Effects of PEGylated scFv antibodies against Plasmodium vivax duffy binding protein on the biological activity and stability in vitro.
Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as dimers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity (KD=1.02 x 10(-7) M). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and play a critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.
D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry / Q000378:metabolism*; D000915:Antibody Affinity; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D010966:Plasmodium vivax / Q000276:immunology*; D011092:Polyethylene Glycols / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014357:Trypsin / Q000378:metabolism
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C066001:Duffy antigen binding protein, Plasmodium; D007128:Immunoglobulin Fragments; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D014357:Trypsin