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Title: Second generation superagonistic anti-PD-1 antibodies
Author: Yu, Susan Yangshan
ISNI:       0000 0004 8508 3035
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Antibodies known as ‘superagonists’, such as those raised against CD28, induce high levels of signalling activity. Based on the kinetic-segregation model, a hypothesis arose: the greater potency of superagonists arises from their ability to more effectively exclude large phosphatases from the vicinity of CD28, favouring its phosphorylation. This hypothesis can be further extrapolated so that any immobilized antibody binding to a membrane-proximal epitope on a receptor with tyrosine phosphorylation motifs should induce signalling by that receptor. Previous studies indicated that antibody epitope positions are related to their signalling induction abilities. Earlier findings demonstrated that strong inhibitory signalling could also be induced by 1st generation anti-PD-1 antibodies that bind close to the membrane with high affinity, or away from the membrane with very low affinity. We have combined the apparently advantageous features of both sets of antibodies into a single 2nd generation anti-PD-1 antibody using a mutagenesis approach and subsequently tested their binding to PD-1 via surface plasmon resonance (SPR). We generated a new model of the superagonistic complex from our mutational SPR data and previous mutational data on PD-1. This model confirmed that it is indeed the position of the epitope, not the chemistry of binding, that dictates whether an antibody is an effective agonist and a blocker of TCR signalling or not. We then devised different in vitro assay formats, plate-bound and in solution, to investigate the effects of these anti-PD-1 antibodies on cytokine generation and cell proliferation. By adding higher levels of anti-PD-1 antibodies, greater IL-2 suppression was observed, regardless of the antibody presentation method. Synergistic inhibitory effects were further observed with combinations of these antibodies. Similarly, we studied the inhibitory effects of our antibodies on germinal centre B cell formation, primary antibody response, antibody affinity maturation and CD8+ T cell response in vivo. Our in vivo assays showed that a higher concentration of anti-PD-1 antibody application led to larger inhibitory effects, therefore dose optimisation will also be key for maximisation of antibody inhibitory potential. Compared to the 1st generation antibodies, the 2nd generation antibodies displayed reduced PD-1 cell surface downregulation in vivo. Our results provided evidence that PD-1 downregulation can be uncoupled from receptor triggering by altering affinity. This could potentially help deliver sustained inhibitory PD-1 signalling. The experiments demonstrated in this thesis provide a foundation for generating a novel class of potentially therapeutic antibodies, the inhibitory superagonists.
Supervisor: Davis, Simon Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Immunology