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 Pam₃Cys 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‐T_N antigen showed a significant cluster effect. It induced in mice prevailing IgG_2a antibodies, which bind to MCF‐7 breast tumor cells and initiate the killing of these tumor cells by activation of the complement‐dependent cytotoxicity complex.     

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 Pam₃Cys 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 Pam₃CSK₄ lipopeptide is described. The resulting fully synthetic vaccines were used for the vaccination of mice, either in a liposome with Freund′s adjuvant or in aqueous PBS buffer. The three‐component vaccines that contained the Tetanus Toxoid P2 T‐cell epitope peptide induced strong immune responses, even when administered just in PBS. By activation of the complement‐dependent cytotoxicity (CDC) complex, the antisera induced the killing of tumor cells.     

Liposomal Antitumor Vaccines Targeting Mucin 1 Elicit a Lipid‐Dependent Immunodominant Response

The tumor‐associated antigen mucin 1 (MUC1) has been pursued as an attractive target for cancer immunotherapy, but the poor immunogenicity of the endogenous antigen hinders the development of vaccines capable of inducing effective anti‐MUC1 immunodominant responses. Herein, we prepared synthetic anti‐MUC1 vaccines in which the hydrophilic MUC1 antigen was N‐terminally conjugated to one or two palmitoyl lipid chains (to form amphiphilic Pam‐MUC1 or Pam₂‐MUC1). These amphiphilic lipid‐tailed MUC1 antigens were self‐assembled into liposomes containing the NKT cell agonist αGalCer as an adjuvant. The lipid‐conjugated antigens reshaped the physical and morphological properties of liposomal vaccines. Promising results showed that the anti‐MUC1 IgG antibody titers induced by the Pam₂‐MUC1 vaccine were more than 30‐ and 190‐fold higher than those induced by the Pam‐MUC1 vaccine and the MUC1 vaccine without lipid tails, respectively. Similarly, vaccines with the TLR1/2 agonist Pam₃CSK₄ as an adjuvant also induced conjugated lipid‐dependent immunological responses. Moreover, vaccines with the αGalCer adjuvant induced significantly higher titers of IgG antibodies than vaccines with the Pam₃CSK₄ adjuvant. Therefore, the non‐covalent assembly of the amphiphilic lipo‐MUC1 antigen and the NKT cell agonist αGalCer as a glycolipid adjuvant represent a synthetically simple but immunologically effective approach for the development of anti‐MUC1 cancer vaccines.     

Synthesis and Immunological Evaluation of Self‐Assembling and Self‐Adjuvanting Tricomponent Glycopeptide Cancer‐Vaccine Candidates

Self‐adjuvanting tricomponent vaccines were prepared and assessed for their self‐assembly and immunological activity in mouse models. The vaccines each consisted of a peptide or glycopeptide antigen that corresponds to a complete copy of the variable‐number tandem repeat (VNTR) of the tumor‐associated mucin 1 (MUC1) glycoprotein, the universal T‐cell helper peptide epitope PADRE, and the immunoadjuvant Pam₃CysSer. The vaccines were shown to spontaneously self‐assemble in water to form isotropic particles varying in size from 17 to 25 nm and elicited robust humoral responses in murine models without the addition of an external adjuvant. The serum antibodies could recognize tumor‐associated MUC1 epitopes on the surface of MCF7 breast‐cancer cells and B16 melanoma cells, which overexpress this tumor‐associated glycoprotein.     

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. To enhance the long‐term availability of these molecules in vivo, the stabilization of the glycosidic bond between the amino acid residue and the carbohydrate is of interest. The described modular approach affords β‐linked C‐glycosyl amino acids by a sequence of Petasis olefination of glyconolactones, stereoselective hydroboration and a mild B‐alkyl‐Suzuki coupling reaction. The coupling products were transformed to C‐glycosyl amino acid building‐blocks suitable for solid‐phase synthesis and successfully incorporated into a partial sequence of the tumor‐associated MUC1‐glycopeptide. The resulting C‐glycopeptides are candidates for the development of long‐term stable mimics of O‐glycopeptide vaccines.     

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

A totally synthetic, self-assembling, adjuvant-free MUC1 glycopeptide vaccine for cancer therapy

In the development of vaccines for epithelial tumors, the key targets are MUC1 proteins, which have a variable number of tandem repeats (VNTR) bearing tumor-associated carbohydrate antigens (TACAs), such as Tn and STn. A major obstacle in vaccine development is the low immunogenicity of the short MUC1 peptide. To overcome this obstacle, we designed, synthesized, and evaluated several totally synthetic self-adjuvanting vaccine candidates with self-assembly domains. These vaccine candidates aggregated into fibrils and displayed multivalent B-cell epitopes under mild conditions. Glycosylation of Tn antigen on the Thr residue of PDTRP sequence in MUC1 VNTR led to effective immune response. These vaccines elicited a high level antibody response without any adjuvant and induced antibodies that recognized human breast tumor cells. These vaccines appeared to act through a T-cell independent pathway and were associated with the activation of cytotoxic T cells. These fully synthetic, molecularly defined vaccine candidates had several features that hold promise for anticancer therapy.     

Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies

Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.     

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.     

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.     

Fast Epitope Mapping for the Anti‐MUC1 Monoclonal Antibody by Combining a One‐Bead‐One‐Glycopeptide Library and a Microarray Platform

Anti‐MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer‐related MUC1 molecules, the O‐glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) “one‐bead‐one‐compound”‐based preparation of bilayer resins carrying glycopeptides on the shell and mass‐tag tripeptides coding O‐glycan patterns in the core, ii) on‐resin screening with an anti‐MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass‐tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O‐glycosylations against anti‐MUC1 mAb clone VU‐3C6. Qualitative mass‐tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray‐based assay. Our screening provides valuable information on O‐glycosylations of epitopes leading to high affinity with mAb.     

Synthetic Antitumor Vaccines Containing MUC1 Glycopeptides with Two Immunodominant Domains-Induction of a Strong Immune Response against Breast Tumor Tissues

A shot in the arm for cancer treatment: Two MUC1 tetanus toxoid vaccines were synthesized and induced a strong immune response in mice. The antibodies elicited by the vaccines show a high selectivity for the tumor cells in mammary carcinoma tissues and also distinguish between tumor tissues at different stages.     

Alum colloid encapsulated inside β-glucan particles enhance humoral and CTL immune responses of MUC1 vaccine

We have developed a MUC1 antigen-based antitumor vaccine loaded on alum colloid encapsulated insideβ-glucan particles(GP-Al). The constructed vaccine induced strong MUC1 antigen specific Ig G antibody titers and enhanced CD8~+ T cells cytotoxic effect to kill tumor cells. These results indicated that GP-Al can be served as an efficient delivery system and adjuvant for the development of cancer vaccines especially small molecule antigens based cancer vaccines.     

Microwave‐Assisted Solid‐Phase Synthesis of Antifreeze Glycopeptides

Microwave‐assisted solid‐phase synthesis allows for the rapid and large‐scale preparation and structure–activity characterization of tandem repeating glycopeptides, namely monodispersed synthetic antifreeze glycopeptides (syAFGPs, H‐[Ala‐Thr(Galβ1,3GalNAcα1→)‐Ala]_n‐OH, n=2–6). By employing novel AFGP analogues, we have demonstrated that of the monodispersed syAFGP_n (n=2–6, degree of polymerization, DP=2–6, M_w=1257–3690 Da), syAFGP₅ (DP=5, M_w=3082 Da) and syAFGP₆ (DP=6, M_w=3690 Da) exhibit the ability to form typical hexagonal bipyramidal ice crystals and satisfactory thermal hysteresis activity. Structural characterization by NMR and CD spectroscopy revealed that syAFGP₆ forms a typical poly‐L ‐proline type II helix‐like structure in aqueous solution whereas enzymatic modification by sialic acid of the residues at the C‐3 positions of the nonreducing Gal residues disturbs this conformation and eliminates the antifreeze activity.     

Synthesis and Characterisation of Self‐Assembled and Self‐Adjuvanting Asymmetric Multi‐Epitope Lipopeptides of Ovalbumin

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host‐cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP‐based vaccine candidates that contained multiple CD4⁺ and CD8⁺ T‐cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave‐assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide–alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self‐assemble in aqueous media. Changes in lipid length and position induced self‐assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism.     

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.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Solid‐Phase Synthesis and Characterization of N‐Terminally Elongated Aβ−3–x‐Peptides

In addition to the prototypic amyloid‐β (Aβ) peptides Aβ_1–40 and Aβ_1–42, several Aβ variants differing in their amino and carboxy termini have been described. Synthetic availability of an Aβ variant is often the key to study its role under physiological or pathological conditions. Herein, we report a protocol for the efficient solid‐phase peptide synthesis of the N‐terminally elongated Aβ‐peptides Aβ_?3–38, Aβ_?3–40, and Aβ_?3–42. Biophysical characterization by NMR spectroscopy, CD spectroscopy, an aggregation assay, and electron microscopy revealed that all three peptides were prone to aggregation into amyloid fibrils. Immunoprecipitation, followed by mass spectrometry, indicated that Aβ_?3–38 and Aβ_?3–40 are generated by transfected cells even in the presence of a tripartite β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. The elongated Aβ peptides starting at Val(?3) can be separated from N‐terminally‐truncated Aβ forms by high‐resolution isoelectric‐focusing techniques, despite virtually identical isoelectric points. The synthetic Aβ variants and the methods presented here are providing tools to advance our understanding of the potential roles of N‐terminally elongated Aβ variants in Alzheimer's disease.     

Simultaneous determination of the novel thiosemicarbazone anti‐cancer agent,Bp4eT,and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats

Novel thiosemicarbazone metal chelators are extensively studied anti‐cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC‐MS/MS method for the simultaneous quantification of 2‐benzoylpyridine 4‐ethyl‐3‐thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1‐E and M1‐Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C₁₈ column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid‐phase extraction on 96‐well plates. This method was validated over the concentration range of 0.18–2.80 μM for Bp4eT, 0.02–0.37 μM for both M1‐E and M1‐Z, and 0.10–1.60 μM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration–time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti‐cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class. Copyright © 2013 John Wiley & Sons, Ltd.     

Syntheses and Functional Studies of Self‐Adjuvanting Anti‐HER2 Cancer Vaccines

The 9‐mer peptide MFCH401 (N: 165–173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self‐adjuvanting anti‐HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam₃CSK₄ via iterative condensation reaction. The in vivo results showed the Pam₃CSK₄‐MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam₃CSK₄‐MFCH401 vaccine conjugate induced a specific immune response to HER2‐overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam₃CSK₄, which provided useful evidence for developing further anti‐HER2 cancer vaccines.