Kim So-Hee, Lee Yong-Seok, Hwang Seung-Young, Bae Gun-Won, Nho Kwang, Kang Se-Won, Kwak Yee-Gyung, Moon Chi-Sook, Han Yeon-Soo, Kim Tae-Yun, Kho Weon-Gyu
Department of Malariology, Paik Institute for Clinical Research, Inje University College of Medicine, Busan, Korea.
J Microbiol Biotechnol. 2007 Oct;17(10):1670-4.
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.
达菲结合蛋白（DBP）在间日疟原虫入侵人类红细胞过程中起着关键作用。我们之前报道了一种对间日疟原虫DBP（PvDBP）具有特异性的单链抗体片段（scFv）。然而，scFv的稳定性和半衰期尚未得到研究。在此，我们研究了聚乙二醇化scFv对其体外生物学活性和稳定性的影响。SDS-PAGE分析表明，三个克隆（SFDBII-12、-58和-92）经聚乙二醇化后形成二聚体（约70 kDa）。克隆SFDBII-58产生的聚乙二醇化scFv产量最高。使用BIAcore进行的DBP与scFv之间的结合分析表明，聚乙二醇化后，SFDBII-12和-58与DBP的结合亲和力水平均下降了约两倍。然而，SFDBII-92克隆仍显示出相对较高的结合亲和力（KD = 1.02×10⁻⁷ M）。结合抑制试验表明，聚乙二醇化scFv仍能够竞争性结合PvDBP，并在抑制Cos-7细胞表面表达的PvDBP蛋白与红细胞表面达菲受体之间的相互作用中发挥关键作用。当scFv及其聚乙二醇化对应物都暴露于胰蛋白酶时，scFv仅在24小时后完全降解，而35%的聚乙二醇化scFv保持完整，直至72小时仍能抵抗胰蛋白酶的蛋白水解攻击，维持其稳定性。综上所述，这些结果表明，聚乙二醇化scFv在体内对蛋白水解酶保持稳定性，对靶抗原DBP的结合亲和力没有显著损失。
