RESUMO
Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands. Fifteen of these scFv-phage, identified using IL-1R(I)-binding assays and dissociation rate ranking, were reformatted as scFv-Fc and IgG(4) molecules. The ease of producing antibodies in the scFv-Fc format permitted rapid identification of four lead clones (C10, C13, C14, C15) that inhibit NF-kappaB nuclear translocation induced by IL-1. Reformatting these clones as IgG(4) molecules increased their inhibition potency by
Assuntos
Anticorpos/química , Região Variável de Imunoglobulina/química , Receptores de Interleucina-1/química , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Ligação Competitiva , Biotinilação , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Clonagem Molecular , DNA/química , Relação Dose-Resposta a Droga , Citometria de Fluxo , Células HeLa , Humanos , Imunoglobulina G/química , Concentração Inibidora 50 , Interleucina-1/metabolismo , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Coelhos , Receptores de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1 , Fatores de TempoRESUMO
We investigated the formation and stability of succinimide, an intermediate of deamidation events, in recombinant monoclonal antibodies (mAbs). During the course of an analytical development study of an IgG1 mAbs, we observed that a specific antibody population could be separated from the main product by cation-exchange (CEX) chromatography. The cell-based bioassay measured a approximately 70% drop in potency for this fraction. Liquid chromatography time-of-flight mass spectrometry (LC-TOF/MS) and tandem mass spectrometry (LC-MS/MS) analyses showed that the modified CEX fraction resulted from the formation of a succinimide intermediate at Asn 55 in the complementarity determining region (CDR) of the heavy chain. Biacore assay revealed a approximately 50% decrease in ligand binding activity for the succinimide-containing Fab with respect to the native Fab. It was found that the succinimide form existed as a stable intermediate with a half-life of approximately 3 h at 37 degrees C and pH 7.6. Stress studies indicated that mildly acidic pH conditions (pH 5) favored succinimide accumulation, causing a gradual loss in potency. Hydrolysis of the succinimide resulted in a further drop in potency. The implications of the succinimide formation at Asn 55, a highly conserved residue among IgG1 (mAbs), are discussed.
Assuntos
Anticorpos Monoclonais/química , Asparagina/química , Regiões Determinantes de Complementaridade/química , Imunoglobulina G/química , Cadeias Pesadas de Imunoglobulinas/química , Succinimidas/síntese química , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Ligantes , Espectrometria de Massas , Papaína/química , Mapeamento de Peptídeos , TripsinaRESUMO
IL-17 is an inflammatory cytokine produced primarily by a unique lineage of CD4 T cells that plays critical roles in the pathogenesis of multiple autoimmune diseases. IL-17RA is a ubiquitously expressed receptor that is essential for IL-17 biologic activity. Despite widespread receptor expression, the activity of IL-17 is most classically defined by its ability to induce the expression of inflammatory cytokines, chemokines, and other mediators by stromal cells. The lack of IL-17 responsiveness in mouse stromal cells genetically deficient in IL-17RA is poorly complemented by human IL-17RA, suggesting the presence of an obligate ancillary component whose activity is species specific. This component is IL-17RC, a distinct member of the IL-17R family. Thus, the biologic activity of IL-17 is dependent on a complex composed of IL-17RA and IL-17RC, suggesting a new paradigm for understanding the interactions between the expanded family of IL-17 ligands and their receptors.