Glucocorticoid-induced TNFR-related protein (GITR) is a member of the tumor necrosis factor receptor (TNFR) superfamily. GITR is expressed constitutively on regulatory T cells (Tregs) and is up-regulated on other T cells following activation. Agonistic antibodies to GITR have demonstrated significant activity in preclinical models of cancer. Here we describe the generation and characterisation of a GITR ligand (GITRL) fusion protein (FP) (MEDI1873), currently in phase 1 clinical trials.
Protein engineering was used to generate a series of GITRL FPs, which were screened using a high throughput reporter gene assay for GITR signalling. The most potent fusion protein resulted in a 20 times greater maximal signal and a 5 times higher EC50 when compared to a GITR targeting antibody. This increased potency was considered to be a result of the enhanced valency achieved by the hexameric format.
Two versions of GITRL FP, MEDI1873 and MEDI5607, bearing an IgG1 and IgG4 Fc respectively, both demonstrated equivalent potency in a reporter assay and were able to enhance T-cell activation, with respect to proliferation and cytokine release, and to overcome the suppressive effect of Tregs, in primary human cell based assays.
Assessment of two surrogate mouse GITRL FPs in the CT26 model of colorectal cancer indicated that the version with increased binding to Fc gamma receptors resulted in increased activity, coincident with an increased depletion of intratumoral Tregs, likely through Fc mediated effector functions.
A comparison of GITR expression on Tregs and effector T cells in mouse and human, via flow cytometry, indicated a similar pattern of expression across species, with significantly higher expression observed on Tregs. Immunohistochemical analysis indicated the presence of high levels of both GITR and FoxP3 in sections from human tumors; suggesting that the intratumoral Treg depletion observed in mice could also occur in humans.
Both MEDI1873 and MEDI5607 demonstrated enhanced binding to Fc gamma receptors when compared to antibody controls of the same isotype, again considered to be a result of their increased valency. However, only MEDI1873 was able to mediate ADCC against activated T cells in vitro; resulting in an increase in the CD8:CD4 ratio within the culture.
As a result of these studies, MEDI1873 was selected as an optimal GITR targeting agent that possessed the ability to both agonise GITR and to modulate Tregs through suppression and/or depletion. MEDI1873 is currently being assessed in a phase 1 clinical study (NCT02583165) in patients with solid tumors.
Citation Format: Ross A. Stewart, Natalie Tigue, Samantha Ireland, James Hair, Lisa Bamber, Michael Oberst, Rebecca Leyland, Amanda Watkins, Maureen Kennedy, Cann Jennifer, Lesley Young, Robert W. Wilkinson. MEDI1873: A novel hexameric GITRL fusion protein with potent agonsitic and immunomodulatory activities in preclinical systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 561.