Influence of terminal residue on adjacent disaccharide immunogenicity

Aberrant O-glycosylation of cell surface mucin antigens is characteristic of epithelial cancer cells. For example, Thomsen-Friedenreich disaccharide (TFD) is a chemically well-defined carbohydrate antigen with a documented link to malignancy. There have been many attempts to improve immune response to carbohydrate antigens, for use in immunotherapy. As part of an alternative strategy to improve carbohydrate immunogenicity, we studied the influence of terminal benzyl (Bzl) or p-nitrophenyl (pNP) residue on immunogenicity of adjacent TFD. Mice immunized with keyhole limpets hemocyanin-TFD (KLH-TFD), KLH-TFD(alpha)Bzl, or KLH-TFD(alpha)pNP produced anti-KLH antibodies, which were analyzed by enzyme-linked immunosorbent assay (ELISA). KLH-TFD did not give significant anti-TFD antibody titer, confirming the poor immunogenicity of TFD. Immunization with KLH-TFD(alpha)Bzl and KLH-TFD(alpha)pNP raised antibody titers against TFD(alpha)Bzl and TFD(alpha)pNP, respectively. KLH-TFD(alpha)Bzl also gave higher anti-TFD antibody response, whereas KLH-TFD(alpha)pNP did not, indicating that terminal Bzl residue improves immune response to adjacent carbohydrate. Analysis of anti-TFD(alpha)Bzl or anti-TFD(alpha)pNP IgG antibodies by competitive ELISA, using carbohydrate-related antigens as inhibitors, demonstrated their high specificity to their respective antigens. Anti-TFD(alpha)pNP antibody was not inhibited by TFD, but was significantly inhibited by GalNAc(alpha)pNP. The fact that p-nitrophenol (pNPol) has more competitive ability that GalNAc indicates that terminal polar residue is the main target antigen. In contrast, anti-TFD(alpha)Bzl antibody was inhibited to a similar degree by GalNAc(alpha)Bzl and TFD, confirming the carbohydrate recognition by antibodies yielded by terminal non-polar modification of the immunogen.