Yoshio A. Teramoto

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. 13We 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 or 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.

The use of lymphocytes from axillary lymph nodes of mastectomy patients to generate human monoclonal antibodies

Abstract Lymphocytes from axillary lymph nodes of breast cancer mastectomy patients were fused with murine non-immunoglobulin-secreting myeloma cells to generate hybridoma cell lines that synthesize human immunoglobulins. Lymph node lymphocytes from 21 patients were used to obtain 505 human-mouse hybrid cultures. From these, 62 cultures were established which synthesized immunoglobulins reactive in radioimmunoassays specific for either human IgG or human IgM. Some of these double-cloned hybrid cell lines produced human monoclonal antibodies for at least 6 months . Sodium dodecylsulfate polyacrylamide gel electrophoresis and immunodiffusion analysis demonstrated that the monoclonal antibodies possessed human heavy and light immunoglobulin chains. Levels of synthesis ranged from 0.1 to 20 μg of human immunoglobulin per ml of culture fluid. The immunoreactivity of some of these human monoclonal antibodies with mammary carcinoma cells is summarized and has been documented elsewhere ( J. Schlom, D. Wunderlich and Y. A. Teramoto . Proc. Natl Acad Sci USA 1980;77: 6841 ); the reactivity of the majority of the immunoglobulins, however, is still unknown at this time. The studies reported here detail the procedures in which axillary lymph nodes from mastectomy patients are used in the generation of human-mouse hybridomas that synthesize human monoclonal antibodies.

Radioimmunoassays That Demonstrate Type-specific and Group-specific Antigenic Reactivities for the Major Internal Structural Protein of Murine Mammary Tumor Viruses

The 28,000-dalton (p28) major structural polypeptide of the mouse mammary tumor virus (MMTV) was isolated and used to develop a highly sensitive and specific radioimmunoassay. Under conditions of limiting antibody in competitive binding assays, as little as 50 pg of purified p28, as well as disrupted MMTV virions and mammary tumor extracts, competed specifically with 125I-labeled MMTV p28. The p28 polypeptide was further shown to contain both group-specific and type-specific antigenic determinants, thus also allowing for further differentiation of various MMTV strains.

Immunological and structural relationships between langur virus and other primate type-D retroviruses

Abstract The major structural proteins of three type-D retroviruses are compared using sodium dodecyl sulfate polyacrylamide slab and tube gel electrophoresis and competitive radioimmunoassays. We report here that the virion-associated polypeptides of the endogenous virus of the langur (LV) and the nongerm line-transmitted Mason-Pfizer monkey virus (MPMV) of the rhesus have indistinguishable electrophoretic mobilities in polyacrylamide gels. Homologous radioimmunoassays (RIAs) for the major 27,000-dalton structural protein, (p27) of LV and for the p27 of MPMV also cannot distinguish these two viruses. LV and MPMV can be differentiated, however, by using either of the RlAs developed for their 10,000- to 12,000-dalton proteins. The major structural protein of the type-D retrovirus of New World squirrel monkeys (SMRV) is more immunoreactive with the endogenous virus of the Old World langur monkey than it is with MPMV. An interspecies RIA has also been developed using antiserum to LV to precipitate 125 I-labeled MPMV p27. This assay is more effective than either of the homologous LV or MPMV RIAs for the recognition of SMRV. These studies further define the structural and immunologic relationships among the type-D retroviruses of primates.

Isolation and characterization of a new mouse mammary tumor virus from BALB/c mice.

Abstract A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of 1%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RIII mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,000-dalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/c) from MMTV(C3H), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL), and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(O). Analysis of Eco RI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease Sac I distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RIII MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.

Noncoordinate expression of murine mammary tumor virus gene products

Abstract The expression of mouse mammary tumor virus (MMTV) gene products was examined in several different murine mammary tissues which showed no production of complete extracellular MMTV B-particles. Analyses by specific competition radioimmunoassay demonstrated that these mammary tissues contained large amounts of protein immunologically related to, or identical to, the 28,000-dalton major nonglycosylated MMTV core protein (p28), but no immunologically detectable 52,000-dalton MMTV envelope glycoprotein (gp52). Such noncoordinate MMTV genome expression was found in primary and transplanted mammary tumors, and in preneoplastic mammary tissues of BALB/c mice, as well as in normal mammary tissue of Swiss albino mice. These mammary tissues exemplify a naturally occurring animal model system for studying noncoordinate expression of MMTV gene products, and its possible significance for MMTV-mediated oncogenesis.

Interspecies radioimmunoassay for the major internal protein of mammary tumor viruses.

Abstract An interspecies radioimmunoassay has been developed which detects antigenic determinants shared by type-B mammary tumor viruses (MTVs). This interspecies assay is specific for antigenic sites which the 28,000-dalton major internal protein of MMTVs of laboratory mice (Mus musculus) has in common with polypeptides of MC-MTV. MC-MTV is a new type-B retrovirus isolated from the Asian rodent, Mus cervicolor ( J. Schlom, P. H. Hand, Y. A. Teramoto, R. Callahan, G. Todaro, and G. Schidlovsky, 1978 , J. Nat. Cancer Inst. 61, 1509–1519). Other retrovirus isolates of Mus cervicolor, i.e., M432, CERV-CI, and CERV-CII, as well as other type-C and type-D retroviruses, do not compete in the interspecies assay. The interspecies assay detected MTV cross-reactive antigenic determinants with equal efficiency in milks, lactating mammary glands, and in spontaneous mammary tumors of three distinct species. Particles morphologically indistinguishable from MMTV and MC-MTV have also been detected in Mus cookii mammary tumor cells. The interspecies MTV p28 radioimmunoassay thus provides a potentially useful tool for the detection of etiologically related viruses or viral translational products in species other than the laboratory mouse.

GENERATION, CHARACTERIZATION, AND UTILIZATION OF MONOCLONAL ANTIBODIES REACTIVE WITH HUMAN MAMMARY CARCINOMA ASSOCIATED ANTIGENS

Publisher Summary This chapter describes an experiment in which splenic lymphocytes of mice, immunized with membrane-enriched fractions of human metastatic mammary carcinoma cells, were fused with murine non- immunog1obulin secretor myeloma cells. Following initial screening and double cloning of hybridoma cultures, 11 monoclonal antibodies were further characterized. These monoclonals could be placed into five major groups based on their differential reactivities with extracts of breast tumor metastases, the surface of live mammary tumor cells in culture, and immunoper-oxidase staining of tissue sections of primary and metastatic mammary tumors. None of the antibodies bound to the surface of 14 human cell lines were derived from a variety of normal tissues, including normal mammary cells. Surface binding to mammary tumor cells by two of the monoclonal antibodies was shown to decrease during density dependent arrest, and further cell-cycle analysis demonstrated differential antibody surface binding at S phase. Prolonged exposure of mammary tumor cells to antibody showed no evidence of antigen capping or internalization.

Viruses and Mammary Carcinoma

Viruses have long been known to be involved in the etiology of a variety of neoplasms of animals ranging from fowl to nonhuman primates. This involvement has been demonstrated by the isolation of these agents from spontaneous tumors, their inoculation into animals of the same species, and the reisolation of the particular virus from the subsequent tumor that developed.