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...

These results support use of an adapted, multiple-challenge, direct conjunctival allergen model to assess efficacy of EBI-005 in late phase AC. In the CAPT, EBI-005 showed statistically significant improvements in clinically meaningful symptoms (ocular itching, tearing, and nasal symptoms) at multiple time points for moderate-to-severe AC subjects.

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.