A CXCR4-Targeted Dual-Acting Nanoconjugate Combines Protein-Toxin and Small-Molecule Warheads for Synergistic Cytotoxicity

Abstract Self-assembling multivalent protein nanoparticles with an architecture based on His-metal coordination and specific binding to cancer stem cells have emerged as promising tools for the selective delivery of microbial toxins in oncology, reaching precise cytotoxic effects. The incorporation of small-molecule drugs aimed at improving drug potency has so far been limited by the loss of stability and especially the loss of selectivity of the final products. Here, we have explored a site-specific conjugation method based on the Staphylococcus aureus sortase A, by which a CXCR4-targeted, engineered form of the diphtheria-toxin-derived protein warhead has been covalently enriched with the anti-tumoral agent monomethyl auristatin E (MMAE). The resulting dual-acting nanoscale conjugate exhibits enhanced cytotoxicity over CXCR4+ cells without compromising selectivity in the intracellular delivery process. Interestingly, the combination of the microbial toxin and MMAE, empowered by the multivalent display of the CXCR4-ligand T22, shows significant synergistic cytotoxicity over the target cells. Therefore, the suggested strategy allows not only the design of dual-acting targeted nanoconjugates that combine protein-toxin and small-molecule warheads in precise stoichiometric ratios but also the enhancement of their cytotoxic effects by allowing a synergistic action.