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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 develop a monoclonal antibody that blocks the KDR of vascular endothelial growth factor (VEGF) receptor and to investigate its in vitro inhibition of the biological activity induced by VEGF165. Methods Balb / c mice were immunized with the prokaryotic expression KDR extracellular domain Ⅲ fusion protein and the anti-KDR extracellular domain III monoclonal antibody was prepared by using the traditional hybridoma technique. The antigen binding specificity was identified by ELISA and FACS. Immunoprecipitation And [3 H] TdR incorporation, the monoclonal antibody was used to block the phosphorylation of KDR tyrosine kinase receptor on the surface of endothelial cells by VEGF165, and to inhibit the activity of VEGF1 65 on the proliferation of endothelial cells. Results Anti-KDR extracellular domain monoclonal antibody Ycom1D3 could not only bind to soluble KDR but also bind to KDR expressed on cell surface and competitively inhibit the interaction between VEGF165 and soluble KDR and block VEGF165-stimulated endothelial KDR Tyrosine kinase receptor phosphorylation significantly inhibited the proliferation of umbilical vein endothelial cells induced by VEGF165. Conclusion Anti-KDR extracellular domain monoclonal antibody Ycom1D3 can inhibit VEGF activity by blocking KDR, which has potential application in the treatment of tumors and other angiogenic diseases.