抗KDR胞外Ⅲ区单抗抑制血管内皮细胞增殖的研究

Effect of monoclonal antibody against extracellular domain Ⅲ of vascular endothelial growth factor receptor KDR on proliferation of vascular endothelial cells

目的 研制能够封闭血管内皮生长因子(VEGF)受体KDR的单克隆抗体,探讨其体外抑制VEGF1 65诱导的生物学活性。方法 以原核表达的KDR胞外Ⅲ区融合蛋白免疫Balb/c小鼠,采用传统杂交瘤技术制备抗KDR胞外Ⅲ区单克隆抗体,采用ELISA和FACS方法鉴定其抗原结合特异性,采用免疫沉淀和[3 H] TdR掺入的方法分析该单克隆抗体阻断VEGF1 65刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,抑制VEGF1 65诱导内皮细胞增殖的活性。结果 抗KDR胞外Ⅲ区单抗Ycom1D3不仅能与可溶性KDR结合,亦可与细胞表面表达的KDR结合,并可竞争性抑制VEGF1 65与可溶性KDR的相互作用,阻断VEGF1 65刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,显著抑制由VEGF1 65诱导脐静脉内皮细胞的增殖。结论 抗KDR胞外Ⅲ区单抗Ycom1D3可通过封闭KDR而抑制VEGF活性,在肿瘤及其他血管新生疾病治疗中具有潜在的应用前景。

Objective To preparea neutralizing monoclonal antibody(McAb)against vascular endothelial growth factor receptor KDR and study its biological activity. Methods Extracellular immunoglobulin (Ig)-like domain Ⅲ of KDR (KDR Ⅲ)was expressed in E.coli and purified by affinity chromatograph. Monoclonal antibody against KDR Ⅲ was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and -TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells. Results McAb Ycom1D3 against KDR Ⅲ was prepared which bound specifically to both the soluble KDR Ⅲ and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells. Conclusion McAb Ycom1D3 against KDR Ⅲ may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.