Detection of Tumor-Associated Autoantibodies in the Sera of Pancreatic Cancer Patients Using Engineered MUC1 Glycopeptide Nanoparticle Probes.

Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin-1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure-guided approach to develop unnatural glycopeptides as model antigens for tumor-associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot-blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best-discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19-9 and carcinoembryonic antigen (CEA).

Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody.

The molecular basis of antibody 5E5, which recognizes the entire GalNAc unit as a primary epitope is disclosed. The antibody's contacts with the peptide are mostly limited to two residues, allowing it to show some degree of promiscuity. These findings open the door to the chemical design of peptide-mimetics for developing efficient anti-cancer vaccines and diagnostic tools.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

Water Sculpts the Distinctive Shapes and Dynamics of the Tumor-Associated Carbohydrate Tn Antigens: Implications for Their Molecular Recognition.

The tumor-associated carbohydrate Tn antigens include two variants, αGalNAc- O-Thr and αGalNAc- O-Ser. In solution, they exhibit dissimilar shapes and dynamics and bind differently to the same protein receptor. Here, we demonstrate experimentally and theoretically that their conformational preferences in the gas phase are highly similar, revealing the essential role of water. We propose that water molecules prompt the rotation around the glycosidic linkage in the threonine derivative, shielding its hydrophobic methyl group and allowing an optimal solvation of the polar region of the antigen. The unusual arrangement of αGalNAc- O-Thr features a water molecule bound into a "pocket" between the sugar and the threonine. This mechanism is supported by trapping, for the first time, such localized water in the crystal structures of an antibody bound to two glycopeptides that comprise fluorinated Tn antigens in their structure. According to several reported X-ray structures, installing oxygenated amino acids in specific regions of the receptor capable of displacing the bridging water molecule to the bulk-solvent may facilitate the molecular recognition of the Tn antigen with threonine. Overall, our data also explain how water fine-tunes the 3D structure features of similar molecules, which in turn are behind their distinct biological activities.

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.

Mucin architecture behind the immune response: design, evaluation and conformational analysis of an antitumor vaccine derived from an unnatural MUC1 fragment.

A tripartite cancer vaccine candidate, containing a quaternary amino acid (α-methylserine) in the most immunogenic domain of MUC1, has been synthesized and examined for antigenic properties in transgenic mice. The vaccine which is glycosylated with GalNAc at the unnatural amino acid, was capable of eliciting potent antibody responses recognizing both glycosylated and unglycosylated tumour-associated MUC1 peptides and native MUC1 antigen present on cancer cells. The peptide backbone of the novel vaccine presents the bioactive conformation in solution and is more resistant to enzymatic degradation than the natural counter part. In spite of these features, the immune response elicited by the unnatural vaccine was not improved compared to a vaccine candidate containing natural threonine. These observations were rationalized by conformational studies, indicating that the presentation and dynamics of the sugar moiety displayed by the MUC1 derivative play a critical role in immune recognition. It is clear that engineered MUC1-based vaccines bearing unnatural amino acids have to be able to emulate the conformational properties of the glycosidic linkage between the GalNAc and the threonine residues. The results described here will be helpful to the rational design of efficacious cancer vaccines.

Deciphering the Non-Equivalence of Serine and Threonine O-Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti-MUC1 Antibody.

The structural features of MUC1-like glycopeptides bearing the Tn antigen (α-O-GalNAc-Ser/Thr) in complex with an anti MUC-1 antibody are reported at atomic resolution. For the α-O-GalNAc-Ser derivative, the glycosidic linkage adopts a high-energy conformation, barely populated in the free state. This unusual structure (also observed in an α-S-GalNAc-Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α-O-GalNAc-Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non-equivalence of Ser and Thr O-glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti-MUC1 antibodies.

Detection of tumor-associated glycopeptides by lectins: the peptide context modulates carbohydrate recognition.

Tn antigen (α-O-GalNAc-Ser/Thr) is a convenient cancer biomarker that is recognized by antibodies and lectins. This work yields remarkable results for two plant lectins in terms of epitope recognition and reveals that these receptors show higher affinity for Tn antigen when it is incorporated in the Pro-Asp-Thr-Arg (PDTR) peptide region of mucin MUC1. In contrast, a significant affinity loss is observed when Tn antigen is located in the Ala-His-Gly-Val-Thr-Ser-Ala (AHGVTSA) or Ala-Pro-Gly-Ser-Thr-Ala-Pro (APGSTAP) fragments. Our data indicate that the charged residues, Arg and Asp, present in the PDTR sequence establish noteworthy fundamental interactions with the lectin surface as well as fix the conformation of the peptide backbone, favoring the presentation of the sugar moiety toward the lectin. These results may help to better understand glycopeptide-lectin interactions and may contribute to engineer new binding sites, allowing novel glycosensors for Tn antigen detection to be designed.

Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins.

The molecular recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn-threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.

Rational design of a Tn antigen mimic.

A novel Tn antigen mimic, in which the natural underlying amino acid has been replaced by the non-natural α-methylserine analogue, is reported. This derivative exhibits a similar affinity for a natural lectin as for the natural Tn and retains the bioactive conformation observed in the Tn-containing glycopeptides with anti-MUC1 antibodies.

The conformation of the C-glycosyl analogue of N-acetyl-lactosamine in the free state and bound to a toxic plant agglutinin and human adhesion/growth-regulatory galectin-1.

The conformational behavior of the C-glycoside analogue of N-acetyl-lactosamine, beta-C-Gal-(1-->4)-beta-GlcNAc-OMe, 1, has been studied using a combination of molecular mechanics calculations and NMR spectroscopy (J and NOE data). It is shown that the C-disaccharide populates three distinctive conformational families in solution, the major one being the anti-psi conformation. Of note, this conformation is only marginally populated for the O-disaccharide. Due to its conspicuous role in the regulation of adhesion, growth and tissue invasion of tumors and its avid binding to N-acetyl-lactosamine human, galectin-1 was tested as a receptor. This endogenous lectin recognizes a local minimum of 1, the syn-PhiPsi conformer, and thus a conformational selection process is correlated with the molecular recognition event.