Antisense oligonucleotides (ASOs) represent a promising therapeutic modality for central nervous system (CNS) disorders, offering highly specific modulation of gene expression. However, their clinical utility is severely limited by their inability to cross the blood-brain barrier (BBB), necessitating effective shuttling strategies. While transferrin receptor (TfR1)-mediated shuttling has shown therapeutic promise, the fundamental mechanisms governing the delivery of antibody-ASO conjugates across the BBB remain poorly understood. This study directly addresses this critical knowledge gap by establishing a mechanistic understanding of how the ASO cargo impacts major cellular interactions during the BrainshuttleTM-mediated transport across the BBB. Using a panel of advanced in vitro assays developed specifically for this purpose, including quantitative transcytosis, detailed imaging-based intracellular trafficking, and binding assays with brain endothelial cells (BECs), the shuttling process was systematically investigated. We demonstrate that ASO conjugation profoundly alters the cellular fate of the BrainshuttleTM. Specifically, conjugation increased the binding to BECs of low-affinity TfR1 shuttles via avidity effects while paradoxically reducing the binding strength of high-affinity shuttles. Functional assays confirmed the biological activity of the delivered ASOs; however, transcytosis of high-to-moderate affinity binders across the BBB model was significantly delayed upon ASO conjugation. Building on these mechanistic insights, we engineered TfR1 BrainshuttlesTM with optimized affinity and explored the shuttling potential of an alternative BBB receptor, CD98hc. These efforts culminated in the development of a novel bispecific BrainshuttleTM targeting both CD98hc and TfR1. This dual-targeting strategy exploits distinct and potentially non-competing trafficking pathways to overcome ASO-induced delays and significantly enhance in vitro transcytosis efficiency. The in vitro findings in this study underscore the necessity of mechanism-driven design to overcome ASO-induced limitations in delivery across the BBB. The bispecific CD98/TfR1 approach presented here provides a promising new strategy for maximizing delivery efficiency and enabling more effective therapeutic outcomes for CNS diseases.