Immunohistochemistry, glycosylation and immunosuppression of glycodelin in human ovarian cancer.

Glycodelins (Gds) are glycoproteins with a gender specific glycosylation. Glycodelin A (GdA) is primarily produced in endometrial and decidual tissue and secreted to amniotic fluid. Glycodelins were also identified in several cancer types, including serous ovarian cancer. Gds act as a T-cell inhibitor and are involved in inactivation of human monocytes. With a Gd peptide antibody, derived from a 15 amino acid sequence of human Gd and in situ hybridization experiments, the expression of Gd in serous, mucinous, endometrioid and clear cell ovarian tumors was identified. In contrast to former investigations with antibodies against GdA, a positive immunohistochemical reaction for Gd was observed in all forms of epithelium ovarian cancer. These results were confirmed with in situ hybridization. In addition, Gd is expressed in granulose cell tumors, a non-epithelial form of ovarian cancer. Furthermore, Gd was purified from ascites fluid of ovarian cancer patients. Ascites Gd showed significant differences in its structure of sialyl Lewis-type oligosaccharides compared to GdA. Additionally, ascites Gd inhibits IL-2 stimulated proliferation of peripheral blood leucocytes and inhibits adhesion of SLe(X)-positive cells to E-selectin. Therefore, Gd could act as an inhibitor of lymphocyte activation and/or adhesion in ovarian cancer.

Effect of sialyl Lewis X-glycoliposomes on the inhibition of E-selectin-mediated tumour cell adhesion in vitro.

The aim of this study was to evaluate the potential of different types of sialyl Lewis X-conjugated liposomes as competitive inhibitors for tumour cell adhesion to endothelial E-selectin. Sterically stabilised liposomes with the sLeX ligand at the terminal end of the polyethyleneglycol (PEG) chain, as well as vesicles that had the ligand embedded within the PEG-layer, were compared to ligand-bearing liposomes without sterical stabilisation. First, 14 different tumour cell lines were characterised for their expression of sialyl Lewis X and/or A. Tumour cell adhesion was characterised in three static assays in vitro using: (i) immobilised E-selectin, (ii) CHO cells, transfected to express E-selectin and (iii) human umbilical vein endothelial cells (HUVEC). Sterically stabilised liposomes with the ligand at the terminal end of the polyethylene chain were the most effective inhibitors in all three assays and inhibited the adhesion of HT29 colon- and Lewis lung (LL) carcinoma cells by about 60-80%. The binding was not affected by a PEG-coating of the liposomes. Sterical stabilisation, on the other hand, completely prevented macrophage uptake (J774 cell line) independently of the presence of the ligand, while plain liposomes were taken up in an amount of 5.4 nmol liposomal lipids/10(6) macrophages.

Human chorionic gonadotropin (hCG) as inhibitior of E-selectin-mediated cell adhesion.

OBJECTIVE: Human chorionic gonadotropin (hCG) is a placental glycoprotein hormone of clinical importance. It is secreted into maternal circulation, amniotic fluid, urine and is also found in the serum and urine of patients with malignant trophoblastic diseases. Depending on the source, we detected a remarkable expression of sialyl Lewisx on this protein. Sialyl Lewis' is known as a minimal ligand recognized by selectins, which are involved in cell adhesion processes in inflammatory and metastatic diseases. In this context, the purpose of the study was to explore the ability of hCG to serve as a selectin antagonist. MATERIALS AND METHODS: We purified hCG from human amniotic fluid, serum and urine of pregnant women and from supernatants of the trophoblastic tumour cell lines Jeg3 and BeWo by immunoadsorption chromatography. The proteins were functionally tested for a specific blockade of the E-selectin-mediated cell adhesion in vitro. The efficiencies of hCG from different origins were compared. RESULTS: We found that hCG isolated from serum, amniotic fluid and supernatant of the cell line Jeg3 were strong inhibitors with at least 10(3)-fold higher potency compared to the monovalent tetrasaccharide sialyl Lewisx. As expected from the carbohydrate expression, hCG isolated from the urine of pregnant women, but even from the supernatant of BeWo cells, showed no inhibitory effect. CONCLUSION: hCG is an effective selectin antagonist. This fact suggests that hCG may play a role in preventing leukocyte adhesion on the foetal syncytiotrophoblast or the metastatic activity of trophoblastic tumour cells.

hCG in trophoblast tumour cells of the cell line Jeg3 and hCG isolated from amniotic fluid and serum of pregnant women carry oligosaccharides of the sialyl Lewis X and sialyl Lewis a type.

Human chorionic gonadotropin (hCG) is a placental hormone and marker for the differentiation process of cytotrophoblast cells to syncytial trophoblasts. It is secreted into the maternal circulation and urine. The glycoprotein hCG is also found in the serum and urine of patients with malignant trophoblastic diseases. For this study hCG was purified from pooled human mid-trimester amniotic fluid, serum and urine of pregnant women and from supernatants of trophoblast tumour cell cultures Jeg3 and BeWo by immunoadsorption chromatography with specific hCG-antibodies. The purity and identity of the isolated hCG were checked by Western blot analysis. Sialylated oligosaccharides of the Lewis type of isolated hCGs were analysed by dot blot and ELISA technique with monoclonal antibodies specific for sialyl Lewis x and sialyl Lewis a-carbohydrate epitopes. BeWo and Jeg 3 cells were additionally investigated by immunofluorescence. HCG isolated from the serum and amnion of pregnant women bear both sialyl Lewis x (sLex)-antigen and sialyl Lewis a (sLea)-antigen. The same result was observed for hCG isolated from the supernatants of the trophoblast tumour cell line Jeg3. On the contrary, hCG isolated from the supernatants of the trophoblast tumour cell line BeWo showed only a very weak expression of these antigens. HCG isolated from the urine of pregnant women was negative for both sialyl Lewis antigens. The results are discussed with respect to a possible relationship to selectin- mediated cell adhesion processes. This could play a role in the prevention of leukocyte adhesion on the foetal syncytiotrophoblast. Glycosylation of hCG in trophoblast tumour cells, on the other hand, could increase the metastatic activity of these cells.

Determination of MUC1 in sera of ovarian cancer patients and in sera of patients with benign changes of the ovaries with CA15-3, CA27.29, and PankoMab.

AIM: Mucin 1 (MUC1) is a high molecular weight transmembrane glycoprotein with unique properties which is used as a tumour marker in sera of ovarian cancer patients. The common test kit for the cancer antigen 15-3 (CA15-3) is not sufficient for the discrimination between sera from healthy individuals and sera from patients with benign changes of the ovaries. In this study, the newly developed anti-MUC1 antibody PankoMab was tested in normal and patient sera with an ELISA, and the obtained data were compared against data from experiments using the commercial kits for CA15-3 and CA27.29. MATERIALS AND METHODS: Sera of 123 patients diagnosed with benign or malignant changes of the ovaries were obtained before surgery. CA15-3 was analysed with an automated ELISA system (Immulite 2000). CA27.29 was measured with the ST AIA-PACK CA27.29 for the AIA-600II-Analyzer (Tosoh Bioscience, Belgium). The release of MUC1 fragments carrying the TA-MUC1 epitope was analysed with an ELISA using the PankoMab antibody. RESULTS: Using the already established markers CA15-3 and CA27.29, significant differences between benign and malignant changes of the ovaries were found. The same result was obtained with the newly developed TA-MUC1 test. In contrast to CA15-3 and CA27.29, however, the median of TA-MUC1 was lower in sera from patients with ovarian cancer compared to sera from patients with benign diseases of the ovary. However, sera of patients with benign ovarian diseases had significantly higher TA-MUC1 values compared to sera of healthy individuals. The risk score of TA-MUC1 achieved an area under the curve (AUC) of 78.4% in receiver operating characteristic (ROC) curves and a sensitivity of 37% for the prediction of ovarian disease, at 95% specificity. CONCLUSION: In this study we employed an additional marker for MUC1 which recognizes a more tumour-specific MUC1 epitope (TA-MUC1). We obtained results showing significant differences between detection in benign and malignant ovarian diseases. Although the mean MUC1 values were elevated in sera of patients with ovarian cancer compared to values of patients with benign cysts, by using all three test systems, a different result was found by analysing the median TA-MUC1 values. PankoMab could be a useful, additional tool for obtaining conclusive information on the transformation process from benign to malignant state in ovarian tissues.

The Thomsen-Friedenreich disaccharide as antigen for in vivo tumor targeting with multivalent scFvs.

The Thomsen-Friedenreich disaccharide (TF(alpha)) is a promising antigen for tumor immunotargeting, since it is almost exclusively expressed on carcinoma tissues. So far, an obstacle preventing the exploitation of TF for immunotargeting has been the lack of suitable (non-IgM) antibodies with high affinity and specificity. Recently we reported on a novel strategy for generating antibodies toward small uncharged carbohydrates and the generation of recombinant antibodies toward TF. Among them, two multivalent scFv antibodies showed sub-micromolar functional affinities and appeared well suited for immunotargeting. In the present study, the trimeric scFv(1aa) and the tetrameric scFv(0aa) have been further developed for radioimmunotargeting. The scFvs were radiolabeled with (111)In using DTPA as chelator without losing binding activity or molecular stoichiometry. Binding affinities as high as 1 x 10(-7) M toward TF displayed on living cells were determined. Antibody biodistribution and tumor targeting efficacy were studied in TF-positive human breast cancer (ZR-75-1) bearing mice. TF was successfully targeted in vivo with tumor uptakes of approximately 11 and 8% ID/g after 24 h for the trimeric and tetrameric scFv, respectively. These results validate TF as a potent antigen for tumor targeting. The biodistribution of the scFvs was comparable to that reported for IgGs. In contrast to the IgGs, the serum clearance of the scFvs was very fast, which could be an advantage in a therapeutic setting. Furthermore, kidney uptake, which is often critical for small recombinant antibodies labeled with radio-metals, was low with the tetramer (11% ID/g). We conclude that the multimeric anti-TF scFvs are promising candidates to be further developed toward therapeutic application.

PankoMab: a potent new generation anti-tumour MUC1 antibody.

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.

Simple separation of DNA in antibody purification.

Producing monoclonal antibodies includes their efficient and simple purification. Growing hybridoma cells in media containing Prolifix, an alternative plant-based substitute for serum, provides supernatants containing large amounts of antibodies and defined low molecular weight additives. Antibodies can easily be separated from these compounds by fast ultrafiltration. However, DNA originating from lysed cells is present in substantial amounts and must be removed for most antibody applications. The present communication provides a fast, cheap, and efficient separation method by precipitating the DNA from a phosphate buffered solution with manganese chloride. Resulting antibodies have a high purity and an unchanged bioactivity. The method is especially valuable for antibodies which lose bioactivity by interactions with chromatographic matrices (as, for example, Sepharose) and can be used for various antibody isotypes.

Glycodelin and amniotic fluid transferrin as inhibitors of E-selectin-mediated cell adhesion.

Human amniotic fluid contains a variety of glycoproteins. Several of these substances have been shown to exert immunomodulatory effects. Glycodelin, previously known as placental protein 14, is one of these glycoproteins. It has a unique carbohydrate configuration, consistent with fucosylated LacdiNAc structures that are very unusual for mammals. Oligosaccharides with fucosylated LacdiNAc antennae have previously been shown to block selectin-mediated cell adhesion. Another glycoprotein, human transferrin, is also present in amniotic fluid in relatively high concentrations. This transferrin shows a different glycosylation compared with serum transferrin. Amniotic fluid transferrin carries sialylated Lewis X antigens. Glycodelin and transferrin were isolated from amniotic fluid and for comparison from serum of pregnant women by chromatographic methods. The purified proteins were used as ligands to block E-selectin-mediated HepG2 cell adhesion. Two types of binding assays with distinct receptor accommodations (immobilised E-selectin and activated HUVECs) were used to quantify inhibition efficiencies of the different proteins. We found that glycodelin is a strong inhibitor with a 10(3)-fold potency compared to the monovalent tetrasaccharide sialyl Lewis X whereas the potency of transferrin is rather low.