Immunoreactivity of the fully humanized therapeutic antibody PankoMab-GEX™ is an independent prognostic marker for breast cancer patients.

BACKGROUND: Mucin-1 (MUC1, CD227), more widely known as CA15-3, is an abundantly expressed epithelial cell surface antigen and has evolved to be the most predictive serum tumour marker in breast cancer. PankoMab-GEX™, which is currently being evaluated for its therapeutic efficacy in a phase IIb clinical trial, is a glyco-optimized anti-MUC1 antibody specifically recognizing a tumour-associated MUC1 epitope (TA-MUC1). The current study aimed to analyse the immunoreactivity of PankoMabGEX™ and its correlation with established clinico-pathological variables including 10-year and overall survival in a large cohort of breast cancer patients. METHODS: Breast cancer tissue sections (n = 227) underwent a standardized immunohistochemical staining protocol for TA-MUC1 by using PankoMab-GEX™ as a primary antibody. The staining was evaluated by two independent observers and quantified by applying the IR-score. RESULTS: TA-MUC1 as detected by PankoMab-GEX™ was identified in 74.9% of breast cancer tissue sections. Patients were subdivided according to the subcellular localisation of TA-MUC1 and cases classified as mem-PankoMab-GEX™ (solely membranous) positive, cyt-PankoMab-GEX™ (solely cytoplasmic) positive, double positive or as completely negative were compared regarding their survival. Herein mem-PankoMab-GEX™-positive patients performed best, while double-negative ones presented with a significantly shortened survival. Positivity for mem-PankoMab-GEX™ as well as a double-negative immunophenotype turned out to be independent prognosticators for survival. CONCLUSIONS: This is the first study to report on PankoMab-GEX™ in a large panel of breast cancer patients. The PankoMab-GEX™ epitope TA-MUC1 could be identified in the majority of cases and was found to be an independent prognosticator depending on its subcellular localisation. Since TA-MUC1 is known to be highly immunogenic cancers staining positive for PankoMab-GEX™ might be more compromised by host anti-tumour immune defence. Further, the observations reported here might be fundamental for selecting patients to undergo PankoMab-GEX™-containing chemotherapy protocols.

What makes cancer stem cell markers different?

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.

Multicentric and multifocal versus unifocal breast cancer: differences in the expression of E-cadherin suggest differences in tumor biology.

BACKGROUND: The aim of this study was to evaluate the expression of the cell adhesion-related glycoproteins MUC-1, ß-catenin and E-cadherin in multicentric/multifocal breast cancer in comparison to unifocal disease in order to identify potential differences in the biology of these tumor types. METHODS: A retrospective analysis was performed on the expression of MUC1, ß-catenin and E-cadherin by immunohistochemistry on tumor tissues of a series of 112 breast cancer patients (total collective) treated in Munich between 2000 and 2002. By matched-pair analysis, 46 patients were entered into two comparable groups of 23 patients after categorizing them as having multicentric/multifocal or unifocal breast cancer. Matching criteria were tumor size, histology grade and lymph node status; based on these criteria, patients were distributed equally between the two groups (p = 1.000 each). Data were analyzed with the Kruskal-Wallis and the Mann-Whitney tests. RESULTS: In the matched groups, we found a significantly down-regulated expression of E-cadherin in multicentric/multifocal breast cancer compared to unifocal disease (p = 0.024). The total collective showed even higher significance with a value of p < 0.0001. In contrast, no significant differences were observed in the expression of ß-catenin between multicentric/multifocal and unifocal tumors (p = 0.636 and p = 0.914, respectively). When comparing the expression of MUC1, E-cadherin and ß-catenin within the unifocal group, we found a significant positive correlation between E-cadherin and ß-catenin (p = 0.003). In the multicentric/multifocal group we observed, in contrast to the unifocal group, a significant decrease of MUC1 expression with increased grading (p = 0.027). CONCLUSION: This study demonstrates that multicentric/multifocal and unifocal breast cancers with identical TNM-staging clearly differ in the expression level of E-cadherin. We suggest that the down-regulation of E-cadherin in multicentric/multifocal breast cancer is causally connected with the worse prognosis of this tumor type.

Specific humoral immune response to the Thomsen-Friedenreich tumor antigen (CD176) in mice after vaccination with the commensal bacterium Bacteroides ovatus D-6.

The tumor-specific Thomsen-Friedenreich antigen (TFα, CD176) is an attractive target for a cancer vaccine, especially as TF-directed antibodies play an important role in cancer immunosurveillance. However, synthetic TF vaccines have not overcome the low intrinsic immunogenicity of TF. Since natural TF-directed antibodies present in human sera are generated in response to microbes found in the gastrointestinal tract, microbial TF structures are obviously more immunogenic than synthetic TF. We recently isolated a new strain (D-6) of the human gut bacterium Bacteroides ovatus, which carries the true TFα antigen. Here, we present experimental data on the immunogenicity of this strain. Mice immunized with B. ovatus D-6 in the absence of adjuvants developed specific anti-TFα IgM and IgG antibodies which also bound to human cancer cells carrying TFα. Our data suggest that B. ovatus D-6 presents a unique TFα-specific immunogenicity based on a combination of several inherent properties including: expression of the true TFα antigen, clustering and accessible presentation of TFα as repetitive side chains on a capsular polysaccharide, and intrinsic adjuvant properties. Therefore, B. ovatus strain D-6 is an almost perfect candidate for the development of the first adjuvant-free TFα-specific anti-tumor vaccine.

Staining of MUC1 in ovarian cancer tissues with PankoMab-GEX detecting the tumour-associated epitope, TA-MUC1, as compared to antibodies HMFG-1 and 115D8.

PankoMab-GEX is a novel humanized and glycooptimized antibody, which recognizes a novel specific tumour epitope of MUC1 (TA-MUC1). The aim of this study was to evaluate PankoMab-GEX binding to a variety of ovarian cancer specimens (n=156) and to normal ovarian tissue. In addition, PankoMab-GEX staining was compared to that of the well-known anti-MUC1 antibodies HMFG-1 and 115D8. PankoMab-GEX showed positive reactivity in serous (100% of cases, mean IRS 8.23), endometrioid (95% of cases, mean IRS 6.40), mucinous (58% of cases, mean IRS 4.17), and clear cell (92% of cases, mean IRS 7.58) carcinomas. In contrast to HMFG-1, healthy ovarian tissue was not recognized by PankoMab-GEX. Staining with antibody 115D8 was increased with staging. Cytoplasmic PankoMab-GEX staining increased with tumour grade, but no correlation was found with staging. Univariate Kaplan-Meier analysis revealed a tendency of reduced survival of patients with high expression of TA-MUC1. The findings are encouraging with respect to a potential use of PankoMab-GEX as a new therapeutic antibody for the treatment of ovarian cancer patients.

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.

Preliminary safety evaluation of a new Bacteroides xylanisolvens isolate.

Besides conferring some health benefit to the host, a bacterial strain must present an unambiguous safety status to be considered a probiotic. We here present the preliminary safety evaluation of a new Bacteroides xylanisolvens strain (DSM 23964) isolated from human feces. First results suggest that it may be able to provide probiotic health benefits. Its identity was confirmed by biochemical analysis, by sequencing of its 16S rRNA genes, and by DNA-DNA hybridization. Virulence determinants known to occur in the genus Bacteroides, such the bft enterotoxin and other enzymatic activities involved in the degradation of the extracellular matrix and the capsular polysaccharide PS A, were absent in this strain. The investigation of the antibiotic susceptibility indicated that strain DSM 23964 was sensitive to metronidazole, meropenem agents, and clindamycin. Resistance to penicillin and ampicillin was identified to be conferred by the ß-lactamase cepA gene and could therefore be restored by adding ß-lactamase inhibitors. The localization of the cepA gene in the genome of strain DSM 23964 and the absence of detectable plasmids further suggest that a transfer of ß-lactamase activity or the acquisition of other antibiotic resistances are highly improbable. Taken together, the presented data indicate that the strain B. xylanisolvens DSM 23964 has no virulence potential. Since it also resists the action of gastric enzymes and low-pH conditions, this strain is an interesting candidate for further investigation of its safety and potential health-promoting properties.

Safety assessment of the commensal strain Bacteroides xylanisolvens DSM 23964.

We recently isolated and characterized the new strain Bacteroides xylanisolvens DSM 23964 and presented it as potential candidate for the first natural probiotic strain of the genus Bacteroides. In order to evaluate the safety of this strain for use in food, the following standard toxicity assays were conducted with this strain in both viable and pasteurized form: in vitro bacterial reverse mutation assay, in vitro chromosomal aberration assay, and 90day subchronic repeated oral toxicity studies in mice. No mutagenic, clastogenic, or toxic effects were detected even at extremely high doses. In addition, no clinical, hematological, ophthalmological, or histopathological abnormality could be observed after necropsy at any of the doses tested. Hence, the NOAEL could be estimated to be greater than 2.3×10(11) CFUs, and 2.3×10(14) for pasteurized bacteria calculated as equivalent for an average 70kg human being. In addition, the absence of any in vivo pathogenic properties of viable B. xylanisolvens DSM 23964 cells was confirmed by means of an intraperitoneal abscess formation model in mice which also demonstrated that the bacteria are easily eradicated by the host's immune system. The obtained results support the assumed safety of B. xylanisolvens DSM 23964 for use in food.

Occurrence of the human tumor-specific antigen structure Galß1-3GalNAcα- (Thomsen-Friedenreich) and related structures on gut bacteria: prevalence, immunochemical analysis and structural confirmation.

The Thomsen-Friedenreich antigen (TF; CD176, Galß1-3GalNAcα-) is a tumor-specific carbohydrate antigen and a promising therapeutic target. Antibodies that react with this antigen are frequently found in the sera of healthy adults and are assumed to play a role in cancer immunosurveillance. In this study, we examined the occurrence of α-anomeric TF (TFα) on a large variety of gastrointestinal bacteria using a novel panel of well-characterized monoclonal antibodies. Reactivity with at least one anti-TF antibody was found in 13% (16 of 122) of strains analyzed. A more in-depth analysis, using monoclonal antibodies specific for α- and ß-anomeric TF in combination with periodate oxidation, revealed that only two novel Bacteroides ovatus strains (D-6 and F-1), isolated from the faeces of healthy persons by TF-immunoaffinity enrichment, possessed structures that are immunochemically identical to the true TFα antigen. The TF-positive capsular polysaccharide structure of strain D-6 was characterized by mass spectrometry, monosaccharide composition analysis, glycosidase treatments and immunoblot staining with TFα- and TFß-specific antibodies. The active antigen was identified as Galß1-3GalNAc-, which was α-anomerically linked as a branching structure within a heptasaccharide repeating unit. We conclude that structures immunochemically identical to TFα are extremely rare on the surface of human intestinal bacteria and may only be identifiable by binding of both antibodies, NM-TF1 and NM-TF2, which recognize a complete immunomolecular imprint of the TFα structure. The two novel B. ovatus strains isolated in this study may provide a basis for the development of TF-based anti-tumor vaccines.

Expression of CD176 (Thomsen-Friedenreich antigen) on lung, breast and liver cancer-initiating cells.

The cancer-initiating capacity of most malignant tumours is considered to reside in a small subpopulation of cells. Therapeutical interventions should target these cells rather than the tumour mass. Numerous studies have shown that the carbohydrate antigen structure CD176 (Thomsen-Friedenreich antigen, core-1) is present in many types of cancer and absent in normal adult human tissues. In this study, we assessed whether CD176 is co-expressed with CD44 or CD133 [markers of cancer-initiating cells (CIC)] in human lung, breast and liver carcinoma. A variety of human cancer cell lines and surgical specimens of these malignancies were examined. It was found that in most cases the majority of tumour cells stained strongly for CD44 by immunohistochemistry and flow cytometry, whereas CD133 expression was found on a smaller, but varying proportion of cells. Co-expression of CD176 with CD44 was found at a surprisingly high percentage of cancer cells in vitro and in vivo. Co-expression of CD176 with CD133 was also detected, although at a lower rate. Tamoxifen treatment of MDA-435 breast cancer cells enhanced the CD44(+) /CD176(+) phenotype. Evidence is provided through a new sandwich solid-phase enzyme-linked immunosorbent assay (ELISA) suggesting that CD44 is a carrier molecule for CD176 not only in colorectal cancer as previously reported, but also in lung, breast and liver cancer. The expression of CD176 in CIC suggests that it may represent an effective target for tumour therapies.

Reactivity of a humanized antibody (hPankoMab) towards a tumor-related MUC1 epitope (TA-MUC1) with various human carcinomas.

Humanized antibodies against tumor-related antigens are now established reagents for in vivo diagnostics and for therapeutic approaches, and are increasingly developed. Humanized PankoMab (hPankoMab), a humanized form of PankoMab directed against a novel tumor-related MUC1 epitope (TA-MUC1), was recently developed for clinical application. In the present study, the reactivity of hPankoMab with various human cancers was systematically studied by immunohistochemistry on 137 surgical specimens, including lung, breast, gastric, colorectal, liver, cervical, kidney, thyroid, and other carcinomas, as well as on several non-epithelial malignancies. The study was performed on paraffin sections without antigen retrieval. hPankoMab reacted strongly with carcinomas originating from glandular or squamous epithelia, weakly with hepatocellular carcinomas, and not with sarcomas. The binding sites of hPankoMab in carcinomas were found around the whole cell surface and, in most cases, also in the cytoplasm of cancer cells.

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells.

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173(+)/CD44(+) cells ranged from 17% to >60% and of CD174(+)/CD44(+) from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.

The human endometrium expresses the glycoprotein mucin-1 and shows positive correlation for Thomsen-Friedenreich epitope expression and galectin-1 binding.

Mucin 1 (MUC1) is a glycoprotein in human endometrium and is abundant at the luminal epithelial surface in the receptive phase. It has a highly glycosylated ecto-domain that contains keratan sulfate chains, that disappears at the time of implantation. In addition, the glycoforms on MUC1 differ in fertile and infertile women. Therefore the aims of this study were investigations on glycosylation of MUC1 with the Thomsen-Friedenreich (TF) epitope on normal human endometrium throughout the menstrual cycle and binding of galectin-1 on the TF epitope in the endometrium and the expression of galectin-1 on the human oocyte. Human endometrial tissue was obtained from 54 premenopausal patients and was immunohistochemically analyzed with monoclonal antibodies against MUC1, TF epitope, galectin-1, and biotinylated galectin-1. In addition, human oocytes were analyzed for TF, galectin-1 expression, and galectin-1 binding. We identified a significant upregulation of MUC1 and TF epitope and, in addition, galectin-1 binding in glandular epithelium and epithelial apical surface tissue from proliferative to secretory phase. With double staining experiments, we identified a coexpression of TF and MUC1 in the early secretory phase and galectin-1 binding to TF during the same period of time. In addition we identified TF epitope and galectin-1 expression plus binding on the human oocyte and irregularly fertilized oocytes. Upregulation of TF epitope on the glandular epithelium and epithelial apical surface tissue in the secretory phase and binding of galectin-1 at the same time show the possibility of galectin-1-mediated trophectoderm binding to the endometrium within the window of implantation.

Evaluation of a novel anti-mucin 1 (MUC1) antibody (PankoMab) as a potential diagnostic tool in human ductal breast cancer; comparison with two established antibodies.

AIM: PankoMab is a novel antibody that recognizes a tumor-specific epitope of Mucin 1 (MUC1). The aim of this study was the evaluation of PankoMab as a potential diagnostic tool and its comparison with two established antibodies against MUC1 in human ductal breast cancer. MATERIALS AND METHODS: Breast carcinomas were obtained from 82 patients. MUC1 expression and hormone receptor status were determined by immunohistochemistry of paraffin-embedded material. RESULTS: PankoMab revealed strong correlation to hormone receptor expression. DF3 showed no correlation with grading, lymph node involvement and/or estrogen receptor (ER) expression. In the subgroup of lymph node-positive and ER-negative tumors, we saw a significantly reduced DF3 staining in G3 tumors compared to G2 tumors. VU-4-H5 showed increased staining intensity in correlation with increased grading. In addition, we also identified a significantly higher expression of the VU-4-H5 epitope in lymph node-positive carcinomas compared to carcinomas without lymph node involvement. CONCLUSION: PankoMab revealed strong correlation to hormone receptor expression in ductal carcinoma of the breast. VU-4-H5 showed increased staining intensity in correlation with increased grading and lymph node involvement. PankoMab and VU-4-H5 staining could be a useful combination in ductal breast cancer prognosis by immunohistochemistry.

Expression of CD175 (Tn), CD175s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells.

The expression of the histo-blood group carbohydrate structures T-nouvelle (Tn, CD175), sialylated Tn (CD175s) and the Thomsen-Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal antibodies in flow cytometry, immunohistology and immunoprecipitation. Expression of sialylated CD176 was evaluated by comparative immunostaining with anti-CD176 antibodies before and after sialidase treatment. While only few cell lines expressed unmasked CD176, sialylated CD176 was present on all hematopoietic cell lines and native lymphocytes examined. CD175 and CD175s are preferentially expressed on erythroblastic leukemia cell lines. CD175s expression in these cells is consistent with the transcription of the gene encoding the key enzyme alpha2,6-sialyltransferase (hST6GalNAc1). The staining intensity was reduced after methanol pretreatment of cells, indicating that these glycans are partially expressed as constituents of glycosphingolipids. Immunoprecipitation and subsequent Western blotting revealed a series of distinct high molecular glycoproteins as carriers for these carbohydrate antigens. CD34 was identified as major carrier of CD176 by immunoprecipitation and microsequencing on a KG-1 subline enriched for CD176 expression. Incubation of several CD176-positive cell lines with anti-CD176 antibodies induced apoptosis of these cells, an effect not observed with anti-CD175/CD175s antibodies. Since the presence of naturally occurring anti-CD176 antibodies may represent a mechanism of immunosurveillance against CD176-positive tumor cells, we propose that sialylation of surface-expressed CD176--among other functions--protects against apoptosis.

Immunomagnetic enrichment of disseminated tumor cells in bone marrow and blood of breast cancer patients by the Thomsen-Friedenreich-Antigen.

PURPOSE: The presence of disseminated tumor cells in the bone marrow (DTC-BM) of breast cancer patients has shown independent prognostic impact. Immunomagnetic enrichment of such cells is an approach to increase the number of detected cells with limited sample volume, especially for circulating tumor cells (CTCs) in blood. The Thomsen-Friedenreich (TF) antigen (CD 176) is a specific oncofetal carbohydrate epitope (Galbeta1-3GalNAcalpha-O) expressed on the surface of various carcinomas. Own studies demonstrated a nearly complete TF expression on DTC-BM, indicating its suitability as marker for immunomagnetic enrichment. METHODS: BM samples of 65 and peripheral blood samples of 11 breast cancer patients were examined immunocytochemically by staining with the anti-Cytokeratin antibody A45-B/B3 before and after immunomagnetic enrichment. Enrichment was done by incubation with the primary antibody TF 2 (IgM), followed by secondary magnetically labelled rat-anti mouse IgM. Cytospin slides were screened manually by bright-field microscopy. RESULTS: 15/65 pts (23%) showed DTC-BM in primary screening with a median of 2/2 mio cells (range 1-10). By enrichment, a median of 23.3 mio cells (0.8-218) could be analysed, increasing positivity to 72% (47/65 pts) with a med. of 4 DTCs (1-105, P < .0001). Blood from 1/11 pts before and 5/11 (45%) after enrichment showed CTCs (med. 2, 1-20), at a med. of 12.4 mio (2.6-38.5) cells analysed. Comparing BM and blood of the same patients after enrichment, 5 were positive in both compartments, 4 showed DTC-BM without presence of CTCs. CONCLUSION: The positive immunomagnetic enrichment technique with TF-antibodies enables to analyse larger sample volumes and increase tumor cell detection rate. This could allow monitoring and characterisation of CTCs as targets for therapies.

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.

Prognostic impact of Thomsen-Friedenreich tumor antigen and disseminated tumor cells in the bone marrow of breast cancer patients.

PURPOSE: The Thomsen-Friedenreich antigen (TF, CD176) is a specific oncofetal carbohydrate epitope (Gal beta1-3GalNAc alpha-O-Ser/Thr) expressed on the surface of various carcinomas. It mediates endothelium adhesion and formation of metastases. As it also causes immune response, its prognostic impact is indeterminate. The presence of disseminated tumor cells in the bone marrow of breast cancer patients (DTC-BM) indicates worse prognosis. We examined the expression of TF in primary breast cancer tissue of 265 patients with known BM status at the time of first diagnosis. METHODS: BM aspiration, cytospin preparation and immunocytochemical staining with the anti-Cytokeratin antibody A45 B/B3 was done following a standardised protocol. TF expression was examined immunohistochemically on Tissue Micro Arrays (TMA) with the anti-TF antibody A78-G/A7. Evaluation was done using the immunoreactive score (IRS). RESULTS: Median IRS for TF expression was 2 (0-12). 68 of 265 patients (25.7%) showed DTC-BM with a median of 2/2 x 10(6) cells (1-1500). There was no correlation between TF expression and DTC-BM. After a median follow up of 60.1 months (7-119), the detection of DTC-BM showed prognostic significance for overall survival (OS, p = 0.034), whereas TF positivity (IRS > 2) indicated prolonged disease-free (p = 0.01), distant disease-free (p = 0.005), and overall survival (p = 0.005). DISCUSSION: Patients with TF-positive tumors had a significantly better prognosis. Dissemination routes, TF-mediated metastasis formation, and the immunogeneity of TF might determine the prognostic impact of TF expression in different tumor entities. Further characterisation of primary tumors and DTC-BM could help to improve the biological understanding of metastases and develop targeted therapies.

Detection, isolation and partial characterization of an immunostimulating glycoprotein from Rhodococcus fascians.

In a search for novel immunostimulating substances we detected that culture supernatants of the gram-positive phytopathogenic bacterium, Rhodococcus fascians, were able to induce cytokine release (TNF(alpha)) from mouse peritoneal macrophages. Monoclonal antibodies were generated against the active principle, and were employed for its isolation and partial characterization as a high molecular (MW>100 kDa) glycoprotein. In addition, methods practicable for its biotechnological preparation and several ELISA variants for its determination were developed.

Binding of galectin-1 (gal-1) to the Thomsen-Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody.

Galectin-1 (gal-1), a member of the mammalian beta-galactoside-binding proteins, recognizes preferentially Galbeta1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen-Friedreich (TF)-disaccharide (Galbeta1-3GalNAc-) conjugated to polyacrylamide (TF-PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galbeta1-4GlcNAc and Galbeta1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.