Biochimica et Biophysica Acta, 1117 (1992) 97-103

97

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4165/92/$05.00 r

BBAGEN 23686

Generation of one set of monoclonal antibodies specific for a-pathway ganglio-series gangliosides Masaharu Kotani a, Hideki Ozawa a, Ikuo Kawashima a, Susumu A n d o b and Tadashi Tai a a The Tokyo Metropolitan Institute of Medical Science, Department of Tumor Immunology, Honkomagome, Bunkyo-ku, Tokyo (Japan) and b The Tokyo Metropolitan Institute of Gerontology, Department of Biochemistry, Sakaecho, Itabashi-ku, Tokyo (Japan)

(Received 26 December 1991)

Key words: Monoclonal antibody; Ganglioside; ELISA; Binding specificity;Glycolipid We established five murine monoclonal antibodies (MAbs) specific for a-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota, followed by fusion with mouse myeloma cells. The binding specificities of these MAbs were determined by enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These five MAbs, designated GMR6, GMB28, GMB16, GMR17, and G M R l l reacted strongly with the gangliosides GM3, GM2, GM1, GDla, and GTla, respectively, that were used as immunogens. Three MAbs, GMB28 (anti-GM2), GMB16 (anti-GM1), and G M R l l (anti-GTla) showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of the other various authentic gangliosides or neutral glycolipids was recognized. On the other hand, the other two MAbs, GMR6 (anti-GM3) and GMR17 (anti-GDla) exhibited broader specificities. MAb GMR6 cross-reacted with GM4, GMlb, GDla, GTlb, and IV3NeuAca-nLcaCer. MAb GMR17 also reacted with GMlb and GTlb. Neither GMR6 nor GMR17 reacted with other gangliosides or neutral glycolipids tested. Using these MAbs, we determined the expression of these gangliosides, especially GM1, GDla, and GTla on mouse, rat and human leukemia cells. GM1 and GDla were expressed on some leukemia cells, whereas GTla was not detected in these cells.

Introduction Gangliosides, in particular, ganglio-series gangliosides are highly expressed on neuroectodermal derived tissues. Monoclonal antibodies (MAbs) against these gangliosides have been shown to be powerful reagents in analyzing biological functions and localization of them on cell surface m e m b r a n e s [1-3]. It would be preferable to establish hybridomas by immunizing mice directly with purified gangliosides. It is, however, still difficult to routinely generate murine MAbs to them. We have recently found that C 3 H / H e N mice demonstrate maximum antibody responses against these gangliosides among the ten different strains of inbred mice

[4]. Based on these results, we have developed an improved method for generating MAbs to gangliosides by immunizing these mice with purified gangliosides adsorbed to bacteria. In fact, we have already generated one set of MAbs specific for b-pathway gangliosides (GD3, GD2, G D l b , G T l b , and G Q l b ) and two MAbs specific for N-glycolylneuraminic acid-containing gangliosides by the procedure [5,6]. In the present paper, we describe the production of a new set of MAbs specific for a-pathway gangliosides (GM3, GM2, GM1, G D l a , and G T l a ) . These MAbs were proved useful for detecting such gangliosides in mouse, rat, and human leukemia cells. Materials and Methods

Correspondence to: T. Tai, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113, Japan. Abbreviations: ELISA, enzyme-linked immunosorbent assay; MAb, monoclonal antibody; TLC, thin-layer chromatography; FITC, fluorescein isothiocyanate; FACS, fluorescence-activatedcell sorter; PBS, phosphate-buffered saline. Sialic acid moiety of the gangliosides is NeuAc unless otherwise noted. Gangliosides are named according to the nomenclature of Svennerholm [31]. Otherwise, the nomenclature used follows the IUPAC-IUB recommendations [32].

Glycosphingolipids

GM1, G D l a , G D l b , and G T l b were prepared from bovine brain. I V 3 N e u A c a - n L c a C e r and I V 3 N e u G c a nLcnCer were isolated from human and bovine erythrocytes, respectively. G M 2 ( N e u G c ) was prepared from mouse ( B A L B / c ) liver. GT3 and G T 2 were prepared from codfish brain [7]. G T l a was isolated from monkey brain. GM2 and G D 2 were prepared from

98 GM1 and GDlb, respectively, by the treatment of bovine testis /3-galactosidase (Boehringer-MannheimYamanouchi, Tokyo, Japan) as described previously [8]. LacCer, Gg3Cer, and GgaCer were prepared from GM3, GM2, and GM1, respectively, by treatment with sialidase as previously described [8]. GM4 (human myelin) was provided by T. Ariga (Virginia Commonwealth University). GMI(NeuGc) and GDla(NeuGc, NeuGc) of mouse (ICR) liver and GD3(NeuGcNeuGc-) of bear erythrocytes were donated by Y. Hashimoto and A. Suzuki (The Tokyo Metropolitan Institute of Medical Science). GMlb was donated by Y. Sanai (The Tokyo Metropolitan Institute of Medical Science). GalCer (bovine brain) and GlcCer (Gaucher's spleen) were purchased from Sigma (St. Louis, MO, USA). GM3 and G Q l b (bovine brain), GD3 and IV3GalNAca-GbaCer (bovine milk), GM3(NeuGc) (horse erythrocytes), and Gb3Cer and GbaCer (porcine erythrocytes) were purchased from Iatron (Tokyo, Japan). The structures of ganglio-series glycolipids used in this study are shown in Table I. Monoclonal antibodies Hybridomas producing MAbs were established as previously described [5]. Briefly, three mice of C 3 H / H e N inbred strain (7 weeks of age) were immunized with purified ganglioside (10 /zg, total amount per mouse) adsorbed to acid-treated S. minnesota (250

/zg, total amount per mouse). Each mouse received the intraveneous injections of antigen-bacteria complex on day 0, 4, 7, 11 and 21. The spleen cells were obtained after 3 days from the last injection and were fused with a mouse myeloma cell line (PAl). The hybridoma fusions were screened against the immunizing gangliosides. The antibody titers in the supernatant of hybridoma cells were determined by ELISA. The purified ganglioside used as antigen in ELISA appeared to be no contamination with any other gangliosides in TLC with resorcinol staining. The positive hybrid was cloned by limiting dilution. The isotype of antibody produced by hybridoma was determined by a mouse monoclonal antibody kit (Amersham, UK). Enzyme-linked immunosorbent assay (ELISA) Solid-phase ELISA was performed as previously described [9]. A 96-well polystyrene microtiter plate (Dynatech, Alexandria, VA, USA) was used. Glycolipid (0.5 nmol) was serially diluted in ethanol. The supernatant of cloning hybridoma cells was diluted in PBS containing 1% human serum albumin. The plates were incubated for 1 h at room temperature. After washing, the second antibody (1:200 diluted), horseradish peroxidase-conjugated goat anti-mouse IgM (/z-chain specific) (Cappel, Malvern, PA, USA), was added to the plates and incubated for another 1 h at room temperature. The absorbance at 405 nm was determined by a

TABLE I

Structures of ganglio-series glycolipids used in this study Glycolipid

Structure

Neutral LacCer Gg3Cer Gg4Cer a-Pathway GM3 GM2 GM1 GDla GTla b-Pathway GD3 GD2 GDlb GTlb GOlb c-Pathway GT3 GT2 Miscellaneous GMlb

Gal~1 GaINAc~l Gal~l ~ 3GaINAc~1

NeuAc~2

GaINAc~l Gal~l ~ 3GaINAc~1 NeuAc~2 ~ 3Gal~l ~ 3GaINAc~1 ~ 8NeuAc~2 ~ 3Gal~l~3GaINAc~1

~ ~ ~ ~

~ 4Gal~l ~ 4Gal~l

NeuAc~2 ~ 3Gal~l 4(NeuAca2 ~ 3)Gal~l 4(NeuAc~2 ~ 3)Gal~1 4(NeuAc~2 ~ 3)Gal~l 4(NeuAc~2~3)Gal~l~

NeuAc~2 ~ 8NeuAc~2 ~ 3Gal~1 GaINAc~l~4(NeuAc~2 ~ 8NeuAc~2 ~ 3)Gal~1~ Gal~l~3GaINAc~1~4(NeuAc~2 ~ 8NeuAc~2~3)Gal~1~ NeuAca2 ~ 3Gal~1 ~ 3GaINAc~l ~ 4(NeuAc~2 ~ 8NeuAca2 ~ 3)Gal~1 NeuAc~2 ~ 8NeuAca2 ~ 3Gal~1 ~ 3GaINAc~1 ~ 4(NeuAc~2 ~ 8NeuAc~2 ~ 3)Gal~1

GaINAc~l

NeuAc~2~SNeuAc~2~SNeuAc~2 ~ 4(NeuAc~2 ~ 8NeuAc~2 ~ 8NeuAca2

4Glc-Cer 4Glc-Cer 4Glc-Cer

4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer

4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer

~ 3Gal~1~4Glc-Cer ~ 3)Gal~1 ~ 4Glc-Cer

NeuAc~2~3Gal~l~3GaINAc~l~4Gal~1~4Glc-Cer

99 Bio-Rad ELISA plate reader. The standard deviation was less than 10% for all values. Backgroud values of absorbance were less than 0.05.

Thin-layer chromatography (TLC) Merck precoated TLC-plastic sheets (Merck, Darmstadt, FRG) were used for fractionation of glycolipids [10]. The solvent system used for developing chromatograms was chloroform/methanol/0.22% CaCI2 in water (55:45:10, v/v). Gangliosides and neutral glycolipids were visualized with resorcinol and orcinol stain, respectively. Enzyme immunostaining on TLC plates Immunostaining on TLC plates was performed as previously described [11]. In brief, after chromatography of the gangliosides, the plates were soaked for 1 h in freshly made PBS containing 1% bovine serum albumin. After drying, the plates were incubated with MAb solution (1 : 10 diluted) for 2 h at room temperature. The chromatogram was washed by dipping in five successive changes of PBS containing 0.05% Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-mouse IgM for 2 h at room temperature. After washing the chromatogram again, this was followed by addition of a solution of 4 0 0 / , g / m l o-phenylenediamine (Sigma, St. Louis, MO, USA) in 80 mM citrate-phosphate buffer, pH 5.0, containing 0.12% H 2 0 2. Color development was stopped after 15 min by washing the plate by dipping it in PBS. Tumor cell lines Mouse leukemia cells (EL4, RBL5, BALBRVE, YAC1, B6RV1, 18-4, 1-29, and RLM1), rat leukemia cells (RBL-2H3), and human leukemia ceils (MOLT-4, MT-1, Jurkat, CCSF-CEM, IM-9, Raji, HL-60, Hutl02, Hut78, and HPB-ALL) were cultured as previously

TABLE II MAbs to a-pathway ganglio-series gangliosides C 3 H / H e N mice were immunized with purified ganglioside (10 /,g, total amount per mouse) coated on acid-treated S. minnesota (250 p.g, total amount per mouse) intraveneously five times on days 0, 4, 7, 11, and 21. After three days from the last injection, the spleen cells were fused with PAl (BALB/c mouse-derived myeloma cells). The hybridoma fusions were screened against the immunizing ganglioside. The antibody titers in the supernatant of hybridoma cells were assayed by ELISA. MAb

Ganglioside used as immunogen

Ig isotype

GMR6 GMB28 GMB16 GMR17 GMRll

GM3 GM2 GM1 GDla GTla

IgM (K) IgM (K) IgM (K) IgM (K) IgM (K)

described [10]. PAl (BALB/c mouse-derived myeloma) was provided by the Japanese Cancer Research Bank, Tokyo, Japan. Glycolipids from tumor cells were extracted by the method of Svennerholm and Fredman [121.

Flow cytofluorometric analysis Cell surface gangliosides were detected by flow cytometry [5]. Cells (5.105 cells) were washed with PBS, incubated at 37°C for 3 min with PBS containing 0.2% trypsin and 1 mM EDTA, and suspended in growth medium (1 ml). After centrifugation, cells were reacted with the supernatant of hybridoma cells for 1 h at 4°C, followed by the addition of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (/z-chain specific, Cappel) and incubated for another 30 min at 4°C. After washing with PBS at 4°C, the ceils were analyzed with a flow cytometer, FACScan (Becton Dickinson, Mountain View, CA, USA). Results

Establishment of MAbs specific for a-pathway ganglioseries gangliosides We finally established five hybridoma clones producing MAbs specific for a-pathway ganglio-series gangliosides (Table II). All of these MAbs were of IgM(k) isotype. ELISA with various authentic glycolipids ELISA was performed with various authentic glycolipids bound to microtiter wells. Among five MAbs, three MAbs, GMB28 (anti-GM2), GMB16 (anti-GM1), and G M R l l (anti-GTla) showed highly restricted binding specificities, detecting only the ganglioside used as immunogen. None of other gangliosides or neutral glycolipids so far tested reacted. On the other hand, the other two MAbs, GMR6 (anti-GM3) and GMR17 (anti-GDla) showed broad binding specificities. MAb GMR6 reacted predominantly with GM3, but moderately with four other gangliosides (GMlb, GDla, GTlb, and IV3NeuAca-nLcnCer) and weakly with GM4. No other gangliosides or neutral glycolipids were recognized. The epitope recognized by the MAb would be the external disaccharide (NeuAca2--+ 3Gal-) sequences in these gangliosides. MAb GMR17 strongly reacted with GDla, but moderately with two other gangliosides (GMlb and GTlb). No other glycolipids were reactive. The epitope detected by the MAb would be the trisaccharide (NeuAca2 ~ 3Gal/31 --+ 3GalNAc-) structures, since none of GM4, GM3, or IV 3 NeuAca-nLcaCer were reactive. Also, fine binding specificities of these five MAbs on sialic acid derivatives were different each other. Three MAbs, GMR6, GMR17, and G M R l l distinguished between different N-acetylneuraminic and N-glycolylneuraminic acid

100 derivatives of gangliosides, whereas the other two MAbs, GMB28 and GMB16 reacted with both types of gangliosides GM2 and GM1, respectively. Since extensive studies on the binding specificities of MAbs to GM3 and GM2 have previously been reported by several groups [13-17], those of the three MAbs, GMB16 (anti-GM1), GMR17 (anti-GDla), and G M R l l (antiGTla) were described in detail (Fig. 1). The reactivities of these five MAbs with various glycolipids determined by ELISA are summarized in Table III.

TLC immunostaining of various authentic glycolipids with the MAbs We also determined the binding reactivities of these three MAbs, GMB16, GMR17, and G M R l l to authentic gangliosides including bovine brain gangliosides and M14 cells (human melanoma cell line) gangliosides that contain GM3, GM2, GD3, and GD2 as major gangliosides by enzyme-immunostaining on TLC (Fig. 2). The TLC immunostaining results agreed well with the ELISA. MAbs GMB16 and G M R l l reacted only with ganglioside GM1 and GTla, respectively. Also, MAb GMB28 reacted only with GM2 (data not shown). In

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Reactiuities of MAbs with L,arious authentic glycolipids

Glycolipid a

GM3 GM3(NeuGc) GM2 GM2(NeuGc) GM1 GMI(NeuGc) GDla GDla(NeuGc, NeuGc) GT1 a GMlb GTlb GM4 IV3NeuAcanLc4Cer IV3NeuGcanLc4Cer

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GMB28

GMB16

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GMRll

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The gangliosides have NeuAc as their sialic acid moiety unless otherwise noted. No other glycolipids, such as b-pathway gangliosids (GD3, GD2, GDlb, and GQlb), or neutral glycolipids (GlcCer, GalCer, LacCer, Gg3Cer, and Gg4Cer, Gb3Cer, GbnCer, and IV3GalNAca-GbaCer) tested were detected. b + + +, strong; + +, moderate; +, weak; - , negative.

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TABLE III

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G@ e~l~.O-,o - o

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1 2-2 2-4 2-e 2-e 2-~o Antibody dilution

Fig. 1. Reactivities of three MAbs, GMB16, GMR17, and G M R l l with various authentic glycolipids in ELISA. A-l, B-I, and C-l, wells were coated with each glycolipid at different concentrations and incubated with MAbs GMB16, GMR17, and GMRll, respectively. The supernatant of hybridoma was used as MAb. A-2, B-2, and C-2, wells were coated with each glycolipid (0.5 nmol/well) and incubated with different concentrations of the MAbs mentioned above, e, GM1; i , GDla; A, GTla; ©, other a-pathway ganglioside (GM3 and GM2); o, b-pathway ganglioside (GD3, GD2, GDlb, and GQlb); D, GMlb and GTlb; zx, neutral glycolipids (GlcCer, GalCer, LacCer, Gg3Cer, Gg4Cer, Gb3Cer, Gb4Cer, and IV3GalNAca-Gb4Cer).

contrast, MAb GMR17 cross-reacted with GMlb and GTlb, but not with any other glycolipids. MAb GMR6 cross-reacted with several gangliosides, such as GM4, GMlb, GDla, GTlb, and IV3NeuAca-nLcaCer (data not shown).

TLC immunostaining of gangliosides from leukemia cell lines with MAbs The gangliosides extracted from a number of leukemia cell lines (mouse, rat, and human) were developed on TLC and stained with these three MAbs, GMB16, GMR17, and G M R l l . GM1 was detected in five cell lines (18-4, RBL-2H3, Jurkat, K562, and MT-1), whereas GDla was detected in three cell lines (18-4, Jurkat, and K562). GTla, in contrast, was not detected in these cell lines tested (data not shown). The reactivities of these three MAbs with gangliosides from these ceils are summarized in Table IV. FACS analysis of leukemia cell lines The results of FACS analysis were quite similar to those of TLC immunostaining (Fig. 3). GM1 was expressed predominantly on 18-4, and slightly on RBL2H3, Jurkat, K562, and MT-1 among these cells. On the other hand, GDla was detected weakly in 18-4, Jurkat, and K562, but not in other cells. In contrast, GTla was not detected in these cells. The FACS analysis of gangliosides on two leukemia cells, 18-4 and Jurkat is shown as a representative (Fig. 3).

101 TABLE IV

Discussion

Summary of ganglioside expression in leukemia cell lines I n this study, we described the g e n e r a t i o n of o n e set of M A b s specific for a-pathway ganglio-series gangliosides. Since we have already established a n o t h e r o n e set of M A b s specific for b - p a t h w a y gangliosides [5], we have b e e n successful in g e n e r a t i n g two sets of MAbs. Thus, o u r results clearly indicate that C 3 H / H e N mice are m o r e suitable t h a n o t h e r strains of mice such as B A L B / c a n d C 5 7 B L / 6 for raising M A b s to these gangliosides. It is n o t e w o r t h y that m o u s e M A b s specific for a-pathway gangliosides have b e e n g e n e r a t e d by the same p r o c e d u r e in spite of that a n t i b o d y responses to these a-pathway gangliosides develop m i n i m u m titers in C 3 H / H e N mice [4]. A few M A b s to a-pathway gangliosides have previously b e e n g e n e r a t e d by several groups with different m e t h o d s [13-17]. M A b s specific for G D l a a n d G T l a have, however, n o t b e e n r e p o r t e d yet. This is the first

A-1

B-1

Ganglioside a Mouse

18-4 EL4 RBL5 BALBRVE YAC1 B6RV1 1-29 RLM1

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GDla + . .

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Rat

RBL-2H3

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Jurkat K562 MT-1 CCSF-CEM IM-9 Raji HL-60 Hutl02 Hut78 HPB-ALL Molt-4

+ + + -

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a Gangliosides GM1, GDla, and GTla were detected with three MAbs, GMB16, GMR17, and GMRll, respectively, by TLC immunostaining. b + + +, strong; + +, moderate; +, weak; - , negative.

1 2 3 4 5 6 1 2 3 4 5 6 Fig. 2. TLC immunostaining of standard gangliosides with three MAbs (GMB16, GMR17, and GMRll). (A) Ganglioside fraction (10 nmol as sialic acid content) from bovine brain and from human melanoma cell line, M14 and the same amount of standard glycolipids were chromatographed with chloroform/methanol/0.22% CaCI2 in water (55 : 45 : 10, v/v) and visualized with resorcinol except lane 6, which was visualized with orcinol. 1, Gangliosides of bovine brain; 2, gangliosides of M14 cells; 3, standard ganglioside GM1; 4, standard ganglioside GDla; 5, standard ganglioside GTla; 6, standard neutral glycolipids(LacCer, Gg3Cer, and Gg4Cer, from top to bottom). (B) The same ganglioside fractions (total 1 nmol/lane) were chromatogrphed similarly and immunostained with three MAbs. 1, GMB16; 2, GMR17; 3, GMRll.

description of a c o m p l e t e set of M A b s specific for a-pathway gangliosides. T h r e e M A b s , GMB28, GMB16, a n d G M R l l showed highly restricted b i n d i n g specificities, reacting only with gangliosides used as i m m u n o gens. N o n e of the o t h e r N e u A c - c o n t a i n i n g gangliosides or n e u t r a l glycolipids reacted. F o r example, the M A b GMB16, which was established by i m m u n i z i n g the mice with purified G M 1 , r e a c t e d only with G M 1 a m o n g the glycolipids thus far tested. Glycolipids that differ by the a d d i t i o n of o n e N - a c e t y l n e u r a m i n i c acid ( G D l a or G D l b ) or the loss of o n e N - a c e t y l n e u r a m i n i c acid ( G g n C e r ) were not recognized by the M A b . F u r t h e r more, glycolipid that only differ from G M 1 by the loss of o n e t e r m i n a l galactose ( G M 2 ) was unreactive. T h e M A b reacted with G M I ( N e u G c ) , b u t not with o t h e r N e u G c - c o n t a i n i n g gangliosides. T h e s e findings suggest that the t e r m i n a l galactose moiety a n d sialic acid residue linked to the i n n e r galactose moiety ( S i a a 2 --+ 3Gal-) s e q u e n c e s are involved in the b i n d i n g specificity. T h e o t h e r two M A b s , G M B 2 8 a n d G M R 1 1 also showed highly restricted b i n d i n g specificities. O n the o t h e r h a n d , we could n o t establish M A b s specific for gangliosides G M 3 a n d G D l a . M A b G M R 6 ( a n t i - G M 3 ) cross-reacted with GM4, G D l a , G M l b , G T l b , a n d I V 3 N e u A c a - n L c 4 C e r , b u t n o t with o t h e r glycolipids i n c l u d i n g c o r r e s p o n d i n g gangliosides having N e u G c as their sialic acid moiety tested. T h e o t h e r M A b G M R 1 7

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Generation of one set of monoclonal antibodies specific for a-pathway ganglio-series gangliosides.

We established five murine monoclonal antibodies (MAbs) specific for a-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purif...
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