ganglio-n-triaosylceramide and Lymphoma

Immunotoxins composed of monoclonal antibodies (mAbs) and various toxins have been developed for the treatment of malignancies. We investigated the efficacy of three ricin toxin A-chain (RTA)-containing immunotoxins (ITs) conjugated from mAbs which recognize glycolipid asialo-GM2 and glycoprotein H-2d. These ITs retained the same immunoreactivity with mAbs. We evaluated the cytotoxicity of these ITs against mouse lymphoma cells L5178Y variants showing high (AA12,CC9) and low (27AV) expression of asialo-GM2. Anti-H-2d-RTA IT had the strongest cytotoxicity for all cell lines. Anti-asialo-GM2 (IgM)-RTA IT had stronger cytotoxicity than anti-asialo-GM2 (IgG3)-RTA IT. Anti-asialo-GM2-RTA ITs had different cytotoxicity against AA12 and CC9 cells. The establishment of appropriate anti-glycolipid mAbs may lead to effective immunotargeting therapy.

Topics: Animals; Antigen-Antibody Reactions; Biomarkers, Tumor; Evaluation Studies as Topic; Gangliosides; Glycoproteins; Glycosphingolipids; Immunotoxins; Lymphoma; Mice; Neoplasm Proteins; Ricin; Tumor Cells, Cultured

YAC-1 cells were propagated in bioreactors in 1 l and 7.5 l volumes. The cells were metabolically labelled with D-[1-14C]galactose and D-[1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was caused by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed.

Topics: Acetylgalactosamine; Animals; Antibody Specificity; Carbohydrate Conformation; Carbohydrate Sequence; Chickens; Chromatography, High Pressure Liquid; Female; G(M1) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Immune Sera; Immunoassay; Lymphoma; Mice; Mice, Inbred CBA; Molecular Sequence Data; Spectrometry, Mass, Fast Atom Bombardment; T-Lymphocytes; Tumor Cells, Cultured

The growth of mouse lymphoma L-5178Y cells with a high degree of gangliotriaosylceramide expression (high-expressor clone AA12) was inhibited by the addition of a biotinylated mouse immunoglobulin M monoclonal antibody (2D4) directed to gangliotriaosylceramide followed by cross-linking with avidin or a second antibody (anti-mouse immunoglobulin M). This growth inhibition was observed in both serum-containing medium and a chemically defined medium containing transferrin as the only growth factor. Cell growth was not inhibited by the addition of biotinylated antibody 2D4 alone, avidin alone, or the biotinylated derivative of an immunoglobulin M monoclonal anti-N-acetyllactosamine antibody (1B2) cross-linked with avidin. The growth of lymphoma L-5178Y 27AV cells, which do not express gangliotriaosylceramide (non-expressor clone), was not inhibited by either of these monoclonal antibodies or their biotinylated derivatives plus avidin. In the presence of biotinylated antibody 2D4 and avidin, cells of the high-expressor clone (L-5178Y AA12) displayed a capping of gangliotriaosyl antigen. In contrast, the transferrin receptor and the major glycoproteins (concanavalin A receptors) were not capped in the presence of biotinylated antibody 2D4 and avidin but were homogeneously distributed on the cell surface. Cells whose growth was inhibited by the addition of biotinylated antibody 2D4 and avidin showed an inhibition of 125I-transferrin internalization, although binding of 125I-transferrin to the cell surface was similar to that of control cells. These results indicate that the tumor antigen gangliotriaosylceramide is functionally associated with the transferrin receptor and may regulate the process of internalization of transferrin.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Avidin; Biotin; Culture Media; Gangliosides; Glycosphingolipids; Killer Cells, Natural; Lymphoma; Mice; Receptors, Cell Surface; Receptors, Transferrin; Transferrin