14 research outputs found
Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab
<div><h3>Background</h3><p>Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling.</p> <h3>Methodology/Principal Findings</h3><p>A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The <em>in vivo</em> efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation <em>in vitro,</em> however this difference was less obvious <em>in vivo.</em> No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists.</p> <h3>Conclusions/Significance</h3><p>The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.</p> </div
Antibody inhibition of <sup>3</sup>H-thymidine incorporation in engineered cell lines.
<p>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. <sup>3</sup>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 <sup>3</sup>H-thymidine. A human anti-CD20 control antibody had no significant effect on <sup>3</sup>H-thymidine incorporation (data not shown).</p
Effect of Anti-IGF1R Antibodies on IGF1R and INSR Autophosphorylation.
<p>The lC<sub>50</sub> for antibody inhibition of ligand-induced receptor activation was extracted from antibody dose titrations. The 95% confidence intervals of the curve fit are given in parentheses. Agonistic potential is the maximum signal obtained with antibody (0.1–1 µM) in the absence of ligand divided by the signal without antibody and ligand.</p
Ligand blocking activity of anti-IGF1R antibodies.
<p>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.</p
Antibody effects on IGF1R and INSR activation by IGF-1 and IGF-2 in COLO 205 cells.
<p>(A–C). Determination of antibody IC<sub>50</sub> 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<sub>50</sub> for antibody inhibition. INSR tyrosine autophosphorylation was determined using the IGF1R/INSR multiplex assay with the same COLO 205 cell extracts used for IGF1R.</p
Analysis of antibody-mediated receptor internalization and degradation.
<p>Levels of IGF1R and INSR-B subunit were determined by direct western and immunoblot quantitation after treatment of COLO 205 cells or tumors with antibodies as indicated. An equal amount (20 µg) of total protein extract was loaded in each lane. (A). COLO 205 cells in 10% FBS growth medium were treated with 250 nM anti-IGF1R antibody, control anti-CD20 antibody, or IGF-1 (100 nM) for 17 hours, and IGF1R levels were determined. (B). COLO 205 cultures were treated for 17 hours with increasing concentrations (0 to 1000 nM) of representative L2, CR, and FnIII-1 domain antibodies and IGF1R levels were determined. (C). COLO 205 cells were grown in the presence of 250 nM antibody over a 2-week period to determine the long-term effects of ganitumab, Mab 391, and F1-B on IGF1R expression. The antibody was replenished when the cells were subcultured. All signals were normalized to the IGF1R signal obtained with the control antibody at each time point. (D). Mice with established (200–300 mm<sup>3</sup>) subcutaneous tumors were treated with ganitumab, Mab 391, or F1-B (300 µg/dose; IP). At the indicated time points, 3 animals were sacrificed and IGF1R levels were determined. % control is the signal obtained for an individual animal divided by the mean for the control antibody multiplied by 100 for each treatment group. Total INSR level was determined in the same cell (E) and tumor (F) extracts used for the long-term analysis of IGF1R (C, D).</p
Analysis of agonistic activity of anti-IGF1R antibodies.
<p>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).</p
Effects of anti-IGF1R antibodies in combination with irinotecan in the COLO 205 xenograft model.
<p>Mice bearing COLO 205 xenografts (∼200 mm<sup>3</sup>) were randomly assigned into treatment groups (n = 10) and treated with antibody (300 µg, twice per week, IP), irinotecan (35 mg/kg, once per week, IV), or the combination of both agents 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. The combination of anti-IGF1R antibodies and irinotecan: (A). ganitumab; (B). L2-B; (C). Mab 391; and (D). F1-B. *<i>p</i><.0003 versus either single agent; **<i>p</i><.001 versus either single agent; <sup>#</sup><i>p</i>>0.004 versus F1-B alone and <i>p</i> = 0.55 versus irinotecan alone.</p
Inhibition of the growth of 32D hIGF1R/IRS-1 tumor xenografts.
<p>(A). TGI by ganitumab, Mab 391, or 1H7. Mice bearing 32D hIGF1R/IRS-1 xenografts (∼200 mm<sup>3</sup>) 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 <i>in vivo</i>. Mice bearing 32D hIGF1R/IRS-1 xenografts (∼400 mm<sup>3</sup>) 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). *<i>p</i><.0003.</p
Effects of Anti-IGF1R Antibodies on Cell Proliferation.
<p>The IC<sub>50</sub> for ligand inhibition of cell proliferation was derived from the antibody dose-titrations curves. The number of replicate experiments is shown parenthesis. ND, not determined.</p