抗人VEGF单抗的研制及对肿瘤生长及转移的抑制作用

Generation and characterization of monoclonal antibodies to human vascular endothelial growth factor 165 and the effect on primary and metastatic tumor growth of breast tumor

目的：为了确定人血管内皮细胞生长因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ，ＶＥＧＦ） 在肿瘤生长及转移中的作用。方法：利用ＤＮＡ 重组技术表达人ＶＥＧＦ，以其为抗原免疫Ｂａｌｂｃ 小鼠，应用杂交瘤技术获得１４ 株稳定分泌抗ＶＥＧＦ单抗的杂交瘤细胞株。结果：ＥＬＩＳＡ 测定所分泌的单抗只与ＶＥＧＦ特异结合，与ＥＧＦ、ＦＧＦ、ＰＤＧＦ、ＴＧＦα及ＴＮＦ 等均无交叉反应。以豚鼠皮肤血管通透性实验（Ｍｉｌｅｓ） 、鸡胚绒毛尿囊膜血管形成实验（ＣＡＭ） 及人脐带静脉血管内皮细胞增殖实验（ＨｕＶＥＣ） 鉴定单抗的中和活性，其中９ 株具有中和活性。在小鼠自发乳癌肺转移模型中进行的抑瘤试验显示，单抗Ｄ１１ 对乳癌原发瘤的抑制率达８３ ．４ ％ ，对肺转移的抑制率为７１９ ％ 。结论：具有中和活性的ＶＥＧＦ单抗能抑制肿瘤的生长和转移。

Objective: Monoclonal antibodies to human vascular endothelial growth factor (VEGF) were established and characterized to determine the role of VEGF in tumor and metastasis. Methods: Balb/c mice were immunized with recombinant hVEGF165 and the immunized spleen cells were fused with SP2/0 Ag14. Hybridomas cells secreting anti VEGF McAbs were screened and characterized by ELISA, Western blot, and RIA. Neutralizing activity of anti VEGF McAbs were identified by miles permeability assay (Miles), chicken chorioallantoic membranes angiogenesis assay (CAM) and human umbilical vein endothelial cell growth assay (HuVEC). The effect of neutralizing McAb D11 on tumor growth was determined in IVT2MA 891 mouse model. Results: 14 hybridomas secreting anti VEGF McAbs were established and 9 of the 14 hybridomas were neutralizing McAbs against hVEGF. Neutralizing McAb D11 significantly inhibited both primary and metastatic tumor growth of mouse breast .Conclusion: These anti VEGF McAbs provide a useful tool to study structure of VEGF and the role in tumor angiogenesis. These neutralizing McAbs against VEGF may be potentially useful for clinical treatment of cancer.