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Ganglioside GM2 As a Human Tumor Antigen (OFA-I-1) (Monospecific Human Antitumor Antibody/Membrane Glycolipid/Fetal Brain/Melanoma) TADASHI TAI*, JAMES C

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Ganglioside GM2 As a Human Tumor Antigen (OFA-I-1) (Monospecific Human Antitumor Antibody/Membrane Glycolipid/Fetal Brain/Melanoma) TADASHI TAI*, JAMES C Proc. Natl. Acad. Sci. USA Vol. 80, pp. 5392-5396, September 1983 Immunology Ganglioside GM2 as a human tumor antigen (OFA-I-1) (monospecific human antitumor antibody/membrane glycolipid/fetal brain/melanoma) TADASHI TAI*, JAMES C. PAULSONt, LESLIE D. CAHANt, AND REIKO F. IRIE*§ Departments of *Surgery/Oncology, tBiological Chemistry, and tSurgery/Neurosurgerv, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024 Communicated by S. Hagiwara, May 23, 1983 ABSTRACT A monospecific antibody produced in vitro by a chemical nature of OFA-I-1 that binds to monospecific anti- B-lymphoblastoid cell line transformed with Epstein-Barr virus body OFA-I-1 produced in vitro. By using immune adherence has been shown to recognize a membrane antigen (OFA-I-1) on inhibition as the assay, the antigen was successfully purified human tumors and fetal brain. This study identifies the chemical and characterized as the ganglioside GM2. nature of OFA-I-1. The glycolipid fraction of antigen-rich spent medium of an OFA-I-1-positive melanoma cell line, M14, was ex- tracted by chloroform/methanol/water, 4:8:3 (vol/vol), and was MATERIALS AND METHODS separated into fractions of neutral glycolipids and gangliosides by OFA-I-1 Antigen Source. A human melanomacell line, UCLA- DEAE-Sephadex followed by base treatment and Bio-sil A column SO-M14 (M14), which expresses OFA-I-1 and OFA-I-2 and sheds elution. OFA-I-1 antigens were found exclusively in the ganglio- both antigens into the culture medium, was maintained in a side fraction when assayed with monospecific anti-OFA-I-1 by an chemically defined medium supplemented with 0.05% human immune adherence inhibition test. The results obtained from thin- serum albumin as described (12). Spent cell culture medium of layer chromatography of the antigenic M14 ganglioside and se- this cell line was collected in plastic flasks when monolayers quential glycosidase digestion suggested that the antigen was a were >80% confluent. As a source of soluble, crude antigen, ganglioside GM2, GalNAcfi1-+4(NeuAca2--*3)Gal81-->4Glc--- this medium was centrifuged at 1,000 x g for 10 min to remove Cer. These results were further supported by testing various au- cell debris and was concentrated 100-fold with an Amicon filter. thentic gangliosides and neutral glycolipids for OFA-I-1 antigen- Fetal Tissue. Human fetal liver and brain tissue were pre- icity. Only GM2 showed positive reactivity. pared as described (8). Adult brain was obtained from post- mortem specimens. Tissues were frozen at -80°C until needed Specific monoclonal antibodies have been used as precise re- for chemical analysis. agents to identify and characterize the molecular properties of Monospecific Antibody to OFA-I-1. Anti-OFA-I-1 antibody various types of membrane antigens of human tumor cells (1- secreted into the spent tissue culture medium of a human lym- 5). Mouse monoclonal antibodies made by hybridoma tech- phoblastoid cell line (L55) was used as the source of antibody. nology are most commonly used in such studies. However, tu- Its immunologic specificities have been described (11). mor-associated antigens defined by xenoantibodies may not be Antigen Assay. The reactivity of OFA-I-1 antibody with sol- as relevant for the recognition of antigens that have immuno- uble antigen was determined by inhibition of immune adher- genicity in man as human antibodies. During the last several ence (7). In brief, serially diluted antigen or test fractions (50 years, we have investigated cell surface antigens that induce ,ul) and 50 ,ul of anti-OFA-I-1 antibody were mixed and in- immune responses in cancer patients (6-8). In 1975, we de- cubated at 4°C overnight. After incubation, 2.5 X 104 M14 cells scribed an antigen expressed on a variety of human tumors that in 25 ,ul of barbital-buffered saline were added to the antigen/ reacted with serum antibodies of cancer patients (9). The an- antibody mixture, and the immune adherence assay was per- tigen was named oncofetal antigen-immunogenic (OFA-I) be- formed. cause it was expressed on both tumors and fetal normal brain Glycolipids. Purified human brain gangliosides (GM3, GM1, (10). However, human serum antibodies, because they are GD1a, GD1b, GT1b) were generously provided by Lars Sven- polyclonal and contain a relatively low level of specific anti- nerholm of The GD3 of hu- bodies, are generally quite difficult to use as reagents for iden- (University Goteborg, Sweden). tification of the precise chemical structure of tumor cell an- man brain was a gift from John Magnani and Victor Ginsburg tigens, particularly for distinguishing between various subsets (National Institutes of Health, Bethesda, MD). GD2 and GM2 of closely related antigenic determinants. were prepared from GD1b and GM1, respectively, by using Recently, we produced human monoclonal antibodies in vi- bovine testes f3-galactosidase as described (12). LacCer, tro against OFA-I by transforming human B lymphocytes with GgOs3Cer, and GgOs4Cer were prepared from GM3, GM2, Epstein-Barr virus (11). This technology enabled us to define and GM1, respectively, by treatment with mild acid hydrolysis two different antigenic specificities on the OFA-I target cells. One Abbreviations: Abbreviations and nomenclature of neutral glycolipids antigen, OFA-I-2, is expressed only on tumors of neu- are according to the recommendation of the Nomenclature Committee, roectodermal origin, whereas the second, OFA-I-1, is found on International Union of the Pure and Applied Chemistry (31). GlcCer = a wide variety of human cancer cells, including melanomas, brain Glc--+Cer; LacCer = Gal,1-4Glc-+Cer; GbOs3Cer = Galal--+ tumors, sarcomas, breast carcinomas, and tumors of several other 4GalP31-*4Glc--+Cer; GbOs4Cer = GalNAcB1-*3Gala1- types. The chemical structure of OFA-I-2 has been identified 4GalP31-4Glc--*Cer; GgOs3Cer = GalNAcl31--4Gal31--4Glc-- Cer; as the ganglioside GD2 [Ga1NAc,81-*4(NeuAca2--8NeuAca2 GgOs4Cer = Gall13GaINAcl14Gall8-4GlcCer. Abbrevi- ations of the ganglio series gangliosides are according to the system of -*3)GalB1-->4Glc-->Cer] (12). In this report, we describe the Svennerholm (32). As an example, the structure of GTlb is Neu- Aca2 -3Gall13GalNAcl14(NeuAca28NeuAca2-3)Gal,81 The publication costs of this article were defrayed in part by page charge 4Glc-+Cer. Other ganglioside structures are given in representational payment. This article must therefore be hereby marked "advertise- form in Fig. 3. OFA-I, oncofetal antigen-immunogenic. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. § To whom reprint requests should be addressed. 5392 Downloaded by guest on October 1, 2021 Immunology: Tai et aL Proc. Natd. Acad. Sci. USA 80 (1983) 5393 (1% acetic acid at 1000C for 1 hr). The products were later pu- of sodium taurocholate (Sigma) at a 3.0 molar ratio of detergent rified by TLC in solvent 1. GlcCer was prepared from GM3 by to substrate and was incubated at 370C overnight. After the re- using sialidase and jack-bean f3-galactosidase as described be- action with an enzyme, products were visualized on TLC in sol- low. GbOs3Cer and GbOs4Cer were purchased from Supelco vent 2, unless otherwise noted. One unit of glycosidase was (Bellefonte, PA). defined as the amount of enzyme required to hydrolyze 1 /Imol TLC. Merck precoated high-performance TLC plates (silica of the substrate per min. gel 60, 200 ,Am in thickness) were employed for fractionation OFA-I-1 (120 nmol) was incubated with 2 units of A. urea- of the glycolipids. Solvents used for developing the chromato- faciens sialidase in 0.05 M sodium acetate buffer (pH 4.5) to a grams were composed of chloroform/methanol/0.22% CaCl2, total volume of 2.0 ml at 37C for 2 days (21). Asialo-OFA-I-1 55:45:10 (vol/vol) (solvent 1), and chloroform/methanol/water, (60 nmol) was incubated with 2 units of N-acetyl-3-hexos- 65:25:4 (vol/vol) (solvent 2). Gangliosides were visualized with aminidase ofjack bean in 0.05 M sodium citrate buffer (pH 5.0) resorcinol stain (13) or orcinol stain (14), and neutral glycolipids (400 sud) (22). After the removal of one sialic acid and one N- were visualized with orcinol stain. Polypeptides were visual- acetylhexosamine, OFA-I-1 (33.3 nmol) was incubated with 2 ized by fluorescamine spray (Supelco) (15). TLC plates pre- units ofjack-bean (galactosidase in 0.05 M sodium citrate buffer coated with silica gel 60 (250 ,m in thickness, Merck) were em- (pH 3.5) (480 p1) (22). ployed when a preparative isolation of the gangliosides was required. Isolation and Purification of Glycolipids. Glycolipids were RESULTS extracted from the OFA-I-1-rich M14 spent medium by using Detection of OFA-I-1 in Ganglioside Fraction of M14 Cells. the method of Svennerholm and Fredman (16) and separated In a previous study, the chloroform/methanol/water extraction into neutral and acidic fractions by DEAE-Sephadex chroma- of OFA-I-2 (ganglioside GD2) from M14 cells that expressed tography (17). Acidic glycolipids were further purified by base both OFA-I-1 and OFA-I-2 antigens suggested that OFA-I-1 treatment and passage over a Bio-sil A column. Briefly, con- was a glycolipid (12). Although the OFA-I-1 antigen was de- centrated M14 spent medium (20 ml) was mixed with 54 ml of tected in the filtrate after the chloroform/methanol/water ex- methanol at room temperature under constant stirring before traction of the cells, its antigenicity could not be destroyed by 27 ml of chloroform was added. The mixture was stirred for 30 treatment with sialidase alone. However, in subsequent studies min and was centrifuged at 3,)00 x g for 30 min. The super- we found that OFA-I-1 could be completely destroyed by sial- natant was clarified by filtration.
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