A trivalent gastric cancer antigen conjugated to a diphtheria toxin epitope synergizes with an NKT cell agonist to boost antitumor immunity

The gastric cancer-associated antigen MG7-Ag is aberrantly expressed in multiple gastrointestinal malignancies and represents a promising broad-spectrum target for pan-gastrointestinal tumor peptide vaccines; however, its clinical application is hampered by low immunogenicity. To overcome this limitation, we covalently conjugated a diphtheria toxin-derived T-helper epitope to the trivalent antigen MG3, which was constructed by tandem repetition of the monovalent MG1 peptide from the MG7-Ag. A liposomal vaccine was subsequently developed by incorporating DT-MG3 together with the natural killer T (NKT) cell agonist α-galactosylceramide (αGalCer). Immunological evaluation demonstrated that DT-MG3 plus αGalCer elicited significantly higher specific IgG titers than DT-MG3 alone or the unconjugated MG3/αGalCer combination. The vaccine also promoted IFN-γ/IL-4 secretion and boosted CD4+/CD8+ T cell responses to elevate antigen immunogenicity. Moreover, the high-titer specific antibodies induced by the vaccine also efficiently bound to EAC tumor cells and induced complement-dependent cytotoxicity. In the EAC ascites model, vaccination with DT-MG3/αGalCer substantially reduced malignant ascites accumulation and prolonged the survival of tumor-bearing mice. Collectively, this novel anti-gastric cancer vaccine based on the covalent conjugation of a diphtheria toxin epitope to the trivalent MG3 antigen, provides valuable insights for the rational design of potent and safe fully synthetic peptide-based anti-tumor vaccines.