Effects of humanized anti⁃ROR1 antibody and its AGAP conjugate on biological characteristics of ovarian cancer
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摘要:
目的:制备酪氨酸激酶样孤儿受体1(tyrosine-kinase-like orphan receptor1,ROR1)的全分子抗体(ROR1-IgG1)及其与东亚钳蝎镇痛抗肿瘤多肽的融合蛋白的全分子抗体(fusion protein of ROR1 and Buthus martensii Karsch analgesic anti-tumor polypeptide IgG1,IgG1-AGAP),检测ROR1-IgG1和IgG1-AGAP对卵巢癌细胞生物学特性的影响,探讨ROR1在卵巢癌中的作用机制。方法:以本实验室筛选并保存的ROR1Fab载体为模板,构建ROR1-IgG1和IgG1-AGAP真核表达载体,并转染中国仓鼠卵巢细胞(CHO-S),收集上清并纯化。利用酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)、Biacore X100、免疫荧光、流式细胞术(fluorescence-activated cell sorting,FACS)等评估IgG1-AGAP和 ROR1- IgG1的免疫学活性。通过CCK-8法、划痕实验、Transwell侵袭实验等比较两者对卵巢癌细胞HO8910生物学特性的影响。用蛋白质印迹法(Western blot)检测上皮细胞-间充质转化(epithelial mesenchymal transition,EMT)相关标志物的表达,评估ROR1-IgG1和IgG1-AGAP对卵巢癌细胞迁移和侵袭能力的影响。结果:成功制备出ROR1-IgG1和IgG1-AGAP,且能够与ROR1蛋白特异性结合;IgG1-AGAP与ROR1-IgG1均可抑制ROR1阳性卵巢癌细胞HO8910的增殖、迁移、侵袭,且IgG1-AGAP组的抑制作用显著高于ROR1-IgG1组,而未观察到对ROR1阴性人正常卵巢上皮细胞IOSE386的抑制作用。Western blot结果表明,在空白对照组、ROR1-IgG1组和IgG1-AGAP组中,Snail的表达量呈现递减趋势,而E-cadherin表达量递增,提示 ROR1-IgG1和IgG1-AGAP可通过调控 Snail和E-cadherin而抑制HO8910细胞发生 EMT,进而抑制其迁移和侵袭能力。结论:IgG1-AGAP可有效靶向表达ROR1的卵巢癌细胞,并具有显著的抗肿瘤活性,IgG1-AGAP可作为ROR1阳性肿瘤患者的潜在候选药物。
Abstract:
Objective:This study aims to prepare anti-tyrosine-kinase-like orphan receptor 1 (ROR1) IgG1(ROR1-IgG1)and the fusion protein of anti-ROR1 and Buthus martensii Karsch recombinant analgesic anti-tumor polypeptide(IgG1-AGAP),and compare their effect on ovarian cancer cells. Methods:ROR1Fab vectors screened and preserved in our laboratory was used as template to construct ROR1-IgG1 and IgG1-AGAP eukaryotic expression vectors;supernatant of CHO-S cells after transfected by ROR1- IgG1 and IgG1-AGAP plasmids was collected and purified,respectively. Their immunological activity was identified and detected by ELISA,Biacore X100,fluorescence-activated cell sorting(FACS)and immunofluorescence. Cell counting kit-8(CCK-8),wound healing,Transwell invasion assays were used to analyze the effects of two antibodies on the ovarian cancer cell HO8910. The expression of relevant markers of epithelial mesenchymal transition(EMT) was detected by Western blot to evaluate the effects of ROR1-IgG1 and IgG1-AGAP on the migration and invasion of ovarian cancer cells. Results:ROR1-IgG1 and IgG1-AGAP were successfully prepared,the binding efficiency of IgG1-AGAP were similarly as ROR1-IgG1. Both IgG1-AGAP and ROR1-IgG1 inhibited proliferation,migration and invasion of ROR1-positive ovarian cancer cell HO8910. Compared with ROR1-IgG1,IgG1-AGAP can significantly inhibit the proliferation and migration of tumor cells(P < 0.05),and these effects were not observed in ROR1-negative cells IOSE386. Western blot results showed that the expression levels of Snail in ROR1-IgG1 group and IgG1-AGAP group decreased compared with the blank control group,while E-cadherin showed the opposite, suggesting that ROR1-IgG1 and IgG1-AGAP can regulate Snail and E-cadherin and inhibit EMT in ovarian cancer cell HO8910,thereby inhibiting their migration and invasion. Conclusion:Our findings demonstrate that IgG1-AGAP can target ROR1 expressing ovarian cancer cells effectively and have obvious antitumor activity. IgG1-AGAP could be as an appropriate and promising therapeutic strategy for ROR1-positive human cancers.