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为了阻断VEGF与KDR的结合,抑制VEGF结合KDR后介导的内皮细胞迁移和血管新生,实验室已经从全人源噬菌体展示抗体库中筛选得到几种抗KDR的单链抗体,从中挑选一种亲和力较高的抗体命名为AK506进行周质表达,纯化后经SDS-PAGE鉴定,可获得纯度为95%的电泳纯抗体,产量可以达到每升发酵液1 mg。经表面等离子共振技术测定,AK506与KDR胞外区相互作用的平衡解离常数为7.99×10-9nmol/L。细胞ELISA分析显示,AK506与内皮细胞表面膜受体KDR的结合呈剂量依赖性。采用细胞划痕损伤修复实验和鸡胚尿囊膜实验证明,AK506可以显著抑制内源性VEGF诱导的血管内皮细胞迁移和新血管生成,具有进一步研究开发用于抗肿瘤血管生成治疗的潜力。
In order to block the binding of VEGF to KDR and to inhibit the endothelial cell migration and angiogenesis mediated by VEGF binding to KDR, several single-chain antibodies against KDR have been screened from the library of whole-human phage display antibodies, and one The higher affinity antibody was named as AK506 for periplasmic expression. After purification, it was identified by SDS-PAGE. The pure antibody with purity of 95% could be obtained and the yield could reach 1 mg per liter of fermentation broth. The equilibrium dissociation constant of the interaction between AK506 and the extracellular domain of KDR was 7.99 × 10-9nmol / L as determined by surface plasmon resonance. Cell ELISA analysis showed that the binding of AK506 to endothelial cell membrane receptor KDR was dose-dependent. Scratch repair experiments using cell scratches and chick chorioallantoic membrane experiments show that, AK506 can significantly inhibit endogenous VEGF-induced vascular endothelial cell migration and neovascularization, with further research and development for anti-tumor angiogenesis potential.