Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT)... Show moreMost lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions. Show less
Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal... Show moreMannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6PoC-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands. Show less
Mannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal... Show moreMannose-6-phosphate (M6P) is recognized by the mannose-6-phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We here describe the assembly of peptide antigen conjugates carrying clusters of mannose-6-C-phosphonates (M6Po). The M6Po's represent stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relying on a "post-assembly" click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C-glycoside amino acid building block that can be used in solid-phase peptide synthesis. The generated conjugates were further equipped with a TLR7-ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR-ligands. Show less
This thesis describes the development of a variety of mannosylated conjugates. Antigen presenting cells bear mannoside recognizing receptors that actively transport antigen into the cell. This... Show moreThis thesis describes the development of a variety of mannosylated conjugates. Antigen presenting cells bear mannoside recognizing receptors that actively transport antigen into the cell. This thesis exploits this feature for the development of improved vaccines. Show less
Dendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation... Show moreDendritic cells (DCs) are armed with a multitude of Pattern Recognition Receptors (PRRs) to recognize pathogens and initiate pathogen-tailored T cell responses. In these responses, the maturation of DCs is key, as well as the production of cytokines that help to accomplish T cell responses. DC-SIGN is a frequently exploited PRR that can effectively be targeted with mannosylated antigens to enhance the induction of antigen-specific T cells. The natural O-mannosidic linkage is susceptible to enzymatic degradation, and its chemical sensitivity complicates the synthesis of mannosylated antigens. For this reason, (oligo)mannosides are generally introduced in a late stage of the antigen synthesis, requiring orthogonal conjugation handles for their attachment. To increase the stability of the mannosides and streamline the synthesis of mannosylated peptide antigens, we here describe the development of an acid-stable C-mannosyl lysine, which allows for the inline introduction of mannosides during solid-phase peptide synthesis (SPPS). The developed amino acid has been successfully used for the assembly of both small ligands and peptide antigen conjugates comprising an epitope of the gp100 melanoma-associated antigen and a TLR7 agonist for DC activation. The ligands showed similar internalization capacities and binding affinities as the O-mannosyl analogs. Moreover, the antigen conjugates were capable of inducing maturation, stimulating the secretion of pro-inflammatory cytokines, and providing enhanced gp100 presentation to CD8+ and CD4+ T cells, similar to their O-mannosyl counterparts. Our results demonstrate that the C-mannose lysine is a valuable building block for the generation of anticancer peptide-conjugate vaccine modalities.. Show less
Li, R.E.; Hogervorst, T.P.; Achilli, S.; Bruijns, S.C.; Arnoldus, T.; Vivès, C.; ... ; Kooyk, Y. van 2019
Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against... Show moreDendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known “high mannose” structures, that we presented in a systematically increasing number of copies (n = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs. Show less
Hendrikse, S.I.S.; Su, L.; Hogervorst, T.P.; Lafleur, R.P.M.; Lou, X.; Marel, G.A. van der; ... ; Meijer, E.W. 2019
Polysaccharides present in the glycocalyx and extracellular matrix are highly important for a multitude of functions. Oligo- and polysaccharides-based biomaterials are being developed to mimic the... Show morePolysaccharides present in the glycocalyx and extracellular matrix are highly important for a multitude of functions. Oligo- and polysaccharides-based biomaterials are being developed to mimic the glycocalyx, but the spatial functionalization of these polysaccharides represents a major challenge. In this paper, a series of benzene-1,3,5-tricarboxamide (BTA) based supramolecular monomers is designed and synthesized with mono- (BTA-β-d-glucose; BTA-Glc and BTA-α-d-mannose; BTA-Man) or disaccharides (BTA-β-d-cellobiose; BTA-Cel) at their periphery or a monosaccharide (BTA-OEG4-α-d-mannose; BTA-OEG4-Man) at the end of a tetraethylene glycol linker. These glycosylated BTAs have been used to generate supramolecular assemblies and it is shown that the nature of the carbohydrate appendage is crucial for the supramolecular (co)polymerization behavior. BTA-Glc and BTA-Man are shown to assemble into micrometers long 1D (bundled) fibers with opposite helicities, whereas BTA-Cel and BTA-OEG4-Man formed small spherical micelles. The latter two monomers are used in a copolymerization approach with BTA-Glc, BTA-Man, or ethylene glycol BTA (BTA-OEG4) to give 1D fibers with BTA-Cel or BTA-OEG4-Man incorporated. Consequently, the carbohydrate appendage influences both the assembly behavior and the internal order. Using this approach it is possible to create 1D-fibers with adjustable saccharide densities exhibiting tailored dynamic exchange profiles. Furthermore, hydrogels with tunable mechanical properties can be achieved, opening up possibilities for the development of multicomponent functional biomaterials. Show less
Covalent linking of immunogenic oligopeptides with synthetic Toll-like receptor ligands is a useful approach to develop self-adjuvanting vaccines. In particular, small-molecule based agonists of... Show moreCovalent linking of immunogenic oligopeptides with synthetic Toll-like receptor ligands is a useful approach to develop self-adjuvanting vaccines. In particular, small-molecule based agonists of Toll-like receptor 7 (TLR7) that are derived from 8-oxo-adenine core are potentially promising because these chemically robust TLR7 ligands can be connected to peptide T-cell epitopes via straightforward solid-phase peptide synthesis. In this contribution we present the synthesis of a Boc-protected 9-benzyl-2-alkoxy-8-oxo-adenine building block and its application in the online solid phase synthesis of three peptide conjugates that differ in the position of the TLR7 ligand within the peptide. The conjugates are able to induce dendritic cell maturation and T cell proliferation while the position of the ligand impacts T cell proliferation potency. Show less
