目的:获得全人源抗人OX40特异性抗体候选株,为相关药物开发奠定基础.方法:依托本实验室构建的大容量全合成全人源噬菌体单链抗体库平台,以OX40为抗原进行固相筛选,经过3轮条件渐进严苛的筛选后,阳性克隆得到富集;将其中富集效果最好的一株单链抗体scFv-1改造成全抗体(IgG1)形式,改造成功的重组质粒按照一定比例转染HEK293-F细胞进行抗体的瞬时表达;基于AKTA系统对全抗体进行纯化,将纯化得到的金抗体重命名为OX40mab-1.通过ELISA、间接免疫荧光、流式细胞术等试验分别验证OX40mab-1与抗原的结合活性、特异性、血清稳定性;通过BIAcore3000初步测定抗体亲和力.结果:3轮筛选后得到1株富集率达95％的单链抗体,改造成全抗体形式后,与抗原OX40的结合EC50为0.015 μmol/L;该抗体与其他无关抗原均不结合,特异性良好;37℃血清放置15 d后,与抗原依具有较好的结合活性,血清稳定性良好;经BlAcore 3000初步测定该抗体亲和力为251 nmol/L.结论:获得1株理化性质良好、特异性较好的抗OX40全人源单克隆抗体OX40mab-1,具有继续开发价值.%Objective:To harvest a fully human anti-human OX40 specific antibody for developing related drug.Methods:We chose OX40 as a target antigen to screen specific antibodies from the fully human single-chain anti-body phage library and got the enriched positive clones after 3 rounds of solid screening.The best enriched single-chain antibody scFv-1 was redesigned to be a fully human antibody,and the corresponding recombinant plasmid was transfected into HEK293-F cells for transient expression.The final prodcut,named as OX40mab-1,was purified based on AKTA system,and the binding activity,specificity,serum stability were verified via ELISA,indirect immunofluorescence,and flow cytometry respectively.The affinity of OX40mab-1 was determined via BIAcore 3000.Results:After 3 rounds of screening,a single chain antibody with an enrichment rate of 95％ was harvested,and was reconstructed into a fully human antibody OX40mab-1 The binding EC50 of OX40mab-1 to OX40 was 0.015 μmol/L.OX40mab-1 did not bind to unrelated antigens,showing its specificity,and its good binding activity to antigen retained after 15 days in serum at 37℃,proving its serum stability and its affinity was 251 nmol/ L determined by BIAcore 3000.Conclusion:An anti-OX40 fully human monoclonal antibody with good binding activity,specificity,and serum stability was obtained,and deserves for further development.
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