Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody

© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.Peer Reviewe

Novel downstream process and analytical tools developed for Influenza VLP vaccine

Vaccination remains the most effective way to prevent the infection with Influenza viruses. However, their constant antigenic drift implies that current human Influenza vaccines need to be annually updated with high inherent costs. Virus-like particles (VLPs) have become widely used as vaccine candidates because of their versatility, immunogenicity, and safety profile. In this iBET project we are attempting to produce a candidate for a universal vaccine for which 35 different VLPs (mono, trivalent and pentavalent) were purified. Here we describe three recent advances on Influenza VLPs bioprocessing: two new analytical tools and the development of an integrated all filtration purification process, inserted in the “anything but chromatography” concept. The first method is a label-free tool that uses Biolayer interferometry technology applied on an Octet platform to quantify Influenza VLPs at all stages of DSP. Human and avian sialic acid receptors were used, in order to quantify hemagglutinin (HA) content in several mono- and multivalent Influenza VLP strains. The applied method was able to detect and quantify HA from crude sample up to final VLP product with high throughput, real-time results and improved detection limits, when compared to traditional approaches, crucial for in-line monitoring of DSP. Using a click-chemistry approach that involves Azidohomoalanine incorporation and functionalization, Influenza VLPs were selectively and fluorescently tagged. Taking into account that this chemical tag does not affect particle size, charge and biological activity we report here a valuable tool to online/at-line product monitoring during DSP optimization of virus related biopharmaceuticals. Moreover, using this tool coupled with FACS we were able to discriminate between VLPs and baculovirus, the major impurity of the system. The proposed all-filtration process will be described, with special focus on the clarification stage, followed by multiple ultrafiltration and diafiltration steps to achieve the needed concentration and purity specifications. Using this all-filtration platform, we are able to speed up the time process, to improve the scale-up and to reduce costs due to the removal of chromatographic steps

Synthesis and study of a new bioorthogonal self-immolative linker based on the Grob fragmentation reaction

Cleavable linkers are designed to release the drug within or in the vicinity of the tumour cells upon a trigger stimulus. In recent years, there have been multiple reports of self-immolative cleavable linkers able to self-degrade in a spontaneous and irreversible manner through a cascade-elimination process. In general, this process is driven by an entropy increase and the formation of thermodynamically stable products and the control of the drug release is achieved by a stimulus such as an enzymatic cleavage of the linker that activates the self-immolative process. [1]We have designed and synthesized a new self-immolative bioorthogonal conditionally cleavable linker based on the Grob fragmentation that allowed the controlled release of sulfonate-containing compounds such as a dansyl group under physiological conditions. We have also tuned conveniently the pKa of the pushing group (amino group) using different substituents, leading to more efficient conversions at physiological pH and in some cases even at acidic pH, which is normally found in tumour environments. In addition, the Grob fragmentation takes place under physiological conditions in living cells, demonstrating the potential bioorthogonal applicability of the reaction. Based on these promising results, research is currently underway to incorporate this type of linker into antibodydrug conjugates for the targeted delivery of cytotoxic drugs and fluorophores

Tn Antigen Mimics Based on sp2-Iminosugars with Affinity for an anti-MUC1 Antibody

The first examples of amino acid (Ser/Thr)-sp-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp-iminosugar and the peptide aglycone structure (Ser/Thr).Peer Reviewe