Thermosensitive elastin-like polypeptides bind pro-inflammatory IL-17A and form local depots following supra-lacrimal injection

Abstract

Purpose : IL-17A is a pro-inflammatory cytokine that is overexpressed in tears of patients with Sjögren’s Syndrome (SjS), an autoimmune disorder that causes chronic inflammation in the lacrimal gland (LG) and is associated with severe dry eye disease. IL-17A targeting therapies have been approved for other autoimmune diseases, but not for SjS. This work characterizes a novel locally administered IL-17A sequestering therapy that incorporates the "HAP" peptide, which binds IL-17A, fused to thermosensitive elastin-like protein-polymers (ELPs).

Methods : HAP was fused to two ELPs to form HAP-A96 and HAP-V96, which are soluble and insoluble respectively at physiological temperature. These fusions were expressed in E. coli, purified through inverse transition cycling, and characterized by MALDI-MS and SDS-PAGE. Transition temperature (Tt) of fusions, above which the fusions coacervate out of solution, was determined by UV spectroscopy and binding affinity was quantified by surface plasmon resonance. The ability of HAP-ELP to sequester IL-17A was determined by competitive ELISA. HAP-ELPs were fluorescently labelled by N-hydroxysuccinimide rhodamine and injected supra-LG in anesthetized Balb/C mice and imaged using an In Vivo Imaging System for 2 weeks and plasma samples were obtained to estimate the depot mean residence time (n= 4 mice per group).

Results : HAP-ELP binds human and mouse IL-17A with equilibrium binding constants, KD of 731 pM and 2.1 nM respectively. HAP-ELPs showed dose-dependent decreases in the apparent concentration of mIL-17A as quantified by ELISA. HAP-A96 and HAP-V96 have Tt of 55°C and 24°C at 50 µM. Since the Tt of HAP-V96 is below body temperature at all tested concentrations, extended depot residence time of 99 hours following supra-LG injection in mice was observed, compared to 7.5 hrs for HAP-A96 (p<0.005), demonstrating how the polypeptide sequence can be used to modulate pharmacokinetics (PK) of the IL-17 binding peptide.

Conclusions : HAP-ELP sequesters IL-17A and can form a depot with ELP-dependent PK. This suggests it may be used to sequester locally overexpressed pro-inflammatory cytokines with infrequent administration of tunable depot, which would be a novel treatment for SjS.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

HAP-ELP binds human and mouse IL-17A, but not bovine serum albumin (BSA) as characterized by SPR

 

IVIS image quantification showing ELP-dependent depot residence time