Markus Glaffig

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.

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. 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.

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. 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.

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.

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. 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.

Synthetic Antitumor Vaccines Through Coupling of Mucin Glycopeptide Antigens to Proteins

The requirements for coupling reactions of carbohydrate molecules very much depend upon the biological recognition processes that should be investigated and upon the target structures of the desired carbohydrate ligand. If the carbohydrate conjugate itself is the recognized ligand, as for example, the binding site of a P-selectin ligand comprising sialyl-LewisX and a specific peptide sequence, the natural glycoside bond must be installed. A stereoselective and regioselective block glycosylation between a sialyl-LewisX trichloroacetimidate and a partially deprotected Thomsen–Friedenreich antigen derivative was developed to achieve this aim. In contrast, the coupling reactions by which glycopeptides from tumor-associated glycoproteins are conjugated to immune stimulating components in order to afford efficient vaccines can entail artificial linkages as long as they do not interfere with the immune reactions. For example, the coupling of glycophorin glycopeptides to bovine serum albumin was successfully achieved by carboxylic activation with a water-soluble carbodiimide in the presence of a supernucleophilic additive. This conjugation method is only recommendable if the glycopeptide does not contain several carboxylic and/or amino functions. The photochemically or radical initiator promoted thiol-ene coupling succeeded in couplings of MUC1 glycopeptide antigens to bovine serum albumin, however, is accompanied by oxidative disulfide formation. The conjugation of glycopeptide antigens from the tandem repeat region of the tumor-associated mucin MUC1 to bovine serum albumin or tetanus toxoid is efficiently accomplished using diethyl squarate as the coupling reagent. The intermediate squaric monoamide esters can be isolated and characterized, and then applied to a mild connecting process to the carrier proteins. The MUC1 glycopeptide-tetanus toxoid conjugates proved to be particularly useful vaccines. They induce extraordinarily strong immune responses in mice. The induced antibodies are prevailingly of the IgG1 isotype and show efficient binding to the glycoproteins exposed on epithelial tumor cells.

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. Immunization with these vaccines in a simple water/oil emulsion produced a strong immune response in mice to which stimulation with complete Freunds 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.