The Development of Vaccines from Synthetic Tumor-Associated Mucin Glycopeptides and their Glycosylation-Dependent Immune Response.

Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood.

Specificity of human natural antibodies referred to as anti-Tn.

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g.

Evaluation of a novel monoclonal antibody against tumor-associated MUC1 for diagnosis and prognosis of breast cancer.

There is still a great unmet medical need concerning diagnosis and treatment of breast cancer which could be addressed by utilizing specific molecular targets. Tumor-associated MUC1 is expressed on over 90 % of all breast cancer entities and differs strongly from its physiological form on epithelial cells, therefore presenting a unique target for breast cancer diagnosis and antibody-mediated immune therapy. Utilizing an anti-tumor vaccine based on a synthetically prepared glycopeptide, we generated a monoclonal antibody (mAb) GGSK-1/30, selectively recognizing human tumor-associated MUC1.

Reduced Breast Tumor Growth after Immunization with a Tumor-Restricted MUC1 Glycopeptide Conjugated to Tetanus Toxoid.

Preventive vaccination against tumor-associated endogenous antigens is considered to be an attractive strategy for the induction of a curative immune response concomitant with a long-lasting immunologic memory. The mucin MUC1 is a promising tumor antigen, as its tumor-associated form differs from the glycoprotein form expressed on healthy cells. Due to aberrant glycosylation in tumor cells, the specific peptide epitopes in its backbone are accessible and can be bound by antibodies induced by vaccination.

Synthetic MUC1 Antitumor Vaccine with Incorporated 2,3-Sialyl-T Carbohydrate Antigen Inducing Strong Immune Responses with Isotype Specificity.

The endothelial glycoprotein MUC1 is known to underlie alterations in cancer by means of aberrant glycosylation accompanied by changes in morphology. The heavily shortened glycans induce a collapse of the peptide backbone and enable accessibility of the latter to immune cells, rendering it a tumor-associated antigen. Synthetic vaccines based on MUC1 tandem repeat motifs, comprising tumor-associated 2,3-sialyl-T antigen, conjugated to the immunostimulating tetanus toxoid, are reported herein.

A Synthetic MUC1 Anticancer Vaccine Containing Mannose Ligands for Targeting Macrophages and Dendritic Cells.

A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose-receptor-positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non-mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4 T cells in the local lymph organs.

Immunization with a Synthetic Human MUC1 Glycopeptide Vaccine against Tumor-Associated MUC1 Breaks Tolerance in Human MUC1 Transgenic Mice.

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen.

Immunogenicity of a Fully Synthetic MUC1 Glycopeptide Antitumor Vaccine Enhanced by Poly(I:C) as a TLR3-Activating Adjuvant.

Fully synthetic MUC1 glycopeptide antitumor vaccines have a precisely specified structure and induce a targeted immune response without suppression of the immune response when using an immunogenic carrier protein. However, tumor-associated aberrantly glycosylated MUC1 glycopeptides are endogenous structures, "self-antigens", that exhibit only low immunogenicity. To overcome this obstacle, a fully synthetic MUC1 glycopeptide antitumor vaccine was combined with poly(inosinic acid:cytidylic acid), poly(I:C), as a structurally defined Toll-like receptor 3 (TLR3)-activating adjuvant.

Polymeric Selectin Ligands Mimicking Complex Carbohydrates: From Selectin Binders to Modifiers of Macrophage Migration.

Novel polymeric cell adhesion inhibitors were developed in which the selectin tetrasaccharide sialyl-Lewis (SLe ) is multivalently presented on a biocompatible poly(2-hydroxypropyl)methacrylamide (PHPMA) backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). For comparison, corresponding polymeric glycomimetics were prepared in which the crucial "single carbohydrate" substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, as they are derived from the same precursor polymer.

Microarray Analysis of Antibodies Induced with Synthetic Antitumor Vaccines: Specificity against Diverse Mucin Core Structures.

Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases.

Protein kinase CK2 governs the molecular decision between encephalitogenic TH17 cell and Treg cell development.

T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance.

Synthetic MUC1 Antitumor Vaccine Candidates with Varied Glycosylation Pattern Bearing R/S-configured Pam3 CysSerLys4.

The Toll-like receptor 2 ligand Pam3 CysSer is of particular interest for the construction synthetic vaccines because of its ability to stimulate of the innate immune system. Such vaccines usually comprise Pam3 CysSer with the natural R-configuration at the glycerol 2-position. Pam3 CysSer peptide vaccines with natural configuration have been shown to be more efficient than the corresponding R/S diastereomers.

Glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin-1 glycoprotein.

We report the preparation of gold nanoparticle (AuNP)-based vaccine candidates against the tumor-associated form of the mucin-1 (MUC1) glycoprotein. Chimeric peptides, consisting of a glycopeptide sequence derived from MUC1 and the T-cell epitope P30 sequence were immobilized on PEGylated AuNPs and the ability to induce selective antibodies in vivo was investigated. After immunization, mice showed significant MHC-II mediated immune responses and their antisera recognized human MCF-7 breast cancer cells.

A Synthetic Glycopeptide Vaccine for the Induction of a Monoclonal Antibody that Differentiates between Normal and Tumor Mammary Cells and Enables the Diagnosis of Human Pancreatic Cancer.

In studies within the realm of cancer immunotherapy, the synthesis of exactly specified tumor-associated glycopeptide antigens is shown to be a key strategy for obtaining a highly selective biological reagent, that is, a monoclonal antibody that completely differentiates between tumor and normal epithelial cells and specifically marks the tumor cells in pancreas tumors. Mucin MUC1, which is overexpressed in many prevalent cancers, was identified as a promising target for this strategy. Tumor-associated MUC1 differs significantly from that expressed by normal cells, in particular by altered glycosylation.

Antibody induction directed against the tumor-associated MUC4 glycoprotein.

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma.

CpG-loaded multifunctional cationic nanohydrogel particles as self-adjuvanting glycopeptide antitumor vaccines.

Self-adjuvanting antitumor vaccines by multifunctional cationic nanohydrogels loaded with CpG. A conjugate consisting of tumor-associated MUC1-glycopeptide B-cell epitope and tetanus toxin T-cell epitope P2 is linked to cationic nanogels. Oligonucleotide CpG complexation enhances toll-like receptor (TLR) stimulated T-cell proliferation and rapid immune activation.

A fully synthetic four-component antitumor vaccine consisting of a mucin glycopeptide antigen combined with three different T-helper-cell epitopes.

In a new concept of fully synthetic vaccines, the role of T-helper cells is emphasized. Here, a synthetic antitumor vaccine consisting of a diglycosylated tumor-associated MUC1 glycopeptide as the B-cell epitope was covalently cross-linked with three different T-helper-cell epitopes via squaric acid ligation of two linear (glyco)peptides. In mice this four-component vaccine administered without external immune-stimulating promoters elicit titers of MUC1-specific antibodies that were about eight times higher than those induced by a vaccine containing only one T-helper-cell epitope.

A fully synthetic glycopeptide antitumor vaccine based on multiple antigen presentation on a hyperbranched polymer.

For antitumor vaccines both the selected tumor-associated antigen, as well as the mode of its presentation, affect the immune response. According to the principle of multiple antigen presentation, a tumor-associated MUC1 glycopeptide combined with the immunostimulating T-cell epitope P2 from tetanus toxoid was coupled to a multi-functionalized hyperbranched polyglycerol by "click chemistry". This globular polymeric carrier has a flexible dendrimer-like structure, which allows optimal antigen presentation to the immune system.

Synthetic multivalent glycopeptide-lipopeptide antitumor vaccines: impact of the cluster effect on the killing of tumor cells.

Multivalent synthetic vaccines were obtained by solid-phase synthesis of tumor-associated MUC1 glycopeptide antigens and their coupling to a Pam3 Cys lipopeptide through click reactions. These vaccines elicited immune responses in mice without the use of any external adjuvant. The vaccine containing four copies of a MUC1 sialyl-TN antigen showed a significant cluster effect.

Water-soluble polymers coupled with glycopeptide antigens and T-cell epitopes as potential antitumor vaccines.

Highly decorated: Tumor-associated MUC1 glycopeptide and tetanus toxoid T-cell epitope P2 can be attached to water-soluble poly(N-(2-hydroxypropyl)methacrylamide) carriers by orthogonal ligation techniques. Fully synthetic vaccine A with additional nanostructure-promoting domains induced antibodies that exhibit high affinity to tumor cells.

Natural product and material chemistries--separated forever?

Chemistry research is an eloquent, yet extremely complex discipline consisting of a diverse range of topics. The complexity of every sub-discipline requires extensive focus, which can limit cross-talk between fields, thus leading to their isolation. In particular, natural product and material chemistries have experienced this trend, and it has led to an ever growing separation between them.

Self-adjuvanting synthetic antitumor vaccines from MUC1 glycopeptides conjugated to T-cell epitopes from tetanus toxoid.

The T-helper epitope peptide P30 (green in the scheme) from tetanus toxoid was used as the immunostimulant in MUC1 glycopeptide antitumor vaccines and apparently also acts as a built-in adjuvant. P30-conjugated glycopeptide vaccines containing three glycans in the immunodominant motifs PDTRP and GSTAP induced much stronger immune responses and complement dependent cytotoxicity mediated killing of tumor cells when applied in plain PBS solution without complete Freund's adjuvant.

C-Glycosyl amino acids through hydroboration-cross-coupling of exo-glycals and their application in automated solid-phase synthesis.

O-Glycosylation is one of the most important post-translational modifications of proteins. The attachment of carbohydrates to the peptide backbone influences the conformation as well as the solubility of the conjugates and can even be essential for binding to specific ligands in cell-cell interactions or for active transport over membranes. This makes glycopeptides an interesting class of compounds for medical applications.

The development of synthetic antitumour vaccines from mucin glycopeptide antigens.

Based on important cell-biological and biochemical results concerning the structural difference between membrane glycoproteins of normal epithelial cells and epithelial tumour cells, tumour-associated glycopeptide antigens have been chemically synthesised and structurally confirmed. Glycopeptide structures of the tandem repeat sequence of mucin MUC1 of epithelial tumour cells constitute the most promising tumour-associated antigens. In order to generate a sufficient immunogenicity of these endogenous structures, usually tolerated by the immune system, these synthetic glycopeptide antigens were conjugated to immune stimulating components: in fully synthetic two-component vaccines either with T-cell peptide epitopes or with Toll-like receptor2 lipopeptide ligands or in three-component vaccines with both these stimulants.

Fully synthetic self-adjuvanting thioether-conjugated glycopeptide-lipopeptide antitumor vaccines for the induction of complement-dependent cytotoxicity against tumor cells.

Glycopeptides of tumor-associated mucin MUC1 are promising target structures for the development of antitumor vaccines. Because these endogenous structures were weakly immunogenic, they were coupled to immune-response-stimulating T-cell epitopes and the Pam(3)Cys lipopeptide to induce strong immune responses in mice. A new thioether-ligation method for the synthesis of two- and three-component vaccines that contain MUC1 glycopeptides as the B-cell epitopes, a T-cell epitope peptide, and the Pam(3)CSK(4) lipopeptide is described.