Periodic Reporting for period 4 - LEGO (Multimodal glycoconjugates: a molecular Lego approach for antitumoral immunotherapy)
Período documentado: 2020-03-01 hasta 2021-02-28
Cancer remains a major cause of mortality worldwide. Despite significant progress in treatment modalities, current therapeutic regimens are still deficient due to intolerable side effects, while stray cancer cells often escape destruction. Immunotherapy-based approaches have been proposed recently and certainly represent the most promising alternatives in this area although very limited approaches are available and major issues remain to be addressed. We have demonstrated in this interdisciplinary program that fully synthetic structures with unprecedented combinations and complexity can immunological properties against cancers. We have developed a “molecular LEGO” approach to construct synthetic molecules capable of redirecting endogenous antibodies present in the human bloodstream against tumors to trigger immune-mediated clearance. By using supramolecular chemistry, molecular engineering, biochemistry, immunochemistry and glycoscience approaches, we have synthesized unprecedented synthetic molecular constructions, namely Antibody Recruiting Glycodendrimers (ARGs) embedded with key parameters that: 1/ recruit natural Abs through clusters of oligosaccharides (antibody binding module) with high affinity by means of multivalent interactions; 2/ target cancer related receptors selectively expressed at the surface of tumors with peptide tumor binding module (TBMs); 3/ promotes the formation of a ternary complex between natural Abs and cancer cells; 4/ promotes up to 70% of cytotoxicity towards cancer cells with human serum as unique source of immune activators.
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).
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).
The aim of the project is to propose an innovative immunochemical approach to fight cancer. By itself, the entire project is unconventional since cancer immunotherapy is dedicated in a large majority to biotechnological approaches. In the LEGO project, we developed a variety of fully synthetic supramolecular constructs and identified a few of them having the ability to redirect endogenous antibodies present in the human bloodstream against tumors. Given that rational design of high affinity multivalent ligands is impossible with carbohydrate binding proteins, we first developed a novel methodology based on glycodendrimer-arrays to identify ligands for a series of compounds varying in their shape, geometry and sugar density, thus allowing the controlled presentation of sugars in a spatially defined arrangement. This approach led to the identification of potent binding modules for endogenous antibodies present in the blood stream of different donors, which were further used in the ARM construction. Also challenging, the utilization of carbohydrate binding domains of lectin has been explored for the first time in a collaborative project. We prepared the first semi-synthetic neolectins by using enzyme-mediated ligation. This supramolecular object is highly innovative and promising in the ARM research field.