A phase I study of PankoMab-GEX, a humanised glyco-optimised monoclonal antibody to a novel tumour-specific MUC1 glycopeptide epitope in patients with advanced carcinomas

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Enhanced binding of antibodies to the DTR motif of MUC1 tandem repeat peptide is mediated by site-specific glycosylation

The epithelial mucin MUC1 is an important tumor marker of breast cancer and other carcinomas. Its immunodominant DTR motif, which is the principal target for immunotherapeutic approaches, has been assumed until recently not to be glycosylated in both normal and tumor MUC1 and to acquire its immunogenic conformation by virtue of a certain number of tandem repeats. We present evidence that the antigenicity of the single repeat toward a considerable number of antibodies to the DTR motif is greatly enhanced if it is glycosylated within this motif, and only in this position. Twenty-eight monoclonal anti-MUC1 antibodies with DTR specificity were tested for binding to synthetic 21-mer (AHG21) or 20-mer (HGV20) tandem repeat peptides O-glycosylated with galactose beta1-3N-acetylgalactosamine alpha or N-acetylgalactosamine alpha at defined Thr or Ser positions. Binding was measured in ELISA experiments using the glycopeptides as plate-immobilized antigens or as inhibitors in solution. At least 12 antibodies revealed significantly enhanced binding to the peptides glycosylated at the DTR motif (Thr-10) as compared to positional isomers glycosylated at Thr-5, Ser-6, Ser-16, or Thr-17 and to the nonglycosylated peptides. Six antibodies (VU-3-C6, A76-A/C7, Ma552, VU-11-D1, VU-12-E1, and VU-11-E2) that were unreactive with the monomeric repeat peptide did bind to the DTR-glycosylated peptide. Several lines of evidence suggest that glycosylation with N-acetylgalactosamine is sufficient for the observed enhancement effect. Our results are of special interest in conjunction with the recent observation that the DTR motif of lactation-associated MUC1 is O-glycosylated in vivo (Müller et al., J. Biol. Chem., 272: 24780-24793, 1997). They may have consequences for the design of efficient tumor vaccines

Blockade of anaesthetic-induced preconditioning in the hyperglycaemic myocardium - The regulation of different mitogen-activated protein kinases

Preconditioning by volatile anaesthetics is blocked by hyperglycaemia. The regulation of mitogen-activated protein kinases during this effect has yet not been investigated. For infarct size measurements, anaesthetized rats were subjected to 25 min coronary artery occlusion followed by 120 min reperfusion. Control animals were not further treated. One group was preconditioned by two 5-min periods of desflurane inhalation (desflurane preconditioning, Des-preconditioning, 1 MAC), each followed by 10-min washout. Four groups received glucose 50% in order to achieve blood glucose concentrations between 22.2 and 33.3 mM/l. Glucose infusion started 40 min before ischaemia (early hyperglycaemia, EH) and stopped with the onset of artery occlusion with (EH+Des-preconditioning) or without (EH) preconditioning. The other two groups received glucose during ischaemia (late hyperglycaemia, LH), again with (LH+Des-preconditioning) or without ((LH) preconditioning. Additional hearts were excised for Western blot of mitogen-activated protein kinases. Infarct size was reduced from 51.7 +/- 9.0% in controls to 28.8 +/- 11.8% after Des-preconditioning (P <0.01 vs Con). Hyperglycaemia alone did not affect infarct size (EH, 51.5 +/- 9.0%, LH, 44.3 +/- 16.9%), but EH as well as LH blocked Des-preconditioning (49.1 +/- 12.3%, P <0.01, 48.1 +/- 17.6%, P <0.05 vs Des-preconditioning). All three mitogen-activated protein kinases showed a similar time course pattern of phosphorylation in the Despreconditioning, EH and EH+Des-preconditioning group. Despite the lack of cardioprotection, mitogen-activated protein kinases are activated in hyperglycaemic myocardium. Therefore, it can be assumed that the hyperglycaemic induced blockade of Des-preconditioning is situated downstream or in parallel of these mitogen-activated protein kinases or involves different signal transduction pathways. (C) 2008 Elsevier B.V. All rights reserve

Chlamydia pneumoniae-induced memory CD4+ T-cell activation in human peripheral blood correlates with distinct antibody response patterns

Chlamydia pneumoniae is a frequent pathogen of the respiratory tract, and persistent infections with this obligate intracellular bacterium have been associated with different severe sequelae. Although T-cell activation during acute C. pneumoniae infections has been described, little is known about the frequency or the role of the C. pneumoniae-specific memory T cells that reside in the human body after the resolution of the infection. In the present study, the C. pneumoniae-induced T-cell responses in peripheral blood mononuclear cells of 56 healthy volunteers were analyzed and compared to the donor's serum antibody reactivity toward whole C. pneumoniae as well as recombinant C. pneumoniae antigens. Following short-term stimulation with C. pneumoniae, both gamma interferon (IFN-gamma)- and interleukin-2 (IL-2)-producing CD4(+) T-cell responses could be detected in 16 of 56 healthy individuals. C. pneumoniae-activated CD4(+) T cells expressed CD154, a marker for T-cell receptor-dependent activation, and displayed a phenotype of central memory T cells showing dominant IL-2 production but also IFN-gamma production. Interestingly, individuals with both IFN-gamma- and IL-2-producing responses showed significantly decreased immunoglobulin G reactivity toward C. pneumoniae RpoA and DnaK, antigens known to be strongly upregulated during chlamydial persistence, compared to IgG reactivity of seropositive individuals with no T-cell response or CD4(+) T-cell responses involving the production of a single cytokine (IFN-gamma or IL-2). Our results demonstrate that memory CD4(+) T cells responding to C. pneumoniae stimulation can be detected in the circulation of healthy donors. Furthermore, among seropositive individuals, the presence or the absence of dual IFN-gamma- and IL-2-producing T-cell responses was associated with distinct patterns of antibody responses toward persistence-associated C. pneumoniae antigens