A training network for the chemical site-selective modification of proteins: Preparation of the next-generation of therapeutic chemically-defined protein conjugates

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Single mutation on Trastuzumab modulates the stability of antibody–drug conjugates built using acetal-based linkers and thiol-maleimide chemistry

Publication . Ferhati, Xhenti; Jiménez-Moreno, Ester; Hoyt, Emily A.; Salluce, Giulia; Cabeza-Cabrerizo, Mar; Navo, Claudio D.; Compañón, Ismael; Akkapeddi, Padma; Matos, Maria J.; Salaverri, Noelia; Garrido, Pablo; Martínez, Alfredo; Laserna, Víctor; Murray, Thomas V.; Jiménez-Osés, Gonzalo; Ravn, Peter; Bernardes, Gonçalo J. L.; Corzana, Francisco

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

2022Journal article

Open access

Structure-based design of potent tumor-associated antigens: modulation of peptide presentation by single-atom O/S or O/SE substitutions at the glycosidic linkage

Publication . Compañón, Ismael; Guerreiro, Ana; Mangini, Vincenzo; Castro-López, Jorge; Escudero-Casao, Margarita; Avenoza, Alberto; Busto, Jesús H.; Castillón, Sergio; Jiménez-Barbero, Jesús; Asensio, Juan L.; Jiménez-Osés, Gonzalo; Boutureira, Omar; Peregrina, Jesús M.; Hurtado-Guerrero, Ramón; Fiammengo, Roberto; Bernardes, Gonçalo J. L.; Corzana, Francisco

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

2019Journal article

Restricted access