Design of a superior cytokine antagonist for topical ophthalmic use

Abstract

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent's size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other β-trefoil family proteins in the IL-1 and FGF families.T he IL-1 cytokines (IL-1α and IL-1β) are master mediators of inflammatory responses (1). IL-1β also regulates immune function through its role in T helper 17 (Th17) cell differentiation and maintenance (2, 3). IL-1 action has been implicated in numerous human diseases, including rheumatoid arthritis, MuckleWells syndrome, gout, type 2 diabetes, and stroke (4). Several natural mechanisms directly oppose the actions of IL-1, including a soluble and cell surface decoy receptor (IL-1R2), a natural antagonist (IL-1Ra), and a soluble signaling receptor (IL-1R1) (5). Therapeutics that block IL-1 based on these mechanisms have been developed (6-8).Recently, a nonoptimized formulation of anakinra (methionyl-IL-1Ra; Kineret) was shown to provide clinical benefit in dry-eye disease (DED) (9). Moderate to severe DED is a chronic inflammatory condition of the corneal surface that results in pain, discomfort, and epitheliopathy (as measured by fluorescein staining). Inability to maintain a proper tear film over the cornea (owing to a variety of etiologies) results in desiccating stress, which drives an inflammatory cascade (10, 11). IL-1 plays a central role in the initiation and maintenance of this cascade, as well as in the pain mediated by the corneal neural plexus. IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease (12), and their mRNA is increased in both humans and in rodent disease models (13,14). Genetic ablation of IL-1R1, the primary receptor for IL-1α and IL-1β, can block the development of corneal staining in a Sjögren syndrome corneal epitheliopathy model (15), and topically administered anakinra can improve surface epithliopathy in a mouse dry-eye model (14). IL-1β is essential for Th17 cell differentiation and maintenance, and Th17 cells are likely the main effector cells that induce epithelial damage (2, 3). Genetic and pharmacologic studies have shown that IL-1β mediates, and IL-1Ra blocks, normal, inflamm...

Molecular Determinants of Agonist and Antagonist Signaling through the IL-36 Receptor

“…Our inability to create superior agonists and antagonists by creating chimeras of the putative binding sites for the D1/2 and D3 domains, in analogy to the strategy used for IL-1b and IL-1Ra (12), indicates that the IL-36 subfamily differs markedly from the IL-1 subfamily in the molecular mechanisms by which they activate and inhibit signaling. Through our mutational and loopswapping experiments, we observed that the affinity of both the agonist IL-36g (by mutation Ala 162 Asp) and the IL-36Ra (by loop swap of b4/5 from IL-36g) can be improved and, at least in the case of IL-36Ra, this also resulted in better antagonism.…”

“…Construction of superior agonists and antagonists chimeras is not directly adoptable from the IL-1b/IL-1Ra system. We created chimeras of IL36g and IL-36Ra in analogy to the superior agonistic and antagonistic IL-1b/IL-1Ra chimeras described by Hou et al (12). Chimera 125:30 corresponds to the superior agonist, chimera 96:63 to the superior antagonist.…”

“…This lack of binding to site II enables a rotation of D3 away from the interface with IL-1RAcP, likely contributing to the antagonistic properties of IL-1Ra (21). Hou et al (12) capitalized on the knowledge that IL-1Ra has a higher affinity for site I and IL-1b for site II to create IL-1b/IL-1Ra chimeras that combine both binding sites and function as either superior agonists or antagonists. To test whether IL-36g and IL-36Ra behave similarly to the IL-1 cytokines, we generated chimeras of IL-36g and IL-36Ra that corresponded to the most agonistic and antagonist chimeric version of IL-1b /IL-1Ra (Supplemental Fig.…”

“…Many chronic inflammatory conditions result from an imbalance in activating versus inhibitory signals but can, in some cases, be treated by restoring molecular brakes to signaling in the form of agonistneutralizing molecules (e.g., Abs or decoy receptors) or engineered antagonists (3,12). Appropriately balanced signaling through IL-36R is a critical determinant of skin and lung health, as epithelial cells secrete IL-36 cytokines.…”

Improvement of Outcome Measures of Dry Eye by a Novel Integrin Antagonist in the Murine Desiccating Stress Model

“…Its potential relevance for the disease is highlighted by the clinical development of an IL-1 receptor antagonist for ocular surface inflammation, EBI-005. 30,31 Matrix metalloproteinase-9 is a protease that has been implicated in the breakage of tight junctions of corneal epithelium and in the corneal barrier disruption in desiccating stress. 8,32,33 Tear levels of MMP-9 have been shown to correlate with corneal staining intensity and other clinical parameters in dry eye patients.…”