The first task of the project was dedicated to the development of novel antibody binding modules (ABMs). For this purpose, we first synthesized functionalized carbohydrates, peptides and other building blocks) that were combined in different manner using orthogonal ligations strategies) to afford a variety of glyco-clusters and dendrimers displaying identical or different sugars. Before screening with human serum, we systematically investigated all glycodendrimers with soluble lectins, enzymes or antibodies which are easier to manipulate. In addition, to avoid time consuming experiments with large quantities of ligands, we developed a microarray-based assay to identify optimal ligands after immobilization using a microspotter. Our approach was first validated with the Helix pomatia agglutinin for which nanomolar ligands have been. The same approach was followed with human serum from healthy patients. We thus identified a series of glycosylated structures as ABM with promising binding ability towards IgG and IgM present in serum of different donors. In parallel, we developed orthogonal processes to synthesize heterovalent glycodendrimers with the perspective to combine several ABMs and TBMs within the same heterotopic molecule.
With the second task, the objective was to identify ligands (TBM) for receptors or biomarkers overexpressed at the surface of tumors. We first used phage display, a new methodology in my group, to discover peptide ligands against several cancer cell lines. The selection process, including DNA sequencing, was highly time consuming but we identified and synthesized a few peptides that have been multimerized onto cyclopeptide scaffold. Because the evaluation of such multimeric compounds is not trivial using standard physicochemical methods, we demonstrated with a model system (GalNAc/HPA lectin) that BioLayer Inteferometry (BLI) could be a reliable method to reach this challenge. Moreover, because cancer cells express specific carbohydrate antigens, we hypothesized that lectins could represent ideal TBM. To this aim, we demonstrated with several groups In Grenoble that recombinant fragments of lectins can be conjugated as a multimer to a peptide scaffold using an enzymatic ligation strategy. The resulting supramolecular construct represents a highly promising and original object to be combined with ABM. Finally, we studied in parallel the well-known cRGD (specific for the αvβ3 integrins) as TBMs. Recognition potency with diverse cancer cell lines was confirmed by ELISA, flow cytometry analysis and confocal microscopy for a tetravalent cRGD derivative.
In the last task of the project, we synthesized a library of ARMs composed of a variety of combinations of ABMs and TBMs. After having demonstrated the recognition potency of ABM and TBM with serum antibodies and cancer cells, it was essential to confirm that ARM can bind antibodies and cells simultaneously. The formation of such ternary complex is indeed an essential step to simulate a selective immune-mediated cytotoxicity. All compounds were tested by flow cytometry and confocal microscopy with human serum when clear binding was evidenced with commercial antibodies. We thus demonstrated that multivalency is an essential requirement to form this complex and we identified a first lead compound. This study was published as the proof of concept of this project in Chem. Eur. J., 2019, 25, 15508. More importantly, we later demonstrated that ABM geometry significantly influences the ternary complex formation. In addition, we showed that ARMs induce high level of immune-mediated cytotoxicity in the presence of human serum as the unique source of immunity effectors and without pre-immunization (Biomater. Sci., 2021, DOI: 10.1039/D1BM00485A).
