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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.

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Related in: MedlinePlus

MEDI1873 is a homogenous hexameric GITRL fusion protein with potent agonistic activity. (A) Schematic of the domain structure of the GITRL FP, consisting of IgG Fc domain, trimerization domain and extracellular domain (ECD) of human GITRL. (B) Percentage-specific binding of GITRL to GITR in an HTRF-based assay in the presence and absence of GITRL FP variants. Error bars represent the standard error of the mean (SEM) from duplicates. (C) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of GITRL FP variants. Error bars represent the SEM from triplicates. (D and E) Combined, deconvoluted mass spectra for tryptic peptide (T42–43) which contains the N-glycosylation consensus sequence at position 161 of hGITRL for hGITRL(wt) FP (coronin 1A) (D) and hGITRL(N161D) FP (coronin 1A) (E). (F) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of hGITRL(wt) FP (coronin 1A) and hGITRL(N161D) FP (coronin 1A). Error bars represent SEM from triplicates. (G) Distribution of sedimentation coefficients (measured in Svedberg) for MEDI1873 following analytical ultracentrifugation analysis.
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f0001: MEDI1873 is a homogenous hexameric GITRL fusion protein with potent agonistic activity. (A) Schematic of the domain structure of the GITRL FP, consisting of IgG Fc domain, trimerization domain and extracellular domain (ECD) of human GITRL. (B) Percentage-specific binding of GITRL to GITR in an HTRF-based assay in the presence and absence of GITRL FP variants. Error bars represent the standard error of the mean (SEM) from duplicates. (C) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of GITRL FP variants. Error bars represent the SEM from triplicates. (D and E) Combined, deconvoluted mass spectra for tryptic peptide (T42–43) which contains the N-glycosylation consensus sequence at position 161 of hGITRL for hGITRL(wt) FP (coronin 1A) (D) and hGITRL(N161D) FP (coronin 1A) (E). (F) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of hGITRL(wt) FP (coronin 1A) and hGITRL(N161D) FP (coronin 1A). Error bars represent SEM from triplicates. (G) Distribution of sedimentation coefficients (measured in Svedberg) for MEDI1873 following analytical ultracentrifugation analysis.

Mentions: Here, we describe the generation and characterization of MEDI1873, a hexameric GITRL fusion protein (GITRL FP) designed on the principles of trimer stabilization, optimal clustering of GITR and Fc receptor engagement. Each MEDI1873 monomer comprises three distinct domains (Fig. 1A):1 a human immunoglobulin G1 (IgG1) fragment crystallizable (Fc) domain,2 an isoleucine zipper trimerization domain from human coronin 1A,3 and the hGITRL extracellular domain (ECD), with a point mutation (N161D) that eliminates the sole occupied glycosylation site. We demonstrate that MEDI1873 can be purified to homogeneity and is a significantly more potent agonist of hGITR than a monoclonal antibody. We show in human primary lymphocyte assays, in vitro, that this agonism results in enhanced T-cell activation and a reduction in regulatory T-cell suppression and that MEDI1873 is capable of mediating ADCC against activated T cells, resulting in an increased CD8+:CD4+ T-cell ratio. Further, we demonstrate that in non-human primates MEDI1873 increases T-cell activation and response to immunization in vivo. Finally, we detail flow cytometric and histological analyses of patient tumor samples that indicate the presence of GITR positive and FoxP3 positive tumor-infiltrating lymphocytes (TILs). Overall these data confirm the translation of previous mouse GITR biology to a human setting and support the targeting of GITR, via high-valency agonists, as a potential therapeutic approach for the treatment of cancer.Figure 1.


MEDI1873, a potent, stabilized hexameric agonist of human GITR with regulatory T-cell targeting potential
MEDI1873 is a homogenous hexameric GITRL fusion protein with potent agonistic activity. (A) Schematic of the domain structure of the GITRL FP, consisting of IgG Fc domain, trimerization domain and extracellular domain (ECD) of human GITRL. (B) Percentage-specific binding of GITRL to GITR in an HTRF-based assay in the presence and absence of GITRL FP variants. Error bars represent the standard error of the mean (SEM) from duplicates. (C) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of GITRL FP variants. Error bars represent the SEM from triplicates. (D and E) Combined, deconvoluted mass spectra for tryptic peptide (T42–43) which contains the N-glycosylation consensus sequence at position 161 of hGITRL for hGITRL(wt) FP (coronin 1A) (D) and hGITRL(N161D) FP (coronin 1A) (E). (F) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of hGITRL(wt) FP (coronin 1A) and hGITRL(N161D) FP (coronin 1A). Error bars represent SEM from triplicates. (G) Distribution of sedimentation coefficients (measured in Svedberg) for MEDI1873 following analytical ultracentrifugation analysis.
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f0001: MEDI1873 is a homogenous hexameric GITRL fusion protein with potent agonistic activity. (A) Schematic of the domain structure of the GITRL FP, consisting of IgG Fc domain, trimerization domain and extracellular domain (ECD) of human GITRL. (B) Percentage-specific binding of GITRL to GITR in an HTRF-based assay in the presence and absence of GITRL FP variants. Error bars represent the standard error of the mean (SEM) from duplicates. (C) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of GITRL FP variants. Error bars represent the SEM from triplicates. (D and E) Combined, deconvoluted mass spectra for tryptic peptide (T42–43) which contains the N-glycosylation consensus sequence at position 161 of hGITRL for hGITRL(wt) FP (coronin 1A) (D) and hGITRL(N161D) FP (coronin 1A) (E). (F) Luminescence signal downstream of an NFκB-luciferase reporter gene in hGITR-transfected Jurkat cells following addition of hGITRL(wt) FP (coronin 1A) and hGITRL(N161D) FP (coronin 1A). Error bars represent SEM from triplicates. (G) Distribution of sedimentation coefficients (measured in Svedberg) for MEDI1873 following analytical ultracentrifugation analysis.
Mentions: Here, we describe the generation and characterization of MEDI1873, a hexameric GITRL fusion protein (GITRL FP) designed on the principles of trimer stabilization, optimal clustering of GITR and Fc receptor engagement. Each MEDI1873 monomer comprises three distinct domains (Fig. 1A):1 a human immunoglobulin G1 (IgG1) fragment crystallizable (Fc) domain,2 an isoleucine zipper trimerization domain from human coronin 1A,3 and the hGITRL extracellular domain (ECD), with a point mutation (N161D) that eliminates the sole occupied glycosylation site. We demonstrate that MEDI1873 can be purified to homogeneity and is a significantly more potent agonist of hGITR than a monoclonal antibody. We show in human primary lymphocyte assays, in vitro, that this agonism results in enhanced T-cell activation and a reduction in regulatory T-cell suppression and that MEDI1873 is capable of mediating ADCC against activated T cells, resulting in an increased CD8+:CD4+ T-cell ratio. Further, we demonstrate that in non-human primates MEDI1873 increases T-cell activation and response to immunization in vivo. Finally, we detail flow cytometric and histological analyses of patient tumor samples that indicate the presence of GITR positive and FoxP3 positive tumor-infiltrating lymphocytes (TILs). Overall these data confirm the translation of previous mouse GITR biology to a human setting and support the targeting of GITR, via high-valency agonists, as a potential therapeutic approach for the treatment of cancer.Figure 1.

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