RESUMEN
Hyperthyroid Graves' disease is a common autoimmune disorder mediated by agonistic antibodies to the TSH receptor, termed thyroid stimulating antibodies (TSAbs). Recently members of the TNF superfamily, B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL), have been identified along with their receptors, B cell maturation antigen and transmembrane activator and calcium-modulator and cyclophilin ligand interactor, and the BAFF-specific receptor. BAFF is a fundamental B cell survival/maturation factor, and both BAFF and APRIL have been implicated in antibody production. We investigated the effect of interfering with BAFF- and APRIL-mediated signals in an induced model of Graves' disease by blockade of these factors using soluble decoy receptors. In a therapeutic setting in mice with established hyperthyroidism, we show that blockade of BAFF or BAFF+APRIL with BAFF-specific receptor-Fc and B cell maturation antigen-Fc, respectively, leads to significant reductions in the induced hyperthyroidism. This was supported by a parallel pattern of declining TSAbs in the responding animals. Histopathological analysis of splenic sections from treated animals revealed marked reductions in the B cell follicle regions, but staining with anti-CD138 revealed the persistence of plasma cells. Thus, the reductions in TSAbs in the treated animals were not related to overall plasma cell numbers in the secondary lymphoid organs. Our results are the first to demonstrate attenuation of established hyperthyroidism by therapeutic intervention aimed at autoreactive B cells and indicate that both BAFF and APRIL appear to play important roles in the development and survival of the autoantibody producing cells in this model.
Asunto(s)
Enfermedad de Graves/tratamiento farmacológico , Proteínas de la Membrana/fisiología , Tiroiditis Autoinmune/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adenoviridae/genética , Animales , Factor Activador de Células B , Linfocitos B/fisiología , Difenilamina/análogos & derivados , Difenilamina/farmacología , Femenino , Citometría de Flujo , Vectores Genéticos , Enfermedad de Graves/patología , Inmunoglobulinas Estimulantes de la Tiroides/metabolismo , Inmunohistoquímica , Ligandos , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Receptores de Tirotropina/genética , Tiroiditis Autoinmune/patología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.
Asunto(s)
Aldehídos/uso terapéutico , Antivirales/uso terapéutico , Interferón beta/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Polietilenglicoles/uso terapéutico , Aldehídos/síntesis química , Aldehídos/farmacocinética , Animales , Antivirales/química , Antivirales/farmacocinética , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Virus de la Encefalomiocarditis/efectos de los fármacos , Femenino , Semivida , Humanos , Interferón beta-1a , Interferón beta/química , Interferón beta/farmacocinética , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/patología , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Polietilenglicoles/síntesis química , Polietilenglicoles/farmacocinética , Ratas , Ratas Endogámicas LewRESUMEN
We report that B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is expressed in the normal human brain at approximately 10% of that in lymphatic tissues (tonsils and adenoids) and is produced by astrocytes. BAFF was regularly detected by enzyme-linked immunosorbent assay in brain tissue lysates and in normal spinal fluid, and in astrocytes by double fluorescence microscopy. Cultured human astrocytes secreted functionally active BAFF after stimulation with interferon-gamma and TNF-alpha via a furin-like protease-dependent pathway. BAFF secretion per cell was manifold higher in activated astrocytes than in monocytes and macrophages. We studied brain lesions with B cell components, and found that in multiple sclerosis plaques, BAFF expression was strongly up-regulated to levels observed in lymphatic tissues. BAFF was localized in astrocytes close to BAFF-R-expressing immune cells. BAFF receptors were strongly expressed in situ in primary central nervous system (CNS) lymphomas. This paper identifies astrocytes as a nonimmune source of BAFF. CNS-produced BAFF may support B cell survival in inflammatory diseases and primary B cell lymphoma.
Asunto(s)
Astrocitos/metabolismo , Neoplasias del Sistema Nervioso Central/metabolismo , Linfoma/metabolismo , Proteínas de la Membrana/metabolismo , Esclerosis Múltiple/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor Activador de Células B , Receptor del Factor Activador de Células B , Linfocitos B , Encéfalo/metabolismo , Humanos , Proteínas de la Membrana/genética , Especificidad de Órganos , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genética , Regulación hacia ArribaRESUMEN
Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest. Recently, effector mechanisms have been shown to play a part in anti-CD154 mAb-induced transplant acceptance in murine models, while this issue remains unresolved for humoral-mediated models. Herein, aglycosyl anti-CD154 mAbs with reduced binding to FcgammaR and complement were used as a novel means to test the role of effector mechanisms in non-human primate and murine models not amenable to gene knockout technology. While aglycosyl hu5C8 mAb was relatively ineffective in rhesus renal and islet allotransplantation, it inhibited primary and secondary humoral responses to a protein immunogen in cynomolgus monkeys. Moreover, an aglycosyl, chimeric MR1 mAb (muMR1) prolonged survival and inhibited pathogenic auto-antibody production in a murine model of SLE. Thus, the mechanisms required for efficacy of anti-CD154 mAbs depend on the nature of the immune challenge.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ligando de CD40/inmunología , Inmunización Pasiva , Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Riñón/inmunología , Lupus Eritematoso Sistémico/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Antígenos CD40/inmunología , Modelos Animales de Enfermedad , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/terapia , Macaca fascicularis , Ratones , Trombocitemia Esencial/inmunología , Trombocitemia Esencial/patología , Trombocitemia Esencial/terapia , Trasplante HomólogoRESUMEN
It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement. In this study we demonstrate that anti-CD154 mAb treatment inhibits EAE development in SJL mice without apparent long-term unresponsiveness or active suppression of disease. To address whether the mechanism of inhibition of EAE by anti-CD154 mAb depends on its Fc effector interactions, we compared an anti-CD154 mAb with its aglycosyl counterpart with severely impaired FcgammaR binding and reduced complement binding activity with regard to their ability to inhibit clinical signs of EAE and report that both forms of the Ab are similarly protective. This observation was largely confirmed by the extent of leukocyte infiltration of the CNS; however, mice treated with the aglycosyl form may display slightly more proteolipid protein 139-151-specific immune reactivity. It is concluded that FcR interactions do not play a major role in the protective effect of anti-CD154 mAb in the context of EAE, though they may contribute to the full abrogation of peripheral peptide-specific lymphocyte responses.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Ligando de CD40/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Receptores Fc/metabolismo , Animales , Linfocitos B/inmunología , Femenino , Glicosilación , Ratones , Proteína Proteolipídica de la Mielina/inmunología , Fragmentos de Péptidos/inmunología , Receptores Fc/inmunología , Linfocitos T/inmunologíaRESUMEN
Peripheral autoantigen binding B cells are poorly competitive with naive B cells for survival and undergo rapid cell death. However, in monoclonal Ig-transgenic mice lacking competitor B cells, autoantigen binding B cells can survive for extended periods. The basis for competitive elimination of autoantigen binding B cells has been unknown. Here we demonstrate that autoantigen binding B cells have increased dependence on BAFF for survival. In monoclonal Ig-transgenic mice, each autoantigen binding B cell receives elevated amounts of BAFF, exhibiting increased levels of NFkappaB p52 and of the prosurvival kinase Pim2. When placed in a diverse B cell compartment, BAFF receptor engagement and signaling are reduced and the autoantigen binding cells are unable to protect themselves from Bim and possibly other death-promoting factors induced by chronic BCR signaling. These findings indicate that under conditions where BAFF levels are elevated, autoantigen-engaged cells will be rescued from rapid competitive elimination, predisposing to the development of autoimmune disease.
Asunto(s)
Autoantígenos/inmunología , Autoinmunidad/fisiología , Linfocitos B/inmunología , Tolerancia Inmunológica , Proteínas de la Membrana/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Traslado Adoptivo , Animales , Factor Activador de Células B , Linfocitos B/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.
Asunto(s)
Interferón Tipo I/metabolismo , Interferón beta/metabolismo , Oligosacáridos/metabolismo , Polietilenglicoles/química , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Glicosilación , Humanos , Interferón Tipo I/genética , Interferón Tipo I/aislamiento & purificación , Interferón beta-1a , Interferón beta/genética , Espectrometría de Masas , Ratas , Proteínas RecombinantesRESUMEN
The cell surface co-stimulatory protein CD154 (CD40L) is a target for monoclonal antibody (mAb) inhibitors of T-cell mediated immune diseases. This protein, like most other members of the TNF ligand family, forms homotrimeric complexes on the cell surface and in solution, with a three-fold axis of symmetry. We find that several different anti-CD154 monoclonal antibodies form distinctive complexes with soluble CD154. These soluble complexes have been analyzed using size exclusion chromatography, static and dynamic light scattering, and electron microscopy and shown to consist of caged structures of various geometries. The cell surface complexes have been analyzed by confocal microscopy and, depending on the mAb, remain as small, separate complexes or form large aggregates. The formation of these complexes in solution is likely to have an impact on measures of affinity, while the cell surface complexes could affect binding potency and provoke other biological effects.