A bioinformatics pipeline for the design of a SART3-targeted cancer vaccine with enhanced immunogenicity

Abstract Background and objectives Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) has emerged as a promising target for cancer immunotherapy, given its overexpression in various malignancies and low or absent expression in non-tumorous tissues. This study aimed to design rationally and in silico evaluate a multi-epitope T cell vaccine targeting SART3, incorporating a TLR4 agonist adjuvant. The vaccine’s predicted immunogenicity, physicochemical properties, structural stability, and interaction with TLR4 were comprehensively assessed. Additional assessments of cytokine-inducing potential, B-cell epitopes, and disulfide engineering opportunities were also executed. Methods Potential T-cell epitopes from SART3 were identified using IEDB and screened for antigenicity (VaxiJen), toxicity (ToxinPred), and MHC-I/II binding affinity. Cytokine-inducing epitopes were evaluated using IL4pred, IL-10Pred, and IFNepitope servers. B-cell epitopes were predicted using ElliPro. The vaccine underwent comprehensive physicochemical, structural (I-TASSER/GalaxyRefine), molecular docking (HDOCK), molecular dynamics simulations, and disulfide engineering (Disulfide by Design 2.0) analyses. Results The optimized 344-residue vaccine demonstrated non-allergenicity, high stability (instability index 17.16), antigenicity (Vaxijen 0.67), and solubility (SOLpro 0.96). HDOCK predicted favorable vaccine–TLR4 binding (ΔG = − 265.61 kcal/mol, confidence 91%). MD simulations confirmed complex stability. Cytokine analysis revealed the potential to induce IL-4 and IL-10. The Val80–Ala123 pair exhibited the lowest bond energy (1.16 kcal/mol), indicating the optimal geometry for disulfide bond formation. The in silico immune simulations demonstrated a robust immune response following vaccine administration. Conclusion This rationally designed SART3-targeted multi-epitope vaccine exhibits promising in silico characteristics across immunogenicity, physicochemical, cytokine-inducing, B-cell epitope, structural, and disulfide engineering profiles, warranting experimental validation for cancer immunotherapy development.