Investigating the therapeutic potential of anti-GITR antibodies

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Re-activating anti-tumour immune responses, and avoiding unwanted autoimmunity, is a primary goal of anti-cancer immunotherapies. Antibodies that block checkpoint inhibitors, to prevent immune inhibition, have been clinically successful. Glucocorticoid-Induced TNFR-Related (GITR) is a cell surface co-stimulatory receptor that belongs to the same tumour necrosis factor (TNFR) superfamily as OX40 and 4-1BB. Constitutive expression of GITR is high on T regulatory cells (T-regs), but low on both naïve and memory effector T cells (T-effs). T-regs are an immune-suppressive cell type that, in multiple cancers, have been implicated in preventing the activation of anti-tumour responses (3). Because of high GITR expression on T-regs, depletion of these cells with therapeutic antibodies has been proposed as an anti-tumour therapy. Activated T-effs dramatically increase GITR surface expression. The stimulation of the GITR pathway increased T-eff proliferation and the expression of pro-inflammatory cytokines like IL-2 and IFNy. Furthermore, tumours with higher levels of GITRL expression had increased numbers of infiltrating CD4+ and CD8+ T cells and delayed growth. Considering the role GITR plays in modulating immune responses, antibodies directed against this surface protein are currently being investigated as anti-tumour immuno-therapeutics.The University of Southampton antibody and vaccine group (AVG) have developed a panel of anti-human GITR (anti-huGITR) antibodies. In this project, we will characterise the function of anti-huGITR antibodies in vitro and in vivo. Research question, aims and objectives Understand how anti-huGITR antibodies influence immune responses using a human GITR knock-in transgenic mouse model (huGITRKI) that expresses human GITR surface protein.Aim 1. Characterise human GITR protein expression and investigate how different anti-huGITR antibodies alter immune responses in vitro. Determine the kinetics of binding affinity as well as the binding epitopes of the anti-GITR antibodies. I. To determine the kinetics of GITR expression, human PBMCs and splenocytes from huGITRKI transgenic mice will be activated in vitro with various stimuli. Changes in surface protein are then measured using flow cytometry. II. Measure cellular proliferation of CD4+ and CD8+ T cells post activation and treatment with each anti-GITR antibody. III. Determine how GITR antibodies influence cell signalling. IV. Determine the kinetics and binding affinity of anti-GITR antibodies using surface plasma resonance.V. Determine the optimal binding epitope of each anti-GITR antibody.