Björn Palitzsch

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. Nanoparticles designed according to this report may become key players in the development of anticancer vaccines.

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.

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. This co-delivery promotes induction of specific MUC1-antibodies binding to human breast tumor cells without external adjuvant.

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. The promising results indicate that multiple activation of different T-helper cells is useful for applications in which increased immunogenicity is required. In personalized medicine, in particular, this flexible construction of a vaccine can serve as a role model, for example, when T-helper-cell epitopes are needed that match human leukocyte antigens (HLA) in different patients.

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. Structurally defined tumor-associated MUC1 cannot be isolated from tumor cells. We synthesized MUC1-glycopeptide vaccines and analyzed their structure-activity relationships in immunizations; a monoclonal antibody that specifically distinguishes between human normal and tumor epithelial cells was thus generated.

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. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.

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. In the current work, three MUC1 antitumor vaccine candidates conjugated with different immune stimulants are evaluated immunologically. For assessment of the influence of the immune stimulant on antibody recognition, a comprehensive library of mucin 1 glycopeptides (>100 entries) is synthesized and employed in antibody microarray profiling; these range from small tumor-associated glycans (TN , STN , and T-antigen structures) to heavily extended O-glycan core structures (type-1 and type-2 elongated core 1-3 tri-, tetra-, and hexasaccharides) glycosylated in variable density at the five different sites of the MUC1 tandem repeat. This is one of the most extensive glycopeptide libraries ever made through total synthesis. On tumor cells, the core 2 β-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched core 2 structures, which favor formation of linear core 1 or core 3 structures, and in particular, truncated tumor-associated antigen structures. The core 2 structures are commonly found on healthy cells and the elucidation of antibody cross-reactivity to such epitopes may predict the tumor-selectivity and safety of synthetic vaccines. With the extended mucin core structures in hand, antibody cross-reactivity toward the branched core 2 glycopeptide epitopes is explored. It is observed that the induced antibodies recognize MUC1 peptides with very high glycosylation site specificity. The nature of the antibody response is characteristically different for antibodies directed to glycosylation sites in either the immune-dominant PDTR or the GSTA domain. All antibody sera show high reactivity to the tumor-associated saccharide structures on MUC1. Extensive glycosylation with branched core 2 structures, typically found on healthy cells, abolishes antibody recognition of the antisera and suggests that all vaccine conjugates preferentially induce a tumor-specific humoral immune response.

Chapter 24:Tumour-associated glycopeptide antigens and their modification in anticancer vaccines

Glycopeptide antigens are obtained by solid-phase glycopeptide synthesis using fluorenylmethoxycarbonyl-(Fmoc)-protected O-glycosyl threonine and serine building blocks representing the tumour-associated mucin carbohydrate antigens. Conjugation of the synthetic mucin glycopeptide antigens with T-cell epitope peptides and/or immune stimulating lipopeptides affords fully synthetic two- and three-component vaccines useful for immunization of mice. Conjugates of the synthetic tumour-associated glycopeptide antigens with carrier proteins, in particular with tetanus toxoid, proved to be potent antitumour vaccines inducing high titres of IgG antibodies, which strongly bind to breast tumour cells. Mimics of the carbohydrate antigens within these glycopeptides also result in efficient vaccines as long as the carbohydrate structure remains closely related to the natural tumour-associated carbohydrate antigen.