Targeted delivery and pro-apoptotic efficacy of an Epstein-Barr virus nuclear antigen 1-specific affibody in EBV-infected cells in vitro

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) sustains viral latency and drives oncogenesis in EBV-driven malignancies such as nasopharyngeal carcinoma and lymphomas. The dimerization of EBNA1 acts as an indispensable molecular switch governing EBV latency and oncogenesis. Disruption of EBNA1 dimerization is a promising strategy, but existing small-molecule inhibitors lack sufficient specificity. We designed Z332-a novel affibody targeting EBNA1's DNA-binding domain (DBD)-with three functional modules: a TAT cell-penetrating peptide for enhanced cellular uptake, a nuclear localization signal (NLS) to drive nuclear import, and an LMP1-derived FWLY motif to improve EBV-specific targeting. Molecular binding kinetics (surface plasmon resonance), dimerization disruption (SDS-PAGE), Apoptosis (Annexin V/PI flow cytometry), and subcellular trafficking (confocal microscopy) were assessed in EBV-positive cells. Z332 showed high-affinity, selective EBNA1 DBD binding without EBV-negative cells cross-reactivity. It dose-dependently inhibited EBNA1 dimerization/ oligomerization and selectively induced Apoptosis in EBV-positive cells. Time-resolved imaging confirmed TAT-mediated uptake and NLS-driven nuclear accumulation in EBV-positive cells, while EBV-negative cells degraded it cytoplasmically. As the first-in-class affibody targeting EBNA1, Z332 combines three essential functions: blocking EBNA1-directed dimerization, promoting pro-apoptotic activity, and enabling precise subcellular delivery. Its high specificity and modular design make it a promising therapeutic strategy for EBV-associated malignancies.