Ph. Hageman

Monoclonal Antibodies against Human Milkfat Globule Membranes Useful in Carcinoma Research

Abstract Murine monoclonal antibodies against antigens of purified membranes from human milkfat globules were generated. The various antigens detectable were partly characterized and studied for their usefulness in tumor classifications. Not only mammary cancers, but a variety of carcinomas from other than mammary gland epithelia showed positive reactions with selected series of these antibodies. Of these antibodies 115D8, against antigens of the Mam-6 group, seems to be the best one for targeting tumors in vivo.

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.

Monoclonal Antibodies Against Human Milkfat Globule Membranes Detecting Differentiation Antigens of the Mammary Gland

Abstract Monoclonal antibodies of mouse origin were raised against antigens of highly purified human milkfat globule membranes. Five groups of antigens, Mam-1 to Mam-5, could be distinguished based on immunoprecipitation followed by determination of the molecular weights, their presence or absence from milk proteins and their tissue distribution in normal and malignant mammary glands.

A radioimmunoassay for mtv-antigens.

The second method of approach resulted in a useful assay by which at least two different sets of antigens can be distinguished. Figure 1 represents regression lines obtained with respectively 4 plasma samples from female tumourbearing mice: (C3Hx020) F1, GR and Balb/c foster C3H, one sample of MTVpositive milk (Balb/c foster C3H), and a mammary tumour extract from (C3H x 020)F1. Plasma and milk samples from Balb/c as well as a RLV (Rauscher Leukemia Virus) preparation extract show no inhibition of binding (average 56% bound at all dilutions). As can

Reactions of Monoclonal Antibodies Against Human Melanoma with Different Tissues and Cell Lines

Abstract A BALB/c mouse was immunized with plasma membranes from the human melanoma cell line MeWo. Spleen cells of the mouse were fused with the IgG-1 producing mouse myeloma cell line P3×63Ag8. Seven hybridomas were selected that produced antibodies reacting against several human melanoma cell lines or shortly cultured melanoma cells. The antibodies did not have a great specificity on cell lines as they reacted with most of the controls. On thin tissue sections the antibodies showed a much higher specificity. On the basis of the reactions on tissue the hybridomas could be grouped according to four reaction patterns. One hybridoma (C-1) produced antibodies with a broad tissue distribution; the reaction was directed against a 88 k protein. Two groups of antibodies (C-2,6,7 and C-5) reacted nearly only with metastatic melanomas. The last group (C-3,4) produced antibodies that reacted with melanoma as well as other tissues, in particular human mammary tumor.

Sweat Glands and Salivary Glands as Model System for the Characterization of Monoclonal Antibodies against Differentiation Antigens of the Human Mammary Gland

Abstract The reaction of monoclonal antibodies against human milkfat globule membranes on thin sections of resting mammary glands and benign and malignant lesions of the human breast is often heterogeneous and not clearly related to morphological structures. We studied the reaction of two antibodies reacting with the Mam-3 antigen and three antibodies reacting with the Mam-6 antigen (Hilkens et al., this volume) on thin sections of formalin-fixed and embedded salivary glands and sweat glands. On these glands a reproducible, though slightly variable reaction pattern in several individuals was observed. Each of the antibodies had its own characteristic reaction with morphologically recognizable glandular structures: the antibodies of the Mam-3 antigen group reacted strongly with the entire surface and the cytoplasm of the cuboid cells of the excretory ducts of sweat glands and the intercalating ducts of salivary glands. Antibodies of the Mam-6 antigen group reacted preferentially with secretory capillaries in the acini of sweat glands and the serous cells of salivary glands; the Mam-3 group reacted only occasionally with these structures. Antibodies that were supposed to react with one antigen molecule showed a different and characteristic reaction pattern with glandular components.

Structure, Processing, Differential Glycosylation and Biology of Episialin

Episialin is a carcinoma associated antigen encoded by the MUC1 gene. We have cloned genomic and cDNA coding for this protein and full length cDNA has been sequenced. The mRNA can be differentially spliced depending on a single nucleotide polymorphism in the second exon. The main part of the coding domain of the MUC1 gene consist of repeated seqences of 60 bp. Numerous allelic forms of the gene have been determined which differ in the number of repeats. Each of the repeated sequences contains several proline, threonine and serine residues; the latter are potential 0-linked glycosylation sites. The large amount of prolines and the 0-linked glycosylation give the episialin molecule a very rigid structure pointing into the extracellular space. Based on the predicted amino acid sequence, episialin contains a transmembrane domain. However, the transmembrane domain is absent in the molecule that is released from carcinoma cells. At least three precursor forms of the molecule can be distinguished. The most mature precursor form of episialin is undersialylated and is present at the cell surface. This premature form is internalized and further sialylated. The glycosylation of episialin is variable both within one cell line and among different cell lineages. This has resulted in the generation of monoclonal antibodies that show a high tissue preference. Episialin cDNA has been transfected into HBL-100 cells under the control of a CMV promotor. Transfectants with high levels of episialin expression showed decreased aggregation properties.

Diversity of Antigenic Expression on Normal and Malignant Human Mammary Cells Recognized by a New Panel of Monoclonal Antibodies

Mouse monoclonal antibodies were generated to membrane preparations of human primary breast carcinomas. The reactivity patterns were studied on frozen sections of malignant and benign mammary tumors and normal breast tissues by immunoperoxidase staining and on cultured mammary tumor cell lines by immunofluorescence reactions. The diversity of reactivities indicated distinct, unique specificities of the monoclonals.

New Approaches to the Pathology of Breast Cancer; Can Immunohistochemistry Add New Information?

If we want to evaluate the contribution of immunohistochemistry to the understanding of breast disease, we must first consider the role of histopathology. The aims of histopathological studies are: 1. differentiation between benign and malignant lesions. 2. classification and grading of malignancy (relevant for prognosis). 3. staging and extent of disease (relevant for prognosis). 4. in case of metastasis of occult primary tumor: differential diagnosis of possible primary tumor. 4. giving insight in histogenesis and tumor biology. Especially in the last decade a large number of polyclonal and monoclonal antibodies and fluorescein- or enzymeconjugated lectins has been developed to define more precisely the composition and functional characteristics of the mammary gland. Some recent developments which may contribute to histopathological studies will be reviewed, without the pretention to be complete. Emphasis will be given to markers that can be applied to routinely fixed, paraffin-embedded material.

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.