RESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD/metabolismo , Glucosa/metabolismo , Receptor de Insulina/metabolismo , Sitio Alostérico , Animales , Péptido C/química , Células CHO , Separación Celular , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/metabolismo , Citometría de Flujo , Humanos , Hiperglucemia/metabolismo , Hiperinsulinismo/metabolismo , Insulina/química , Resistencia a la Insulina , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Biblioteca de Péptidos , Fosforilación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Hiperinsulinismo Congénito/inmunología , Receptor de Insulina/antagonistas & inhibidores , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/inmunología , Células CHO , Hiperinsulinismo Congénito/tratamiento farmacológico , Hiperinsulinismo Congénito/patología , Cricetinae , Cricetulus , Glucosa/inmunología , Resistencia a la Insulina/inmunología , Ratones , Ratas , Receptor de Insulina/inmunología , Anticuerpos de Cadena Única/farmacologíaRESUMEN
Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.
Asunto(s)
Técnicas de Visualización de Superficie Celular , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Receptor de Insulina/inmunología , Receptor TIE-2/inmunología , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistemas de Lectura Abierta , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor TIE-2/genética , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genética , TransfecciónRESUMEN
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.