Human antibodies against the polymorphic epithelial mucin in ovarian cancer patients recognise a novel sequence in the tandem repeat region.

The humoral immune response to the polymorphic epithelial mucin (PEM) was studied by characterising the reactivity of human antibodies generated by EBV-immortalised B-cells from tumour-draining lymph nodes of ovarian cancer patients. All the human antibodies, selected in ELISA for their reactivity to the protein tandem core repeat sequence, reacted with PEM-expressing tumour cells. Aberrant glycosylation of the peptide core of the PEM molecule in cancer cells leads to the exposure of peptide epitopes that can be considered tumour specific. The epitope mapping of six human antibodies revealed that only one of them contained the PDTR sequence, shown to be the immunodominant epitope in the mouse. Four of the six human antibodies recognised a novel common immunogenic sequence (APPAH) in the tandem repeats. The binding of these human antibodies did not appear to be modulated by the length of the carbohydrate side chains, as shown by O-glycosylation inhibition studies. These results indicate that distinct sequences within the tandem repeat of PEM are target for a humoral immune response in humans. The presence of antibodies directed against different epitopes within the same antigenic region may modulate the antigen presentation process and the ongoing immune response. This data may help in clarifying the mechanisms of the immune response to PEM in cancer patients for the development of PEM-based immunotherapy.

Integrin Beta-4 expression in colorectal-cancer.

Integrin alpha 6 beta 4 plays an important role in the interaction of epithelia with basement membranes, and its expression appears to be profoundly altered during tumor progression. Using a quantitative immunochemical assay, we investigated the expression of the beta 4 subunit associated with alpha 6 in 25 primary carcinomas, and in matching normal mucosae. alpha 6 beta 4 was expressed in all the carcinoma and mucosa samples. The highest beta 4 levels were detected in tumors at high clinical stage (Dukes' stage C). Furthermore, beta 4 reactivity inversely correlated with the degree of differentiation. By immunohistochemistry,beta 4 expression was particularly strong in the epithelium lining the upper third of the crypts and the absorbing surface of normal mucosa. In villous adenomas, beta 4 immunostaining tended to be enhanced in the epithelium lining the outer surfaces of neoplastic villi, but only 5 of 8 samples tested scored positive. In carcinomas, beta 4 expression was detected in 18 of 21 samples tested, and was strongly influenced by the pattern of tumor growth and by the type and level of differentiation. Carcinomas, or areas of carcinomas, with cohesive and differentiated growth pattern demonstrated weak beta 4 expression at the tumor-stroma interface. Carcinoma cells at the lumenal surface of the intestine, and carcinomas, or areas of carcinomas, composed of small clusters of cells surrounded by stroma, demonstrated strong beta 4 expression. Altogether, our observations indicate that in colorectal tumors the expression of the beta 4 subunit is strongly influenced by microenvironmental factors and tends to increase in high stage, poorly differentiated lesions.

Human B cell immune response to selected epitopes of the polymorphic epithelial mucin (PEM) in cancer patients.

Human antibodies were generated by Epstein-Barr Virus (EBV) immortalization of B cells derived from tumor draining lymph nodes of cancer patients. Antibodies were screened for reactivity in ELISA against a synthetic peptide corresponding to the protein core of the Polymorphic Epithelial Mucin (PEM). Epitopes within this region are in fact considered to be tumor specific since they are selectively exposed on tumor cells due to aberrant glycosylation. Human antibodies thus selected react in ELISA and immunohistochemistry with PEM-expressing tumor cells. This is the first demonstration of the existence of B cell immune response against selected epitopes of PEM and, in association with the cytotoxic T cell (CTL) response already demonstrated, represents the basis for the use of synthetic peptides as vaccines in cancer patients.

Human B-cell immune response to the polymorphic epithelial mucin.

Human antibodies generated by Epstein-Barr virus immortalized B-cells from tumor-draining lymph nodes of an ovarian cancer patient were screened for reactivity in enzyme-linked immunosorbent assay with a synthetic peptide corresponding to the protein core of the polymorphic epithelial mucin. Epitopes within this region are in fact considered tumor specific since they are selectively exposed in tumor cells due to aberrant glycosylation. Human antibody BB5, thus selected, reacts in enzyme-linked immunosorbent assay and immunohistochemistry with polymorphic epithelial mucin-expressing tumor cells. This is the first demonstration of the existence of a B-cell immune response to selected epitopes of polymorphic epithelial mucin and, together with the cytotoxic T-cell response already demonstrated, constitutes the basis for the use of synthetic peptides as a vaccine in cancer patients.

Immunohistochemical analysis of the distribution of a breast tumor associated antigen recognized by a monoclonal antibody.

A new monoclonal antibody (MoAb), MM 1-80, recognizing a tumor associated epitope of a breast high molecular weight mucin molecule was tested, using the avidin biotin immunoperoxidase method on normal and pathological mammary tissues. The normal mammary ducts and lobules were negative. Fibroadenomas showed a strong intracytoplasmic staining. In apocrine metaplasia, adenosis, and papillomatosis, scattered cells showed intracytoplasmic, luminal border or secretion reactivity. In lobular and ductal hyperplasia the cells showed intracytoplasmic immunoreactivity which, however, became more intense and homogeneous in atypical lesions, i.e. lobular and ductal in-situ carcinomas. The infiltrating carcinomas of different histotype expressed positivity on 98% of the cases (113/115) and axillary metastatic lymph nodes were always positive (20/20). The MoAb was tested on 175 human neoplasias of different origin which were in the majority of the cases negative with the exception of adenocarcinomas of the lung, ovary and bladder. MM1-80 appears to react preferentially with mammary cells undergoing hyperplastic, metaplastic and neoplastic processes. The 1-80 epitope distribution is different in these lesions starting with a predominant luminal expression in benign lesions and becoming strong and cytoplasmic in the malignant breast cell.

Immunological properties of a radioiodinated monoclonal antibody and its F(ab')2mu fragments directed against the polymorphic epithelial mucin of human breast cancer.

The purification of the IgM monoclonal antibody 436 against a breast tumor antigen from mouse ascitic fluid is reported. The purified immunoglobulin was radioiodinated and the resulting product assessed for its binding capacity and binding specificity. Purified IgM-436 served for F(ab')2 mu preparation which was tested for its antigen binding capacity. Radioiodinated IgM-436 and its F(ab')2 mu retained their immunological activity which was never lower than those of the corresponding cold products.

N-methylformamide effects on cell proliferation and antigenic pattern in HT-29 colon carcinoma cell line.

The effects of the differentiating agent N-methylformamide (NMF) on cell proliferation and antigenic pattern of HT-29 colon carcinoma cells have been investigated. The cell line was cultured in the presence, or absence, of 1% NMF and tested for the above mentioned characteristics, both in vitro and after injection into nude mice. The percentage of cells in the various cell cycle compartments was estimated by flow cytometry. The presentation on the cell surface of molecules such as tumour associated antigens (TAAs), HLA class I molecules and epidermal growth factor receptor (EGF-R) was analysed by ELISA, flow cytometry and immunohistochemistry. Results demonstrate that NMF impairs HT-29 cell proliferation with a remarkable accumulation in the G0/G1 phases, as well as inducing a modification of the membrane antigenic pattern. The presence of NMF in the culture medium decreases the TAAs and EGF-R whereas HLA antigen maintains the same level of positivity in the two cell lines. These alterations are consistent with a different behaviour in vivo of the tumours originated from NMF treated and untreated cells. Tumours derived from NMF treated cells show a delay in the appearance and low levels of immunodetectable carcinoembryonic antigen (CEA) molecules.

Characterization of monoclonal antibody 436 recognizing the Arg-Pro-Ala-Pro sequence of the polymorphic epithelial mucin (PEM) protein core in breast carcinoma cells.

Epithelial mucins have obtained increasing clinical relevance since they were found in the serum of cancer patients and were shown to be elevated in metastatic disease. We report here the characterization of the monoclonal antibody (MAb) 436 which recognises the protein core of the polymorphic epithelial mucin (PEM) of the human breast. MAb 436 was generated by immunizing Balb/c mice with membrane-enriched fractions prepared from metastatic lesions in the axillary lymph nodes. The antigenic determinant recognized by the MAb 436 is expressed on the surface of breast cancer cells and was measured by ELISA on all of 50 cytosol preparations of primary breast tumors. Immunohistochemistry showed 98% of primary and 100% of metastatic breast cancer lesions to be positive with the 436 antigenic determinant expressed both in the cytoplasm and at the plasma membrane level of the tumor cells. Moreover, the antigen was expressed in a homogeneous fashion (80-100% of the total number of tumor cells) in more than 60% of the tumors. Reactivity with normal tissues was rare and scattered and restricted to glandular structures particularly at the luminal border level except for the distal and collecting tubules of adult and fetal kidney, where a cytoplasmic 436 antigen distribution was observed. Other cancers proved positive but the reactivity was always variable and heterogeneous. The antigen recognized by MAb 436 appears in Western Blotting as a M(r) of more than 200,000 daltons protein resolved in two bands. Epitope mapping experiments using overlapping octapeptides in the repeat unit of the PEM identified in the RPAP (Arg-Pro-Ala-Pro) sequence the binding site of the 436 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic expression changes occurring in adriamycin resistant MCF-7 mammary carcinoma cells.

The antigenic phenotypic repertoire of MCF-7 human breast carcinoma cell line variants that display different sensitivity to adriamycin (Adr) was analyzed using monoclonal antibodies (MoAbs) recognizing five different tumor associated antigens (TAAs) and the external domain of the epidermal growth factor receptor (EGF-R). ELISA and cytofluorimetry determinations were used and results indicate a diminished expression of one antigenic determinant of the carcinoembryonic antigen (CEA) molecule, the disappearance of all the other TAAs and the de novo expression of the EGF-R in the MCF-7 AdrR (IC50/Adr:10 uM). Treatment with recombinant alfa-Interferon (alfa-IFN) did not enhance antigenic expression in MCF-7 AdrR cells.

High frequency production of hybridomas secreting antibodies to cell antigens.

The efficiency of various immunization protocols for the production of hybridomas secreting immunoglobulins specific to cell antigens was evaluated in 15 different fusion experiments. Some of these experiments were performed with splenic B-lymphocytes from mice at different stages of immunization. This approach allowed a dynamic analysis of immunocompetent splenic B-lymphocyte production during the immunization cycle.