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Splenic lymphocytes of mice immunized with membrane enriched fractions of human mammary carcinomas were fused with the NS-1 nonsecretory++ myeloma cell line. The resulting hybridomas were screened for the synthesis of immunoglobulins reactive with human mammary tumor associated antigens, and two IgG monoclonal antibodies (B1.1 and F5.5) were identified as being reactive with purified carcinoembryonic antigen (CEA). These antibodies were shown to bind to different epitopes on CEA based on their differential reactivities to five different purified CEA preparations, and their differential binding to the surface of tumor cells derived from various organ sites. Monoclonal B1.1 bound equally to the surface of human breast, colon, and melanoma cell lines. Monoclonal F5.5, on the other hand, did not react with the surface of melanoma cell lines, and showed a differential binding to breast carcinoma versus colon carcinoma cells. Monoclonals F5.5 and B1.1 were both used in immunoperoxidase studies with fixed tissue sections of a variety of histologic types of human mammary carcinomas and were shown to be reactive with 55% and 66%, respectively, of tumor masses.

Monoclonal antibodies (MAbs) of predefined specificity have been generated by utilizing a synthetic peptide reflecting amino acid positions 10-17 of the Hu-rasT24 gene product as immunogen. These MAbs, designated RAP-1 through RAP-5 (RA, ras; P, peptide), have been shown to react with the ras gene product p21. Since the Hu-ras reactive determinants (positions 10-17) have been predicted to be within the tertiary structure of the p21 molecule, it was not unexpected that denaturation of cell extracts or tissue sections with Formalin or glutaraldehyde enhanced binding of the RAP MAbs. When paraffin-embedded Formalin-fixed tissue sections and the avidin-biotin complex immunoperoxidase method were used, the RAP MAbs clearly defined enhanced ras p21 expression in the majority of human colon and mammary carcinomas. The majority of all abnormal ducts and lobules from fibroadenoma and fibrocystic disease patients were negative, as were all normal mammary and colonic epithelia examined. The findings reported here form the basis for quantitative radioimmunoassays for a ras translational product and provide a means to evaluate ras p21 expression within individual cells of normal tissues and benign, "premalignant," and malignant lesions.

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.

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a "pancarcinoma" reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Eight different human breast carcinoma cell lines, as well as clonally derived cell lines, were used to study the expression of four different monoclonal antibody (MAb)-defined tumor-associated antigens (TAAs) and the ability of recombinant human (rHu)-interferon (IFN)-alpha A and rHu-IFN-gamma to increase tumor-associated and/or normal cell-surface antigen expression. The different breast tumor cell lines expressed a wide range of antigenic phenotypes with respect to four different cell-surface TAAs. The cell lines could be divided into high and low antigen-expressing groups based on their constitutive levels of the four TAAs. In general, those breast tumor cell lines that expressed high levels of carcinoembryonic antigen, the MAb DF-3-defined 290-kD antigen, and the 90-kD antigen reactive with MAb B6.2 were poor candidates for antigen augmentation by rHu-IFN-alpha A or rHu-IFN-gamma. In contrast, breast cell lines that constitutively express low levels of these TAAs were found to be highly responsive to the ability of either of the rHu-IFNs to enhance MAb binding to the cell surface. Treatment with either of the rHu-IFNs increased the level of surface binding of MAbs as much as fourfold. The relative abilities of the IFNs to increase MAb binding to the surface of human breast tumor cells thus appeared to depend on the degree of constitutive surface antigen expression of the breast tumor cells. The high-molecular-weight TAA, TAG-72, is known not to be expressed on most cell lines but is expressed on the majority of human carcinoma biopsies. Most breast tumor cell lines employed in this study did not express the TAG-72 high-molecular-weight TAA. However, rHu-IFN-alpha A did substantially increase the level of expression of TAG-72 on the surface of breast tumor cells that were isolated from a pleural effusion. These studies may thus provide an important insight into the criteria used in the selection of carcinoma patients for IFN-mediated antigen augmentation when employing MAb-guided radioimmunolocalization or MAb-guided therapy.

Molecular studies have demonstrated increased expression of the Harvey (Ha) ras oncogene in human breast and colon carcinomas. With the use of a direct-binding liquid competition radioimmunoassay (RIA), capable of providing truly quantitative analysis of the 21,000-dalton (p21) ras oncogene and protooncogene products, absolute levels of Ha-ras p21 have been determined in human breast and colon carcinomas, benign lesions, and/or their respective normal tissues. Enhanced Ha-ras expression was documented in 66% of breast and 100% of colon carcinomas as compared with their normal counterparts, with levels in breast carcinomas ranging from 10.1 to 50.4 pg ras p21/micrograms protein and those in colon carcinomas ranging from 18.4 to 51.7 pg ras p21/micrograms protein. Some dysplastic lesions of the breast and colon also contained elevated Ha-ras p21. Relative levels of Ha-ras p21 expression, detected by competition RIA, correlated with percent Ha-ras p21-positive cells as determined by immunohistochemical assays. By use of liquid competition RIA and immunohistochemical assays, it has been shown that levels of ras p21 expression did not always correlate between primary and metastatic colon lesions of the same patient. The use of the quantitative RIA and semiquantitative immunohistochemical assays, in concert with cDNA probes for identification of specific ras point-mutated oncogenes or protooncogenes, may now provide the means for definitive quantitative analyses of ras p21 in human carcinomas and benign lesions.

Monoclonal antibodies RAP-5 and Y13-259, directed against the ras gene product [a protein with a molecular weight of 21,000 (p21)] have been used to evaluate ras p21 expression in malignant and benign mammary tissues as well as in the lesions of intermediate stature such as atypical hyperplasia using immunohistochemical assays. Invasive carcinoma demonstrated enhanced expression of ras p21, with generally decreasing expression in carcinoma in situ, atypical hyperplasia, and nonatypical hyperplasia, respectively. Heterogeneous expression of ras p21 was observed among primary as well as metastatic mammary carcinomas. Carcinomas from postmenopausal patients generally demonstrated higher levels of ras p21 than those from premenopausal patients, but no significant difference in ras p21 expression in carcinomas between estrogen-receptor rich and estrogen-receptor poor patients was found. Normal mammary epithelium in terminal duct lobular units from patients with hyperplasia generally demonstrated higher levels of ras p21 expression than did epithelium in large ducts. This demonstration of enhanced ras p21 expression by the epithelium of peripheral lobular portion of the breast is consistent with the previous hypothesis that these areas preferentially undergo malignant transformation. Analyses of the limited number of specimens available from patients with 15-yr follow-up revealed a generally higher level of ras p21 in hyperplasia from patients who subsequently developed carcinoma, as compared to those from patients without carcinoma development. However, no conclusions regarding the potential for malignant transformation could be drawn for any individual patient on the basis of ras p21 expression. Concomitant analyses of ras p21 expression in mammary carcinomas and benign lesions using liquid competition radioimmunoassay and immunohistochemical assay demonstrated the complementary nature of these alternative approaches. These results suggest that enhanced ras p21 expression may be involved in the early stages of mammary carcinogenesis but is probably not involved in the maintenance of the transformed phenotype.

Monoclonal antibodies reactive with the surface of human breast carcinoma cells have been generated and characterized. The immunogens used were membrane-enriched fractions of metastatic carcinoma lesions. The various monoclonals were shown to react with previously known as well as with novel tumor-associated antigens (TAAs). The most specific of the latter group is monoclonal B72.3, which is reactive with a 220,000 to 400,000 high-molecular-weight glycoprotein complex found in 50% of human mammary carcinomas and 80% of human colon carcinomas. Monoclonal antibody B6.2, which recognizes a 90,000-d glycoprotein, was radiolabeled and shown to efficiently localize human carcinoma transplants in athymic mice via gamma imaging without the use of second antibody or background subtraction manipulations. F(ab')2 and Fab' fragments were shown to be more efficient for tumor localization than intact immunoglobulin. Whereas the phenomenon of antigenic heterogeneity of tumor cell populations has long been known to exist, this phenomenon was also shown to manifest itself as antigenic modulation, in which specific TAAs can modulate their expression on the cell surface concurrent with different phases of the cell cycle. A phenomenon known as antigen evolution, in which a specific cloned tumor cell population can gradually drift in antigenic phenotype, has also been demonstrated. Recombinant interferon has been employed to (1) enhance the expression of specific TAAs on the surface of tumor cells already expressing the antigen; and (2) induce the expression of specific TAAs on the surface of carcinoma cells not previously expressing the antigen. The clinical implications of such phenomena in gamma scanning for the detection of tumor masses and for tumor immunotherapy are discussed. Methods for circumvention of problems inherent in the clinical use of monoclonal antibodies are also addressed.

Murine monoclonal antibodies, prepared against human metastatic mammary tumor cells, were used to demonstrate differential expression of several tumor-associated antigens (TAAs) among various mammary carcinomas and within a given tumor mass. Using the immunoperoxidase technique on serial sections of 39 human primary mammary carcinomas, a spectrum of antigenic phenotypes of TAAs was observed: 13% of the tumors reacted with all of a panel of four monoclonal antibodies; while 10% of the mammary tumors scored negative with all four antibodies. The remaining 30 tumors could be divided into several additional groups based on their differential reactivity with some, but not all, of the monoclonal antibodies. Furthermore, variation among mammary carcinomas was also observed in the cellular localization of antigens. Antigenic phenotypic diversity of mammary tumor cell populations within a given tumor mass was also observed; this was noted with respect to (a) antigenic expression in one area of a tumor mass and not another and (b) a "patchwork" effect in which antigens were expressed on cells immediately adjacent to cells which scored negative. Antigenic phenotypic diversity was also observed in established mammary tumor cell lines grown in vitro. A differential loss of some cell surface TAAs was observed as a function of continued cell passage; consistent with this finding, MCF-7 mammary tumor cell lines obtained from four sources could be differentiated from each other by their pattern of cell surface TAA expression. Single-cell clones derived from the MCF-7 mammary tumor cell line exhibited at least four distinct antigenic phenotypes; a change in cell surface phenotype of some of the clones was seen during subsequent passage. This definition of phenotypic variation and modulation of TAA expression among, and within, human mammary carcinomas has implications towards both the design and the outcome of studies involving the in situ immunodetection and therapy of breast cancer.

Treatment of human breast or colon carcinoma cells with recombinantly derived human leukocyte (clone A) interferon (IFN-alpha A) increases the surface expression of specific tumor-associated antigens (TAAs) recognized by monoclonal antibodies (MAbs). The MAbs used, B1.1, B6.2, and B72.3, recognize three distinct TAAs, i.e., the Mr 180,000 carcinoembryonic antigen, a Mr 90,000, and a Mr 220,000 to 400,000 glycoprotein, respectively. The binding of the MAbs to the surface of tumor cells increased in a dose-dependent manner, with optimal levels of TAA enhancement at 100 to 1,000 units IFN-alpha A/ml. Higher concentrations of IFN-alpha A that were cytostatic or cytotoxic were also less effective in enhancing TAA expression. Human melanoma (A375) cells and normal fibroblasts (WI-38 and Flow 4000) do not express any of the three TAAs, either before or after interferon treatment. The ability of IFN-alpha A to increase the expression of TAAs on human carcinoma cells was also temporally dependent, with optimal enhancement occurring after 16 to 24 hr. The enhancement of specific TAAs at the surface of the carcinoma cells by IFN-alpha A was confirmed, using fluorescence-activated cell sorter analysis. These data demonstrate that the IFN-alpha A-mediated increase of surface antigen is a result of both an accumulation of more antigen per cell, and an increase in the percentage of cells expressing the antigen. The ability of recombinant interferon to enhance specific TAAs on human carcinoma cells may be exploited in designing protocols for the in situ detection and therapy of human carcinoma lesions by MAbs, as well as in further defining the role of specific TAAs in the expression of the transformed phenotype.