Carbohydrate-derived molecules as ligands for immune cell receptors

The macrophage inducible C-type lectin (Mincle) is a pattern recognition receptor on myeloid cells that recognises both pathogen-associated and danger-associated molecular patterns. Trehalose dimycolate (TDM), the most abundant glycolipid in the cell wall of Mycobacterium species, and its synthetic analogue trehalose dibehenate (TDB), were among the first non-proteinaceous potent Mincle agonists to be identified. Further studies have uncovered the potential of Mincle ligands as adjuvants in vaccine formulations due to the ability of the agonists to elicit a TH1-polarising immune response. There has been much recent interest in glucose-based Mincle ligands as they are capable of inducing potent Mincle-dependent immune responses similar to those elicited by TDM and TDB. However, much is still unknown about the structural requirements for glucose ligands to induce optimal Mincle signalling. Thus, the first section of this thesis focuses on the synthesis of rationally designed α-branched and aromatic glucose monoesters and glucosides, followed by biological evaluation of the derivatives using murine and human cell-based assays. From this work, it was determined that docosyl α-glucoside induces potent human Mincle activity and superior immune responses than TDB by human monocytes, thereby warranting further research as a potential adjuvant.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that play an important role in many pathologies ranging from viral and bacterial infections, to autoimmune disorders and cancer. Accordingly, MAIT cells represent an attractive target for immunotherapeutics. MAIT cells are activated via the presentation of ligands by major histocompatibility complex (MHC) class I-related molecule, MR1, to their invariant T cell receptor (TCR). In 2012, the first class of agonist for MAIT cells was identified as the ribityllumazines, followed by the discovery of a more potent but unstable agonist, 5-(2-oxopropylideneamino)-6-ᴅ-ribitylaminouracil (5-OP-RU), in 2014. To date, few studies have explored the effects of systematic changes to the ligand structure on MAIT cell activation. Additional insight into the structural motifs required for regulating MAIT cell activity would contribute greatly towards the design of future MAIT cell agonists and antagonists. Accordingly, the next section of this thesis focusses on investigating how changes to the sugar motif of MR1 ligands 7-hydroxy-6-methyl-8-ᴅ-ribityllumazine (RL-6-Me-7-OH) and 5-OP-RU influence MAIT cell activation. A highly efficient protecting group-free three step synthesis of lumazines was developed, which facilitated the substitution of different sugar residues to afford the target lumazine and 5-OP-RU glyco-analogues. Biological assays and docking studies revealed the importance of the 2′-OH group on the sugar side chain for MAIT cell activity.

To further examine the effect of gross structural changes to the 6-amino side chain of 5-OP-RU, uracil analogues containing monohydroxyalkylamine or unfunctionalised alkylamine side chains were synthesised and assessed for their ability to bind MR1 and activate MAIT cells. In doing so, a new simplified MAIT cell agonist with a functional profile similar to 5-OP-RU was identified, and a new structural class of MAIT cell antagonist was discovered. Furthermore, for the first time, it was demonstrated that changing the 6-amino side chain of 5-OP-RU can switch the function of the MAIT cell ligand from agonist to antagonist.

The unstable nature of 5-OP-RU in aqueous conditions makes it challenging to prepare samples of this compound for biological assays, thereby requiring the generation of 5-OP-RU immediately before testing. In order to harness MAIT cells as a potential target for vaccines, the development of a stable MAIT cell ligand is crucial to allow for its incorporation into vaccine formulations. Accordingly, the last section of this thesis focusses on the generation of stable MAIT cell agonists, followed by studies into their ability to bind MR1 and activate MAIT cells. A series of amide and carbamate analogues of 5-OP-RU were synthesised and biologically assessed using a murine MAIT cell line, which resulted in the identification of three new amide MAIT cell agonists. Two amide analogues were found to be more stable than 5-OP-RU in aqueous conditions, with preliminary studies demonstrating their ability to induce a functional response by both murine and human MAIT cells. Accordingly, the stable amine agonists represent attractive targets for further MAIT cell research.