Pioneering of Antibody-Assisted Structure-Based Drug Discovery for PD-L1

The principle of antibody-assisted drug discovery aims to use a diverse panel of single domain antibodies (VHH) to identify rare conformational states and/or novel allosteric sites that could modulate function, which may facilitate the discovery of new therapeutics. Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment, however, limitations remain in patient response and off-target toxicity. The work reported in this thesis aimed to use antibody-assisted drug discovery applied to PD-L1 using heavy chain only antibodies (VHH).

A diverse panel of VHHs specific against either the functional (D1) or membrane proximal (D2) domain of PD-L1 were identified and characterised. The panel of VHHs identified in this work demonstrated affinities of 0.7 nM to 8.3 μM, with all anti-PD-L1 D1 VHHs being completely inhibitory. The binding site for the VHHs on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1 binding site for the anti-PD-L1 D1 VHHs. Crystal structures of two representative VHHs in complex with PD-L1 revealed unique binding modes. In contrast, the anti-PD-L1 D2 VHHs demonstrated a range of binding sites. In addition, the binding site of a second functional partner of PD-L1, CD80 was determined using NMR chemical shift perturbation mapping experiments and revealed that CD80 and PD-1 shared a highly overlapping binding site on PD-L1. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, with potential therapeutic benefits.