Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1.

A monoclonal antibody (MAb), VU-2-G7, was generated against a synthetic 60-mer MUC1 triple tandem repeat peptide with N-acetyl-galactosamine (GalNAc) O-linked to the threonine in the PDTR region of each repeat (3M GalNAc). VU-2-G7 and 8 MUC1 MAbs (VU-3-C6, VU-4-H5, 139H2, A76-A/C7, VU-12-E1, BCP9, MF11 and BW835) were tested against various glycosylated and nonglycosylated MUC1 tandem repeat peptides. VU-2-G7 showed strong reactivity with its immunogen, 3M GalNAc, and much lower reactivity with the nonglycosylated 60-mer MUC1 triple tandem repeat peptide. VU-2-G7 showed no reactivity with a 60-mer MUC1 triple tandem repeat peptide modified at the PDTR region or with a 60-mer MUC1 triple tandem repeat peptide with 3 GalNAc per repeat outside the PDTR region (9M GalNAc). In ELISA and flow cytometry, VU-2-G7 ubiquitously reacted with 4 MUC1-expressing breast cancer and 2 ovarian cancer cell lines and with a MUC1-gene-transfected Chinese hamster ovary cell line. The reactivity of VU-2-G7 was always higher than that of VU-4-H5, raised against a nonglycosylated 60-mer MUC1 triple tandem repeat peptide. Immunohistochemical staining of paraffin sections of breast and ovarian tumor tissues showed strong binding of VU-2-G7 predominantly at the cell membrane. The dominant epitope of VU-2-G7 is in the glycosylated PDTR motif of the MUC1 tandem repeat, and this epitope is abundantly present on the surface of tumor cell lines and breast and ovarian tumor tissues. Given the ubiquitously aberrant glycosylation of MUC1 in malignant cells, the production of MAbs against highly purified glycosylated MUC1 tandem repeat peptides may yield MAbs better suited for the immunotherapy of carcinomas than those available at the moment.

Reactivity of natural and induced human antibodies to MUC1 mucin with MUC1 peptides and n-acetylgalactosamine (GalNAc) peptides.

Antibodies (Abs) to MUC1 occur naturally in both healthy subjects and cancer patients and can be induced by MUC1 peptide vaccination. We compared the specificity of natural and induced MUC1 Abs with the objective of defining an effective MUC1 vaccine for active immunotherapy of adenocarcinoma patients. Serum samples, selected out of a screened population of 492 subjects for their high levels of IgG and/or IgM MUC1 Abs, were obtained from 55 control subjects and from 26 breast cancer patients before primary treatment, as well as from 19 breast cancer patients immunized with MUC1 peptides coupled to keyhole limpet hemocyanin (KLH) and mixed with QS-21. The samples were tested with enzyme-linked immunoassays for reactivity with (1) overlapping hepta- and 20-mer peptides spanning the MUC1 tandem repeat sequence; (2) two modified 60-mer peptides with substitutions in the PDTR (PDTA) or in the STAPPA (STAAAA) sequence of each tandem repeat; and (3) four 60-mer glycopeptides with each 1, 2, 3 and 5 mol N-acetylgalactosamine (GalNAc) per repeat. More than one minimal epitopic sequence could be defined, indicating that Abs directed to more than one region of the MUC1 peptide core can coexist in one and the same subject. The most frequent minimal epitopic sequence of natural MUC1 IgG and IgM Abs was RPAPGS, followed by PPAHGVT and PDTRP. MUC1 peptide vaccination induced high titers of IgM and IgG Abs predominantly directed, respectively, to the PDTRPAP and the STAPPAHGV sequences of the tandem repeat. Natural MUC1 Abs from breast cancer patients reacted more strongly with the N-acetylgalactosamine (GalNAc) peptides than with the naked 60-mer peptide, while reactivity with the GalNAc-peptides was significantly reduced (2-tailed p < 0.0001) in the MUC1 IgG and IgM Abs induced by MUC1 peptide vaccination. Whereas in cancer patients glycans appear to participate in epitope conformation, the epitope(s) recognized by MUC1 Abs induced by peptide vaccination are already masked by minimal glycosylation. Therefore, our results indicate that a MUC1 glycopeptide would be a better vaccine than a naked peptide.

Aberrant expression of MUC1 mucin in ductal hyperplasia and ductal carcinoma In situ of the breast.

MUC1 mucin is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of normal glandular epithelia. In many human adenocarcinomas, this protein is up-regulated and/or underglycosylated, and its expression changes from apical to the entire cell membrane. It is thought that entire cell membrane expression of MUC1 reduces cell-cell and cell-extracellular matrix interactions and therefore may facilitate invasive growth and development of metastases. In this study, we determined immunohistochemically the expression of normal and underglycosylated MUC1 in normal breast tissue (n = 8) and in a spectrum of breast lesions, including usual ductal hyperplasia (n = 23), atypical ductal hyperplasia (n = 7), and ductal carcinoma in situ (DCIS) (n = 22). We used 4 monoclonal antibodies; 115D8 is directed to a glycopeptide, the other 3 to the peptide core of the molecule, of which 139H2 is not affected by the degree of glycosylation of MUC1, whereas SM3 and VU-4-H5 stain only underglycosylated forms. All cases showed apical positivity for 115D8 and 139H2. Entire cell membrane expression of fully (normal) glycosylated MUC1 was mainly found in DCIS lesions. Apical staining of SM3 was found in 38% of normal cases and 60% of the ductal lesions with no difference between the different subgroups. Apical staining of VU-4-H5 was found more often in DCIS (27%) than in normal tissue or ductal hyperplasia (3%). Membrane expression of underglycosylated MUC1 was found only in poorly differentiated DCIS. In conclusion, aberrant expression of MUC1, i.e., on the entire cell membrane and/or underglycosylated forms, can be found in ductal hyperplasia with atypia and especially in DCIS of the breast. This finding implies that these lesions with aberrant expression are at higher risk for developing subsequent invasive breast carcinoma.

'Epitope fingerprinting' using overlapping 20-mer peptides of the MUC1 tandem repeat sequence.

The ISOBM TD-4 Workshop antibodies 122-177 were tested for reactivity with 20 overlapping MUC1 tandem repeat 20-mer peptides by an ELISA, in order to determine the complete amino acid sequences of the epitopes. Of the 56 antibodies studied, 30 showed specific binding and thus the epitopes were characterized. The epitopes appear to be 'broader' when compared to those deduced from studies using smaller peptides. Interassay variation is remarkably small, allowing for precise grouping of clusters with very similar epitope patterns. Five groups of antibodies show remarkable similarity: BC3 and VU-4-H5; BC4W154, C595 and Mc5; MF06 and B27.29; VU-11-D1 and VU-11-E2; Ma552, VU-3-C6, 7540MR and BC4E549. We have used the term 'epitope fingerprinting' to refer to the 'fine structure' of the epitope with all its essential and flanking amino acids. We believe this method is more precise than the usual epitope mapping with short peptides.

Establishment of immunological probes to study human gonadotropin-releasing hormone receptors.

Monoclonal antibodies were raised to a synthetic peptide corresponding to amino acids 1-29 of the human gonadotropin-releasing hormone (GnRH) receptor. One of the two antibodies was found to recognise GnRH receptors on human pituitary gonadotrophs as determined by immunohistochemistry and supported by Western blotting. The antibody also bound to T47D human breast carcinoma cell line as determined by flow cytometric analysis.

Tenascin expression in basal cell carcinoma.

Tenascin (hexabrachion, cytotactin) is an extracellular matrix glycoprotein whose expression is strongly increased in hyperproliferative skin diseases, as shown by immunohistochemistry with polyclonal sera. In this study we describe a new monoclonal antibody (T2H5) against human tenascin. The specificity of T2H5 was validated by sequential immunoprecipitation of tenascin with polyclonal sera. T2H5 was used to analyse the presence of tenascin in basal cell carcinoma. Using Western blotting, at least two forms of tenascin were found, with approximate molecular weights of 210 and 300 kDa. In cultured human skin fibroblasts only the high molecular weight form was found.

Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212.

Monoclonal antibodies (MAbs) 123C3 and 123A8 generated against a membrane preparation of a small cell lung carcinoma (SCLC) specimen recognize not only SCLC and bronchial carcinoids but also a significant portion of non-small cell lung carcinomas (non-SCLC) of various histological types. Together with 13 other monoclonal antibodies, which show preference for SCLC, they have been ranked as SCLC cluster 1 (SC-1) Mabs. In this study we show that SC-1 MAbs are directed against a restricted number of epitopes, and that SC-1 MAbs and a polyclonal antiserum directed against the neural cell adhesion molecule (NCAM) recognize identical glycoproteins, indicating that SC-1 antigens are closely related to or identical with NCAM. Long polysialic acid units composed of alpha-(2,8)-linked N-acetylneuraminic acid units, which in mammals are found exclusively on NCAM, were present on SC-1 antigens in SCLC. This provides further evidence that SC-1 MAbs recognize NCAM. The SC-1 antigens in the SCLC cell line H69 were present in two forms, NCAM-containing alpha-(2,8)-polysialic acid units identified by antiserum 735, the NCAM-H form, and the less sialylated NCAM-L form. The NCAM-H form consisted of diffusely migrating sialoglycoproteins with a molecular weight of 200,000-250,000, which resolved after neuraminidase treatment into two proteins with molecular weights of 140,000 and 180,000. Since the NCAM-H form is expressed in the lung tumor type with a poor prognosis, our results suggest that NCAM might be implicated in the invasive behavior of these NCAM-positive lung tumors.

Effect of sodium butyrate and other differentiation inducers on poorly differentiated human ovarian adenocarcinoma cell lines.

We have studied the effects of sodium butyrate, retinoic acid, and dimethyl sulfoxide on two human ovarian carcinoma cell lines PE04 and PE01. PE04 cells, after treatment with sodium butyrate at cytostatic doses (2-3 mM for 4 days), exhibited phenotypic changes including induction of alkaline phosphatase and determinants recognized by the monoclonal antibodies 123C3 and 123A8. These effects are not simply the result of cytostasis as they were not produced by dimethyl sulfoxide or retinoic acid. Other markers are also modified by sodium butyrate including lipid, acid mucin, and glycogen. Retinoic acid modulated expression of lipid and CA125, while dimethyl sulfoxide reduced expression of CA125. Other short chain fatty acids such as propionic acid and valeric acid (in addition to butyric acid) also induced alkaline phosphatase and the determinants recognized by 123C3 and 123A8 in PE04 cells. Other differentiation inducers and cytotoxic agents studied did not induce these markers at cytostatic concentrations. The effects of sodium butyrate (and related short chain fatty acids) thus appear to be relatively specific for this cell line.

Characterization and properties of nine human ovarian adenocarcinoma cell lines.

Four series of cell lines have been derived from patients with ovarian adenocarcinoma. Nine cell lines have been established at one from a solid metastasis. Six lines were derived from the ascites or pleural effusion of patients with poorly differentiated adenocarcinoma: PEO1, PEO4, and PEO6 from one patient, PEA1 and PEA2 from a second, and PEO16 from a third. Three lines (PEO14 and PEO23 from ascites and TO14 from a solid metastasis) were derived from a patient with a well-differentiated serous adenocarcinoma. Each set of cell lines was morphologically distinct. The five cell lines PEO1, PEO4, PEO6, PEA1, and PEA2 had cloning efficiencies on plastic of 1-2% and only a few cells in these lines expressed alkaline phosphatase or vimentin. Only a low percentage of these cells reacted with the monoclonal antibodies 123C3 and 123A8 but most reacted with OC125. Conversely the cell lines PEO14, TO14, PEO23, and PEO16 were characterized by low cloning efficiency values (less than 0.05%), marked expression of alkaline phosphatase and vimentin, and good reaction with 123C3 and 123A8 but not OC125. These four cell lines also exhibited dome formation. Four of the cell lines, PEO1, PEO4, PEO6, and PEO16, have been xenografted into immune-deprived mice and found to be tumorigenic.

Generation of monoclonal antibodies against human carcinosarcoma cell line for identification of myoepithelium and basement membrane.

An immunohistochemical method is described for identification of myoepithelial cells and basement membrane for cryostat tissue sections of normal, benign, and in situ carcinomas of the breast using two monoclonal antibodies 155C1 and 155D10 generated against human breast carcinosarcoma cell line HS578T. In the majority of infiltrating ductal carcinomas of the breast, there was a discontinuity in the myoepithelial cell layer, as a result an intact basement membrane could not be visualized. The reactivity of these two monoclonal antibodies might prove useful in the study of myoepithelial differentiation antigens and in the delineation of basement membrane. Among the other types of tissues studied, prominent staining was present with soft tissue tumors like leiomyosarcoma and synovial sarcoma.

Monoclonal antibody 123C3, identifying small cell carcinoma phenotype in lung tumours, recognizes mainly, but not exclusively, endocrine and neuron-supporting normal tissues.

Monoclonal antibody (MAb) 123C3 was raised against a membrane preparation of a small cell lung carcinoma (SCLC) specimen and its reactivity on normal tissues was tested. For the endocrine system, positive tissues included: pituitary and adrenal glands, thyrocytes and C-cells of the thyroid, the parathyroids, testis Leydig cells and pancreatic islets. In bronchioles and intestinal epithelium occasional cells, resembling Kultchitsky and enterochromaffin cells, were also positive. Epithelia like rete testis, mammary epithelium and gastric mucosa were positive in all or a significant proportion of cells. The positive cells in mammary epithelium and gastric mucosa were too numerous to represent the endocrine cells only. Neurons were usually negative or weakly positive. Their supportive cells such as glial, Schwann and ganglionic satellite cells were positive. Mesenchymal cell types, such as smooth muscle cells in most organs, cardiac muscle cells, the pia-arachnoid and ovarian stroma cells were positive, indicating that 123C3 reactivity is not confined to epithelial and neuron-supporting tissues. In Western blots of tumour specimens 123C3 recognized a 29 kDa band in reducing conditions, shifting to approximately 150 kDa in non-reducing conditions. Immunofluorescence on live tissue culture cells demonstrated presence of the antigen on the cell surface.