Syngeneic Monoclonal Antibody against Melanoma Antigen with

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Lauter, C. J., and Trams, E. G. (1961) J. Lipid Res. 3, 136-138. 12. Hakomori, S. ... proteins and Proteoglycans (Lennarz, W. J., ed) pp. 1-34,. Plenum Publishing ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 24, Issue of October 25, pp. 13328-13333,1985 Printed in U.S.A.

Syngeneic Monoclonal Antibody against Melanoma Antigen with

(Received for publication, February 28, 1985)

Yoshio Hirabayashi, AkiraHamaoka, and Makoto Matsumoto From the Department of Biochemistry, Shizuoka College of Pharmacy, Shizuoka-shi, Shizuoka 422, Japan

Toshiko Matsubara From the Department of Chemistry, Faculty of Science and Technology, Kinki University, Kowakae, Higashi-Osaka 589, Japan

Masatoshi Tagawa, Seiji Wakabayashi, and Masaru Taniguchi From the Department of Immunology, Chiba University School of Medicine, Chiba-shi, Chiba 280, Japan

It has previously been reported thata mouse (C57BL/ 6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to reactonly with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). Inthe presentstudy,the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAca2-3Galfll-4Glc (or -GlcNAc))of gangliosides by threedifferentassay systems including enzyme immunostaining on thin layer plates,sandwich radioimmunoassay, andenzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosideshaving NeuGc terminus,including NeuGca23Galfl1- 4Glc - ceramide and NeuGca2 - [email protected], nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was .isolated from B16melanoma cells, and its structure was determined to be NeuAca2-3Galfl1-4Glc-ceramideby fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:l as its long-chain base and C16:0, C24:1, and C24:O as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.

pacity for glycoconjugates containing sialic acids. In the present study, the melanoma antigen recognized by the M2590 monoclonal antibody was successfullypurified from B16 melanoma cells and characterized as the ganglioside NeuAcot23Gal~l-4Glc-ceramide(GM3(NeuAc)’).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations for gangliosides are listed in Table I. Other abbreviations used are: ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FAB-MS, fast atom bombardment mass spectrometry.

MATERIALS AND METHODS

Celk-P3/X63-Ag8-U1 (P3U1) derived from the BALB/c myeloma cell line and B16 melanoma cell line derived from C57BL/6 mice were maintained in Dulbecco’s modified minimal essential medium (Nissui Co., Tokyo, Japan) supplemented with 5% fetal calf serum. Momclonal Antibody-The monoclonal antibody M2590 was obtained by the fusion of P3U1 cells and C57BL/6 spleen cells hyperimmunized with mitomycin C-treated B16 melanoma as described previously (3). The M2590 antibody reacts with melanoma cells of mouse, human, and hamster origin, but not with other tumor cells or normal tissues (2,3). Standard Gangliosides-Gangliosides were obtained from the following sources and purified as described elsewhere (4,5): GM3(NeuAc) and GM~(N~uGc) were from human liver and horse erythrocyte membrane, respectively; GM2(NeuAc)and GM2(NeuGc)were from TaySachs brain and C57BL/6 mouse liver, respectively; GMls,Gol. and G D were ~ ~ from human brain; GHlb was from rat ascites hepatoma AH 7974F cells; sialylparagloboside(NeuAc)and sialylparagloboside(NeuGc) were from human erythrocyte membrane and bovine erythrocyte membrane, respectively; GD3 and GMr were from chicken egg yolk. Extraction and Purification of the Melanoma Antigen-Cultured melanoma cells or culture supernatants were lyophilized. The lyophilized materials were then homogenized with 20 volumes of chloroform/methanol (2:1, v/v), followed by stirring overnight, and filtered. The residue was re-extracted twice. The combined filtrate was evapMouse monoclonal antibodies to defined carbohydrate orated to dryness, redissolvedin chloroform/methanol/water (30:60:8, structures have been isolated and proved to be very useful v/v/v), and subjected to DEAE-Sephadex A-25 column cbromatogprobes in the characterization of carbohydrate structures of raphy as described previously (6). Theacidic fraction eluted with 0.1 the cell surface membranes (for a review see Ref.1).Recently, M Na acetate in methanol was evaporated, dialyzed against water, two types of monoclonal antibodies specific to two distinct and lyophilized. The final purification of antigenic gangliosides was determinants of melanoma antigens were produced (2, 3) by achieved by Iatrobeads (Iatron Chemical Co., Tokyo) column chroimmunizing C57BL/6 mice with the syngeneic melanoma cells matography (7, 8). The purity of the gangliosides was monitored by (B16). One monoclonal antibody (M622) recognized the spe- thin layer chromatography (TLC) using precoated Silica Gel 60 plates (Merck) with solvent systems of chloroform/methanol/water (60:35:8, cies-specific melanoma antigenic determinants, and the other v/v/v, solvent I) and chloroform/methanol/2.5 N ammonia (60:35:8, (M2590) recognized the interspecies cross-reactive melanoma v/v/v, solvent 11). The gangliosides were visualized by spraying the antigenic determinants widely shared in various mammalian plates with the resorcinol-HC1reagent. species. The latter antibody was shown to have binding caThe yields of the GM3(NeuAc)ganglioside were about 780 and 440

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Monoclonal Antibody to Murine Melanoma Ganglioside GM13 @gfrom 7 x lo8 cells and 2.1 liters of the culture supernatants, respectively. Structural Analysis-Neutral sugars and amino sugars in melanoma ganglioside were analyzed by gas-liquid chromatography after methanolysis, N-acetylation, and trimethylsilylation as described by Bhatti etal. (9). The long-chain base was also analyzed by gas-liquid chromatography according to themethod of Gaver and Sweeley (10). The sphingosine base was also determined colorimetrically (11).The ganglioside was permethylated by the method of Hakomori (12). The partiallymethylatedalditolacetates were analyzed by gas-liquid chromatography as described previously (13). Negative FAB-MS analysis of the intact ganglioside was taken with a JEOL JMS-HX100 mass spectrometer (JEOL Ltd., Tokyo) ~ with a JMA-DA5000 data system (JEOL). About 1pg of G Mganglioside dissolved in chloroform/methanol (2:1, v/v) at a concentration of 1 pg/pl was mixed with 1 pl of triethanolamine, 0.3 pl of tetramethylurea, and 0.1 pl of 0.3%NaI aqueous solution. The targetwas bombarded with xenon atoms having a kinetic energy of 6 keV. Emission current used was 10 mA and accelerating voltage was 5 keV. The sequence and theanomeric configuration of the sugar units of the ganglioside were determined by sequential hydrolysis using jack bean &galactosidase and Clostridiumperfringens neuraminidase (14). Immunological Methods-A sensitive sandwich radioimmunoassay was performed by the method of Purcell et al. (15),with a modification as described previously (16). The enzyme-linked immunosorbent assay (ELISA) was performed by the method of Higashi et al. (17) with the following modifications. The concentration of glycolipid antigens in chloroform/methanol(l:4,v/v) was adjusted with the same solvent to a concentration of 10 ng/pl. Aliquots (50 pl) were pipetted into each microtiter well and evaporated to dryness at 37 "C. The wells were treated with 100 pl of PBS containing 1%egg albumin, 1% polyvinylpyrrolidone, and 0.02% sodium azide (solution A), and incubated for 30 min at 30 "C; then the wells were emptied, and 100 pl of the antibody solution diluted with solution A to the desired concentration was added to each well. The plates were incubated for 2 h at 30 T , and washed three times with PBS containing 0.1% Tween 20 (washing solution), and then 100 pl of horseradish peroxidase-conjugated anti-mouse IgG (heavy and light chains, Lot 20784, Cappel Laboratories, Cochranville, PA) solution diluted to 1:lOOO with PBS containing 3%polyvinylpyrrolidone (solution B) was added as the second antibody. After incubation for 2 h at 30 "C, the wells were washed five times with PBS and incubated for 15 min at 37 "C with 125 p1 ofthe substrate, 0.1% o-phenylenediamine in 0.1 M sodium citrate buffer, pH 7.0. The absorbance of the reaction produced was measured at 405 nm with a microplate spectrophotometer. Enzyme immunostaining of gangliosides on thin layer plates was performed by our recently developed method (18). In brief, after chromatography of the gangliosides, the TLCplates (Polygram Si1 G, Macherey-Nagel, Postfach, West Germany) were soaked with solution A, then with the monoclonal antibody (7 pg/ml), and horseradish peroxidase-conjugated rabbitanti-mouse immunoglobulins diluted (1:lOOO) with solution B. Quantity of the reaction product was measured a t 578 nm by means of a Dual-Wavelength TLC Scanner CS910 (Shimadzu, Kyoto, Japan). RESULTS

Specificity of M2590 Monoclonal Antibody-In the present study, monoclonal antibody M2590 produced by the fusion of P3U1 and spleen cells of C57BL/6 primed with syngeneic B16 melanoma cells has been shown to be specifically reactive to G Mcontaining ~ NeuAc (GM3(Nedc)), but not toG Mcontain~ ing NeuGc (GM3(NeuGc)).Specific binding of the antibody to GM3(Nedc)has been demonstrated by sandwich radioimmunoassay (Fig. LA), ELISA assay (Fig. l B ) , andTLC/ enzyme immunostaining (Fig. 2). Serially twice-diluted antibody failed to bind GM3(NeuGc)(Fig. lB), and no reactivity of the M2590 antibody was observed at various concentrations of G M ~ ( N ~ u G(Fig. c ) IA). The reactivity of the antibody to GM3(NeuAc)was abolished by treatment with clostridial neuraminidase (see also Table I). Therefore, the reactivity of the antibody to G Mwas ~ highly restricted to the N-acetylneuraminyl residue of the ganglioside. As shown in Fig.2, the binding of M2590 antibody to

m I

2

X

t

a

U

0 10

GM,

1.0 ( n m o l0.1e / m l

0,Ol

B

1 t

Ollution of monoclonal antibody M 2590 ( x 7 ~ g l w e l l l

FIG. 1. Specificreactivity of monoclonalantibody with Gm(NeuAc) and not with GM3(NeuGc)nor lactosylceramide. A, quantitative reactivity of GM3(NeuAc)antigen with M2590 antibody in sandwich radioimmunoassay. A plasticplate was coated with M2590 antibody andthen 50-pl aliquots of antigen solution (0, G ~ ~ ( N e d 0, c ) GM3(NeuGc) ; or lactosylceramide) at various concentrations in PBS were reacted with the antibody-coated plate, followed by incubation with lSI-labeled M2590 (80,000 cpm/well).B, titration of M2590 antibody by ELISA. The plastic plate was coated with the or lactosylceramide). antigen, 500 ng (0,Ghl,(NeuAc); 0,G~a(NeuGc) Other conditions were as described under "Materials and Methods."

G ~ ~ ( N e don c ) the thin layer plate was dependent on the c) on the plate. By this method, amounts of G ~ ~ ( N e dapplied aslittle as 2 pmol of GM3(NeuAc)could be detected and determined. The specific binding of the M2590 antibody to sialylparagloboside(NeuAc) was also observed by TLC/enzyme immunostaining(TableI). The density of sialylparagloboside(NeuAc)-antibody complex on TLC was almost equal to that of GM3(NeuAc).Here again, NeuGc-containing sialylparagloboside could not be stained with the M2590 antibody. GM4(NeuAc)was less reactive to the antibody. Interestingly, GMlb and GD~,,which are structurally related to sialylparagloboside(NeuAc),were hardly stained by the antibody. From the results described above, the epitope defined by the M2590 antibody was determined to beNeuAca2-3Gal/31-4Glc (or -GlcNAc). Chemical Characterization of M2590-reactiveGanglioside from B16 Melanoma Cells-Preliminary TLC/enzyme immunostaining of gangliosides extracted from B16 melanoma cells showed that the cells possessed a single ganglioside,

Monoclonal Antibody to Murine Melanoma Ganglioside GM3

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TABLE I Specificity of monoclonal antibody M2590 revealed by TLClenzyme immunostaining Peak Glycosphingolipids area' Name Structure of carbohydratemoiety GM, GM3(NeuAc) GM3(NeuGc) GM2(NeuAc)

J

2.7 53.0 0 0

NeuAca2-3Gal NeuAca2-3Galpl-4Glc NeuGca2-3Galpl-4Glc [email protected] 3

I

NeuAca2 GM2(NeuGc) GalNAcpl-4Galpl-4Glc 3

0

I

2

10

20

100 200 1000

NeuGca2 GMI~

Galpl-3GalNAcpl-4Gal~1-4Glc

0

3

I

p mole

NeuAca2 GMlb Sialylparagloboside(NeuAc) Sialylparagloboside(NeuGc) GDI.

Traceb NeuAca2-3Gal~l-3GalNAc~1-4Gal~l-4Glc NeuAc~2-3Galpl-4GlcNAcpl-3Gal~l-4Glc49.6

NeuGca2-3Gal~l-4GlcNAc~l-3Gal~l-4Glc0 Traceb NeuAca2-3Gal~l-3GalNAc~1-4Gal~l-4Glc 3

I NeuAca2 Gmb

Gal~1-3GalNAc~l-4Gal~l-4Glc

0

3

I

NeuAca2-8NeuAca2 NeuAca2-8NeuAca2-3Gal~l-4Glc Galpl-4Glc

0 GD~ 0 Lactosylceramide' 0 ParagloGalpl-4GlcNAcpl-3Gal~1-4Glc boside' =Fifty pmol of each glycolipid was applied on a TLC plate and analyzed by enzyme immunostaining using the M2590 antibody. Peak areas were measured by integration (17). Under the present assay condition,50 pmol of each compound could not be stained. However, when 0.5 nmol of each was analyzed, slight staining could be observed. Lactosylceramide and paragloboside were prepared from GM~(N~uAc) and sialylparagloboside(NeuAc), respectively, by clostridial neuraminidase treatment.

Analysis of the permethylated sugars from melanoma GM3 showed it contained 2,4,6-tri-O-methylgalactitol and 2,3,6-triGM3 , n mole 0-methylglucitol. The anomeric configuration and sugar sequence were determined by using exoglycosidases. LactosylFIG. 2. TLC/enzyme immunostaining of GM3(NeuAc)by using M2590 antibody and densitometric estimation of immu- ceramide produced from the ganglioside by hydrolysis with clostridial neuraminidase was.converted to glucosylceramide of G~s(NeuAc)were nostained spot. A , theindicatedamounts applied to a TLC plate and enzyme immunostained. The conditions by jack bean /3-galactosidase (Fig. 5). As shown in Table 11, were those described under "Materials and Methods." B, integrated C16:0, C240, and C241were dominant fatty acids. The longA . G M ~ chain base was mainly composed of d181. densitometric response of (h3(NeuAc) (0) onTLCin (NeuGc) (0)was also analyzed by TLC/enzyme immunostaining in The underivatized G Mganglioside ~ from B16 melanoma was the sameway as for A . characterized by FAB-MS analysis (Fig. 6). Molecular ion species, (M - H)-, weregiven by a series of strong peak Grur3(NeuAc),as M2590 antibody-reactive ganglioside (Fig. 3). signals a t mle 1179 to 1263. These peaks showed that the The active ganglioside was then isolated and purified to ganglioside molecule contained one N-acetylneuraminic acid, homogeneity as judged by thin layer chromatography employ- two hexoses, and a ceramide containing a series of normal ing two different solvent systems (Fig. 4). (.& from the cells fatty acids (160 at mle 1151, 180 at mle 1179, 200 at mle migrated tothe sameposition asthat of thestandard 1207, 22:O a t mle 1235, 241 at mle 1261, and 240 at mle GM~(N~uAc) in both of the solvent systems (I and 11). The 1263). This result is consistent with the fact that ceramide is ganglioside contained glucose, galactose, N-acetylneuraminic composed of the sphingosine of d l 8 1 long-chain base and the acid, and sphingosine in molar ratios of 1:l:l:l (Table 11). fatty acids of C160 to C24:l as shown in Table 11. FAB-MS

0

0,l

02

1,O

Monoclonal Antibody to Murine Melanoma Ganglioside G M ~

B

A

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TABLE I1 Chemical composition of G m ganglioside from B16 melanoma cells Ratios of carbohydrate and long-chain base

1

2

3

1

FIG.3. M2590 antibody-reactivegangliosides of B16 mel6.0 TLC by enzyme immunostaining. A , anoma cells detected on gangliosides reacting with M2590 and horseradish peroxidase-conjugated anti-mouse IgG. Lune 1, standard GM3(NeuAc)(100 ng); lanes 2 and 3, M2590-reactive gangliosides in total ganglioside fraction isolated from B16 cells, 75 and 150 ng, respectively. B, standard GM3(NeuAc)visualized with resorcinol-HC1 reagent. Solvent system I1 was used.

M

Glucose Galactose N-Acetylhexosamines N-Acetylneuraminic acid Long-chain base

1.0 0.85 0 0.95 0.98

Fatty acid composition

%

C160 C161 C180 C181 c200 c22:o C23:O c240 c241

21.6 Trace 8.1 Trace 5.4 11.4 21.5 19.9 %

Sphingosine base composition

dl80 dl81 d200 d20:l

0.6 95.6 Trace 3.8

GI u-cer t-

Lac-cer +

GM3

1 a s 4 1 2 3 4 FIG.4. TLC of M2590-reactive gangliosides purified from B16 cells and culture supernatants. Lunes 1, standard ~ B16 cells; lanes 3, G M from ~ B16 GM3(NeuAc);lanes 2, G M from culture supernatants; lanes 4, standard GM3(NeuGc).The plates of A and B were developed with solvent systems I and 11, respectively. Detection was by resorcinol-HC1reagent

also yielded the fragment ions at m/e 860 and 970 for lactosylceramide containing the C16:O and C241 fatty acids, respectively. However, the intense peaks of fragment ions corresponding to monoglycosylceramide and ceramide could not be observed under the present analytical conditions. These combined results indicate that the ganglioside recognized by the M2590 antibody has the structure of NeuAca2-3Gali314Glc-ceramide. The melanoma antigen was also isolated and purified from the cell-cultured supernatants. As shown in Fig. 4, GMS(NeuAc) could bedetected in the culture supernatants,showing the antigen was shed or secreted from the cell surface (2). The same ganglioside could not be detected with TLC-resorcinol reagent in cell-free culture medium. FAB-MS analysis of underivatized GM~(N~uAc) derived from B16 culture supernatants showed fragment ions quite similar to those indicated in Fig. 6 (not shown).

1

2

3

4

5

FIG.5. Stepwise hydrolysis of GMs(NeuAc) byexoglycosidases. Lanes 1 and 5, standard glycolipids: glucosylceramide (Glucer), lactosylceramide (Lac-cer), and GM3(NeuAc)(GM~);lane 2, GM3(NeuAc)from B16 melanoma cells; lane 3,lane 2 + neuraminidase; lane 4, lane 3 + P-galactosidase. Arrows indicate spots derived from Na taurodeoxycholate. DISCUSSION

In 1981, Wakabayashi et al. (19) demonstrated that murine cytotoxic T lymphocytesspecific for syngeneic melanoma cells killed human as well as mouse melanoma cells. From this result, they suggested that melanoma cells may have tumor antigens widely shared in mammalian species which are recognized by the immune system of the syngeneic host. In order to characterize the novel melanoma antigen described in the present report, a stable mouse hybridoma was established which produced a monoclonal antibody (M2590) reactive to cross-species determinants of the melanoma antigen. Detection of the sugar antigen with cross-species determinants which was expressed exclusively on various mammalian melanoma cells was successfully performedby using this monoclonal antibody (2). Although several monoclonal antibodies to the human melanoma-associated gangliosides such as G D ~

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Monoclonal Antibody to Murine Melanoma Ganglioside

h3

CHtOH c-0

trr~u

lor C Z I H ~ ~ I

OH

M.W.

1152 l o r 1 2 6 2 I

FIG. 6. Negative FAB-MS spectra of native GMs(NeuAc) ganglioside purified from B16 cells and fragmentation diagram of Gm(NeuAc). The negative spectra were recorded according to the method described under “Materials and Methods.” The main signals were assigned as described in text. The signals at mle 591, 632, and 744 were probably due to contaminants.

100

50 *5.0

580 400 I E0 100

50 -

308

200

fi00

700

e00

f

860

970

i *5.0 988

1000

(20, 21), GDz (22), and G D with ~ 0-acetylated sialic acid (23) have been described, the melanoma antigen recognized bythe syngeneic monoclonal antibody, M2590, is different from these gangliosides. The results of ELISA, sandwich assay, and TLC/enzyme immunostaining suggested that the antibody possessed high affinity for trisaccharide head group of G ~ ~ ( N e d especially c), for its terminal a-N-acetylneuraminyl residue. G Mcontaining ~ N-glycolylneuraminic acid could not be recognized bythe M2590 antibody. Chemically synthesized GM3(NeuAc)(d18:1/C24:0) was found to be equally well reactive to theantibody.’ Although NeuAc-containing G”3 is widely distributed on a various normal or malignant tissuesas the common precursor for ganglio series gangliosides, the M2590 monoclonal antibody reacted with only melanoma cells from various species including human, hamster, and mouse, but not with normal tissues or other tumor cells lines so far examined (2). There may be several possibilities to be considered as to why common GM3ganglioside on the melanoma cell surface behaves as theantigen for the antibody. First, the fattyacid moiety of glycosphingolipids may affect the organization and orientation of the saccharide chain in cell surface membranes (21, 24-26). For example, it has been suggested that human melanoma GD3antigen defined by a monoclonal antibody, 4.2, has thesame saccharide structure asthat of human brainGD3, butits ceramide was characterized by a predominance of longer chain fatty acids (C22:O to C24:O) in contrast to the brain GD3 (mainly C180fatty acid) (21). In our case, GM3(NeuAc)from B16 cells was demonstrated to contain a relatively high proportion of long-chain fatty acids, like GD3 ganglioside in the human melanoma case (21), whereas enzymic hydrolysis, methylation analysis, and FAB-MS analysis d l -indicated that .the sugar structure 0% bhe antigen was identical to usual GM3(NeuAc)(27). Therefore, the results suggest that ceramide composition may affect the generation Y. Hirabayashi and T. Ogawa, manuscript in preparation.

1100

11500 2 8 8 1408 1300

1688

of the unique antigenicity of the ganglioside on the cell surface. The second possibility is that G~3(NeuAc) in normal cells may be cryptic, while that in melanoma membrane is highly exposed. It has been suggested that there are several factors which control the degree of exposure on the cell surface membranes, i.e. other glycosphingolipids (25), sialylated glycoconjugates (26), and proteins in adjacent domains (28, 29). In fact, the M2590 antibody did not react with human red blood cells expressing GM3(NeuAc)on their cell surface, but from the cells? It has also been with purified GMM3(N,euAc) found that a 31,000-dalton glycoprotein (2, 3) was alwayscoimmunoprecipitated with GM3(NeuAc)by the M2590 antibody, indicating that the 31,000-dalton protein is associated with G M ganglioside. ~ The most typical association of G M ~ ganglioside with a membrane protein(s)has been demonstrated with Paul-Bunnell antigen: the protein with PaulBunnell antigenic structure has a specific affinity for G M ~ ganglioside, and a protein-ganglioside complex givesits maximum antigenicity (30). Thus, in thepresent case, the 31,000comdalton protein (or other unknown factors)-G~~(NeuAc) plex possibly generates the melanoma antigenic structure. No data concerning the structural relationship between GM3(NeuAc)and the 31,000-dalton protein in melanoma antigen have so far been obtained. However, it is possible that the 31,000-dalton protein of melanoma cells may have the or siasame terminal residue as the G M ~ ( N ~ ~ganglioside Ac) lylparagloboside(NeuAc), because the nonreducing terminal trisaccharide of [email protected] distributed widely in the sugar side chain of asparagine-linked complex type structure in a variety of glycoproteins (for a review, see Ref. 31)and also because the 31,000-daltonprotein was always precipitated by the M2590 antibody. In any event, the purification andbiochemical characterization of the 31,000-dalton Y. Hirabayashi, A. Hamaoka, M. Matsumoto, T. Matsubara, M. Tagawa, S. Wakabayashi, and M. Taniguchi, unpublished observation.

Monoclonal Antibody to Murine Melanoma

Ganglioside G3

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15. Purcell, R. H., Wong, D.C., Moritsugu, Y., Dienstag, J. L., Routenberg, J. A,, and Boggs, J. D. (1976) J. Immunol. 1 1 6 , 349-356 16. Wakabayashi, S., Okamoto, S., and Taniguchi, M. (1984) Gann 75,427-432 17. Higashi, H., Fukui, Y., Ueda, S., Kato, S., Hirabayashi, Y., Matsumoto, M., and Naiki, M. (1984) J. Biochem. (Tokyo) 9 5 , 1517-1520 18. Higashi, H., Ikuta, K., Ueda, S., Hirabayashi, Y., Matsumoto, M., and Naiki, M. (1984) J. Biochem. (Tokyo) 9 5 , 785-794 19. Wakabayashi, S., Taniguchi, M., Tokuhisa, H., and Okamoto, S. (1981) Nature 2 9 4 , 748-750 20. Pukel, C. S., Lloyd, K. O., Travassos, L.R., Dippold, W. G., Oettgen, H. F., and Old, L. J. (1982) J. Exp. Med. 155,11331147 REFERENCES 21. Nudelman, E., Hakomori, S., Kannagi, R., Levery, S., Yeh, M.Y., Hellstrom, K. E., and Hellstrom, I. (1982) J. Biol. Chem. Hakomori, S. (1984) Annu. Rev. Immunol. 2 , 103-126 257,12752-12756 Taniguchi, M., and Wakabayashi, S. (1984) Gann 7 5 , 418-426 Wakabayashi, S., Saito, T., Shinohma, N., Okamoto, S., To- 22. Cahan, L. D., Irie, R. F., Singh, R., Cassidenti, A., and Paulson, J. C. (1982) Proc. Natl. Acud. Sci. U. S. A. 7 9 , 7629-7633 mioka, H., and Taniguchi, M. (1984) J. Inuest. Dermatol. 8 3 , 23. Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., 128-133 Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259,7453Laine, R., Stellner, K., and Hakomori, S. (1974) Methods Membr. 7459 Biol. 2,205-244 24. Symington, F. W., Bernstein, I. D., and Hakomori, S. (1984) J. Ledeen, R. W., and Yu, R. K. (1982) Methods Enzymol. 83,139Biol. Chem. 259,6008-6012 191 Ledeen, R. W., Yu, R. K., and Eng, L. F. (1973) J. Neurochem. 25. Kannagi, R., Stroup, R., Cochran, N. A., Urdal, D. L., Young, W. W., Jr., and Hakomori, S.(1983) Cancer Res. 4 3 , 4997-5005 21,829-839 Momoi, T., Ando, S., and Nagai, Y. (1976) Biochim. Biophys. 26. Urdal, D. L., and Hakomori, S. (1983) J. Biol. Chem. 258,68696874 Acta 441,488-497 Matsumoto, M., Taki, T., Samuelsson, B., Pascher, I., Hirabay- 27. Kinoshita, Y., Makita, A., and Takeuchi, T. (1982) J. Biochem. (Tokyo) 92,801-808 ashi, Y., Li, S.-C., and Li, Y.-T. (1981) J. Biol. Chem. 2 5 6 , 28. Gahmbern. C.G.. and Hakomori.. S. (1975) J. Biol. Chem. 250. 9737-9741 . 2438-2446 Bhatti, T., Chambers, R. E., and Clamp, J. R. (1970) Biochim. 29. Stein. K. N.. Schwartine. G.A.. and Marcus. D.M. (1978) J. Biophys. Acta 222,339-347 ImAunol. i20,676-6% Gaver, R.C., and Sweeley,C. C. (1966) J. Am. Oil Chem. SOC. 30. Watanabe, K., Hakomori, S., Powell, M. E., and Yokota, M. 88,3643-3647 (1980) Biochem. Biophys. Res. Commun. 92, 638-646 Lauter, C. J., and Trams, E. G. (1961) J. Lipid Res. 3, 136-138 31. Kornfeld, R., and Kornfeld, S. (1980) in Biochemistry of GlycoHakomori, S. (1964) J. Biochem. (Tokyo) 55,205-208 proteins and Proteoglycans (Lennarz, W. J., ed) pp. 1-34, Hirabayashi, Y., Taki, T., and Matsumoto, M. (1978) Biochim. Plenum Publishing Corp., New York Biophys. Acta 529,96-105 Chien, J.-L., Li, S.-C., Laine, R, A., and Li, Y.-T. (1978) J . Bid. 32. Wakabayashi, S., Okamoto, S., and Taniguchi, M. (1984) Gann 75,707-713 Chem. 253,4031-4035

glycoprotein are required to demonstrate the whole structure of the melanoma antigen. The biological significance of the tumor antigen(s)defined by the syngeneic monoclonal antibody M2590 remains to be clarified. Since the monoclonal antibody, M2590, reacts equally with human and mouse melanomas, the application of this antibody should be valuable for establishing experimental models of diagnosis or therapyfor human melanoma. Indeed, we could demonstrate that intravenous injection of minute amountsof 1311coupled with thismonoclonal antibody into tumor-bearing mice strikingly inhibited melanoma cell growth in vivo (32). 1. 2. 3.

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