184 m(’lYY2 Elsevier
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Science
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53796
Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides Hideki Ozawa, Masaharu
Kotani,
Ikuo Kawashima
and Tadashi
Tai
The Tokyo Metropolitan Instifute of’Medicu1 Science. Depur~mer~t CJ~Twnor /nznmr~do~~. Honk(,mrr,sor,~~,,Hunkyo-ku. Tokyo iJuparr I (Received
Key words:
Monoclonal
Zh June
19Yl)
antibody:
Ganglioside
We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGBl9, GMR2, GMR7, GGR12, GMRS, and GGR13 reacted strongly with the gangliosides GD3, O-AC-GD3, GD2, GDlb, GTlb, and GQlb, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-AC-GD3, GQlb, and GTla, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GDlb, GTlb, and GQlb on mouse, rat, and human leukemia ceils. GDlb was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GTlb nor GQlb was detected in these cell lines.
Introduction Ganglio-series gangliosides are highly expressed on neuroectodermal origined tissues. Recently, some of them have also been identified as tumor-associated antigens expressed on the surface of these tissue-derived tumors such as melanomas, neuroblastomas, and gliomas [ 1,2]. Monoclonal antibodies (MAbs) against these gangliosides have been shown to be powerful reagents not only in the diagnosis and treatment of cancer patients, but also in analyzing biological functions of gangliosides on cell surface membranes [3-61. In the past decade, these MAbs have generally been
Abbreviations: ELISA, enzyme-linked immunosorbent assay: MAb, monoclonal antibody: TLC, thin-layer chromatography. FITC; fluorescein isothiocyanate, FACS; fluorescence activated cell sorter, PBS: phosphate-buffered saline. Sialic acid moiety of the gangliosides was NeuAc unless otherwise noted. Gangliosides are named according to the nomenclature of Svennerholm 1311. Otherwise. the nomenclature used follows the IUPAC-IUB recommendations [321. Correspondence: T. Tai. The Tokyo Metropolitan Institute cal Science, Honkomagome, Bunkyo-ku, Tokyo 113. Japan.
of Medi-
established by immunizing BALB/c or C57BL/6 mice with neuroectoderm-derived tumor cells [7]. It is. however, still difficult to routinely generate murine MAbs to gangliosides by immunizing mice directly with purified gangliosides. In order to develop a Tethod for generating MAbs with high efficiency, we recently compared antibody responses to ganglio-series gangliosides in different strains of inbred mice, including BALB/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, etc. We have found that (i) b-pathway gangliosides (GD3, O-AC-GD3, GD2, GDlb, GTlb, and GQlb) induced high-titer antibody responses, whereas a-pathway gangliosides (GM3, GM2, GMl, GDla, and GTla) induced low-titer antibody responses; (ii) C3H/HeN and A/J mice demonstrated maximum antibody responses against these gangliosides among these mice, although the pattern of reactivity to the various ganglioside was similar in all the strains tested [33]. These findings suggest that C3H/HeN or A/J mice are more suitable than other strains of mice for raising MAbs to these gangliosides. The present study described the production of one set of MAbs specific for b-pathway ganglio-series gangliosides. These MAbs are expected to be powerful reagents in analyzing the biological
185
functions and localization of these gangliosides on cells and tissues, in particular, neuroectoderm-derived cells.
tron. The structure of ganglio-series gangliosides used in this study are shown in Table I. Tumor cell lines
Materials
and Methods
Glycosphingolipids
GMl, GDla, GDlb, and GTlb were prepared from bovine brain. GM3 and GQlb (bovine brain), and GD3 (bovine milk) were purchased from Iatron (Tokyo, Japan). GM2 and GD2 were prepared from GM1 and GDlb, respectively, by the treatment of bovine testis P-galactosidase (Boehringer-Mannheim-Yamanouchi, Tokyo, Japan) as described previously [S]. 9-0Acetylated GD3 (0-AC-GD3) was isolated from a human melanoma cell line, Ml4 transplanted in nude mice (nu/nu BALB/c) and the carbohydrate structure was confirmed with FAB-MASS and NMR spectroscopy by M. Suzuki and A. Suzuki, and F. Inagaki (The Tokyo Metropolitan Institute of Medical Science), respectively. Codfish brain gangliosides (mixtures of GT3, GT2, and other polysialosyl gangliosides) [9] and GTla (monkey brain) were gifts from S. Ando (The Tokyo Metropolitan Institute of Gerontology). GM4 (human myelin) was provided by T. Ariga (Virginia Commonwealth University). Sialylparagloboside (SPG) was isolated from human erythrocytes. GM3(NeuGc), GM2(NeuGc), and GMl(NeuGc) of mouse liver and GD3(NeuGc-NeuGc-) of bear erythrocytes were donated by Y. Hashimoto and A. Suzuki (The Tokyo Metropolitan Institute of Medical Science). LacCer, GgOse,Cer, and GgOse,Cer were prepared from GM3, GM2, and GMl, respectively, by treatment with sialidase as previously described [S]. GbOse,Cer, GbOse,Cer, and GbOse,Cer were purchased from Ia-
Murine leukemia cells (EL& RBLS, BALBRVE, YACl, B6RV1, RLF8, 18-4, I-29, and RLMl) and human leukemia cells (MOLT-4, MT-l, Jurkat, CCSFCEM, IM-9, Raji, HL-60, Hut102, Hut78, and HPBALL) were supplied by Dr. N. Tada (Tokai University) and Dr. Y. Yamamoto (The Tokyo Metropolitan Institute of Medical Science), respectively, as previously described [lo]. The following cell lines were provided by the Japanese Cancer research Resources Bank, Tokyo: RBL-2H3 (rat leukemia) and PA1 (BALB/c mouse-derived myeloma). These cell lines were cultured in RPM11640 with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, U.S.A.). Glycolipids from tumor cells were extracted by the method of Svennerholm and Fredman [ 111. Immunization Salmonella minnesota mutant R595 was kindly sup-
plied by T. Yasuda (Okayama University School of Medicine) and T. Tomita (Institute of Medical Science, University of Tokyo). Female mice of C3H/HeN were purchased from Japan Clea (Tokyo, Japan). Mice of the inbred strain (7 weeks of age) were immunized with purified ganglioside (10 pg, total amount per mouse) adsorbed to acid-treated S. minnesota (250 pg, total amount per mouse) by the procedure originally described by Galanos et al. [12] and later modified by Young et al. [13]. Each mouse received the intraveneous injections of antigen-bacteria complex on day 0, 4, 7, 11, and 21. The spleen cells were obtained 3 days after the last injection and were fused with a myeloma
TABLE I Structures of ganglio-series gangliosides used in this study
Ganglioside (a-pathway) GM3 GM2 GM1 GDla GTla (b-pathway) GD3 0-AC-GD3 GD2 GDlb GTlb GQlb k-pathway) GT3 GT2
Structure
GalNAcPl
NeuAccu2 + 3GalPl+ 4Glc-Cer * 3)Galpl--) 4Glc-Cer 4(NeuAccu2 -t 3)Galfil+ 4Glc-Cer 4(NeuAca2 + 3)GalPl+ 4Glc-Cer 4(NeuAca2 + 3)GalPl+ 4Glc-Cer
+ 4(NeuAca2
Gal/31 --f 3GalNAcpl* NeuAca2 + 3GalPl--) 3GalNAcPl+ NeuAccr2 + 8NeuAca2 + 3Galfil--) 3GalNAc@l+
NeuAca 2 + 8NeuAca 2 + 3GalP 1 + 0-Ac-NeuAca2 + BNeuAccu2 --t 3GalPl + GalNAcPl * 4(NeuAca2 --f BNeuAccu2 + 3Xial/31+ Galpl + 3GalNAc$l+ 4(NeuAcm2 + BNeuAca2 + 3jGal/31+ NeuAccu2 + 3Galpl-+ 3GalNAcPl --f 4(NeuAccY2 + BNeuAca2 + 3)Galpl+ NeuAccu2 -P BNeuAccu2 + 3Galpl+ 3GalNAcPl + 4(NeuAca2 + BNeuAca2 + 3)Gal@l-+ NeuAca2 + 8NeuAm2 + BNeuAcLu2 + 3GalPl-+ GalNAcpl
+ 4cNeuAccu2 + 8NeuAca2 + BNeuAca2 + 3)Galpl+
4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer
186 cell line (PAI) by a routine cell hybridization procedure as previously described [lo]. The hybridoma fusions were screened against the immunizing ganglioside. The antibody titers in the supernatant of hybridoma cells were determined by ELISA. The positive hybrid was cloned by limiting dilution. The isotype of antibody produced by hybridoma was determined by a mouse monoclonal antibody kit (Amersham, U.K.). Murine MAb Al-201 (IgM, K) was established as prcviously described [14]. MAb R24 (IgG3. K) was a kind gift from Dr. K.O. Lloyd (Memorial Sloan-Kettering Cancer Institute) [ 151. Enzyme-linked immunosorbent assay (ELISA) Solid-phase ELISA was performed as previously described [16]. A 96 well polystyrene microtiter plate (Dynatech, Alexandria, VA, U.S.A.) was used. Glycolipid (0.5 nmol) was serially diluted in ethanol and applied to the plate in a vol of 50 pi/well. 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 IgG (y-chain specific) or IgM (p-chain specific) (Cappel, Malvern, PA, U.S.A.), was added to the plates and incubated for another 1 h at room temperature. The absorbance at 405 nm was determined by a Bio-rad ELISA plate reader. Three samples of each experiment were tested. The standard deviation was less than 10% for all values. Background values of absorbance at 405 nm were less than 0.05. Thin-layer chromatography (TLC) Merck precoated TLC-plastic sheets (Merck, Darmstadt, F.R.G.) were used for fractionation of gangliosides [lo]. The solvent system used for developing chromatograms was chloroform-methanol-0.22% CaCl 2 in water (55 : 45 : 10, v/v). Gangliosides were visualized with resorcinol stain and neutral glycolipids were visualized with orcinol stain. En,yme immunostaining on TLC plates Immunostaining on TLC plates was performed as previously described [17]. 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% Tween20 and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or IgM for 2 h at room temperature. After washing the chromatogram again, a solution of 400 pg/ml o-phenylenediamine (Sigma, St. Louis, MO, U.S.A.) in 80 mM citrate-phosphate buffer, pH
5.0, containing O.l2%H ,O, was added. Color Jcvelopment was stopped after 15 min by washing the plate by dipping it in PBS. Flow cytofluorometric analysis Cell surface gangliosides were detected by l-low cytometry [18]. Cells (5 X IO” 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 (FIT(Z)-conjugated goat anti-mouse IgG (ychain specific. Cappel) or IgM (p-chain specific, Cappel) and incubated for another 30 min at 4°C. After washing with PBS at 4°C. the cells were analyzed with a flow cytometer, FACS IV (Becton Dickinson FACS Systems, Sunnyvale, CA, U.S.A.). Results
Establishment of M2bs specific for b-pathway ganglioseries gangliosides We finally established six hybridoma clones producing MAbs specific for b-pathway ganglio-series gangliosides (Table II). Three MAbs (GMR2, GMR7, and GMRS) were of IgM (K) isotype. On the other hand, MAbs GGB19, GGR12, and GGR13 were of IgG3 (K). IgG2b (~1, and IgG2a (~1. respectively. ELISA with L,arious authentic glycolipids ELISA was performed with 23 authentic glycolipids bound to microtiter wells. Among six MAbs, five MAbs tGMR2, GMR7. GGR12, GMRS, and GGR13) showed highly restricted binding specificities, detecting only the ganglioside used as immunogen. None of other gangliosides or neutral glycolipids so far tested reacted.
TABLE II
MAb
Ganglioside used as immunogen
Ig isotype
GGBI’) GMRZ GMR7 GGR12 GMR5 GGR13
CD3 O-AC-CD3 CD2 GDlb GTlh GQlb
IgG3 (~1 IgM (~1 IgM (~1 IgG2h (K) IgM (~1 IgG’a (K)
C3H/HeN mice were immunized with purified ganglioside (10 pg. total amount per mouse) coated on acid-treated S. minnrsotu (250 pg. total amount per mouse) intraveneously five times on day 0. 4. 7. 1I and 21. After 3 days from the last injection. the spleen cells were fused with PA1 (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. Details are described in Materials and Methods.
187 A-2
‘\.\
B-l
B-2
c-2
1
zs3 Antigen
1s
2-’
2”
2-s 1” z-l3 dilution (nmol/well)
On the other hand, MAb GGB19 showed broad binding specificities, reacting predominantly with GD3, but slightly with three other gangliosides CO-AC-GD3, GQlb, and GTla). No other gangliosides were recognized. The epitope of the MAb GGB19 would be an external tri-saccharide (NeuAccu2 + 8NeuAcrr2 + 3Gal-) sequence in these gangliosides. Since extensive studies on the binding specificities of MAbs to GD3, 0-AC-GD3, and GD2 have previously been reported by several groups including our laboratory, those of the three MAbs GGR12 (anti-GDlb), GMRS (anti-GTlb), and GGR13 (anti-GQlb), were described in detail (Fig. 1). The reactivities of these six MAbs with various glycolipids determined by ELISA are summarized in Table III. TLC immunostaining of various authentic gangliosides with the hUbs We also determined the binding reactivities of these MAbs to authentic gangliosides including bovine brain gangliosides and Ml4 cells (human melanoma cell line)
2., 2-e 2-a 24O 2-‘1 1’ Antibody dilution
Fig. 1. Reactivities of three MAbs (GGR12, GMR5 and GGR13) with various authentic glycolipids in ELISA. A-l, B-l and C-l, wells were coated with each ganglioside at different concentrations and incubated with MAbs GGR12, GMR5 and GGR13, respectively. The supernatant of hybridoma was used as MAb. A-2, B-2 and C-2, wells were coated with each ganglioside (0.5 nmol/well) and incubated with different concentrations of the MAbs mentioned above. l, GDlb; W, GTlb; A, GQlb; 0, other b-pathway ganglioside (GD3, O-AC-GD3 and GD2); 0, a-pathway ganglioside (GM3, GM2, GMl, GDla and GTla); A, neutral glycolipid (LacCer. GgOssCer, GgOs,Cer, GbOssCer, GbOs,Cer and GbOs,Cer).
B-l
A-2
B-2
GM1 w GDla * GDlb * GTlb ) Origin +
TABLE
A-l
. _._.-
III
Reactit,ities Gangliosides
of MAbs with rbarious authentic
’
B-3
MAbs GGB19
GD3 O-AC-GD3 GD2 GDlb GTlb GQlb GTla
A-3
glycolipids
+++z + _ _ + ++
GMR2 +++ _ _ _ _ _
GMR7
GGR12
GMRS
GGR13
_
_
_ _
+++ _
_
_ _ _
+++ _ -
+++ -
+++ _ _
’ All of the gangliosides have NeuAc as their sialic acid moiety. No other glycolipids, such as a-pathway gangliosides (GM3, GM2, GM1 and GDla), c-pathway gangliosides (GT3 and GT2), gangliosides having NeuGc as their sialic acid moiety (GM3, GM2, GM1 and GD3). or the neutral glycolipids (LacCer, GbOsesCer, GbOse,Cer. GbOsesCer, GgOsesCer, GgOse,Cer) tested were detected. ’ + + +. strong: + +, moderate: + , weak or trace; - , negative.
GM1
*
e”e m
--
Origin * 1
2
3’
4
‘5‘
1
2
3
4
5
Fig. 2. TLC immunostaining of standard gangliosides with three MAbs (GGRl2, GMRS and GGR13). (A) Ganglioside fraction (10 nmol as sialic acid content) from bovine brain and from human melanoma cell line, Ml4 and the same amount of standard gangliosides were chromatographed with chloroform-methanol-0.22%CaClz in water (55:45: 10, v/v) and visualized with resorcinol. 1, gangliosides of bovine brain; 2, gangliosides of Ml4 cells; 3, standard ganglioside GDlb; 4, standard ganglioside GTlb; 5, standard ganglioside GQlb. (B) The same ganglioside fractions (total 1 nmol/lane) were chromatographed similarly and immunostained with three MAbs. 1, GGR12; 2. GMRS; 3, GGR13.
188 gangliosides that contain GM3, GM2, GD3, and GD2 as major gangliosides by enzyme-immunostaining on TLC (Fig. 2). The results agreed well with those obtained by an ELISA. MAbs GGR12, GMRS. and GGR13 reacted only with ganglioside GDlb, GTlb, and GQlb, respectively. MAbs GMR2 and GMR3 also reacted only with 0-AC-GD3 and GD2, respectively. In contrast, MAb GGB19 cross-reacted with O-AC-GD3, GQlb, and GTla, but not with any other glycolipids tested. The fine binding specificities of MAbs GGBI9 and GMR7 were similar to those of MAbs R24 and Al-201, respectively (data not shown).
TLC immunostaining of gangliosides from leukemia cell lines with h44bs The gangliosides from several leukemia cell lines (murine and rat) were developed on TLC and stained with these three MAbs (GGR12, GMRS, and GGR13). GDlb was detected in a cell line, RBL-2H3, but not in nine mouse leukemia cell lines tested (Fig. 3). Neither GTlb nor GQlb was detected in these cell lines. Also, none of GDlb, GTlb, and GQlb were detected in ten human leukemia cells (data not shown). On the other hand, GD2 was highly expressed on these several murine, rat, and human leukemia cells as previously described [ 101.
GDlb) Origin
>
:
Fig. 4. FACS analysis of gangliosides on leukemia cells. RBL-2H3 cells were incubated with MAh for 1 h at 4°C. followed by the addition of FITC-lab~lled goat anti-mouse IgG or IgM and incubated for another 30 min at 3°C. After washing, the cells were analyzed with a Beckton-Dickinson FACS IV. (A), (B) and (0 show the profiles of ganglioside expression stained with MAhs GGRI2, GMR5 and GGRl3. respectively. Dotted line. control; solid line stained with MAh.
““-
FACS analysis of leukemia cell lines The results of FACS analysis were quite similar to those of TLC immunostaining (Fig. 4). Onfy GDlb was expressed on rat leukemia cells, RBL-2H3. Neither GTlb. nor GQlb was detected in the cells. None of GgOse,Cer group such as GDlb, GTlb, and GQlb were expressed on these mouse and human leukemia cell lines (data not shown).
C
Discussion
GQlb~ Origint 1
2
3
4
Fig. 3. lmmunostaining on TLC of gangliosides from mouse, rat, and human leukemia cell lines. Ganglioside fractions equivaient to 5 mg of tissue (wet weight) from tumor cell lines were chromatographed with chloroform-methanol-0.22% CaCI, in water (55 : 45 : 10, v/v) and immunostained with three MAbs (GGR12, GMR5, and GGR13). The details are as described in Materials and Methods. (A), (B) and (C) were stained with MAbs GGR12, GMRS, and GGR13, respectively. 1, gangliosides of bovine brain; 2, mixtures of standard ganghosides GDlb, GTlb and GQlb; 3, RBL-2H3; 4, EIA. Left ordinate, migration of ganglioside standards. The patterns of other leukemia cell lines (RBW, BALBRVE, YACl, B6RV1, RLFS, 18-4, I-29, RLMI, Jurkat, MOLT-4, MT- I, CCSF-CEM, IM-9, Raji, HL-60, HutiO2. Hut78 and HPB-ALL) were similar to that of EL4.
We have been successful in generating one set of MAbs specific for b-pathway ganglio-series gangliosides by immunizing the mice with these purified gangliosides adsorbed to S. minnesota. C3H/HeN mice were chosen instead of BALB/c or C57BL/6 since we have recently found that these mice produced maximum antibody titiers to gangliosides among the ten different strains of inbred mice. Our results presented in this paper have proved that these mice are more suitable than other strains of mice for raising MAbs to these gangliosides. Small amount of purified ganglioside (IO pg) was enough for the generation of these MAbs in our protocol. Several different immunization protocols to generate murine MAbs to gangliosides have been reported
189 [7]. In general,.BALB/c
or C57BL/6 mice have been immunized with either tumor cells, purified gangliosides adsorbed to bacteria, or those-containing liposomes. It has been assumed that cells rich in ganglioside are better immunogens than purified gangliosides for producing anti-ganglioside MAbs [ 191. A number of MAbs to b-pathway gangliosides have already been generated by several groups with different methods [20-241. MAb specific for ganglioside GTlb has, however, not been reported yet. This is the first description of one set of MAbs specific for b-pathway gangliosides including GTlb. Five MAbs (GMR2, GMR7, GGR12, GMRS, and GGR13) showed restricted binding specificities, reacting only with gangliosides used as immunogens. None of the other gangliosides or neutral glycolipids reacted. For example, the MAb GMRS, which was established by immunizing the mice with purified GTlb, reacted only with GTlb among the gangliosides thus far tested. Gangliosides that differ by the addition of one Nacetylneuraminic acid (GQlb) or the loss of one Nacetylneuraminic acid (GDlb or GDla) were not recognized by the MAb. These findings suggest that the terminal disaccharide (NeuAca2 + 3Gal) sequence and the disialosyl residue (NeuAccu2 -+ 8NeuAc-1 linked to the inner galactose moiety are involved in the binding specificity. Other four MAbs also showed highly restricted binding specificities. In contrast, we could not establish a MAb specific for ganglioside GD3. MAb GGB19 weakly cross-reacted with O-AC-GD3, GQlb, and GTIa, although none of other gangliosides tested were recognized. The fine binding specificity of MAb GGB19 was quite similar to that of MAb R24 [15]. It may be difficult to generate a MAb specific for GD3, since a number of gangliosides share the terminal structure of ganglioside GD3, i.e., NeuAca2 + 8NeuAccu2 + 3Gal-. Previously, we reported that GD2 was expressed on mouse and human leukemia cells, in particular, T-cell lines using murine MAbs specific for GD2 [lo]. In this study, we determined whether GDlb, GTlbl and GQlb are expressed on these leukemia cells. None of these gangliosides were expressed on these cells except a rat leukemia cell line (RBL-2H3). This cell line expressed both GD2 and GDlb, but not GTlb or GQlb. These results suggested that a galactosyl transferase synthesizing GDlb from GD2 is active in the rat leukemia cell line, but not in other GD2-positive cells. Although we also determined the expression of GDlb, GTlb, and GQlb on a number of human neuroectoderm-derived tumor cells such as melanomas, neuroblastomas and gliomas, none of these gangliosides were expressed on these human cells (Tai, T., et al., unpublished observation). To the best of our knowledge, this is the first description of cells that express high amounts of GDlb. The precise ganglioside composition ex-
pressed on the cells remains to be studied. On the other hand, it has previously been reported that a few mouse and rat neuroectoderm-derived tumor cells such as Neuro2a and PC12 express GTlb and/or GQlb [24,25]. Gangliosides have been proposed not only to be cell surface receptors for certain bacterial toxins and viruses, but also to regulate cellular functions including neuronal cell adhesion, neurite outgrowth, and differentiation [26]. GTlb and GQlb have been reported as the receptors for tetanus toxins [27,28]. However, a precise relationship between the gangliosides and toxins remains unclear. Recently, Tiemeyer et al. reported that rat brain membranes contain a high affinity, structurally specific and saturable binding protein (ganglioside receptor) for major brain gangliosides related to GTlb [29]. The MAbs specific for GTlb and GQlb developed in this study would be a powerful reagent for elucidating the biological functions of these gangliosides. Also, they would be useful for the investigation of the localization of these gangliosides in the mammalian central nervous system, since developmental changes in ganglioside composition in rat brain have been reported by Yu et al. [30]. An immunohistochemical study on the localization of these gangliosides in rat brain is now in progress. The method used in this study may be generally applicable to the generation of murine MAbs to other gangliosides. In fact, we have recently generated hybridomas producing MAbs specific for gangliosides having N-glycolylneuraminic acid such as GM3 (NeuGc-1, GM2(NeuGc), and GD3(NeuGc-NeuGc-) by immunizing the mice with these purified gangliosides (H. Ozawa, et al., manuscript in preparation). Acknowledgements
The authors thank Prof. T. Yamakawa (Tokyo College of Pharmacy) and Prof. Y. Nagai (The Tokyo Metropolitan Institute of Medical Science) for their valuable suggestions and support. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant of The Naito Foundation. References Hakomori, S. (1985) Cancer Res. 45, 2405-2415. Feizi, T. (1985) Houghton, A.N. 165-179. Reisfeld, A. and Herlyn. M. and 283-308. Lloyd, K.O. and
Nature 314, 53-57. and Scheinberg, D.A. (1986) Seminars
Oncol.
13,
Cheresh, D.A. (1987) Adv. Immunol. 4, 323-377. Koprowski, H. (1988) Annu. Rev. Immunol. 6, Old, L.J. (1989) Cancer
Res. 49, 3445-3451.
7 Karma& R. and Hakomori, S. (1986) in Handbook of Experimental Immunology, (Weir. D.M. Herzenberg. L.A. and Blackwell, C.C. eds) Vol. 4, I17 pp.. Blackwell, Oxford. X Tai. T., Paulson. J.C., Cahan, L.D. and Irir. R.F. (19X3) Proc. Natl. Acad. Sci. U.S.A. 80, 5397-5396. 9 Yu, R.K. and Ando. S. (1980) in Structure and Functions of Gangliosides (Svennerholm, L.. Dreyfus, H. and Urban, P.F.. rdal pp. 33-45. Plenum. New York. 10 Kawashima. I., Tada, N., Ikegami, S.. Nakamura. S.. Ueda. R. and Tai. T. (19881 Int. J Cancer 31. 267-774. 1 I Svennerholm, L. and Fredman, P. (1980) Biochim. Biophys. Acta 617,97-109. 11 Cialanos, C.. Luderitz. 0. and Westphal. 0. (1971) Eur. J Biochem. 24. 116-121. 13 Young Jr.. W.W., Macdonald, E.M.S., Nowinski. R.C. and Hakomori. S. (1979) J. Exp. Med. 150. 100%11119. I4 Tai. T.. Kawashima. I., Tada. N. and Dairiki. K. (19Xx) J Biochem. 103. 682-687. I5 Tai. T.. Kawashima. I., Furukawa. K. and Lloyd, K.O. tI9XXl Arch. Biochem. Biophys. 260. 5 I-55. 16 Tai. T.. Cahan, L.D.. Paulson. J.C.. Saxton. R.E. and Irie. R.F. (19841 J. Natl. Cancer Inst. 73. 627-633. 17 Tai. T., Sze. L.. Kawashima. I.. Saxton. R.E. and Irie, R.F. ( IYX7l J. Biol. Chem. 256. 6X03-6807. IX Ozawa. H., Iwaguchi. T. and Kataoka. T. ( IYXh)Cancer Immunol. Immunother. 23, 73-77. 1Y He. X., Wikstrand. C.J., Fredman. P., Mansson. J.E.. Svennerhelm, L. and Bigner, D.D. (1989) Acta Neuropathol. 79. 317-325.
20 Pukel. C.S., Lloyd. K.O.. Travassoa. L.R., Dippold, W.ti., Oettgen. H.F. and Old. L.J. (19x2) J. Exp. Med. 155. 113%1147. 71 Chcung, N.H.. Saarincn. U.M.. Neely. J.E.. Landmeier. B.. Donovan. D. and Coccia. P.F. (19X5) Cancer Res. 45, 764?-264Y. 27 Cherrsh. D.A.. Varki. A.P.. Varki. N.M.. Stallcup. W.B., I.evine. J. and Reisfeld. R.A. (19X-t) J. Biol. Chcm. 759, 735%745s. 73 Fredman. P.. Jeansson. S.. Lyckc. E. and Svennerholm. 1.. llYX5l FEBS Lett. 1X9. 23-26. 14 Yamamoto. II.. Tsuji, S. and Nagai. Y. (IYYO) J. Neurochcm. 54. 513-519. 75 Walton. K.M.. Sandhrrg, K., Rogers. T.B. and Schnaar. R.L.. ( 19XXl J. Biol. Chem. 263. 1055-2063. 26 Hakomori. S. (10X1) Annu. Rev. Biochem. 50. 733-76-1. 27 Rogers. T.B. and Snyder. S.H. (19x1) J. Biol. Chcm. 2%. 24022107. 2X Holmgren. J.H.. Elwing, P.. Fredman, P. and Svenncrholm. L. (19X0) Eur. J. Biochem. 106, 371-379. 29 Tiemeyer. M., Yasuda. Y. and Schnaar, R.L. (19X9) J. Biol. Chem. 264. 1671-16X1. 30 Yu. R.K.. Macala, L.J.. Taki. T.. Weinfeld. I1.M. and Yu. F.S. (19Xx) J Neurochem. 50. 1X?- 1x20. 31 Svcnnerholm. L. (1961) J. Lipid Res. 5. 145-155. 31 ILJPAC-IUB Commission on Biochemical Nomenclature (1977) Eur. J. Biochem. 79. I l-21. 33 Kawashima. I.. Nakamura, 0. and Tai. T. (IWI ) Mol. Immunol.. in press.
BBALIP
Science
Niodl/,rlrc~a C’f Hir~ph\‘riclr :lc~/a. I 12.3( I YY.2) I s-l- I’)11 Publishers B.V. All rights rrselxxxi 01105-77h1~/‘Y2,~Ro.5.0
53796
Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides Hideki Ozawa, Masaharu
Kotani,
Ikuo Kawashima
and Tadashi
Tai
The Tokyo Metropolitan Instifute of’Medicu1 Science. Depur~mer~t CJ~Twnor /nznmr~do~~. Honk(,mrr,sor,~~,,Hunkyo-ku. Tokyo iJuparr I (Received
Key words:
Monoclonal
Zh June
19Yl)
antibody:
Ganglioside
We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGBl9, GMR2, GMR7, GGR12, GMRS, and GGR13 reacted strongly with the gangliosides GD3, O-AC-GD3, GD2, GDlb, GTlb, and GQlb, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-AC-GD3, GQlb, and GTla, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GDlb, GTlb, and GQlb on mouse, rat, and human leukemia ceils. GDlb was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GTlb nor GQlb was detected in these cell lines.
Introduction Ganglio-series gangliosides are highly expressed on neuroectodermal origined tissues. Recently, some of them have also been identified as tumor-associated antigens expressed on the surface of these tissue-derived tumors such as melanomas, neuroblastomas, and gliomas [ 1,2]. Monoclonal antibodies (MAbs) against these gangliosides have been shown to be powerful reagents not only in the diagnosis and treatment of cancer patients, but also in analyzing biological functions of gangliosides on cell surface membranes [3-61. In the past decade, these MAbs have generally been
Abbreviations: ELISA, enzyme-linked immunosorbent assay: MAb, monoclonal antibody: TLC, thin-layer chromatography. FITC; fluorescein isothiocyanate, FACS; fluorescence activated cell sorter, PBS: phosphate-buffered saline. Sialic acid moiety of the gangliosides was NeuAc unless otherwise noted. Gangliosides are named according to the nomenclature of Svennerholm 1311. Otherwise. the nomenclature used follows the IUPAC-IUB recommendations [321. Correspondence: T. Tai. The Tokyo Metropolitan Institute cal Science, Honkomagome, Bunkyo-ku, Tokyo 113. Japan.
of Medi-
established by immunizing BALB/c or C57BL/6 mice with neuroectoderm-derived tumor cells [7]. It is. however, still difficult to routinely generate murine MAbs to gangliosides by immunizing mice directly with purified gangliosides. In order to develop a Tethod for generating MAbs with high efficiency, we recently compared antibody responses to ganglio-series gangliosides in different strains of inbred mice, including BALB/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, etc. We have found that (i) b-pathway gangliosides (GD3, O-AC-GD3, GD2, GDlb, GTlb, and GQlb) induced high-titer antibody responses, whereas a-pathway gangliosides (GM3, GM2, GMl, GDla, and GTla) induced low-titer antibody responses; (ii) C3H/HeN and A/J mice demonstrated maximum antibody responses against these gangliosides among these mice, although the pattern of reactivity to the various ganglioside was similar in all the strains tested [33]. These findings suggest that C3H/HeN or A/J mice are more suitable than other strains of mice for raising MAbs to these gangliosides. The present study described the production of one set of MAbs specific for b-pathway ganglio-series gangliosides. These MAbs are expected to be powerful reagents in analyzing the biological
185
functions and localization of these gangliosides on cells and tissues, in particular, neuroectoderm-derived cells.
tron. The structure of ganglio-series gangliosides used in this study are shown in Table I. Tumor cell lines
Materials
and Methods
Glycosphingolipids
GMl, GDla, GDlb, and GTlb were prepared from bovine brain. GM3 and GQlb (bovine brain), and GD3 (bovine milk) were purchased from Iatron (Tokyo, Japan). GM2 and GD2 were prepared from GM1 and GDlb, respectively, by the treatment of bovine testis P-galactosidase (Boehringer-Mannheim-Yamanouchi, Tokyo, Japan) as described previously [S]. 9-0Acetylated GD3 (0-AC-GD3) was isolated from a human melanoma cell line, Ml4 transplanted in nude mice (nu/nu BALB/c) and the carbohydrate structure was confirmed with FAB-MASS and NMR spectroscopy by M. Suzuki and A. Suzuki, and F. Inagaki (The Tokyo Metropolitan Institute of Medical Science), respectively. Codfish brain gangliosides (mixtures of GT3, GT2, and other polysialosyl gangliosides) [9] and GTla (monkey brain) were gifts from S. Ando (The Tokyo Metropolitan Institute of Gerontology). GM4 (human myelin) was provided by T. Ariga (Virginia Commonwealth University). Sialylparagloboside (SPG) was isolated from human erythrocytes. GM3(NeuGc), GM2(NeuGc), and GMl(NeuGc) of mouse liver and GD3(NeuGc-NeuGc-) of bear erythrocytes were donated by Y. Hashimoto and A. Suzuki (The Tokyo Metropolitan Institute of Medical Science). LacCer, GgOse,Cer, and GgOse,Cer were prepared from GM3, GM2, and GMl, respectively, by treatment with sialidase as previously described [S]. GbOse,Cer, GbOse,Cer, and GbOse,Cer were purchased from Ia-
Murine leukemia cells (EL& RBLS, BALBRVE, YACl, B6RV1, RLF8, 18-4, I-29, and RLMl) and human leukemia cells (MOLT-4, MT-l, Jurkat, CCSFCEM, IM-9, Raji, HL-60, Hut102, Hut78, and HPBALL) were supplied by Dr. N. Tada (Tokai University) and Dr. Y. Yamamoto (The Tokyo Metropolitan Institute of Medical Science), respectively, as previously described [lo]. The following cell lines were provided by the Japanese Cancer research Resources Bank, Tokyo: RBL-2H3 (rat leukemia) and PA1 (BALB/c mouse-derived myeloma). These cell lines were cultured in RPM11640 with 10% heat-inactivated fetal calf serum (Hyclone, Logan, UT, U.S.A.). Glycolipids from tumor cells were extracted by the method of Svennerholm and Fredman [ 111. Immunization Salmonella minnesota mutant R595 was kindly sup-
plied by T. Yasuda (Okayama University School of Medicine) and T. Tomita (Institute of Medical Science, University of Tokyo). Female mice of C3H/HeN were purchased from Japan Clea (Tokyo, Japan). Mice of the inbred strain (7 weeks of age) were immunized with purified ganglioside (10 pg, total amount per mouse) adsorbed to acid-treated S. minnesota (250 pg, total amount per mouse) by the procedure originally described by Galanos et al. [12] and later modified by Young et al. [13]. Each mouse received the intraveneous injections of antigen-bacteria complex on day 0, 4, 7, 11, and 21. The spleen cells were obtained 3 days after the last injection and were fused with a myeloma
TABLE I Structures of ganglio-series gangliosides used in this study
Ganglioside (a-pathway) GM3 GM2 GM1 GDla GTla (b-pathway) GD3 0-AC-GD3 GD2 GDlb GTlb GQlb k-pathway) GT3 GT2
Structure
GalNAcPl
NeuAccu2 + 3GalPl+ 4Glc-Cer * 3)Galpl--) 4Glc-Cer 4(NeuAccu2 -t 3)Galfil+ 4Glc-Cer 4(NeuAca2 + 3)GalPl+ 4Glc-Cer 4(NeuAca2 + 3)GalPl+ 4Glc-Cer
+ 4(NeuAca2
Gal/31 --f 3GalNAcpl* NeuAca2 + 3GalPl--) 3GalNAcPl+ NeuAccr2 + 8NeuAca2 + 3Galfil--) 3GalNAc@l+
NeuAca 2 + 8NeuAca 2 + 3GalP 1 + 0-Ac-NeuAca2 + BNeuAccu2 --t 3GalPl + GalNAcPl * 4(NeuAca2 --f BNeuAccu2 + 3Xial/31+ Galpl + 3GalNAc$l+ 4(NeuAcm2 + BNeuAca2 + 3jGal/31+ NeuAccu2 + 3Galpl-+ 3GalNAcPl --f 4(NeuAccY2 + BNeuAca2 + 3)Galpl+ NeuAccu2 -P BNeuAccu2 + 3Galpl+ 3GalNAcPl + 4(NeuAca2 + BNeuAca2 + 3)Gal@l-+ NeuAca2 + 8NeuAm2 + BNeuAcLu2 + 3GalPl-+ GalNAcpl
+ 4cNeuAccu2 + 8NeuAca2 + BNeuAca2 + 3)Galpl+
4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer 4Glc-Cer
186 cell line (PAI) by a routine cell hybridization procedure as previously described [lo]. The hybridoma fusions were screened against the immunizing ganglioside. The antibody titers in the supernatant of hybridoma cells were determined by ELISA. The positive hybrid was cloned by limiting dilution. The isotype of antibody produced by hybridoma was determined by a mouse monoclonal antibody kit (Amersham, U.K.). Murine MAb Al-201 (IgM, K) was established as prcviously described [14]. MAb R24 (IgG3. K) was a kind gift from Dr. K.O. Lloyd (Memorial Sloan-Kettering Cancer Institute) [ 151. Enzyme-linked immunosorbent assay (ELISA) Solid-phase ELISA was performed as previously described [16]. A 96 well polystyrene microtiter plate (Dynatech, Alexandria, VA, U.S.A.) was used. Glycolipid (0.5 nmol) was serially diluted in ethanol and applied to the plate in a vol of 50 pi/well. 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 IgG (y-chain specific) or IgM (p-chain specific) (Cappel, Malvern, PA, U.S.A.), was added to the plates and incubated for another 1 h at room temperature. The absorbance at 405 nm was determined by a Bio-rad ELISA plate reader. Three samples of each experiment were tested. The standard deviation was less than 10% for all values. Background values of absorbance at 405 nm were less than 0.05. Thin-layer chromatography (TLC) Merck precoated TLC-plastic sheets (Merck, Darmstadt, F.R.G.) were used for fractionation of gangliosides [lo]. The solvent system used for developing chromatograms was chloroform-methanol-0.22% CaCl 2 in water (55 : 45 : 10, v/v). Gangliosides were visualized with resorcinol stain and neutral glycolipids were visualized with orcinol stain. En,yme immunostaining on TLC plates Immunostaining on TLC plates was performed as previously described [17]. 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% Tween20 and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or IgM for 2 h at room temperature. After washing the chromatogram again, a solution of 400 pg/ml o-phenylenediamine (Sigma, St. Louis, MO, U.S.A.) in 80 mM citrate-phosphate buffer, pH
5.0, containing O.l2%H ,O, was added. Color Jcvelopment was stopped after 15 min by washing the plate by dipping it in PBS. Flow cytofluorometric analysis Cell surface gangliosides were detected by l-low cytometry [18]. Cells (5 X IO” 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 (FIT(Z)-conjugated goat anti-mouse IgG (ychain specific. Cappel) or IgM (p-chain specific, Cappel) and incubated for another 30 min at 4°C. After washing with PBS at 4°C. the cells were analyzed with a flow cytometer, FACS IV (Becton Dickinson FACS Systems, Sunnyvale, CA, U.S.A.). Results
Establishment of M2bs specific for b-pathway ganglioseries gangliosides We finally established six hybridoma clones producing MAbs specific for b-pathway ganglio-series gangliosides (Table II). Three MAbs (GMR2, GMR7, and GMRS) were of IgM (K) isotype. On the other hand, MAbs GGB19, GGR12, and GGR13 were of IgG3 (K). IgG2b (~1, and IgG2a (~1. respectively. ELISA with L,arious authentic glycolipids ELISA was performed with 23 authentic glycolipids bound to microtiter wells. Among six MAbs, five MAbs tGMR2, GMR7. GGR12, GMRS, and GGR13) showed highly restricted binding specificities, detecting only the ganglioside used as immunogen. None of other gangliosides or neutral glycolipids so far tested reacted.
TABLE II
MAb
Ganglioside used as immunogen
Ig isotype
GGBI’) GMRZ GMR7 GGR12 GMR5 GGR13
CD3 O-AC-CD3 CD2 GDlb GTlh GQlb
IgG3 (~1 IgM (~1 IgM (~1 IgG2h (K) IgM (~1 IgG’a (K)
C3H/HeN mice were immunized with purified ganglioside (10 pg. total amount per mouse) coated on acid-treated S. minnrsotu (250 pg. total amount per mouse) intraveneously five times on day 0. 4. 7. 1I and 21. After 3 days from the last injection. the spleen cells were fused with PA1 (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. Details are described in Materials and Methods.
187 A-2
‘\.\
B-l
B-2
c-2
1
zs3 Antigen
1s
2-’
2”
2-s 1” z-l3 dilution (nmol/well)
On the other hand, MAb GGB19 showed broad binding specificities, reacting predominantly with GD3, but slightly with three other gangliosides CO-AC-GD3, GQlb, and GTla). No other gangliosides were recognized. The epitope of the MAb GGB19 would be an external tri-saccharide (NeuAccu2 + 8NeuAcrr2 + 3Gal-) sequence in these gangliosides. Since extensive studies on the binding specificities of MAbs to GD3, 0-AC-GD3, and GD2 have previously been reported by several groups including our laboratory, those of the three MAbs GGR12 (anti-GDlb), GMRS (anti-GTlb), and GGR13 (anti-GQlb), were described in detail (Fig. 1). The reactivities of these six MAbs with various glycolipids determined by ELISA are summarized in Table III. TLC immunostaining of various authentic gangliosides with the hUbs We also determined the binding reactivities of these MAbs to authentic gangliosides including bovine brain gangliosides and Ml4 cells (human melanoma cell line)
2., 2-e 2-a 24O 2-‘1 1’ Antibody dilution
Fig. 1. Reactivities of three MAbs (GGR12, GMR5 and GGR13) with various authentic glycolipids in ELISA. A-l, B-l and C-l, wells were coated with each ganglioside at different concentrations and incubated with MAbs GGR12, GMR5 and GGR13, respectively. The supernatant of hybridoma was used as MAb. A-2, B-2 and C-2, wells were coated with each ganglioside (0.5 nmol/well) and incubated with different concentrations of the MAbs mentioned above. l, GDlb; W, GTlb; A, GQlb; 0, other b-pathway ganglioside (GD3, O-AC-GD3 and GD2); 0, a-pathway ganglioside (GM3, GM2, GMl, GDla and GTla); A, neutral glycolipid (LacCer. GgOssCer, GgOs,Cer, GbOssCer, GbOs,Cer and GbOs,Cer).
B-l
A-2
B-2
GM1 w GDla * GDlb * GTlb ) Origin +
TABLE
A-l
. _._.-
III
Reactit,ities Gangliosides
of MAbs with rbarious authentic
’
B-3
MAbs GGB19
GD3 O-AC-GD3 GD2 GDlb GTlb GQlb GTla
A-3
glycolipids
+++z + _ _ + ++
GMR2 +++ _ _ _ _ _
GMR7
GGR12
GMRS
GGR13
_
_
_ _
+++ _
_
_ _ _
+++ _ -
+++ -
+++ _ _
’ All of the gangliosides have NeuAc as their sialic acid moiety. No other glycolipids, such as a-pathway gangliosides (GM3, GM2, GM1 and GDla), c-pathway gangliosides (GT3 and GT2), gangliosides having NeuGc as their sialic acid moiety (GM3, GM2, GM1 and GD3). or the neutral glycolipids (LacCer, GbOsesCer, GbOse,Cer. GbOsesCer, GgOsesCer, GgOse,Cer) tested were detected. ’ + + +. strong: + +, moderate: + , weak or trace; - , negative.
GM1
*
e”e m
--
Origin * 1
2
3’
4
‘5‘
1
2
3
4
5
Fig. 2. TLC immunostaining of standard gangliosides with three MAbs (GGRl2, GMRS and GGR13). (A) Ganglioside fraction (10 nmol as sialic acid content) from bovine brain and from human melanoma cell line, Ml4 and the same amount of standard gangliosides were chromatographed with chloroform-methanol-0.22%CaClz in water (55:45: 10, v/v) and visualized with resorcinol. 1, gangliosides of bovine brain; 2, gangliosides of Ml4 cells; 3, standard ganglioside GDlb; 4, standard ganglioside GTlb; 5, standard ganglioside GQlb. (B) The same ganglioside fractions (total 1 nmol/lane) were chromatographed similarly and immunostained with three MAbs. 1, GGR12; 2. GMRS; 3, GGR13.
188 gangliosides that contain GM3, GM2, GD3, and GD2 as major gangliosides by enzyme-immunostaining on TLC (Fig. 2). The results agreed well with those obtained by an ELISA. MAbs GGR12, GMRS. and GGR13 reacted only with ganglioside GDlb, GTlb, and GQlb, respectively. MAbs GMR2 and GMR3 also reacted only with 0-AC-GD3 and GD2, respectively. In contrast, MAb GGB19 cross-reacted with O-AC-GD3, GQlb, and GTla, but not with any other glycolipids tested. The fine binding specificities of MAbs GGBI9 and GMR7 were similar to those of MAbs R24 and Al-201, respectively (data not shown).
TLC immunostaining of gangliosides from leukemia cell lines with h44bs The gangliosides from several leukemia cell lines (murine and rat) were developed on TLC and stained with these three MAbs (GGR12, GMRS, and GGR13). GDlb was detected in a cell line, RBL-2H3, but not in nine mouse leukemia cell lines tested (Fig. 3). Neither GTlb nor GQlb was detected in these cell lines. Also, none of GDlb, GTlb, and GQlb were detected in ten human leukemia cells (data not shown). On the other hand, GD2 was highly expressed on these several murine, rat, and human leukemia cells as previously described [ 101.
GDlb) Origin
>
:
Fig. 4. FACS analysis of gangliosides on leukemia cells. RBL-2H3 cells were incubated with MAh for 1 h at 4°C. followed by the addition of FITC-lab~lled goat anti-mouse IgG or IgM and incubated for another 30 min at 3°C. After washing, the cells were analyzed with a Beckton-Dickinson FACS IV. (A), (B) and (0 show the profiles of ganglioside expression stained with MAhs GGRI2, GMR5 and GGRl3. respectively. Dotted line. control; solid line stained with MAh.
““-
FACS analysis of leukemia cell lines The results of FACS analysis were quite similar to those of TLC immunostaining (Fig. 4). Onfy GDlb was expressed on rat leukemia cells, RBL-2H3. Neither GTlb. nor GQlb was detected in the cells. None of GgOse,Cer group such as GDlb, GTlb, and GQlb were expressed on these mouse and human leukemia cell lines (data not shown).
C
Discussion
GQlb~ Origint 1
2
3
4
Fig. 3. lmmunostaining on TLC of gangliosides from mouse, rat, and human leukemia cell lines. Ganglioside fractions equivaient to 5 mg of tissue (wet weight) from tumor cell lines were chromatographed with chloroform-methanol-0.22% CaCI, in water (55 : 45 : 10, v/v) and immunostained with three MAbs (GGR12, GMR5, and GGR13). The details are as described in Materials and Methods. (A), (B) and (C) were stained with MAbs GGR12, GMRS, and GGR13, respectively. 1, gangliosides of bovine brain; 2, mixtures of standard ganghosides GDlb, GTlb and GQlb; 3, RBL-2H3; 4, EIA. Left ordinate, migration of ganglioside standards. The patterns of other leukemia cell lines (RBW, BALBRVE, YACl, B6RV1, RLFS, 18-4, I-29, RLMI, Jurkat, MOLT-4, MT- I, CCSF-CEM, IM-9, Raji, HL-60, HutiO2. Hut78 and HPB-ALL) were similar to that of EL4.
We have been successful in generating one set of MAbs specific for b-pathway ganglio-series gangliosides by immunizing the mice with these purified gangliosides adsorbed to S. minnesota. C3H/HeN mice were chosen instead of BALB/c or C57BL/6 since we have recently found that these mice produced maximum antibody titiers to gangliosides among the ten different strains of inbred mice. Our results presented in this paper have proved that these mice are more suitable than other strains of mice for raising MAbs to these gangliosides. Small amount of purified ganglioside (IO pg) was enough for the generation of these MAbs in our protocol. Several different immunization protocols to generate murine MAbs to gangliosides have been reported
189 [7]. In general,.BALB/c
or C57BL/6 mice have been immunized with either tumor cells, purified gangliosides adsorbed to bacteria, or those-containing liposomes. It has been assumed that cells rich in ganglioside are better immunogens than purified gangliosides for producing anti-ganglioside MAbs [ 191. A number of MAbs to b-pathway gangliosides have already been generated by several groups with different methods [20-241. MAb specific for ganglioside GTlb has, however, not been reported yet. This is the first description of one set of MAbs specific for b-pathway gangliosides including GTlb. Five MAbs (GMR2, GMR7, GGR12, GMRS, and GGR13) showed restricted binding specificities, reacting only with gangliosides used as immunogens. None of the other gangliosides or neutral glycolipids reacted. For example, the MAb GMRS, which was established by immunizing the mice with purified GTlb, reacted only with GTlb among the gangliosides thus far tested. Gangliosides that differ by the addition of one Nacetylneuraminic acid (GQlb) or the loss of one Nacetylneuraminic acid (GDlb or GDla) were not recognized by the MAb. These findings suggest that the terminal disaccharide (NeuAca2 + 3Gal) sequence and the disialosyl residue (NeuAccu2 -+ 8NeuAc-1 linked to the inner galactose moiety are involved in the binding specificity. Other four MAbs also showed highly restricted binding specificities. In contrast, we could not establish a MAb specific for ganglioside GD3. MAb GGB19 weakly cross-reacted with O-AC-GD3, GQlb, and GTIa, although none of other gangliosides tested were recognized. The fine binding specificity of MAb GGB19 was quite similar to that of MAb R24 [15]. It may be difficult to generate a MAb specific for GD3, since a number of gangliosides share the terminal structure of ganglioside GD3, i.e., NeuAca2 + 8NeuAccu2 + 3Gal-. Previously, we reported that GD2 was expressed on mouse and human leukemia cells, in particular, T-cell lines using murine MAbs specific for GD2 [lo]. In this study, we determined whether GDlb, GTlbl and GQlb are expressed on these leukemia cells. None of these gangliosides were expressed on these cells except a rat leukemia cell line (RBL-2H3). This cell line expressed both GD2 and GDlb, but not GTlb or GQlb. These results suggested that a galactosyl transferase synthesizing GDlb from GD2 is active in the rat leukemia cell line, but not in other GD2-positive cells. Although we also determined the expression of GDlb, GTlb, and GQlb on a number of human neuroectoderm-derived tumor cells such as melanomas, neuroblastomas and gliomas, none of these gangliosides were expressed on these human cells (Tai, T., et al., unpublished observation). To the best of our knowledge, this is the first description of cells that express high amounts of GDlb. The precise ganglioside composition ex-
pressed on the cells remains to be studied. On the other hand, it has previously been reported that a few mouse and rat neuroectoderm-derived tumor cells such as Neuro2a and PC12 express GTlb and/or GQlb [24,25]. Gangliosides have been proposed not only to be cell surface receptors for certain bacterial toxins and viruses, but also to regulate cellular functions including neuronal cell adhesion, neurite outgrowth, and differentiation [26]. GTlb and GQlb have been reported as the receptors for tetanus toxins [27,28]. However, a precise relationship between the gangliosides and toxins remains unclear. Recently, Tiemeyer et al. reported that rat brain membranes contain a high affinity, structurally specific and saturable binding protein (ganglioside receptor) for major brain gangliosides related to GTlb [29]. The MAbs specific for GTlb and GQlb developed in this study would be a powerful reagent for elucidating the biological functions of these gangliosides. Also, they would be useful for the investigation of the localization of these gangliosides in the mammalian central nervous system, since developmental changes in ganglioside composition in rat brain have been reported by Yu et al. [30]. An immunohistochemical study on the localization of these gangliosides in rat brain is now in progress. The method used in this study may be generally applicable to the generation of murine MAbs to other gangliosides. In fact, we have recently generated hybridomas producing MAbs specific for gangliosides having N-glycolylneuraminic acid such as GM3 (NeuGc-1, GM2(NeuGc), and GD3(NeuGc-NeuGc-) by immunizing the mice with these purified gangliosides (H. Ozawa, et al., manuscript in preparation). Acknowledgements
The authors thank Prof. T. Yamakawa (Tokyo College of Pharmacy) and Prof. Y. Nagai (The Tokyo Metropolitan Institute of Medical Science) for their valuable suggestions and support. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant of The Naito Foundation. References Hakomori, S. (1985) Cancer Res. 45, 2405-2415. Feizi, T. (1985) Houghton, A.N. 165-179. Reisfeld, A. and Herlyn. M. and 283-308. Lloyd, K.O. and
Nature 314, 53-57. and Scheinberg, D.A. (1986) Seminars
Oncol.
13,
Cheresh, D.A. (1987) Adv. Immunol. 4, 323-377. Koprowski, H. (1988) Annu. Rev. Immunol. 6, Old, L.J. (1989) Cancer
Res. 49, 3445-3451.
7 Karma& R. and Hakomori, S. (1986) in Handbook of Experimental Immunology, (Weir. D.M. Herzenberg. L.A. and Blackwell, C.C. eds) Vol. 4, I17 pp.. Blackwell, Oxford. X Tai. T., Paulson. J.C., Cahan, L.D. and Irir. R.F. (19X3) Proc. Natl. Acad. Sci. U.S.A. 80, 5397-5396. 9 Yu, R.K. and Ando. S. (1980) in Structure and Functions of Gangliosides (Svennerholm, L.. Dreyfus, H. and Urban, P.F.. rdal pp. 33-45. Plenum. New York. 10 Kawashima. I., Tada, N., Ikegami, S.. Nakamura. S.. Ueda. R. and Tai. T. (19881 Int. J Cancer 31. 267-774. 1 I Svennerholm, L. and Fredman, P. (1980) Biochim. Biophys. Acta 617,97-109. 11 Cialanos, C.. Luderitz. 0. and Westphal. 0. (1971) Eur. J Biochem. 24. 116-121. 13 Young Jr.. W.W., Macdonald, E.M.S., Nowinski. R.C. and Hakomori. S. (1979) J. Exp. Med. 150. 100%11119. I4 Tai. T.. Kawashima. I., Tada. N. and Dairiki. K. (19Xx) J Biochem. 103. 682-687. I5 Tai. T.. Kawashima. I., Furukawa. K. and Lloyd, K.O. tI9XXl Arch. Biochem. Biophys. 260. 5 I-55. 16 Tai. T.. Cahan, L.D.. Paulson. J.C.. Saxton. R.E. and Irie. R.F. (19841 J. Natl. Cancer Inst. 73. 627-633. 17 Tai. T., Sze. L.. Kawashima. I.. Saxton. R.E. and Irie, R.F. ( IYX7l J. Biol. Chem. 256. 6X03-6807. IX Ozawa. H., Iwaguchi. T. and Kataoka. T. ( IYXh)Cancer Immunol. Immunother. 23, 73-77. 1Y He. X., Wikstrand. C.J., Fredman. P., Mansson. J.E.. Svennerhelm, L. and Bigner, D.D. (1989) Acta Neuropathol. 79. 317-325.
20 Pukel. C.S., Lloyd. K.O.. Travassoa. L.R., Dippold, W.ti., Oettgen. H.F. and Old. L.J. (19x2) J. Exp. Med. 155. 113%1147. 71 Chcung, N.H.. Saarincn. U.M.. Neely. J.E.. Landmeier. B.. Donovan. D. and Coccia. P.F. (19X5) Cancer Res. 45, 764?-264Y. 27 Cherrsh. D.A.. Varki. A.P.. Varki. N.M.. Stallcup. W.B., I.evine. J. and Reisfeld. R.A. (19X-t) J. Biol. Chcm. 759, 735%745s. 73 Fredman. P.. Jeansson. S.. Lyckc. E. and Svennerholm. 1.. llYX5l FEBS Lett. 1X9. 23-26. 14 Yamamoto. II.. Tsuji, S. and Nagai. Y. (IYYO) J. Neurochcm. 54. 513-519. 75 Walton. K.M.. Sandhrrg, K., Rogers. T.B. and Schnaar. R.L.. ( 19XXl J. Biol. Chem. 263. 1055-2063. 26 Hakomori. S. (10X1) Annu. Rev. Biochem. 50. 733-76-1. 27 Rogers. T.B. and Snyder. S.H. (19x1) J. Biol. Chcm. 2%. 24022107. 2X Holmgren. J.H.. Elwing, P.. Fredman, P. and Svenncrholm. L. (19X0) Eur. J. Biochem. 106, 371-379. 29 Tiemeyer. M., Yasuda. Y. and Schnaar, R.L. (19X9) J. Biol. Chem. 264. 1671-16X1. 30 Yu. R.K.. Macala, L.J.. Taki. T.. Weinfeld. I1.M. and Yu. F.S. (19Xx) J Neurochem. 50. 1X?- 1x20. 31 Svcnnerholm. L. (1961) J. Lipid Res. 5. 145-155. 31 ILJPAC-IUB Commission on Biochemical Nomenclature (1977) Eur. J. Biochem. 79. I l-21. 33 Kawashima. I.. Nakamura, 0. and Tai. T. (IWI ) Mol. Immunol.. in press.
