Synthetically-defined glycoconjugate vaccine candidates against Influenza

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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

Sequential dual site-selective protein labelling enabled by lysine modification

Publication . Matos, Maria J.; Brown, Libby; Bernardim, Barbara; Guerreiro, Ana; Jiménez-Osés, Gonzalo; Bernardes, Gonçalo J. L.

Methods that allow for chemical site-selective dual protein modification are scarce. Here, we provide proof-of-concept for the orthogonality and compatibility of a method for regioselective lysine modification with strategies for protein modification at cysteine and genetically encoded ketone-tagged amino acids. This sequential, orthogonal approach was applied to albumin and a therapeutic antibody to create functional dual site-selectively labelled proteins.

2020Journal article

Restricted access

Quaternization of Vinyl/Alkynyl Pyridine enables ultrafast cysteine‐selective protein modification and charge modulation

Publication . Matos, Maria J.; Navo, Claudio D.; Hakala, Tuuli; Ferhati, Xhenti; Guerreiro, Ana; Hartmann, David; Bernardim, Barbara; Saar, Kadi L.; Compañón, Ismael; Corzana, Francisco; Knowles, Tuomas P. J.; Jiménez‐Osés, Gonzalo; Bernardes, Gonçalo J. L.

Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody-drug conjugate that features a precise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl- and alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.

2019Journal article

Open access

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment

Publication . Bermejo, Iris A.; Navo, Claudio D.; Castro-López, Jorge; Guerreiro, Ana; Jiménez-Moreno, Ester; Sánchez Fernández, Elena M.; García-Martín, Fayna; Hinou, Hiroshi; Nishimura, Shin-Ichiro; García Fernández, José M.; Mellet, Carmen Ortiz; Avenoza, Alberto; Busto, Jesús H.; Bernardes, Gonçalo J. L.; Hurtado-Guerrero, Ramón; Peregrina, Jesús M.; Corzana, Francisco

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

2020Journal article

Open access