Systematic Dual Targeting of Dendritic Cell C-Type Lectin Receptor DC-SIGN and TLR7 Using a Trifunctional Mannosylated Antigen

Abstract

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 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.

Targeting of the C-Type Lectin Receptor Langerin Using Bifunctional Mannosylated Antigens

“…Fmoc-based solid phase peptide synthesis and conjugated to propargyl-functionalized B6, C6, D6, E6 clusters to generate bifunctional mannosylated antigens ( Figure 1B; Li et al, 2019). Four natural occurring amino acids gp100 276−279 were used as spacer between the two moieties.…”

“…Five (oligo)mannoside structures (Man; Manα1,2Man; Manα1,3Man; Manα1,6Man; and Manα1,3Man1,6Man saccharides), each representing a substructure of the high affinity Man 9 oligosaccharide, were used to build the library. The library members systematically vary in saccharide structure (coded A-E) and number of copies on the peptide scaffold (n = 1, 2, 3, 6, Figure 1A, and Supplementary Figure 1) (Li et al, 2019).…”

Molecular Recognition in C‐Type Lectins: The Cases of DC‐SIGN, Langerin, MGL, and L‐Sectin

“…[137] To exemplify, dimannoside-containing clusters have been noticed to be preferentially shuttled inside DCs than the trimannoside-based analogues. [138] Similarly, DC-SIGN promotes uptake of fucosylated oligolysine scaffolds, but not of the mannosylated ones. [139] In all these cases, it is always important to consider the different responses that might be driven by similar CLRs, like MR or langerin.…”

“…[137,139,140] Frequently, a common strategy to trigger an efficient DCmediated immune response consists of combining sugar epitopes for CLRs and other ligands to co-stimulate Toll-like receptors, achieving for instance effective antitumor responses. [138,141] DC-SIGN internalization capabilities have been also exploited for intracellular delivery of therapeutic agents, [142] and for this purpose, sugar-coated liposomes are ideal platforms. In general, micelles and liposomes are quick pathways to afford enormous multivalent structures from suitable function-alized lipids.…”

Synthesis of C‐Glycosyl Amino Acid Building Blocks Suitable for the Solid‐Phase Synthesis of Multivalent Glycopeptide Mimics

“…in cancer immunotherapeutic strategies. [3][4][5][6] Such multivalent conjugates can not only be tailored to effectively mimic complex glycan structures, [7][8][9] but also their physical properties can be changed and tuned. [4,10,11] Another example is the exploitation of the abundance of the circulating anti-L-rhamnose antibodies in human blood, [12,13] that can be recruited using multivalent rhamnose conjugates.…”