抗IL-5纳米抗体筛选及活性检测

Anti-IL-5 Nanobody Screening and Activity Detection

白介素-5(IL-5)是一种同源二聚体细胞因子,是嗜酸性粒细胞(eosinophilic,EOS)增殖、活化和成熟的重要调节因子。抗IL-5单克隆抗体能阻断IL-5与IL-5受体亚单位α(IL-5Rα)结合,已成功用于治疗嗜酸性粒细胞哮喘。目前上市的单克隆抗体药物需要反复注射给药,严重影响了患者的依从性,而且注射给药的全身暴露率高。为获得适合吸入给药的抗体,在羊驼天然库中通过3轮淘选,挑取单克隆通过Phage ELISA初筛,共获得461个阳性克隆,其中50个为序列独特的分子,最终选择30个分子进行重组表达纯化。通过ELISA结合、ELISA阻断、FACS阻断、TF-1增殖抑制等实验对候选抗体进行体外活性检测,成功获得一个具有阻断IL-5和IL-5Rα结合的纳米抗体AIL-A96-Fc。通过与人和猴IL-5的ELISA结合实验表明,该分子具有良好人猴交叉活性,而且在FACS阻断实验和ELISA阻断实验中,AIL-A96-Fc表现出良好的阻断效果。该开发方法不仅提供了一个靶向IL-5的候选纳米抗体AIL-A96-Fc,也为后续开发更多靶向IL-5的候选纳米抗体提供了指导意义。

Interleukin-5(IL-5),a homodimeric cytokine,is an important regulator of eosinophil(EOS)proliferation,activation and maturation.Anti-IL-5 monoclonal antibodies block the binding of IL-5 to the IL-5 receptor subunit alpha(IL-5Rα)and have been used successfully in the treatment of eosinophilic asthma.Currently available monoclonal antibody drugs require repeated administration by injection,which has a significant impact on patient compliance,and the systemic exposure rate of injection is high.To obtain nanobodies suitable for inhalation administration,monoclonal clones were selected through three rounds of panning in the natural alpaca library by phage ELISA screening.A total of 461 positive clones were obtained,of which 50 clone sequences were unique,and 30 molecules were selected for recombinant expression and purification of nanobodies.The in vitro activity of the candidate antibodies was tested by ELISA binding,ELISA blocking,FACS blocking and TF-1 proliferation inhibition assays,and a nanobody AIL-A96-Fc with the ability to block the binding of IL-5 and IL-5Rα was successfully obtained.ELISA binding assays with human and cynomolgus IL-5 showed that the molecule has good human-cynomolgus cross-species activity,and AIL-A96-Fc showed good blocking effects in FACS and ELISA blocking assays.This study not only provides a candidate nanobody(AIL-A96-Fc),but the development methodology also provides guidance for the subsequent development of additional candidate nanobodies targeting IL-5.