The sialic acid-Siglec immune checkpoint: an opportunity to enhance immune responses and therapy effectiveness in melanoma.

Modulation of immune responses through immune checkpoint blockade has revolutionized cutaneous melanoma treatment. However, it is still the case that not all patients respond successfully to these therapies, indicating the presence of as yet unknown resistance mechanisms. Hence, it is crucial to find novel targets to improve therapy efficacy. One of the described resistance mechanisms is regulated by immune inhibitory Siglec receptors, which are engaged by the carbohydrates sialic acids expressed on tumour cells, contributing to programmed cell death protein-1 (PD1)-like immune suppression mechanisms. In this review, we provide an overview on the regulation of sialic acid synthesis, its expression in melanoma, and the contribution of the sialic acid-Siglec axis to tumour development and immune suppressive mechanisms in the tumour microenvironment. Finally, we highlight potential sialic acid-Siglec axis-related therapeutics to improve the treatment of melanoma.

Recent advances on smart glycoconjugate vaccines in infections and cancer.

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen-specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency virus or to induce cellular T-cell immunity in the fight against cancer. The recent SARS-CoV-2 outbreak has reinforced the relevance of designing smart therapeutic vaccine modalities to ensure public health. Indeed, academic and private companies have ongoing joint efforts to develop novel vaccine prototypes for this virus. Many pathogens are covered by a dense glycan-coat, which form an attractive target for vaccine development. Moreover, many tumor types are characterized by altered glycosylation profiles that are known as "tumor-associated carbohydrate antigens". Unfortunately, glycans do not provoke a vigorous immune response and generally serve as T-cell-independent antigens, not eliciting protective immunoglobulin G responses nor inducing immunological memory. A close and continuous crosstalk between glycochemists and glycoimmunologists is essential for the successful development of efficient immune modulators. It is clear that this is a key point for the discovery of novel approaches, which could significantly improve our understanding of the immune system. In this review, we discuss the latest advancements in development of vaccines against glycan epitopes to gain selective immune responses and to provide an overview on the role of different immunogenic constructs in improving glycovaccine efficacy.

Emerging glyco-based strategies to steer immune responses.

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell-cell communications and in the regulation of immune responses. Through the interaction with glycan-binding receptors, glycans are able to affect the activation status of antigen-presenting cells, leading either to induction of pro-inflammatory responses or to suppression of immunity and instigation of immune tolerance. This unique feature of glycans has attracted the interest and spurred collaborations of glyco-chemists and glyco-immunologists to develop glycan-based tools as potential therapeutic approaches in the fight against diseases such as cancer and autoimmune conditions. In this review, we highlight emerging advances in this field, and in particular, we discuss on how glycan-modified conjugates or glycoengineered cells can be employed as targeting devices to direct tumor antigens to lectin receptors on antigen-presenting cells, like dendritic cells. In addition, we address how glycan-based nanoparticles can act as delivery platforms to enhance immune responses. Finally, we discuss some of the latest developments in glycan-based therapies, including chimeric antigen receptor (CAR)-T cells to achieve targeting of tumor-associated glycan-specific epitopes, as well as the use of glycan moieties to suppress ongoing immune responses, especially in the context of autoimmunity.

Self-Adjuvanting Cancer Vaccines from Conjugation-Ready Lipid A Analogues and Synthetic Long Peptides.

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.

Chemically engineered glycan-modified cancer vaccines to mobilize skin dendritic cells.

Dendritic cell (DC)-targeting vaccines show great promise in increasing antitumor immunity. Glycan-engineered vaccines facilitate both DC targeting and increased uptake by DCs for processing and presentation to CD4+ and CD8+ T cells to induce tumor-specific T-cell responses. However, the complexity of various DC subsets in skin tissues, expressing different glycan-binding receptors that can mediate vaccine uptake or drainage of vaccines via lymphatics directly to the lymph node-resident DCs, complicates the success of vaccines. Moreover, the influx of inflammatory immune cells to the site of vaccination, such as monocytes that differentiate to DCs and coexpress glycan-binding receptors, may contribute to the strength of DC-targeting glycovaccines for future clinical use.

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin-1.

The core fucose, a major modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin-1, a known anti-ß-glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.

Biocompatible single-chain polymer nanoparticles loaded with an antigen mimetic as potential anticancer vaccine.

The "pancarcinoma" Tn antigen (αGalNAc-O-Ser/Thr) is a tumor-associated carbohydrate antigen (TACA) overexpressed on the surface of cancer cells and suitable target for anticancer vaccines. However, TACAs commonly show weak immunogenicity, low in vivo stability, and poor bioavailability. To address these issues, the development of physiologically stable TACA synthetic mimetics and novel nanocarriers for multivalent display are object of intense research. Nanomaterials represent suitable scaffolds to multimerize antigens, but absence of toxicity, easy functionalization and capability to incorporate biomolecules are compulsory characteristics for vaccine nanocarriers. Here, we report on the conjugation of a synthetic Tn-antigen mimetic to biocompatible and water-dispersible dextran-based single-chain nanoparticles (DXT-SCPNs). In vitro stimulation of PBMCs and analysis of interleukins production indicated a specific innate immune modulation mediated by the multivalent presentation of the Tn mimetic at the nanoparticle surface. These preliminary results pave the way for the development of Tn-mimetic clusters on biocompatible DXT-SCPN for TACA-based vaccines.

Synthesis and evaluation of fluorescent Pam3Cys peptide conjugates.

Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.

Effective targeting of DC-SIGN by α-fucosylamide functionalized gold nanoparticles.

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-ß-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.

Galactofuranose-coated gold nanoparticles elicit a pro-inflammatory response in human monocyte-derived dendritic cells and are recognized by DC-SIGN.

Galactofuranose (Galf) is the five-membered ring form of galactose exclusively found in nonmammalian species, among which several are pathogens. To determine the putative role of this carbohydrate in host-pathogen interactions, we synthesized multivalent gold nanoparticles carrying Galf (Galf-GNPs) and show that they are recognized by the EB-A2 antibody, which is widely used to detect Galf-containing galactomannan in the serum of Aspergillosis patients. We demonstrated that human monocyte-derived dendritic cells bound Galf-GNPs via interaction with the lectin DC-SIGN. Moreover, interaction of dendritic cells with Galf-GNPs resulted in increased expression of several maturation markers on these cells and induced secretion of the pro-inflammatory cytokines IL-6 and TNF-α. These data indicate that Galf is able to modulate the innate immune response via dendritic cells. In conclusion, Galf-GNPs are a versatile tool that can be applied in multiple functional studies to gain a better understanding of the role of Galf in host-pathogen interaction.

High sensitive detection of carbohydrate binding proteins in an ELISA-solid phase assay based on multivalent glyconanoparticles.

Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or Streptococcus pneumoniae were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against S. pneumoniae respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells.

Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells in order to achieve an appropriate uptake, processing, and presentation to Ag-specific T cells. C-type lectins have shown to be ideal receptors for the targeting of antigens to dendritic cells and allow the use of their natural ligands - glycans - instead of antibodies. Amongst them, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is an interesting candidate due to its biological properties and the availability of its natural carbohydrate ligands. Using Le(b)-conjugated poly(amido amine) (PAMAM) dendrimers we aimed to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16-32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases.