摘要
目的:制备可特异识别人表皮生长因子受体-2(human epidermal growth factor receptor-2,HER2)膜抗原的单克隆抗体,筛选出亲和力较高并对胃癌细胞有明显抑制作用的克隆。方法:以高表达HER2膜抗原的人胃癌细胞系NCI-N87免疫BABL/c小鼠,免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2/0融合,ELISA法筛选出HER2反应阳性克隆株,活细胞免疫荧光法检测获得膜阳性克隆株,使用抗体亚型鉴定试剂盒鉴定抗体亚型,MTT法检测抗体对NCI-N87细胞增殖的抑制,采用与群司珠单抗的夹心ELISA和竞争抑制ELISA鉴定单抗结合HER2抗原的表位,免疫组化、Westernblotting进一步检测抗HER2抗体在不同条件下与HER2抗原反应情况。结果:细胞融合产生1442个单个集落的杂交瘤,重组HER2抗原ELISA筛选出HER2反应阳性克隆79株,活细胞免疫荧光检测获得较强膜阳性克隆7株,并测定其亚型,其中命名为15C15的1株属IgG1κ类单抗,免疫竞争及结合实验显示15C15与群司珠人源化抗体识别不同的表位。细胞增殖筛选显示15C15能显著抑制人胃癌NCI-N87细胞的增殖,在80μg/ml时抑制率可达34.8%。免疫组化显示15C15能识别部分石蜡包埋的HER2抗原,Westernblotting显示15C15不能结合电泳条件下变性为线形结构的HER2抗原。结论:建立了高通量制备、筛选和鉴定靶向HER2抑制胃癌细胞生长的功能性单抗技术,获得1株可特异识别HER2抗原的明显抑制胃癌细胞增殖的IgG1类单抗15C15,具有人源化改造后用于治疗的应用潜力。
Objective： To prepare and identify monoclonal antibodies that can specifically react with HER2 and suppress gastric tumor growth. Methods： Gastric carcinoma cell line NCI-N87 highly expressing HER2 was used to immunize 6 BABL/c mice. The spleen ceils of the immunized mice were fused with SP2/0 ceils and cultured with methyl cellulose. Antibodies of each clone were identified by ELISA to screen for those could positively react with HER2 antigen. Immuno- fluorescence of viable ceils was performed to see whether the HER2-directed monoclonal antibodies could react with membrane integrated HER2. The subtypes of these monoclonal antibodies were identified by related kit purchased from Southern Biotech. The epitope of the monoclonal antibody was identified by sandwich ELISA and competition ELISA with trastuzumab. MTT cell proliferation assay was used to select functional antibody with therapeutic potential from HER2-directed monoclonal antibodies. Immunohistochemistry and Western blotting assay were performed to identify the reaction of the antibodies with HER2 antigen. Results： A total of 1 442 monoclonal antibody clones were obtained by the fusing mouse spleen cells with SP2/0 cells. Seventy-nine clones positively reacted with recombined HER2 protein in ELISA assay. Seven of these antibodies recognized membrane integrated HER2 protein in immunofluorescence detection of viable cells. The subtypes of these monoclonal antibodies were further identified. One of these 7 antibodies, 15C15, which was identified as IgG1 kapa, significantly suppressed NCI-N87 cell proliferation in MTT assay. The epitope of 15C15 was different from trastuzumab according to sandwich ELISA and competition ELISA identification. The 15C15 antibody only reacted with part of the HER2 antigen in immunohistochemistry assay and did not react with HER2 antigen （denaturated into linear structure） in Western blotting analysis. Conclusion： Using functional antibody library screening technique we have successfully obtained functional monoclonal antibodies which can suppress the gastric carcinoma cell proliferation by specially neutralizing HER2 protein. One of these antibodies, 15C15 may be applied in anti-tumor therapy of gastric carcinoma.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2008年第1期14-19,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No30570818)
国家高技术研究发展计划(863)项目(No20060102Z4124)
国家重点基础研究发展规划(973)资助项目(No2002CB513106)~~
