Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: Binding assays with hIGF1R(ECD)-mFc and Ru-labeled IGF-1 (A, C) and IGF-2 (B, D) were used to characterize the ligand blocking by ganitumab and other antibodies as indicated. (A, B). Antibody and ligand competition assays. Binding curves were generated by incubating hIGF1R(ECD)-mFc, Ru-labeled ligands (IGF-1/2) and increasing concentrations of antibodies or unlabeled IGF-1/2 (positive control). (C, D). The effect of excess antibody on binding of Ru-labeled ligand to hIGF1R(ECD)-mFc. Binding curves were generated with increasing concentrations of Ru-labeled IGF-1 and IGF-2 in the presence of 1 µM antibody. Background signal was subtracted before data analysis. Abbreviation: Ab, antibody.

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Binding Assay, Labeling, Blocking Assay, Generated, Positive Control

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: 32D hIGF1R/IRS-1 cells (A–C) and Balb/C 3T3 hIGF1R cells (D–F) were treated with increasing concentrations of the indicated domain-specific antibodies in the presence or absence of 2 nM IGF-1 or 8 nM IGF-2. (A–C). 32D hIGF1R/IRS-1 cells treated for 48 hours with antibody and growth factors in 5% FBS, RPMI. 3 H-thymidine labeling occurred between 24–48 hours. (D–F). Balb/C 3T3 hIGF1R cells were serum-starved in DMEM +0.1% BSA overnight before treatment with antibody and growth factor for an additional 24 hours in the presence of 3 H-thymidine. A human anti-CD20 control antibody had no significant effect on 3 H-thymidine incorporation (data not shown).

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Labeling

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: Effect of Anti-IGF1R Antibodies on IGF1R and INSR Autophosphorylation.

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: (A–C). Determination of antibody IC 50 for IGF1R inhibition. Serum-starved COLO 205 cells were treated for 20 minutes simultaneously with either 2 nM IGF-1 or 8 nM IGF-2 and antibody as indicated. Total (t) and tyrosine autophosphorylation (p) IGF1R after αIR3 and Mab 391 treatment were determined in duplicate using an MSD assay with F1-B as the capture agent. All other receptor measurements were obtained using an MSD multiplex in which IGF1R and INSR were simultaneously determined. Titration with a non-specific anti-CD20 antibody increased receptor autophosphorylation to maximum of about 110% of control (data not shown). (D). Titrations with IGF-1 and IGF-2 in the presence of excess antibody. Balb/C 3T3 hIGF1R cells were treated for 5 minutes with increasing concentrations of IGF-1 or IGF-2 in the presence of anti-IGF1R or control antibody (1 µM). Total (no change, not shown) and pIGF1R were assayed by immunoprecipitation and western blotting. The control extract included in each immunoblot was prepared from cells stimulated with 100 nM IGF-1 or IGF-2 in the presence of 1 µM anti-CD20 antibody. (E–G). Determination of INSR IC 50 for antibody inhibition. INSR tyrosine autophosphorylation was determined using the IGF1R/INSR multiplex assay with the same COLO 205 cell extracts used for IGF1R.

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Inhibition, Multiplex Assay, Titration, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: Serum-starved cells were treated with antibody for 20 minutes without the addition of IGF-1 or IGF-2 under conditions identical to those used for the analysis of receptor inhibition. Total and phosphorylated (p) IGF1R and INSR were determined; the fold change relative to background is plotted against antibody dose. (A–D). Determination of IGF1R tyrosine phosphorylation with COLO 205, MCF-7, Balb/C 3T3 hIGF1R, and 32D hIGF1R/IRS-1 cells. E,F. Analysis of INSR phosphorylation in response to antibody treatment in COLO 205 and MCF-7 cells. Partial IGF1R agonism was also observed for F1-A, F1-C, 24-57, and L2-B in a qualitative survey with Balb/C 3T3 hIGF1R cells (data not shown).

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: Effects of Anti-IGF1R Antibodies on Tumor Growth Inhibition of 32D higf1r/IRS-1 Xenografts.

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

doi: 10.1371/journal.pone.0055135

Figure Lengend Snippet: (A). TGI by ganitumab, Mab 391, or 1H7. Mice bearing 32D hIGF1R/IRS-1 xenografts (∼200 mm 3 ) were randomly assigned into treatment groups (n = 10) and treated with antibody (300 µg, twice per week, IP) for the duration of the experiment. Tumor volumes and body weights were measured twice per week using calipers and an analytical scale, respectively. Data are presented as mean tumor volume ± standard error of the mean. (B). Relationship between ganitumab serum concentration and TGI. Ganitumab serum concentration (µg/mL) was determined 2 hours after the final dose and plotted as the mean ± standard deviation. (C–F). Inhibition of IGF1R signaling in vivo . Mice bearing 32D hIGF1R/IRS-1 xenografts (∼400 mm 3 ) were randomly assigned into treatment groups (n = 6) and treated with ganitumab or IgG1 control for 6 hours. Three animals from each group were then challenged with 5 µg IGF-1 or PBS IV for 15 minutes. Total and phosphorylated IGF1R, IRS-1, Akt, and p70S6K in tumor extracts (100 µg) were determined with MSD multiplex assays. No significant changes in GSK3β were observed (data not shown). * p <.0003.

Article Snippet: Each binding assay used 75 ng hIGF1R(ECD)-mFc preloaded on anti-mouse IgG-coated MA 6000 96-well plates (Mesoscale Discovery, Gaithersburg, MD) as recommended by the manufacturer.

Techniques: Concentration Assay, Standard Deviation, Inhibition, In Vivo, Multiplex Assay