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MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential

View Article: PubMed Central - PubMed

ABSTRACT

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.

No MeSH data available.


Related in: MedlinePlus

Functional outcome of MEDI1873 in primary T cells. (A) Thymidine incorporation in CD3+ human T cells restimulated with anti-CD3 and anti-CD28 antibodies in the presence of MEDI1873, anti-GITR (A18) or isotype control in solution of plate bound. Error bars represent the SEM from triplicates. (B) Expression of GITR (solid histograms) on CD4+ FoxP3+ Tregs (purple), CD4+ FoxP3− helper cells (red) and CD8+ cytotoxic T cells (blue) within the TIL of a NSCLC tumor. Semi-transparent, dotted line histograms represent isotype control staining. Data is representative of three independent samples. (C) Percentage lysis of anti-CD3 and anti-CD28 activated CD3+ T cells by allogeneic NK cells in the presence of MEDI1873 and IgG4 GITRL FP. Error bars represent the SEM from triplicates. Two-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. ****p < 0.0001, **p = 0.0015, +p = 0.0138, ns = not significant. (D) Percentage of CD4+ and CD8+ T cells present within the total CD3+ population following treatment with no drug or with MEDI1873 at 3.6 nM at the end of the assay illustrated in (C). (E) Percentage of divided CD4+ FoxP3− effector T cells, measured via CFSE dilution, following activation with anti-CD3 and anti-CD28 in the presence or absence of CD4+ FoxP3+ Tregs with or without MEDI1873 at the concentrations indicated. Error bars, where present, represent the SEM from duplicates. One-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. p values, where significant are indicated as follows ***p = 0.0005, **p < 0.006.
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f0003: Functional outcome of MEDI1873 in primary T cells. (A) Thymidine incorporation in CD3+ human T cells restimulated with anti-CD3 and anti-CD28 antibodies in the presence of MEDI1873, anti-GITR (A18) or isotype control in solution of plate bound. Error bars represent the SEM from triplicates. (B) Expression of GITR (solid histograms) on CD4+ FoxP3+ Tregs (purple), CD4+ FoxP3− helper cells (red) and CD8+ cytotoxic T cells (blue) within the TIL of a NSCLC tumor. Semi-transparent, dotted line histograms represent isotype control staining. Data is representative of three independent samples. (C) Percentage lysis of anti-CD3 and anti-CD28 activated CD3+ T cells by allogeneic NK cells in the presence of MEDI1873 and IgG4 GITRL FP. Error bars represent the SEM from triplicates. Two-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. ****p < 0.0001, **p = 0.0015, +p = 0.0138, ns = not significant. (D) Percentage of CD4+ and CD8+ T cells present within the total CD3+ population following treatment with no drug or with MEDI1873 at 3.6 nM at the end of the assay illustrated in (C). (E) Percentage of divided CD4+ FoxP3− effector T cells, measured via CFSE dilution, following activation with anti-CD3 and anti-CD28 in the presence or absence of CD4+ FoxP3+ Tregs with or without MEDI1873 at the concentrations indicated. Error bars, where present, represent the SEM from duplicates. One-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. p values, where significant are indicated as follows ***p = 0.0005, **p < 0.006.

Mentions: The ability of MEDI1873 to enhance the proliferation of human T cells was assessed in a restimulation assay in which CD3+ T cells were activated with anti-CD3, subsequently rested and then restimulated. In this context, both MEDI1873 and A18, were able to minimally enhance the proliferation of T cells. The maximal proliferative signal reached by the anti-GITR antibody A18 (7290) was not significantly (p = 0.09) greater than that seen with isotype control (4272). In contrast, MEDI1873 generated a significantly (p = 0.03) greater proliferative signal (8474) as compared with isotype control, though this was not significantly different to A18 (p = 0.6). Similarly the EC50s for MEDI1873 and A18 (4.7 and 7.9 nM, respectively) were not significantly different (p = 0.5). When cross-linked via plate binding the EC50 of MEDI1873 was similar to the soluble form (2.6 nM), and not significantly different to cross linked A18 (p = 0.8). However, the maximal proliferative signal generated increased significantly (p = 0.0009) by 2.5-fold to 21,048. In contrast, the activity of the anti-GITR antibody A18 was not notably affected (Fig. 3A).Figure 3.


MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential
Functional outcome of MEDI1873 in primary T cells. (A) Thymidine incorporation in CD3+ human T cells restimulated with anti-CD3 and anti-CD28 antibodies in the presence of MEDI1873, anti-GITR (A18) or isotype control in solution of plate bound. Error bars represent the SEM from triplicates. (B) Expression of GITR (solid histograms) on CD4+ FoxP3+ Tregs (purple), CD4+ FoxP3− helper cells (red) and CD8+ cytotoxic T cells (blue) within the TIL of a NSCLC tumor. Semi-transparent, dotted line histograms represent isotype control staining. Data is representative of three independent samples. (C) Percentage lysis of anti-CD3 and anti-CD28 activated CD3+ T cells by allogeneic NK cells in the presence of MEDI1873 and IgG4 GITRL FP. Error bars represent the SEM from triplicates. Two-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. ****p < 0.0001, **p = 0.0015, +p = 0.0138, ns = not significant. (D) Percentage of CD4+ and CD8+ T cells present within the total CD3+ population following treatment with no drug or with MEDI1873 at 3.6 nM at the end of the assay illustrated in (C). (E) Percentage of divided CD4+ FoxP3− effector T cells, measured via CFSE dilution, following activation with anti-CD3 and anti-CD28 in the presence or absence of CD4+ FoxP3+ Tregs with or without MEDI1873 at the concentrations indicated. Error bars, where present, represent the SEM from duplicates. One-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. p values, where significant are indicated as follows ***p = 0.0005, **p < 0.006.
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f0003: Functional outcome of MEDI1873 in primary T cells. (A) Thymidine incorporation in CD3+ human T cells restimulated with anti-CD3 and anti-CD28 antibodies in the presence of MEDI1873, anti-GITR (A18) or isotype control in solution of plate bound. Error bars represent the SEM from triplicates. (B) Expression of GITR (solid histograms) on CD4+ FoxP3+ Tregs (purple), CD4+ FoxP3− helper cells (red) and CD8+ cytotoxic T cells (blue) within the TIL of a NSCLC tumor. Semi-transparent, dotted line histograms represent isotype control staining. Data is representative of three independent samples. (C) Percentage lysis of anti-CD3 and anti-CD28 activated CD3+ T cells by allogeneic NK cells in the presence of MEDI1873 and IgG4 GITRL FP. Error bars represent the SEM from triplicates. Two-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. ****p < 0.0001, **p = 0.0015, +p = 0.0138, ns = not significant. (D) Percentage of CD4+ and CD8+ T cells present within the total CD3+ population following treatment with no drug or with MEDI1873 at 3.6 nM at the end of the assay illustrated in (C). (E) Percentage of divided CD4+ FoxP3− effector T cells, measured via CFSE dilution, following activation with anti-CD3 and anti-CD28 in the presence or absence of CD4+ FoxP3+ Tregs with or without MEDI1873 at the concentrations indicated. Error bars, where present, represent the SEM from duplicates. One-way ANOVA, with Dunnett's correction for multiple comparisons was used to compare treatment values to control values. p values, where significant are indicated as follows ***p = 0.0005, **p < 0.006.
Mentions: The ability of MEDI1873 to enhance the proliferation of human T cells was assessed in a restimulation assay in which CD3+ T cells were activated with anti-CD3, subsequently rested and then restimulated. In this context, both MEDI1873 and A18, were able to minimally enhance the proliferation of T cells. The maximal proliferative signal reached by the anti-GITR antibody A18 (7290) was not significantly (p = 0.09) greater than that seen with isotype control (4272). In contrast, MEDI1873 generated a significantly (p = 0.03) greater proliferative signal (8474) as compared with isotype control, though this was not significantly different to A18 (p = 0.6). Similarly the EC50s for MEDI1873 and A18 (4.7 and 7.9 nM, respectively) were not significantly different (p = 0.5). When cross-linked via plate binding the EC50 of MEDI1873 was similar to the soluble form (2.6 nM), and not significantly different to cross linked A18 (p = 0.8). However, the maximal proliferative signal generated increased significantly (p = 0.0009) by 2.5-fold to 21,048. In contrast, the activity of the anti-GITR antibody A18 was not notably affected (Fig. 3A).Figure 3.

View Article: PubMed Central - PubMed

ABSTRACT

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is part of a system of signals involved in controlling T-cell activation. Targeting and agonizing GITR in mice promotes antitumor immunity by enhancing the function of effector T cells and inhibiting regulatory T cells. Here, we describe MEDI1873, a novel hexameric human GITR agonist comprising an IgG1 Fc domain, a coronin 1A trimerization domain and the human GITRL extracellular domain (ECD). MEDI1873 was optimized through systematic testing of different trimerization domains, aglycosylation of the GITRL ECD and comparison of different Fc isotypes. MEDI1873 exhibits oligomeric heterogeneity and superiority to an anti-GITR antibody with respect to evoking robust GITR agonism, T-cell activation and clustering of Fc gamma receptors. Further, it recapitulates, in vitro, several aspects of GITR targeting described in mice, including modulation of regulatory T-cell suppression and the ability to increase the CD8+:CD4+ T-cell ratio via antibody-dependent T-cell cytotoxicity. To support translation into a therapeutic setting, we demonstrate that MEDI1873 is a potent T-cell agonist in vivo in non-human primates, inducing marked enhancement of humoral and T-cell proliferative responses against protein antigen, and demonstrate the presence of GITR- and FoxP3-expressing infiltrating lymphocytes in a range of human tumors. Overall our data provide compelling evidence that MEDI1873 is a novel, potent GITR agonist with the ability to modulate T-cell responses, and suggest that previously described GITR biology in mice may translate to the human setting, reinforcing the potential of targeting the GITR pathway as a therapeutic approach to cancer.

No MeSH data available.


Related in: MedlinePlus