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.

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

doi:10.1021/jacs.8b13503

Abstract

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.

Highlights

MUC1 mucin is an O-glycoprotein overexpressed in many tumor tissues.[1,2,3,4] in healthy cells the backbone of this protein is decorated with complex glycans, in cancer cells this backbone carries rather simple and truncated oligosaccharides
(C) Geometry and flexibility at the glycosidic linkage and peptide backbone for the unnatural synthesis of the amino acid thiothreonine (SThr) residue of glycopeptide 2* in solution derived from 20 ns experimentguided molecular dynamics (MD) simulations
The yellow circles correspond to the conformation found in the crystal structure of glycopeptide 1* bound to a single-chain variable fragment of the SM3 antibody. (D) Structural ensembles derived from 20 ns experiment-guided MD simulations for compounds 1*, 2* and 3* in solution, together with the conformation of the peptide backbone of 1* found by X-ray crystallography bound to scFv-SM3 antibody (PDB ID: 5A2K). (E) Circular dichroism (CD) spectra of compounds 1* and 2* (0.25 mM in sodium phosphate buffer, pH = 7.5, 20 oC)

Summary

Introduction

MUC1 mucin is an O-glycoprotein overexpressed in many tumor tissues.[1,2,3,4] in healthy cells the backbone of this protein is decorated with complex glycans, in cancer cells this backbone carries rather simple and truncated oligosaccharides. Compounds 2* and 3* showed conformations characterized by a folded structure around the unnatural residues SThr[4] or SeThr[4], respectively (Figures 2, S4 and S5).

Results

Conclusion