D. Wunderlich

Monoclonal Antibodies Reactive With Breast Tumor-Associated Antigens

Publisher Summary Monoclonal antibodies (MAbs) led to the identification of novel tumor-associated antigens (TAAs) from various human carcinomas, melanomas, leukemias, and lymphomas. There are approximately two dozen characterized monoclonals reactive with human breast tumors. Several of these are chosen to elaborate those phenomena that are most relevant for the use of MAbs in the management of human breast cancer as well as in the study of the biology of human mammary carcinoma cell populations. This chapter describes numerous MAbs that are reactive with human mammary carcinomas. They can be classified into four groups based on the immunogen used to generate the MAb. These include using breast tumor cell lines, milk fat globule membrane, and membrane-enriched extracts of breast carcinoma metastases as immunogen, and lymph nodes from mastectomy patients. Each of the MAbs, including those prepared by several different groups to milk fat globule membrane, appears to be unique with respect to percentage of reactive mammary tumors, percentage of reactive cells within tumors, location of reactive antigen within the tumor cell, or degree of reactivity with nonmammary tumors as well as normal tissues.

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.

[89] Radioimmunoassay for detection of changes in cell surface tumor antigen expression induced by interferon

Publisher Summary This chapter describes radioimmunoassay for the detection of changes in cell surface tumor antigen expression induced by interferon. The interferons are multifaceted biological modifiers that exert antiviral, antiproliferative, and several different immunomodulatory actions. Their ability to increase the amount of tumor antigens on the surface of human carcinoma cells would have potential utility in enhancing the in vivo detection and/or therapy of tumors with monoclonal antibodies in clinical trials. These antigens are recognized by a panel of monoclonal antibodies that are generated in laboratory with membrane-enriched fractions from liver metastases from patients with primary breast carcinoma. Partially purified preparations of human interferon have been shown to increase the amount of HLA-A,B,C and β 2 -microglobulin on the surface of several different human cell types. A radioimmunoassay with live cells is used to determine whether human recombinant leukocyte (alpha) A interferon (IFN-αA) could increase the presentation of tumor antigens on the surface of human breast and colon carcinomas as detected by monoclonal antibodies. The results suggest that recombinant human leukocyte interferon can increase the expression of tumor antigens on the surface of human breast and colon carcinoma cells.

Generation of Monoclonal Antibodies Reactive with Human Colon Carcinomas

Monoclonal antibodies have been generated that are reactive with human colon carcinomas. The rationale for the studies was to utilize extracts from patient biopsy material (and not colon cancer cell lines) as immunogen to increase the probability that any monoclonal antibody generated be reactive with colon carcinomas in a clinical setting. Five immunization protocols were used employing extracts and membrane enriched fractions from both primary and metastatic colon carcinoma lesions. Twenty nine monoclonal antibodies from double-cloned hybridoma cultures have been characterized; all are of the IgG isotope. Preliminary results indicate that the monoclonal antibodies can be placed into 15 groups on the basis of their differential reactivities to six colon carcinoma extracts, the surface of three colon carcinoma cell lines and five partially purified CEA preparations from bloods of colon cancer patients. Some of the monoclonal antibodies were shown to bind from one to five of the five CEA preparations, while others showed no anti-CEA reactivity. None of the 29 monoclonal antibodies reacted with extracts of 21 normal human tissues including livers, spleens, kidneys, red blood cells, (of several blood groups), or polymorphonuclear leucocytes. All the 29 monoclonal antibodies could be distinguished, on the basis of differential reactivities to various tumors and normal human tissues, from several monoclonal antibodies (B1.1, B6.2, B72.3) reactive with colon carcinomas that have been previously generated in our laboratory. Further immunohistochemical and radiolocalization studies will further define the potential clinical utility of the monoclonals described.

Monoclonal Antibodies Generated to a Synthetic Peptide Define Ras Gene Expression at the Single Cell Level in Human Colon and Mammary Carcinomas

Ras oncogenes were first recognized as the transforming genes of two rat-derived viruses, the Harvey (Ha) and Kirsten (Ki) strains of murine sarcoma virus (MuSV) (1). Molecular cloning studies have identified Ha-ras, Ki-ras, and subsequently, N-ras, as members of a gene family present in a wide range of species including humans (2). At least two mechanisms have been identified by which ras activation can mediate transformation of NIH 3T3 cells: (a) a point mutation in the ras gene resulting in an alteration of a single amino acid in the 21,000 dalton (p21) ras gene product (3), or (b) increased expression of the normal cellular p21 ras gene product (1,4,5).

Biology and Immunology of Human Carcinoma Cell Populations

The rationale for the studies overviewed here was to utilize membrane-enriched extracts of human metastatic mammary tumor cells as immunogens in an attempt to generate and characterize monoclonal antibodies (MAbs) reactive with determinants that would be maintained on primary as well as metastatic human mammary carcinoma cells. Multiple assays have been employed to reveal the range of reactivities and diversity of the various antibodies.

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.