Accueil UNamur > Agenda > Défense de thèse de doctorat en Sciences chimiques - Léa CHARTIER

Catégorie : défense de thèse
Date : 01/07/2022 09:00 - 01/07/2022 12:00
Lieu : CH11
Orateur(s) : Léa CHARTIER
Organisateur(s) : Stéphane VINCENT

Antibodies (Abs) can be usually functionalised with molecular probes, drugs or fluorophores by stochastic conjugation protocols involving cysteine or lysine residues naturally present on the polypepetide framework. These modifications give novel properties such as functions or reactivity useful for diagnostic or therapeutic purposes. Nevertheless, these stochastic protocols lead to antibody batches that are heterogeneous in terms of number and spacial distribution of the payloads. Moreover, the resulting Abs are known to have lower activity compare to their unmodified counterparts. Indeed, the stochastic functionalisation can occur on the variable regions, which are located in the functional site responsible for the antigen recognition. This is why it is interesting to use site-selective antibody modifications, which are known to retain high structural homogeneity, biological activity, and a control of the degree of labelling. Abs functionalised by these approaches are ideal tools for diagnostic and therapeutic uses. Among the most widely used sites for site-selective antibody modification, the N-glycan moiety on the Fc region is attracting increasing interest.

In this contest, this thesis proposes a new protocol for the modification of antibody N-glycans, which aims to be as universal as possible (e.g applicable to a broad range of mammalian Abs). To that end, the glycosylation profile of many Abs from different species was analysed using complementary techniques including HRMS. Both F(6)M3 and F(6)A2 common glycoforms were found aboundant and selected for further functionalisation. The F(6)M3 glycan was obtained by the action of several hydrolases and the reaction was optimized to a yield of over 80 %. The modified N-acetylglucosamine (GlcNAc) with an azide group (GlcNAz) was added to both glycoforms thanks to glycosyltransferases MGAT1 and MGAT3, respectively. The reaction was particularly optimized on F(6)A2 to obtained the F(6)A2BGlcNAz bisecting glycan with 82 % yield. Methods were developed to functionalise and purify this Ab, and its activity was quantified through the determination of the ImmunoReactive Fraction (IRF). These values were compared with Abs modified by commercial kits allowing site-selective N-glycan biotinylation and with Abs with a payload (juorophore, biotin) added in a stochastic manner. The results of these experiments show that the proposed glycan modification induces IRF higher than the ones of stochastically modified Abs, and comparable to that of the unmodified Abs. Furthermore, the procedure has a yield and an easiness of implementation comparable to the procedure required by the commercial kits. These results are very encouraging for future development of this technology and the related diagnostic and therapeutic applications.