Biolayer Interferometry-based FcγRIIa binding assay for a therapeutic antibody with strong effector function.

FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency.

Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays.

Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight.

Preclinical Development of EBI-005: An IL-1 Receptor-1 Inhibitor for the Topical Ocular Treatment of Ocular Surface Inflammatory Diseases.

Objective: Topical interleukin (IL)-1 receptor (R)1 blockade is therapeutically active in reducing signs and symptoms of dry eye disease. Herein, we describe in vitro and in vivo nonclinical Investigational New Drug (IND)-enabling studies of EBI-005, a novel protein chimera of IL-1β and IL-1 receptor antagonist (IL-1Ra or anakinra) that potently binds IL-1R1 and blocks signaling. These studies provide an assessment of receptor affinity, drug bioavailability, immunogenic response, safety, and tolerability in mice and rabbits.

A simple enzyme-substrate localized conjugation method to generate immobilized, functional glutathione S-transferase fusion protein columns for affinity enrichment.

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor.

Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein.

Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale.

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.

Design of a superior cytokine antagonist for topical ophthalmic use.

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.

Macrophage-specific chemokines induced via innate immunity by amino acid copolymers and their role in EAE.

The random amino acid copolymer poly(Y,E,A,K)(n) (Copaxone®) is widely used in multiple sclerosis treatment and a second generation copolymer poly(Y,F,A,K)(n) with enhanced efficacy in experimental autoimmune encephalomyelitis in mice has been described. A major mechanism through which copolymers function to ameliorate disease is the generation of immunosuppressive IL-10-secreting regulatory T cells entering the CNS. In addition, the antigen presenting cell to which these copolymers bind through MHC Class II proteins may have an important role.

Safety, pharmacokinetic, and pharmacodynamic evaluations of PI-2301, a potent immunomodulator, in a first-in-human, single-ascending-dose study in healthy volunteers.

PI-2301 is an amino acid copolymer acting as an immunomodulator for the treatment of autoimmune diseases. The present study evaluated the safety, pharmacokinetics (PK), and pharmacodynamics of PI-2301 in a single ascending dose, first-in-human study involving healthy, male adult volunteers. A total of 56 subjects were given a subcutaneous injection of PI-2301 ranging from 0.