The fine specificity analysis of two human monoclonal antibodies (AbFCM1 and AbHJM1) reacting with gangliosides is described and their specificities are compared with analogous mouse monoclonal antibodies (mAbs). These two antibodies were generated from lymphocytes of melanoma patients by Epstein-Barr virus transformation followed by fusion with mouse myeloma NS-1. Using a wide variety of gangliosides, including N-glycolylneuraminic acid (NeuGc)-containing compounds, the precise structures recognized by these two antibodies were elucidated by enzyme-linked immunosorbent assay and immunostaining of thin-layer chromatograms. AbFCM1 reached with N-acetylneuraminic acid (NeuAc)-type GM3, GD1a, sialylparagloboside, and GT1b in decreasing order of intensity. This antibody also reacted with (NeuAc-NeuGc-)-GD3 and -disialylparagloboside, but did not react with NeuGc-type GM3, GM2, sialylparagloboside, (NeuGc)2-GD3 and -disialylparagloboside. The main epitope structures recognized by AbFCM1 are, therefore, NeuAcα2→3Galβ1- and NeuAcα2→8NeuGcα2→Galβ1-. These results are similar to the specificity of mouse mAb M2590. AbHJM1 reacted with NeuActype GD3 and disialylparagloboside, GD2, GD1b, GM3, and GT1b, in decreasing order of intensity. Among NeuGc-type gangliosides, this antibody reacts with (NeuAc-NeuGc-)-GD3 and -disialylparagloboside, but did not react with gangliosides containing only NeuGc. Consequently the epitope structure recognized by AbHJM1 is probably (R)-(NeuAcα2→8Sialic acidα2→3)Galβ1-. Mouse anti-GD3 mAbR24, in contrast, showed strong reactivity only with GD3 and -disialylparagloboside among NeuAc-type gangliosides, but showed a similar pattern to AbHJM1 in its reactivity with NeuGc-containing gangliosides. Although these two human monoclonal antibodies are not highly restricted in their specificities, they reacted best with the major gangliosides, GM3 and GD3, present in the majority of human melanomas.
This work was supported by grants from the American Cancer Society (IM-429) and the National Cancer Institute (CA 47427 and CA 08478).