Paul A.W. Edwards

Monoclonal antibodies to the human mammary gland

A range of primary and metastatic human breast carcinomas has been examined with respect to the staining by four monoclonal antibodies which were raised to the human milk fat globule membrane. Within the normal breast the luminal epithelial cells expressing the antigens detected by the monoclonal antibodies were heterogeneous in their distribution. The heterogeneity was not only confined to single cells, but also to regions within the breast. The breast carcinomas also expressed the antigens in a variable manner. Morphological differentiation and functional differentiation, defined by the monoclonal antibodies, were not invariably coincident. Lymph node metastases gave similar results to the primary carcinomas. The monoclonal antibodies have revealed a heterogeneity, with respect to surface antigenic expression, within the normal and neoplastic breast epithelium. This cellular heterogeneity of breast carcinomas, may have significant prognostic and therapeutic implications in the management of primary breast cancer.

Antigenic subsets of human breast epithelial cells distinguished by monoclonal antibodies

Three monoclonal antibodies raised to the human milk fat globule membrane bind, within the normal breast, to the surface of the luminal epithelial cells but not to the surrounding myoepithelial, connective tissue, or blood vessel cells. These antibodies distinguish three subsets of the epithelial cells that are not distinguishable by conventional histology. To show the arrangement of the cells in two dimensions over the sheet of epithelium, ducts were dissected out of normal breast tissue, opened up and laid flat as sheets of epithelium. The apical faces of the cells were strained, unfixed, using two-color immunofluorescence to contrast the subsets of cells stained by the different antibodies. The epithelium was then seen to be a mosaic of cells that express different surface antigens. The grouping and appearance of the cells stained by the different antibodies was characteristic. This may be just a random heterogeneity of antigen expression but alternatively the different cells may be in different physiological states. Regardless of its biological significance, the observation has practical consequences for the use of such antibodies in identifying cells and the study of antigenic heterogeneity in tumors.

Two mouse hybridoma antibodies against human milk-fat globules recognise the I(MA) antigenic determinant β-d-Galp-(1→4)-β-d-GlcpNAc-(1→6)

Abstract Two mouse hybridoma antibodies (LICR-LON-M39 and LICR-LON-M18) against the human-milk-fat globules were found to resemble human autoantibodies of anti-I type in their cold agglutinating property and their preferential reactions with erythrocytes of I- rather than i-type. From inhibition of binding assays with glycoproteins having known A, B, H, Lea, Leb, I, and i activities, and oligosaccharides of the Type 1 and Type 2 lacto-N-glycosyl series, it was established that these antibodies are directed at Type 2 structures, and that the I(Ma) determinant, β- d -Galp-(1→4)-β- d -GlcpNAc-(1→6), which is usually found on branched oligosaccharides, is the preferred sequence. The hybridoma antibodies as well as anti-I Ma were shown to react well with the β- d -Galp-(1→4)-β- d -GlcpNAc-(1→6)- d -Gal or - d -Man sequence. Studies of the reactions of these antibodies with glycolipids on thin-layer plates showed that the two hybridoma antibodies differ from anti-I Ma in reacting weakly with the unbranched i-type sequence β- d -Galp-(1→4)-β- d -GlcpNAc-(1→3)-β- d -Galp-(1→4)- β- d -GlcpNAc-(1→3)-β- d -Galp-(1→4) as found on lacto-N-norhexaosylceramide. Furthermore, they differ from anti-I Ma but resemble anti-I Woj and Sti, and a hybridoma antibody 1B2 in their failure to react with their determinant in the presence of α- d -(1→3)-linked galactosyl groups. From their lack of reactions with blood-group-A and -H active glycoproteins, and their reactions with neuraminidase-treated erythrocytes, it was deduced that the determinants recognised by the two hybridoma antibodies are also masked in the presence of α- l -(1→2)-linked focosyl groups and sialic acid.

Selective culture of epithelioid cells from a human squamous carcinoma using a monoclonal antibody to kill fibroblasts.

Monoclonal antibody LICR LON/FIB86 is cytotoxic for human fibroblasts from at least some sources, with rabbit complement. Explants of human squamous carcinomas of the head and neck were put into culture and fibroblasts and epithelioid cells began to grow out. The cultures were treated regularly with antibody plus complement: the fibroblasts were killed consistently, as they emerged, leaving sheets of epithelioid cells. We conclude that this is a practical method for controlling human fibroblast growth in culture.

A New Human Hybridoma System (Licr-Lon-HMγ2) and Its use in the Production of Human Monoclonal Antibodies

Publisher Summary This chapter discusses a new human hybridoma system (LICR-LON-HMy2) and its use in the production of human monoclonal antibodies. This system is a HAT-sensitive variant of ARH-77 that is capable of reproducibly giving rise to stable hybrids when fused with human lymphocytes from a variety of sources. A series of cloned human hybrids have been characterized to formally demonstrate that true hybrids are formed and that some produce new immunoglobulins. Success with hybridoma systems involves the reproducible production of stable, clonable cell-lines secreting useful quantities of monoclonal antibodies to specified antigens. The identity of the HAT-insensitive cells generated from HMy2 after polyethylene glycol-induced fusion with human lymphocytes have been examined using the criteria of morphology, karyology and DNA content, and Ig secretion patterns and HLA typing. The various HAT-sensitive myeloma/lymphoblastoid lines that are available could be fused with one another using this method, thus generating a whole series of new HAT-sensitive human lines with novel properties.

The selective culture of keratinocytes using a cytotoxic antifibroblast monoclonal antibody

A technique is described for the selective killing of fibroblasts in primary cultures and subcultures of human keratinocytes using a monoclonal antibody raised against human fibroblasts. The antibody kills fibroblasts by complement mediated cytotoxicity but is not toxic to keratinocytes.

Micrometastases in Breast Cancer

Publisher Summary This chapter discusses micrometastases in breast cancer. Single tumor cells are readily distinguished from normal marrow cells by the greater intensity of their epithelial membrane antigen staining combined with their morphological characteristics; namely a nuclear size at least that of a blast cells with a lower nucleo-cytoplasmic ratio. The value of this test in patients with primary breast cancer will remain uncertain until its prognostic significance is established. However, it does appear that the presence of these cells predicts the development of bone metastases in patients with recurrent disease. In addition, the percentage of patients with primary breast cancer found to have positive marrows corresponds with a similar percentage of patients known to have a poor prognosis by other means. The screening of these bone marrow aspirates is cumbersome and time-consuming and it may be that an automated system will be found to replace manual examination.

Generating human monoclonal antibodies

Present methods and systems for the generation of human monoclonal antibodies are briefly reviewed. The specificities of the available reagents are outlined. It would appear that the generation using hybridoma methods of human monoclonal antibodies to human tumor cell surface antigens is a rare event and that methods ofin vitro immunostimulation may have to be used if such antibodies are to be reliably produced.