Synthetic oligosaccharides and glycoconjugates are increasingly used as probes for biological research and as lead compounds for drug and vaccine discovery. These endeavors are, however, complicated by a lack of general methods for the routine preparation of this important class of compounds. Recent development such as one-pot multi-step protecting group manipulations, the use of unified monosaccharide building blocks, the introduction of stereoselective glycosylation protocols, and convergent strategies for oligosaccharide assembly, are beginning to address these problems. Furthermore, oligosaccharide synthesis can be facilitated by chemo-enzymatic methods, which employ a range of glycosyl transferases to modify a synthetic oligosaccharide precursor. Glycosynthases, which are mutant glycosidases, that can readily form glycosidic linkages are addressing a lack of a wide range glycosyltransferases. The power of carbohydrate chemistry is highlighted by an ability to synthesize glycoproteins.There is a growing appreciation that posttranslational modifications, such as glycosylation, dramatically increase protein complexity and function. [1][2][3][4][5][6] For example, almost all cell surface and secreted proteins are modified by covalently-linked carbohydrate moieties and the glycan structures on these glycoproteins have been implicated as essential mediators in processes such as protein folding, cell signaling, fertilization, embryogenesis, neuronal development, hormone activity, and the proliferation of cells and their organization into specific tissues. In addition, overwhelming data supports the relevance of glycans in pathogen recognition, inflammation, innate immune responses, and the development of autoimmune diseases and cancer. [7][8][9][10] The importance of protein glycosylation is also underscored by the developmental abnormalities observed in a growing number of human disorders known as Congenital Disorders of Glycosylation caused by defects in the glycosylation machinery. 11Polysaccharides are major constituents of the microbial cell surfaces and, for example, the bacterial cell wall can contain relatively large amounts of capsular polysaccharides (CPS) or lipopolysaccharides (LPS). 12 These components are important virulence factors by promoting bacterial colonization, blocking phagocytosis, and interfering with leukocyte migration and adhesion. CPS and LPS can be recognized by receptors of the innate immune system leading to the production of cytokines, chemokines, and cellular adhesion molecules.13 -16 With a few exceptions, bacterial polysaccharides can induce an adaptive immune response and, not surprisingly, bacterial saccharides have been employed for the development of vaccines for several pathogens.17 -20

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 mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam 3 CysSK 4 , a peptide T helper epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.cancer vaccine | multicomponent | chemical synthesis | Tn antigen

A Lewis(y) (Le(y)) tetrasaccharide modified by an artificial aminopropyl spacer was synthesized by a highly convergent approach that employed a levulinoyl ester and a 9-fluorenylmethoxycarbonate for temporary protection of the hydroxy groups and a trichloroethyloxycarbonyl as an amino protecting group. The artificial aminopropyl moiety was modified by a thioacetyl group, which allowed efficient conjugation to keyhole limpet hemocyanin (KLH) modified by electrophilic 4-(maleimidomethyl)cyclohexane-1-carboxylate (MI). Mice were immunized with the KLH-MI-Le(y) antigen. A detailed analysis of sera by ELISA established that a strong immunoglobulin G (IgG) antibody response was elicited against the linker region. The use of a smaller and more flexible 3-(bromoacetamido)propionate for the attachment of Le(y) to KLH not only reduced the IgG antibody response against the linker but also led to a significantly improved immune response against the Le(y) antigen. This study shows that highly antigenic linkers suppress antibody responses to weak antigens such as self-antigens.

Aberrant glycosylation of glycoproteins and glycolipids of cancer cells, which correlates with poor survival rates, is being exploited for the development of immunotherapies for cancer. In particular, advances in the knowledge of cooperation between the innate and adaptive system combined with the implementation of efficient synthetic methods for assembly of oligosaccharides and glycopeptides is providing avenues for the rationale design of vaccine candidates. In this respect, fully synthetic vaccine candidates show great promise because they incorporate only those elements requires for relevant immune responses, and hence do not suffer from immune suppression observed with classical carbohydrate-protein conjugate vaccines. Such vaccines are chemically well-defined and it is to be expected that they can be produced in a reproducible fashion. In this review article, recent advances in the development of fully synthetic sub-unit carbohydrate-based cancer vaccines will be discussed.