Functional properties of FC-2.15, a monoclonal antibody that mediates human complement cytotoxicity against breast cancer cells

Abstract

FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A 51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1-50 micrograms/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8 +/- 2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15-. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with 125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9 x 10(7) M-1 and 2.8 x 10(6) antigenic sites/cell. 125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells.

Selection and characterization of HER2/neu-binding affibody ligands

“…Upon interaction with cell-specific markers, antibodies can show direct cytotoxic effects by several mechanisms of action, e.g. blocking of growth factor receptors (Baselga and Mendelsohn, 1994), induction of apoptosis (Trauth et al, 1989), and formation of anti-idiotypic antibodies (Fagerberg et al, 1994), or indirect effects, such as antibody-dependent cellular cytotoxicity (ADCC) (Steplewski et al, 1983) and complement-mediated cytotoxicity (Ballare et al, 1995). As the cytotoxic actions induced by antibodies often are insufficient, they have been coupled to various toxic substances, including radioactive isotopes (Wilder et al, 1996), toxins (Kreitman, 1999) and chemotherapeutic drugs (Garnett et al, 1985), to enhance their potency.…”

Monoclonal Antibodies Against Tumour-Associated Carbohydrate Antigens

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Inflammatory Changes after Cryosurgery-Induced Necrosis in Human Melanoma Xenografted in Nude Mice

“…The immuno¯uorescence assay was performed as described (Ballare  et al, 1995). Brie¯y, 4% formalin-®xed cells were blocked with 10% normal goat serum.…”