Soutenance de thèse : Design of innovative tools for the NMR studies of glycosylated therapeutic antibodies at atomic resolution

Par Béatrice Vibert (IBS/Groupe RMN des grands assemblages & Sanofi)

Thanks to their high specificity and the few associated side effects, monoclonal antibodies are excellent candidates for the treatment of numerous diseases including cancers, infectious diseases, and neurodegenerative disorders. This class of biopharmaceuticals has expanded considerably in recent years and now represents an increasingly important proportion of approved therapeutic molecules. Monoclonal antibodies must undergo extensive quality controls throughout their development before going on the market, and new techniques are required to reliably and precisely characterize these emerging biopharmaceuticals. In particular, knowing the three-dimensional structure of these proteins is essential to ensure their efficacy and safety. The structure of therapeutic antibodies can be affected at different development stages by changes in the production protocol, the formulation buffer, or the storage conditions. It is also critical to detect any structural modification between two production batches. However, the methods routinely used today in the pharmaceutical industry do not provide detailed information about the structure of antibodies in solution. Nuclear magnetic resonance (NMR) spectroscopy is a technique capable of delivering structural information at atomic resolution. Although biomolecular NMR has long been limited to the study of low-molecular-weight proteins, methyl-based NMR has recently emerged as a powerful tool for monitoring the structure of therapeutic antibodies under formulation conditions. Nevertheless, significant methodological developments are still required to make NMR a quality-control tool capable of translating each observed spectral change into the localisation of impacted residues.

The work presented in this thesis manuscript focuses on developing innovative tools to precisely characterize therapeutic antibodies in solution using NMR. To simplify spectrum analysis and mprove their quality, the crystallizable fragment (Fc) and the antigen-binding fragment (Fab) were studied separately. A first essential step in analysing these fragments by NMR is the assignment of signals to their corresponding amino acids. By combining cell-free expression of antibody fragments enriched in ²H, ¹³C et ¹⁵N with high-quality NMR spectra acquisition, nearly all signals were assigned. The main structural difference between fragments produced in a cell-free system and therapeutic antibodies produced in mammalian cells is the presence of glycans at asparagine 297 of the Fc fragment, which induces changes in biological activity. Faced with the impossibility of implementing a technique of enzymatic glycosylation of the Fc fragment produced in a cell-free system, isotopically enriched antibodies produced in eukaryotic cells were employed. Thus, the spectrum of a glycosylated Fc was assigned by comparison with that of the non-glycosylated Fc. By combining these results with the work performed on the Fab fragment of the anti-LAMP1 antibody, the methyl groups resonances of the full antibody could be assigned. A method was also developped to accelerate methyl resonance assignment of IgG1 Fab fragments. This work therefore transformed methyl-based NMR into a tool capable of locating any structural modification of an antibody by overlaying simple 2D spectra acquired under formulation conditions. This tool made it possible to characterize the structural impact of glycosylation, of methionine oxidation in the Fc, of Fab interaction with its antigen, as well as the dynamics of a full antibody and its fragments.

Mots clés : Anticorps monoclonaux, RMN, Biologie structurale, Marquage isotopique, Fragment cristallisable``