| Resumo: | In this thesis, unnatural MUC1 glycopeptides were developed according to a structure-guided design by utilizing already reported data to enhance their affinity in vitro toward well-characterized antibodies by implementing synthetic Tn surrogates at key positions in the amino acid sequence. The main objectives of this thesis are to exploit the biological properties of MUC1 to develop novel cancer vaccine candidates and cancer diagnostic tools. In this regard, although MUC1 glycopeptides hold great promise as potential cancer vaccine candidates, MUC1-based vaccines usually result in a weak immune response since the antigen is a self-antigen. To tackle this problem, we have carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate a robust immune response in mice, artificial antigens must mimic the structure of the natural antigen in solution and have an equivalent or higher binding affinity towards anti-MUC1 antibodies. As a proof of concept, we have developed a glycopeptide that contains a noncanonical amino acid (2S,3R)- 3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. Despite an enthalpy-entropy balance, this synthetic glycopeptide had a slightly higher binding affinity than its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulated the formation of specific anti-MUC1 IgG antibodies in mice and showed efficacy comparable to that of the natural derivative. The antibodies also exhibited cross-reactivity selectively targeting human breast cancer cells. These results underscore the promise of structure-based rational design to advance cancer vaccine development. Furthermore, we developed a novel vaccine candidate utilizing an unnatural MUC1 glycopeptide and a protein carrier. This glycopeptide contains a (4S)-4-fluoro-L-proline and an α-S-GalNAc-Thr at key positions in the amino acid sequence and is specifically conjugated to the immunogenic protein carrier CRM197. The conjugation method involves the selective reduction and re-bridging of one of the disulfide bridges in CRM197, allowing the attachment of a single copy of the non-natural MUC1 glycopeptide. This strategy provides a chemically defined and homogeneous vaccine while maintaining the structural integrity of the carrier protein as well as its immunogenicity. The resulting vaccine elicits a robust Th1-like immune response in mice and generates antibodies capable of recognizing human cancer cells expressing tumor-associated MUC1 on their surface. When tested in mouse models of colon adenocarcinoma and pancreatic cancer, the vaccine is effective both prophylactically and therapeutically, significantly delaying tumor growth. In therapeutic applications, improved outcomes are observed when the vaccine was combined with an anti-PD1 checkpoint inhibitor. In parallel, we also have attempted to develop diagnostic assays based on unnatural MUC1 glycopeptides. For that reason, we modified the acetamide of the GalNAc moiety in unnatural Tn surrogates to enhance their binding affinity to the 5E5 antibody using a structure-guided design These Tn mimics were placed in short peptides showcasing the 5E5 epitope and were tested in terms of binding affinity against the respective antibody, exhibiting showing significantly enhanced binding affinity. Then, the best peptide candidates were modified accordingly and were implemented in two state-of-the-art diagnostic assays for detecting cancer. Even though the experiments are ongoing, and only limited data is available, the first results seem promising.
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