抗SIRPα单克隆抗体的制备及其体外活性

Preparation and in vitro Activity of Anti-SIRPαMonoclonal Antibody

利用分子生物学技术构建可表达带有小鼠抗体Fc端(mFc)的信号调节蛋白α(SIRPα)的质粒,并转染至中国仓鼠卵巢癌细胞(CHO细胞)中来制备SIRPα-mFc蛋白。将获得的SIRPα-mFc蛋白作为抗原免疫BALB/c小鼠,并将免疫后的小鼠脾脏细胞与SP2/0骨髓瘤细胞进行细胞融合。经过酶联免疫吸附反应(ELISA)和流式细胞仪的多轮筛选后,得到7株靶向SIRPα的单克隆抗体。其中,40F3和57B10这2株单克隆抗体结合活性较高,半数效应浓度(EC_(50))分别为5.866、5.457ng/mL;半数抑制浓度(IC_(50))分别为65.31、63.29 ng/mL,均优于商业化抗体SE5A5(抗SIRPα/β抗体,EC_(50)为22.47 ng/mL,IC_(50)为121.2 ng/mL)。并且,这2株抗体的亲和力数量级在1×10^(–11)且不与SIRPg结合,为后续的抗体人源化以及临床前研究提供了参考。

The signal regulatory proteinα(SIRPα)-mFc expression plasmid constructed by molecular biological technique was transfected into Chinese hamster ovary(CHO)cell lines in order to express SIRPα-mFc protein.The BALB/c mice were immunized with SIRPα-mFc protein antigens,then the spleen cells of the immunized BALB/c mice were fused with SP2/0 myeloma cells.After multiple rounds screening through enzyme linked immunosorbent assay(ELISA)and flow cytometry,seven monoclonal antibodies targeting SIRPαprotein were obtained.Two of the seven clones named 40F3 and 57B10 showed higher binding activity,the median effective concentration(EC_(50))of 40F3 and 57B10 were 5.866 and 5.457 ng/mL,and the half maximal inhibitory concentration(IC_(50))were 65.31 and 63.29 ng/mL,both were better than the commercial product SE5A5(anti-SIRPα/βantibody,EC_(50)=22.47 ng/mL,IC_(50)=121.2 ng/mL).The affinity activity level tested of these two clones were 1×10^(–11),and the antibodies 40F3 and 57B10 could not bind to SIRPg protein,which provided a reference for the subsequent antibodies humanization and preclinical studies.