Chang-Chun Ling

Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity

As an approved vaccine adjuvant for use in humans, alum has vast health implications, but, as it is a crystal, questions remain regarding its mechanism. Furthermore, little is known about the target cells, receptors, and signaling pathways engaged by alum. Here we report that, independent of inflammasome and membrane proteins, alum binds dendritic cell (DC) plasma membrane lipids with substantial force. Subsequent lipid sorting activates an abortive phagocytic response that leads to antigen uptake. Such activated DCs, without further association with alum, show high affinity and stable binding with CD4+ T cells via the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function–associated antigen-1 (LFA-1). We propose that alum triggers DC responses by altering membrane lipid structures. This study therefore suggests an unexpected mechanism for how this crystalline structure interacts with the immune system and how the DC plasma membrane may behave as a general sensor for solid structures.

Receptor-independent, direct membrane binding leads to cell surface lipid sorting and Syk kinase activation in dendritic cells

Binding of particulate antigens by antigen-presenting cells is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. By using atomic force microscopy as a tool for real-time single-cell activation analysis, we report that uric acid crystals could directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein-based cellular contacts. Binding of particulate substances activated Syk kinase-dependent signaling in dendritic cells. These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell-surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation, and adjuvanticity.

Concise and Efficient Synthesis of 2-Acetamido-2-deoxy-β-d-hexopyranosides of Diverse Aminosugars from 2-Acetamido-2-deoxy-β-d-glucose

The furanose acetonide derivative 1 is readily prepared from 2-acetamido-2-deoxy-D-glucose on a large scale without the need for chromatography. Mesylation of 1 provides an efficient, concise, synthetic route to rare 2-acetamido-2-deoxy-beta-D-hexopyranosides (2 and 3) via the corresponding methyl 2-acetamido-2-deoxy-3-O-methanesulfonyl-beta-D-glucopyranoside and subsequent inversion of configuration by direct displacement or formation of a 3,4-epoxide. Opening of this epoxide by azide provided a direct route to methyl 2-acetamido-4-amino-2,4,6-trideoxy-beta-D-gulopyranoside 4. Benzylation of 1 followed by ring expansion to the glucopyranoside, deoxygenation at C-6, and subsequent displacement of a C-4 triflate permitted the synthesis of methyl 2-acetamido-4-amino-2,4,6-trideoxy-beta-D-galactopyranoside 5. Methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside available from 1 in quantitative yield was readily converted to methyl 2-acetamido-2-deoxy-beta-D-galactopyranoside 6 (>60%) by inversion of configuration at C-4. Introduction of a lactyl substituent at C-3 of oxazoline 1 also provides a facile synthesis of the biologically important muramic acid beta-glycoside 7. An interesting reaction to convert 2-acetamido-2-deoxyhexopyranosides to the corresponding 2-deoxy-2-tetrazole is also reported.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins—ConA, LcH, and PSA—that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Recent advances in developing synthetic carbohydrate-based vaccines for cancer immunotherapies

Cancer cells can often be distinguished from healthy cells by the expression of unique carbohydrate sequences decorating the cell surface as a result of aberrant glycosyltransferase activity occurring within the cell; these unusual carbohydrates can be used as valuable immunological targets in modern vaccine designs to raise carbohydrate-specific antibodies. Many tumor antigens (e.g., GM2, Le(y), globo H, sialyl Tn and TF) have been identified to date in a variety of cancers. Unfortunately, carbohydrates alone evoke poor immunogenicity, owing to their lack of ability in inducing T-cell-dependent immune responses. In order to enhance their immunogenicity and promote long-lasting immune responses, carbohydrates are often chemically modified to link to an immunogenic protein or peptide fragment for eliciting T-cell-dependent responses. This review will present a summary of efforts and advancements made to date on creating carbohydrate-based anticancer vaccines, and will include novel approaches to overcoming the poor immunogenicity of carbohydrate-based vaccines.

Synthesis of P-Triazole Dithienophospholes and a Cyclodextrin-Based Sensor via Click Chemistry

The synthesis of a series of highly luminescent, functional dithienophospholes via a click reaction is reported. Slight modification of the lateral aromatic substituents leads to a significant difference in their solid-state organization. In addition, a novel water-soluble β-cyclodextrin hybrid is demonstrated to be an effective sensor for picric acid.

Controlled Synthesis of Linear α-Cyclodextrin Oligomers Using Copper-Catalyzed Huisgen 1,3-Dipolar Cycloaddition

The design and efficient synthesis of a novel class of linear oligomers based on cyclodextrins are described. These supramolecules have relatively rigid structures with well-defined topology and sizes, which could provide them with the ability to be used as scaffolds to present bioactive molecules to their receptors as well as host molecules.

Reducing Epitope Spread during Affinity Maturation of an Anti-Ganglioside GD2 Antibody

Ab affinity maturation in vivo is always accompanied by negative selection to maintain Ag specificity. In contrast, in vitro affinity maturation can lead to epitope spread, resulting in loss of specificity. Anti-ganglioside-GD2 mAbs are clinically effective against neuroblastoma; pain and neuropathy are major side effects. We used structural relatives of GD2 to define epitope spread during in vitro affinity maturation of an anti-GD2 single-chain variable fragment (scFv) called 5F11-scFv. Clonal dominance identified by polyclonal sequencing was confirmed by analyzing individual clones. Affinity-matured mutations were introduced into scFv-streptavidin for functional studies. Without a negative selector, 19-fold affinity improvement (clone Q, where Q is the symbol for glutamine) was associated with strong cross-reactivity with GM2 and GD1b and moderate cross-reactivity with GD3, resulting in positive immunohistochemical staining of all 13 non-neural normal human tissues, in contrast to none of 13 tissues with parental clone P. With GM2 as a negative selector, clone Y (where Y is the symbol for tyrosine) was generated with only weak cross-reactivity with GD1b, adrenal and thyroid glands, and no staining of other non-neural normal tissues. Even though there was only a 3-fold affinity improvement, clone Y showed significantly higher tumor uptake over parental clone P (134%, p = 0.04), whereas clone Q was inferior (54% of clone P; p = 0.05) as confirmed by tumor-to-normal tissue ratios across 16 organs (41% of clone P; p < 0.0001). Using the less efficient negative selector GD3, a clone mixture (Q, V, and Y, where V is the symbol for valine) emerged. We conclude that epitope spread during affinity maturation can be reduced by negative selection. Furthermore, efficiency of the negative selector depends on its cross-reactive affinity with the matured scFv.

First Per-6-O-tritylation of Cyclodextrins

Because of the large dimension of the trityl group and the truncated conical geometry of cyclodextrin (CD) molecules, it is unclear if there is enough space at the narrower end of CDs to permit a per-6-O-tritylation. This work demonstrates that it is indeed possible to simultaneously install a trityl group at the O6-position of every glucopyranosyl unit in a CD. A novel per-6-substitution method has been developed for CD chemistry.

Synthesis of a novel class of cyclodextrin-based nanotubes.

The synthesis and characterization of a novel class of structurally well-defined nanotubes from β-cyclodextrin are described. These new hosts were formed using disulfide linkages that substitute all the primary hydroxyl groups of a β-cyclodextrin. A deep and rigid hydrophobic channel with a size of more than 1.5 nm is found in the molecules. Because of their unique geometry and the potential biodegradability of the disulfide bond, this class of molecules could find broad applications in biology and other areas of research.