A novel monoclonal antibody (7B10) with differential reactivity between human mammary carcinoma and normal breast

A monoclonal antibody (7B10) which displays differential reactivity with breast carcinomas compared to benign lesions or normal breast tissue was selected by fusion of spleen cells from BALB/c mice immunized with the T47D human mammary carcinoma cell line. The antigen, recognized by 7B10 on T47D cells, appeared to be both surface and cytoplasm localized, as demonstrated by indirect immunofluorescence, immunoperoxidase, and electron microscopy studies. This antibody (IgG1) bound with four human breast cancer cell lines (T47D, MCF7, ZR-75-1, and HSL53) which express estrogen receptors. No binding was observed with cancer cell lines of other origin or with normal cells. In vivo, by immunoperoxidase staining of frozen sections of normal breast, the antigen recognized by 7B10 appeared to be located on epithelial cell membranes, whereas in benign and malignant mammary disorders, staining also involved the cytoplasm, as confirmed by electron microscopy on fresh cancer tissue. On formalin-fixed, paraffin-embedded sections, cytoplasmic staining was detected in breast cancer, but no immunostaining was observed with benign lesions or normal breast. In paraffin sections, most normal tissues investigated did not react with 7B10 antibody. However, ducts in the parotid gland, tubules in the kidney and some biliary ducts, and apocrine glands in the skin showed irregular, diffuse weak staining. 7B10 was unreactive with adenocarcinomas of origin other than breast, except for some cells in ovarian clear cell carcinoma. No reactivity was observed with squamous carcinomas, lymphomas, or melanomas. The antigen recognized by 7B10 appeared to be a Mr 32,000 protein, as identified by immunoprecipitation from extracts of T47D after labeling with [35S]methionine. Since the antigen was present only on the membrane of differentiated normal mammary epithelial cells, and was also expressed in the cytoplasm of tumor cells, it may be of interest in immunological studies of mammary epithelial cell differentiation. Moreover, since in formalin-fixed tissues immunostaining is virtually confined to mammary carcinomas, monoclonal antibody 7B10 may have diagnostic applications in breast cancer.     

The immunohistochemical reactivity of a human monoclonal antibody with tissue sections of human mammary tumors

Human monoclonal antibodies have been generated following the fusion of human lymphocytes (obtained from axillary lymph nodes of mastectomy patients) with a murine nonimmunoglobulin secreting myeloma cell line. We report a detailed analysis of the reactivity of a human IgM monoclonal antibody secreted by one of these double cloned hybridoma cell lines. Tissue sections of both malignant and benign human mammary tumors, as well as apparently normal tissues, were tested using the immunoperoxidase technique and the human monoclonal antibody. Eighty-one per cent (54/67) of primary malignant mammary tumors, 100% (20/20) of metastatic breast lesions, and 14% (3/22) of benign breast lesions reacted positively with a moderate for strong intensity. The per cent of mammary carcinoma cells that stained and the pattern of staining varied among different tumor samples. While reactivity was observed with selected carcinomas of nonbreast origin, little or no reactivity was observed with apparently normal human tissues including normal mammary epithelium. The antibody reactivity observed was shown to be clearly distinct from those of both anti-T and anticarcinoembryonic antigen sera.     

Two monoclonal antibodies selective for human mammary carcinoma

Mouse myeloma cells were fused with spleen cells from BALB/c mice immunized with the MCF-7 human mammary carcinoma cell line. Among hybridomas, two (3B18 and 15A8) were selected and cloned. Hybridoma 3B18 produces kappa-IgG1 antibodies that react with a cytoplasmic component of MCF-7 cells. In immunoperoxidase assays, 3B18 reacts with 27 of 31 specimens of human mammary carcinoma. It reacts most consistently with poorly differentiated and infiltrating ductal breast cancers, but it also reacts with isolated cells in 3 of 5 benign mammary pathological lesions with a variable distribution. The antibody does not react with normal mammary epithelium. It does not react with any normal human tissues, and it reacts with only one of 19 other cancers tested. Hybridoma 15A8 produces kappa-IgG1 antibodies that react with the surface membranes of the cells of two human breast cancer cell lines but not with a human fibroblast cell line. In immunoperoxidase assays, the antibody reacted with 28 out of 31 human mammary carcinomas. The antibody also reacts more weakly with normal human epithelial cells of breast, renal proximal tubule, skin, esophagus, and salivary gland, but no other normal tissue. The antibody was unreactive with 14 of 18 other malignant tissues tested. Since 3B18 and 15A8 detect antigens found predominantly in human mammary carcinomas and, possibly, distinguish overlapping categories of human mammary carcinomas, they may prove useful in determining the cellular lineage from which human mammary carcinomas arise, or they may have other clinical applications in breast cancer.     

Biological behavior of human breast carcinoma-associated antigens expressed during cellular proliferation

The cell surface-binding properties of two murine monoclonal antibodies reactive with human mammary tumor cells are described. Fluorescence-activated cell sorter analyses demonstrate that both monoclonal antibodies, B6.2 and B38.1, are reactive with the surface of the majority of human breast tumor cell lines tested but are unreactive with a variety of normal human cell lines, melanomas, sarcomas, and lymphoid tumors. Antibody B6.2 was also reactive with selective carcinomas, while antibody B38.1 showed even broader reactivity. The two monoclonal antibodies were unreactive with the surface of a variety of normal human tissues obtained at biopsy, including lymph nodes, bone marrow, and spleen, but were reactive with mammary tumor cells obtained from four of six pleural effusions. Surface binding to mammary tumor cells by both monoclonal antibodies was shown to decrease during density-dependent arrest; further cell cycle analysis demonstrated differential antibody surface binding during S phase. Prolonged exposure of mammary tumor cells to antibody showed no evidence of antigen capping or internalization. Both monoclonal antibodies were shown to lyse mammary tumor cells in antibody-dependent cell-mediated cytotoxicity.     

A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations

We report here both the range and patterns of reactivity of an IgG1 monoclonal antibody, B72.3, prepared against human, metastatic mammary carcinoma cells. When the avidin-biotin complex (ABC) immunoperoxidase technique was used on tissue sections, monoclonal B72.3 reacted with 19 of 41 (46%) primary mammary carcinomas and 13 of 21 (62%) metastatic lesions, either in axillary lymph nodes or at distal sites. Variable concentrations of antigen, recognized by B72.3, were observed among mammary tumors, as well as among different cell populations of a given tumor mass. Several patterns of antigen distribution were observed: membrane, diffuse cytoplasmic, focal and marginal. No reactivity was observed to normal mammary epithelium, stroma, or lymphocytes of the breast, nor to any cell types in a variety of other normal human tissues, melanomas, and sarcomas. Reactivity with all of four colon carcinomas was also observed. Assay of serial sections of mammary carcinomas with B72.3 and a monoclonal antibody directed against carcinoembryonic antigen demonstrated that these antigens were both distinct and non-coordinately expressed.     

Generation and immunohistological characterization of human monoclonal antibodies to mammary carcinoma cells

The purpose of this study was to identify human antibodies generated against autologous breast tumor cells by the host's immune response. Accordingly, lymphocytes from lymph nodes of seven different patients with metastatic breast carcinomas were immortalized by fusing them with a nonsecreting variant of murine myeloma cells. The screening for binding of antibodies to tumor cells was performed by indirect immunoperoxidase staining of paraffin-embedded tissue sections of the autologous tumor. The selected hybrid cells, after being cloned three times, were stable for the secretion of immunoglobulins for over 2 years. A total of 81 human immunoglobulin-producing clones was obtained from an initial 595 wells with hybrid growth. Nine of these clones produced immunoglobulin M, none of which showed detectable binding to tissue antigens in breast. Seventy-two clones produced immunoglobulin G monoclonal antibodies, and 15 of these showed preferential binding to breast carcinoma cells. Three of these immunoglobulin G monoclonal antibodies were subjected to detailed immunohistological evaluations. Using these antibodies at concentrations ranging from 10 to 100 ng/tissue section, the morphologically normal mammary epithelial cells could be discriminated from their malignant counterparts. The antibodies showed diffuse staining of cytoplasmic components in the malignant counterparts. Under these conditions, lymphocytes, erythrocytes, and stromal cells in breast tissues were unstained. The antibodies showed variable reactivity with malignant epithelial cells of colon and stomach, and with normal epithelial cells lining the renal tubules and sebaceous glands in skin. Antigenic heterogeneity of malignant mammary epithelial cells was revealed. The antibodies may have value in the characterization of tumor-associated antigens responsible for inducing autologous immune responses.     

Biochemical and immunological characterizations of antigens recognised by human monoclonal antibodies

The lymphocytes from lymph nodes of six patients with metastatic mammary carcinomas were hybridised by fusion with a non-secreting variant of murine myeloma cells. Hybrid cells producing human immunoglobulin were detected by screening of culture supernatants using a solid-phase enzyme-linked immunosorbent assay for human IgG or IgM. Reactivity of human immunoglobulins to breast tumour cells was assessed by an indirect immunoperoxidase staining of fresh-frozen breast carcinoma sections. In the initial screening, the tissues used were those removed from the patients who acted as source of lymphocytes for fusion. The hybrid-cells, after repeated cloning, were stable for secretion of immunoglobulins. A total of 14 immunoglobulin G and 51 immunoglobulin M human monoclonal antibodies, showing variable reactivity to mammary carcinoma cells in tissue sections by an indirect immunoperoxidase staining method, were obtained. Two immunoglobulin G monoclonal antibodies (designated HMA-29 and HMA-31) were selected on the basis of their strong reactivity to the tumour cells and utilised to identify their corresponding antigens. The antibodies quantitatively discriminated, as expressed by the degree of staining, malignant from normal or benign mammary epithelia in freshly frozen or formalin-fixed breast tissues. The antibodies also showed reactivity to malignant cells of colon, stomach and lung and to normal cells lining the renal tubules and surface epithelium of colon. As revealed by blocking experiments, the epitopes recognised by these antibodies were not expressed on carcinoembryonic antigens, erythrocytes, lymphocytes, glycoproteins from milk-fat-globule membrane or keratins. The antibody HMA-29 immunoprecipitated a phosphoprotein (Mr = 29,000), and antibody HMA-31 two protein components (Mr = 31,000 and 34,000), from lysates of intrinsically labelled human mammary carcinoma cell line (MCF7). Neither of these proteins were present in detectable amounts in an intrinsically labelled melanoma cell line. Immunoblocking and immunoprecipitation experiments suggested that epitopes recognised by these two antibodies are dissimilar and are expressed on different molecules. The antibodies appear to be useful for functional characterisation of those antigens which are present in elevated levels in malignant compared with normal mammary epithelia.     

Proliferative rates of cloned malignant mammary epithelial cells as a measure of clonal heterogeneity in human breast carcinomas

Malignant mammary epithelial cells (MMECs) were isolated from 8 human breast carcinomas and 1 adenoma as single cells or organoids and established in vitro. Depending on the cellularity of the tumor, between 9 X 10(4) and 4 X 10(6) cells were released per gram of tumor tissue. With the use of conditioned media and growth-promoting agents, a high proportion of cells (ranging from 0.5 to 11.4%) could be established in culture. A high degree of tumor cell heterogeneity in breast carcinomas was suggested by the observation that significantly different proliferative rates were found for 50 mammary epithelial cells cloned from 2 different tumors during the first subpassage in vitro prior to significant expansion of the cell colonies. The computed doubling times of these clones varied between 16 hours and more than 48 hours. The mammary epithelial nature of the cells was confirmed by their surface reactivity with monoclonal antibodies specific for MMECs. Studies on clones from 2 tumors revealed a positive correlation between proliferative rates of MMECs, their lactate production, and specific proteins synthesized as analyzed by two-dimensional macromolecular protein maps.     

Monoclonal antibodies (F36/22 and M7/105) to human breast carcinoma

Cloned hybridoma cell lines were obtained from fusions of murine myeloma cells with lymphocytes of mice immunized against human breast cancer cells. Hybridomas F36/22 and M7/105 produced antibodies whose binding to breast cancer cells could not be inhibited by prior absorptions with fibroblasts, lymphoblastoid cells, or erythrocytes. Results from cell surface binding assays using a panel of tumor cell lines indicated that antibodies F36/22 and M7/105 recognized determinants expressed maximally on breast cancer cells. Antibody F36/22 reacted with normal mammary epithelial membranes and milk fat globule membranes, whereas antibody M7/105 produced no detectable binding to these specimens. Antigens carrying these epitopes each showed reactivity with concanavalin A lectin. The determinant corresponding to antibody F36/22 was detectable in histological sections of a subset of breast tumors obtained at surgery.     

Normal epithelial antigens recognized by GB3 and GB5 are diminished in intraductal and lost in infiltrating human breast carcinomas

GB3 and GB5 are mouse monoclonal antibodies raised against human amnion. GB3 detects the epidermal basement membrane, and GB5 reacts with the junctional substances between the epithelial cells. These two antibodies were studied on breast tissues by indirect immunofluorescence. All (n = 8) of the normal mammary glandular basement membranes and epithelia reacted strongly with GB3 and GB5. In the intraductal carcinomas (n = 10), although nine out of ten tissues were reactive, their reactivities were greatly reduced. More strikingly, in the infiltrating carcinomas (n = 15), none of the tumor specimens were recognized by GB3 and only one out of fifteen biopsies was moderately reactive with GB5, indicating that the syntheses of the antigens of GB3 and GB5 were discontinued in the metastatic breast adenocarcinomas. These data suggest that these antigens may contribute to the interaction of the epithelium with the extracellular matrix and the intercellular binding between the epithelial cells; the loss of these antigens may facilitate the wide dissemination of tumor cells.     

High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: A preventive mechanism of malignant tumor cells against complement attack?

Summary GB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.     

Characterization of monoclonal antibodies reactive with normal resting, lactating and neoplastic human breast

Mouse monoclonal antibodies reacting with human mammary gland constituents have been generated and characterized in an attempt to raise breast- or breast-cancer-specific antisera. The immunogens used in these studies included fractionated human milk fat globule membrane, the human breast cancer cell line MCF7 and a crude membrane preparation derived from an axillary nodal metastasis from a patient with breast cancer. Of the antibodies obtained, 8 were characterized and found to bind to different structures in the normal breast. The antibody LICR-LON-LC28 recognizes secretory component and binds strongly to normal resting and lactating breast, but only focally to a minority of breast carcinomas. The antibodies LICR-LON-14.1 and 32.2 react strongly with the lactating breast and recognize kappa- and beta-casein, respectively. Caseins are not produced by breast tumors. The antibodies LICR-LON-TW19.5, H10A and 39.8 all react with carbohydrate epitopes and bind heterogeneously to normal resting breast luminal epithelium and cellular subsets of breast carcinomas. LICR-LON-59.2 and 19.2 react with normal breast myoepithelial cells and the basement membrane, respectively. LICR-LON-59.2 is unusual as a myoepithelial marker in that it stains cells in the majority of breast carcinomas. LICR-LON-19.2 shows extensive reactivity to tumor cell lines in culture but has no reactivity with carcinoma cells from breast biopsies.     

Normal tissue reactivity of four anti-tumor monoclonal antibodies of clinical interest

Normal tissue reactivity on frozen sections was examined with 4 monoclonal antibodies (MAbs) that were reported previously to be negative or weakly reactive with normal tissues, and strongly reactive with some types of carcinoma. All 4 antibodies reacted strongly with certain normal epithelial cells. The antibodies tested include 2 antibodies to ovarian cancer, MOv18 and MOv19, one antibody to breast cancer, H23, and one antibody reactive with a range of carcinomas, B72.3. MOv18, MOv19 and H23 reacted with many normal glandular and ductal epithelial cells, while B72.3 reacted most strongly with secretions of the small intestine epithelium and the suprabasal squamous epithelial cells of the esophagus. Since the tissue distribution of MOv19 was very similar to that of another antibody described previously, MW207, these two antibodies were compared by competitive binding inhibition, and found to recognize the same epitope. Our data emphasize the importance of repeated, independent tests of antibody specificity.     

Expression of two antigens defined by monoclonal antibodies in normal, benign and malignant human mammary tissues

We examined the reactivity of two monoclonal antibodies (MAbs), MBrI and MBr8, on sections from normal breast tissues, benign lesions, primary infiltrating carcinomas and distant breast cancer metastases and compared the expression of the recognized antigens (CaMBr1 and CaMBr8 respectively) in these different normal and pathologic conditions. The expression of both antigens was found to significantly decrease in the neoplastic tissues, in comparison to normal breast tissue (34% vs 61% for MBr1 and 52% vs 86% for MBr8; p less than 0.001 for both). Atypical epithelial hyperplasia was found to be reactive on the same level (31%) of the tumors when tested with MBr1, whereas its reactivity was similar to that of normal tissues (78%) when MBr8 was used. Nonatypical epithelial hyperplasia showed the same reactivity with both MAbs (positive staining on about 50% of the cells). 65% of the "in situ" ductal carcinoma cases was MBr1-positive, whereas 50% was MBr8-positive; these values, when compared with the reactivity on normal glands, were significantly lower for MBr8, but not for MBr1. When testing sections from premenopausal women who had biopsies performed on different days during their menstrual cycle a correlation was found between the expression of CaMBr1 in normal breast epithelial cells and the stimulatory phase of the cycle. These findings suggest that both antigens are typical of normal breast and their expression could possibly decrease in pathologic tissues.     

Monoclonal antibodies against human milk-fat globule membranes detecting differentiation antigens of the mammary gland and its tumors

Mouse monoclonal antibodies have been raised against human milk-fat globule membranes (HMFGM) to obtain reagents for mammary tumor diagnosis. A panel of 17 anti-HMFGM antibodies was selected for further investigation. Antibody-blocking studies indicated that with these antibodies at least nine different non-overlapping epitopes could be distinguished on six differend molecules, MAM-1 to MAM-6. Electron microscopic studies of the cellular localization of the antigens detected by some of these antibodies revealed that they were present on the cell membrane mainly, on the microvilli, lining intercellular and intracytoplasmic lumina. The reactivity of the antibodies was studied on normal and tumor tissues and on in vitro cell lines. All antibodies reacted with the resting mammary gland while eight antibodies also bound to breast tumors. None of the antibodies was specific for the mammary gland or its tumors only, but most antibodies also reacted with other epithelial cells, especially of secretory tissues. When tested on a variety of cell lines a distribution reflecting the tissue distribution could be demonstrated. One of the antibodies reacted with nearly all carcinomas and their metastases and did not react with lymphomas, sarcomas, neuroblastomas, melanomas or nervous system tumors. The specificity of the antibodies, tested individually, was not sufficient for further differential diagnosis of the carcinomas, but when some of these antibodies were used in a panel they contribute to an important improvement of the diagnosis.     

A monoclonal antibody (AB/3) reactive with human breast cancer

Monoclonal antibody AB/3 was produced from a fusion of spleen cells of a human breast cancer cell-primed BALB/c mouse with the murine myeloma cell line P3-NS1-Ag4-1. The antibody reacted strongly with the plasma membrane of human breast cancer cells. Tissue sections of both malignant and benign human mammary carcinomas and tumors of non-breast origin as well as apparently normal tissues were tested with immunoperoxidase. Ninety-six of 124 (77%) primary human breast cancers, 12 of 14 (86%) metastatic breast lesions, and 12 of 44 (27%) benign breast lesions reacted positively. Little or no appreciable reactivity was observed with apparently normal human tissues and carcinomas of non-breast origin, with the exception of colon carcinoma. Antibody AB/3 did not immunoprecipitate any identifiable protein from radiolabeled extracts of the immunizing cell line.     

Highly tumor-reactive, internalizing, mouse monoclonal antibodies to Le(y)-related cell surface antigens.

Two monoclonal antibodies, designated BR64 (IgG1) and BR96 (IgG3), were generated that, according to immunohistology, bind selectively to carcinomas of the colon, breast, ovary, and lung. They have no or very low reactivity with normal human tissues, except for the fact that BR64 strains capillaries in the hearts from certain normal donors and that both monoclonal antibodies stain some epithelial cells from the gastrointestinal system, including stomach. Preliminary studies indicate that at least a portion of the epitope recognized by BR64 and BR96 is a Le(y) carbohydrate chain. Both monoclonal antibodies can be "internalized" by antigen-positive tumor cells, since immunoconjugates with ricin A-chain are cytotoxic. BR96 has the additional properties of being cytotoxic by itself, and it can mediate antibody-dependent cellular cytotoxicity and complement dependent cytotoxicity.     

Comparison of spontaneous mutagenesis in early-passage human mammary cells from normal and malignant tissues.

Spontaneous mutant frequencies were determined in early-passage epithelial-cell strains derived from either normal or malignant human breast tissues, as well as a non-tumorigenic immortalized human mammary epithelial cell line (184B5) derived from normal cells. Mutations at the hypoxanthine-guanine phophoribosyltransferase (HPRT) locus were quantified by determining the 6-thioguanine resistance. Mutant frequencies in human mammary epithelial cells (HMEC) from 4 normal and 5 carcinoma tissue samples did not differ significantly. In contrast, the mutant frequency in the immortalized HMEC was approximately 10 times higher than in average normal HMEC and normal non-immortalized cells from early-passage cultures of the same cell lineage. Our data suggest that the progression of normal breast cells to invasive carcinoma cells does not necessarily involve the establishment of a general genetic instability during this progression.     

A murine monoclonal antibody to human breast cancer cells associated with DNA ploidy status

A murine monoclonal antibody, 5D10, raised against the human breast cancer cell line MCF7 reacted preferably with mammary carcinomas and weakly with normal epithelial cells. The antigens recognised by the antibody had molecular weights of about 28 and 90 kD. The reactivity of the antibody to human breast carcinomas correlated with the DNA ploidy status of the tumour cells. Upon analysis of 54 breast carcinoma specimens, the percentage of antibody positive cells was significantly higher in tumours with an aneuploid stemline than in those with a diploid DNA content (P < 0.001). This antibody therefore could be a useful tool in evaluating the prognosis of breast carcinomas.