RESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Interleucina-1beta/fisiologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Bioengenharia , Fibroblastos/citologia , Fibroblastos/fisiologia , Células HeLa/efeitos dos fármacos , Células HeLa/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Interleucina-1/fisiologia , Interleucina-1beta/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/fisiologia , Cinética , Ligantes , Luciferases/genética , Pulmão/citologia , Pulmão/fisiologia , NF-kappa B/fisiologia , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Receptores de Interleucina-1/efeitos dos fármacos , Receptores de Interleucina-1/fisiologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling â¼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory hyperinsulinism in insulin resistant conditions.
Assuntos
Anticorpos Monoclonais/química , Antígenos CD/metabolismo , Glucose/metabolismo , Receptor de Insulina/metabolismo , Sítio Alostérico , Animais , Peptídeo C/química , Células CHO , Separação Celular , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Citometria de Fluxo , Humanos , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Insulina/química , Resistência à Insulina , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Biblioteca de Peptídeos , Fosforilação , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.
Assuntos
Anticorpos Monoclonais/imunologia , Hiperinsulinismo Congênito/imunologia , Receptor de Insulina/antagonistas & inibidores , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/imunologia , Células CHO , Hiperinsulinismo Congênito/tratamento farmacológico , Hiperinsulinismo Congênito/patologia , Cricetinae , Cricetulus , Glucose/imunologia , Resistência à Insulina/imunologia , Camundongos , Ratos , Receptor de Insulina/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Many patients with diabetes mellitus (both type 1 and type 2) require therapy to maintain normal fasting glucose levels. To develop a novel treatment for these individuals, we used phage display technology to target the insulin receptor (INSR) complexed with insulin and identified a high affinity, allosteric, human monoclonal antibody, XMetA, which mimicked the glucoregulatory, but not the mitogenic, actions of insulin. Biophysical studies with cultured cells expressing human INSR demonstrated that XMetA acted allosterically and did not compete with insulin for binding to its receptor. XMetA was found to function as a specific partial agonist of INSR, eliciting tyrosine phosphorylation of INSR but not the IGF-IR. Although this antibody activated metabolic signaling, leading to enhanced glucose uptake, it neither activated Erk nor induced proliferation of cancer cells. In an insulin resistant, insulinopenic model of diabetes, XMetA markedly reduced elevated fasting blood glucose and normalized glucose tolerance. After 6 weeks, significant improvements in HbA(1c), dyslipidemia, and other manifestations of diabetes were observed. It is noteworthy that hypoglycemia and weight gain were not observed during these studies. These studies indicate, therefore, that allosteric monoclonal antibodies have the potential to be novel, ultra-long acting, agents for the regulation of hyperglycemia in diabetes.