Etudes structurales de complexes anticorps-antigène multispécifiques par cryo-microscopie électronique

Cryo-electron microscopy (cryo-EM) has experienced a surge in capability and popularity in recent years. In particular, advances in hardware and software have allowed the imaging of the structures at progressively higher resolutions (<4 Å). This has enabled the exploration of cryo-EM as a tool for elucidating antibody-antigen binding interactions in near-native conditions. This would be an invaluable step forward in drug discovery. In the field of antibody therapeutics, multispecific antibodies, which are engineered to bind to two or more targets, are currently of the highest interest. Unfortunately, there are currently no reports of high resolution cryo-EM studies of whole antibodies, or of antibody-antigen interfaces, within the full-antibody context. Thus, the main goals of this thesis were two-fold: 1) The establishment of a pipeline, from sample preparation to structure, for whole antibody cryo-EM studies, that will serve as a starting point for future projects in the field; 2) The use of cryo-EM to elucidate, at close to atomic level, details of multispecific antibody-antigen interfaces within the full-antibody context. This work targeted three antibodies: CODV (bispecific), TBTI (bispecific) and TDT (trispecific), with a strong focus on CODV and its complexes with the small antigen ligands IL4 and IL13.Initial work (Chapter 1) concentrated on the preparation, purification and biophysical characterization of individual antibodies and of CODV-antigen complexes. Here, SPR studies showed that CODV binds to both of its IL4 and IL13 antigens with picomolar affinity and that this affinity is independent of binding order or positional effects.Preliminary negative-stain electron microscopy (NSEM) experiments (Chapter 2) provided information concerning sample quality and dispersion and allowed the production of low-resolution reconstructions for potential use in cryo-EM analysis. The NSEM experiments suggested a high level of flexibility and conformational heterogeneity of the antibodies, especially at the Fc level. Intriguingly, the reconstructions obtained for CODV-antigen complexes did not show any density corresponding to the antigens.Due to the therapeutic advantages of the format and its priority within the antibody development efforts of Sanofi, the CODV system was then used for cryo-EM pipeline development (Chapter 3). Grid preparation strategies were first explored in order to ameliorate on-grid aggregation of CODV antibodies and their aversion for conventional supports. Cryo-EM datasets for apo-CODV, CODV-IL4 and CODV-IL4/IL13 were then collected at the Titan Krios 300 keV microscope at the ESRF CM01 beamline. Processing of these datasets confirmed both the apparent absence of antigens bound to CODV and the high conformational heterogeneity of the antibody observed in NSEM experiments. The latter meant that only very low resolution, and sometimes incomplete, reconstructions of CODV and its antigen complexes could be obtained.A strategy to reduce the conformational flexibility was therefore proposed. This comprised labelling the IL13 antigen with Tralokinumab, an anti-IL13 antibody (Chapter 3). A ternary CODV-IL13-Tralokinumab complex was therefore prepared and purified. NSEM experiments revealed density for IL13 and cryo-EM experiments based on the pipeline developed was therefore carried out. These resulted in a medium-resolution reconstruction (3.93 Å) of the CODV-IL13-Tralokinumab interface, suggesting that this labelling strategy can be used in other studies. This work therefore paves the way for future cryo-EM studies of antibody-antigen complexes, especially in those where the presence of Fc is key for the correct biological context, such as opsonization or cell degranulation.La microscopie cryoélectronique (cryo-EM) a connu une montée en puissance et en popularité ces dernières années. En effet, les progrès du matériel et des logiciels ont permis la détermination de structures à des résolutions élevées (