Effects of PEGylated scFv Antibodies against Plasmodium vivax DuffyBinding Protein on the Biological Activity and Stability In Vitro

Dufy binding protein (DBP) plays a critical rolein Plasmodium vivax invasion of human red blood cells. Wepreviously reported a single-chain antibody fragment (scFv)that was specific to P. vivax DBP (PvDBP). However, theHere, we investigated the effect of PEGylated scFvs on theirbiological activity and stability in vitro. SDS-PAGE analysisshowed that three clones (SFDBII-12, -58, and -92) were formedas monomers (about 70 kDa) with PEGylation. Clone SFDBI-58 gave the highest yield of PEGylated scFv. Binding analysisusing BIAcore between DBP and scFv showed that bothSFDBII-12 and -58 were decreased approximately by twofolds at the level of binding affinity to DBP after PEGylation.However, the SFDBII-92 clone still showed a relatively high levelof binding affinity (KD=1.02× 10-7M). Binding inhibition assaybind the PvDBP and play a critical role in inhibiting theinteractions between PvDBP protein expressed on the surface ofCos-7 cells and Dufy receptor on the surface of erythrocytes.When both scFvs and their PEGylated counterparts were exposedto trypsin, scFv was completely degraded only after 24h,whereas 35% of PEGylated scFvs remained intact, maintainingtheir stability against the proteolytic attack of trypsin until 72 h.Taken together, these results suggest that the PEGylated scFvsretain their stability against proteolytic enzymes in vivo, withno significant loss in their binding affinity to target antigen, DBP. KCI Citation Count: 5