人源化抗组织因子(TF)抗体的制备及杀伤结肠癌细胞和抑制细胞迁移的机制

Preparation of humanized anti-tissue factor antibody and its mechanism of killing colon cancer cells and inhibiting cell migration

目的探讨靶向组织因子(TF)的人源化抗体对结肠癌细胞杀伤和迁移作用。方法体外培养人源化TF抗体高表达CHO-5G4.1细胞,经蛋白A(Protein A)和凝胶层析柱纯化,利用毛细管SDS-PAGE检测抗体纯度,采用凝血酶原时间(PT)法测定抗凝血活性,采用抗体依赖细胞介导的细胞毒作用(ADCC)分析抗体对SW620和SW480结肠癌细胞的杀伤效应,Transwell^(TM)检测抗体对细胞迁移的影响,明胶酶谱和Western blot法检测人源化TF抗体处理后,迁移相关蛋白基质金属蛋白酶2(MMP2)、MMP9、黏着斑激酶(FAK)和磷酸化的FAK(p-FAK)蛋白水平的变化。结果通过两步法纯化人源化TF抗体毛细管SDS-PAGE检测纯度可达96.9%,并具有抗凝血活性。人源化TF抗体可通过ADCC杀死结肠癌细胞并明显抑制结肠癌细胞的迁移,抑制率达到99%,并可降低MMP2的表达及p-FAK的表达。结论获得了高纯度的人源化TF抗体,该抗体可通过ADCC杀伤结肠癌细胞,并可抑制FAK信号通路降低MMP2的表达,抑制结肠癌细胞的迁移。

Objective To investigate the killing effect of humanized antibodies targeting tissue factors on colon cancer cells as well as migration-inhibiting effect.Methods Humanized anti-tissue factor antibody was purified by Protein A and gel filtration chromatography from cultured CHO-5G4.1 cells that highly express the antibody.The purity was detected by capillary SDS-PAGE.Anticoagulant activity was assessed using the prothrombin time test.Killing effect of the antibody on SW620 and SW480 colorectal cancer cells was tested using antibody-dependent cell-mediated cytotoxicity(ADCC).The effect of antibodies on cell migration was investigated using Transwell^(TM)assay.Gelatin zymography and Western blotting were used to detect the changes of matrix metalloproteinase 2(MMP2),MMP9,focal adhesion kinase(FAK)and phosphorylated FAK(p-FAK)after treatment with humanized anti-tissue factor antibody.Results The purity of the humanized anti-tissue factor antibody was estimated 96.9%by the two-step method.The purified antibody showed an obvious anticoagulant activity.The antibody treatment had a significant killing effect on colorectal cancer cells through ADCC and a significant inhibiting effect(99%)on cell migration.The antibody significantly inhibited expression of MMP2 and p-FAK.Conclusion The humanized anti-tissue factor antibody can effectively kill tumor cells through ADCC and inhibit cancer cell migration,which is possibly mediated by MMP2 expression through suppression of FAK signaling.