From the Laboratory to the Clinic: A Retrospective on Fully Synthetic Carbohydrate-Based Anticancer Vaccines Frequently used abbreviations are listed in the appendix

This review provides an account of our explorations into oligosaccharide and glycoconjugate construction for the creation and evaluation of vaccines based on carbohydrate-centered tumor antigens. Our starting point was the known tendency of transformed cells to express selective carbohydrate motifs in the form of glycoproteins or glycolipids. Anticancer vaccines derived from carbohydrate-based antigens could be effective targets for immune recognition and attack. Obtaining significant quantities of such structures from natural sources is, however, extremely difficult. With the total synthesis of tumor-associated carbohydrate antigens accomplished, we began to evaluate at the clinical level whether the human immune system can respond to such fully synthetic antigens in a focused and useful way. Toward this goal, we have merged the resources of chemistry and immunology in an attack on the problem. The synthesis and immunoconjugation of various tumor-associated carbohydrate antigens and the results of such constructs in mice vaccinations will be described. For fashioning an effective vaccine, conjugation to a suitable immunogenic carrier was necessary and conjugates of KLH (keyhole limpet cyanin) have consistently demonstrated the relevant immunogenicity. Preclinical and clinical studies with synthetic conjugate carbohydrate vaccines show induction of IgM- and IgG-antibody responses. Another approach to anticancer vaccines involves the use of clustered glycopeptides as targets for immune attack. Initial attention has been directed to mucin related O-linked glycopeptides. Synthetic trimeric clusters of glycoepitopes derived from the Tn-, TF- and Lewis(y)-antigens, appropriately bioconjugated, have been demonstrated to be immunogenic. The hope is that patients immunized in an adjuvant manner with synthetic carbohydrate vaccines would produce antibodies reactive with cancer cells and that the production of such antibodies would mitigate against tumor spread, thereby enabling a more favorable survival and "quality of life" prognosis.     

Pursuit of optimal carbohydrate-based anticancer vaccines: preparation of a multiantigenic unimolecular glycopeptide containing the Tn, MBr1, and Lewis(y) antigens.

A novel preparation of nonnatural glycoamino acids starting from n-pentenyl glycosides is described. The approach involves a Horner-Emmons olefination with a suitably protected glycine-derived phosphonate, followed by catalytic asymmetric hydrogenation, which proceeds with excellent diastereomeric selectivity. The synthetic methodology was useful for the preparation of glycoamino acids containing the Tn antigen, the MBr1 antigen (Globo-H), the Le(y) antigen, and lactose. These glycoamino acids can also serve as units for peptide synthesis. The synthesis of polyvalent glycopeptides containing three different antitumor antigens is described (28 and 29), and these have been prepared for conjugation to carrier protein in order to access the immunogenicity for tumor immunotherapy applications.     

A Cancer Therapeutic Vaccine based on Clustered Tn‐Antigen Mimetics Induces Strong Antibody‐Mediated Protective Immunity

Tumor‐associated carbohydrate antigens (TACAs) are key components of cancer vaccines. A variety of vaccines based on native TACAs such as α‐Tn have shown immunogenicity and protection in preclinical animal studies, however, their weak immunogenicity, low in vivo instability, and poor bioavailability, have discouraged their further evaluations in clinical studies. A new improved vaccine prototype is reported. It is composed of four clustered Tn‐antigen mimetics and a immunogenic peptide epitope that are conjugated to a cyclopeptide carrier. The immunization of mice with this vaccine 1) was safe, 2) induced a strong and long‐lasting Tn‐specific response with IgM/IgG antibodies able to recognize native carbohydrate antigens; 3) produced high titers of IgG1, IgG2a, and IgG3 antibodies; and 4) produced a significant antibody‐dependent regression of tumors and conferred protection. Altogether, these findings pave the way for the clinical development of safe and effective therapeutic vaccines against Tn‐expressing cancers.     

Immunology of O-glycosylated proteins: approaches to the design of a MUC1 glycopeptide-based tumor vaccine

Until about 1990 there was general consent about the assumption that only protein and peptide antigens have the capacity of CD4(+) or CD8(+) T-cell stimulation. Since about ten years evidence is now accumulating that carbohydrate-peptide epitopes do play a role in classical MHC-mediated immune responses. This holds true for glycopeptides, where the glycan chain is short and not located at an "anchor residue" needed for MHC interaction. T-cell recognition of O-glycosylated peptides is potentially of high biomedical significance, because it can mediate the immune protection against microorganisms, the vaccination in anti-tumor therapies, but also some aspects of autoimmunity. The epithelial type 1 transmembrane mucin MUC1 is established as a marker for monitoring recurrence of breast cancer and is a promising target for immunotherapeutic strategies to treat cancer by active specific immunization. Natural human immune responses to the tumor-associated glycoforms of the mucin indicate that antibody reactivities are more directed to glycopeptide than to non-glycosylated peptide epitopes. To overcome the weak immunogenicity of the natural target, heavily O-glycosylated MUC1, the question was addressed whether O-linked glycans remain intact during processing in the MHC class II pathway and interfere with endosomal processing and peptide presentation. Attempts were made to define on a biochemical level the structural requirements for an efficient endosomal proteolysis catalyzed by cathepsin L in antigen-presenting cells. Evidence based on work with CD4(+) T-hybridomas confirms that O-glycopeptides can be effectively presented to T-cells and that glycans can form integral parts of the TCR defined epitopes. Similar approaches are currently followed in the MHC class I pathway which aim at the identification of immunogenic glycopeptides generated by immunoproteasomes.     

Injectable microencapsulated islet cells as a bioartificial pancreas

Rat islets encapsulated in semipermeable membranes remained viable in culture for 4 months. Multiple allotransplants of islets encapsulated in alginate-polylysine-polyethyleneimine membranes restored normoglycemia in recipient diabetic rats for most of a 90-day experimental period. Each individual transplant restored normal fasting plasma glucose levels for 15–20 d. The failure of the encapsulated islets was caused by an inflammatory response induced by polyethyleneimine. In contrast a single transplant of islets encapsulated in a biocompatible alginate-polylysine-alginate membrane restored normoglycemia in recipient animals for up to 10 months. Capsules with intact membranes and containing viable islets were recovered from the abdominal cavity 5 months post-transplantation. SEM studies on capsule membranes revealed essentially smooth surfaces. Differences between wet and dry wall thicknesses indicated that the membrane is a hydrogel, 4.00±0.28 μm thick in an aqueous environment. The clinical potential of transplanting cells encapsulated in biocompatible semipermeable hydrogel membranes is demonstrated by this study.     

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response–Inducing Efficacy of Sustained Antigen Release Alone

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy‐to‐use, non‐immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real‐life infections. Recombinant elastin‐like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross‐linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd‐containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next‐generation vaccines.     

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases.     

Emulsion membrane systems for preconcentration of uranium

Various double emulsion systems with liquid membranes containing tri-n-octylphosphine oxide /TOPO/, tri-n-butylphosphate /TBP/, KELEX 100^® and di-2-ethylhexylphosphoric acid /DEHPA/, as carriers for uranium/VI/ pertraction, were studied. The conditions were found at which the systems are most efficient, comparing with solvent extraction. The use of DEHPA as a membrane carrier with solutions of H₂SO₄ and H₃PO₄ encapsulated in the membrane was substanfiated.     

Recent Development in Carbohydrate Based Anticancer Vaccines

The development of carbohydrate-based anticancer vaccines is of high current interest. Herein, the latest development in this exciting field is reviewed. After a general introduction about tumor-associated carbohydrate antigens and immune responses, the review focuses on the various strategies that have been developed to enhance the immunogenicity of these antigens. The results from animal studies and clinical trials are presented.     

A novel linker methodology for the synthesis of tailored conjugate vaccines composed of complex carbohydrate antigens and specific TH-cell peptide epitopes

Pathogenic organisms or oncogenically transformed cells often express complex carbohydrate structures at their cell surface, which are viable targets for active immunotherapy. We describe here a novel, immunologically neutral, linker methodology for the efficient preparation of highly defined vaccine conjugates that combine complex saccharide antigens with specific TH-cell peptide epitopes. This novel heterobifunctional approach was employed for the conjugation of a (1-->2)-beta-mannan trisaccharide from the pathogenic fungus Candida albicans as well as the carbohydrate portion of tumor-associated ganglioside GM2 to a TH-cell peptide epitope derived from the murine 60 kDa self heat-shock protein (hsp60). Moreover, the linkage chemistry has proven well suited for the synthesis of more complex target structures such as a biotinylated glycopeptide, a three component vaccine containing an immunostimulatory peptide epitope from interleukin-1 beta (IL-1 beta), and for the conjugation of complex carbohydrates to carrier proteins such as bovine serum albumin.     

Chemical and chemoenzymatic synthesis of S-linked ganglioside analogues and their protein conjugates for use as immunogens

Analogues of the tumor-associated gangliosides GM(3) and GM(2) containing terminal S-linked neuraminic acid residues and an amino terminated, truncated ceramide homologue have been synthesized and conjugated to a protein. The synthesis involved coupling of a S-linked sialyl alpha(2-->3) galactose disaccharide with a glucosyl sphingosine analogue, followed by elaboration and deprotection to give amino-terminated glycosyl ceramide 1. Glycosyltransferase-catalyzed extension of the trisaccharide 1 provided access to the modified GM(2) tetrasaccharide 2 or sulphur-containing GD(3) analogue 30. Owing to their potentially enhanced resistance to endogenous exo-glycoside hydrolases and their inherent non-self character, carbohydrate antigens containing non-reducing terminal thioglycosidic linkages may be more immunogenic than O-linked antigens and may stimulate the production of antibodies capable of recognizing naturally occurring oligosaccharides. Our initial results suggest that in fact these antigens are viable immunogens and furthermore, that immune sera cross reacts with O-gangliosides in the context of a heterologous glycoprotein conjugate.     

Recent progress of fully synthetic carbohydrate-based vaccine using TLR agonist as build-in adjuvant

This review highlights recent advances in developing full synthetic carbohydrate antigen based vaccines, with an emphasis on the structure-activity relationships that provide a primary basis for future vaccine design and immunotherapy developing.     

Identification of Potential Vaccine Candidates Against Streptococcus pneumoniae by Reverse Vaccinology Approach

In the past few decades, genome-based approaches have contributed significantly to vaccine development. Our aim was to identify the most conserved and immunogenic antigens of Streptococcus pneumoniae, which can be potential vaccine candidates in the future. BLASTn was done to identify the most conserved antigens. PSORTb 3.0.2 was run to predict the subcellular localization of the proteins. B cell epitope prediction was done for the immunogenicity testing. Finally, BLASTp was done for verifying the extent of similarity to human proteome to exclude the possibility of autoimmunity. Proteins failing to comply with the set parameters were filtered at each step. Based on the above criteria, out of the initial 22 pneumococcal proteins selected for screening, pavB and pullulanase were the most promising candidate proteins.     

Chemoenzymatic synthesis of sialylated glycopeptides derived from mucins and T-cell stimulating peptides.

The Tn, T, sialyl-Tn, and 2,3-sialyl-T antigens are tumor-associated carbohydrate antigens expressed on mucins in epithelial cancers, such as those affecting the breast, ovary, stomach, and colon. Glycopeptides carrying these antigens are of interest for development of cancer vaccines and a short, chemoenzymatic strategy for their synthesis is reported. Building blocks corresponding to the Tn (GalNAc alpha-Ser/Thr) and T [Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens, which are relatively easy to obtain by chemical synthesis, were prepared and then used in the synthesis of glycopeptides on the solid phase. Introduction of sialic acid to give the sialyl-Tn [Neu5Ac alpha(2-->6)GalNAc alpha-Ser/Thr] and 2,3-sialyl-T [Neu5Ac alpha(2-->3)Gal beta(1-->3)GalNAc alpha-Ser/Thr] antigens is difficult when performed chemically at the building block level. Sialylation was therefore carried out with recombinant sialyltransferases in solution after cleavage of the Tn and T glycopeptides from the solid phase. In the same manner, the core 2 trisaccharide [Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc] was incorporated in glycopeptides containing the T antigen by using a recombinant N-acetylglucosaminyltransferase. The outlined chemoenzymatic approach was applied to glycopeptides from the tandem repeat domain of the mucin MUC1, as well as to neoglycosylated derivatives of a T cell stimulating viral peptide.     

Synthesis of N-modified sTn analogs and evaluation of their immunogenicities by microarray-based immunoassay

A series of sTn derivative-keyhole limpet hemocyanin (KLH) conjugates were synthesized, and their immunogenicities were evaluated by corresponding IgG production. To achieve a high-throughput screening immunoassay, a glycan microarray with sTn and its analogs was used to detect the production of corresponding antibodies in mouse sera. The immunoassay results revealed that the derived sTn antigens are generally more immunogenic than the parent sTn antigen. The N-propionyl sTn antigen was the most immunogenic among the sTn derivatives investigated, and its antiserum was cross-reactive with natural sTn. These results indicate that N-propionyl sTn may serve as a viable vaccine candidate to produce antibody for detection of sTn antigen.     

Formation of lactones from sialylated MUC1 glycopeptides

The tumor-associated carbohydrate antigens TN, T, sialyl TN and sialyl T are expressed on mucins in several epithelial cancers. This has stimulated studies directed towards development of glycopeptide-based anticancer vaccines. Formation of intramolecular lactones involving sialic acid residues and suitably positioned hydroxyl groups in neighboring saccharide moieties is known to occur for glycolipids such as gangliosides. It has been suggested that these lactones are more immunogenic and tumor-specific than their native counterparts and that they might find use as cancer vaccines. We have now investigated if lactonization also occurs for the sialyl TN and T antigens of mucins. It was found that the model compound sialyl T benzyl glycoside , and the glycopeptide Ala-Pro-Asp-Thr-Arg-Pro-Ala from the tandem repeat of the mucin MUC1, in which Thr stands for the 2,3-sialyl-T antigen, lactonized during treatment with glacial acetic acid. Compound gave the 1'--> 2' lactone as the major product and the corresponding 1'--> 4' lactone as the minor product. For glycopeptide the 1'--> 4' lactone constitued the major product, whereas the 1'--> 2' lactone was the minor one. When lactonized was dissolved in water the 1'--> 4' lactone underwent slow hydrolysis, whereas the 1'--> 2' remained stable even after a 30 days incubation. In contrast the corresponding 2,6-sialyl-TN glycopeptide did not lactonize in glacial acetic acid.     

Biomimetic synthesis of the tumor-associated (2,3)-sialyl-T antigen and its incorporation into glycopeptide antigens from the mucins MUC1 and MUC4

Glycoproteins on epithelial tumor cells often exhibit aberrant glycosylation profiles. The incomplete formation of the glycan side chains resulting from a down-regulated glucosamine transfer and a premature sialylation results in additional peptide epitopes, which become accessible to the immune system in mucin-type glycoproteins. These cancer-specific structure alterations are considered to be a promising basis for selective immunological attack on tumor cells. Among the tumor-associated saccharide antigens, the (2,3)-sialyl-T antigen has been identified as the most abundant glycan, found in several different carcinoma cell lines. According to a linear biomimetic strategy, the (2,3)-sialyl-T antigen was synthesized by a stepwise glycan chain extension of a protected galactosamine-threonine precursor. For the construction of immunostimulating antigens combining both peptide and saccharide motifs, this antigen was incorporated into glycopeptide partial structures from the mucins MUC1 and MUC4 by sequential solid-phase synthesis.     

Design and synthesis of trivalent Tn glycoconjugate polymers by nitroxide-mediated polymerization

A new synthetic method for preparing Tn glycoconjugate polymers, containing tumor-associated carbohydrate antigens, by controlled living radical polymerization is reported. To mimic the authentic structures of Tn glycopeptide antigens and to explore the controlled living radical polymerization, three tumor-associated carbohydrate antigens (GalNAc, GalNAcα1-O-Ser, and GalNAcα1-O-Thr) were attached to a styrene-type monomer through a diethylene glycol spacer. Under nitroxide-mediated polymerization, controlled living radical polymerization proceeded to afford defined glycopeptide polymers with different Tn densities and compositions. The polydispersity index (PDI) and molecular weights were increased and conversions were decreased upon increasing the concentration of Tn glycoconjugate monomers. The resulting Tn glycoconjugate polymers were characterized by NMR and IR. The spectral data indicate that the Tn glycoconjugate moiety did attach to the polymer chain and Tn glycoconjugate density could be adjusted through the nitroxide-mediated polymerization conditions. The number of Tn units containing in the polymer chains could be estimated by NMR integration. This synthetic approach provides a new and efficient tool for constructing novel Tn glycoconjugate polymers.     

Preparation and characterization of polyvinyl alcohol/β‐cyclodextrin/GO‐Ag nanocomposite with improved antibacterial and strength properties

In this work, new hydrogel films based on polyvinyl alcohol (PVA) and β‐cyclodextrin (β‐CD) were prepared with the aim of studying their ability as an antibacterial and drug carrier system. Gallic acid (GA) was used as an antibacterial drug which was encapsulated into the β‐CD cavity, and finally, β‐CD inclusion complex (GA/βCD‐IC) was prepared. On the other hand, silver nanoparticles (AgNPs) were synthesized on the graphene oxide (GO) surface (GO‐Ag), and the obtained GO‐Ag was used to enhance the antibacterial properties and mechanical strength of their films. FT‐IR and DSC analysis approved the formation of cross‐linking with glutaraldehyde between the PVA and β‐CD. Hydrogel films were characterized using XRD and SEM. The disc diffusion method showed the antibacterial activity for the films containing GO‐Ag and GA. Due to the good strength, elasticity, WVP, and swelling ability, PVA/GA/βCD‐IC/GO‐Ag can be proposed as a potential antibacterial drug delivery system.     

Antigenic and immunogenic phage displayed mimotopes as substitute antigens: applications and limitations

The most exciting potential of phage displayed peptide libraries is to obtain small peptide molecules that mimic an antigen, at least with respect to a particular epitope. In addition to their interest as research tools, such mimotopes could in principle be useful as diagnostic tools or for eliciting antibodies to a predefined epitope. However, the reduction of the phage insert sequence to a short peptide that can compete with the antigenic and in particular with the immunogenic properties of the natural antigen faces considerable difficulties. This review assesses critically the antigenicity of phage displayed peptides as free peptides and in different molecular environments. The difficulties to use mimotopes to induce antibodies that bind to the natural antigen (crossreactive immunogenicity) and the considerable discrepancy between antigenicity and immunogenicity of phage-derived peptides are discussed. Peptides selected with antibodies from phage displayed random peptide libraries have raised considerable expectations as low molecular weight substitutes of the natural antigen. This review will focus on the results of phage displayed random peptide libraries screened with antibodies specific for proteins, carbohydrates and nucleic acids and critically examine how the above expectations have been met.