Novel immunogenicity of Thomsen-Friedenreich disaccharide obtained by a molecular rotation on its carrier linkage

The a-anomeric Gal beta 1-3CalNAc, called Thomsen-Friedenreich disaccharide (TFD), is overexpressed in epithelial cancer cells by aberrant O-glycosyla tion. TFD is also the main ligand of Agaricus bisporus lectin (ABL), a reve rsible noncytotoxic inhibitor of proliferation of epithelial cell lines. In order to obtain anti-TFD antibody response with a fine carbohydrate-bindin g specificity similar to that of ABL, we designed an immunogen of TFD with a molecular rotation on its carrier linkage that exposes more GalNAc than G al, since ABL recognizes GalNAc more than Gal in TFD, The synthesis was acc omplished by C-6 oxidation of Gal from TFD or its a-benzyl derivative (Bzl alpha TFD), followed by reductive amination between the C-6 aldehyde yielde d and the available amine of protein. Mice immunized with TFD-KLH (keyhole limpet hemocyanin) or Bzl alpha TFD-KLH produced antibodies which were then analyzed by ELISA against several target antigens, Both immunogens raised anti-KLH antibody titers; however, TFT)-KLH did not raise anti-TFD antibodi es showing low TPD immunogenicity. In contrast, Bzl alpha TFD-KLH gave much higher anti-TFD antibody response, indicating that benzyl residue helps im prove anti-carbohydrate immune response, When IgG and IgM anti-TFD antibodi es were analyzed by competitive ELISA using TFD-related carbohydrates as in hibitors, a high specificity to TFD as well as an enhanced binding to GalNA c over Gal were observed. The axial C-4 hydroxyl group of GalNAc interacted with IgG anti-TFD antibody, as evidenced by the lack of inhibitory activit y of GlcNAc in contrast to GalNAc, These findings indicate that the anti-TF D antibodies have fine carbohydrate-binding specificity more similar to ABL than to other TFD-binding proteins that stimulate proliferation of epithel ial cell lines.