一株抗人OX40激发型单克隆抗体的研制及其生物学功能的初步研究

Preparation of anti-human OX40 agonist monoclonal antibody and its biological function

鼠抗人OX40分子激发型单克隆抗体(mAb)的研制及其生物学特性的鉴定。以高表达人OX40分子的基因转染细胞L929-OX40为免疫原,常规免疫6～8周龄的雌性BALB/c小鼠;采用B淋巴细胞融合技术,将小鼠脾脏细胞与骨髓瘤细胞SP2/0融合,并以L929-OX40细胞为阳性筛选细胞,L929-Mock细胞为阴性筛选细胞,采用免疫荧光技术对杂交瘤进行反复筛选及多次克隆化培养;采用快速定性试纸法检测抗体亚型;竞争抑制实验分析抗原结合表位;Western blot检测抗体特异性;采用MTT法分析单抗在体外对T细胞增殖的促进作用,并采用ELISA检测培养上清中的细胞因子。结果:获得一株稳定、高效分泌鼠抗人OX40单克隆抗体的杂交瘤细胞株(克隆号:9H8),该株mAb能够特异性识别U937、Jurkat、Thp-1等血液来源肿瘤细胞株表面OX40分子,并且能够有效促进T细胞活化、增殖及细胞因子的分泌。成功研制了一株分泌鼠抗人OX40分子激发型单克隆抗体的杂交瘤细胞株,该抗体能够特异性识别人OX40分子并能够介导OX40正性协同刺激效应。

To prepare the anti-OX40 agonist monoclonal antibody and to characterize its biological function,routine immunization of 6～8 weeks female BALB/c mice by using the transfected cell L929-OX40 as antigen was performed,and then the spleen cells of the immunized mice were fused with SP2/0 cells.The positive clones were to be cloned by FCM,in which L929- OX40 cell and L929-Mock cell were used as the positive and negative cells respectively.Fast-strip analysis was used to identify the Ig subclass of this monoclonal antibody（mAb）.The epitope of this mAb was studied by competitive inhibition test;Investigation of its specificity was performed by Western-blot,and the promotion of mAb in proliferation and cytokines secretion of T cells in vitro in the early stage were studied by MTT and ELISA respectively,One hybridoma cell secreting specific anti-OX40 monoclonal antibody continuously and efficiently,was obtained and named as 9H8 cell.This mAb could recognize the OX40 molecule on several hematological tumor cell lines,such as U937,Jurkat,Thp-1.Moreover it promoted proliferation of T cells and cytokine secretion in vitro efficiently.One hybridoma cell line secreting agonist anti-OX40 monoclonal antibody has been established successfully.It can recognize human OX40 specifically and mediate positive OX40 co-stimulation effect.