A Monoclonal Antibody-defined Antigen Associated with ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No 23, Issue of December 10, pp 14365-14369, 1982 Prlnted in U.S.A.

A Monoclonal Antibody-defined Antigen Associatedwith Gastrointestinal Cancer Is a Ganglioside Containing Sialylated Lacto-N-fucopentaose 11* (Received for publication, May 5, 1982)

John L. MagnaniS, Bo Nilssong, Manfred BrockhausSV, DavidZopfl, Zenon SteplewskiJI, Hilary Koprowskill, and Victor Ginsburg$ From the $National Instituteof Arthritis, Diabetes, a n d Digestive a n d Kidney Diseases a n d the §National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 a n d the (1 Wistar Institute of Anatomy a n d Biology, Philadelphia, Pennsylvania 19104

Two monoclonal antibodies produced by hybridomasbe a sialylated Le“-activepentasaccharide (sialylated 1acto-Nobtained from a mouse immunized with a colorectal fucopentaose 11, IV~--ol-NeuNAc-1114-a-Fuc-LcOse4) with the carcinoma cell line bind specifically to human gastroin- following structure’: testinal cancer cells. The antigen of this antibody in the NeuNAca2-3Ga1~1-3GlcNAcpl-3Galpl-4Glc carcinoma cell line, a monosialoganglioside,has been 4 isolated. Its carbohydrate structureis probably

I

NeuNAcaZ-3Gal/ll-3GlcNAc~1-3Ga~1-4Glc

Fucal

4 EXPERIMENTAL PROCEDURES

I Fucal

Muterials-Monoclonal antibodies 19-9 and 52a are produced by obtained from a mouse immunized with human colon This oligosaccharide is a sialyl derivative of 1acto-N- hybridomas carcinoma cell line SW 1116 (2). The two antibodies areof the IgGl fucopentaose 11, a haptenof the human Le” blood groupisotype and detect the same ganglioside antigen as determined by antigen. About 30 pg of ganglioside is obtained from 1 autoradiography (4).Serum-free supernatant fluids from hybridoma cell culture (5) containing approximately 10 pg/ml of antibody were g of cells, wet weight. The ganglioside was detected by autoradiography in used for the experimentsdescribed here. Trifluoroacetic acid and trifluoroacetic anhydride were obtained lipid extracts of some carcinomas and of meconium, but from Aldrich SP-2340 (3%) onSupelcoport was purchased from not in lipid extracts of normal adult tissues. Antigen Supelco, Inc., Bellefonte,PA; and fused silica OV-1 W.C.O.T. capillary was detected by solid phase radioimmunoassayin lipid columns from Hewlett-Packard, Rockville, MD. extracts from12 out of 21 adenocarcinomas of the Asialylated lacto-N-fucopentaose 111 ceramide (1V’-a-NeuNAccolon, from 4 out of 5 gastric adenocarcinomas, and IIl’-a-Fuc-LcnOse4Cer) isolated from human kidney (6) was kindly from 4 out of 7 pancreatic carcinomas. Antigen was not supplied by Dr. Heiki Rauvala(University of Helsinki,Helsinki, detected in lipid extracts from 5 esophageal carcinomas Finland). LS-tetrasaccharide a isolated from human milk (7) was a gift from Dr. David F. Smith (Virginia Polytechnical Institute,Blacksorfromnormal colon and gastric mucosa, pancreas, burg, VA). Frozen specimens of normal and cancer tissue were obkidney, liver, and bone marrow. tained from the Biological Carcinogenesis Branch of the National Cancer Institute. Autoradiography of Glycolipid Antigens-Glycolipid antigens were detected on thin layer chromatograms by autoradiography as Many monoclonal antibodies produced by hybridomas ob- previously described (4, 8) with minor modifications as follows. Glytained from mice immunized with a human colon adenocar- colipids were chromatographed on aluminum-backed high perform(Silica Gel 60, E. Merck, cinoma cell line have an apparentspecificity for human intes- ancethinlayerchromatographyplates Darmstadt, West Germany; American Supplier, Applied Analytical tinal tumors (2). The binding of two of these antibodies to chloroform/methanol/0.2% CaCIL Wilmington, NC) in tumor cells is inhibited by serum from most patients with Services, gastrointestinal cancer, but not by the serum of normal indi- (60409 by volume). The dried chromatogram was soaked for 30 s in a saturated solution of polyisobutylmethacrylate (Polysciences, Inc., viduals, patients with inflammatory bowel diseases, or most Warrington, PA) in hexane. After drying in air, the chromatogram patients with other malignancies (3). The antigen for these was sprayed with phosphate-buffered saline (0.15 M NaC1. 0.01 M antibodies in the cell line used for immunization is a monosi- sodium phosphate,pH 7.4) containing 1% bovine serumalbumin aloganglioside (4). It has been isolated and its carbohydrate (buffer A) and immediately soaked in buffer A until all of the silica has been characterized by the data in this paper to probably gel was wet (about 10 min). The plate was then removed and overlayed with monoclonal antibody solution diluted 1:4 with buffer A (about 55 pl/cmL) and incubated for 3 h a t 4 “C. The chromatogram * This work was supported in part by Research Grants CA-10815 was washed by dipping in four successive changes of cold phosphateand CA-21124 from the National Cancer Institute and Grant RRbuffered saline a t I-min intervals and overlayed with buffer A con05540 from the Division Research Resources. Part of this work was taining lo6 cpm/ml of ’“I-labeled F ( a b ) 2 of rabbit anti-mouse impresented at the67th Annual Meetingof the Federationof American munoglobulin G antibodies (about 40 pCi/pg; Amersham Corp., ArSocieties for Experimental Biology (I). The costs of pubIication of lington Heights, IL). After 3 h a t 4 “C, the chromatogramwas washed this article were defrayed in part by the payment of page charges. as before in cold phosphate-buffered saline, dried, and exposed to This article must therefore be herebv marked “advertisement” in accordance with 18 U.S.C. Section 17g4 solely to indicate this fact. fi Recipient of a Fellowship from the DeutscheForschungsgemeinAll monosaccharides mentioned in this paper are assumed to be schaft. Present address, Basel Institutefor Immunology, Basel, Swit- in the pyranose form and to have a o-configuration except for fucose zerland. which has an L-configuration. ~~~

‘

14365

14366

Gastrointestinal Cancer-associated Ganglioside

XR-5 x-ray film (Eastman Kodak, Rochester, NY) against an intensifying screen (Cronex, Lightning Plus AH, DuPont,Wilmington, DE) for 2-3 h a t -90 "C. SolidPhaseRadioimmunoassay-The binding of antibodyto glycolipid was measured by solid phase radioimmunoassay as previously described (8):glycolipid in 20 p1 of methanol was addedto wells of a roundbottom pol.yvinylchloride microtiterplate(Dynatech, Alexandria, VA) and the solutions dried by evaporation. The wells were then filled with buffer A. After 30 min, the wells were emptied and to each was added20 pI of monoclonal antibody solution diluted 1:4 with buffer A. The wells were covered with parafilm. incubated for 3 h at 22 "C, washed once with buffer A, and then to each was added about 20,000 cpm of anti-mouse Fab from rabbit, "51-labeled F(ab')? in 20 p1of buffer A. After 3 h, the wells were washed six times with cold phosphate-buffered saline, cut from the plate, and assayed for lZ5I in an Auto-Gamma spectrometer. For testingin solid phase radioimmunoassay,glycolipidswere extracted from lyophilized tissues and phase-partitioned as described by Folch et al. (9). T o each well was added upper phase glycolipids equivalent to 1 mg of tissue wet weight. Isolation of the Ganglioside Antigen-The ganglioside antigen was isolatedfrom thehumanadenocarcinoma cell line SW 1116 originally used for immunization of the mouse from which the two hybridomas were prepared (2). Purificationwas followed by solid phase radioimmunoassay and by autoradiography asdescribed above. Ten gramsof human adenocarcinoma cells, wet weight, were treated by a method devised for the quantitative extraction of gangliosides from brain (10).The cells were homogenizedin a Dounce homogenizer with 30 ml of H20 at 4 "C. The homogenate was added to 108 ml of methanol with constant stirring. Chloroform ( 5 4 ml) was added and the mixture was stirred a t room temperature for 30 min. The homogenate was centrifuged a t 1500 X g for 20 min. The pellet was rehomogenized in 20 ml of H20, added to 80 ml of chloroform/methanol (1:2 by volume), stirred, and centrifuged as before. The supernatant solutions were combined in a separatory funnel with 52 ml of H?O. After phaseseparation,the lower phase was re-extracted by the addition of 30 ml of methanol and 20 ml of 10 mM KC1 in HrO. The combined upper phases (about 300 ml) were evaporated to dryness under vacuum, the residue was resuspended in 15 ml of HIO, dialyzed against three changes of cold distilled H20, and lyophilized. The dry residue (45 mg) was dissolved in chloroform/methanol/water (30508 by volume) and applied to a column of DEAF,-Sepharose CL-6B (0.9 X 10 cm) equilibrated in the same solvent. The columnwas washed with 10 column volumesof methanol. Neutralglycolipids pass through the column and the antigen was eluted in the monosialoganglioside fraction by 10 columnvolumes of 0.01 M ammoniumacetatein methanol. After lyophilization, the dried residue (12 mg) was subjected to silicic acid chromatography by passage through a column of Bio-Si1 HA -325 mesh (0.9 X 100 cm) in chloroform/methanol (4:l by volume) which was eluted with a concave gradient from chloroform/methanol (4:l by volume) to methanol/0.2% CaCI2 in H 2 0 ( 5 0 3 by volume). Those fractions containing antigen and exhibiting only one band migrating between GMIand GIX.standards upon thin layer chromatography were pooled for structural analysis. Approximately 300 pg of the monosialoganglioside of interest was obtained by this procedure. Desialylation of the Ganglioside-Purified ganglioside (100 pg) was dissolved in 200 p1 of 0.1 M sodium acetate buffer, pH 5.5. containing 0.18 CaCI?. Clostridium perfringens neuraminidase (40 milliunits. Rethesda Research Laboratories, Gaithersburg, MD) was added and thereaction mixture was incubated for 24 h a t 37 "C. The reaction was stopped by the additionof 2 ml of chloroform/methanol (2:l. by volume). After evaporation underN1, the samplewas dissolved in 5 ml of 0.1 M KC1 and desalted on a reverse phase matrix (SepPak C18, Waters Associates, Milford, MA) as described in Ref. 11. The solution was passed through the cartridge 5 times. After washing with 10 ml of H20, bound glycolipid was eluted with 20 ml of chloroform/methanol (2:l by volume). Recovery of the desialylated glycolipid was nearly quantitative. Gas-Liquid Chromatographyand MassSpectrometry-Monosaccharides were identified as alditol acetates by combined gas-liquid chromatography/massspectrometry (12) using a Hewlett-Packard 5992A instrument equipped with a glass column(1 m X 2 mm) packed with 3% SI'-2340 on Supelcoport 100/200 and run a t 190-240"C. Quantitation was performed by integration of GLC peaks obtained using a flame ion detector. Analysis of partially methylated alditol acetates (13) and N-trifluoroacetylated permethylated oligosaccha-

ride alditols (14) was carried out on a Hewlett-Packard 5958B instrument equipped with a fused silica OV-1 W.C.O.T. capillary column (12 m X 0.2 mm) passed from the oven through heated transferblocks directly into the ion source. The injector and transfer blocks were maintained at 350 "C. The samplewas injected in chloroform in splitless mode with the column a t 40 "C. After 2 min. the column temperature was raised a t 30 "C/min to 200 "C and then at8 "C/min to 350 "C. Mass spectra wererecorded a t 70 eV at an ion source temperature of 200 "C and pressure of 5 X 10 -Ii Torr. Quantitation of partially methylated alditol acetates was performed on a HewlettI'ackard 5840 gas chromatograph equipped with the same capillary column described above and a flame ionimtion detector. Preparation of Permethylated a n d N-Trifluoroacetylated Oligosaccharide Alditol-Desialylated glycolipid (80 pg) was dissolved in 2 ml of trifluoroacetic acid/trifluoroacetic anhydride (1:100 by volume) and heated to 100 "C for 48 h in athick-walledglass tube (caution, corrosive mixture under pressure). After cooling to room temperature, the mixture was concentrated to dryness and the residue was evaporated from p.yridine, reduced with sodium borodeuteride, and permethylated as previouslydescribed (14). The material obtained by partitioning into chloroform after permethylation was further purified on a column of Silica Gel G (Merck. Darmstadt. West Germany) (2.5 X 80 mm) eluted first with one column volume of ethyl acetate to remove impurities and then chloroform/acetone (1:2 by volume) to recover the product. RESULTS AND DISCUSSION

Isolation of the Ganglioside Antigen-Thin layer chromatograms of ganglioside fractions during purification of the antigen from the adenocarcinoma cell line are shown in Fig. 1, lanes 1-3. The antigen is a major component of the monosialoganglioside fraction. Autoradiography of the purified antigen using monoclonal antibody is shown in Fig. 1, lanes 4-7. The antigen revealed by autoradiography coincides with the Resorcinol

CMH

-

CDH

-

Autoradiograohy

CTH Globoside

-

GUS

Origin

-

4

5

6

7

FIG. 1. Thin layer chromatography of ganglioside fractions during purification of the antigen. The fractions are described under "Experimental Procedures." The gangliosides in lanes 1-3 were visualized with resorcinol reagent (15). Lane I , upper phase gangliosides from 20 mg of cells, wet weight; lane 2, monosialogangliosides from 20 mg of cells, wet weight; and lane3, 1 pg of ganglioside antigen (from 33 mg of cells, wet weight) after silicic acid chromatography. Lanes 4-7 contain varying amounts of purified ganglioside antigen visualized by autoradiography with antibody 19-9 as described under "Experimental Procedures": lane 4, 5 ng; lane 5, 1 ng; lane 6, 0.5 ng; and lane 7, 0.1 ng. The positions of some standard gangliosides and neutral glycolipids are shown on the left. CMH, Gal/Il-lCeramide; CDH, Galpl-4Glc~l-1Ceramide; CTH, Galal-4Gal~l-4Glc~lICeramide; Gal~1-3GalNAcB1-4[NeuNaca2-3]Gal~l-4Glc~lICeramide; GI,I", NeuNAca2-3Galpl-3GalNAcpl-4[NeuNAca2-3] Galpl-4Glcpl-1Ceramide.

Ganglioside

Gastrointestinal Cancer-associated

14367

A

w

bMe:

6

i

“l

R

I 1

C

H

FIG. 2. Mass spectra of NTF permethylated oligosaccharide alditols. A, authentic lacto-N-fucopentaose111; B , authentic lacto-Nfucopentaose 11; and C, unknown pentasaccharideobtained bytrifluoroacetolysis of the desialylatedganglioside as describedunder “Experimental Procedures.”The origin of sequence ions fromthe two authentic oligosaccharides are indicated in the structures.

purified ganglioside revealed with resorcinol reagent. About 0.5ngof ganglioside is easily detected by autoradiography (Fig. 1, lane 6). Carbohydrate Structureof the Purified Ganglioside-The isolated monosialylganglioside contains neutral monosaccharides in the following molar ratios: 1.0 fucose, 1.7 galactose, 1.0 glucose, 1.2 N-acetyl-glucosamine, determined as alditol acetatesby gas chromatography as described under “Experimental Procedures.” The sialic acid released byneuraminidase co-chromatographed with authentic N-acetylneuraminic acid. A neutral oligosaccharide was obtained from the desialylated ganglioside by trifluoroacetolysis. The oligosaccharide was reduced with sodium borodeuteride, permethylated, and analyzed by gas-liquid chromatography/mass spec-

trometry as a permethylated NTF’ oligosaccharide alditol. The retention time and mass spectrumof the unknown pentasaccharide (Fig. 2C) are identical with that of authentic lacto-N-fucopentaose I1 (Fig. 2B) (16) and different from that of authentic lacto-N-fucopentaose 111 (Fig. 2 A ) . The sequence ions fromthe nonreducing ends of the oligosaccharide indicate two nonreducing termini, a deoxyhexose ( m / z 125, m / z 157, m / z 189) and a hexose ( m / z 155, m / z 187, m / z 219). The ion m / z 692, consideredwith the previousions, indicatesthe sequence: hex-hexNTF

I

deoxyhex The abbreviation used is: NTF, N-trifluoroacetyl,


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Substitution of the hexNTF residue is determined by com3r m / z 456 paring relative intensities of the secondary fragments and m / z 486 which originate from m / z 692 (see Fig. 3). As shown previously (16), eliminationfrom position 3 of hexNTF is favored over elimination from position 4 following primary cleavage of the N-trifluorohexosaminidic linkage. Therefore, the intensityof m / z 456 greater thanm / z 486 in the unknown spectrum (Fig. 2C) indicates thathexose is linked to position 3 and deoxyhexose to position 4 of N-trifluoroacetyl hexosamine. This structure is further supported by the presence of m / z 913 (Fig. 2, B and C ) which is formed by cleavage of the GLYCOLIPID (ngl 3-substituent leaving a positivecharge at C-3 of N-trifluoroacetylhexosamine. The C-3 carbonium ion is probably stabilized FIG.4. Binding of antibody to the purified ganglioside antiby formation of an oxonium ion involving the carbonyloxygen gen. Solid phase radioimmunoassays were performed as described of the N-trifluoroacetyl group. The corresponding ion from under “Experimental Procedures.” Binding of antibody to purified is not present in ganglioside (o.“o) and to the same ganglioside treated with neurlacto-N-fucopentaose 111, m / z 943 (Fig. U), the mass spectrum of the unknown (Fig. 2C). Sequence ions aminidase as described under “Experimental Procedures” (0-- -0). containing the alditol ( m / z 236, m / z 376, m / z 408, m / z 440) NeuNAca2-3Gal~l-3GIcNAcpl-3Gal~l-4Glc indicate the sequence -hex-hexitol-1-dinall three spectra 4 (Fig. 2, A , B, and C). I In addition to the pentasaccharide, material with retention Fuca I times and mass spectra identical with lacto-N-tetraose derivAlthough the anomeric configuration of the neutral sugars atives were detected. These derivatives were also detected cannot be deduced from methylation analysis andmass specduring analysis of authentic lacto-N-fucopentaose11. Methylation analysis of the monosialoganglioside gave ter- trometry, the pentasaccharide released from the desialylated minal fucose (1.0 residue), 3-0-substituted galactose (1.9 res- ganglioside by trifluoroacetolysis is probably lacto-N-fucopentaose 11, which is found in a glycolipid antigen of the human idues),4-0-substituted glucose (1.0 residue),and3,4-di-0substituted N-acetylglucosamine (not quantitated). The ab- Lewis blood group(17). Support for thisstructure is the sence of a monosubstituted hexosamine in the methylation finding that theganglioside antigen was not found ingastroinanalysis suggests that the lacto-N-tetraose detected by gas- testinaltumors from patients belonging tothe Le(a-b-) liquid chromatography/mass spectrometry derives from de- blood group (18). Le(a-b-) individuals lack the fucosyltransfucosylation of the pentasaccharide during work up of the ferase specified by the Le gene (19) which is responsible for sample after trifluoroacetolysis as observedwith authentic the synthesisof lacto-N-fucopentaose I1 from lacto-N-tetraose and therefore cannotsynthesize a sialyl derivativeof 1acto-Nlacto-N-fucopentaose 11. Combined data from the monosaccharide composition, sus- fucopentaose 11. Solid Phase Radioimmunoassay-Binding of antibody to ceptibility to neuraminidase, methylation analysis, and mass the purified ganglioside in solid phase radioimmunoassay is spectrometry of the derivatized oligosaccharide suggest that the ganglioside antigen has thefollowing carbohydrate struc- shown in Fig. 4. About 2 ng of ganglioside is requiredfor halfmaximal binding. Binding is specific for sialylated 1acto-Nture which has not been described before: fucopentaose I1 as 10 ng of other gangliosides including the isomeric sialylated lacto-N-fucopentaose 111 whose carbohyCH,OMe CH,OMe CHIOMe drate structure is

0 0

Deoxyhex 0

-

MeNTF

Hex 0 MeNTF

NeuNAca2-3Gal~l-4GIcNAc~l-3Galpl-4Glc 3 Deoxyhex

0

1

MeNTF

486

do not bind antibody under the same assay conditions. The sialyl residue is necessary for binding as desialylation with neuraminidase(see“ExperimentalProcedures”) abolishes binding nearly completely(Fig. 4). The fucosyl residue is probably also required for binding as the milk oligosaccharide LS-tetrasaccharide a whose structure is

456

I

I CHzOMe

1

I

I

I Fucal

rwz

CHIOMe Deoxyhex-0

MeNTF

692

h FIG.3. Elimination of hexose (Hex) and deoxyhexose (Deoxyhex) from m / z 692, an ion derived fromhomolytic cleavage of the hexosaminidic linkage of the permethylated NTF alditois of lacto-N-fucopentaose 111 (top) and lacto-N-fucopentaose I1 (bottom)(see Fig. 2, A and B). Elimination of hexose from the 3,4di-0-substituted hexNTF residue gives m / z 456, whereas elimination of deoxyhexose gives m / z 486. Since elimination from position 3 is favored, the relative positions of hexose and deoxyhexose can be inferred from the relative abundances of the secondary fragments.

NeuNAca2-3Gal~1-3G1cNAcpl-3Galpl-4Glc

does not inhibit binding of antibody to antigenin solid phase radioimmunoassay at concentrations up to1 mg/ml. mixOccurrence of Ganglioside Antigens-Ganglioside tures from various tissues were chromatographed and subjected to thin layer chromatography and autoradiography. As shown in Fig. 5, antigen with the same mobility as the ganglioside described here (lane 4) was detected in extracts of two carcinomas (lanes 2 and 3) and in extracts of meconium (lane 5). In addition, a small amount of antigen with a lower mobility was detected in extracts of the colon carcinoma cell line and with a highermobility in extracts of meconium. Antigen, possibly glycoprotein, was detected at the origin of both carcinoma extracts. No antigenwas detected in extracts


CMH -

CDH CTH Globoside -

Origin

-

”.

1 2 3 4 5 6 7 8 9 FIG. 5. Distribution of antigen among human tissues. Autoradiography of antigens on thin layer chromatogramswas performed as described under “Experimental Procedures.” Antibody 19-9 was used. The amount of extract chromatographedexpressed as the volume of packed cells from which it was obtained is as follows: lane 1, 2 1-11 normal intestinal mucosa; lane 2, 2 pl of primary colon adenocarcinoma; lane 3, 0.5 pl of liver metatasis of pancreatic carcinoma; lane 4, 1 pl of colorectal carcinoma cell line SW 1116; lane 5, 1 1-11 of meconium; lane 6, 1 pl of melanoma cell line WM 9; lane 7, 1 pg of brain gangliosides; lane 8,1 p1 of normal spleen; and lane 9, 1 pl of erythrocytes. The positions of some standard gangliosides and neutral glycolipids are shown on the left. The abbreviations used are the same as those in Fig. 1. of normal intestinal mucosa, normal spleen, erythrocytes, a melanoma cell line, or in human brain gangliosides (Fig. 5). Meconium, the firstfeces of the newborn, is a rich sourceof fetal glycolipids (20). The occurrence of the ganglioside in meconium suggests that it maybe a tumor-associated embryonic antigen (21). In support of this notion is the finding that antigen is detected by immunofluorescence microscopy using antibody 19-9 in some human embryonic tissues but not in the corresponding adult tissues.:’ Antigen was assayed in lipid extracts of various normal and malignant tissues by solid phase radioimmunoassay as described under “Experimental Procedures.” By this method, antigen was found in 12 out of 21 colorectal adenocarcinomas, in4 out of 5 gastric adenocarcinomas,and in 4 out of 7 pancreatic adenocarcinoma^.^ Antigenwas not detected in extracts from 5 esophageal carcinomas or from normal colon and gastric mucosa, pancreas, kidney, liver,and bonemarrow. By immunoperoxidase assay in another study (22), antigen was detected in 29 out of 53 colorectal adenocarcinomas, 13 out of 13 gastric adenocarcinomas, and14 out of 17 pancreatic adenocarcinomas. Antibodies 19-9 and 52a may be useful diagnostically. In a recent study using antibody 52a (23), antigen was detected in sera from 163 out of 255 patients with colorectal adenocarcinomas, 8 out of 11 with gastric adenocarcinomas, and 45 out of 49 with pancreatic adenocarcinomas. The antigen in serum appears to be a mucin, not a ganglioside.5 Why a ganglioside containing sialylated lacto-N-fucopentaose I1 is made by some cancer cells and by some fetal cells, but not by normal adult tissue, remains to be determined. A

:’P. Burtin, unpublished results. Glycolipid extracts of tissueswhichbindless than 10% of the antibody boundby glycolipid extracts of the colon carcinomacell line are scored negative. ’J. L. Magnani, unpublished results.

14369

fucosyltransferase that adds fucose to lacto-N-tetraose, Gal~l-3GlcNAc~l-3Gal/31-4Glc, to formlacto-N-fucopentaose I1 has been purified from human milk (24) and a sialyltransferase that adds sialic acid to lacto-N-tetraose to form LS-tetrasaccharide a has been purified from porcine submaxillarygland(25).However, the purifiedfucosyltransferase does not add fucose to LS-tetrasaccharide a and the purified sialyltransferase does not add sialic acid to lacto-N-fucopentaose 11.” Possibly, fetaltissue and some adenocarcinomas have a fucosyltransferase or a sialyltransferase with a broader acceptor specificity than the adultform of the enzyme($ and are thus able to synthesize the sugar sequence found in the ganglioside antigen whereas adult tissue cannot. In addition to the antibodies reported here, other antibodies produced by hybridomas obtainedfrom mice immunized with human cancers arealso directed againstglycolipids (8,26,27). Acknoulledgments-We thank Jo Anne Cashel and Urner Chase for technical assistance and Julie Maltagliati for typing the manuscript. REFERENCES 1. Magnani, J. L.,Nilsson, B., Brockhaus, M., Zopf. 11.. Steplewski, Z.,Koprowski, H., and Ginsburg, V. (1982) Fed. Proc. 41,897 2. Koprowski, H., Steplewski, Z.,Mitchell, K., Herlyn, M.. Herlvn, D., and Fuhrer, J . P. (1979) Somat. Cell Genet. 5, 957-972 3. Koprowski, H., Herlyn, M., and Steplewski,Z.(1981)Science 212, 53-55 4. Magnani, J. L., Brockhaus, M., Smith, D. F., Ginsburg, V., Blaszczyk, M.. Mitchell,K.F.,Steplewski, A,, andKoprowski, H. (1981)Science 212.55-56 5.Chang, T.H., Steplewski, Z.,andKoprowski.H. (1980) J. Zmmunol. Methods 39,369-375 6. Rauvala, H. (1976) FEBS Lett. 62. 161-164 7. Smith. D. F... Zonf. - V. (1978)Anal. Biochem. . .D. A.. and Ginsbura. 85,602-608 8.Brockhaus. M.. Maanani. J. L.. Blaszczvk.M..Stenlewski. . Z.. . Koprowski, H., Karlsson, K. A., Larson, G., and Ginsburg, V. (1981) J. Biol. Chem. 256, 13223-13225 9.Folch, J., Lees, M.,and Sloane Stanley, G. H. (1957) J . Biol. Chem. 226,497-509 10. Svennerholm, L., and Fredman. P. (1980)Biochim. Biophys.Acta 617.97-109 11. Williams, M.A.,andMcCluer, R. H. (1980) J. Neurochem. 35, 266-269 12. Golovkina, L. S., Chizov, 0. S., andWulsson, N. S. (1966) Zzu.

Akad. Nauk. SSR. Ser. Khin. 1915-1926 S. 13. Bjorndd, H., Hellerqvist, C. G.,Lindberg,B.,andSvenson, (1970)Angew. Chem. Znt. Ed. Engl. 9,610-619 14. Nilsson, B., and Zopf, D. (1982)Methods Enzymol. 82, 46-58 15. Svennerholm, L. (1957)Biochim. Biophys. Acta 24,604-61 1 16. Nilsson, R.,and Zopf. D. (1982)Arch. Biochem. Biophys.. in press 17. Hakomori, S.,and Andrews, H. D. (1970) Biochim. Biophys. Acta 202,225-228 18. Koprowski, H., Brockhaus. M., Blaszczyk, M., Magnani, J., Steplewski, Z..and Ginsburg, V. (1982)Lancet 1, 1332-1333 19.Grollman,E.F., Kobata, A., andGinsburg, V. (1969) J . Clin. Znuest. 48, 1489-1494 20. Karlsson, K.-A., and Larson, G. (1978) FEBS Lett. 87.283-287 21. Hakomori, S. (1981)Ann. Reu. Biochem. 50, 733-764 22. Atkinson, B. F., Ernst, C. S., Herlyn, M. Steplewski, Z., Sears, H. S., and Koprowski, H. (1982)Cancer Res., 42,4820-4823 23. Herlyn, M., Sears, H. F., Steplewski, Z., andKOprowski, H. (1982) Clin. Immunol. 2, 135-140 24. Prieels, J.-P., Monnom, D., Dolmans, M., Beyer, T. A., and Hill, R. L. (1981)J. Biol. Chem. 256, 10456-10463 25. Paulson, J. C., Hearick, J. I., and Hill, H. L. (1977) J. Biol. Chem. 252,2363-2371 26. Brockhaus, M., Magnani, J. L., Herlyn, M., Blaszczyk. M.. Steplewski, Z.Koprowski, H., and Ginsburg, V. (1982)-4rch. Biochem. Biophys., 217,647-651 27.Pukel, C. S.. Lloyd, K. 0..Travassos, L. H., Dippold, W . G., Oettgen, H. F., and Old. L. J. (1982) J. Exp. Med. 155. 11331147

“ J . C. Paulson, T. A. Reyer, J. L. Magnani,and unpublished results.

V. Ginsburg.