In Silico Design and Structural Characterization of 4D5mocB-PE24: A Novel Immunotoxin for EpCAM-Targeted Cancer...

Antibody-based immunotoxins are a promising novel therapeutic approach aimed at targeted cancer therapy, utilizing the target specificity of antibody fragments attached to a potent cytotoxic molecule. Oportuzumab monatox, an EpCAM-specific immunotoxin, has shown clinical activity in Non-Muscle-Invasive Bladder Cancer (NMIBC) but is limited by its high immunogenicity. To address this limitation, we developed a novel construct comprising the 4D5mocB antibody fragment fused to a truncated, deimmunized Pseudomonas aeruginosa exotoxin A.

Various bioinformatics methods were employed to assess physicochemical characteristics and analyze structural stability using Molecular Dynamics (MD) simulations. The predicted epitope content for B cells and T cells, as well as the binding affinity for the EpCAM receptor, were also evaluated and compared with that of the parental immunotoxin.

Molecular dynamics analysis showed that the 4D5mocB-PE24 immunotoxin rapidly stabilized within 10 ns, maintaining RMSD values of 0.5-0.7 nm, whereas the PE40-based complex exceeded 2.0 nm after 22 ns. Moreover, computational modeling of the truncation and deimmunization process reduced the predicted number of B- and T-cell epitopes by 56% and 43%, respectively, and markedly decreased MHC class II binding.

These findings demonstrate that epitope deletion and rational mutagenesis substantially reduce predicted immunogenicity while preserving high target-binding affinity; however, these findings will require experimental validation.

This study identifies 4D5mocB-PE24 as a promising anti-EpCAM immunotoxin candidate with reduced immunogenicity, supporting further preclinical and clinical evaluation for EpCAM-positive cancers.