用CDR3导向抗体库法对鼠抗人膀胱癌单抗进行人源化

Humanization of mouse anti-human bladder cancer McAb by CDR3 directed phage antibody library

目的 构建含鼠单抗CDR3的人源噬菌体抗体库 ,对膀胱癌鼠单抗进行人源化。方法通过重叠PCR及DNA重组技术 ,将抗膀胱癌鼠单抗BDI的CDR3区与人淋巴细胞来源的多样化的VH和Vκ组合 ,构建含BDICDR3的人源噬菌体抗体库 ;利用loxp cre定位重组系统 ,增加轻、重链的组合配对 ,获大容量抗体库 ;用膀胱癌细胞 (EJ)从中筛选人源化抗膀胱癌单抗Fab段 ,ELISA方法对所筛克隆进行特异性鉴定 ,同时运用竞争抑制实验对所筛特异性克隆进行抗原表位的核实。结果 首先构建了库容为 2× 10 7的含鼠CDR3区的人源噬菌体抗体库 ;经过在cre+ 细菌胞内的定位重组后 ,获得库容为 10 1 0 的大容量噬菌体抗体库 ;用EJ细胞筛选到可与其特异结合的人源性Fab段 ;竞争抑制实验表明所获人源抗体片段与亲本鼠源单抗结合同一抗原表位。结论 在没有亲本鼠单抗基因 ,仅了解其CDR3序列的条件下 ,可通过CDR3导向 ,构建大容量抗体库 ,筛选到相应的人源化抗体基因。

Objective To humanize an murine anti-human bladder cancer monoclonal antibody BDI. Methods Using overlap PCR and DNA recombination technology, the murine V H and Vκ CDR3 of BDI were spliced into human diverse Fd and κ genes and cloned into a phage antibody expression vector pAL, in which the Fd is flanked by two non-homologous loxp sites, forming a CDR3-incorporated human phage antibody library. Through cre-loxp mediated recombination, the Fd and κ chains at different vectors were shuffled and a large human antibody library was constructed. Panning of the phage antibody library against bladder cancer EJ cells were conducted to select humanized BDI antibodies. The antigen binding activity was determined by ELISA. Results A primary BDI CDR3-incorporated human phage antibody library 2×10 7 of in size was constructed, and a large antibody library of 1×10 10 in size was obtained usingcre-loxp recombination system. Human anti-bladder cancer antibodies were successfully cloned by panning of the large phage antibody library against bladder cancer cells. Binding competition studies demonstrated that they bound the same epitope of the Ag as the parental Ab. Conclusion Using CDR3 directed phage antibody library technique, human counterpart of murine McAb could be selected without the parental murine V region as a template.