A fully human, allosteric monoclonal antibody that activates the insulin receptor and improves glycemic control

Abstract

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.

FIG. 1.

XMetA binds to the INSR at an allosteric site. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (■) or isotype control antibody (○) and antibody binding measured by flow cytometry (n = 4). B: Increasing concentrations of CHO-hINSR cells were incubated with 80 pmol/L insulin and either 70 nmol/L XMetA (■) or isotype control antibody (○). Insulin binding to the INSR was determined by KinExA (n = 3). mAb, monoclonal antibody.

FIG. 2.

XMetA is a partial agonist of the INSR that selectively activates the PI3K/Akt pathway. A: CHO-hINSR cells were incubated with increasing concentrations of either XMetA (■), isotype control antibody (○), or insulin (▲), and INSR autophosphorylation was measured by ELISA (n = 3). B: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. INSR autophosphorylation was then measured (n = 3). C: CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA (n = 3). D: CHO-hINSR cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. Akt phosphorylation was then measured (n = 3). E: CHO-hINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and extracellular signal–related kinase (Erk)1/2 phosphorylation was measured by ELISA (n = 3). mAb, monoclonal antibody.

FIG. 3.

XMetA does not activate the IGF-IR. CHO-hIGF-IR cells were incubated with either 33 nmol/L XMetA or isotype control antibody in the presence or absence of 100 nmol/L IGF-I. IGF-IR autophosphorylation was measured by ELISA (n = 3). p, phosphorylation.

FIG. 4.

XMetA promotes glucose uptake, but not cell growth. A: 3T3 cells expressing hINSR were incubated with increasing concentrations of either XMetA (■), isotype control antibody (○), or insulin (▲), and 2-deoxy-d-glucose uptake was measured (n = 5). B: MCF-7 cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and cell proliferation was determined by CellTiter Glo assay (n = 6). C: MCF-7 cells were incubated with either 33 nmol/L XMetA (■) or isotype control antibody (○) with increasing concentrations of insulin. Cell proliferation was then measured (n = 3). mAb, monoclonal antibody.

FIG. 5.

XMetA improves hyperglycemia and other metabolic markers of disease in diabetic mice. A: CHO-mINSR cells were incubated with increasing concentrations of XMetA (■), isotype control antibody (○), or insulin (▲), and Akt phosphorylation was measured by ELISA (n = 3). B: Fasting blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). C: After 3 weeks of treatment, fasting blood glucose was measured in control mice treated with 10 mg/kg isotype control antibody (white bar), diabetic mice treated with 10 mg/kg isotype control antibody (gray bar), and diabetic mice treated with the indicated doses of XMetA (black bars). D: Nonfasted blood glucose measurements were obtained weekly for 6 weeks from control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). After 6 weeks of treatment, blood hemoglobin A_1c (E) and nonfasted plasma β-hydroxybutyrate (F) were measured in control mice treated with 10 mg/kg isotype control antibody (white bar) and diabetic mice treated with either 10 mg/kg isotype control antibody (gray bar) or XMetA (black bar). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control; n = 8 mice/group. mAb, monoclonal antibody.

FIG. 6.

XMetA improves glucose tolerance in diabetic mice. A: After 3 weeks of treatment, glucose was administered intraperitoneally (IP) at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). B: After 3 weeks of treatment, glucose was administered orally at 1 g/kg to fasted mice. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.

FIG. 7.

XMetA improves insulin tolerance in diabetic mice. A: After 5 weeks of treatment, insulin was administered intraperitoneally at 0.75 units/kg. Blood glucose levels were measured for 120 min in control mice treated with 10 mg/kg isotype control antibody (○) and diabetic mice treated with either 10 mg/kg XMetA (■) or isotype control antibody (●). B: Data normalized to preinsulin glucose values. Values shown are mean ± SEM. *P < 0.05 for diabetic mice treated with XMetA compared with isotype control antibody; n = 8 mice/group.