Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.

Inhibitory role of Annexin A1 in pathological bone resorption and therapeutic implications in periprosthetic osteolysis.

There is currently no therapy available for periprosthetic osteolysis, the most common cause of arthroplasty failure. Here, the role of AnxA1 in periprosthetic osteolysis and potential therapeutics were investigated. Reducing the expression of AnxA1 in calvarial tissue was found to be associated with increased osteolytic lesions and the osteolytic lesions induced by debris implantation were more severe in AnxA1-defecient mice than in wild-type mice. AnxA1 inhibits the differentiation of osteoclasts through suppressing NFκB signaling and promoting the PPAR-γ pathway. Administration of N-terminal-AnxA1 (Ac2-26 peptide) onto calvariae significantly reduced osteolytic lesions triggered by wear debris. These therapeutic effects were abrogated in mice that had received the PPAR-γ antagonist, suggesting that the AnxA1/PPAR-γ axis has an inhibitory role in osteolysis. The administration of Ac2-26 suppressed osteolysis induced by TNF-α and RANKL injections in mice. These findings indicate that AnxA1 is a potential therapeutic agent for the treatment of periprosthetic osteolysis.

Structure-based Design of Anti-cancer Vaccines: The Significance of Antigen Presentation to Boost the Immune Response.

Immunotherapy, alone or in combination with other therapies, is widely used against cancer. Glycoprotein Mucin 1 (MUC1), which is overexpressed and aberrantly glycosylated in tumor cells, is one of the most promising candidates to engineer new cancer vaccines. In this context, the development of stable antigens that can elicit a robust immune response is mandatory. Here, we describe the design and in vivo biological evaluation of three vaccine candidates based on MUC1 glycopeptides that comprise unnatural elements in their structure. By placing the Tn antigen (GalNAcα-O-Ser/Thr) at the center of the design, the chemical modifications include changes to the peptide backbone, glycosidic linkage, and carbohydrate level. Significantly, the three vaccines elicit robust immune responses in mice and produce antibodies that can be recognized by several human cancer cells. In all cases, a link was established between the conformational changes induced by the new elements in the antigen presentation and the immune response induced in mice. According to our data, the development of effective MUC1-based vaccines should use surrogates that mimic the conformational space of aberrantly glycosylated MUC1 glycopeptides found in tumors.

Amplified Detection of Breast Cancer Autoantibodies Using MUC1-Based Tn Antigen Mimics.

In many human carcinomas, mucin-1 (MUC1) is overexpressed and aberrantly glycosylated, resulting in the exposure of previously hidden antigens. This generates new patient antibody profiles that can be used in cancer diagnosis. In the present study, we focused on the MUC1-associated Tn antigen (α-O-GalNAc-Ser/Thr) and substituted the GalNAc monosaccharide by a glycomimic to identify MUC1-based glycopeptides with increased antigenicity. Two different glycopeptide libraries presenting the natural Tn antigen or the sp2-iminosugar analogue were synthesized and evaluated with anti-MUC1 monoclonal antibodies in a microarray platform. The most promising candidates were tested with healthy and breast cancer sera aiming for potential autoantibody-based biomarkers. The suitability of sp2-iminosugar glycopeptides to detect anti-MUC1 antibodies was demonstrated, and serological experiments showed stage I breast cancer autoantibodies binding with a specific unnatural glycopeptide with almost no healthy serum interaction. These results will promote further studies on their capabilities as early cancer biomarkers.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Synthesis, conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment.

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp2-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

Further applications of classical amide coupling reagents: Microwave-assisted esterification on solid phase.

Ester linkage (s) is a key chemical connector in organic chemistry, including natural products, peptides, and synthetic polymers. We herein describe a straightforward method for the efficient formation of ester linkage (s) on solid-phase. This method simply involves the use of amide coupling reagents under microwave irradiation. The robustness of this method relies on the use of classical solid-phase coupling reagents, heating by microwave irradiation, and a short time period, which results in high yields and the minimization of racemization.

CLEC10A Is a Specific Marker for Human CD1c+ Dendritic Cells and Enhances Their Toll-Like Receptor 7/8-Induced Cytokine Secretion.

Dendritic cells (DCs) are major players for the induction of immune responses. Apart from plasmacytoid DCs (pDCs), human DCs can be categorized into two types of conventional DCs: CD141+ DCs (cDC1) and CD1c+ DCs (cDC2). Defining uniquely expressed surface markers on human immune cells is not only important for the identification of DC subpopulations but also a prerequisite for harnessing the DC subset-specific potential in immunomodulatory approaches, such as antibody-mediated antigen targeting. Although others identified CLEC9A as a specific endocytic receptor for CD141+ DCs, such a receptor for CD1c+ DCs has not been discovered, yet. By performing transcriptomic and flow cytometric analyses on human DC subpopulations from different lymphohematopoietic tissues, we identified CLEC10A (CD301, macrophage galactose-type C-type lectin) as a specific marker for human CD1c+ DCs. We further demonstrate that CLEC10A rapidly internalizes into human CD1c+ DCs upon binding of a monoclonal antibody directed against CLEC10A. The binding of a CLEC10A-specific bivalent ligand (the MUC-1 peptide glycosylated with N-acetylgalactosamine) is limited to CD1c+ DCs and enhances the cytokine secretion (namely TNFα, IL-8, and IL-10) induced by TLR 7/8 stimulation. Thus, CLEC10A represents not only a candidate to better define CD1c+ DCs-due to its high endocytic potential-CLEC10A also exhibits an interesting candidate receptor for future antigen-targeting approaches.

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

Glycopeptides as Targets for Dendritic Cells: Exploring MUC1 Glycopeptides Binding Profile toward Macrophage Galactose-Type Lectin (MGL) Orthologs.

The macrophage galactose-type lectin (MGL) recognizes glycan moieties exposed by pathogens and malignant cells. Particularly, mucin-1 (MUC1) glycoprotein presents an altered glycosylation in several cancers. To estimate the ability of distinct MGL orthologs to recognize aberrant glycan cores in mucins, we applied evanescent-field detection to a versatile MUC1-like glycopeptide microarray platform. Here, as binding was sequence-dependent, we demonstrated that not only sugars but also peptide region impact the recognition of murine MGL1 (mMGL1). In addition, we observed for all three MGL orthologs that divalent glycan presentation increased the binding. To assess the utility of the glycopeptide binders of the MGL orthologs for MGL targeting, we performed uptake assays with fluorescein-MUC1 using murine dendritic cells. A diglycosylated MUC1 peptide was preferentially internalized in an MGL-dependent fashion, thus showing the utility for divalent MGL targeting. These findings may be relevant to a rational design of antitumor vaccines targeting dendritic cells via MGL.

Synthetic Mucin-Like Glycopeptides as Versatile Tools to Measure Effects of Glycan Structure/Density/Position on the Interaction with Adhesion/Growth-Regulatory Galectins in Arrays.

Functional pairing of cellular glycoconjugates with tissue lectins is a highly selective process, whose determinative factors have not yet been fully delineated. Glycan structure and modes of presentation, that is, its position and density, can contribute to binding, as different members of a lectin family can regulate degrees of responsiveness to these factors. Using a peptide repeat sequence motif of the glycoprotein mucin-1, the principle of introducing synthetic (glyco)peptides with distinct variations in these three parameters to an array-based screening of tissue lectins is illustrated. Interaction profiles of seven adhesion/growth-regulatory galectins cover the range from intense signals with core 2 pentasaccharides and core 1 binding for galectins-3 and -5 to a lack of binding for galectin-1 and also the galectin-related protein, which was included as a negative control. Remarkably, the two tandem-repeat-type galectins-4 and -8 were distinguished by core 1 sialylation, as the two separated domains were. These results encourage further synthetic elaboration of the glycopeptide library and testing of the network of natural galectins and rationally engineered variants of the lectins.

The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

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.

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 Ca(2+) . 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.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides.

BACKGROUND: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. METHODS: A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. RESULTS: Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galß1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. CONCLUSION: We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. GENERAL SIGNIFICANCE: The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.

Glycoblotting-based high throughput protocol for the structural characterization of hyaluronan degradation products during enzymatic fragmentation.

Increasing interests have been focused on the functional roles of hyaluronan degradation products, namely hyaluronan oligosaccharides, as signal molecules regulating cell growth, differentiation, malignancy, and inflammatory responses. It is clear that molecular size of hyaluronan oligosaccharides might be crucial for defining possible and dynamic roles in supporting and suppressing homeostatic cellular processes. The present paper communicates a facile and efficient approach based on glycoblotting method for the characterization of hyaluronan fragments liberated from three different sources of hyaluronan (rooster comb, bovine vitreous humor, and Streptococcus) by in vitro degradation using two typical hyaluronidases of bovine testicular (EC 3.2.1.35) and Streptomyces hyalurolyticus (EC 4.2.2.1). It was demonstrated that glycoblotting method allows for high throughput and quantitative analysis of hyaluronan fragments within a wide dynamic range (1 ~ 1,000 pmole) when 5 µg of hyaluronan digests were applied for this enrichment protocol. Molecular size and distribution of hyaluronan fragments were proved to be influenced strongly by conditions and hyaluronidases employed while source of hyaluronan did not affect the degradation profiles. Strikingly, the present method uncovered the existence of the smallest and unusual hyaluronan degradation fragments such as a disaccharide GlcAß1-3GlcNAc during the digestion by bovine hyaluronidase and a trisaccharide GlcAß1-3GlcNAcß1-4GlcA derivative by Streptomyces hyaluronidase. Bovine testis hyaluronidases afforded hyaluronan tetra- and hexasaccharides as major products. On the other hand, it was demonstrated that Streptomyces hyaluronidase can produce odd number fragments from three to nine sugar residues while even number fragments from four to fourteen sugar residues were major products.

An efficient protocol for the solid-phase synthesis of glycopeptides under microwave irradiation.

A standardized and smooth protocol for solid-phase glycopeptides synthesis under microwave irradiation was developed. Double activation system was proved to allow for highly efficient coupling of Tn-Ser/Thr and bulky core 2-Ser/Thr derivatives. Versatility and robustness of the present strategy was demonstrated by constructing a Mucine-1 (MUC1) fragment and glycosylated fragments of tau protein. The success of this approach relies on the combination of microwave energy, a resin consisting totally of polyethylene glycol, a low excess of sugar amino acid and the "double activation" method.

Design and synthesis of FAJANU: a de novo C(2) symmetric cyclopeptide family.

A novel cyclic peptide has been designed from several potent marine cytotoxic peptides, including IB-01212, luzopeptin, triostin, and thiocoraline. The FAJANU scaffold maintains C 2 symmetry, cyclic structure, and the construction of aromatic and aliphatic character at the N- and C-terminal extremes. A first six-member family was previously synthesized and evaluated biologically. Several analogues presented greater activity than IB-01212. Furthermore, on the basis of the most active candidate, we have performed a more exhaustive synthetic and structural analysis: (i) structure-activity relationship provided clues about the key elements in the framework, (ii) NMR assignment confirmed C 2 symmetry, and (iii) confocal images revealed its penetration and cellular localization.

Synthesis of oligonucleotide derivatives using ChemMatrix supports.

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.

The synergy of ChemMatrix resin and pseudoproline building blocks renders RANTES, a complex aggregated chemokine.

Traditionally, solid-phase synthesis has relied on polystyrene-based resins for the synthesis of all kinds of peptides. However, due to their high hydrophobicity, these resins have certain limitations, particularly in the synthesis of complex peptides, and in such cases, poly(ethylene glycol) (PEG)-based resins are often found to give superior results. Another powerful strategy for expediting the assembly of complex peptides is to employ pseudoproline dipeptides. These derivatives disrupt the interactions among chains that are usually the cause of poor coupling yields in aggregated sequences. Here we report on an efficient stepwise solid-phase synthesis of RANTES (1-68) by combining the advantages of the totally PEG-based ChemMatrix resin and pseudoproline dipeptides.