Customized Protein Nanoreactors for Affibody-Directed Activation of 5-Fluorcytosin in HER2-Positive Cells

The development of targeted therapies for HER2-positive cancers remains critical due to resistance and toxicity challenges in current treatments. Here, we present the rational engineering of protein-based encapsulin nanocompartments for selective catalytic prodrug activation. Encapsulins provide precise cargo loading, exceptional stability, and versatile engineerability, making them ideal nanoreactors for therapeutic applications. Our encapsulin constructs encapsulate tandem cytosine deaminase enzymes and display HER2-specific affibodies on their exterior, enabling precise cellular targeting. These engineered nanoreactors catalyze the efficient conversion of the prodrug 5-fluorocytosine (5-FC) into the cytotoxic agent 5-fluorouracil (5-FU), yielding an 83% reduction in viability of HER2-overexpressing SKOV3 cells. Structural characterization using native gel electrophoresis confirms stable assembly with functional affibody presentation. This enzyme-prodrug approach showcases how supramolecular protein architectures can serve as customizable platforms for affibody-directed, enzyme-mediated therapy, offering a promising strategy to enhance therapeutic specificity and minimize systemic side effects in HER2-positive cancer treatment. Significance StatementThis work establishes encapsulins as a new class of programmable therapeutic nanoreactors by integrating selective affibody-mediated targeting with enzyme-prodrug catalysis in HER2-positive cancer cells. Unlike virus-like particles, which often lack precise cargo loading and structural robustness, encapsulins enable dual engineering of both interior and exterior domains for stable, multifunctional assemblies. Compared to antibody-drug conjugates, these nanoreactors achieve amplified drug generation through localized prodrug activation, overcoming payload limitations and reducing systemic toxicity. This platform introduces a versatile supramolecular strategy for targeted cancer therapy that exceeds current delivery technologies.