Interplay between Affinity and Valency in Effector Cell Degranulation: A Model System with Polcalcin Allergens and Human Patient-Derived IgE Antibodies.

This is the accepted, uncopyedited version of the manuscript. The definitive version was published in The Journal of Immunology August 28, 2019, ji1900509; DOI: https://doi.org/10.4049/jimmunol.1900509Originally published in The Journal of Immunology. Bucaite G, Kang-Pettinger T, Moreira J, et al. Interplay between Affinity and Valency in Effector Cell Degranulation: A Model System with Polcalcin Allergens and Human Patient-Derived IgE Antibodies. J Immunol. 2019;203(7):1693-1700. doi:10.4049/jimmunol.1900509 The American Association of Immunologists, Inc.An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor FcεRI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.This work was supported by Medical Research Council Grant G1100090. G.B. was supported by a studentship from the King’s Bioscience Institute and by the Guy’s and St. Thomas’ Charity Prize Ph.D. Program in Biomedical and Translational Science. We acknowledge the support of the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust (Grant 085944)

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.</p