Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3x10(-9 )towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4x10(6 )for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except approximately 7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

Since the cancer stem cell concept has been widely accepted, several strategies have been proposed to attack cancer stem cells (CSC). Accordingly, stem cell markers are now preferred therapeutic targets. However, the problem of tumor specificity has not disappeared but shifted to another question: how can cancer stem cells be distinguished from normal stem cells, or more specifically, how do CSC markers differ from normal stem cell markers? A hypothesis is proposed which might help to solve this problem in at least a subgroup of stem cell markers. Glycosylation may provide the key.

Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides, Oglycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C-or N-terminal fragments registered for the mono-to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser 5 ,Thr 6 (GSTA) and Thr 14 (VTSA) but contrary to previous in vitro glycosylation studies also at Thr 19 and Ser 15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo and in vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.Post-translational modification of proteins by glycosylation has extensively been studied in the case of N-linked glycans, and accordingly, rules for the substitution of asparagine residues by dolichol phosphate-linked glycans are well established (1). While N-glycosylation is directed by the consensus peptide motif Asn-X-(Ser/Thr), no strict sequence dependence is known for the initiation of O-glycosylation. Currently, there are several approaches to the identification of O-glycosylation sites: studies on in vitro O-glycosylation of synthetic peptides (2-4) or studies on in vivo processed mucin-type glycoproteins (5-7).The relative merits of both approaches have been discussed (8).The results obtained so far agree with the statement that there are no clear-cut motifs for the addition of GalNAc at Ser or Thr residues; however, the non-random patterns of O-glycosylation suggest influences of flanking sequences (2). Wang et al. (3) proposed different motifs for threonine and serine glycosylation, and the studies of O'Connell et al. (2) revealed critical positions in the vicinity of putative O-glycosylation sites. The obviously less specific GalNAc addition to Ser/Thr residues compared with N-glycosylation is reflected also in the existence of at least four distinct species of UDP-GalNAc/peptide Nacetylgalactosaminyltransferase(s) (GalNAc-transferase) for which different substrate specificities have been established (9). Accordingly, the differentiation and organ localization of a cell should determine its characteristic equipment ...