The over-expression of saccharides such as Globo-H, Lewis Y and Tn antigen is a common feature of oncogenic transformed cells. Endeavors to exploit this aberrant glycosylation for cancer vaccine development has been complicated by difficulties of eliciting high titers of IgG antibodies against classical conjugates of tumor-associated carbohydrates to carrier proteins. We have designed, chemical synthesized and immunologically evaluated a number of fully synthetic vaccine candidates to establish strategies to overcome the poor immunogenicity of tumor-associated carbohydrates and glycopeptides. We have found that a three-component vaccine composed of a TLR2 agonist, a promiscuous peptide T-helper epitope and a tumor-associated glycopeptide, can elicit in mice exceptionally high titers of IgG antibodies that can recognize cancer cells expressing the tumor-associated carbohydrate. The superior properties of the vaccine candidate are attributed to the local production of cytokines, upregulation of co-stimulatory proteins, enhanced uptake by macrophages and dendritic cells and avoidance of epitope suppression.A broad and expanding body of preclinical and clinical studies [1][2][3][4] demonstrates that naturally acquired, passively administered or actively induced antibodies against carbohydrate-associated tumor antigens are able to eliminate circulating tumor cells and micro-metastases in cancer patients. Tumor-associated saccharides are, however, of low antigenicity, because they are self-antigens and consequently tolerated by the immune system. In addition, foreign carrier proteins such as keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) and the linker that attach the saccharides to the carrier protein can elicit strong B-cell responses, which may lead to the suppression of antibody responses against the carbohydrate epitope 5,6 . It is clear that the successful development of carbohydrate-based cancer vaccines requires novel strategies for the more efficient presentation of tumor-associated carbohydrate epitopes to the immune system, resulting in a more efficient class switch to IgG antibodies 7-17 .We reasoned that a three-component vaccine composed of a tumor-associated carbohydrate B-epitope, a promiscuous peptide T-helper (Th) epitope and a Toll-like receptor (TLR) ligand will circumvent immune suppression caused by a carrier protein or the linker region of a classical conjugate vaccine. Such a vaccine candidate contains, however, all mediators required for eliciting a strong IgG immune response. In the first instance, vaccine candidates 1 and 2 were designed, which contain as a B-epitope a tumor-associated glycopeptide derived from MUC1 1,18 and the well-documented murine MHC class II restricted Th (Fig. 1). Furthermore, compound 1 contains as an built-in adjuvant the lipopeptide Pam 2 CysSK 4 , which is a potent activator of TLR2/6, whereas compound 2 contains Pam 3 CysSK 4 , which induces cellular activation through TLR1/2 20 .Compound 1 was prepared by a solid-phase peptide synthesis ...

The lipid A moiety of lipopolysaccharides (LPS) initiates innate immune responses by interacting with Toll-like receptor 4 (TLR4) which results in the production of a wide range of cytokines. Derivatives of lipid A show potential for use as immuno-modulators for the treatment of a wide range of diseases and as adjuvants for vaccinations. Development to these ends requires a detailed knowledge of patterns of cytokines induced by a wide range of derivatives. This information is difficult to obtain by using isolated compounds due to structural heterogeneity and possible contaminations with other inflammatory components. To address this problem, we have developed a synthetic approach that provides easy access to a wide range of lipid As by employing a common disaccharide building block functionalized with a versatile set of protecting groups. The strategy was employed for the preparation of lipid As derived from E. coli and S. typhimurium. Mouse macrophages were exposed to the synthetic compounds and E. coli 055:B5 LPS and the resulting supernatants examined for tumor necrosis factor alpha (TNF-α), interferon beta (IFN-β), interleukin 6 (IL-6), interferon-inducible protein 10 (IP-10), RANTES, and IL-1β It was found that for each compound, the potencies (EC 50 values) for the various cytokines differed by as much as 100-fold. These differences did not follow a bias towards a MyD88-or TRIF-dependent response. Instead, it was established that the observed differences in potencies of secreted TNF-α and IL-1β were due to differences in the processing of respective pro-proteins. Examination of the efficacies (maximum responses) of the various cytokines showed that each synthetic compound and E. coli 055:B5 LPS induced similar efficacies for the production of IFN-β, and IP-10. However, lipid As 1-4 gave lower efficacies for the production of RANTES and IL-6 compared to LPS. Collectively, the presented results demonstrate that cytokine secretion induced by LPS and lipid A is complex, which can be exploited for the development of immuno-modulating therapies.

Recent studies have indicated that lipopolysaccharides (LPS) isolated from particular bacterial strains can bias innate immune responses toward different signal transduction pathways thereby eliciting unique patterns of cytokines. Heterogeneity in the structure of lipid A (the active component of LPS) and possible contaminations with other inflammatory components have made it difficult to confirm these observations and dissect molecular motifs that may be responsible for modulatory properties. To address these issues, we have examined, for the first time, the ability of a range of well defined synthetic lipid As and isolated LPS and lipid A preparations to induce the production of a wide range of cytokines in three different mouse cell types. It was found that, for a given compound, the potencies of production of the various cytokines differed significantly. An additive model, in which a chemical change in the structure of a compound effects the potencies of all cytokines in the same manner, could describe the potencies of the cytokines for all compounds. Thus, no evidence was found that the structure of lipid A can modulate the pattern of cytokine production. In addition, the statistical analysis showed that the relative ordering of the potencies of the compounds was identical in the different cell types and that structural features such as the presence of a 3-deoxy-D-manno-octulosonic acid moiety, anomeric phosphate, lipid length, and acylation pattern were important for pro-inflammatory activity. Finally, it was found that transcriptional and post-transcription control mechanisms determine potencies and efficacies of cytokine production in cell-specific manners.The innate immune system is an evolutionarily ancient system designed to detect the presence of microbial invaders and activate protective reactions (1). It responds rapidly to unique molecules that are integral parts of pathogens and are perceived as danger signals by the host. Recognition of these molecular patterns is mediated by sets of highly conserved receptors (2), whose activation results in acute inflammatory responses. These responses include the production of a diverse set of cytokines and chemokines, direct local attack against the invading pathogen, and initiation of responses that activate and regulate the adaptive immune system (3-7).Evidence is emerging that innate immune responses can be exploited for therapeutic purposes such as the development of adjuvants for vaccines and the treatment of a wide range of diseases, including asthma, infections, and cancer (8, 9). An important concern of such therapies is, however, that overactivation of innate immune responses may lead to the clinical symptoms of septic shock (10). Thus, an important issue for the design of safe immune modulators is detailed knowledge of structure-activity relationships to harness beneficial effects without causing toxicity.LPSs 2 are structural components of the outer surface membrane of Gram-negative bacteria that trigger innate immune responses through Toll-like r...

SummaryTetra-acylated lipid As derived from Porphyromonas gingivalis LPS have been synthesized using a key disaccharide intermediate functionalized with levulinate (Lev), allyloxycarbonate (Alloc) and anomeric dimethylthexylsilyl (TDS) as orthogonal protecting groups and 9-fluorenylmethoxycarbamate (Fmoc) and azido as amino protecting groups. Furthermore, an efficient cross metathesis has been employed for the preparation of the unusual branched R-(3)-hydroxy-13-methyltetradecanic acid and (R)-3-hexadecanoyloxy-15-methyl-hexadecanoic acid of P. gingivalis lipid A. Biological studies have shown that the synthetic lipid As can not activate human and mouse TLR2 and TLR4 to produce cytokines. However, it has been found that the compounds are potent antagonist of cytokine secretion by human monocytic cells induced by enteric LPS.

Differences in the pattern and chemical nature of fatty acids of lipid A of Neisseria meningitides lipooligosaccharides (LOS) and Escherichia coli lipopolysaccharides (LPS) may account for differences in inflammatory properties. Furthermore, there are indications that dimeric 3-deoxy-Dmanno-oct-2-ulosonic acid (KDO) moieties of LOS and LPS enhance biological activities. Heterogeneity in the structure of lipid A and possible contaminations with other inflammatory components have made it difficult to confirm these observations. To address these problems, a highly convergent approach for the synthesis of a lipid A derivative containing KDO has been developed, which relies on the ability to selectively remove or unmask in a sequential manner an isopropylidene acetal, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonate (Alloc), azide, and thexyldimethylsilyl (TDS) ether. The strategy was employed for the synthesis of N. meningitidis lipid A containing KDO (3). Mouse macrophages were exposed to the synthetic compound and its parent LOS, E. coli lipid A (2), and a hybrid derivative (4) that has the asymmetrical acylation pattern of E. coli lipid A, but the shorter lipids of meningococcal lipid A. The resulting supernatants were examined for tumor necrosis factor alpha (TNF-α) and interferon beta (IFN-β) production. The lipid A derivative containing KDO was much more active than lipid A alone and just slightly less active than its parent LOS, indicating that one KDO moiety is sufficient for full activity of TNF-α and IFN-β induction. The lipid A of N. meningitidis was a significantly more potent inducer of TNF-α and IFN-β than E. coli lipid A, which is due to a number of shorter fatty acids. The compounds did not demonstrate a bias towards a MyD88-or TRIF-dependent response.