RESUMEN
It has been proposed that monoclonal antibodies may become therapeutics for metabolic diseases such as diabetes mellitus. We have previously characterized an allosteric monoclonal antibody to the human insulin receptor (IR), XMetA, that activated metabolic signaling leading to enhanced glucose transport in cultured cells, and chronically reduced fasting blood glucose levels in mouse models of diabetes mellitus. Under acute dosing conditions, the large size of an IR-binding antibody like XMetA (â¼ 150 kDa) could lead to a more rapid access into liver, an insulin sensitive tissue with well-fenestrated capillaries, when compared to other insulin sensitive tissues with non-fenestrated capillaries, such as muscle and adipose. Thus, in the present study we administered XMetA (10 mg/kg) and insulin (0.5 U/kg) via IV injection, and for 90 min compared their effects on blood glucose lowering and IR activation in three of the major insulin-sensitive tissues of the normal fasted mouse: liver, adipose, and muscle. Like insulin, XMetA lowered blood glucose levels, although the effect was less rapid. Insulin activated IR autophosphorylation and Akt phosphorylation in liver, fat, and muscle. In contrast, IR activation by XMetA was primarily observed in the liver. Both insulin and XMetA lowered ß-hydroxybutyrate levels in plasma; however, only insulin reduced both non-esterified fatty acids (NEFA) and glycerol concentrations. These data indicate that, in normal mice, acute glucose regulation by XMetA is largely mediated by its action on the liver.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Glucemia/efectos de los fármacos , Insulina/administración & dosificación , Hígado/metabolismo , Receptor de Insulina/agonistas , Ácido 3-Hidroxibutírico/sangre , Tejido Adiposo/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Células CHO , Cricetulus , Humanos , Inyecciones Intravenosas , Insulina/farmacología , Masculino , Ratones , Músculos/metabolismo , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Receptor de Insulina/metabolismoRESUMEN
The monoclonal antibody XMetA is an allosteric partial agonist of the insulin receptor (IR), which activates the metabolic Akt kinase signaling pathway while having little or no effect on the mitogenic extracellular signal-regulated kinase (ERK) signaling pathway. To investigate the nature of this selective signaling, we have conducted a detailed investigation of XMetA to evaluate specific phosphorylation and activation of IR, Akt, and ERK in Chinese hamster ovary cell lines expressing either the short or long isoform of the human IR. Insulin activated both pathways, but the phosphorylation of Akt was more sensitive to the hormone than the phosphorylation of ERK. Maximally effective concentrations of XMetA elicited phosphorylation patterns similar to 40-100 pM insulin, which were sufficient for robust Akt phosphorylation, but had little effect on ERK phosphorylation. These data indicate that the preferential signaling of XMetA is due to an innate difference in pathway sensitivity of Akt versus ERK responses to IR activation and partial agonism by XMetA, rather than a separate pathway-biased mechanism. The metabolic selectivity of partial IR agonists like XMetA, if recapitulated in vivo, may be a desirable feature of therapeutic agents designed to regulate blood glucose levels while minimizing undesirable outcomes of excessive IR mitogenic activation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor de Insulina/metabolismo , Regulación Alostérica , Animales , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetulus , Agonismo Parcial de Drogas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacología , Fosforilación , Unión Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/agonistas , Receptor de Insulina/inmunología , Transducción de SeñalRESUMEN
Interleukin-1ß (IL-1ß) is a proinflammatory cytokine that is implicated in many autoinflammatory disorders, but is also important in defense against pathogens. Thus, there is a need to safely and effectively modulate IL-1ß activity to reduce pathology while maintaining function. Gevokizumab is a potent anti-IL-1ß antibody being developed as a treatment for diseases in which IL-1ß has been associated with pathogenesis. Previous data indicated that gevokizumab negatively modulates IL-1ß signaling through an allosteric mechanism. Because IL-1ß signaling is a complex, dynamic process involving multiple components, it is important to understand the kinetics of IL-1ß signaling and the impact of gevokizumab on this process. In the present study, we measured the impact of gevokizumab on the IL-1ß system using Schild analysis and surface plasmon resonance studies, both of which demonstrated that gevokizumab decreases the binding affinity of IL-1ß for the IL-1 receptor type I (IL-1RI) signaling receptor, but not the IL-1 counter-regulatory decoy receptor (IL-1 receptor type II). Gevokizumab inhibits both the binding of IL-1ß to IL-1RI and the subsequent recruitment of IL-1 accessory protein primarily by reducing the association rates of these interactions. Based on this information and recently published structural data, we propose that gevokizumab decreases the association rate for binding of IL-1ß to its receptor by altering the electrostatic surface potential of IL-1ß, thus reducing the contribution of electrostatic steering to the rapid association rate. These data indicate, therefore, that gevokizumab is a unique inhibitor of IL-1ß signaling that may offer an alternative to current therapies for IL-1ß-associated autoinflammatory diseases.
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Receptores de Interleucina-1/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/inmunología , Células HeLa , Humanos , Interleucina-1beta/antagonistas & inhibidores , Unión Proteica/inmunología , Receptores de Interleucina-1/metabolismoRESUMEN
Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Interleucina-1beta/fisiología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Bioingeniería , Fibroblastos/citología , Fibroblastos/fisiología , Células HeLa/efectos de los fármacos , Células HeLa/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Interleucina-1/fisiología , Interleucina-1beta/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiología , Cinética , Ligandos , Luciferasas/genética , Pulmón/citología , Pulmón/fisiología , FN-kappa B/fisiología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Receptores de Interleucina-1/efectos de los fármacos , Receptores de Interleucina-1/fisiología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Interleukin-1ß (IL-1ß) is a potent mediator of inflammatory responses and plays a role in the differentiation of a number of lymphoid cells. In several inflammatory and autoimmune diseases, serum levels of IL-1ß are elevated and correlate with disease development and severity. The central role of the IL-1 pathway in several diseases has been validated by inhibitors currently in clinical development or approved by the FDA. However, the need to effectively modulate IL-1ß-mediated local inflammation with the systemic delivery of an efficacious, safe and convenient drug still exists. To meet these challenges, we developed XOMA 052 (gevokizumab), a potent anti-IL-1ß neutralizing antibody that was designed in silico and humanized using Human Engineering™ technology. XOMA 052 has a 300 femtomolar binding affinity for human IL-1ß and an in vitro potency in the low picomolar range. XOMA 052 binds to a unique IL-1ß epitope where residues critical for binding have been identified. We have previously reported that XOMA 052 is efficacious in vivo in a diet-induced obesity mouse model thought to be driven by low levels of chronic inflammation. We report here that XOMA 052 also reduces acute inflammation in vivo, neutralizing the effect of exogenously administered human IL-1ß and blocking peritonitis in a mouse model of acute gout. Based on its high potency, novel mechanism of action, long half-life, and high affinity, XOMA 052 provides a new strategy for the treatment of a number of inflammatory, autoimmune and metabolic diseases in which the role of IL-1ß is central to pathogenesis.