Design and immunogenic evaluation of multi-epitope vaccines for colorectal cancer: insights from molecular dynamics and

Background: This study aimed to identify cytotoxic T lymphocyte (CTL)-specific epitopes from three tumor-associated antigens (TAAs)-Dickkopf-like 1 (DKKL1), F-box protein 39 (FBXO39), and Opa-interacting protein 5 (OIP5)-which are overexpressed in colorectal cancer (CRC), as potential candidates for CTL-mediated immunotherapy. Methods: The amino acid sequences of DKKL1, FBXO39, and OIP5 were analyzed to predict high-affinity CTL epitopes using the NetCTL server. Their antigenicity, allergenicity, conservation, and glycosylation potential were assessed for safety and effectiveness. Cross-reactivity and binding affinities were evaluated through molecular docking. Two multi-epitope vaccine constructs were designed incorporating the CTL epitopes, GM-CSF and IL-2 adjuvants, and a PADRE sequence. Docking studies with Toll-like receptor 4 (TLR-4) were performed. Results: High-affinity CTL-specific epitopes were successfully identified from DKKL1, FBXO39, and OIP5, showing strong binding potential to HLA class I molecules. The selected epitopes were predicted to be non-allergenic, non-glycosylated, and conserved across species. Molecular docking confirmed stable binding interactions between the epitopes and HLA alleles. Conclusion: This study identified CTL-specific epitopes from DKKL1, FBXO39, and OIP5 as potential targets for colorectal cancer immunotherapy. The multi-epitope vaccine constructs exhibited significant immunogenic potential, providing a foundation for future clinical validation. These findings underscore the promise of these TAAs as key targets for CTL-based vaccine development in colorectal cancer.