Biochemical and immunological characterizations of antigens recognised by human monoclonal antibodies

Abstract

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.