
Establishment and Characterization of Monoclonal Antibodies
Carbohydrate Antigens on Peanut Agglutinin Receptor Glycoprotein
of Gastric Cancer KATO-III
UETSUKI,1 AKIRA KATO,1 HITOMI NAGAKURA,1 FUJIMOTO,1 YASUO KATO,1 YUKIKO ITSUKI,1 MASAKAZU ADACHI,2 and YASUO NAKAYAMA'
'Formulation Research Institute, Otsuka Pharmaceutical, Tokushima 771-01, Japan 2Japan Immunoresearch Laboratories, Takasaki, Gunma 370, Japan ABSTRACT mouse monoclonal antibodies, GOM-1, GOM-2, GOM-3, GOM-5, GOM-6, GOM-7, GOM-8 and GOM-9 were established that recognized carbohydrate antigens on the human gastric cancer cell line KATO-M Their binding specificities were studied by enzyme- 1 inked immunosorbent assay, cellular enzyme- 1 inked immunosorbent assay, flow cytometry analysis and thin layer chromatography immunostaining. All these monoclonal antibodies bound to peanut agglutinin receptor glycoproteins and neutral glycolipids extracted from KATO-M cells, but they could be divided into three groups, namely GOM-1, -3, -9 group, GOM-5 and GOM-2, -6, -7, -8 group. GOM-3 specifically bound to the Le" structure, Gal/3 l-3(Fuca l-4)GlcNAc/j 1-, and GOM-5 specifically bound to the Lec structure, Ga 1 ß 1-3G1cNAcß -. GOM-2 showed specific binding to KATO-M. but little or no binding to various other cell lines examined or to normal human leukocytic cells. It also did not bind to the synthetic glycoconjugates tested, carrying 10 different terminal sugar chains including T, Tn, Le' Le c and Le* structures. The binding specificity of GOM-2 was also different from those of the monoclonal antibodies anti-Le*, anti-Le' and anti-le' These results suggest that GOM-2 recognizes a new carbohydrate antigen on KATO-M cells that is distinct from Lea, Leb, Lec, Le", Ley, T and Tn
Many monoclonal antibodies against tumor associated antigens have been widely used as diagnostics (1). Recently some have also been used clinically as anticancer agents, and also as ligands for drug targeting (2,3,4). However for these purposes new antibodies with the desired specificity and higher affinity are required to obtain more satisfactory results. KATO-M is a gastric signet ring cancer cell line established by Sekiguchi et al. (5) that expresses many types of carbohydrate antigens (6,7). Miyauchi et al. found that carbohydrate rich, high molecular weight glycoproteins from the surface of KATO-M cells could be purified by peanut aggulutinin (PNA) (8) affinity column chromatography (9). Furthermore, in binding assays using
carbohydrate antigens as well as T-antigen (Gal/8 l-3GalNAca 1-) are expressed in purified KATO-M PNA receptor glycoproteins (10). Thus we expected that there are many as yet unidentified-carbohydrate antigens on KATO-M PNA receptor glycoproteins. With the aim of obtains monoclonal antibodies that recognized these novelcarbohydrate antigens, we immunized a balb/c mouse with KATO-M cells, and established many hybridomas producing monoclonal antibody. In this paper we describe the development and characterization of these monoclonal antibodies and studies on their antigen structures. MATERIALS AND METHODS
KATO-M cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% C02 in air. The cells (3 x 10') were washed 3 times with phosphate buffered saline (PBS) and then injected i.p. into an 8-week-old male balb/c mouse 3 times at 3-week intervals. Fusion and Three
cloning days after
the final immunization, spleen cells were fused to mouse myeloma P3-NS1/1-Ag4-1 (NS1) cells (11). The fusion was performed at a 5:1 (sp 1 een:myeloma) cell ratio using PEG1500 (Beoringer-Mannheim). Cells were cloned by HAT selection and limiting dilution in 96-well plates. This limiting dilution process was repeated more than 2 times. Hybridomas were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 °C under 5% CO¡ in air.
PNA receptor glycoproteins PNA receptor glycoproteins were purified from SDS extracts of the membrane fraction of KATO-M cells by PNA affinity chromatography (10). They were then reduced with dithiothreito 1 and alkylated with iodoacetamide, and purified again by PNA affinity chromatography, and finally heated at 100 °C for 10 minutes. Protein concentrations were determined by the method of Lowry et
(12) with bovine
Screening Enzyme-1 inked immunosorbent assay (ELISA) was used for screening for hybridomas producing monoclonal antibodies with specificity for KATO-M PNA receptor glycoproteins. Samples of 10 ng of KATO-M PNA receptor glycoproteins were adsorbed on the wells of 96-well plates at 37 °C overnight. Then the wells were washed with PBS and blocked with 1% BSA in PBS. The culture supernatants of the hybridomas were added and the plates were incubated 4 °C for 30 minutes. Then the wells were washed 5 times with PBS, and incubated with peroxidase conjugated goat anti-mouse Ig(A+G+M) antibody (Cappel) diluted with 1% BSA in PBS at 4 °C for 2 hours. After washing, they were incubated with 0.1% o-phenylenediamine solution in 0.1M citrate-phosphate buffer containing 0.01% hydrogen peroxide at room temperature for 30 minutes. Finally 2N H¡ SO» was added to stop the enzyme reaction, and the absorbances of the solutions at 492/690nm were measured with a Titertek Multiskan MCC
The isotypes of mouse monoclonal antibodies Ab-ID EIA kit (Zymed).
Plates coated with KATO-M PNA receptor glycoproteins were prepared as described above, and washed 5 times with PBS containing 0.05% Tween20 (washing buffer). Twofold serially diluted culture supernatants in washing
buffer were added and incubated at 4 °C overnight. The wells were then washed 5 times with washing buffer, and treated with peroxidase conjugated goat anti-mouse Ig(A+G+M) antibody at 4 °C for 4 hours. The plates were then washed, and peroxidase activity was determined as described above. The culture supernatant of the fusion partner NS1 was used as a control. Inhibition assay with KATO-M PNA receptor glycoproteins with or without periodate treatment KATO-M PNA receptor glycoproteins were treated with 5mM periodate for 1 hour at room temperature. The reaction was stopped by the addition of polyethyleneglyco1. The test monoclonal antibody and KATO-M PNA receptor glycoproteins with or without periodate treatment as inhibitors were added simultaneously to wells precoated with 1 ng of PNA receptor glycoproteins at 4 °C overnight. After incubation, the plates were washed and treated with peroxidase conjugated goat anti-mouse Ig(A+G+M) antibody at 4 "C for 4 hours.
Flow cytometry (FCM) assay with sialidase treated KATO-M cells KATO-M cells immobilized with 10% formalin were treated with sialidase (Sigma) at a final concentration of 20 U/ml in 0.IM acetate buffer, pH 5.0, containing 0.1% BSA at 3 7°C overnight. The reaction was stopped by washing with PBS. Then treated or untreated cells (1 x 10l cells) were incubated with 0.2 ml of the monoclonal antibody solution at 4 °C for 1 hour, washed with PBS and treated with 0.2 ml fluorescein i sothiocyanate (FITC) conjugated goat anti-mouse IgG antibody (Cappel) or FITC conjugated goat anti-mouse IgM antibody (Cappel) at 4 °C for 1 hour. After the reaction, the cells were washed with PBS and their fluorescence was determined in a flow cytometer (Spectrum-M Ortho). Anti-sialyl SSEA-1 mouse IgM monoclonal antibody (FH6) (13), anti-Le" mouse IgM monoclonal antibody (FH-2) (14) and the NS1 culture supernatant were used as reference antibodies. FH-2 and FH-6 were kindly donated by Dr. S.Hakomori, of the Biomembrane Institute. .
(55:25:20 by volume)
described by Kannagi et al. (15), and the extract was partitioned by the method of Folch et al. (16). The aqueous phase was evaporated to a small volume, and dialyzed in Spectrapor membrane tubing, water
with a molecular weight cut off of 3,000 (Spectrum Medical). After dialysis, ch 1oroform: methano1 (1:2 by volume) was added to give a final ratio of chloroform : methano1 : water of 30:60:8, and insoluble material was removed by centrifugation. The glycolipids were subjected to column chromatography on DEAE-Sephadex A-25. The flow-through fraction was collected as the neutral glycolipid fraction, and acidic glycolipids were eluted with chloroform: methanol: 0.45M ammonium acetate (30:60:8 by volume). Each glycolipid fraction was concentrated to a small volume.
layer chromatography (TLC) immunostaining Immuno sta ining of glycolipids on TLC plates was performed by a modification of the method of Magnani et al. (17). Glycolipids were chromatographed on high performance thin layer chromatography (HPTLC) plates (J.T.Baker) with chloroform: methano1 : wat er (60:35:8 by volume) as solvent. Then the TLC plates were dried and soaked in 1% BSA in PBS for 1 hour to prevent nonspecific adsorption of antibody. The plates were then incubated
20- to 50- fold diluted culture supernatant containing monoclonal antibody at 4°C overnight, and then with peroxidase conjugated goat antimouse IgM antibody (Cappel) or peroxidase conjugated Protein A (Zymed). The plates were then washed and glycolipids were stained with 0.05% 4-chloro-lnaphthol solution in 50mM Tris-HCl buffer containing 0.01% hydrogen peroxide. Glycolipids on TLC plates were also detected by spraying with orcinolsurfuric acid reagent and then heating at 110°C with
ELISA of fractioned neutral glycolipids Neutral glycolipids were fractionated by chromatography through a column of Iatrobeads 6RS-8010 of 4.0 x 600 mm size (Dia-Iatron) in an LKB apparatus. The glycolipid solution was injected into the column equilibrated with solvent A, [isopropano1 :hexane : water (IHW), 55:40:5 by volume]. Then gradient elution was carried out with stepwise increase in the proportion of solvent B, (IHW, 55:20:25 by volume). The gradient program was 07.B for 10 minutes, 0%B to 30%B from 10-20 minutes, 30%B to 37%B from 20-80 minutes, 37%B to 50%B from 80-100 minutes, 50%B to 100%B from 100-110 minutes, 100%B from 110-130 minutes, and then 0%B from 130-140 minutes. Elution was carried out at 0.5 ml/min and fractions of 0.3 ml were collected. Samples of 50 ß I of each fraction were dried in the wells of 96-well plates, and incubated with 1% BSA in PBS for 30 minutes. After washing with PBS, the plates were incubated with monoclonal antibodies, washed and incubated with peroxidase conjugated goat anti-mouse IgM antibody or peroxidase conjugated Protein A. Peroxidase activity was determined as described above. Anti-Le" (Biotest) and anti-Leb (Biotest) were used as reference monoclonal antibodies.
enzyme-linked immunosorbent assay (CELISA) to various cultured cells 3LL (Lewis lung following cell lines were used: 14 mouse cell lines carcinoma), B16 BL6 (melanoma), B16 Fl (melanoma), EL4 (lymphoma). Ehrlich (ascites carcinoma), Meth A (sarcoma), MH134 (hepatoma), L929 (connective tissue), L1210 (lymphocytic leukemia), L5178Y (Lymphoma), P388 (lymphoid neoplasm), S180 (sarcoma), X5563 (Plasmacytoma) and YAC-1 (lymphoma); 2 rat cell lines AH66 (ascites hepatoma) and DL (Dunning leukemia), and 25 human cell lines A375 (malignant melanoma), A498 (carcinoma, kidny), ACHN (renal adenocarcinoma), Balll (acute lymphocytic leukemia), Daudi (Burkitt lymphoma), FL (amnion), Flow7000 (embrionic foreskin fibroblast), HL-60 (promyelogeneous leukemia), H0C21 (ovarian carcinoma), K-562 (chronic myelogenous leukemia), KATO-M (gastric cancer), KB (epidermoid carcinoma), KU-1 (bladder carcinoma), MKN45 (gastric cancer), MKN74 (gastric cancer), M0LT-4 (acute 1ymphoblastic leukemia), NB-1 ( neurob 1 astoma ) OS-RC-2 (renal cell carcinoma), PC7 (lung cancer), PC9 (lung cancer), Raji (Burkitt lymphoma), U-87MG ( g 1iob1 astoma, astrocytoma), U-937 (histocytic lymphoma), WiDr (colon, adenocarcinoma) and -
(breast carcinoma). Adherent cells
were treated with 0.25% trypsin. 5x10* cells were washed 3 times with PBS and seeded into wells precoated with polylysine (0.5 mg/ml). After cell adhesion, the cells were immobilized by treatment with 0.1% glutaraldehyde. Before use, the wells were washed with PBS and blocked with 2% BSA in PBS at 37 °C for 1 hour. Then the cells were treated with diluted culture supernatants containing monoclonal antibodies at 4 °C overnight. They were then washed and treated with
peroxidase conjugated goat anti-mouse IgG antibody (Cappel) or peroxidase conjugated goat anti-mouse IgM antibody (Cappel) at 4 °C overnight. Peroxidase activity was then determined as described above. FCM assay of normal human blood cells Heparinized human peripheral blood was collected from a normal human volunteer. After lysis of red blood cells, white blood cells were washed 3 times with PBS. The cells were incubated with each monoclonal antibody at 4 °C for 1 hour, and then with FITC conjugated goat anti-mouse IgG antibody or FITC conjugated goat anti-mouse IgM antibody at 4°C for 1 hour. They were then washed and the fluorescence intensities of the lymphocytes, monocytes and granulocytes were determined separately with a Spectrum-M Anti-Le" mouse IgM monoclonal antibody (AH-6) (18), kindly donated by Dr. S.Hakomori, was used as a reference. •
Assays of binding to synthetic glycoconjugates (Gala l^Galß -0-2-( 2-carbome t hoxye thy 1 thio ) ethy 1 (CETE) )n-BSA, (Glca l-6Glc a l-4Glc a 1-4G1C/3 -0-CETE)n-BSA, (GlcNAc ß 1-4 (Fue a l-6)GlcNAc/3 -0CETE)n-BSA, (Galß l-3GalNAc/3 -O-CETE ) n-B SA (Mana l-6(Mana l-3)Mana -0CETE)n-BSA, (Gal/3 l-3GlcNAc ß -O-CETE)n-BSA, (GalNAc ß l-3Gala -O-CETE)n-BSA ,
and (GalNAcß l-4Gal ß -O-CETE)n-BSA were purchased from CHI. Galß 1-3G1cNAcß l-3Gal ß l-4(Glc)-Phenyl-Alkyl (PA),,, Gal ß l-4GlcNAc ß l-3Galß 1-4(Glc)-PA,. Gal/3 l-3(Fuca l-4)GlcNAcy3 l-3Gal/3 1-4 ( G 1 c )-P A, » and Gal/S l-4(Fuca 1-3) GlcNAcß l-3Galß l-4(Glc)-PA. were purchased from BioCarb. The wells of 96-well plates were coated with 10 ng of each synthetic glycocon jugate. The wells were then blocked with 1% BSA in PBS, and incubated with diluted culture supernatants at 4 °C overnight. They were then washed and treated with peroxidase conjugated goat anti-mouse Ig(A+G+M) antibody at 4 °C for 4 hours. Peroxidase activity was then determined as described above. ,
Isotypes We established nine hybridomas producing mouse monoclonal antibodies named GOM-l, GOM-2, GOM-3, GOM-4, GOM-5, GOM-6, GOM-7 , GOM-8 and GOM-9, and studied their binding specificities. The isotypes of these monoclonal antibodies were IgG3 K or IgM K (TABLE 1). Titration
The activities of the culture supernatants of GOM-l~GOM-9 and NS1 were examined by ELISA in plates coated with the KATO-M PNA receptor glycoprotein. As shown in FIGURE 1., the binding activity was detected in highly diluted culture supernatants (more than 211) of GOM-1, -2, -3, -5, -6, -7 and -9. The supernatant of GOM-8 showed a lower titer than the others, and GOM-4 showed no
A-A-a-&\&-A-a\ a- a a û, -"
10' Dilution of
GOM-5 GOM-6 GOM-7 GOM-8 GOM-9 NS1 GOM-4
receptor glycoproteins by ELISA. lOng of KATO-M PNA receptor glycoproteins were adsorbed on 96-well plates and treated with the diluted culture supernatants. Inhibition assay To obtain the structural information on the epitopes, we examined the effects of periodate treatment of the antigens on their binding to these antibodies by inhibition assay (TABLE 1). The bindings of all the antibodies except GOM-4 were inhibited dose-dependent1 y by the intact KATO-M PNA receptor glycoproteins, but not by the periodate-treated glycoproteins. Reactivities with neutral and acidic carbohydrates on KATO-M cells The effects of sialidase treatment of KATO-M cells on their binding the each monoclonal antibodies were examined by FCM (TABLE 2).
Isotypes and inhibitory effects of peri odate-1reated on the binding of antibodies. Plates coated with KATO- M
glycoproteins were treated with periodate-treated or untreated antigens. Monoclonal
the monoclonal antibodies and
untreated PNA receptor
periodate-treated PNA receptor
GOM-1 GOM-2 GOM-3 GOM-4 GOM-5 GOM-6 GOM-7 GOM-8 GOM-9
IgM IgM IgM IgM IgM IgG3
n K K K, /c
inhibited inhibited inhibited not tested inhibited inhibited inhibited inhibited inhibited
inhibited inhibited not inhibited not tested not inhibited not inhibited not inhibited not inhibited not inhibited not not
2. Effect of sialidase treatment of KATO-M cells their reactivity with monoclonal antibodies determined flow cytometry.
Mean channel of
Sialidase(-) GOM-1 GOM-2 GOM-3 GOM-5 GOM-6 GOM-7 GOM-8 GOM-9 FH-2 FH-6
21.1 109.4 29.5 107.7 165.1 160.4 75.8 28.7 11.4 96.2
Sialidase(+) 28.9 100.2 32.5 100.6 162.7 158.7 71.2 21.6 98.6 4.6
Sialidase treatment did not affect the binding of any of the antibodies to cells. On the other hand, it greatly increased the binding of the reference antibodies FH-2 (specific to Le") and greatly decreased that of FH-6 (specific to sialyl SSEA-1). We found by TLC immunostaining that all these antibodies bound to glycolipids extracted from KATO-M cells (FIGURE 2), but that no antibody bound to acidic glycolipids (data not shown). The binding patterns of the monoclonal antibodies to neutral glycolipids fractionationed on an Iatrobeads column are summarized in FIGURE 3. From their binding patterns, the monoclonal antibodies were divided into two main groups, group A and group B. Group A (GOM-1, GOM-3, GOM-5 and GOM-9) gave a main peak at a retention time of 40 to 47 minutes, whereas group B (GOM-2, GOM-6, GOM-7 and GOM-8) gave a doublet peak at 46 to 53 minutes and broad reactivity after 60 minutes. The binding pattern of anti-Le" was similar to that of group A, but anti-Leb showed a different pattern with a peak at 44 to 49 minutes.
o FIGURE 2.
immunostaining by monoclonal antibodies of extracted from KATO-M cells: lane 0, orcinol-H,SO,; lane 1, GOM-1; lane 2, GOM-3; lane 3, GOM-5; lane 4, GOM-9; lane 5, GOM-2; lane 6, GOM-6; lane 7, GOM-7, and lane 8, GOM-8. TLC
GOM-9 GOM-2 GOM-6