RESUMO
Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)-mediated T cell activation. The movement of calcium is mediated by calcium release-activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca(2+) sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca(2+) influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited ICRAC current, store-operated Ca(2+) influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/imunologia , Equorina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/biossíntese , Anticorpos Monoclonais/biossíntese , Western Blotting , Quimera , Citocinas/sangue , Mapeamento de Epitopos , Epitopos/efeitos dos fármacos , Citometria de Fluxo , Genes Reporter , Células HEK293 , Humanos , Células Jurkat , Cinética , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/genética , Proteína ORAI1 , Técnicas de Patch-Clamp , Polimorfismo de Nucleotídeo Único , RatosRESUMO
Passive therapy with neutralizing human monoclonal antibodies (mAbs) could be an effective therapy against severe acute respiratory syndrome coronavirus (SARS-CoV). Utilizing the human immunoglobulin transgenic mouse, XenoMouse, we produced fully human SARS-CoV spike (S) protein specific antibodies. Antibodies were examined for reactivity against a recombinant S1 protein, to which 200 antibodies reacted. Twenty-seven antibodies neutralized 200TCID(50) SARS-CoV (Urbani). Additionally, 57 neutralizing antibodies were found that are likely specific to S2. Mapping of the binding region was achieved with several S1 recombinant proteins. Most S1 reactive neutralizing mAbs bound to the RBD, aa 318-510. However, two S1 specific mAbs reacted with a domain upstream of the RBD between aa 12 and 261. Immunoglobulin gene sequence analyses suggested at least 8 different binding specificities. Unique human mAbs could be used as a cocktail that would simultaneously target several neutralizing epitopes and prevent emergence of escape mutants.