Conformational analysis of MUC1 derivatives: Finding the key elements required for an optimal interaction antigen-antibodyMucin MUC1 is an O-glycoprotein strongly overexpressed in various tumors. In healthy tissues, the MUC1 backbone is decorated with complex oligosaccharides, while in cancer cells it shows simple and truncated carbohydrates. As a result, different tumor-associated carbohydrate antigens (TACAs), such as the Tn determinant (α-O-GalNAc-Ser/Thr), are exposed to the immune system and can be recognized by different antibodies. Due to this unique characteristic, MUC1 derivatives are attracting great interest as a potential tool in developing therapeutic vaccines for the treatment of cancer. Nevertheless, the structural bases for the design of these vaccines remain unclear. From the view of molecular recognition, the sequence Ala-Pro-Asp-Thr-Arg-Pro (PDTRP) comprises the minimal epitope for most of anti-MUC1 antibodies. It is observed that the glycosylation of the threonine residue of this epitope with GalNAc enhances the binding affinity towards the antibodies, resulting in a stronger immune response. Therefore, we consider of pivotal importance to determinate the key elements responsible for the molecular recognition between MUC1-like glycopeptides and anti-MUC1 antibodies. Our group has dedicated a considerable effort to understand the conformational behavior of MUC1 derivatives in both the free state and bound to biological targets. We have found differences between the α-O-GalNAc-Ser and the α-O-GalNAcThr antigens, with implications for the molecular recognition processes in which these compounds are involved. In this regard, we have recently reported the X-ray structures of short glycopeptides bound to the anti-MUC1 antibody SM3. The analysis of these structures reveals that for glycopeptides bearing the α-O-GalNAcThr antigen, the sugar moiety interacts with the antibody through a hydrogen bond between its hydroxymethyl group and a tyrosine of the protein and establishing a hydrophobic contact through the N-acetyl group. In turn, the methyl group of the threonine residue is engaged in a hydrophobic contact and forces the sugar unit to be exposed to the solvent, a particular orientation that is not possible for the analogue derivative with serine.
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Design of cancer vaccines
Tumor-associated carbohydrate antigens (TACAs), such as Tn antigen, are crucial components of cancer vaccines. Although vaccines based on native TACAs show immunogenicity and protection in preclinical animal studies, their weak immunogenicity, low stability and poor bioavailability have discouraged their further evaluations in clinical studies.
To overcome these issues, we postulate that TACA-based vaccines displaying mimics instead of native Tn antigens can be more resistant to enzymatic degradation, which might translate into increased in vivo bioavailability and stronger immune response. Moreover, we consider that the mode of presentation of the Tn antigens is also a determinant factor. In this regard, we have recently reported that engineered MUC1-based vaccines having unnatural amino acids have to be capable to emulate the conformational and dynamics properties of the glycosidic linkage GalNAc-threonine present in the natural Tn antigen.
Bearing these ideas in mind, we are now focus on developing novel vaccines based on the use of unnatural antigens presented on the surface of nanoparticles loaded with different immunostimulants.
To overcome these issues, we postulate that TACA-based vaccines displaying mimics instead of native Tn antigens can be more resistant to enzymatic degradation, which might translate into increased in vivo bioavailability and stronger immune response. Moreover, we consider that the mode of presentation of the Tn antigens is also a determinant factor. In this regard, we have recently reported that engineered MUC1-based vaccines having unnatural amino acids have to be capable to emulate the conformational and dynamics properties of the glycosidic linkage GalNAc-threonine present in the natural Tn antigen.
Bearing these ideas in mind, we are now focus on developing novel vaccines based on the use of unnatural antigens presented on the surface of nanoparticles loaded with different immunostimulants.
Anti-MUC1 antibodies conjugates as potential biosensors for detecting tumors in vivo
Taking into account the data inferred from the X-ray structures, we propose to develop a new generation of anti- MUC1 antibodies (by using site-directed mutagenesis) that present higher affinity as well as high selectivity for MUC1 glycopeptides found in tumors. The designed antibodies will be able to recognize not only the peptide epitope but also the carbohydrate moiety. For each designed antibody, we will choose three or four accessible residues to be replaced by a cysteine. The extensive molecular dynamics (MD) simulations will help us to select the most accessible amino acids. Next, we will perform site-selective chemical incorporation of suitable near-infra red (NIR) dyes at cysteine using cutting edge methodology developed in the Bernardes lab. The accessible free cysteine present on the new anti-MUC1 antibodies will be chemically converted to dehydroalanine (Dha) which will then serve as a tag for thio- or aza-Michael addition conjugation . The resulting fluorescently labelled antibodies will be tested in vitro and in vivo for their ability to bind and accumulate at tumor sites. This project has the potential to generate a new class of antibodies to be used in drug delivery to tumor sites.
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