D1.123 - Inhibition of the Interleukin Cytokines using novel Peptide Inhibitors for Allergy and Asthma Treatment

The Interleukin Cytokine signalling pathways play an integral role in the pathology of allergic disease. Inhibition of these cytokines by antibodies, including Dupilumab, Tralokinumab and Benralizumab have yeilded highly effective therapeutics for allergies and asthma, however they are limited by the inherent properties of antibody based therapeutics, notably high costs of production, invasive administration and possible immunological side effects. To address these limitations, we have created a low molecular weight (<1.4kDa) peptide capable of inhibiting a key cytokine in asthma and allergies, Interleukin 4 (IL-4) in cell based studies and Biolayer Interferometry (BLI) Experiments.

We created a unique form of Phage Display Biopanning to yield peptide sequences which not only bind to the pro-inflammatory interleukin cytokines, but also inhibit their interactions with their cell surface receptors. Traditional Phage Display was performed for three rounds using a 12mer M13 peptide Phage Library against human IL-4 protein. A fourth round of inverse inhibitory biopanning was performed with the enriched phage library and IL-4 against IL-4Ra. Following synthesis of the recombinant consensus peptides, they were screened for inhibition in a HEK293 IL-4/IL-13 Reporter cell line. The inhibition of the cytokine/receptor interactions by these peptides was also confirmed using Bio Layer Interfereometry experiments.

Three rounds of Traditional Phage Display Biopanning was performed against either recombinant IL-4 protein or IL-13 protein. An additional fourth round of inverse biopanning utilising either IL-4Ra or IL-13Ra1 was used to isolate peptides which inhibited the proteins interactions. Consensus sequences were identified from phage DNA sequencing and then the recombinant peptides were synthesised. Screening of the consensus sequences in the HEK293 reporter cells identified an IL-4 inhibitor that achieved over 81% pathway inhibition over 20hrs. Similarly the IL-13 consensus sequence inhibited IL-13 signalling by 76% over the 20hr experimental period. Real time experiments were performed on the IL-4 inhibitor peptide using BLI, where full inhibition of the IL-4/IL-4Ra interaction was observed with an IC50 of 30.08µM (±4.01µM). 

Using a unique biopanning method, we have identified low molecular weight peptides which can inhibit IL-4 and IL-13 signalling in cell line studies and BLI interaction experiments. These peptides currently possess high concentrations for the effect, however with sequence studying and optimisation these can be greatly improved. This study proves that peptides can inhibit cytokine signalling and lays the foundation for future development of smaller, less invasive and easier to produce cytokine inhibitor therapeutics.