Anion Binding and Controlled Aggregation of Human Interleukin-1 Receptor Antagonist

Raibekas, Andrei A.; Bures, Edward J.; Siska, Christine C.; Kohno, Tadahiko; Latypov, Ramil F.; Kerwin, Bruce A.

doi:10.1021/bi050388g

Cited by 40 publications

(76 citation statements)

References 36 publications

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“…Along with their increased T onset , the chimeras lack three lysines in IL-1Ra (K6, K93, and K96) that form a positively charged cluster on the surface and drive the earliest steps in aggregation (39). This may further enhance both aggregation stability and solubility.…”

Section: Discussionmentioning

confidence: 99%

Design of a superior cytokine antagonist for topical ophthalmic use

Hou¹,

Townson²,

Kovalchin³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

confidence: 99%

Design of a superior cytokine antagonist for topical ophthalmic use

Hou¹,

Townson²,

Kovalchin³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Long-term stability studies were performed for IL1RA WT and mutants in CSE buffer, at a protein concentration of 50 mg ml À 1 (ref. 39). For experiments at 30°C, 10 ml of protein sample was used and 20 ml for 40°C.…”

Section: Methodsmentioning

confidence: 99%

“…IL1RA was chosen as a test example for the stabilization of a protein drug as it is biopharmaceutically characterized by an aggregation propensity linked to poor thermal stability 38,39 . Therefore, extensive efforts have been made to improve the stability of IL1RA by reformulation 40 in conjunction with studies of longer-term storage and thermal stability 38,39,41 .…”

Section: Evolution Of Protein Thermostability For Structural Biologymentioning

confidence: 99%

“…New mutations in this area might allow the selection of further stabilized variants with retention or improvement of affinity. In light of these results and the potential therapeutic application of these variants, accelerated stability studies were performed using buffer conditions and concentrations similar to the formulation of the clinical version of IL1RA, Anakinra 39 . The best first-and second-cycle mutants (IL1RA-2 and IL1RA-8, respectively) were compared with WT protein in a thermal challenge in CSE buffer, at a concentration of 50 mg ml À 1 over 1 week at 30°C, measuring aggregation spectrophotometrically at 405 nm (Fig.…”

Section: Evolution Of Protein Thermostability For Structural Biologymentioning

confidence: 99%

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Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

et al. 2013

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Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several biotechnologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins.

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“…Although conformational stability is generally a good indicator of protein stability in the context of non-native aggregation, it provides an incomplete picture of the downstream processes contributing to aggregation, e.g., the association of aggregation-prone intermediates into higher-order species (15). Moreover, there are instances where unfolding does not necessarily correlate with the propensity towards aggregation, and there are examples of proteins that unfold reversibly (16,17). Here, we report a novel high-throughput approach using ThT (to follow progression of amyloid forming aggregates) in parallel with SYPRO Orange (to follow protein unfolding) to rank-order formulation conditions.…”

Section: Introductionmentioning

confidence: 99%

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

et al. 2013

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Abstract. A high-throughput thermal-scanning method to rank-order formulation conditions for therapeutic proteins is described. Apparent transition temperatures for unfolding and aggregation of four different proteins are determined using the dyes SYPRO Orange and thioflavin T (ThT) under a variety of buffer conditions. The results indicate that the ThT-based thermal scanning method offers several advantages over the previously described SYPRO Orange-based thermal scanning and allows rapid rank ordering of solution conditions relevant toward long-term storage of therapeutic molecules. The method is also amenable to high protein concentration and does not require sample dilution or extensive preparation. Additionally, this parallel use of SYPRO Orange and ThT can be readily applied to the screening of mutants for their unfolding and aggregation propensities.

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Anion Binding and Controlled Aggregation of Human Interleukin-1 Receptor Antagonist

Cited by 40 publications

References 36 publications

Design of a superior cytokine antagonist for topical ophthalmic use

Design of a superior cytokine antagonist for topical ophthalmic use

Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell

Orthogonal High-Throughput Thermal Scanning Method for Rank Ordering Protein Formulations

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