ALT-P7

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.

THIOMAB drug conjugate (TDC) technology provides site-specific conjugation of linker drugs to antibodies, allowing for targeted delivery of the payload. While a direct measurement of TDC cytotoxic potency allows efficient screening and confirmation that new drugs conjugated to antibodies result in proper processing in cells, additional mechanistic characterization is often needed to provide information-rich data to guide further optimization of TDC design. For example, a quantitative understanding of how TDCs are processed intracellularly can help determine which processing step is impacting payload delivery and thereby inform the basis of the TDC efficacy. Here, we measure the cellular accumulation of two different TDC drug payloads: MAPK (mitogen-activated protein kinase) pathway inhibitor targeting ETbR-expressing tumor cells and an antibiotic active against Staphylococcus aureus with an in vitro cell-based drug release LC-MS/MS assay in a 96-well format. This assay allowed us to correlate the cellular potency of each unconjugated molecule with the amount of payload that accumulated inside the cell. In the case of the pathway inhibitor drug, the biochemical characterization of TDC processing by cathepsin B and purified human liver enzyme extract demonstrated a correlation between the efficiency of the linker drug cleavage and intracellular payload accumulation. For the antibody-antibiotic conjugate, kinetic analysis of intracellular free drug retention provided valuable insight into the chemistry modifications needed for an efficient TDC. Taken together, we demonstrated the utility of quantitative LC-MS/MS assays as one tool in guiding the design of more effective TDCs via the mechanistic release characterization of two distinct payloads.