Synthesis of asparagine derivatives harboring a Lewis X type DC-SIGN ligand and evaluation of their impact on immunomodulation in multiple sclerosis

The protein myelin oligodendrocyte glycoprotein (MOG) is a key component of myelin and an autoantigen in the disease multiple sclerosis (MS). Post‐translational N‐glycosylation of Asn31 of MOG seems to play a key role in modulating the immune response towards myelin. This is mediated by the interaction of Lewis‐type glycan structures in the N‐glycan of MOG with the DC‐SIGN receptor on dendritic cells (DCs). Here, we report the synthesis of an unnatural Lewis X (LeX)‐containing Fmoc‐SPPS‐compatible asparagine building block (SPPS=solid‐phase peptide synthesis), as well as asparagine building blocks containing two LeX‐derived oligosaccharides: LacNAc and Fucα1‐3GlcNAc. These building blocks were used for the glycosylation of the immunodominant portion of MOG (MOG31‐55) and analyzed with respect to their ability to bind to DC‐SIGN in different biological setups, as well as their ability to inhibit the citrullination‐induced aggregation of MOG31‐55. Finally, a cytokine secretion assay was carried out on human monocyte‐derived DCs, which showed the ability of the neoglycopeptide decorated with a single LeX to alter the balance of pro‐ and anti‐inflammatory cytokines, inducing a tolerogenic response.Bio-organic Synthesi

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

Langerhans cells (LCs) are antigen-presenting cells that reside in the skin. They uniquely express high levels of the C-type lectin receptor Langerin (CD207), which is an attractive target for antigen delivery in immunotherapeutic vaccination strategies against cancer. We here assess a library of 20 synthetic, well-defined mannoside clusters, built up from one, two, and three of six monomannosides, dimannosides, or trimannosides, appended to an oligopeptide backbone, for binding with Langerin using surface plasmon resonance and flow cytometric quantification. It is found that Langerin binding affinity increases with increasing number of mannosides. Hexavalent presentation of the mannosides resulted in binding affinities ranging from 3 to 12 mu M. Trivalent presentation of the dimannosides and trimannosides led to Langerin affinity in the same range. The model melanoma gp100 antigenic peptide was subsequently equipped with a hexavalent cluster of the dimannosides and trimannosides as targeting moieties. Surprisingly, although the bifunctional conjugates were taken up in LCs in a Langerin-dependent manner, limited antigen presentation to cytotoxic T cells was observed. These results indicate that targeting glycan moieties on immunotherapeutic vaccines should not only be validated for target binding, but also on the continued effects on biology, such as antigen presentation to both CD8(+)and CD4(+)T cells.Bio-organic Synthesi

MPLA incorporation into DC-targeting glycoliposomes favours anti-tumour T cell responses

Dendritic cells (DC) are attractive targets for cancer immunotherapy as they initiate strong and long-lived tumour-specific T cell responses. DC can be effectively targeted in vivo with tumour antigens by using nanocarriers such as liposomes. Cross-presentation of tumour antigens is enhanced with strong adjuvants such as TLR ligands. However, often these adjuvants have off-target effects, and would benefit from a DC-specific targeting strategy, similar to the tumour antigen. The goal of this study was to develop a strategy for specifically targeting DC with tumour antigen and adjuvant by using glycoliposomes. We have generated liposomes containing the glycan Lewis(Le)X which is highly specific for the C-type lectin receptor DC-SIGN expressed by DC. LeX-modified liposomes were taken up by human monocyte-derived DC in a DC-SIGN-specific manner. As adjuvants we incorporated the TLR ligands Pam3CySK4, Poly I:C, MPLA and R848 into liposomes and compared their adjuvant capacity on DC. Incorporation of the TLR4 ligand MPLA into glycoliposomes induced DC maturation and production of pro-inflammatory cytokines, in a DC-SIGN-specific manner, and DC activation was comparable to administration of soluble MPLA. Incorporation of MPLA into glycoliposomes significantly enhanced antigen cross-presentation of the melanoma tumour antigen gp100280–288 peptide to CD8+ T cells compared to non-glycosylated MPLA liposomes. Importantly, antigen cross-presentation of the gp100280–288 peptide was significantly higher using MPLA glycoliposomes compared to the co-administration of soluble MPLA with glycoliposomes. Taken together, our data demonstrates that specific targeting of a gp100 tumour antigen and the adjuvant MPLA to DC-SIGN-expressing DC enhances the uptake of peptide-containing liposomes, the activation of DC, and induces tumour antigen-specific CD8+ T cell responses. These data demonstrate that adjuvant-containing glycoliposome-based vaccine

Intradermal Delivery of TLR Agonists in a Human Explant Skin Model: Preferential Activation of Migratory Dendritic Cells by Polyribosinic-Polyribocytidylic Acid and Peptidoglycans

Experimental cancer immunology and therap

C-Mannosyl Lysine for Solid Phase Assembly of Mannosylated Peptide Conjugate Cancer Vaccines

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 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..Bio-organic Synthesi