Enhanced Targeted Drug Delivery System to Control Avidity and Drug Encapsulation Using E2 Nanocages and SpyTag/SpyCatcher.

Although antibody-drug conjugates offer advanced targeted anticancer therapy that overcomes the limitations of conventional chemotherapy and therapeutic antibodies, they are restricted in their capacity to carry multiple hydrophobic payloads. Protein nanocages have emerged as versatile therapeutic platforms for targeted drug delivery, offering advantages like precise molecular assembly, biocompatibility, and multivalent targeting. This study presents the development of engineered E2 nanocages functionalized with anti-HER2 single-chain variable fragments (scFv) using the SpyTag/SpyCatcher ligation system to achieve controlled scFv display valency. The results demonstrate that increasing anti-HER2 scFv valency enhances HER2 binding affinity via avidity effects, with the highest valency nanocages showing the highest binding avidity. Furthermore, cysteine residues were introduced into the E2 nanocages to enable conjugation with monomethyl auristatin E (MMAE) through maleimide chemistry, achieving efficient drug loading. The resulting MMAE-conjugated nanocages displayed potent, subnanomolar cytotoxicity in HER2-positive SKBR3 and BT-474 cell lines while sparing HER2-negative MDA-MB-231 cells at concentrations up to 1 nM. These results underscore the critical role of scFv valency in enhancing HER2 targeting and highlight the potential of E2 protein nanocages as specific, potent platforms for targeted cancer therapy. In this study, we developed an enhanced targeted drug delivery system using E2 nanocages and scFv with SpyCatcher/SpyTag ligation to regulate binding avidity and encapsulate hydrophobic drugs. The modular design and pH-sensitive dissociation of these nanocages establish a foundation for next-generation precision medicine strategies.