Joyce Taylor-Papadimitriou

TARGETING OF IODINE-123-LABELLED TUMOUR-ASSOCIATED MONOCLONAL ANTIBODIES TO OVARIAN, BREAST, AND GASTROINTESTINAL TUMOURS

Two tumour-associated monoclonal antibodies, HMFG1 and HMFG2, were labelled with iodine-123 and used to detect primary and metastatic ovarian, breast, and gastrointestinal neoplasms by external body scintigraphy in twenty patients with advanced disease. Tumours became visible 3 min to 18 h after injection of labelled antibody. The presence of antibody in the tumours was confirmed by autoradiography and immunoperoxidase staining of surgically removed tissues. The mean tumour uptake of radiolabel was 0.6% of the injected amount. These antibodies can therefore localise specifically to tumours and successful imaging can thus be achieved. This method can complement existing diagnostic techniques and also provide a basis for a selective therapeutic approach to malignant disease.

Comparison of O-Linked Carbohydrate Chains in MUC-1 Mucin from Normal Breast Epithelial Cell Lines and Breast Carcinoma Cell Lines: DEMONSTRATION OF SIMPLER AND FEWER GLYCAN CHAINS IN TUMOR CELLS

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcα2-3Galβ1-3GalNAc-ol, Galβ1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcα2-3Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3]GalNAc-ol and NeuAcα2-3Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

A Novel Gene (PLU-1) Containing Highly Conserved Putative DNA/Chromatin Binding Motifs Is Specifically Up-regulated in Breast Cancer

A novel human gene (PLU-1) has been identified which shows a highly restricted expression in normal adult tissues but which is consistently expressed in breast cancers. A fragment of the PLU-1 cDNA was identified by differentially screening a fetal brain library with cDNAs prepared from ce-1 cells (a human mammary epithelial cell line overexpressing c-ErbB2) treated or untreated with the antibody 4D5, which inhibits c-ErbB2 phosphorylation. Clones covering the full cDNA sequence of 6.4 kilobases were isolated from a breast cancer cDNA library. Although expression of PLU-1 in ce-1 cells is regulated by signaling from c-ErbB2, the gene is expressed in all the breast cancer cell lines examined, in cells cultured from primary breast cancers, and in the invasive and in situ components of primary breast cancers. Translation of the open reading frame predicts a protein of 1544 amino acids, which contains three PHD/LAP motifs, a specific DNA-binding domain found in aDrosophila protein (dri) and novel domains showing extensive homology with other human and non human gene products. Transient transfection of cell lines with MYC-tagged PLU-1 showed the protein to be localized in the nucleus and associated with discrete foci. The presence of the dri motif and PHD/LAP fingers together with the clear nuclear localization and consistent expression in breast cancers, suggest a role for PLU-1 in regulating gene expression in breast cancers.

Differential expression of keratin 19 in normal human epithelial tissues revealed by monospecific monoclonal antibodies

SummaryThree monospecific monoclonal antibodies (BA16, BA17 and A53—B/A2) recognizing different epitopes of the human keratin 19 were used to determine tissue distribution of this 40 kDa keratin polypeptide. Immunohistochemical methods revealed four different staining patterns among normal human epithelial tissues: firstly, complete negativity of the epidermis, sebaceous glands, hepatocytes and other tissues; secondly, homogeneous positivity as seen for example in the gall bladder and urinary bladder epithelium, endometrium and many other epithelia; thirdly, a mosaic of positive and negative cells among mammary gland luminal cells, prostate epithelia and some other epithelia and fourthly, a more complex heterogeneous pattern found in non-keratinizing squamous epithelia and hair follicles with generally the basal layer being the most strongly or sometimes exclusively stained. The pattern seen in non-keratinizing squamous epithelia varied considerably according to the fixation method and the antibody used as well as among different donors and in different areas of the same organ. The other three staining patterns were on the other hand nearly identical with all three antibodies on both frozen sections and sections of methacarn-fixed paraffinembedded tissues. Our results provide evidence for differential expression of the human keratin 19 at the single cell level, an observation which could be exploited in the study of epithelial differentiation and pathology.

Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals

An antiserum against the carboxy-terminal seventeen amino acids of the human MUC1 mucin has been raised and extensively characterized. This antiserum, CT1, immunoprecipitates two high molecular weight polymorphic bands (greater than 200 kDa) from a metabolically labelled breast cancer cell line corresponding to the two alleles which have previously been shown to contain different numbers of a twenty amino acid repeat. The CT1 antiserum reacted with tissues from many mammalian species and immunoprecipitated large polymorphic proteins, suggesting that the cytoplasmic portion of the molecule is well conserved. The cell and tissue distribution of Muc-1 mucin in the mouse has been studied by immunocytochemistry. This protein is abundant at the apical surfaces of epithelial tissues and is found expressed in the stomach, kidney, mammary gland, pancreas, salivary gland, lung, trachea, uterus, cervix and vagina.

Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: Effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene

The immune response of CAF1 mice to various synthetic peptides (SP) related to the amino acid sequence (PDTRPAPGSTAPPAHGVTSA) of the tandem repeat of the MUC1 human breast mucin core peptide was evaluated. The most immunogenic preparations of the synthetic peptides were those conjugated to keyhole limpet hemocyanin (KLH) or clustered in a dendritic multiple antigenic peptide (MAP-4) configuration. The mice were immunized subcutaneously with synthetic peptides emulsified in RIBI adjuvant, employing various immunization protocols. Equivalently high IgG responses were induced using SP-KLH conjugates (GVTSAPDTRPAPGSTA-KLH) or an SP — MAP-4 chimeric configuration (SP1-6), which also included a universal malarial CST-3 T-helper epitope (SP1-6 = SAPDTRPAEKKIAKMEKASSVFNVVNS — MAP-4). These IgG antibodies bound both the appropriate MUC1 synthetic peptides and the cell surface expressed MUC1 mucin on murine mammary cells that had been transfected with the human MUC1 gene and a human breast cancer cell line that expresses cell-surface MUC1. A MAP-4 molecule, which included the entire 20-aminoacid sequence of the MUC1 tandem repeat (SP1-5 = PDTRPAPGSTAPPAHGVTSA—MAP-4) induced a poor IgG response. In contrast, all three types of molecule: SP-KLH, SP1-6 and SP1-5, were found to be good immunogens for the induction of specific delayedtype hypersensitivity (DTH) reactions measured using either synthetic peptides or MUC1-transfected cells. In addition, immunization with irradiated MUC1-transfected cells induced strong DTH reactions measured using synthetic peptides that expressed the PDTRP sequence, which has been shown to be, or to overlap, a T cell epitope in humans and a B cell epitope in mice. Finally, it was demonstrated that synthetic MUC1 peptide “vaccines” could be used both prophylactically and therapeutically to inhibit the growth of MUC1-transfected tumor cells and prolong the survival of tumor-bearing mice.

USE OF TWO EPITHELIUM-SPECIFIC MONOCLONAL ANTIBODIES FOR DIAGNOSIS OF MALIGNANCY IN SEROUS EFFUSIONS

Two monoclonal antibodies, HMFG2 and AUA1, raised against epithelial-cell determinants were used to detect carcinoma cells in smears of serous effusions. Both antibodies react strongly with malignant epithelial cells but not with normal mesothelial or endothelial cells. A third antibody, 11-4.1, which does not react with any human tissue, was used as a negative control. An immunoperoxidase-staining technique was applied to smears of cells from serous effusions. The immunohistochemical diagnosis was in agreement with the cytological diagnosis. In cases with equivocal cytology in which the diagnosis became clear in later samples, the immunohistochemical diagnosis gave the correct result. With this combination of tumour-associated monoclonal antibodies a confident diagnosis of malignancy in serous effusions can be made.

Exploiting altered glycosylation patterns in cancer: Progress and challenges in diagnosis and therapy

In this review, we describe the changes in glycosylation and expression of mucins, in particular, polymorphic epithelial mucin (PEM), the product of the MUC1 gene in tumours and normal tissues. In addition, some of the areas where exploitation of altered glycosylation patterns in tumour mucins can be used for the better understanding of the disease process and be applied for in vivo diagnosis and therapy are addressed.

Intramuscular immunisation with MUC1 cDNA can protect C57 mice challenged with MUC1-expressing syngeneic mouse tumour cells.

Much interest is currently being shown in immunotherapy as a treatment for cancer since several tumour‐associated antigens have been identified and the genes encoding them cloned. One such molecule is the tumour‐associated human MUC1 gene product. In this report we describe tumour rejection studies in a C57BI murine model system with syngeneic MUC1‐expressing tumour cells designed to examine the efficacy of MUC1 cDNA as an immunogen. Intra‐muscular immunisation with 100 μg MUC1 cDNA 3 times at 3‐weekly intervals resulted in tumour protection in approximately 80% of mice. Tumour protection was dose‐dependent, with 50–100 μg being the most effective dose. Both humoral and cell‐mediated MUC1‐specific immune responses were detected. Anti‐MUC1 antibodies were detected after immunisation with DNA alone, indicating that the injected DNA was expressed. Humoral immune responses did not correlate with tumour rejection. Tumour challenge with syngeneic tumour cells expressing MUC1 appeared to be a pre‐requisite for the generation of MUC1‐specific cytotoxic T lymphocytes.

The polymorphic epithelial mucin : potential as an immunogen for a cancer vaccine

Abstract The identification and cloning of several tumour antigens together with an improvement in the understanding of the mechanisms involved in antigen presentation and immune recognition has opened up the possibility of using active specific immunotherapy as a treatment for certain cancers. This review discusses the tumour-associated MUC1 gene product of the polymorphic epithelial mucin (PEM), as a potential target molecule for cancer treatment. PEM is both over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule. Furthermore, immune responses specific for PEM have been detected in cancer patients. Both syngeneic and transgenic murine model systems have been developed in order to compare the efficacy and toxicity of various PEM-based immunogens in tumour rejection studies, and to further improve the understanding of antigen presentation and the mechanisms underlying tumour rejection. Such models also allow the examination of MUC1-based immunogens as a treatment for existing tumours. Clinical trials in progress using immunogens based on the MUC1 gene product are briefly discussed.