Tn Hitchhiker Vaccines for High-Affinity Antibody Production and Targeted Cancer Cell Binding

The development of effective cancer vaccines relies on the ability to generate a strong and specific humoral immune response against tumor-associated antigens. The Tn antigen, a truncated O-glycan overexpressed by various carcinomas, is a potent immunotherapeutic target. In this study, we evaluate the antibody specificity elicited in mice by the Tn-DSPE-PEG-2000 amphiphilic vaccine, along with initial insights into its cellular processing using a lysosomal assay.The vaccine is found to undergo lysosomal escape, thereby facilitating cytoplasmic antigen delivery, which is a crucial step for effective antigen presentation. ELISA studies show that the individual components of Tn antigen construct are essential for the antibody recognition. Specific binding of the serum-anti-Tn antibodies to Tn-positive MCF-7 cells is assessed by confocal microscopy, whereas negligible binding is observed in Tn-negative HEK293 cells, thereby supporting the high specificity of the immune response. Flow cytometry validates the strong reactivity of serum antibodies toward Tn-expressing cells and surface plasmon resonance (SPR) analysis enables quantification of the high-affinity interactions between serum anti-Tn antibodies and the Tn antigen. The study establishes (i) the specificity of antibody generated by Tn-DSPE-PEG-2000 and (ii) the structural requirement of the antigen for the anti-Tn antibody recognition.