Differential T cell reactivation by two PD-L1 nanobodies through blockade alone or blockade with internalization

Programmed death-ligand 1 (PD-L1) plays a key role in tumor immune evasion by suppressing T cell activity through its interaction with programmed death-1 (PD-1). Although PD-L1 antibodies have significantly advanced cancer immunotherapy, clinical responses remain limited, associated with tumor heterogeneity, acquired resistance, and the structural and functional constraints of full-length immunoglobulin G formats. These limitations highlight the need for alternative PD-L1–targeting scaffolds that offer improved modularity and functional versatility. In this study, two PD-L1-specific nanobodies (variable domains of heavy-chain-only antibodies, VHHs; K113.1 and K113.2) were isolated using phage display and reformatted as Fc-fusion antibodies (VHH-Fc; K113.1-Fc and K113.2-Fc). Both VHH-Fc antibodies exhibited high-affinity binding to human PD-L1 (KD < 3.4 nM) and effectively blocked PD-1/PD-L1 interactions. Notably, K113.2-Fc also induced PD-L1 internalization and surface downregulation, which was associated with enhanced T cell reactivation in vitro compared with blockade alone. Collectively, these findings indicate that closely related VHH-Fc antibodies can display distinct functional profiles, enabling direct comparison between blockade-only and blockade-plus-internalization mechanisms within the same scaffold. These VHH-based formats may provide useful tools for studying PD-L1 regulation and offer potential advantages in molecular size, engineering flexibility, and future multispecific design.