Tumor‐Associated MUC1 Tandem‐Repeat Glycopeptide Microarrays to Evaluate Serum– and Monoclonal–Antibody Specificity

Abstract

The membrane-bound mucin glycoprotein MUC1 is extensively overexpressed on epithelial tumor cells and considered an attractive target in the development of cancer immunotherapy and immunodiagnostics. In epithelial tumors downregulation of glycosyltransferases, in particular the core 2 b-1,6-N-acetylglucosaminyltransferase, and up-regulation of sialyltransferases results in the formation of short saccharides with premature sialylation of the extracellular domain of mucins, [1][2][3][4] exposure of the peptide backbone of these otherwise heavily glycosylated proteins, and formation of a tumor-associated epitope consisting of both the saccharide and the peptide structure.[5]The MUC1 extracellular domain consists of numerous tandem repeats of the sequence HGVTSAPDTRPAPG-STAPPA which embodies five potential glycosylation sites (Scheme 1 a). The development of synthetic vaccines based on the exposed MUC1-tandem-repeat peptide backbone, the tumor associated glycans, such as T N , T, sialyl-T N , sialyl-T, and recent tandem-repeat glycopeptides has been the subject of intensive research efforts. [6][7][8][9] Such vaccines need to induce a specific immune response, which leads to the formation of antibodies that differentiate between normal and cancer epithelial cells. This requirement and the substantial effort required to synthesize and conjugate the carbohydrate and glycopeptide antigens call for the development of miniaturized analytical technologies which allow rapid and reliable mapping of antibody specificity. The immobilization of biomolecules such as peptides, [10][11][12][13] oligonucleotides, [14,15] carbohydrates, [16][17][18] and proteins [10,[19][20][21] in microarray formats and their use in bioassays has proven to be a very valuable, time-and material-saving methodology; it is also a viable alternative to the established solution-phase bioassays, such as the ELISA-based formats. Herein we report on the development of a microarray platform for mapping the specificity of antibodies raised against tumor-associated mucin MUC1 glycopeptides. (for previous work on glycopeptide microarrays see reference [22]).Recently, we synthesized vaccine constructs consisting of sialyl-T N -and T N -20-mer tandem-repeat MUC1 glycopeptides connected to a T H -cell peptide epitope from ovalbumin (OVA [323][324][325][326][327][328][329][330][331][332][333][334][335][336][337][338][339] ). Immunization of transgenic mice whose T cells express a receptor recognizing the OVA 323-339 T-cell epitope, induced a humoral immune response, [6] thereby leading to the formation of antibodies directed against the MUC1 glycopeptide B-cell epitope. To evaluate the specificity of these antibodies we have now synthesized a number of MUC1 glycopeptides having variations in the glycan structure, the glycan position on the tandem-repeat peptide, and the number of tandem repeats or shorter peptides excluding parts of the repeat (Scheme 1 b and Scheme 2); for a description of the synthesis see the Supporting Information). These glycopeptides were equ...

Resolving the α-glycosidic linkage of arginine-rhamnosylated translation elongation factor P triggers generation of the first ArgRha specific antibody

“…The resulting BSA-glycoconjugate was injected into rabbits to raise polyclonal antibodies targeting the Arg Rha moiety. 39,40 After the third immunization, the crude anti -sera were collected and their specificity was monitored by employing an enzyme-linked immunosorbent assay (ELISA) analysis with horseradish peroxidase linked anti -rabbit IgG. The antibodies from two batches of crude anti -sera from two immunized rabbits were found to bind robustly and specifically to the BSA-glycoconjugate with high titers, showing strong immune reactivity even after 128 000 fold dilution (Fig.…”

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

“…Binding studies with MUC1 glycopeptides bound to microchips [10] showed that the recognition profile of the antibodies induced by the aforementioned vaccines is different from that found for the biologically optimized, tumorselective antibody SM3 [11] . It also differs from those of autoantibodies isolated from serum of tumor patients.…”

“…The MUC1 tetanus toxoid vaccines [9] described above carried the T antigen or the sialyl-T N antigen side chains at threonine-6 of 1, whereas the SM3 antibody [10,11] as well as the autoantibodies in the sera of patients [12] showed intensive binding to MUC1 glycopeptides glycosylated in the GSTA region (Ser 17 , Thr 18 ). NMR spectroscopic analyses had shown that glycan side chains in the STAPPA peptide sequence of MUC1 influence the conformation of this peptide segment.…”

“…[13] As the conformation is apparently decisive for the tumor selectivity of the MUC1 glycopeptides, [14] the investigation of MUC1 tetanus toxoid vaccines glycosylated at serine-17 or threonine-18 is of particular interest. To include the conformational influence of the STAPPA sequence, the peptide sequence was extended to the 22mer glycopeptides displayed in 1 incorporating both the APDTRP motif [11] and the second binding epitope [10,11] of the tumor-selective anti-MUC1 antibody.…”

Glycan Microarrays for Decoding the Glycome

“…They observe high IgG titers to unique glycoforms of MUC1 glycopeptides in various cancers that are completely absent in healthy individuals (150). Westerlind and colleagues have also generated a MUC1 glycopeptide array for similar studies (151). These initial results suggest that this technology will become an important tool for unraveling the “glycopeptidome”.…”