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. Immunization with these vaccines in a simple water/oil emulsion produced a strong immune response in mice to which stimulation with complete Freund's adjuvant (CFA) was not superior. In both cases, high levels of IgG1 and IgG2a/b were induced in C57BL/6 mice. Additional glycosylation in the immunodominant PDTRP domain led to improved binding of the induced antisera to MCF-7 breast tumor cells, compared with that of the monoglycosylated peptide vaccine.

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. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4+ T-, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor-associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines.

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. This vaccine preparation elicited extraordinary titers of IgG antibodies which strongly bound human breast cancer cells expressing tumor-associated MUC1. Beside the humoral response, the poly(I:C) glycopeptide vaccine induced a pro-inflammatory environment, very important to overcome the immune-suppressive mechanisms, and elicited a strong cellular immune response crucial for tumor elimination.

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. Comparison of di- and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose-coupled vaccine and the non-mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells.

Enhancing the immunogenicity of an antitumour vaccine still poses a major challenge. It depends upon the selected antigen and the mode of its presentation. We here describe a fully synthetic antitumour vaccine, which addresses both aspects. For the antigen, a tumour-associated MUC1 glycopeptide as B-cell epitope was synthesised and linked to the immunostimulating T-cell epitope P2 derived from tetanus toxoid. The MUC1-P2 conjugate is presented multivalently on a hyperbranched polyglycerol to the immune system. In comparison to a related vaccine of lower multivalency, this vaccine exposing more antigen structures on the hyperbranched polymer induced significantly stronger immune responses in mice and elicited IgG antibodies of distinctly higher affinity to epithelial tumour cells.

Recent efforts towards the development of synthetic glycopeptide vaccines, which aim at the active immunization of patients against their own tumor tissues, are outlined. To achieve sufficient tumor selectivity, glycopeptides of the tandem repeat region of tumor-associated mucin, MUC1, have been synthesized. Since the endogenous structures usually exert low immunogenicity, these glycopeptide antigens, as B-cell epitopes, were conjugated with immunostimulating components. In the present short review, work is outlined in which the MUC1 B-cell epitope peptides are conjugated with bovine serum albumin ( BSA), keyhole limpet hemocyanin ( KLH), or tetanus toxoid ( TTox). In particular, the synthetic vaccines based on tetanus toxoid induce very strong tolerance-breaking immune responses in mice. The induced antibodies of the IgG type indicate the installation of an immunological memory. In addition, these antibodies strongly bind to human breast tumor cells in culture, demonstrated by flow cytometry experiments, and also to the tumor cells in mammary carcinoma tissues.

AbstractIn einem neuen Konzept für vollsynthetische Vakzine wird die Rolle von T‐Helferzellen hervorgehoben. In einer solchen synthetischen Antitumor‐Vakzine wurde ein zweifach glycosyliertes tumorassoziiertes MUC1‐Glycopeptid als B‐Zellepitop mit drei verschiedenen T‐Helferzell‐Epitopen durch Quadratsäurekonjugation zweier linearer (Glyco)Peptide kovalent verknüpft. In Mäusen löste die Impfung mit dieser Vier‐Komponenten‐Vakzine ohne zusätzliche Immunstimulantien etwa achtmal höhere MUC1‐spezifische Antikörpertiter aus als eine Vakzine, die nur ein T‐Helferzell‐Epitop enthielt. Diese ermutigenden Ergebnisse zeigen, dass die gleichzeitige Aktivierung von T‐Helferzellen verschiedener Spezifität nützlich für Anwendungen ist, die eine gesteigerte Immunogenität von Epitopen erfordern. Besonders in der personalisierten Medizin kann der flexible Aufbau der Vakzine als Vorbild dienen, wenn z. B. T‐Helferzell‐Epitope benötigt werden, die zum humanen Leukozytenantigen‐Typ (HLA) verschiedener Patienten passen.

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.

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.

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.

ChemInformVolume 45, Issue 34 Reviews ChemInform Abstract: Tumor-Associated Glycopeptide Antigens and Their Modification in Anticancer Vaccines Sebastian Hartmann, Sebastian Hartmann Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorBjoern Palitzsch, Bjoern Palitzsch Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorMarkus Glaffig, Markus Glaffig Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorHorst Kunz, Horst Kunz Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this author Sebastian Hartmann, Sebastian Hartmann Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorBjoern Palitzsch, Bjoern Palitzsch Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorMarkus Glaffig, Markus Glaffig Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this authorHorst Kunz, Horst Kunz Inst. Org. Chem., Johannes-Gutenberg-Univ., D-55128 Mainz, GermanySearch for more papers by this author First published: 07 August 2014 https://doi.org/10.1002/chin.201434266AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume45, Issue34August 26, 2014 RelatedInformation

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. The resulting fully synthetic vaccine induced strong immune responses in mice and IgG antibodies recognizing human breast-cancer cells.