Identification and Characterization of a Novel IL-4 Receptor α Chain (IL-4Rα) Antagonist to Inhibit IL-4 Signalling

Abstract

Background/Aims: In recent times, allergy has become a financial, physical and psychological burden to the society as a whole. In allergic cascades, cytokine IL-4 binds to IL-4 receptor (IL-4R), consequently producing allergen-specific IgE antibodies by B cells. In addition, among other functions, IL-4 is also responsible for B and T cell proliferation and differentiation. Hence, characterization of novel antagonists that inhibit IL-4 signalling forms the overall aim of this study. Methods: Phage display was used to screen a random 12-mer synthetic peptide library with a human IL-4Rα to identify peptide candidates. Once identified, the peptides were commercially synthesized and used for in vitro immunoassays. Results: We have successfully used phage display to identify M13 phage clones that demonstrated specific binding to IL-4Rα. The peptide N1 was synthesized for use in ELISA, demonstrating significant binding to IL-4Rα and inhibiting interaction with cytokine IL-4. Furthermore, the peptide was tested in a transfected HEK-Blue IL-4 reporter cell line model, which produces alkaline phosphatase (AP). QUANTI-Blue, a substrate, breaks down in the presence of AP producing a blue coloration. Using this colorimetric analysis, >50% inhibition of IL-4 signalling was achieved. Conclusion: We have successfully identified and characterised a synthetic peptide antagonist against IL-4Rα, which effectively inhibits IL-4 interaction with the IL-4Rα in vitro. Since IL-4 interaction with IL-4Rα is a common pathway for many allergies, a prophylactic treatment can be devised by inhibiting this interaction for future treatment of allergies.

Macrophage Susceptibility to Emactuzumab (RG7155) Treatment

“…To further investigate the contribution of STAT1 and STAT6 signaling to the nonresponsiveness of M(IL4) to RG7155, we employed a potent low molecular weight STAT1 antagonist S14-95 and sIL4Ra. sIL4Ra has been shown to efficiently and selectively inhibit IL4 while maintaining IL13 signaling in contrast to an IL4R-blocking antibody (27). Although sIL4Ra as well as RG7155 efficiently blocked STAT6 signaling, both had negligible influence on M(IL4) viability (Fig.…”

Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

“…We initially tested all isolated Abs for the biological activity because biological potency of Abs is often determined by epitope binding rather than binding affinity 17 . For this, we used a HEK-Blue TM IL-4/IL-13 cell line, in which HEK293 cells are engineered to produce secreted embryonic alkaline phosphatase (SEAP) in the supernatant in response to IL-4 or IL-13 stimulation 18,19 . Thus, IL-4/IL-13-dependent type I receptor activation can be monitored by quantification of the enzymatic activity of SEAP using QUANTI-Blue TM (Fig.…”

Identification of a BACE1 Binding Peptide Candidate for the Prevention of Amyloid Beta in Alzheimer’s Disease

“…To address this, we utilised the phage display and biopanning technique to identify a 12 amino acid peptide that could not only bind to BACE1 specifically but prevent the enzymatic ability to cleave APP. Biopanning and phage display has been successful in acquiring positive binding peptides to numerous targets like interleukin-4 receptor, alkaline phosphatase, hepsin, tissue metalloproteinases and tyrosinase (28)(29)(30)(31)(32). Defining a construct with effective binding properties is the proactive direction of the current study to refining an effective new candidate for AD.…”