RESUMO
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. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Receptores Fc/química , Albumina Sérica/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ligação Competitiva , Feminino , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Cinética , Ligantes , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Ratos , Homologia de Sequência , Albumina Sérica/genética , Microglobulina beta-2/químicaRESUMO
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-1ß 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 â¼100-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 ß-trefoil family proteins in the IL-1 and FGF families.
