Nouvelles synthèses d'analogues de galactosylceramides pour l'étude de la réponse des lymphocytes iNKT humains – GALCERDEO

The project involves 2 CNRS groups of chemists from Nantes and Le Mans, and a group of immunobiologists (Inserm U892-CRCNA). The collaboration has already been ongoing for three years, supported by the «Cancéropôle Grand Ouest» program, the regional research grant 2006 «CIMATH» and ARC (Ass. Against Cancer). The preliminary results obtained have led to international patents PCT/IB2006/003929 and PCT/IB2007/004270. The aim of the present project is to pursue the development of this chemists/biologists collaboration and in particular to confirm several original results that may not fit with current paradigms. Project objectives: CD1 restricted T lymphocytes represent a subset of αβ TCR+T cells which recently appeared to be major players of the immune response. A major subpopulation of the CD1d restricted lymphocytes, called iNKT cells, bears an invariant TCR and reacts against α-galactosylceramides (αGal-Cer) extracted from a marine sponge. Recognition of αGal-Cer bound to CD1d molecules of an antigen presenting cell, leads to the fast and strong secretion of a large panel of cytokines. These cytokines can stimulate the maturation of dendritic cells, activate the proliferation of IFNγ synthesis and stimulate cytotoxic CD8 lymphocytes. These various mechanisms contribute to the control of tumor progression. iNKT cells can also act as suppressors of the immune response through their secretion of Th2 cytokines such as IL-4, IL-10, IL-13. A therapeutic effect has been documented after injection of αGal-Cer analogue KRN7000 to tumor-bearing mice and so far it did not appear to be toxic in Humans. Our biological aims will be two fold. First, we will evaluate the ability of newly designed glycolipids prepared by the teams of chemists within our network to stimulate human iNKT cells in terms of proliferation, cytotoxic activity and cytokine secretion. Since many active synthetic analogues of αGal-Cer have already been described, the discovery of novel structures with potent activities at this stage remains challenging. However, considering the poor availability until now of such analogues and the uncertainties regarding the optimal agonists that should be used in vivo, obtaining molecules with distinct biological properties, that can be tested later in preclinical in vivo models, represents a critical step toward the development of immunotherapeutic approaches against infectious, tumor or autoimmune processes associated with iNKT cell activation. We thus expect to design new glycolipid compounds able to trigger activation/expansion of iNKT cells, in order to set up new immunotherapeutic approaches against infectious, tumor or autoimmune processes associated with iNKT cell activation. Second, from a more basic standpoint, we aim at getting insights into the nature of the endogenous glycolipid antigens recognized by human iNKT cells and into the mechanisms underlying the autoreactive/nonautoreactive iNKT dichotomy. Characterization of such antigens should provide insights into the central selection of iNKT cells and the mechanisms underlying their selection in broad physiopathological contexts. To this end, the availability of synthetic β-anomers will be of outmost importance to pursue from the preliminary data mentioned above, which suggest that natural β-anomers can serve as endogenous ligands depending on the cellular context of presentation. To demonstrate that simple glycolipids with a terminal sugar unit in β-linkage constitute the endogenous ligands recognized by autoreactive iNKT clones, it will be necessary to block their synthesis from tumor cell lines that are efficiently recognized by autoreactive iNKT cells. We will try to achieve this task by knocking down the expression of the glycosyltransferases genes by an shRNA approach that has been recently set up in our laboratory. The chemical aims challenge the access to original α- and β-GalCer analogues by optimized synthetic pathways. Preliminary investigations have allowed to perform the synthesis of a variety of novel deoxy αGal-Cers derivatives by an original and efficient strategy based on a metathesis process. We have already shown that a 4-deoxy-KRN7000 analogue is at least as efficient as parent KRN7000 presently in clinical trial for anticancer therapy. Furthermore, 4-deoxy-KRN7000 derivatives are much more easier to produce by our simplified synthetic strategy (patented). We are now in a position to straignthforward design new chemical targets with selective and complementary modifications in order to help elucidate the autoimmune response against cancer and determinate the parameters that control the selection of secreted cytokines.