Preparation of engineered extracellular vesicles with full-length functional PD-1 membrane proteins by baculovirus expression system.

Extracellular vesicles (EVs) facilitate intercellular communication by transporting functional molecules. The modification of EVs for clinical use as drug delivery systems is of considerable interest because of their biocompatibility and molecular transport ability. Programmed cell death ligand 1 (PD-L1) is an effective target molecule for drug delivery to cancer tissues and binds the single-transmembrane protein, Programmed cell death protein 1 (PD-1), an immune checkpoint that guards against autoimmunity. In this study, EVs were modified in a new surface engineering strategy to incorporate recombinant full-length functional PD-1 using a baculovirus system and newly designed PD-1 mutant with higher PD-L1 affinity. The insect cell line Spodoptera frugiperda 9 was infected with recombinant baculoviruses incorporating the PD-1 mutant gene to express the target membrane proteins. To ensure an effective insertion into the membrane, the native signal peptide of PD-1 was also replaced with that of the baculovirus envelope glycoprotein. Engineered EVs expressing the high-affinity PD-1 mutants (PD-1 EVs) were then isolated and characterized. Immunostaining and confocal laser scanning microscopy results confirmed the presence of full-length functional PD-1 mutants expressed by viral infection on both infected Spodoptera frugiperda 9 cell membrane surfaces and released EV membranes. Furthermore, the signal peptide substitution drastically increased the binding between PD-1 EVs and PD-L1. PD-1 EVs effectively bound PD-L1 and PD-L1-expressing cancer cells, showing potential as a candidate in new therapy approaches targeting PD-L1 EVs.