FGF2 人源化单抗E12 及其联合顺铂/ 紫杉醇体外抑制乳腺癌细胞增殖作用的研究

目的:研究人源化FGF2 单抗体E12 体外联合化疗药物抑制乳腺癌肿瘤细胞增殖的作用。方法:分析人源化FGF2 单抗可变区人源氨基酸序列比率;SDS-PAGE 和HPLC 检测单抗纯度;ELISA 测定单抗效价;BSA 法检测单抗浓度;CCK-8 法检测单抗联合化疗药物对乳腺癌肿瘤细胞株的细胞增殖抑制作用;流式细胞术检测FGF2 单抗联合化疗药物对乳腺癌细胞株的促凋亡作用;使用Discovery Studio 4.5 软件对单抗的表位进行分析。结果:单抗可变区人源氨基酸序列比率重链为98.89%,轻链为100.00%;单抗纯度为99.02%;单抗ELISA 效价为1/243 000;单抗浓度为8.74 mg/ ml;CCK-8 结果表明顺铂为10、5、2.5、0.625 μg/ ml 时,单抗+顺铂组比顺铂组对MCF-7 细胞抑制率提高了14.5%、10.4%、1.5%、0.5%;对MDA-MB453 细胞抑制率提高了15.5%、14.8%、13.0%、6.8%,紫杉醇为1.0、0.1、0.01 μg/ ml 时,单抗+紫杉醇组比紫杉醇组对MCF-7 细胞抑制率提高了14.5%、11.1%、9.3%,紫杉醇为0.1、0.01、0.001 μg/ ml 时,单抗+紫杉醇组比紫杉醇组对MDA-MB453 细胞的抑制率提高了18.6%、19.9%、6.8%;流式结果表明,单抗、紫杉醇及单抗+紫杉醇组的MCF-7 细胞在AnnexinV/ PI 双阳性区所占比例提高了6.13%、7.36%和12.83%,同理MDA-MB453 细胞所占比例提高了4.86%、7.1%和14.1%,单抗、顺铂及单抗+顺铂组MCF-7 细胞在Annexin V/ PI 双阳性区所占比例提高了6.39%、9.46% 和17.76%,同理MDA-MB453细胞所占比例提高了5.38%、9.06%和19.39%;表位分析结果表明FGF2 单抗有效封闭FGF2 与受体结合的部分位点。结论:FGF2 人源单抗E12 人源化程度较高;FGF2 人源化单抗联合化疗药物相对单药组可较明显地提高肿瘤细胞增殖抑制作用以及促细胞凋亡作用;本研究为FGF2 抗体药物研发提供了前期基础。

Inhibitory effect of novel FGF2 humanized monoclonal antibody E12 combined with cisplatin / paclitaxel on breast cancer cell proliferation in vitro

Objective:To study the effect of humanized FGF2 single antibody E12 in combination with chemotherapeutic agents on breast cancer cell proliferation.Methods:Humanized amino acid sequence ratio of humanized FGF2 McAb variable region was analyzed;SDS-PAGE and HPLC were used to detect the purity of monoclonal antibody;ELISA was used to determine the titer of monoclonal antibody;BSA method was used to detect the concentration of monoclonal antibody;CCK-8 method was used to detect the combination of monoclonal antibody inhibitory effect of cell proliferation in breast cancer cell lines;Flow cytometry was used to detect the pro-apoptotic effects of FGF2 monoclonal antibody and chemotherapeutic drugs on breast cancer cell lines;the epitope of monoclonal antibody were analyzed using Discovery Studio 4.5 software.Results:The ratio of the human amino acid sequence of the variable region of the monoclonal antibody was 98.89% for the heavy chain and 100.00% for the light chain;the purity of the monoclonal antibody was 99.02%;the titer of the monoclonal antibody was 1/243 000;the concentration of the monoclonal antibody was 8.74 mg/ ml;CCK-8 results showed that when cisplatin was 10,5,2.5 and 0.625 μg/ ml,the inhibition rate of MCF-7 cells was increased by 14.5%, 10.4%,1.5% and 0.5% in the monoclonal antibody + cisplatin group compared with the cisplatin group.The inhibition rate of MDA-MB453 cells was increased by 15.5%,14.8%,13.0%,6.8%.When paclitaxel was 1.0,0.1,and 0.01 μg/ ml,the inhibition rate of MCF-7 cells was increased in the monoclonal antibody + paclitaxel group compared with the paclitaxel group at 14.5%,11.1%,and 9.3%.When paclitaxel was 0.1,0.01,and 0.001 μg/ ml,the inhibition rate of MDA-MB453 cells by the monoclonal antibody + paclitaxel group was 18.6%,19.9%,and 6.8% higher than that of the paclitaxel group;the results showed that the percentage of MCF-7 cells in the monoclonal antibody,paclitaxel,and monoclonal antibody+paclitaxel group increased by 6.13%,7.36%,and 12.83% in the Annexin V/ PI double positive region,and the proportion of MDA-MB453 cells in the same way.The percentages of MCF-7 cells in the Annexin V/ PI double-positive regions increased by 6.39%,9.46%,and 17.76%,respectively,with the increase of 4.86%, 7.1%,and 14.1%.The proportion of MDA-MB453 cell increased by 5.38%,9.06%,and 19.39%.Epitope analysis showed that the FGF2 monoclonal antibody effectively blocked sites of FGF2 binding to the receptor.Conclusion:Humanized human FGF2 monoclonal antibody E12 has a higher degree of humanization;FGF2 humanized monoclonal antibody combined with chemotherapeutic drugs can significantly increase the inhibition of tumor cell proliferation and promote apoptosis.This study provides an early basis for FGF2 monoclonal antibody drug development.