Investigating the therapeutic potential of monoclonal antibodies targeting human GITR

Abstract

The goal of this project was to investigate the immunomodulatory and anti-tumour activity of a
series of novel monoclonal antibodies (mAbs) targeting human Glucocorticoid-induced TNFRrelated protein (GITR). GITR is a cell surface co-stimulatory receptor constitutively expressed at high
levels on T regulatory cells (Tregs) and at low levels on naïve and memory T cells. Its activation
results in the increased survival, proliferation and function of effector T cells as well as inhibiting
the suppressive capacity of Tregs. GITR is therefore an attractive target for anti-tumour
immunotherapies. To explore this, a panel of novel anti-human GITR mAbs were generated and
investigated. To characterise these mAbs, both in-vitro and in-vivo methodologies were utilised.
Surface plasmon resonance identified the binding affinities and binding domains of different
antibody clones. Human GITR (hGITR) expressing Jurkat/NF-κB/GFP reporter cells were generated
to understand how these mAbs influenced intracellular signalling pathways. Furthermore, CFSElabelled human peripheral blood mononuclear cells (PBMCs) were co-cultured with sub-optimal
concentrations of anti-hCD3, alongside anti-hGITR mAbs, to determine the ability of anti-hGITR
mAbs to modulate human T cell proliferation in-vitro. To investigate these mAbs in-vivo, a novel
hGITR knock-in (hGITRKI) transgenic mouse was developed and characterised, followed by studies
in mouse tumour models.
The generated mAbs bound to either hGITR domain 1 or 2 with a range of affinities, with the
majority increasing intracellular NF-κB. Two of the mAb clones reduced the proliferation of hCD3-
stimulated T cells, demonstrating their ability to influence cellular function in human T cells in-vitro.
However, this reduction was only observed when these clones were of the mIgG2a isotype, which
suggested a potential mechanism of action through the mAb-mediated depletion of hGITR
expressing T cells. In-vivo impacts were explored in newly generated hGITRKI mice. Insertion of the
chimeric hGITR gene into exon 1 of the mGITR gene resulted in the simultaneous expression of cell
surface hGITR as well as disruption of mGITR expression. Furthermore, hGITRKI mice displayed a
hGITR expression pattern similar to that of human PBMCs. These mice were then used to determine
the therapeutic efficacy of anti-hGITR mAbs in-vivo. One of the anti-hGITR mAbs (84-9 mIgG2a) was
shown to reduce tumour size and increase survival in EG7-OVA tumour-bearing hGITRKI mice. This
therapeutic benefit was dependent on antibody isotype. A potential mechanism of action was that
84-9, in the mIgG2a format, depleted intratumoural Tregs which altered the ratio of effector to
regulatory T cells within the tumour microenvironment. Future work will seek to further define
mechanisms of action and the underlying properties that mediate its therapeutic activity in
comparison to other mAbs directed to hGITR or other immune stimulatory receptors.

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Identifiers

ORCID for Shaun Maguire: orcid.org/0000-0001-9221-4601

ORCID for Mark Cragg: orcid.org/0000-0003-2077-089X

ORCID for Jane Willoughby: orcid.org/0000-0002-6326-4519

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