Diphtheria Toxoid-Derived T‑Helper Epitope\nand α‑galactosylceramide Synergistically Enhance the\nImmunogenicity of Glycopeptide Antigen

The\ntumor-associated antigen MUC1 is an attractive target for immunotherapy,\nhowever, its weak immunogenicity limits the induction of antitumor\nimmune responses. To overcome this limitation, in this study, MUC1\nglycopeptide was covalently linked with a diphtheria toxin-derived\nT-helper epitope (DT<sub>331–345</sub>). Subsequently, the\nresulting DT-MUC1 glycopeptide was physically mixed with natural killer\nT cell agonist αGalCer to explore their immunomodulatory synergy.\nBiological results demonstrated that compared to MUC1+αGalCer\nand DT-MUC1 groups, the specific IgG antibody titer of DT-MUC1+αGalCer\ngroup increased by 189- and 3-fold, respectively, indicating that\nthe diphtheria toxin-derived T-helper epitope synergistically enhanced\nMUC1 immunogenicity with αGalCer. Moreover, the DT-MUC1+αGalCer\nvaccine induced potent cellular immune responses and significantly\ninhibited the growth of B16-MUC1 tumors in vivo. Furthermore, it was\nfound that the <i>anti</i>-MUC1 IgG antibody titer induced\nby DT-MUC1+αGalCer was equivalent to that induced by palmitoylated\nDT-MUC1+αGalCer (P1-DT-MUC1+αGalCer) and significantly\nhigher than that induced by doubly palmitoylated DT-MUC1+αGalCer\n(P2-DT-MUC1+αGalCer), suggesting that the easily synthesized\nDT-MUC1 may not require lipid chain modification and already possess\ngood amphiphilicity. This is the first time that a diphtheria toxin-derived\nhelper T-helper epitope was covalently linked to a glycopeptide antigen\nto enhance its immunogenicity, and this study may provide an effective\nvaccine design strategy for MUC1-targeted antitumor vaccines and offer\nnovel insights into the design of fully synthetic peptide vaccines.