Preclinical development of a long-acting trivalent bispecific nanobody targeting IL-5 for the treatment of eosinophilic asthma

Abstract

Background: Eosinophilic asthma is a common subtype of severe asthma with high morbidity and mortality. The cytokine IL-5 has been shown to be a key driver of the development and progression of disease. Although approved monoclonal antibodies (mAbs) targeting IL-5/IL-5R have shown good safety and efficacy, some patients have inadequate responses and frequent dosing results in medication nonadherence.

Results: We constructed a novel trivalent bispecific nanobody (Nb) consisting of 3 VHHs that bind to 2 different epitopes of IL-5 and 1 epitope of albumin derived from immunized phage display libraries. This trivalent IL-5-HSA Nb exhibited similar IL-5/IL-5R blocking activities to mepolizumab (Nucala), an approved targeting IL-5 mAb. Surprisingly, this trivalent Nb was 58 times more active than mepolizumab in inhibiting TF-1-cell proliferation. In primate studies, the trivalent IL-5-HSA Nb showed excellent pharmacokinetic properties, and peripheral blood eosinophil levels remained significantly suppressed for two months after a single dose. In addition, the trivalent IL-5-HSA Nb could be produced on a large scale in a P. pastoris X-33 yeast system with high purity and good thermal stability.

Conclusions: These findings suggest that the trivalent bispecific IL-5-HSA Nb has the potential to be a next-generation therapeutic agent targeting IL-5 for the treatment of severe eosinophilic asthma.

Keywords: Eosinophilic asthma; IL-5; Long-acting; Trivalent nanobody.

Fig. 1

Schematic depicting the immunization and screening strategy used to isolate IL-5 Nbs. PBLs, peripheral blood lymphocytes. The cropping gel of purified IL-5-Fc antigen is displayed

Fig. 2

The generation of heterobivalent Nb21/Nb66. A The affinity of 11 IL-5 Nbs was detected by biofilm interferometry (BLI). B The recognition specificity of 11 IL-5 Nbs to different species antigens was determined by ELISA, with IgG1 as a negative control. C The competition binding activity of Nb66 or Nb21 towards the binding between Nb21-biotin and IL-5 or Nb66-biotin and IL-5 was detected by ELISA. D The recognition specificity of Nb21/Nb66 towards Nb21-biotin-IL-5 or Nb66-biotin-IL-5 binding sites, with IL33-Nb biotin as a negative control. The experiments were performed in triplicate. The data were presented as mean ± SD. ***P < 0.001. E The blocking effect of the Nb21/Nb66 or Nucala analogue on the interaction between IL-5 and IL-5R was determined by flow cytometry. MFI: median fluorescence intensity. F The inhibitory effect of the Nb21/Nb66 or Nucala analogue on TF-1-cells proliferation was determined by the CCK8 assay. The IC₅₀ was calculated

Fig. 3

Construction of the trivalent bispecific IL-5-HSA Nb. A The binding activity of HSA Nb towards human albumin and cynomolgus monkey albumin (CSA) at pH 5.5 and pH 7.4 was determined by ELISA. B The structure of trivalent bispecific IL-5-HSA Nb. C-D The affinity of trivalent IL-5-HSA Nb and Nucala analogue to IL-5 was detected by Fortebio

Fig. 4

The functional activity determination of the trivalent IL-5-HSA Nb. A The binding activity of the trivalent IL-5-HSA Nb towards human IL-5 and cyno IL-5. B The binding activity of the trivalent IL-5-HSA Nb towards HSA and CSA at pH 5.5 and pH 7.4. The binding activity was detected by ELISA. C The blocking effect of trivalent IL-5-HSA Nb or Nucala analogue on the IL-5/IL-5R interaction was determined by flow cytometry. D The inhibitory effect of trivalent IL-5-HSA Nb or Nucala analogue on TF-1-cell proliferation was determined by the CCK8 assay

Fig. 5

The pharmacokinetic and pharmacodynamic effects of the trivalent IL-5-HSA Nb in cynomolgus monkeys. A Schematic representation of its pharmacokinetic and pharmacodynamic study. B The pharmacokinetic effect of the trivalent IL-5-HSA Nb in cynomolgus monkeys with the primary doses of 5 mg/kg and 2 mg/kg. The plasma concentration of trivalent IL-5-HSA Nb was measured within 56 days. C The inhibitory effect of the trivalent IL-5-HSA Nb on eosinophil (EOS) numbers was determined within 84 days

Fig. 6

Large-scale production of the trivalent IL-5-HSA Nb and its preliminary developability analysis. A The protein expression of the trivalent IL-5-HSA Nb was detected through SDS-PAGE, followed by affinity chromatography, hydrophobic chromatography and molecular sieve chromatography. The cropping gel of purified trivalent IL-5-HSA Nb is displayed. B The protein titer and wet cell weight in the fermentation tank were both measured at the indicated times. C The purity of the trivalent IL-5-HSA Nb was determined through SEC-HPLC analysis. D The stability of the trivalent IL-5-HSA Nb under different temperatures and 3 freeze‒thaw cycles was detected by SEC-HPLC and CEX-HPLC