AbstractMany human carcinomas overexpress the Lewisy (Ley) blood-group epitope [Fuc12Gal14 (Fuc13)GlcNAc13Gal-]. With a view to developing Ley based vaccines we have examined the immunogenicity of Ley-protein conjugates in mice. Ley pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Ley oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Ley oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Ley epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Ley (MCF-7). These experiments suggest that Ley-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients.