一株新型鼠抗人PD-L2单克隆抗体的研制及其生物学特性的鉴定

Preparation and characterization of a novel monoclonal antibody against human PD-L2(B7-DC)

为了研制特异性识别人PD-L2(B7-DC,CD273)的鼠单克隆抗体,并对其生物学特性和PD-L2分子的表达特性进行初步分析。以高表达人PD-L2分子的基因转染细胞L929/PD-L2作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L2作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-L2单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法和竞争结合抑制实验对单抗进行生物学特性的分析,继而利用该单抗进行免疫荧光标记和流式细胞术检测PD-L2在肿瘤细胞株和免疫细胞上的表达特性。结果显示,通过多次融合和反复筛选,成功获得一株特异性鼠抗人PD-L2(B7-DC)的杂交瘤,该杂交瘤分泌的单克隆抗体能特异识别人PD-L2分子。继而利用上述研制获得的单克隆抗体8F2进行免疫荧光标记和流式细胞术检测发现,PD-L2上调性表达在成熟的树突状细胞和调节性T细胞上。提示,成功地获得一株特异性鼠抗人PD-L2单克隆抗体,并对其生物学特性和表达谱进行初步分析,证明其识别抗原表位不同于商品化抗体,是一株新型鼠抗人PD-L2单抗,这为进一步研究PD-1/PD-L2信号通路在免疫应答中的生物学作用提供了有价值的物质基础。

A functional monoclonal antibody against human PD-L2（B7-DC,CD273） molecule was prepared and their biological characteristics was analyzed in the present study.The highly expressed human PD-L2 was transfected to L929/PD-L2 cell line and the expressed molecule was used as the immunogen to immunize BALB/c mice.By means of hybridoma cell fusion technique,LD29/PD-L2 cells was used as the target cells,while L929/mock cell was used as the negative control.The hybridoma specifically secreting mouse anti-human PD-L2 monoclonal antibody was generated after indirect fluorescence labeling,flow cytometry（FCM）,repeated screening and multiple subcloning.Their biological characterization was investigated by Western blotting,rapid murine Ig subclass typing,indirect immunofluorescene assay,and mutual competitive inhibition test.After multiple fusion and repeated screening,one hybridoma cell line secreting mouse anti-human PD-L2 monoclonal antibody was obtained successfully,and this monoclonal antibody secreted（named as 8F2） could bind to human PD-L2 specially.Through the indirect fluorescence labeling and FCM,it was demonstrated that the PD-L2 molecule was expressed on the mature dendritic cells and regulatory T cells by up-regulation and it could recognize a new epitope on the PD-L2 molecule.By means of the FCM analysis,the expression patterns of PD-L2 on human different cell lines was determined,indicating the increased expression of PD-L2 in mature dendritic cells and activated T cells.From these observations,it is evident that a novel mouse anti-human PD-L2 monoclonal antibody was generated successfully,which recognize different epitopes on PD-L2 and appears to be different to the commercial products.This PD-L2 monoclonal antibody may provide the initial material for further study on the role of human PD-1/PD-L2 signaling pathway in the biological role of immune response.