Bispecific antibody-based cancer vaccines are designed to deliver peptide cargoes to professional antigen-presenting cells (APCs) and activate targeted anti-tumor immune responses via CD8+ T cells. However, the mechanism by which these peptides enter APCs and escape the endosomal compartment remains unclear. A current hypothesis proposes that, after internalization, the peptides dissociate from the antibody scaffold and undergo endosomal escape, enabling their presentation via MHC I/HLA I to CD8⁺ T cells. In parallel, dendritic cells can also take up peptides and present them via MHC II/HLA II to CD4⁺ T cells, thereby engaging both arms of the adaptive immune response. This project aims to validate this hypothesis and assess the functional potential of the vaccine platform.
This project examined several potential factors that may influence the release of the antigenic pTag from the antibody platform, including the secondary structure of the pTag, its stability in plasma, and the pH changes during uptake by dendritic cells (DCs). ELISA results indicated that pH did not have a significant effect on the binding affinity between the pTag and the antibody scaffold. The membrane permeabilization assay did not reveal any noticeable membrane disruption, nor did the control peptide, raising questions about the reliability of this method. Circular dichroism (CD) analysis also showed no significant differences among the three pTag variants. Flow cytometry (FACS) data confirmed that the antibody facilitated cellular uptake of the peptide; however, no significant differences were observed between the different pTag sequences.