Synthesis of Glycocluster−Tumor Antigenic Peptide Conjugates for Dendritic Cell Targeting

Abstract

The use of dendritic cells (DC) for the development of therapeutic cancer vaccines is attractive because of their unique ability to present tumor epitopes via the MHC class I pathway to induce cytotoxic CD8+ T lymphocyte responses. C-Type membrane lectins, DC-SIGN and the mannose receptor (MR), present on the DC surface, recognize oligosaccharides containing mannose and/or fucose and mediate sugar-specific endocytosis of synthetic oligolysine-based glycoclusters. We therefore asked whether a glycotargeting approach could be used to induce uptake and presentation of tumor antigens by DC. To this end, we designed and synthesized glycocluster conjugates containing a CD8+ epitope of the Melan-A/Mart-1 melanoma antigen. These glycocluster-Melan-A conjugates were obtained by coupling glycosynthons: oligosaccharyl-pyroglutamyl-beta-alanine derivatives containing either disaccharides, a dimannoside (Manalpha-6Man) or lactoside, or a Lewis oligosaccharide, to Melan-A 16-40 peptide comprising the 26-35 HLA-A2 restricted T cell epitope, extended with an oligolysine stretch at the C-terminal end. We showed by confocal microscopy and flow cytometry that fluorescent-labeled Melan-A glycoclusters containing either dimannoside or Lewis oligosaccharide were taken up by DC and concentrated in acidic vesicles; conversely lactoside glycopeptides were not at all taken up. Furthermore, using surface plasmon resonance, we showed that dimannoside and Lewis-Melan-A conjugates bind MR and DC-SIGN with high affinity. DC loaded with these conjugates, but not with the lactose-Melan-A conjugate, led to an efficient presentation of the Melan-A epitope eliciting a CD8+ T-lymphocyte response. These data suggest that synthetically designed glycocluster-tumor antigen conjugates may induce antigen cross-presentation by DC and represent a promising tool for the development of tumor vaccines.

Dendritic Cells and their Receptors in Antitumor Immune Response

“…Owing to the carbohydrate specificity of DC-SIGN, Srinivas et al showed that the synthetic glycocluster conjugates containing a CD8 + epitope of the Melan-A/Mart-1 melanoma Ag binds to DC-SIGN followed by internalization and Ag presentation to induce antitumor immune response. In this study DCs loaded with these conjugated Ags but not with the lactose-Melan-A conjugate, induced CD8 + T cell specific responses, which further confirmed that DC-SIGN can serve as a potential candidate for carbohydrate based Ag deliveries [52].…”

Targeting Antigens to Dendritic Cell Receptors for Vaccine Development

“…The melanoma antigen, Melan-A/Mart-1 (peptide 16-40, containing the CD8+ HLA-A2 restricted T-cell epitope, amino acids [26][27][28][29][30][31][32][33][34][35], was coupled to either Manalpha-6 Man or lactoside, or a Lewis oligosaccharide [88]. The glycoconjugates containing Lewis oligosaccaride bound with high affinity to DC-SIGN were taken up by DCs into acidic vesicles and presented by MHC class I and stimulated CD8+ T-cell responses [88]. However, glycoconjugates containing lactoside were not taken up by DCs.…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…For dendritic cell targeting (Srinivas, et al, 2007) Peptides Gal-GalNAc TOF Sequential segment coupling using Nalkylcysteine-assisted thioesterification PEG-poly-Llysine.…”