Compromising the immunogenicity of diphtheria toxin-based immunotoxins through epitope engineering: An in silico approach.

Immunotoxins are genetically engineered recombinant proteins consisting of a targeting moiety, such as an antibody, and a cytotoxic toxin moiety of microbial origin. Pseudomonas exotoxin A and diphtheria toxin (DT) have been abundantly used in immunotoxins, with the latter applied as the toxin moiety of the FDA-approved drug Denileukin diftitox (ONTAK®). However, the use of immunotoxins provokes an adverse immune response in the host body against the toxin moiety, limiting their efficacy. In silico approaches have received increasing attention in protein engineering. In this study, the epitopes responsible for immunogenicity were identified through multiple platforms. By subtracting conserved and ligand-binding residues, K33, T111, and E112 were identified as common epitopes across all platforms. Substitution analysis evaluated alternative residues regarding their impact on protein stability, considering 19 different amino acid substitutions. Among the mutants explored, the T111A-E112G mutant exhibited the most destabilizing substitution for DT, thereby reducing immunogenicity. Finally, a 3D model of the mutant was generated and verified. The model was then docked with its native ligand NADH, and the complex's molecular behavior was simulated using molecular dynamics.