Development and characterization of anti-IL-5 monoclonal antibody Fab fragment for blocking IL-5/IL-5Rα binding.

Interleukin-5 (IL-5) is a homodimeric cytokine that is a crucial regulator of the proliferation, activation, and maturation of eosinophils. Anti-IL-5 monoclonal antibodies, which block the binding of IL-5 to the IL-5 receptor subunit alpha (IL-5Rα), have been successfully used to treat eosinophilic (EOS) asthma. The currently marketed monoclonal antibody drugs require repeated injections for administration, which seriously affect patient compliance and high systemic exposure for injectable drug delivery. Here we successfully screened and developed the Fab (fragment of antigen binding), which is 1/3rd the molecular weight of IgG, favoring inhalation-mediated delivery to the lungs, making it more effective for asthma treatment. The 20A12-Fab-H12L3 can bind to IL-5 with a binding constant of 1.236E-09 M while significantly inhibiting the IL-5/IL-5Rα complex formation. We found that the light chain amino acids (S46 and F71) significantly affected the antibody expression during humanization. The 20A12-Fab-H12L3 significantly inhibited the proliferation of TF-1 cells and blocked the IL-5 binding to the IL-5Rα-overexpressing human embryonic kidney (HEK)-293 cells in vitro. Therefore, based on the mutant IL-5 binding with Fab, we explained why antibodies blocked IL-5 binding to IL-5Rα. Thus, this study provided a candidate pharmaceutical antibody for inhalation drug delivery.