12 research outputs found
Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms
Improved glucose metabolism in vitro and in vivo by an allosteric monoclonal antibody that increases insulin receptor binding affinity.
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 ∼15-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
XMetS binds to the A and B isoforms of the INSR with high affinity.
<p>CHO cells expressing either the B or A isoform of the hINSR, and parental CHO cells were preincubated for 10 minutes at 4°C in the presence of 100 nM insulin followed by a 120 minute incubation at 15°C with increasing concentrations of XMetS. XMetS binding to the INSR was measured by FACS. Mean of duplicate determinations are shown.</p
XMetS potentiates insulin-mediated 2-deoxy-D-glucose uptake in L6 muscle cells, but does not enhance the growth of cancer cells.
<p><b>A.</b> L6 cells expressing both isoform B of the hINSR and GLUT-4 were preincubated with either XMetS or control antibody for 60 minutes at 37°C followed by a 10 minute incubation with increasing concentrations of insulin. [<sup>3</sup>H]-2-deoxy-D-glucose (2DG) was added and uptake was measured after 20 minutes. <b>B.</b> MCF-7 human breast cancer cells were incubated for 48 hours at 37°C with increasing concentrations of insulin in the presence of either XMetS or control antibody, and cell proliferation was determined by CellTiter Glo® assay. <b>C.</b> Saos-2 human osteosarcoma cells were incubated for 72 hours at 37°C with increasing concentrations of insulin in the presence of either XMetS or control antibody, and cell proliferation was determined by CellTiter Glo® assay. The mean ± SD from triplicate determinations are shown.</p
Metabolic profile of diet induced obesity mice treated with XMetS and controls.
<p>At the end of the 4-week study with diet induced obesity mice, the animals were fasted 14 hours, weighed and plasma was obtained to determine the above parameters. Mice (n = 10/group) were treated with antibody at 10 mg/kg, twice weekly. Values are shown as mean ± SEM. *p<0.05 vs. diabetic+control IgG.</p
Metabolic profile of multi-low dose, streptozotocin/high-fat diet mice treated with XMetS and controls<b>.</b>
<p>At the end of the 6-week study with multi-low dose, streptozotocin/high-fat diet mice, the animals were fasted 14 hours, weighed and plasma was obtained to determine the above parameters. Mice (n = 8/group) were treated with antibody at 10 mg/kg, twice weekly. Values are shown as mean ± SEM. *p<0.05 vs. diabetic+control IgG.</p
XMetS improves glucose metabolism in multi-low dose, streptozotocin/high-fat diet mice.
<p><b>A–C.</b> ICR multi-low dose, streptozotocin/high-fat diet mice were treated twice weekly with either control antibody (10 mg/kg) or XMetS (10 mg/kg). Age-matched non-diabetic ICR mice were treated twice weekly with control antibody (10 mg/kg). <b>A</b>. Blood glucose levels were obtained weekly for six weeks following a 14-hour fast. <b>B</b>. After five weeks of treatment and following a 4-hour fast, insulin was administered intraperitoneally (0.75 U/kg) and blood glucose levels were obtained for an additional 120 minutes. <b>C</b>. After three weeks of treatment and following a 14-hour fast, a glucose bolus was administered intraperitoneally (1 g/kg) and blood glucose levels were measured for 120 minutes. Mean ± SEM are shown (n = 8 mice/group).</p
XMetS improves glucose metabolism in diet induced obesity mice.
<p>C57BL/6 diet induced obesity mice were treated twice weekly with either control antibody (10 mg/kg) or XMetS (10 mg/kg). Age-matched, lean C57BL/6 mice were treated twice weekly with control antibody (10 mg/kg). After one week of treatment and following a 14-hour overnight fast, a glucose bolus was administered intraperitoneally (1 g/kg) and blood glucose levels were measured for 120 minutes. Mean ± SEM are shown (n = 10 mice/group).</p
XMetS potentiates insulin-dependent Akt and Erk phosphorylation.
<p><b>A.</b> CHO-hINSR cells were preincubated for 10 minutes at 37°C with either the IgG2 form of XMetS, the Fab form of XMetS, or control antibody followed by a 10 minute incubation with increasing concentrations of insulin. Phosphorylation of Akt was then determined. <b>B</b>. CHO-hINSR cells were preincubated for 10 minutes at 37°C with a wide range of XMetS concentrations followed by a 10 minute incubation with increasing concentrations of insulin. Schild analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088684#pone.0088684-Schild1" target="_blank">[30]</a> of these data was carried out using a curve fit algorithm (Prism, allosteric EC<sub>50</sub> shift). <b>C.</b> EC<sub>50</sub> values were determined from each curve fit (of the data presented in B) and are plotted as a function of XMetS concentration. <b>D</b>. CHO-hINSR cells were preincubated for 10 minutes at 37°C with either XMetS or control antibody followed by a 10 minute incubation with increasing concentrations of insulin and phosphorylation of Erk was then determined. The mean of duplicate determinations are shown.</p