Synthesis of Lectin Targeting Agents For Use In Vaccines

Abstract

T–cells are a population of lymphocytes which are able to destroy cells infected with pathogens and cancer cells. For a T–cell immune response to be initiated, antigens need to be presented on a major histocompatibility complex (MHC) by an antigen presenting cell (APC) to a T–cell receptor. At the same time, the T–cell response is regulated by a balance of co–stimulatory and inhibitory signals which are referred to as immune checkpoints. These inhibitory pathways are crucial formaintaining self tolerance and modulating the duration and amplitude of physiological immune responses. Therapeutic vaccines need to stimulate CD8+ T–cells to destroy cancer cells. New vaccine strategies are required to achieve this as it has proved challenging in the past. Current vaccines can stimulate CD4+ expressing T–cells by delivering antigens to MHC II expressing APC but are not effective at stimulating CD8+ T–cells, which requires the presentation of antigens on MHC I. There are very few therapeutic vaccines which are able to stimulate CD8+ T–cells. Those which do have limited results when treating cancer. Cancer cells express inhibitory ligands and receptors that regulate T–cell effectors function and cause immunosuppression. When CD8+ expressing T–cells are no longer inhibited, they are able to mature to become cytotoxic T–cells which can kill cancer cells. Important recent advances in cancer treatment have used drugs such as Ipilimumab to overcome immunosuppression by targeting immune checkpoints. These immune checkpoint blockers reduce the inhibition of CD8+ T–cells which allows the immune system to fight the cancer. However, these drugs do not work if the patient has never had an immune response to generate the CD8+ T–cells. Therefore therapeutic vaccines which can stimulate CD8+ T–cell responses are needed to be used in conjunction with drugs such as Ipilimumab to increase the efficacy of these cancer treatments. In order to design new therapeutic vaccines which can generate CD8+ T–cells, antigen presenting cells (APCs) need to be targeted. Dendritic cells (DCs) are the most important APC. Macrophage galactose–type lectin (MGL) is a protein receptor found on the surface of DCs, which are involved in the cross presentation of antigens onto MHC I. MGL is known to bind GalNAc specifically, a carbohydrate which is part of the TN antigen. This project aimed to design a molecule which could deliver a cargo directly to the MGL receptor on DCs. After processing the molecule, its antigens can be presented on the surface of MHC I and MHC II proteins to T–cells which activates an immune response. A library of TN antigen mimic peptide dendrimers was synthesised with the goal of selective targeting of the MGL lectin commonly found as trimeric clusters on DCs. TN antigen mimics containing N–acetylgalactosamine (GalNAc) were synthesised to include an alkyne handle attached via a linker to the anomeric position. Through the alkyne functionality, the carbohydrate could be conjugated to branched polylysine dendrimers which contained azide moieties via the Cu(I)–catalysed Huisgen azide–alkyne cycloaddition reaction (Scheme 1). A library of dendrimers was synthesised with varying size, strength of fluorescence and number of GalNAc residues. Scheme 1: Fmoc solid phase peptide synthesis of a lectin targeting dendrimer and GalNac glycosylation using the Cu(I)–catalysed Huisgen azide–alkyne cycloaddition reaction. The library of synthetic compounds was tested in a thermalmelt assay using differential scanning fluorimetry to measure the strength and availability of GalNAc for binding to a series of truncated monomeric MGL proteins containing the carbohydrate binding domain. The library of compounds was also tested using flow cytometry in a cell binding assay for the selectivity and quantity of binding to MGL–expressing monocyte dendritic cells (MoDC). The two tests were compared to see if differential scanning fluorometry could be used as a pre–screening tool to predict which compounds would bind selectively and have high loading to MGL–expressing MoDCs. Finally, a cell binding assay using peripheral blood mononucleated cells (PBMC) was conducted using the best performing compounds from the thermal melt and cell binding assays. The PBMC study included a wider variety of cells than the previous assays and was more representative of the system found in the human body. To measure the selectivity and quantity of loading, the lead compounds identified in the preliminary assays were compared in a PBMC cell binding assay which included different cells which had varying degrees of MGL expression. The TN antigen mimic dendrimers were shown to be an effective delivery system for a fluorophore cargo when targeting MGL–expressing cells.