Anti-IL-5 Nanobody Screening and Activity Detection
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.
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