Occurrence of the human tumor-specific antigen structure Galβ1-3GalNAcα- (Thomsen-Friedenreich) and related structures on gut bacteria: Prevalence, immunochemical analysis and structural confirmation

The Thomsen-Friedenreich antigen (TF; CD176, Gal beta 1-3GalNAc alpha-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of alpha-anomeric TF (TF alpha) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for alpha- and beta-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TF alpha antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TF alpha- and TF beta-specific antibodies. The active antigen was identified as Gal beta 1-3GalNAc-, which was alpha-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TF alpha are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TF alpha structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.