Structurally defined synthetic cancer vaccines: analysis of structure, glycosylation and recognition of cancer associated mucin, MUC-1 derived peptides

Abstract

Translation of an immune response into therapy is probably the toughest task in designing vaccines for cancer due to the heterogeneity of the cell surface antigens which display tremendous variations in glycoforms. Consequently, a small segment (antigen) of cancer-associated mucin, in spite of generating antigen-specific immune responses, may be limited in therapeutic value. It is important that the synthetic segment resembles the native cancer-associated mucin in both structure and conformation. Synthetic cancer associated mucin derived 16 amino acid peptide GVTSAPDTRPAPGSTA and its partially glycosylated forms have demonstrated specific binding to two monoclonal antibodies, B27.29 and BCP8, raised against the native cancer associated mucin, MUC-1 and a MUC-1 derived synthetic peptide, respectively. In spite of the structural similarities at the core peptide level of both glycosylated and unglycosylated peptides, it appears that partial glycosylation does not inhibit and even slightly enhances binding to the MAb B27.29 indicating that the glycosylated synthetic peptide more closely resembles the native mucin epitope recognized by MAb B27.29. From molecular dynamic simulations using NMR derived distance constraints, both glycosylated and unglycosylated peptides have shown a type 1 beta turn involving the same amino acids in both glycosylated and unglycosylated peptides. The alpha GalNAc attached to the threonine (T3) and serine (S4) in the 16 amino acid sequence has not imposed any conformational changes to the peptide backbone nor has offered severe steric resistance to the binding of either antibody to the glycopeptides as indicated by hapten inhibition studies. Nevertheless, all peptides have displayed glycosylation dependent specificities in binding to these antibodies, i.e. the glycosylated peptides demonstrated relative higher affinities to the native mucin antibody B27.29 while the unglycosylated peptide is more specific to the MAb BCP8. Immune responses generated by these synthetic glycopeptides are highly specific in recognizing the native cancer associated mucin.

Probing cell-surface architecture through synthesis: An NMR-determined structural motif for tumor-associated mucins

“…Peptide flexibility is reduced on monoglycosylation with GalNAc (12,18) and further restricted on addition of a second GalNAc (3,9). These low degrees of glycosylation result in modest ␤-turn formation usually in the vicinity of the glycodomain and appear to be sequence sensitive.…”

“…Altered expression of cell-surface mucin character is often characteristic of malignant cells (1,3,5). Accordingly, tumor-associated mucins are good targets for a vaccination strategy (3,(5)(6)(7)(8)(9). We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials.…”

“…We have surmounted the synthetic challenges of constructing polypeptides bearing clustered glycodomains (7,8), and one such construct is currently in human clinical trials. Given this access, we probed the effects of clustered glycosylation patterns on peptide conformation and recognition (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We have conducted extensive NMR and restrained molecular dynamics calculations (19,20) on fully synthetic clustered carbohydrate tumor antigens (1)(2)(3)(4) corresponding to a fragment of CD43, a glycoprotein aberrantly expressed on the surface of acute myelogenous leukemia cells (21)(22)(23)(24).…”

Analysis of the Role of Type 1 Core O-Glycans in the Binding of Anti-MUC1 Antibodies by Cytofluorometry and Synthetic Peptide/Glycopeptide Binding Inhibition Studies

“…Rates for no inhibitor controls are used to calculate the percent inhibition of binding for each MAb. The synthetic peptides and glycopeptides were prepared as previously described [17,18].…”

“…Synthetic glycopeptide binding inhibition studies were conducted essentially as previously described [15,17]. The test solid phase was prepared using a semipurified MUC1 mucin preparation that was isolated from a pool of 15 individual patient ascites fluids [16].…”