Glycobiology Advance Access published online on January 31, 2006
Glycobiology, doi:10.1093/glycob/cwj082
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1 Unité de Chimie Organique URA CNRS 2128, Institut Pasteur, Paris, France
* To whom correspondence should be addressed. In cancer, mucins are aberrantly O-glycosylated and consequently, they express tumor-associated antigens such as the Tn determinant (
Received August 31, 2005
Revised January 25, 2006
Accepted January 25, 2006
Article
Enzymatic large-scale synthesis of MUC6-Tn glycoconjugates for anti-tumor vaccination
Teresa Freire 1,
Richard Lo-Man 2,
Friedrich Piller 3,
Véronique Piller 3,
Claude Leclerc 2,
and
Sylvie Bay 1 *
2 Unité de Biologie des Régulations Immunitaires INSERM E352, Institut Pasteur, Paris, France
3 Centre de Biophysique Moléculaire, CNRS UPR 4301 affiliated to INSERM and the Université d’Orléans, Orléans, France
Sylvie Bay, E-mail: sbay{at}pasteur.fr
Abstract 
-GalNAc-O-Ser/Thr). They also exhibit a different pattern of expression as compared to normal tissues. In particular, MUC6, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal and breast carcinomas. Recently, we have shown that the MCF7 breast cancer cell line expresses MUC6-Tn glycoproteins in vivo. Cancer-associated mucins show antigenic differences from normal mucins and, as such, they may be used as potential targets for immunotherapy. In order to develop anti-cancer vaccines based on the Tn antigen, we prepared several MUC6-Tn glycoconjugates. To this end, we performed the GalNAc enzymatic transfer to two recombinant MUC6 proteins expressed in E. coli by using UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), which catalyze in vivo the Tn antigen synthesis. We used either a mixture of ppGalNAc-Ts from MCF7 breast cancer cell extracts or a recombinant ppGalNAc-T1. In both cases, we achieved the synthesis of MUC6-Tn glycoconjugates at a semi-preparative scale (mg amounts). These glycoproteins displayed a high level of Tn antigens, although the overall density depends on both enzyme source and protein acceptor. These MUC6-Tn glycoconjugates were recognized by two anti-Tn monoclonal antibodies which are specific for human cancer cells. Moreover, the MUC6-Tn glycoconjugate glycosylated using MCF7 extracts as the ppGalNAc-T source was able to induce IgG antibodies that recognized a human tumor cell line. In conclusion, the production in large amounts of MUC6 with tumor-relevant glycoforms holds considerable promise for developing effective anti-cancer vaccines and further studies of their immunological properties are warranted.
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