Trophoblast cell-surface antigen 2-directed enhancement of gene delivery using a nanobody-engineered recombinant adeno-associated virus for bispecific T-cell engager-mediated T-cell cytotoxicity in triple-negative breast cancer cells.
TL;DR
RAAV-Nb was used to deliver the BiTE transgene, resulting in robust T-cell activation and potent BiTE-mediated T-cell cytotoxicity against TROP2-positive TNBC cells in vitro.
Abstract
Triple-negative breast cancer (TNBC) poses a significant therapeutic challenge because it has limited actionable molecular targets. Trophoblast cell-surface antigen 2 (TROP2) is a promising therapeutic target, overexpressed in approximately 80% of TNBC cases. However, conventional recombinant adeno-associated virus (rAAV) vectors exhibit broad natural tropism, which limits selective gene delivery to TROP2-positive tumor cells. In this study, we developed a TROP2-directed capsid-engineered rAAV platform designed to incorporate a TROP2-specific nanobody through genetic insertion into the VP1 capsid protein. Through phage display selection, we identified K118.1, a nanobody with nanomolar affinity, efficient internalization following TROP2 binding, and cross-reactivity with both human and mouse TROP2. We then generated a TROP2-directed capsid-engineered vector (rAAV-Nb) by genetically inserting K118.1 into the GH2/GH3 loop of the rAAV6 VP1 subunit. Compared with the parental rAAV6 vector, rAAV-Nb increased BiTE transgene expression by approximately 3- to 6-fold in TROP2-positive TNBC cells. GFP reporter analyses further demonstrated that TROP2 engagement contributes to the enhanced transduction mediated by rAAV-Nb. As a proof-of-concept therapeutic application, rAAV-Nb was used to deliver the BiTE transgene, resulting in robust T-cell activation and potent BiTE-mediated T-cell cytotoxicity against TROP2-positive TNBC cells in vitro. Epitope mapping and structural modeling indicated that K118.1 recognizes a conformation-dependent epitope within hTROP2, providing molecular insight into target recognition. Collectively, these findings suggest that nanobody-guided capsid engineering represents a promising strategy to enhance rAAV-mediated transgene delivery to TROP2-expressing cancer cells.