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.

In vivo delivery of heat shock protein 70 accelerates wound healing by up-regulating macrophage-mediated phagocytosis.

Injury causes tissue breakdown, which releases large quantities of intracellular contents into the extracellular space. Some of these materials are well-established activators of the immune system and include heat shock proteins (HSPs), uric acid, nucleotides, High Mobility Group Box-1 protein (HMGB-1), and DNA. Here, we show that in vivo delivery of HSPs into BALB/cJ mice with full-thickness wounds accelerates the rate of wound closure by 60% as compared with control-treated mice.

In vivo treatment of mice with heat shock protein, gp 96, improves survival of skin grafts with minor and major antigenic disparity.

Immunization with high doses of heat shock protein, gp 6 given in vivo, has been shown to mediate the activation of CD4+ T cell, which in turn suppresses an ongoing CD8+ immune response. Here we demonstrate that high doses of gp 6 (HDgp 6) (100 microg given subcutaneously on days 0 and 7 following skin graft transplantation) improve survival of skin grafts with both minor and major histocompatibility. First, skin from male C57BL/6 donors was grafted onto female C57BL/6 recipients that were subsequently treated with either HDgp 6 or buffer and graft survival was monitored.

Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and stimulates phagocytosis, processing, and MHC-II presentation of antigens.

The extracellular presence of endotoxin-free heat shock protein 70 (HSP70) enhances the rate and capacity of macrophage-mediated phagocytosis at 6 times the basal rate. It is protein-specific, dose- and time-dependent and involves the internalization of inert microspheres, Gram-positive and -negative bacteria and fungi. Structurally, exogenous HSP70 binds the macrophage plasma membrane, specifically on its lipid raft-microdomain.

Glycoprotein 96 can chaperone both MHC class I- and class II-restricted epitopes for in vivo presentation, but selectively primes CD8+ T cell effector function.

The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the dual function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8(+) T cell effector responses.

Immune modulation with high-dose heat-shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis.

Immunization with heat-shock protein (HSP) gp96 elicits protective immunity to the cancer or virus-infected cells from which it is derived. Low doses of gp96 generate immunity, while doses 10 times the immunizing dose do not. We show here that injection of high doses of gp96 generates CD4(+) T cells that down-regulate a variety of ongoing immune responses.