激活型人源化抗人CD137单克隆抗体的研制与生物学活性鉴定

Preparation and biological activity of humanized monoclonal antibody against human CD137

目的制备鼠抗人CD137抗体,并对该抗体进行人源化改造。方法构建h CD137-p CDNA3.4真核表达质粒,经293F细胞表达纯化及鉴定后,获得CD137蛋白。制备p HAGE-CMV-MCS-IRES-Zs Green-h CD137的慢病毒,感染HEK-293FT细胞,获得用来筛选杂交瘤的CD137转基因细胞株。将CD137蛋白免疫小鼠,检测小鼠血清抗体效价,将免疫小鼠的脾脏和骨髓瘤细胞SP2/0融合,采用ELISA方法及流式细胞术对小鼠杂交瘤进行筛选。将筛选后的杂交瘤细胞接种小鼠,获得腹水。对抗体亚型和抗体效价进行测定,并通过竞争结合抑制实验等方法对所获的CD137单抗的生物学特性进行分析鉴定。提取该m Ab杂交瘤细胞株的总RNA逆转录成c DNA,进行鼠抗人CD137抗体可变区序列克隆,构建人源化抗体重链和轻链表达载体,瞬时转染HEK-293FT,检测分析各重组人源化抗体与抗原的亲和力。结果获得1株CD137单抗(6F5),该抗体与CD137L有竞争作用。蛋白结合表位在A.A 30-100。6F5能够激活NF-κB信号通路,对外周血单个核细胞增殖具有显著促进作用,且人源化后抗体的亲和力不变。结论成功制备1株CD137人源化抗体,该人源化抗体为下一步开展体内肿瘤治疗奠定了基础。

Objective To prepare mouse anti-human CD137 antibody and humanize this antibody. Methods The h CD137-PCDNA3. 4 eukaryotic expression plasmid was constructed,followed by the expression,purification and identification using 293 F cells,and a CD137 transgenic cell line was obtained for screening hybridoma. Furthermore,the experimental mice were immunized with CD137 protein to detect the serum titer of mice. Spleens of immunized mice were fused with myeloma cells SP2/0,besides,enzyme-linked immunosorbent assay（ ELISA） and flow cytometry were utilized to screen the hybridoma of mice. Simultaneously,the hybridoma ascites of mice were prepared and purified. Then,the total RNA of the m Ab hybridoma cell line was extracted,reversely transcripted to c DNA,and the variable region sequence of mouse anti-human CD137 antibody was cloned. Subsequently,the heavy chain and light chain expression vectors of humanized antibody were obtained accordingly,and HEK-293 FT was transfected instantaneously. The affinity of recombinant human antibody to antigen was detected and analyzed later. Results The results displayed that the affinity of humanized antibodies of combined was not reduced,and6 F5 had a competitive role with its ligand CD137 L. Corresponding protein binding epitope was located at a. a 30-100. Conclusion We successfully prepares a humanized CD137 antibody,which lay the foundation for the next step in tumor treatment in vivo.