抗OX40/EGFR双特异性抗体的制备和活性鉴定
Construction and activity identification of anti-OX40/anti-EGFR bispecific antibody
晋瑞娜 1边海波 2张晓敏 1杨帆 1王晚萍2
作者信息
- 1. 长治市人民医院中心实验室,山西长治 046000
- 2. 长治市人民医院呼吸与危重症学科,山西长治 046000
- 折叠
摘要
目的 构建同时靶向表皮生长因子受体(epidermal growth factor receptor,EGFR)和OX40 的双特异性抗体,并在体外初步验证其对T细胞的特异性激活作用.方法 构建抗OX40/EGFR双特异性抗体的真核表达载体,转染 293F细胞进行表达和纯化.构建外源表达OX40 和EGFR的HEK293T细胞,利用流式细胞术分析双抗与靶蛋白表达细胞的结合活性.并利用Jurkat-OX40-NF-κB-GFP报告细胞,验证双特异性抗体对报告细胞的激活活性.通过酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的白细胞介素-2(interleukin-2,IL-2)和γ-干扰素(interferon-γ,IFN-γ)分泌,进一步验证抗OX40/EGFR双抗对原代T细胞的激活.结果 成功构建并且纯化了抗OX40/EGFR双特异性抗体,并且验证了该双抗可以特异性地结合表达EGFR和OX40 的HEK293T细胞.在表达EGFR的A549 肺癌细胞介导的交联作用下,该双特异性抗体较OX40 单抗更强地激活OX40-NF-κB报告细胞,并且促进PBMC分泌IL-2 和IFN-γ细胞因子.结论 抗OX40/EGFR双特异性抗体构建成功,可同时识别OX40 和EGFR受体并激活肿瘤特异性T细胞.
Abstract
Objective To construct a bispecific antibody targeting epidermal growth factor receptor(EGFR)and OX40 and evaluate the function for tumor-specific T cell activation.Methods The gene of anti-OX40/anti-EGFR bispecific antibody was cloned into eukaryotic expression vector,and then the constructed vector were transfected to 293F cells for the bispecific antibody purification.The binding activity of anti-OX40/anti-EGFR bispecific antibody with the cells expressing target proteins were detected by flow cytometry.To identify the activation of T cells mediated by anti-OX40/anti-EGFR bispecific antibody,the activation of NF-κB signal activation was evaluated by Jurkat-OX40-NF-κB-GFP reporter cells and the activation of primary T cells was detected by interleukin-2(IL-2)and interferon-γ(IFN-γ)secretion of peripheral blood mononuclear cell(PBMC).Results Anti-OX40/anti-EGFR bispecific antibody was successfully constructed and purified,and its binding ability to HEK293 cells expressing OX40 and EGFR was verified.Jurkat-OX40-NF-κB-GFP reporter cells were activated by the bispecific antibody with the crosslinking of A549 cells.Further,the anti-OX40/anti-EGFR bispecific antibody promoted the secretion of IL-2 and IFN-γ of PBMC.Conclusion Anti-OX40/anti-EGFR bispecific antibody was successfully constructed which could specifically recognize OX40 and EGFR and activate tumor specific T cells.
关键词
OX40/表皮生长因子受体/双特异性抗体/癌症/T细胞激活Key words
OX40/epidermal growth factor receptor(EGFR)/bispecific antibody(BsAb)/cancer/T cell activation引用本文复制引用
基金项目
山西省自然科学基金面上项目(20210302124004)
山西省卫生健康委项目(2022024)
出版年
2024
NETL
NSTL
维普
万方数据